Professional Documents
Culture Documents
Microbiology
www.ebook3000.com
Essentials of
Microbiology
Surinder Kumar
MD DNB MNAMS
Director Professor
Department of Microbiology
Maulana Azad Medical College
New Delhi, India
www.ebook3000.com
Jaypee Brothers Medical Publishers (P) Ltd
Headquarters
Jaypee Brothers Medical Publishers (P) Ltd
4838/24, Ansari Road, Daryaganj
New Delhi 110 002, India
Phone: +91-11-43574357
Fax: +91-11-43574314
Email: jaypee@jaypeebrothers.com
Overseas Offices
J.P. Medical Ltd Jaypee-Highlights Medical Publishers Inc Jaypee Medical Inc
83, Victoria Street, London City of Knowledge, Bld. 237, Clayton The Bourse
SW1H 0HW (UK) Panama City, Panama 111 South Independence Mall East
Phone: +44-2031708910 Phone: +1 507-301-0496 Suite 835, Philadelphia, PA 19106, USA
Fax: +44(0)20 3008 6180 Fax: +1 507-301-0499 Phone: +1 267-519-9789
Email: info@jpmedpub.com Email: cservice@jphmedical.com Email: jpmed.us@gmail.com
Jaypee Brothers Medical Publishers (P) Ltd Jaypee Brothers Medical Publishers (P) Ltd
17/1-B Babar Road, Block-B, Shaymali Bhotahity, Kathmandu
Mohammadpur, Dhaka-1207 Nepal
Bangladesh Phone: +977-9741283608
Mobile: +08801912003485 Email: kathmandu@jaypeebrothers.com
Email: jaypeedhaka@gmail.com
Website: www.jaypeebrothers.com
Website: www.jaypeedigital.com
The views and opinions expressed in this book are solely those of the original contributor(s)/author(s) and do not necessarily represent those
of editor(s) of the book.
All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical,
photocopying, recording or otherwise, without the prior permission in writing of the publishers.
All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective
owners. The publisher is not associated with any product or vendor mentioned in this book.
Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject
matter in question. However, readers are advised to check the most current information available on procedures included and check
information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of
administration, adverse effects and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions.
Neither the publisher nor the author(s)/editor(s) assume any liability for any injury and/or damage to persons or property arising from or
related to use of material in this book.
This book is sold on the understanding that the publisher is not engaged in providing professional medical services. If such advice or services
are required, the services of a competent medical professional should be sought.
Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce copyright material. If any have
been inadvertently overlooked, the publisher will be pleased to make the necessary arrangements at the first opportunity.
Essentials of Microbiology
First Edition: 2016
ISBN 978-93-5152-380-2
Printed at
Dedicated to
My father
Late Shri Lachhman Das
My mother
Late Smt Bal Kaur
My wife
Dr (Prof) Savita Kumari
and my sons
Dr Sourabh Kumar and Sanchit Kumar
whose love and affection make everything I do possible.
www.ebook3000.com
Preface
Microbiology is an extremely diverse discipline and can be a bewildering field to the novice. The
traditional books, seem to be exhaustive of microbiologic facts, are too voluminous and detailed to be
read by a typical medical student, who is trying to keep up with simultaneously several classes of other
subjects. One of the ways to fulfill the goals of the book is to concentrate on understanding concepts—
important fundamental ideas or themes that form a framework of the subject. Essentials of Microbiology
is the result of the author’s 28 years of experience in teaching medical students. The book is readable and
complete enough to meet the students’ needs. The overall objective of this book is to provide students
the basics of medical microbiology as well as a complete coverage of the subject. It contains all of the
information that is pertinent to the medical students who are studying microbiology keeping in mind
their examination.
Although the text has been designed to teach undergraduate and postgraguate medical students, it
would also serve as a review tool for the individuals who are taking medical examinations and persons
working in health-related professions, physicians, and infectious disease scientists.
Microbiology has expanded beyond recognition with various medical specialty, and it is not possible
for any one book to cover all aspects of medical microbiology in depth. This book is divided into
the following seven sections, based on the major disciplines included within microbiology: General
Bacteriology, Immunology, Systemic Bacteriology, Virology, Medical Mycology, Miscellaneous, and
Diagnostic Medical Microbiology. The chapters themselves are comprehensive yet free of unnecessary
detail and provide the reader with a framework for understanding. Mycology and parasitology have
continued to flourish and have blossomed into fields of study of their own rights. Therefore, parasitology
has not been included in this book which has a sturdy independence.
I would be thankful for any comments or suggestions from students, teachers, and all the readers of
this book for further improvements in its future editions.
Surinder Kumar
www.ebook3000.com
Acknowledgments
This book took years in writing but a lifetime in preparation. I would like to thank those whose example,
teaching, and prodding helped me develop a thirst for knowledge as well as methods for quenching
that thirst. I am greatly indebted to a number of my mentors, friends and colleagues who encouraged
me and gave valuable suggestions for improving the text. I am always indebted to my late brother Sant
Swaran Dev whose advice, guidance and true life philosophy gave me strength and courage to continue
my work in all odds. I would like to thank my family for patiently enduring the writing of this book,
which seemed at times to be an endless process. I am especially grateful to my wife Dr Savita Kumari,
Professor, Department of Internal Medicine, Post Graduate Institute and Medical Education and Research
(PGIMER), Chandigarh, for her support and encouragement and to my two sons, Dr Sourabh Kumar
and Mr Sanchit Kumar (medical student) who gave me their valuable moments without any complaint
for completing this book, so I could retain my sanity.
My special thanks is to Dr Sanjeev R Saigal, my PhD student and now my Research Associate Indian
Council of Medical Research (ICMR) for his constant help for this book. Sincere appreciation also goes
to all staff of M/s Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, for their guidance and support
in this project.
www.ebook3000.com
Contents
www.ebook3000.com
xii | Essentials of Microbiology
∙∙ Classification 56
∙∙ Classification Systems 56
10. Bacterial Genetics 58
∙∙ Structure and Functions of the Genetic Material 58
∙∙ Extrachromosomal Genetic Elements 60
∙∙ Genotypic and Phenotypic Variations 61
∙∙ Transmission of Genetic Material (Gene Transfer) 63
∙∙ Genetic Mechanisms of Drug Resistance in Bacteria 68
∙∙ Transposable Genetic Elements 68
∙∙ Molecular Genetics 68
∙∙ Genetic Probes 70
∙∙ Blotting Techniques 71
∙∙ Polymerase Chain Reaction (PCR) 71
∙∙ Gene Therapy 72
11. Infection 75
∙∙ Microorganisms and Host 75
∙∙ Infection and Infectious Disease 75
∙∙ Classification of Infections 75
∙∙ Sources of Infection 76
∙∙ Modes of Transmission of Infection 77
∙∙ Factors Predisposing to Microbial Pathogenicity 78
∙∙ Determinants of Virulence 78
∙∙ Types of Infectious Diseases 80
∙∙ Epidemiological Terminologies 80
Section 2: Immunology
12. Immunity 81
∙∙ Definition 81
∙∙ Classification 81
∙∙ Mechanisms of Innate Immunity 82
∙∙ Local Immunity 86
∙∙ Herd Immunity 86
13. Antigens 87
∙∙ Types of Antigens 87
∙∙ Antigenic Determinant or Epitome 87
∙∙ Determinants of Antigenicity 87
∙∙ Superantigens 89
14. Antibodies—Immunoglobulins 90
∙∙ Antibody Structure 90
∙∙ Immunoglobulin Classes 92
15. Complement System 96
∙∙ Principle Pathways of Complement Activation 96
∙∙ Quantitation of Complement (C) and its Components 99
∙∙ Biosynthesis of Complement 99
∙∙ Complement Deficiencies 99
16. Antigen–Antibody Reactions 101
∙∙ Antigen–Antibody Interactions 101
∙∙ General Characteristics of Antigen–Antibody Reactions 102
∙∙ Antigen and Antibody Measurement 102
∙∙ Parameters of Serological Tests 102
∙∙ Serological Reactions 102
∙∙ Types of Antigen and Antibody Reactions 102
∙∙ Applications of Agglutination Reaction 106
∙∙ ELISA 112
17. Structures and Functions of the Immune System 117
∙∙ Types of Immune Response 117
∙∙ Organs and Tissues of the Immune System 117
∙∙ Cells of the Lymphoreticular System 119
∙∙ Major Histocompatibility Complex 122
∙∙ Mhc Restriction 123
Contents | xiii
18. Immune Response 125
∙∙ Type of Immune Response 125
∙∙ Humoral Immunity 125
∙∙ Fate of Antigen in Tissues 126
∙∙ Production of Antibodies 127
∙∙ Cell-mediated Immune Responses 130
∙∙ Cytokines 131
∙∙ Immunological Tolerance 134
∙∙ Theories of Antibody formation 135
19. Immunodeficiency Diseases 137
∙∙ Classification of Immunodeficiency Diseases 137
∙∙ Primary Immunodeficiencies 137
∙∙ Disorders of Specific Immunity 137
∙∙ Disorders of Complement 140
∙∙ Disorders of Phagocyte 140
∙∙ Secondary Immunodeficiencies 140
20. Hypersensitivity Reactions 142
∙∙ Classification of Hypersensitivity Reactions 142
∙∙ Type I Hypersensitivity (IgE Dependent) 143
∙∙ Type II Hypersensitivity: Cytolytic and Cytotoxic 146
∙∙ Type III Hypersensitivity: Immune Complex-mediated 147
∙∙ Type IV Hypersensitivity—Delayed Hypersensitivity 148
∙∙ Type V: Hypersensitivity (Stimulatory Type) Jones–Mote Reaction or
Cutaneous Basophil Hypersensitivity 149
∙∙ Schwartzman Reaction 149
21. Autoimmunity 151
∙∙ Mechanisms of Autoimmunity 151
∙∙ Classification of Autoimmune Diseases 152
22. Immunology of Transplantation and Malignancy 154
∙∙ Definition 154
∙∙ Types of Transplants 154
∙∙ Allograft Reaction 154
∙∙ Histocompatibility Testing 155
∙∙ Fetus as an Allograft 156
∙∙ Graft-versus-host Reaction 156
∙∙ Immunology of Malignancy 156
∙∙ Tumor Antigens 156
∙∙ Immune Response in Malignancy 157
∙∙ Immunological Surveillance 157
∙∙ Immunotherapy of Cancer 157
23. Immunohematology 159
∙∙ Other Blood Group Systems 160
∙∙ Medical Applications of Blood Groups 160
∙∙ Complications of Blood Transfusion 160
∙∙ Hemolytic Disease of the Newborn 160
www.ebook3000.com
xiv | Essentials of Microbiology
27. Neisseria and Moraxella 188
∙∙ Neisseria meningitidis (Meningococcus; Diplococcus intracellularis meningitidis) 188
∙∙ Neisseria gonorrhoeae (Gonococcus) 191
∙∙ Nongonococcal (Nonspecific) Urethritis 193
∙∙ Commensal Neisseriae 194
∙∙ Moraxella 194
∙∙ Kingella 195
28. Corynebacterium 197
∙∙ Corynebacterium diphtheriae 197
∙∙ Other Medically Important Corynebacteria 202
∙∙ Diphtheroids 203
∙∙ Other Coryneform Genera 203
29. Bacillus 205
∙∙ General Characterstics of Bacillus 205
∙∙ Bacillus anthracis 205
∙∙ Anthracoid Bacilli 208
30. Clostridium 211
∙∙ General Features of Clostridia 211
∙∙ Classification 212
∙∙ Clostridium tetani 215
∙∙ Clostridium botulinum 218
∙∙ Clostridium difficile 219
31. Nonsporing Anaerobes 221
∙∙ Anaerobic Cocci 221
∙∙ Gram-negative Anaerobic Cocci 222
∙∙ Anaerobic, Nonspore-forming, Gram-positive Bacilli 222
∙∙ Anaerobic Gram-negative Bacilli 223
∙∙ Anaerobic Infections 224
32. Mycobacterium tuberculosis 227
∙∙ M. tuberculosis Complex or MTC 227
∙∙ Mycobacterium tuberculosis 228
33. Mycobacterium leprae 240
∙∙ Mycobacterium leprae 240
∙∙ Mycobacterium lepraemurium 245
34. Nontuberculous Mycobacteria 247
∙∙ Properties of Nontuberculous Mycobacteria 247
∙∙ Classification 248
∙∙ Saprophytic Mycobacteria 249
35. Actinomycetes 252
∙∙ Actinomyces 252
∙∙ Nocardia 253
∙∙ Actinomycotic Mycetoma 254
36. Enterobacteriaceae: Escherichia, Klebsiella, Proteus and Other Genera 256
∙∙ Characteristics of the Family Enterobacteriaceae 256
∙∙ Classification of Enterobacteriaceae 256
∙∙ Classification of Enterobacteriaceae by Tribes 257
∙∙ Escherichia coli 257
∙∙ Infections 263
∙∙ Edwardsiella 263
∙∙ Citrobacter 263
∙∙ Klebsiella 264
∙∙ Enterobacter 265
∙∙ Hafnia 265
∙∙ Serratia 265
37. Tribe Proteeae: Proteus, Morganella and Providencia 267
∙∙ Proteus 267
∙∙ Morganella 269
∙∙ Providencia 269
∙∙ Erwinia 269
38. Shigella 270
Contents | xv
39. Enterobacteriaceae III: Salmonella 274
∙∙ Salmonella 274
∙∙ Diagnosis of Carriers 283
∙∙ Prophylaxis 283
∙∙ Drug Resistance 284
∙∙ Salmonella Gastroenteritis 284
∙∙ Salmonella Septicemia 285
∙∙ Multiresistant Salmonellae 285
40. Vibrio, Aeromonas and Plesiomonas 287
∙∙ Vibrio 287
∙∙ Vibrio cholerae 287
∙∙ Resistance 288
∙∙ Halophilic Vibrios 294
∙∙ Aeromonas 295
∙∙ Plesiomonas 295
41. Campylobacter and Helicobacter 297
∙∙ Campylobacter 297
∙∙ Helicobacter 298
42. Pseudomonas, Stenotrophomonas, Burkholderia 301
∙∙ Pseudomonas aeruginosa 301
∙∙ Burkholderia cepacia (Formerly Pseudomonas cepacia) 304
∙∙ Burkholderia mallei (Formely Pseudomonas mallei) 304
∙∙ Burkholderia pseudomallei 305
43. Legionella 307
∙∙ Legionella pneumophila 307
44. Yersinia, Pasteurella, Francisella 309
∙∙ Yersinia pestis (Formerly Pasteurella pestis) 309
∙∙ Yersiniosis 313
∙∙ Pasteurella multocida (Formerly Pasteurella septica) 314
∙∙ Francisella tularensis (Pasteurella tularensis, Brucella tularensis) 315
45. Haemophilus 317
∙∙ Haemophilus Influenzae 317
∙∙ Haemophili Other Than H. influenzae 320
46. Bordetella 323
∙∙ Bordetella pertussis 323
∙∙ Bordetella parapertussis 326
∙∙ Bordetella bronchiseptica (Bord. bronchicanis) 326
47. Brucella 327
48. Spirochetes 333
∙∙ Classification 327
∙∙ Treponema 334
∙∙ Nonvenereal Treponematoses 341
∙∙ Nonpathogenic Treponemes 341
∙∙ Borrelia 342
∙∙ Leptospira 344
49. Mycoplasma and Ureaplasma 348
∙∙ Classification 348
∙∙ Morphology 349
∙∙ Cultural Characteristics 349
∙∙ Pathogenicity 350
∙∙ Mycoplasma pneumoniae 350
∙∙ Laboratory Diagnosis 351
∙∙ Mycoplasmas and L Forms of Bacteria 352
∙∙ Ureaplasma urealyticum 353
50. Miscellaneous Bacteria 355
∙∙ Listeria monocytogenes 355
∙∙ Erysipelothrix rhusiopathiae 356
∙∙ Alcaligenes faecalis 357
∙∙ Chromobacterium violaceum 357
www.ebook3000.com
xvi | Essentials of Microbiology
∙∙ Flavobacterium meningosepticum 357
∙∙ Donovania granulomatis (Calymmatobacterium granulomatis) or Klebsiella granulomatis 357
∙∙ Acinetobacter (Mima polymorpha; Bacterium anitratum 358
∙∙ Rat-bite fever (Streptobacillus moniliformis and Spirillum minus) 358
∙∙ Spirillum minus 359
∙∙ Eikenella corrodens 359
∙∙ Cardiobacterium hominis 360
∙∙ Capnocytophaga 360
∙∙ Gardnerella vaginalis 360
51. Rickettsiaceae, Bartonellaceae and Coxiella 362
∙∙ Genus Rickettsia 362
∙∙ Ehrlichia, Anaplasma and Neorickettsia 366
∙∙ Genus Coxiella: Q Fever 367
∙∙ Bartonella 368
52. Chlamydia and Chlamydophila 371
∙∙ Classification 371
Section 4: Virology
53. General Properties of Viruses 378
∙∙ Main Properties of Viruses 378
∙∙ Morphology of Viruses 378
∙∙ Structure and Chemical Composition of the Viruses 379
∙∙ Susceptibility to Physical and Chemical Agents 380
∙∙ Viral Hemagglutination 381
∙∙ Viral Replication 381
∙∙ Eclipse Phase 383
∙∙ Abnormal Replicative Cycles 383
∙∙ Cultivation of Viruses 383
∙∙ Detection of Virus Growth in Cell Culture 385
∙∙ Viral Assay 386
∙∙ Viral Genetics 387
∙∙ Classification of Viruses 388
∙∙ Viroids 390
∙∙ Prions 390
54. Virus–Host Interactions: Viral Infections 392
∙∙ Interactions between Viruses and Host Cells 392
∙∙ Pathogenesis of Viral Diseases 393
∙∙ Transmission of Human Virus Infections 393
∙∙ Spread of Virus in the Body 394
∙∙ Significance of the Incubation Period 394
∙∙ Host Response to Virus Infections 395
55. Laboratory Diagnosis, Prophylaxis and Chemotherapy of Viral Diseases 397
∙∙ Laboratory Diagnosis of Viral Infections 397
∙∙ Immunoprophylaxis of Viral Diseases 399
∙∙ Chemoprophylaxis and Chemotherapy of Virus Diseases 400
56. Bacteriophages 402
∙∙ Role of Bacteriophages 402
∙∙ Morphology 402
∙∙ Life Cycle 402
∙∙ Significance of Phages 404
57. Poxviruses 406
∙∙ Morphology 406
∙∙ Physical and Chemical Properties 407
∙∙ Other Poxvirus Diseases 408
58. Herpesviruses 409
∙∙ Structure 409
∙∙ Classification 409
∙∙ Herpes Virus Simiae: B Virus 411
∙∙ Varicella-Zoster Virus 411
Contents | xvii
∙∙ Herpes Zoster (Shingles, Zona) 412
∙∙ Cytomegalovirus 413
∙∙ Epstein–Barr Virus 413
∙∙ Human Herpesviruses 6 (Hhv6) 415
∙∙ Human Herpesvirus 7 (Hhv7) 415
∙∙ Human Herpesvirus 8 (Hhv8) 416
59. Adenoviruses 418
∙∙ Morphology 418
∙∙ Resistance 418
∙∙ Pathogenesis 418
∙∙ Laboratory Diagnosis 419
∙∙ Treatment, Prevention and Control 420
60. Papovaviruses 421
∙∙ Papillomaviruses 421
∙∙ Polyomaviruses 422
61. Parvoviruses 424
∙∙ Parvovirus 424
∙∙ Dependovirus 424
∙∙ Erythrovirus 424
62. Picornaviruses 426
∙∙ Important Properties of Picornaviruses 426
∙∙ Enteroviruses 426
∙∙ Poliovirus 427
∙∙ Coxsackievirus 429
∙∙ Echoviruses 430
∙∙ Other Enterovirus Types 431
∙∙ Rhinoviruses 432
63. Orthomyxovirus 434
∙∙ Influenza Viruses 434
64. Paramyxoviruses 440
∙∙ Morphology and Structural Proteins of Paramyxoviruses 440
∙∙ Classification 440
∙∙ Parainfluenza Viruses 441
∙∙ Genus Rubulavirus 442
∙∙ Genus Morbillivirus 443
∙∙ Nipah and Hendra Viruses 444
∙∙ Genus Pneumovirus 444
∙∙ Metapnemovirus 445
65. Arboviruses 446
∙∙ Classification 446
∙∙ Properties 446
∙∙ Laboratory Diagnosis 447
∙∙ Pathogenesis 447
∙∙ Families of Arboviruses 447
∙∙ Ungrouped Arboviruses 455
∙∙ Arbovirus Known to Be Prevalent in India 455
66. Rhabdoviruses 457
∙∙ Rabies Virus 457
∙∙ Rabies-related Viruses 463
67. Hepatitis Viruses 465
∙∙ Hepatitis A Virus (Infectious Hepatitis) 465
∙∙ Hepatitis B Virus—Serum Hepatitis 467
∙∙ Hepatitis C Virus (HCV) 471
∙∙ Hepatitis D Virus (HDV) (Delta Agent) 472
∙∙ Hepatitis E Virus (HEV) 473
∙∙ Hepatitis G Virus 474
68. Retroviruses: Human Immunodeficiency Virus (HIV) 476
∙∙ Retroviruses 476
∙∙ Human Immunodeficiency Virus 476
www.ebook3000.com
xviii | Essentials of Microbiology
69. Slow Virus and Prion Diseases 488
∙∙ Characteristics of Slow Viruses 488
∙∙ Classification 488
70. Miscellaneous Viruses 492
∙∙ Rubivirus 492
∙∙ Rubella (German Measles) 492
∙∙ Viral Hemorrhagic Fevers 493
∙∙ Arenaviruses 493
∙∙ Filoviruses 494
∙∙ Coronaviruses 494
∙∙ Reoviridae 496
∙∙ Norwalk Virus 497
71. Oncogenic Viruses 499
∙∙ Properties of Cells Transformed by Viruses 499
∙∙ Types of Tumor Viruses 499
∙∙ Oncogenic Viruses 499
∙∙ Viruses Associated with Human Cancer 501
∙∙ Oncogenes 501
∙∙ Antioncogenes 502
∙∙ Mechanisms of Viral Oncogenesis 502
Section 6: Miscellaneous
77. Normal Microbial Flora of the Human Body 530
∙∙ Role of Normal Microbial Flora 530
78. Infective Syndrome 533
∙∙ Bacteremia, Septicemia and Infective Endocarditis 533
∙∙ Bacteremia and Septicemia 533
∙∙ Meningitis 535
∙∙ Purulent Meningitis (Acute Pyogenic Meningitis) 535
∙∙ Aseptic Meningitis 537
∙∙ Tuberculous Meningitis 538
Contents | xix
∙∙ Urinary Tract Infection 538
∙∙ Types of UTI 538
∙∙ Sore Throat 541
∙∙ Pneumonia 542
∙∙ Diarrhea and Dysentery 544
∙∙ Diarrhea 544
∙∙ Dysentery 546
∙∙ Food Poisoning 547
∙∙ Sexually Transmitted Diseases (STDs) 547
∙∙ Wound Infection 550
∙∙ Pyrexia of Unknown Origin (PUO) 551
79. Hospital-acquired Infection 555
∙∙ Sources of Infections 555
∙∙ Factors Influencing Hospital-associated Infections 555
∙∙ Microorganisms Causing Hospital Infection 555
∙∙ Routes of Transmission 556
∙∙ Common Hospital-acquired Infection 556
∙∙ Diagnosis and Control of Hospital Infection 557
∙∙ Infection Control Policy 557
∙∙ Prevention 557
80. Laboratory Control of Antimicrobial Therapy 559
∙∙ Antibiotic Sensitivity Tests 559
∙∙ Kirby–Bauer Disk Diffusion Method 560
∙∙ Stokes Disk Diffusion Method 561
∙∙ Dilution Methods 563
∙∙ Antibiotic Assays in Body Fluids 564
81. Antimicrobial Chemotherapy 565
∙∙ Antimicrobial Agent 565
∙∙ Antibiotic 565
∙∙ Chemotherapeutic Agents 565
∙∙ Antibacterial Agents 565
∙∙ Mechanisms of Action of Antibacterial Drugs 566
∙∙ Antibiotic Resistance 569
82. Immunoprophylaxis 571
∙∙ Vaccines 571
∙∙ Immunization 572
83. Bacteriology of Water, Milk and Air 575
∙∙ Bacteriology of Water 575
∙∙ Bacterial Flora in Water 575
∙∙ Factors Determining the Number of Bacteria in Water 575
∙∙ Water-borne Pathogens 576
∙∙ Collection of Water Samples 576
∙∙ Bacteriological Examination of Water 576
∙∙ Bacteriology of Milk 577
∙∙ Examination of Air 578
84. Hospital Waste Management 580
∙∙ Universal Precautions 580
∙∙ Definition of Biomedical Waste (BMW) 580
∙∙ Categories of Biomedical Waste 581
∙∙ Waste Segregation 581
∙∙ Treatment and Disposal Technologies for Healthcare Waste 581
∙∙ Disposal 582
85. Vehicles and Vectors 584
86. Emerging and Re-emerging Infectious Diseases 586
∙∙ Re-emerging, or Resurging Diseases 586
www.ebook3000.com
xx | Essentials of Microbiology
88. Staining Methods 592
∙∙ Preparing Film or Smear for Staining 592
∙∙ Types of Stain 592
∙∙ Stained Preparations 592
∙∙ Simple Stains 593
∙∙ Differential Stains 593
∙∙ Special Stains 594
89. Practical Microbiology for MBBS Students 596
∙∙ Spots 596
∙∙ Staining for Practical Examination 598
∙∙ Reagents 600
∙∙ Gram-positive and Gram-negative Bacteria 601
∙∙ Identification of Bacterial Culture 601
∙∙ Staphylococcus 605
∙∙ Bacillus, Proteus sp. 606
∙∙ Lactose Fermenter (LF) 607
∙∙ Stool/Feces Examination 609
Index 615
Section 1: General Bacteriology
1
Chapter
Learning Objectives
www.ebook3000.com
Chap-01.indd 1 15-03-2016 11:32:50 AM
2 | Section 1: General Bacteriology
formation of microbes. Franz Schulze (1815–1873), Louis Pasteur (Fig. 1.1) is known as ‘Father
Theodore Schwann (1810–1882), Georg Friedrich of Microbiology’ because his contribution led to
Schroder and Theodor von Dusch attempted to the development of microbiology as a separate
counter such arguments. Louis Pasteur of proved scientific discipline.
conclusively that all forms of life, even microbes,
arose only from their like and not de novo. Contributions of Louis Pasteur in
John Tyndall (1820–1893), the English Microbiology (Box 1.1)
physicist finally in 1877 proved and was able
∙∙ Coined the term ‘Microbiology’: For the study
to explain satisfactorily the need for prolonged
of living organnisms of microscopic size.
heating to eliminate microbial life from infusions.
∙∙ Proposed germ theory of disease: He
Intermittent heating, now called tyndallization,
established that putrefaction and fermentation
killed both heat-stable form and a heat-sensitive
was the result of microbial activity and that
form of bacteria.
different types of fermentations were associated
with different types of microoganisms (1857).
ROLE OF MICROORGANISMS IN DISEASEs
∙∙ Disapprove d the or y of sp ontan e ous
Augustino Bassi (1773–1856) demonstrated in generation: He disapproved the theory of
1835 that a silkworm disease called ‘muscardine’ spontaneous generation in 1860–1861 in public
was due to a fungal infection. MJ Berkeley (1845) controversy with Pouchet who was a proponent
proved that the great potato blight of Ireland was of spontaneous generation. In a series of
caused by a fungus. Following his success with the classic experiments in the swan-necked flasks,
study of fermentation, Pasteur imeestigated the Pasteur proved conclusively by demonstrating
pebrine disease of silueosm. the the ubiquity of microorganisms that all
Indirect transmission was recognized forms of life, even microbes, arose only from
in the 1840s, when American poet-physician their like and not de novo.
Oliver Wendell Holmes (1843) in Boston, USA ∙∙ Developed sterilization techniques and
and Ignaz Semmelweis in Vienna (1846) had developed the steam sterilizer, hot-air oven and
independently concluded that puerperal sepsis was autoclave in the course of these studies.
contagious. Semmelweis also identified its mode ∙∙ Developed methods and techniques for
of transmission by doctors and medical students cultivation of microorganisms.
attending on women in labor in the hospital and ∙∙ Studies on pebrine (silkworm disease),
had prevented it by the simple measure of washing anthrax, chicken cholera and hydrophobia.
hands in an antiseptic solution. Semmelweis was ∙∙ Pasteurization: He deviced the process of
persecuted by medical orthodoxy and driven destroying bacteria, known as pasteurization.
insane for the service to medicine and humanity. ∙∙ Coined the term ‘Vaccine’: It was Pasteur who
coined the term vaccine for such prophylactic
SCIENTIFIC DEVELOPMENT OF preparations.
MICROBIoLOGY ∙∙ Discovery of the process of attenuation
and chicken cholera vaccine: An accidental
The development of microbiology as a scientific
observation that chicken cholera bacillus
discipline dates from Louis Pasteur, perfection
cultures left on the bench for several weeks lost
on microbiological studies by Robert Koch, the
their pathogenic property but retained their
introduction of antiseptic surgery by Lord Lister
ability to protect the birds against subsequent
and contributions of Paul Ehrlich in chemotherapy.
infection by them, led to the discovery of the
process of attenuation and the development
LOUIS PASTEUR (1822–1895)
of live vaccines.
Louis Pasteur (1822– ∙∙ Developed live attenuated anthrax vaccine:
1895) (Fig. 1.1) was born He attenuated cultures of the anthrax bacillus
in the village of Dole, by incubation at high temperature (42–43°C)
France on December 27, and proved that inoculation of such cultures
1822, the son of humble in animals induced specific protection against
p a re nt s. Hi s f at h e r anthrax. The success of such immunization
was a tanner. He was was dramatically demonstrated by a public
originally trained as a experiment on a farm at Pouilly-le-Fort (1881)
chemist, but his studies during which vaccinated sheep, goats and cows
on fermentation led him were challenged with a virulent anthrax bacillus
to take interest in micro- Fig. 1.1: Louis Pasteur culture. All the vaccinated animals survived
organisms. His discoveries revolutionized medical the challenge, while an equal number of
practice, although he never studied medicine. unvaccinated control animals succumbed to it.
www.ebook3000.com
Chap-01.indd 3 15-03-2016 11:32:51 AM
4 | Section 1: General Bacteriology
2. Reported acid-fastness of tubercle bacillus difficult. Larger viruses could be seen under
3. Introduced methods of standardizing toxin light microscope after appropriate staining but
and antitoxin their detailed morphology could only be studied
4. Proposed ‘side chain theory’ of antib ody by electron microscope by Ruska (1934).
production 4. Cultivation of viruses
5. Salvarsan introduction: He introduced The technique of growing them on chick
salvarsan, an arsenical compound, sometimes embryos was developed by Goodpasture in
called the ‘magic bullet’. It was capable of 1930s. The use of living human and animal
destroying the spirochaete of syphilis. Later tissue cells for the in-vitro culture of viruses was
on, he discovered neosalvarsan. Thus, he developed by John Enders (1949) and others.
created a new branch of medicine known as 5. Virus infection and malignancy
chemotherapy. i. Leukemia: Vilhelm Ellerman and Oluf
Bang (1908) in Copenhagen put forth the
DISCOVERY OF VIRUSES possibility that virus infection could lead to
As a science, virology evolved later than malignancy.
bacteriology. Although the physical nature of ii. Sarcoma in fowls: Peyton Rous (1911)
viruses was not fully revealed until the invention three years later isolated a virus causing
of the electron microscope, the infections they sarcoma in fowls. Several viruses have been
cause have been known and feared since the dawn blamed to cause natural and experimental
of history. tumors in birds and animals. Viruses also
1. Infectious Agents Smaller than Bacteria cause malignant transformation of the
The infectious agents of numerous diseases infected cells in tissue culture.
were being isolated and many infectious 6. Viral oncogenesis: The discovery of viral and
diseases had been proved to be caused by cellular oncogenes have put forth the possible
bacteria. But there remained a large number mechanisms of viral oncogenesis. Positive
of diseases for which no bacterial cause could proof of a virus causing human malignancy
be established until it was realized that the was established when the virus of human T-cell
responsible agents were smaller than bacteria. leukemia was isolated in 1980.
2. Various Infections 7. Bacteriophages: Frederick W Twort (1915)
i. Rabies in dogs: Pasteur had suspected and Felix D Herelle (1917) independently
that rabies in dogs could be caused by a discovered a lytic phenomenon in bacterial
microbe too small to be seen under the cultures. The agents responsible were termed
microscope. bacteriophages (viruses that attack bacteria).
ii. Tobacco mosaic disease: Iwanowski
(1892), Russian scientist and Martinus IMMUNITY AND IMMUNIZATION
Beijrinck (1898) in Holland attributed the
cause of tobacco—mosaic disease to the 1. Ancient knowledge
infectious agents in bacteria-free filtrates to The practice of producing a mild form of
be living but fluidcontagium vivum fluidum smallpox intentionally (variolation) was
and introduced the term virus (Latin for prevalent in India, China and other ancient
‘poison’) for such filterable infectious agents. civilizations from time immemorial.
iii. Foot-and-mouth disease of cattle: Friedrich 2. Edward Jenner (1749–1823)
Loeffler and Paul Frosch at the same time in The first scientific attempts at artificial
1898 in Germany found that foot-and-mouth immunizations in the late eighteenth century
disease of cattle was also caused by a similar were made by Edward Jenner (1749–1823)
filter-passing virus. from England. He introduced the technique of
iv. Yellow fever: The discovery of first human vaccination using a related but mild live virus
disease proved to have a viral etiology was of cowpox (1796). Edward Jenner is known as
yellow fever. The US Army Yellow Fever the ‘Father of Immunology’.
Commission under Walter Reed in Cuba 3. Live vaccines
(1902) showed that this human disease Further work on immunization was carried
(yellow fever) was not only a filterable virus out by Louis Pasteur and derived attenuated
but also transmitted through the bite of (reduced in virulence) live vaccines for fowl
infected mosquitoes. cholera, anthrax, swine erysipelas and rabies.
3. Electron microscope: Viruses could not be 4. Vaccine for hydrophobia
visualized under the light microscope or grown Pasteur’s development of a vaccine for hydro
in culture media. So investigation of viruses phobia made the greatest impact in medicine.
and the disease caused by them were rendered This was acclaimed throughout the world.
Contd...
www.ebook3000.com
Chap-01.indd 5 15-03-2016 11:32:51 AM
6 | Section 1: General Bacteriology
Contd...
www.ebook3000.com
Chap-01.indd 7 15-03-2016 11:32:51 AM
2
Chapter
Microscopy
Learning Objectives
After reading and studying this chapter, you should ∙∙ E xplain the principles and describe the uses of the
be able to: following: darkfield microscopy; phase-contrast;
∙∙ Discuss microscopic methods microscopy; fluorescent microscopy and electron
microscopy.
www.ebook3000.com
10 | Section 1: General Bacteriology
Types of Electron Microscopes Phase-contrast microscopy: It allows the detailed
There are two types of electron microscopes in observation of the living organisms.
general use: Fluorescence microscopy: Fluorescence microscopy
is used primarily in a diagnostic procedure
called fluorescent-antibody (FA) technique, or
(i) Transmission Electron Microscope (TEM) immunofluorescence.
In transmission electron microscope (TEM), Electron microscopy: Electron microscopes use
electrons like light pass directly through the electromagnetic lenses instead of glass lenses,
electrons and fluorescent screens to produce a
specimen that has been prepared by thin magnified image. There are two types (i) Transmission
sectioning, freeze fracturing, or freeze etching. It electron microscopes (TEMs); and (ii) Scanning
is used to observe fine details of cell structure. electron microscopes
Scanned-probe microscopy: Their resolving power is
(ii) Scanning Electron Microscope (SEM) much greater than the electron microscope.
Morphology of Bacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should be ∙∙ Discuss capsule or bacterial capsule
able to: ∙∙ Describe bacterial flagellae
∙∙ Differentiate between prokaryotes and eukaryotes ∙∙ Describe fimbriae or pili
∙∙ Describe anatomy of bacterial cell ∙∙ Discuss bacterial spores or endospores
∙∙ Describe cell envelope ∙∙ Explain L-forms of bacteria.
∙∙ Describe bacterial cell wall
www.ebook3000.com
Chap-03.indd 11 15-03-2016 11:09:37
12 | Section 1: General Bacteriology
1 Angstrom unit (A) = one-tenth of a nanometer. 4. Spirilla: Spirilla are rigid spiral or helical forms.
The diameter of the smallest body that can be 5. Spirochaetes: Spirochaetes are flexuous spiral
resolved and seen clearly with naked eye is 200 forms.
µm. Bacteria of medical importance generally 6. Mycoplasma: Mycoplasma are cell wall
measure 0.2–1.5 µm in diameter and about 3–5 deficient bacteria and, hence, do not possess
µm in length. To see bacteria, a light microscope a stable morphology. They occur as round or
must be used. The best light microscope, using the oval bodies and interlacing filaments.
most advanced optics, is capable of magnifications
of 1000–2000 times. The electron microscope ARRANGEMENT OF BACTERIAL CELLS
provides superb resolving power. Following types
of microscopes are used for the examination of Pathogenic bacterial species appear as sphere
bacteria: (cocci), rods (bacilli), and spirals. Bacteria sometimes
show characteristic cellular arrangement or
STUDY OF BACTERIA grouping (Fig. 3.2). The type of cellular arrangement
is determined by the plane through which binary
Stained Preparations fission takes place and by the tendency of the
Live bacteria do not show much structural detail daughter cells to remain attached even after division.
under the light microscope due to lack of contrast.
Hence, it is customary to use staining techniques Cocci Arrangement
to produce color contrast. Various staining i. Diplococci: Cocci may be arranged in pairs
techniques are commonly used in bacteriology (diplococci), when cocci divide and remain
(See Chapter 87). together;
Shape of Bacteria
Depending on their shape, bacteria are classified
into several varieties (Fig. 3.1):
1. Cocci: Cocci (from kokkos meaning berry) are
spherical, or nearly spherical.
2. Bacilli: Bacilli (from baculus meaning rod)
are relatively straight, rod-shaped (cylindrical)
cells. In some of the bacilli, the length of the
cells may be equal to the width. Such bacillary
forms are known as coccobacilli and have to be
carefully differentiated from cocci.
3. Vibrios: Vibrios are curved or comma-
shaped rods and derive the name from their
characteristic vibratory motility.
www.ebook3000.com
Chap-03.indd 13 15-03-2016 11:09:40
14 | Section 1: General Bacteriology
ii. Microdissection thick), elastic and can only be seen with electron
iii. Exposure to specific antibody microscope. It is a typical ‘unit membrane’,
iv. Mechanical rupture of the cell composed of phospholipids and proteins.
v. Differential staining procedures Chemically, the membrane consists of
vi. Electron microscopy. lipoprotein with small amounts of carbohydrate.
Enzymes that attack cell walls With the exception of Mycoplasma, bacterial
i. Lysozyme: The enzyme lysozyme, which is cytoplasmic membrane lacks sterols.
found in animal secretions (tears, saliva, nasal Functions of cytoplasmic membrane:
secretions) as well as in egg white, is a natural i. Semipermeable membrane: Controlling the
body defence substance which lyses bacteria inflow and outflow of metabolites to and from
of many species. the protoplasm.
ii. Autolysins: Bacteria themselves possess ii. Housing enzymes : Involved in outer
enzymes, called autolysins, able to hydrolyse membrane synthesis, cell wall synthesis,
their own cell wall substances. and in the assembly and secretion of extra
Protoplasts and spheroplasts cytoplasmic and extracellular substances.
Protoplasts iii. Housing many sensory and chemotaxis
These are derived from gram-positive bacteria. proteins
The gram-positive cell wall is almost completely iv. Generation of chemical energy (i.e. ATP)
destroyed by lysozyme. They contain cytoplasmic v. Cell motility
membrane and cell wall is totally lacking. Typically, vi. Mediation of chromosomal segregation
a protoplast is spherical and is still capable of carry during replication
ing on metabolism. Protoplasts cannot revert to
normal bacterial morphology.
b. Cellular Appendages
Spheroplasts I. Bacterial capsule or slime layer
When lysozyme is applied to gram-negative cells, Structure: Many bacteria synthesize large amount of
usually the wall is not destroyed to the same extent extracellular polymer in their natural environments.
as in gram-positive cells. Spheroplasts retain outer When the polymer forms a condensed, well-defined
membrane and entrapped peptidoglycan from layer closely surrounding the cell, it is called the
gram-negative cell. It differs from the protoplast in capsule as in the Pneumococcus. If the polymer
that some cell wall material is retained. It is also a is easily washed off and does not appear to be
spherical structure. They are capable of reverting associated with the cell in any definite fashion, it
to parent bacterial form when cell wall inhibitor is is referred to as a slime layer as in leuconostoc.
removed from the culture medium. A glycocalyx is a network of polysaccharide
2. Cytoplasmic (plasma) membrane extending from the surface of bacteria and other
Structure: The cytoplasmic (plasma) membrane cells. Capsules too thin to be seen under the light
limits the bacterial protoplast. It is thin (5–10 nm microscope are called microcapsules.
www.ebook3000.com
Chap-03.indd 15 15-03-2016 11:09:42
16 | Section 1: General Bacteriology
Composition of capsules and slime layers: for the typing of pneumococci in the pre
Capsules and slime layers usually are composed sulfonamide days.
of polysaccharide (for example, Pneumococcus)
Functions of capsule
or of polypeptide in some bacteria (for example,
i. Virulence factor: Capsules often act as a
Bacillus anthracis and Yersinia pestis). Some
virulence factor by protecting the bacterium
bacteria may have both a capsule and a slime layer
from ingestion by phagoc ytosis and
(for example, Streptococcus salivarius). Bacteria
noncapsulate mutant of these bacteria are
secreting large amounts of slime produce mucoid
nonvirurlent.
growth on agar, which is of a stringy consistency
ii. Protection of the cell wall: In protecting
when touched with the loop.
the cell wall attack by various kinds of
Capsulated bacteria: Streptococcus pneumoniae, antibacterial agents.
several groups of streptococci, Neisseria menin iii. Identification and typing of bacteria.
gitidis, Klebsiella, Haemophilus influenzae,
II. Flagella
Yersinia and Bacillus.
Motile bacteria, except spirochetes, possess one or
more unbranched, long, sinuous filaments called
Demonstration of Capsule flagella, which are the organs of locomotion.
i. Gram stain: Capsules and slime are usually
not visible in films stained by ordinary Structure: They are long, hollow, helical filaments,
methods except as clear haloes surrounding usually several times the length of the cell. They are
the stained smear. 3–20 μm long and are of uniform diameter (0.01–
ii. Special capsule-staining techniques : 0.013 μm) and terminate in a square tip. It originates
Usually employing copper salts as mordants. in the bacterial protoplasm and is extruded through
iii. India ink staining (negative staining): The the cell wall. Flagella consists of largely or entirely
capsule appears as a clear halo around the a protein, flagellin.
bacterium, against a dark background in the Flagella are highly antigenic and flagellar
film (Fig. 3.7). antigens induce specific antibodies in high titers.
iv. Electron microscope. Flagellar antibodies are not protective but are useful
v. Serological methods: Capsular material in serodiagnosis.
is antigenic and may be demonstrated by Parts and Composition
serological methods. When a suspension Each flagellum consists of three parts (Fig. 3.8):
of a capsulated bacterium is mixed with its i. Filament: The filament is the longest and most
specific anticapsular serum and examined obvious portion which extends from the cell
under the microscope, the capsule becomes surface to the tip.
very prominent and appears ‘swollen’ due to ii. Hook: The hook is a short, curved segment
an increase in its refractivity. It is known as which links the filament to its basal body and
the capsule-swelling reaction or Quellung functions as universal joint between the basal
reaction (Quellung-Ger swelling). It was body and the filament.
described by Neufeld (1902), hence called iii. Basal body: The basal body is embedded
Neufeld reaction. It was widely employed in the cell (cytoplasmic membrane). In the
Fig. 3.7: Pneumococci negatively stained with India ink to Fig. 3.8: The structure of bacterial flagellum
show capsule
3. Mesosomes (Chondroids)
These are convoluted or multilaminated
membranous bodies formed as invaginations of
the plasma membrane into the cytoplasm. These
are of two types:
Fig. 3.9: Arrangement of flagella i. Septal mesosomes; ii. Lateral mesosomes.
www.ebook3000.com
Chap-03.indd 17 15-03-2016 11:09:43
18 | Section 1: General Bacteriology
Functions of mesosomes: progeny either through conjugation or the agency
i. Compartmenting of DNA; ii. Sites of the of bateriophages.
respiratory enzymes. Functions of plasmids: Plasmids are not essential
for host growth and reproduction they inhibit, but
4. Intracytoplasmic Inclusions may confer on it certain properties such as drug
These are not permanent or essential structures, resistance, and toxigencity which may constitute a
and may be absent under certain conditions of survival advantage.
growth. These bodies are usually sources of storage
of energy. They consist of volutin (polyphosphate), 6. Bacterial Spore
lipid, glycogen, starch or sulfur.
A number of gram-positive bacteria, such as those
of the genera Clostridium and Bacillus can form a
5. Bacterial Nucleus
special resistant dormant structure called an endo
The genetic material of a bacterial cell is contained spore or, simply, spores. Sporulation in bacteria, is
in a single, long molecule of double-stranded not a method of reproduction but of preservation.
deoxyribonucleic acid (DNA) which can be
extracted in the form of a closed circular thread Sporulation: Spore formation, sporogenesis or
about 1 mm (1000 μm) long. The bacterial sporulation normally commences when growth
chromosome is haploid and replicates by simple ceases due to lack of nutrients, depletion of the
fission instead of by mitosis as in higher cells. nitrogen or carbon source (or both) being the most
significant factor.
Plasmids Stages: It may be divided into several stages
Bacteria may possess extranuclear genetic (Fig. 3.10).
elements in the cytoplasm consisting of DNA ∙∙ Spore septum: In the first observable stage
termed plasmids or episomes (see Chapter 10). of sporulation, a newly replicated bacterial
These can exist and replicte independently of the chromosome and a small portion of cytoplasm
chromosome or may be integrated with it. In either are isolated by an ingrowth of the plasma
case, they normally are inherited or passed on the membrane called a spore septum.
Germination
Germination is the process of conversion of a spore
into vegetative cells under suitable conditions. It
occurs in three stages: activation, initiation and Fig. 3.12: Types of spores. 1. Central, bulging; 2. Central, not
outgrowth. Once activated, a spore will initiate bulging; 3. Subterminal, bulging; 4. Subterminal, not bulging;
germination if the environmental conditions are 5. Terminal, spherical; 6. Terminal, oval
favorable.
ii. Modified Ziehl-Neelsen (ZN) staini ng:
Shape and Position of the Spore Spores are slightly acid-fast. Ziehl-Neelsen
The shape and position of the spore and its size staining with 0.25–0.5% sulfuric acid (instead
relative to the parent cell are species’ characteristics. of 20% sulfuric acid as used in conventional
Spores may be central (equatorial), subterminal method) as decoloring agent is used for spore
(close to one end), or terminal (Fig. 3.12). The staining.
appearance may be spherical, ovoid or elongated,
and being narrower that the cell, or broader and Uses of Spores
bulging it. The diameter of spore may be same 1. Importance in food, industrial, and medical
or less than the width of bacteria (Bacillus), or microbiology
may be wider than the bacillary body producing a 2. Sterilization control: For proper sterilization,
distension or bulge in the cell (Clostridium). spores of certain species of bacteria are
e mp l oye d a s i n d i cat o r, e. g. B a cillus
Resistance stearothermophilus which is destroyed at a
These structures are extraordinary resistant to temperature of 121°C for 10–20 minutes. Proper
environmental stresses. Spores of all medically sterilization is indicated by the absence of the
important species are destroyed by autoclaving at spores after autoclaving.
120°C for 15 minutes. Endospore heat resistance 3. Research
probably is due to several factors: calcium-
dipicolinate and acid-soluble protein stabilization
PLEOMORPHISM AND INVOLUTION FORM
of DNA, protoplast dehydration, the spore coat,
DNA repair, the greater stability of the cell proteins During growth, bacteria of a single strain may show
in bacteria adapted to growth at high temperatures considerable variation in size and shape. This is
and others. known as pleomorphism and occurs most readily
in certain species. The abnormal cells are generally
Demonstration regarded as degenerate or involution forms.
i. Gram-staining: Spores appear as an unstained Pleomorphism and involution forms are often
refractile body within the cell. due to defective cell wall synthesis. Involution
www.ebook3000.com
Chap-03.indd 19 15-03-2016 11:09:44
20 | Section 1: General Bacteriology
forms may also develop due to the activity of 3. Write short notes on:
autolytic enzymes. a. Bacterial cell wall
b. Cytoplasmic membrane
c. Plasmids or episomes
L-FORMS OF BACTERIA (CELL-WALL- d. Capsule or bacterial capsule
DEFECTIVE ORGANISMS) e. Bacterial flagellae
f. Bacterial spores or endospores
The first isolation of a naturally occurring L-form g. L-forms of bacteria
took place when Kleineberger-Nobel, studying
Streptobacillus moniliformis in the Lister MULTIPLE CHOICE QUESTIONS (MCQs)
Institute, London, observed abnormal forms of the
bacteria and named them L-forms after the Lister 1. Which of the following is not a distinguishing
Institute, London (hence, the “L”). characteristic of prokaryotic cells?
These are abnormal growth forms that may a. They usually have a single, circular chromo-
arise spontaneously or by the inhibition of cell wall some
b. They lack membrane-enclosed organelles
synthesis in bacteria of normal morphology (in the
c. They have cell walls containing peptidogycan
presence of penicillin or other agents that interfere
d. They lack a plasma membrane
with cell wall synthesis).
2. Peptidoglycan layer of cell wall is thicker in:
L-forms may be unstable. They lack a rigid cell
a. Gram-positive bacteria
wall. They are capable of growing and multiplying b. Gram-negative bacteria
on a suitable nutrient medium unlike protoplasts. c. Fungi
L-forms are difficult to cultivate. Colonies of d. Parasites
L-phase organisms on agar media are small and 3. All the following statements are true for
have a characteristic ‘fried egg’ appearance, rather lipopolysaccharide except:
like mycoplasma, L-forms are nonpathogenic a. It consists of three components: Lipid A, core
to laboratory animals. L-forms in the host may polysaccharide and O polysaccharide.
produce chronic infections and are relatively b. Lipid A functions as an endotoxin.
resistant to antibiotic treatment. They present c. It is an integral part of the cell wall of the gram-
special problems in chemotherapy and explain positive bacteria.
relapses after treatment. d. Polysaccharide represents a major surface
antigen of the bacterial cell.
Key Points 4. A tuft of flagella present at one or both the ends of
Bacteria are unicellular, and most of them multiply
bacterial cell is known as:
by binary fission. They are prokaryotes. a. Monotrichous
The structure of the prokaryotic cell b. Amphitrichous
The bacterial cell consists of (A) cell envelope c. Lophotrichous
outer layer (B) cell interior (i) cell wall and (ii) d. Peritrichous
plasma membrane—beneath cell wall and cellular 5. Which of the following is not true about fimbriae?
appendages—capsule, fimbriae, and flagella, and a. They originate in the cytoplasmic membrane
cell interior (and include cytoplasm, cytoplasmic b. They are composed of protein
inclusions (mesosomes, ribosomes, inclusion
c. They may be used for attachment
granules, vacuoles) and a single circular chromosome
d. They may be used for motility
of deoxyribonucleic acid (DNA).
Gram-positive cell wall: The gram-positive cell wall 6. Which of the following bacteria is cell-wall
contains a relatively thick layer of peptidoglycan. deficient?
Teichoic acids stick out of the peptidoglycan layer. a. Escherichia coli
Gram-negative cell wall: Gram-negative bacteria have b. Streptococcus aureus
a lipopolysaccharidelipoprotein-phospholipid outer c. Mycoplasma
membrane surrounding a thin peptidoglycan layer. d. Treponema pallidum
Endospores are a dormant stage produced by
7. All of the following are spore-forming bacteria
members of Bacillus and Clostridium for survival
except:
during adverse environmental conditions.
a. Clostridium botulinum
b. Bacillus anthracis
IMPORTANT QUESTIONS c. Bacillus subtilis
d. Vibrio cholerae
1. Describe briefly the anatomy of a bacterial cell.
2. Draw a labelled diagram of a bacterial cell. Write ANSWERS (MCQs)
briefly on the cell wall of bacteria. Ans: 1. d; 2. a; 3. c; 4. c; 5. d; 6. c; 7. d
Physiology of Bacteria
Learning Objectives
www.ebook3000.com
22 | Section 1: General Bacteriology
of carbon, a source of nitrogen and some inorganic
salts. The water content of bacterial cells can vary
from 75 to 90% of the total weight and is the vehicle
for the entry of all cells and for the elimination of
all waste products. It participates in the metabolic
reactions and also forms an integral part of the
protoplasm.
www.ebook3000.com
24 | Section 1: General Bacteriology
Oxidation–Reduction (O–R) Potential 2. What are the heterotrophic bacteria? Discuss the
nutritional and physical requirements for the
(Redox Potential) growth of the bacteria.
Oxidation is the removal of electrons, and reduction 3. Write short notes on:
is the addition of electrons. In other words, each a. Bacterial growth curve/growth phases of bacteria.
time one substance is oxidized, another one is b. Redox potential.
simultaneously reduced. The pairing of these
reactions is called oxidation-reduction or a redox Muliple choice questions (MCQs)
reaction.
The ability of a substance to take up or part 1. Generation time for Mycobacterium tuberculosis
is:
with electrons is known as oxidation-reduction
a. 20 seconds
or redox or Eh-potential.
b. 20 minutes
Toxic derivatives of oxygen: Aerotolerant c. 20 hours
microorganisms may lack catalase but almost have d. 20 days
superoxide dismutase. All strict anerobes lack both 2. Bacteria which can grow at temperature between
enzymes or have them in very low concentrations 20°C and 40°C are known as:
and, therefore, cannot tolerate O2. It is suggested a. Mesophiles
that in the presence of oxygen, hydrogen peroxide b. Psychrophiles
accumulates in the media and inhibits the growth c. Thermophiles
d. None of the above
of anerobes. Another reason might be that anerobes
3. The bacteria which require much higher level of
possess essential enzymes that are active only in the
carbon dioxide for their growth are known as:
reduced state. The enzyme catalase, which splits
a. Microaerophilic bacteria
hydrogen peroxide, is present in aerobic bacteria, b. Capnophilic bacteria
but is absent in the anerobes. c. Aerobic bacteria
Key Points d. Phototrophs
4. Which of the following acidophilic bacteria can
Bacterial growth curve: The bacterial growth curve grow in acidic conditions?
can be divided into four major phases: lag phase,
a. Escherichia coli
exponential or log (logarithmic) phase, stationary
phase, and decline phase. b. Lactobacilli
The requirements for microbial growth: Chemical and c. Pseudomonas aeruginosa
physical. d. Vibrio cholerae
Chemical requirements: All organisms require a 5. The enzyme catalase is present in:
carbon source, nitrogen, and other chemicals a. Aerobic bacteria
required for microbial growth. b. Obligate anerobic bacteria
c. Aerobic and obligate anerobic bacteria
Important Questions d. None of the above
1. Draw a typical bacterial growth curve and describe Answers (MCQs)
it. 1. c; 2. a; 3. b; 4. b; 5. a
5
Chapter
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Describe filtration—uses and types
able to: ∙∙ Discuss types of radiations and their uses
∙∙ Define the following terms—sterilization, dis ∙∙ Give the mechanism of action for each type of
infection and antisepsis chemical agent commonly used in antiseptics and
∙∙ Describe various agents used in sterilization disinfectants
∙∙ Describe sterilization by moist heat ∙∙ Describe the following—aldehydes as disinfectants,
∙∙ Describe the different heat methods and their uses of formaldehyde and glutaraldehyde as
respective applications disinfectants
∙∙ Describe the following—pasteurization, tyndaliz ∙∙ Explain vapor-phase disinfectants or gaseous
ation or intermittent sterilization or fractional sterilization and discuss the role of ethylene oxide
sterilization, inspissation or serum inspissator, hot in sterilization of disposable items
air oven ∙∙ Describe various tests used for testing of
∙∙ Explain the principle and functioning of autoclave disinfectants.
www.ebook3000.com
26 | Section 1: General Bacteriology
Table 5.1 Methods of sterilization and disinfection
A. Physical agents
1. Sunlight
2. Drying
3. Heat
a. Dry heat
b. Moist heat
4. Filtration
5. Radiation
6. Ultrasonic and sonic vibrations
B. Chemical agents
a. Agents that damage the cell membranes
1. Surface-active disinfectants
2. Phenolic compounds
3. Alcohols
b. Agents that damage proteins
1. Acids and alkalies
2. Alcohols
c. A
gents that modify functional groups of proteins and
nucleic acids
1. Heavy metals
2. Oxidizing agents
3. Dyes Fig. 5.1: Hot air oven
4. Alkylating agents
www.ebook3000.com
28 | Section 1: General Bacteriology
days is called tyndallization or intermittent Principle of Autoclave
sterilization. This is a fractional method of The principle of the autoclave or steam sterilizer is
sterilization. The instrument commonly that water boils when its vapor pressure equals that
used is Koch and Arnold steamer. of the surrounding atmosphere. When pressure
Principle: Vegetative cells and some spores inside a closed vessel increases, the temperature at
are killed during the first heating and that which water boils also increases. Saturated steam
the more resistant spores subsequently has penetrative power and is a better sterilizing
germinate and are killed during either agent than dry heat.
the second or the third heating. Though Steam condenses to water and gives up its latent
generally adequate, this method may heat to that surface when it comes into contact with a
fail with spores of certain anerobes and cooler surface. The energy available from this latent
thermophiles. heat is considerable, e.g. 1600 mL steam at 100°C
Uses: This method is useful in sterilizing and at atmospheric pressure condenses into 1 mL of
heat-sensitive culture media containing water at 100°C and releases 518 calories of heat. The
such materials as carbohydrates, egg or large reduction in volume sucks in more steam to the
serum, which are damaged by higher area and the process continues till the temperature of
temperature of autoclave. that surface is raised to that of the steam. The water
C. At temperature above 100°C of condensation ensures moist conditions for killing
Steam under pressure: Steam above 100°C or of the exposed microorganisms.
saturated steam is a more efficient sterilizing
agent than hot air. Procedure
1. Water: Sufficient water is put in the cylinder.
Autoclave Above this is a perforated shelf on which
Autoclaving is the process of sterilization by articles to be sterilized are placed, and the
saturated steam under high pressure above 100°C. autoclave is heated.
Steam sterilization is carried out in a pressure 2. Lid: The lid is screwed tight with the discharge
chamber called an autoclave (a device somewhat tap open and the safety valve is adjusted to the
like a fancy pressure cooker). required pressure.
3. Air removal: The steam-air mixture is allowed
Various components of autoclave: In its simplest to escape freely till all the air has been displaced.
form, the laboratory autoclave consists of a vertical To know when all the air inside the autoclave
or horizontal cylinder of gunmetal or stainless has escaped the discharge tap is connected with
steel, in a supporting sheet iron case. The lid or one end of a rubber tube and the other end of
door is fastened by screw clamps and made airtight it is placed in water. When the air bubbles stop
by a suitable washer. The autoclave has on its lid coming, it indicates that all the air from inside
or upper side a discharge tap for air and steam, a the autoclave has been removed.
pressure gauge and a safety valve that can be set to 4. The discharge tap is now closed.
blow off at any desired pressure. Heating is done by 5. Holding period: The steam pressure rises
gas or electricity (Fig. 5.3). The domestic pressure inside and when it reaches the desired set level
cooker serves as a miniature autoclave and may (15 psi), the safety valve opens and the excess
be used for sterilizing small articles in clinics and steam escapes. From this point, the holding
similar establishments. period (15 minutes) is calculated.
6. Autoclave cooling: When the holding period
is over, the heater is turned off and the
autoclave allowed to cool till the pressure gauge
indicates that the pressure inside is equal to the
atmospheric pressure.
7. Air entry in the autoclave: The discharge tap
is opened slowly and air is allowed to enter the
autoclave.
8. Removal of articles: The lid is now opened and
the sterilized articles removed.
Precautions
i. Air escape from the chamber : Since
temperature of air-steam mixture is lower than
that of pure steam, the air must be allowed to
Fig. 5.3: A simple autoclave escape from the chamber.
Chapter 5: Sterilization and Disinfection | 29
ii. Arrangement of the materials: This should been used widely for purification of water for
be done in such a manner which ensures free industrial and drinking purposes. They are of
circulation of steam inside the chamber. two types:
Uses a. Unglazed ceramic filters, e.g. Chamberland,
i. For sterilizing culture media and other and Doulton filters.
laboratory supplies, aqueous solutions, b. Compressed diatomaceous earth filters,
rubber material, dressing materials, gowns, e.g. the Berkefeld and Mandler filters.
dressing, linen, gloves, instruments and ii. Asbestos filters (Seitz filter): They are made
pharmaceutical products. up of a disc of asbestos (magnesium trisilicate).
ii. For all materials that are water-containing, It is supported on a perforated metal disc
permeable or wettable and not liable to be within a metal funnel. It is then fitted on to a
damaged by the process. sterile flask through a silicone rubber bung.
iii. Particularly useful for materials which cannot Sterilized fluid is collected from the flask and
withstand the higher temperature of hot air filter disc is discarded after use. These discs are
oven. available with different grades of porosity.
Examples: Seitz filter, Carlson and Sterimat
Sterilization Controls filters.
A. Biological control (Bacterial spores): iii. Sintered glass filters: They are prepared
An envelope containing a filter paper strip by size grading powdered glass followed by
impregnated with 10 6 spores of Bacillus heating.
stearothermophilus is placed with the iv. Membrane filters: Membrane filters consist
load sterilization is over, the strip is removed of a variety of polymeric materials such
and inoculated into. No growth of B. stea as cellulose nitrate, cellulose diacetate,
rothermophilus indicates proper sterilization. polycarbonate and polyester. They are
Spores of this organism withstand 121°C for up manufactured as discs from 13 to 293 mm
to 12 minutes and this has made the organism diameter and with porosities from 0.015 to
ideal for testing autoclaves. 12 mm. They come in a wide range of average
B. Chemical control: A Browne’s tube containing pore diameters (APD), the 0.22 mm size being
red solution changes to green when exposed most widely used for sterilization because the
to temperature of 121°C for 15 minutes in pore size is smaller than that of bacteria.
autoclave. It indicates proper sterilization.
Uses
C. Autoclave tapes
1. They are used routinely in water analysis
D. Thermocouples: These may also be used which
and purification.
record the temperature by a potentiometer.
2. Sterilization and sterility testing.
4. Filtration 3. For preparing sterile solutions for
Filtration is the principal method used in the parenteral use.
laboratory for the sterilization of heat labile 4. Bacterial counts of water:
materials, e.g. sera, solutions of sugars or antibiotics v. Syringe filters: Membrane of different
used for the preparation of culture media. diameters are commonly fitted in syringe-like
holders of stainless steel or polycarbonate.
Uses
For sterilization, the fluid is forced through
1. Heat-sensitive solutions: For sterilization of
the disc (membrane) by pressing the piston
pharmaceuticals, ophthalmic solutions, culture
of the syringe.
media, oils, antibiotics and other heat-sensitive
vi. Vacuum and ‘in-line’ filters: They are
solutions.
suitable for the sterilization or disinfection of
2. For separation of bacteriophages and
large volumes of liquid or air.
bacterial toxins from bacteria.
3. Isolation of organisms which are scanty in vii. Pressure filtration: It may be used for the pro
fluids. duction of very pure water for laboratory use.
4. Concentration of bacteria from liquids, e.g. viii. Air filters: They are widely used in air
in testing water samples for cholera vibrios or filtration.
typhoid bacilli.
5. For virus isolation. 5. Radiation
Two types of radiations are used:
Types of Filters I. Non-ionizing radiations : Infrared and
i. Earthware filters: These are manufactured in ultraviolet rays are of non-ionizing type.
several different grades of porosity and have The effectiveness of UV light as a lethal and
www.ebook3000.com
30 | Section 1: General Bacteriology
mutagenic agent is closely correlated with its Factors that determine the potency of dis
wavelength. The most effective bactericidal infectants
wavelength is in the 240–280 nm range, with the 1. The concentration and stability of the agent
optimum being about 260 nm, the wavelength 2. Nature of the organism
most effectively absorbed by DNA and this 3. Time of action
interfers with DNA replication. 4. pH
Microbial sensitivity to UV radiation 5. Temperature
i. Bacterial spores are generally more resistant 6. The presence of organic (especially protein) or
to UV light than are vegetative cells. other interfering substances
ii. Viruses are also inactivated. 7. Nature of the item to be disinfected.
iii. To disinfect drinking water.
iv. Disinfection of enclosed areas such Categories of Disinfectants
as entryways, hospital wards, operating Disinfection processes have been categorized as
theatres, laboratories and in ventilated high level, intermediate level and low level.
safety cabinets in which dangerous 1. High-level disinfection: High-level disinfections
microorganisms are being handled. can generally approach sterilization in
II. Ionizing radiations: These include X-rays, g effectiveness, whereas spore forms can survive
(gamma) rays and cosmic rays. These have very intermediate-level disinfection, and many
high penetrative power and are highly lethal to microbes can remain viable when exposed to
all cells including bacteria. Ionizing radiations low-level disinfection.
damage the DNA by various mechanisms. High-level disinfectants are used for items
involved with invasive procedures that cannot
Applications withstand sterilization procedures (e.g. certain
i. For sterilization in pharmacy and medicine. types of endoscopes, surgical instruments with
ii. Sterilization of packaged disposable plastic or other components that cannot be
articles such as plastic syringes, intravenous autoclaved).
lines, catheters and gloves that are unable to
withstand heat. Since there is no appreciable Examples:
increase in temperature in this method, it is i. Treatment with moist heat.
known as cold sterilization. ii. Use of liquids such as glutaraldehyde,
iii. Use for antibiotics, hormones, sutures, and hydrogen peroxide, peracetic acid, chlorine
vaccines and to prevent food spoilage. dioxide, and other chlorine compounds.
2. Intermediate-level disinfectants: Intermediate-
B. Chemical Agents level disinfectants are used to clean surfaces
Germicidal chemicals can be used to disinfect and, or instruments in which contamination with
in some cases, sterilize. bacterial spores and other highly resilient
organisms is unlikely. These include flexible
Characteristics of a Disinfectant fiberoptic endoscopes, laryngoscopes, vaginal
An ideal antiseptic or disinfectant should: specula, anesthesia breathing circuits, and other
∙∙ Have a wide spectrum of activity and must be items. These have been referred to as semi
effective against a wide variety of infectious critical instruments and devices.
agents (Gram-positive and gram-negative Examples: Alcohols, iod ophor compounds,
bacteria, acid-fast ; bacteria, bacterial phenolic compounds.
endospores, fungi, and viruses) 3. Low-level disinfectants: Low-level disinfectants
∙∙ Be active at high dilutions and in the presence are used to treat noncritical instruments
of organic matter; and devices such as blood pressure cuffs,
∙∙ Be effective in acid as well as alkaline media; electrocardiogram electrodes and stethoscopes.
∙∙ Have speedy action; They do not penetrate through mucosal surfaces
∙∙ Have high penetrating power; or into sterile tissues, although these items come
∙∙ Be stable; into contact with patients.
∙∙ Be compatible with other antiseptics and
disinfectants; Examples: Quaternary ammonium compounds.
∙∙ Not corrode metals;
∙∙ Not cause local irritation or sensitization; Mechanisms of Antimicrobial Action
∙∙ Not interfere with healing; The main modes of action are follows:
∙∙ Not be toxic if absorbed into circulation; A. Agents that damage the cell membrane
∙∙ Be cheap and easily available; 1. Surface active disinfectants
∙∙ Be safe and easy to use. 2. Phenolic compounds
Such an ideal chemical is yet to be found. 3. Alcohols
Chapter 5: Sterilization and Disinfection | 31
B. Agents that denature proteins active at acid pH and are active against gram-
1. Acids and alkalies positive organisms but are relatively ineffective
2. Alcohols against gram-negative species.
C. Agents that modify functional groups of c. Ampholytic (amphoteric) compounds:
proteins and nucleic acids: Known as ‘Tego’ compounds, these possess
1. Heavy metals and their compounds detergent properties of anionic and antimi
2. Oxidizing agents—Halogens crobial activity of cationic compounds. They
– Hydrogen peroxide are active over a wide range of pH but organic
3. Dyes—Aniline dyes matter markedly reduces their activity.
– Acridine dyes Uses: They are effective against a wide range
4. Alkylating agents—Aldehydes-formal of gram-positive and gram-negative organisms
dehyde, glutaraldehyde and some viruses at a concentration of 1% in
– Ethylene oxide water.
2. Phenols and phenolics
A. Agents that Damage the Cell Membrane Phenol: Phenols are obtained by distillation of coal
1. Surface-active agents tar between temperatures of 170°C and 270°C. It
Substances that alter the energy relationships is now rarely used as an antiseptic or disinfectant
at interfaces, producing a reduction of surface because it irritates the skin and has a disagreeable
or interfacial tension, are referred to as surface- odor. Phenol is bactericidal at a concentration of
active agents or surfactants. They possess both 1%.
hydrophobic (water-repelling) and hydrophilic Phenolics: Derivatives of phenol are called
(water-attracting) groups. phenolics.
Classification: These surfactants are classified Phenol derivatives: Certain phenol derivatives
into anionic, cationic, nonionic and ampholytic like cresol, chlorhexidine, chloroxylenol
(amphoteric). Of these, the cationic and anionic and hexachlorophane are commonly used as
compounds have been the most useful antibacterial antiseptics.
agents. i. Cresols: Cresols, obtained industrially by the
a. Cationic agents: These act on phosphate distillation of coal tar, are emulsified with green
groups of cell membrane phospholipids soap and sold under the trade names of Lysol
and also enter the cell. This leads to loss of and Creolin. ‘White fluids’ such as Lysol are
membrane semipermeability and leakage from effective but are irritant to the skin. They are
the cell of nitrogen and phosphorus-containing active against a wide range of organisms. They
compounds. The agent which enters the cell are most commonly used for sterilization of
denatures its proteins. Quaternary ammonium infected glasswares, cleaning floors, disinfection
compounds (quats) are the most important of excreta. They are not readily inactivated by
cationic compounds. Examples of quaternary the presence of organic matter.
ammonium compounds include cetrimide ii. Chlorhexidine: Chlorhexidine is a member
(cetavalon), benzalkonium chloride (Zephiran, of the biguanide group with a broad spectrum
a brand name) and cetylpyrimidium chloride of activity. They are more active against
(Cepacol, a brand name). Antimicrobial activity gram-positive than gram-negative bacteria.
is affected greatly by organic matter and by pH, They are biocidal against most vegetative
most active at alkaline pH and acid inactivates bacteria and fungi; mycobacteria are relatively
them. They are inactivated by hard water and resistant; endospores and protozoan cysts
soap. are not affected. The only viruses affected are
Uses certain enveloped (lipophilic) types.
i. They are primarily active against gram- Uses: Savlon (chlorhexidine and cetrimide)
positive, non-sporing bacteria; lethal is widely used in wounds, preoperative
to gram-negative organisms at high disinfection of skin, as bladder irrigant, etc.
concentrations. However, contact with the eyes can cause
ii. They are also fungistatic and active against damage.
viruses with lipid envelopes (e.g. herpes iii. Chloroxylenol: It is an active ingredient of
and influenza) and much less against dettol. It is less toxic and less irritant.
nonenveloped viruses (e.g. enteroviruses). iv. H e x a c h l o r o p h a n e : G r a m - p o s i t i v e
b. Anionic agents: These include soap and fatty staphylococci and streptococci, which
acids. Anionic surfactants such as common can cause skin infections in newborns, are
soaps usually have strong detergent but weak particularly susceptible to hexachlorophane.
antimicrobial properties. These agents are most So it is used notably for prophylaxis against
www.ebook3000.com
32 | Section 1: General Bacteriology
staphylococcal infection in nurseries. However, a. Iodine: Iodine compounds are the
it can cause neurotoxicity (brain damage), most effective halogens available for
especially in infants, and its use is now severely disinfection. It is actively bactericidal, with
restricted. moderate action against spores. It is active
against the tubercle bacteria and viruses.
B. Agents that Denature Proteins Uses
Acids and alkalies: Many aliphatic and aromatic i. Skin disinfectant: Iodine in aqueous and
acids are employed as preservatives, especially alcoholic solution has been used widely
in the food industry, and, to some extent, in as a skin disinfectant. Iodine often has
pharmaceutical and cosmetic products. They are been applied as tincture of iodine, 2% or
not sporicidal. more iodine in a water-ethanol solution of
potassium iodide.
Alcohols ii. Iodophors: Mixtures of iodine with various
Ethyl alcohol (ethanol) and isopropyl alcohol surface-active agents that act as carriers
are the most frequently used. They rapidly kill for iodine are known as iodophors (iodo,
bacteria including tubercle bacilli but they have ‘iodine’; phor ‘carrier’). Povidine-iodine
no action on spores and viruses. However, human (Betadine) for wounds and Wescodyne for
immunodeficiency virus (HIV) is susceptible to skin and laboratory disinfection are some
ethyl alcohol (70%) and isopropyl alcohol (35%) in popular brands.
the absence of organic matter. They must be used at b. Chlorine: In addition to chlorine itself, there
a concentration of 60 to 70% in water to be effective. are three types of chlorine compounds—
They are most frequently used as skin disinfectants hypochlorites and inorganic and organic
and act by denaturing bacterial proteins. chloramines. The disinfectant action
Isopropyl alcohol is preferred. of all chlorine compounds is due to
Methyl alcohol is effective against fungal the liberation of free chlorine. When
spores and is used for treating cabinets and elemental chlorine or hypochlorites are
incubators affected by them. Methyl alcohol added to water, the chlorine reacts with
vapour is toxic and inflammable. water to form hypochlorous acid (HOCL),
which in neutral or acidic solution is a
C. Agents that Modify Functional Groups of strong oxidizing agent and an effective
Proteins and Nucleic Acids disinfectant.
1. Heavy metals: For many years the ions of heavy The activity of chlorine is markedly
metals such as mercury, silver, arsenic, zinc influenced by the presence of organic
and copper were used as germicides. matter.
i. Mercuric chloride: It is very toxic and at Uses: The usual disinfectant for water
present has limited use supplies, swimming pools, dairy product
ii. Silver nitrate: The most commonly and food industries.
employed of the silver salts. c. Hypochlorites: The hypochlorites have
Use a bactericidal, fungicidal, virucidal and
1. Highly bactericidal for the gonococcus. rapidly sporicidal action.
2. Routinely used for the prophylaxis of i. Bleaching powder or hypochlorite
the ophthalmic neonatorum in newborn solution is most widely used for human
infants in a 1% solution. immunodeficiency virus (HIV) infected
3. To prevent infection of burns. material. Hypochlorite solution decays
iii. Copper derivatives are used as algicides, rapidly and should be prepared daily.
fungicides, wood, paint, cellulose and Chloramines are used as antiseptics for
fabric preservation. dressing wounds.
ii. Hydrogen peroxide: It is used to
2. Oxidizing Agents disinfect plastic implants, contact lenses
The most useful antimicrobial agents in this group and surgical prostheses.
are the halogens and hydrogen peroxide. They
inactivate enzymes by converting functional-SH 3. Dyes
groups to the oxidized S-S form. Aniline dyes and acridine dyes are two groups of
i. Halogens: Chlorine and iodine are among dyes which are used extensively as skin and wound
our most useful disinfectants. They are antiseptic. Both are bacteriostatic in high dilution
bactericidal and sporicidal. They are active but are of low bactericidal activity.
in very high dilutions and their action is very i. Aniline dyes: Of the aniline dyes, derivatives
rapid. of triphenylmethane, especially brilliant
Chapter 5: Sterilization and Disinfection | 33
green, malachite green and crystal violet Vapor-phase disinfectants
have many uses. 1. Ethylene oxide: This is a colorless liquid
They are highly selective for gram-positive with a boiling point of l0.7°C and is a highly
than against gram-negative organisms and penetrating gas with a sweet ethereal smell. It
have been used in the laboratory in the is highly inflammable and in concentrations
formulation of selective culture media. They in air greater than 3%, highly explosive. It is
have no activity against tubercle bacilli, and, unsuitable for fumigating rooms because of its
hence, the use of malachite green in the explosive property.
Lowenstein-Jenson medium. It is highly lethal to all kinds of microbes
ii. Acridine dyes: They are more active against including spores and tubercle bacilli.
gram-positive organisms than against gram- Uses
negative but are not as selective as the aniline a. Sterilization of articles liable to be
dyes. The more important dyes are proflavine, damaged by heat: It is specially used for
acriflavine, euflavine and aninacrine. They sterilizing heart-lung machines, respirators,
show no significant differences in potency. sutures, dental equipment, books and
clothing.
4. Alkylating Agents
b. Sterilization of a wide range of materials
i. Formaldehyde: Formaldehyde is lethal to
such as glass, metal and paper surfaces,
bacteria and their spores, viruses and fungi.
clothing, plastics, soil, some foods and
It is employed in the liquid and vapor states.
tobacco.
Formaldehyde is commercially available in
Disadvantages of ethylene oxide
aqueous solutions containing 37% formaldehyde
i. It is irritant, and personnel working with it
(formalin) or as paraformaldehyde, a solid
have to take strict precautions.
polymer that contains 91% to 99 % formaldehyde.
ii. Its use as a disinfectant presents a potential
Uses
toxicity to human beings, including
a. Formalin
mutagenicity and carcinogenicity. Baci
i. Used for preserving fresh tissues for
llus globigi, a red-pigmented variant of
histological examination and is the
B. subtilis, has been used to test ethylene
major component of embalming fluids.
oxide sterilizers.
ii. Used extensively to inactivate viruses in
2. Formaldehyde gas: It is used for fumigation of
the preparation of vaccines.
complex heat-sensitive equipment, including
b. Formaldehyde
anaesthetic machine and baby incubators and
i. Used for preser ving anatomical
for periodic decontamination of laboratory safety
specimens.
cabinets.
ii. Used for destroying anthrax spores in
Fumigation of operation theaters and other
hair.
rooms: This is also widely employed for
iii. Used as an antiseptic mouthwash.
fumigation of operation theaters and other
iv. Used for the disinfection of membranes
rooms (such as isolation rooms). After sealing
in dialysis equipment.
the windows and other outlets, formaldehyde
v. Used as a preservative in hair shampoos.
gas is generated by adding KMnO4 to formalin.
ii. Glutaraldehyde: This has an action similar The reaction produces considerable heat, and
to formaldehyde. It has a broad-spectrum so heat-resistant vessels should be used. After
action against vegetative bacteria including starting generation of formaldehyde vapour, the
mycobacteria, fungi and viruses, but acts more doors should be sealed and left unopened for
slowly against spores. It is more active and 48 hours.
less toxic than formaldehyde. It is used as 2% Formaldehyde has an extremely unpleasant
buffered solution. It can be used for delicate odor and is irritant to mucus membranes.
instruments having lenses. It is available 3. B e ta p r o p i o l a c t o n e ( B P L ) : T h i s i s a
commercially as ‘cidex’. condensation product of ketane and formal
Uses dehyde with a boiling point of 163°C. It also
i. Cold sterilant: It has been used increasingly destroys microorganisms more readily than
as a cold sterilant for surgical instruments ethylene oxide but does not penetrate materials
and endoscopes such as cystoscopes, well and may be carcinogenic. For sterilization
endoscopes and bronchosope. of biological products, 0.2% BPL is used. It is
ii. Used safely to sterilize corrugated rubber capable of killing all micro-organisms and is
anesthetic tubes and face-masks, plastic very active against viruses.
endotracheal tubes, metal instruments and Use: In the liquid form, it has been used to
polythene tubing. sterilize vaccines and sera.
www.ebook3000.com
34 | Section 1: General Bacteriology
RECOMMENDED CONCENTRATIONS OF Table 5.3 Various procedures of sterilization/
VARIOUS DISINFECTANTS disinfection of some important materials
www.ebook3000.com
36 | Section 1: General Bacteriology
4. Sterilization at 100°C for 20 minutes on three 10. The most widely used disinfectant for human
successive days is known as: immunodeficiency virus (HIV) infected material is:
a. Tyndallization b. Inspissation a. Phenol b. Lysol
c. Pasteurization d. Vaccine bath c. Hypochlorite solution d. Silver nitrate
5. Which bacterial spores are used as sterilization 11. Glutaraldehyde is used as a cold sterilant for
control in autoclave? sterilization of:
a. Bacillus cereus a. Cystoscopes b. Endoscopes
b. Bacillus stearothermophilus c. Bronchosope d. All of the above
c. Clostridium perfingens 12. All the following statements are true for ethylene
d. Pseudomonas aeruginosa oxide except:
6. Autoclave is not useful for sterilization of: a. It diffuses through many types of porous
a. Disposable plastic Petri dishes materials
b. It is used for sterilizing heart-lung machines,
b. Surgical dressings
respirators, books and clothing
c. Metallic instruments
c. It is used for sterilizing glass, metal and paper
d. Liquid paraffin
surfaces, clothing, plastics, some foods
7. Which of following is most effective for sterilizing d. It is suitable for fumigating rooms
mattresses and Petri dishes: 13. Which test is used to simulate the natural conditions
a. Chlorine b. Autoclaving under which the disinfectants are used in the
c. Ethylene oxide d. Glutaraldehyde hospitals?
8. Which of the these disinfectants does not act by a. Kelsey-Sykes capacity test
disrupting the plasma membrane? b. Rideal-Walker test
a. Phenolics b. Ethylene oxide c. In-use test
c. Halogens d. Phenol d. Chick-Martin test
9. Ionizing radiation can be used for sterilization of: Answers (MCQs)
a. Plastic syringes b. Gloves 1. b; 2. a; 3. c; 4. a; 5. b; 6. d; 7. c; 8. c; 9. d; 10. c; 11. d;
c. Catheters d. All of the above 12. d; 13. a
6
Chapter
Culture Media
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Differentiate between the following: enriched
able to: media and enrichment media; indicator media and
∙∙ Describe classification of media differential media; selective media and differential
media with suitable examples.
www.ebook3000.com
38 | Section 1: General Bacteriology
in broth’s appearance from clear to turbid (i.e. and used as a general purpose media, e.g.
cloudy). peptone water, nutrient broth and nutrient agar
(Tables 6.2 and 6.3).
2. Solid (Agar) Media 2. Complex media: Media that contain some
ingredients of unknown chemical composition
Solid media are made by adding a solidifying
are called complex media. One common
agent to the nutrients and water. Agarose is the
ingredient is peptone. Extracts, which are the
most common solidifying agent. The Petri dish
water-soluble components of a substance, are
containing the agar is referred to as agar.
also used.
3. Semisolid Media Nutrient broth: A commonly used complex
medium, nutrient broth (in liquid form). It is a
For special purposes where agar is added to media simple basal liquid medium, supports growth of
in concentrations that are too low to solidify them. many organisms.
www.ebook3000.com
40 | Section 1: General Bacteriology
v. Differential Media
A medium, which has substances incorporated in
it, enabling it to bring out differing characteristics
of bacteria and thus helping to distinguish between
them, is called a differential medium. Fig. 6.6: MacConkey agar
Example
MacConkey agar (Fig. 6.6): MacConkey agar is
both differential and selective. It contains peptone,
meat extract, NaCl, bile salt, lactose and neutral
red indicator.
Lactose fermenters (LF) form pink or red color
colonies and nonlactose fermenters (NLF) form
colorless or pale colonies.
www.ebook3000.com
42 | Section 1: General Bacteriology
acts as a reducing agent and creates an anaerobic
Selective media: By inhibiting unwanted organisms
environment deeper in the tube, and allows selective media allow growth of only the desired
anaerobic bacteria to grow. Glucose and microbes, e.g. deoxycholate citrate agar (DCA);
thioglycollate maintain the anerobic condition. Lowenstein Jensen medium (L.J medium)
Agar (0.05%) prevents convection currents of air. Differential media or indicator media distinguish one
Methylene blue or resazurin act as an oxidation- microorganism from one another growing on the
same media, e.g. MacConkey medium
reduction potential indicator, which should show
Sugar media usually consist of 1% sugar in peptone
that the medium is anerobic except in the surface water along with an appropriate indicator
layer in addition to a reducing agent and semi Transport media are used to maintain the viability of
solid agar. certain delicate organisms during their transport to
the laboratory e.g. Stuart’s transport medium
Anaerobic media: Reducing media chemically
ii. Cooked Meat Broth or RCM Broth
remove molecular oxygen that might interfere with
(Robertson’s Cooked Meat Broth) for more the growth of anaerobes.
details, refer to Chapter 7
Important questions
Media to Test Special Properties
1. Distinguish between a selective medium and a
Various media to test special properties like urease differential medium.
production, and composite media for simultane 2. Write short notes on:
ous demonstration of different features have been a. Enriched media
devised. They are dealt with in the appropriate b. Enrichment media
chapters. c. Indicator media
d. Differential media
f. Transport media
Process of Media Making g. Anaerobic media
It is essential to monitor the quality of culture h. Cooked meat broth (CMB) or RCM broth.
media at all stages in their preparation and use.
Culture media used to be prepared in laboratories Multiple choice questions (MCQs)
themselves, starting with basic ingredients. Not only
was this laborious, but it also led to considerable 1. The important source of nutrition for bacteria to
batch variation in the quality of media. The process grow is:
a. Agar b. Electrolytes
of media making has become simpler and its
c. Inorganic salts d. Peptone
quality more uniform with the ready availability of
2. All the following are examples of enriched media
commercial dehydrated culture media. except:
Key Points a. Blood agar b. Chocolate agar
c. Loeffler’s serum slope d. Bile salt agar
A culture medium is any material prepared for the
growth of bacteria in a laboratory 3. All the following are examples of selective media
Agar is a common solidifying agent for a culture except:
medium a. Potassium tellurite medium
Simple media include the nutrient broth and peptone b. Deoxycholate citrate agar
water, which form the basis of other media (e.g., c. Lowenstein-Jensen medium
nutrient broth, nutrient agar, etc.) d. Nutrient agar
Enriched media are solid media supplemented with
4. Which enrichment medium is preferred to grow
blood, serum, etc. (e.g. blood agar, chocolate agar,
Vibrio cholerae?
Loffler’s serum slope, Lowenstein-Jensen medium)
Enrichment media: An enrichment culture is used a. Tetrathionate broth b. Selenite F broth
to encourage the growth of a particular micro- c. Alkaline peptone water d. All of the above
organism in a mixed culture and are the liquid media Answers (MCQs)
(e.g. selenite F broth or tetrathionate broth)
1. d; 2. d; 3. d; 4. c.
7
Chapter
Culture Methods
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Explain the principle and describe uses of the
able to: following: Mclntosh and Fildes anaerobic jar;
∙∙ Discuss anaerobic culture methods cooked meat broth (CMB).
Introduction
In the clinical laboratory, the indications for
culture are mainly to:
1. Isolate bacteria in pure culture
2. Demonstrate their properties
3. Obtain sufficient growth for preparation of
antigens and for other tests.
4. Type isolates
5. Determine sensitivity to antibiotics
6. Estimate viable counts
7. Maintain stock cultures.
www.ebook3000.com
44 | Section 1: General Bacteriology
soaked in the bacterial culture or suspension. 6. Liquid Culture
After incubation, lawn culture provides a uniform Liquid cultures in tubes, bottles or flasks may be
growth of the bacterium. inoculated by touching with a charged loop or by
adding the inoculum with pipettes or syringes.
Uses
i. Antibiotic susceptibility testing Uses
ii. Bacteriophage typing i. Blood culture and for sterility.
iii. For preparation of bacterial antigens and ii. Dilution in the medium: For inocula containing
vaccines—when a large amount of growth is antibiotics and other inhibitory substances.
required on solid media. iii. Large yields.
www.ebook3000.com
46 | Section 1: General Bacteriology
the inlet tube connected to a hydrogen supply. iii. Sulfhydryl compounds (present in cysteine)
Hydrogen is drawn in rapidly. As soon as this inrush also contribute for a reduced oxidation-
of gas has ceased, the inlet tap is also closed and the reduction (OR) potential.
jar is held on the bench for 10 minutes.
Method
Catalyst Liquid media should be prereduced by holding
After the jar is filled with hydrogen, the electrical in a boiling water bath for 10 minutes to drive off
terminals are connected to a current supply to heat dissolved oxygen, then quickly cooled to 37°C just
the catalyst and if room temperature catalyst is before use. The surface of the CMB medium may
used, heating is not required. The catalyst will help be covered with a layer of sterile liquid paraffin for
to combine hydrogen and residual oxygen to form strict anaerobiosis.
water. The jar is then incubated at 37°C.
Interpretation
Indicator It permits the growth of even strict anaerobes and
An indicator should be employed for verifying the indicates their saccharolytic or proteolytic activities.
anaerobic condition in the jars. Reduced methylene With growth of saccharolytic anaerobes (C. welchi),
blue is generally used as indicator (mixture of color of meat pieces turns red while it becomes
NaOH, methylene blue and glucose). It becomes black in case of proteolytic anaerobes (C. tetani).
colorless anaerobically but regains blue color on
exposure to oxygen. Uses of CMB
i. CMB is suitable for growing anaerobes in air
Disadvantage ii. Also for the preservation of stock cultures of
Any anaerobic jar system has the major disadvantage aerobic organisms.
that the plates have to be removed from the jar to be The inoculum is introduced deep in the
examined and this, of course, exposes the colonies medium in contact with the meat.
to oxygen, which is especially hazardous to the
anaerobes during their first 48 hours of growth. F. Other Anaerobic Culture Systems
a. Anaerobic cabinets: The advantage of anaerobic
E. By Reducing Agents cabinets is that all of the processing, including
periodic examination of plates and preparation
Oxygen in culture media can be reduced by various
of subcultures, can be done without exposure
agents and these reducing agents include glucose,
to oxygen.
ascorbic acid, cysteine, sodium mercaptoacetate
or thioglycollate, or the particles of meat in cooked b. Anaerobic bags or pouches: Anaerobic bags are
meat broth. available commercially.
i. Thioglycollate broth: (See Chapter 6: Culture
Methods of isolating pure cultures
Media)
ii. Cooked meat broth (CMB): Cooked meat 1. Surface plating: It is routinely employed in
broth (CMB) original medium known as clinical bacteriology.
‘Robertson’s cooked meat (RCM) medium’) 2. Use of selective, enrichment or indicator media:
has a special place in anaerobic bacteriology. are widely used for the isolation of pathogens
It contains nutrient broth and pieces of fat- from specimens, such as feces, with varied flora.
free minced cooked meat of ox heart. Meat 3. Selective treatment of the specimen before
particles are placed in 30 mL bottles to a depth culture
of about 2.5 cm and covered with about 15 i. Heating at 65°C for 30 minutes or at higher
mL broth. If test tubes are used, the surface temperatures for shorter periods.
medium may be covered with a 1 cm layer of iii. Pretreatment of specimens with appropriate
sterile liquid paraffin, but this is not essential. bactericidal substances: This method is
the standard practice for the isolation of
Principle tubercle bacilli from sputum and other
i. Unsaturated fatty acids present in meat utilize clinical specimens.
oxygen for auto-oxidation, the reaction is 4. Use of selective growth conditions
being catalyzed by hematin in the meat. i. Separation of bacteria with different
ii. Certain reducing substances, such as gluta temperature optima
thione and cysteine present in meat also ii. Cultivation under aerobic or anaerobic
utilize oxygen. conditions.
Chapter 7: Culture Methods | 47
5. Separation of motile from nonmotile bacteria 3. Write short notes on:
can be effected using Craigie’s tube. a. Streak culture
6. Animal inoculation: Laboratory animals are b. Lawn culture
highly susceptible to certain organisms; for c. Stroke culture
example, the mouse to the pneumococcus. d. Pour-plate culture
7. Filters: Used for separating viruses from e. Anaerobic culture methods/culture of anaerobic
bacteria. bacteria.
f. McIntosh-Fields’ anaerobic jar
Key Points
g. Cooked meat broth (CMB) or/RCM broth
The methods of bacterial culture used in the clinical h. Methods of isolating pure cultures.
laboratory include streak culture, lawn culture, stroke
culture, stab culture, pour-plate culture, shake culture
and liquid culture. Special methods are employed for MulTiple choice questions (MCQs)
culturing anaerobic bacteria.
Streak culture (surface plating): This method is 1. The most useful method for obtaining discrete
routinely employed for the isolation of bacteria in colonies of the bacteria is by:
pure culture from clinical specimens. a. Lawn culture b. Stab culture
Anaerobic culture methods: Anaerobic bacteria c. Pour-plate culture d. Streak culture
require incubation without oxygen. 2. The most useful method for obtaining a uniform
Anaerobic jars: Anaerobiosis obtained by Mclntosh
layer of bacterial growth on a solid medium is by:
and Fildes’ anaerobic jar is the most dependable and
widely used method.
a. Lawn culture b. Stab culture
Cooked meat broth (CMB): Original medium known as c. Pour-plate culture d. Streak culture
‘Robertson’s cooked meat (RCM) medium’ has a special 3. All the following are used for anaerobic culture
place in anaerobic bacteriology. except:
a. Robertson’s cooked meat broth
Important questions b. Thioglycollate broth
c. Nutrient broth
1. Discuss in detail anaerobic culture methods. d. McIntosh and Fildes’ anaerobic jar.
2. Enumerate various methods of bacterial culture.
Describe in detail the anaerobic methods of Answers (MCQs)
cultivation. 1. d; 2. a; 3. c
www.ebook3000.com
8
Chapter
Identification of Bacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should reduction test; 7. Urease test; 8. Catalase production;
be able to: 9. Oxidase test; 10. Phenylalanine deaminase test;
∙∙ Explain principle and discuss interpretation of 11. Hydrogen sulfide production; 12. Triple-sugar
the following biochemical reactions: 1. Sugar Iron (TSI)
fermentation; 2. Indole production; 3. Methyl ∙∙ List various examples of bacteria giving positive
red (MR) test; 4. Voges-Proskauer (VP) test for biochemical reactions for mentioned above.
acetoin production; 5. Citrate utilization; 6. Nitrate
METHODS USED TO IDENTIFY BACTERIA nature of the culture medium, temperature and
time of incubation, age of the culture and the
Once a bacterium has been obtained in pure culture, number of subcultures it has undergone. The
it has to be identified. Identification schemes are characteristics noted are shape, size, side ends,
classified into one of the two categories (Table 8.1): arrangement, and irregular forms, motility, flagella
1. Phenotypic characteristics fimbriae, spores, capsule and staining.
2. Genotypic characteristics.
B. Staining Reactions
1. PHENOTYPIC CHARACTERISTICS
A number of staining techniques for the identifi
A. Microscopic Morphology cation of bacteria, are available. Of these, Gram
The morphology of the bacterium depends on stain and Ziehl-Neelson stain are most important.
a number of factors, such as the strain studied, Gram stain: The Gram stain divides bacteria into
gram-positive and gram-negative.
Table 8.1 Methods used to identify bacteria
Ziehl-Neelsen staining: With Ziehl-Neelsen stain
Phenotypic characteristics Genotypic characteristics ing divides bacteria into acid fast and non-acid fast.
A. Microscopic Nucleic acid hybridization Numerous other stains are used for special
morphology PCR-amplifying specific purposes, such as demonstration of flagella,
B. Staining reactions DNA sequences capsule, spores, and metachromatic granules. The
C. Metabolism differences Sequencing rRNA genes
fluorescent antibody technique enables one to
i. Macroscopic
morphology identify them according to their surface antigens.
or cultural
characteristics C. Metabolic Differences
ii. Fermentation and
The requirements of oxygen, the need for carbon
other biochemical
reactions dioxide, the capacity to form pigments, and the
D. Serology production of hemolysis help in classification.
E. Antibiotic tolerance
(resistance) tests, dye i. Cultural Characterstics or Macroscopic
tolerance, and other Morphology
inhibition tests
F. Bacteriophage and These provide additional information for the
bacteriocin typing identification of the bacterium. The characteristics
G. Pathogenicity revealed in different types of media are noted.
www.ebook3000.com
Chap-08.indd 49 15-03-2016 11:10:58
50 | Section 1: General Bacteriology
Gas production can be seen as bubbles in
Durham’s tube.
2. Indole Production
Principle: This test demonstrates the ability of
certain bacteria to decompose the amino acid
tryptophane to indole, which accumulates in the
medium. Tryptophan is decomposed by an enzyme
tryptophanase produced by certain bacteria.
Fig. 8.5: Indole test
Method: Indole production is detected by
inoculating the test bacterium into peptone water
(tryptophan rich) and incubating it at 37°C for 48–96
hours. Add 0.5 mL Kovac’s reagent and shake gently.
Tryptophan Tryptophanase
→ Indole
Kovac’s reagent consists of:
∙∙ Paradimethylaminobenzaldehyde 10 g
∙∙ Amyl or isoamyl alcohol 150 mL
∙∙ Concentrated hydrochloric acid 50 mL.
Fig. 8.7: Voges-Proskauer (VP) test Fig. 8.8: Citrate utilization test
www.ebook3000.com
Chap-08.indd 51 15-03-2016 11:11:01
52 | Section 1: General Bacteriology
www.ebook3000.com
Chap-08.indd 53 15-03-2016 11:11:02
54 | Section 1: General Bacteriology
and lactose or sucrose fermentatively and forms the slant acidic (yellow), provided the reaction
hydrogen sulfide (H2S). TSI medium facilitates is read in 18–24 hours. Consequently, when the
preliminary identification of gram-negative bacilli. tube is examined at the end of 18–24 hours, acid
production from fermentation of lactose is still
Media occurring and both the slant and the deep appear
TSI is a composite medium and contains 10 parts yellow, resulting in an acid-slant-acid d
eep reaction.
lactose: 10 parts sucrose: 1 part glucose and Reactions in TSI should not be read beyond 24
peptone. Phenol red and ferrous sulfate serve hours of incubation, because aerobic oxidation
as indicators of acidification and H2S formation, of the fermentation products from lactose and/or
respectively. The medium is distributed in tubes, sucrose does proceed and the slant will eventually
with a butt and slant. revert to the alkaline state.
H2S producing bacteria: Certain bacteria produce
Procedure
H2S which is a colorless gas. H2S combines with
Medium is inoculated with bacterial culture by a ferric ions (from ferric salts) to form ferous
straight wire pierced deep in the butt (stab culture). sulfide as black precipitate and is manifested by
Incubate the tube at 35° C in ambient air for 18–24 blackening of the butt of the medium in the tube
hours.
Glucose-fermenting organism: If the tube is Interpretation (Table. 8.3)
inoculated with a glucose-fermenting organism Red color (alkaline): No fermentation.
that cannot utilize lactose, only a relatively small
Yellow color (acidic): Fermentation of carbo
quantity of acid can be obtained from the 0.1%
hydrate.
concentration of glucose in the medium. Initially
the entire medium becomes acidic (yellow) in Bubbles in the butt: Gas is also produced during
8–12 hours. However, within the next few hours, fermentation of carbohydrate.
the glucose supply is completely exhausted and
Blackening of the medium: H2S production.
the bacteria begin oxidative degradation of the
To determine the ability of an organism to
amino acids within the slant portion of the tube
attack specific carbohydrates incorporated in a
where oxygen is present. This results in the release
growth medium, with or without the production
of amines that soon counteract the small quantities
of gas, along with the determination of possible
of acid present in the slant. The entire slant reverts
hydrogen sulfide (H2S) production.
to an alkaline pH (color returns to red) by 18–24
hours. The deep (anaerobic portion) of the tube
remains acidic (yellow) because amino acid degra D. Serology
dation is insufficient to counteract the acid formed. By using specific sera we can identify organisms
by agglutination or other suitable serological
Lactose-fermenting organism: If the tube is
reactions.
inoculated with a lactose-fermenting organism,
then fermentation continues as the organism
is able to use lactose (present in 10 times the E. Antibiotic Tolerance Tests, Dye
concentration of glucose), even though the glucose Tolerance, and Other Inhibition Tests
is completely used up after the first 8–12 hours. These range from disk tests of resistance to
Therefore, when, in addition to glucose, lactose antibiotics, such as penicillin, bacitracin,
and/or sucrose are fermented, the large amount gentamicin, novobiocin and metronidazole, to
of fermentation products formed on the slant will special tests that demonstrate tolerance of dyes
more neutralize the alkaline amines and render and other chemicals.
www.ebook3000.com
Chap-08.indd 55 15-03-2016 11:11:02
9
Chapter
Bacterial Taxonomy
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Discuss bacterial classifications.
www.ebook3000.com
10
Chapter
Bacterial Genetics
Learning Objectives
After reading and studying this chapter, you should and conjugation in the transfer of genetic material
be able to: from one bacterium to another
∙∙ Explain Lac operon ∙∙ Discuss resistance transfer factor (RTF)
∙∙ Discuss regulation or control of gene expression ∙∙ Differentiate between mutational and plasmid-
mediated drug resistance
∙∙ Discuss structure and functions of the plasmids
∙∙ Discuss transposons
∙∙ Describe mutations ∙∙ Describe principle and clinical applications
∙∙ Discuss various methods of gene transfer of the following: Nucleic acid probes; genetic
∙∙ Differentiate among the mechanisms of trans engineering; polymerase chain reaction
formation, transduction, lysogenic conversion ∙∙ Describe gene therapy.
www.ebook3000.com
60 | Section 1: General Bacteriology
A. Phenotypic Variation
The phenotype (‘Phaeno’; display) is the physical
expression of various characters by bacterial cells
in a given environment. These properties are
determined not only by its genome (genotype), Fig. 10.4: Frameshift mutation
but also by its environment. Phenotypic variations
are reversible. Also important, but not part of the operon, is a
distant regulatory gene (in this case is LacI) which
codes for a repressor protein. It is a protein molecule
Examples of Environmental Influence on
which can combine with either operator region on
Bacteria
the chromosome or with the inducer (lactose).
1. Synthesis of flagella: The typhoid bacillus is
normally flagellated. But the flagella are not Effect of Lactose on the Control of the Lactose
synthesized when grown in phenol agar and is Operon
reversed when subcultured from phenol agar For transcription to occur as the first stage in
into broth. protein synthesis, RNA polymerase has to attach to
2. Synthesis of enzyme: Another example of DNA at a specific promoter region and transcribe
environmental influence is the synthesis the DNA in a fixed direction. In resting stage when
by Escherichia coli of the enzyme beta- lactose (inducer) is not present in the medium,
g a l a c t o s i d a s e, n e c e s s a r y f o r l a c t o s e repressor molecule is bound to the operator,
fermentation but the actual synthesis takes preventing the passage of RNA polymerase from
place only when it is grown in a medium promoter to the structural genes. The repressor
containing lactose. molecule has an affinity for lactose, in the presence
Such enzymes which are synthesized only of which it leaves the operator region free enabling
when induced by the substrate are called the transcription to take place. When lactose
induced enzymes. The enzymes which are present is completely metabolized, the repressor
synthesized irrespective of the presence or again attac hes to the operator, switching off
absence of the substrate are called constitutive transcription. Lactose acts both as an inducer and
enzymes. the substrate for the enzyme.
www.ebook3000.com
62 | Section 1: General Bacteriology
Mutagens
1. Physical agents: (i) UV rays; (ii) lonizing
radiation, e.g. X-rays; (iii) Visible light; (iv) Heat
2. Chemical agents: (i) Alkylating agents; (ii)
Acridine dyes ; (iii) 5-Bromouracil; (iv)
2-aminopurine; (v) Nitrous acid.
C. Point mutations: As the name suggests, point
mutations affect just one point (base pair)
Fig. 10.5: Examples of types of mutation. A portion of the wild in a gene. Such mutations may be a change
type chromosome is shown on the top sequence from which to or substitution of a different base pair.
different mutational rearrangements are derived
Alternatively, a point mutation can result in
the deletion or addition of a base pair. It is, in
Mutation is a natural event, taking place all the general, reversible and is of two classes:
time at its particular frequency in all the dividing 1. Base pair substitution: This comprises those
forms of life. Particular mutations occur at fairly mutants in which a single base pair (nucleotide)
constant rates, normally between once per 104 and has been substituted for another pair, and can
once per 1010 cell divisions. be subdivided into transition (one purine is
Though mutations are taking place all the time, replaced by other purine or a pyrimidine is
most mutants go unrecognized as the mutation replaced by other pyrimidine) and transversion
may involve minor function or it may be lethal. (substitution of a purine for a pyramidine and
Mutation is best appreciated when it involves a vice versa in base pairing).
function which can be readily observed. Mutants
can be detected by selecting or testing for an altered Normal sequence: The Fat Cat Ate The Rat.
phenotype. For example, an E. coli mutant that Substitution: The Fat Can Ate The Rat.
loses its ability to ferment lactose can be readily
Depending on the placement of the substituted
detected on MacConkey agar but is unrecognizable
base, when the mRNA is translated this may cause
on nutrient agar.
no change (silent mutation), lead to the insertion
Lethal mutation: Some mutations involve vital of the wrong amino acid (missense mutation)
functions, and such mutants are nonviable (lethal or generate a stop codon (nonsense mutation),
mutation). An important type of lethal mutation prematurely terminating the polypeptide.
is conditional mutation.
Conditional mutation: A conditional lethal mutant Missense mutation: If the triplet code is altered
may be able to live under certain permissive so as to specify an aminoacid different from that
conditions but not under other or nonpermissive normally located at a particular position in the
conditions. The most common type of conditional protein. This is called missense mutation.
mutant is the temperature sensitive (ts) mutant, Nonsense mutation: Deletion of a nucleotide within
which is able to live at the permissive temperature a gene may cause premature polypeptide chain
(say, 35°C), but not at the restrictive temperature termination by generating a nonsense codon (UAG,
(say, 39°C). UAA or UGA). This is known as nonsense mutation.
Recognition of mutation: Mutation can be best
recognized when it involves a function which can 2. Base pair deletion or insetion
be readily observed by experimental methods like Frameshift mutations: If the number of bases
alteration in colonial morphology, pigmentation, inserted or deleted is not a multiple of three, there
alteration in cell surface antigens, sensitivity to will be shift in the reading frame, i.e. frameshift
bacteriophages or bacteriocins, loss of ability to mutations. This shifts the normal ‘reading frame’ of
produce capsule or flagella, loss of virulence and the coded message forming newest of triplet codon.
change in biochemical characters. The coded message is read correctly up to the point
of addition or deletion, but the subsequent codons
Types of Mutation will specify the incorrect amino acids (Fig. 10.4).
Mutations can be divided conveniently into:
A. Spontaneous mutation: Many mutations occur Survival advantage of mutation: When mutation
spontaneously in nature in the absence of any confers a survival advantage, it is of vital importance.
mutation-causing agents. For example, if a streptomycin resistant mutant of
B. Induced mutation: The frequency of mutation is the tubercle bacillus develops in a patient under
greatly enhanced by exposure of cells to several treatment with the drug, it multiplies selectively
agents (mutagens) which may be physical or and ultimately replaces the original drug sensitive
chemical. population of bacteria.
Chapter 10: Bacterial Genetics | 63
Importance of Bacterial Mutation
1. Drug resistance and developm ent of live
vaccines: The practical importance of bacterial
mutation is mainly in the field of drug resistance
and development of live vaccines.
www.ebook3000.com
64 | Section 1: General Bacteriology
Griffith’s Experiment Demonstrating Genetic them genetically so that their progeny were
Transformation encapsulated and therefore virulent.
i. Griffith (1928) found that injections of living The nature of the transforming principle was
encapsulated bacteria killed the mouse (Fig. identified as DNA by Oswald T. Avery and
10.9). his associates Colin M MacLeod and Maclyn
ii. Injections of live nonencapsulated bacteria McCarty in the United States in 1944.
or dead encapsulated bacteria did not kill the Transformation and Bacteria: Transformation
mouse. occurs naturally among very few genera of bacteria,
iii. When the dead encapsulated bacteria were including Bacillus, Haemophilus, Neisseria,
mixed with live nonencapsulated bacteria and Acinetobacter, and certain strains of the genera
injected into the mice, many of the mice died. Streptococcus and Staphylococcus.
In the blood of the dead mice, Griffith found
living, encapsulated bacteria. Hereditary B. Transduction
material (genes) from the dead bacteria
had entered the live cells and changed The transfer of a portion of the DNA from one
bacterium to another by a bacteriophage is known
as transduction. Bacteriophages are viruses that
parasitise bacteria and consist of a nucleic acid
core and a protein coat. Most bacteriophages carry
their genetic information (the phage genome) as a
length of double-stranded DNA coiled up inside
a protein coat. When bacteriophages multiply
inside an infected bacterial cell, each phage head is
normally filled with a copy of the replicated phage
genome. During the assembly of bacteriophage
progeny inside infected bacteria, ‘packaging
errors’ may occur occasionally. A phage particle
may have at its core a segment of the host DNA
besides its own nucleic acid. When this particle
infects another bacterium, DNA transfer is affected
and the recipient cell acquires new characteristic
coded by the donor DNA. Bacterial genes have
been transduced by the phage into the second cell
Fig. 10.8: The mechanism of genetic transformation in
bacteria
(Fig.10.10).
www.ebook3000.com
66 | Section 1: General Bacteriology
www.ebook3000.com
68 | Section 1: General Bacteriology
Genetic mechanisms of drug Table 10.1 Comparison of mutational and trans
resistance in bacteria ferable drug resistance
Mutational drug Transferable drug
Mechanisms of Drug Resistance in resistance resistance
Bacteria 1. Resistance to one 1. Multiple drug resistance
A. By mutation (chromosomal) drug at a time at one time
B. By genetic exchange. 2. Low degree resistance 2. High degree resistance
3. Can overcome by high 3. High dose ineffective
A. By Mutation (Chromosomal) drug dose
Mutational resistance is mainly of two types: 4. Development of 4. Development of drug
1. Stepwise mutation: The stepwise mutation, drug resistance resistance cannot be
as seen with penicillin, where high levels of can be prevented prevented treatment
by treatment with with combination of
resistance are achieved only by a series of combination of drug drugs
small-step mutations. 5. Resistance is not 5. Resistance is transferable
2. Single large-step mutation or ‘one-step’ mutation: transferable to other organisms
As seen with streptomycin, the drug target is 6. Mutants may 6. Not defective
altered by mutation so that it is totally unable be metabolically
to bind a drug. defective
7. Virulence may be low 7. Virulence not decreased
Clinical importance in tuberculosis: Mutational
resistance is of clinical importance in tuberculosis.
If a patient is treated with streptomycin alone are larger (4–25 Kb) composite elements
initially, the bacilli die in large numbers but soon containing genes for movement as well as
resistant mutants appear and multiply unchecked. genes that encode for various functions, such
If two or more antituberculous, drugs are used for as drug resistance and toxin production. In the
combined treatment, repopulation by resistant 1950s, American geneticist Barbara McClintock
mutants does not occur, as a mutant resistant to discovered transposons in corn during her studies
one drug will be destroyed by the other drug. on maize genetics, for which she was awarded the
Inadequate or inappropriate treatment over the Nobel prize for Medicine in 1983.
years has caused extensive resistance in tubercle
bacilli, leading to a pandemic of multidrug resistant
Structure of Transposons
tuberculosis (MDR TB) across the world. The simplest transposable elements are insertion
sequences (IS), or IS elements. It is a short
B. By Genetic Exchange sequence of DNA that contains a gene that
Transferable antibiotic resistance codes for enzyme. The more complex is called a
1. Transformation: Its significance in nature is not composite transposon.All transposons contain the
known. information for their own transposition.
2. Transduction: Acquisition of resistance by A transposon is a segment of DNA with
transduction is common in staphylococci. one or more genes in the center, and the two
The penicillinase plasmids are transmitted by ends carrying ‘inverted repeat’ sequences of
transduction. nucleotides-nucleotide sequences complementary
3. Conjugation: Plasmids conferring resistance to each other but in the reverse order. Because of
to one or more unrelated groups of antibiotics this feature, each strand of the transposon can
(R plasmids) can be transferred rapidly by form a single-stranded loop carr ying the gene,
conjugation. and a double-stranded stem formed by hydrogen
Transferable drug resistance mediated by the bonding between the terminal inverted repeat
R factor is the most important method of drug sequences (Fig. 10.15).
resistance. Table 10.1 compares mutational and
Significance of Transposons
transferable drug resistance.
1. Mechanism for amplifying genetic transfers
Transposable genetic elements 2. Spread from one organism-or species-to another
3. Antibiotic resistance
Certain structurally and genetically discrete
4. Gene manipulations.
segments of DNA which move around between
chromosomal and extrachrom osomal DNA Molecular Genetics
molecules within cells are called transposons
(‘jumping genes’). This mode of genetic transfer Genetic Engineering
is called transposition. They vary from simple Genetic engineering is a method of inserting foreign
(insertion sequences) to complex. Transposons genes into a bacterium and obtaining chemically
Chapter 10: Bacterial Genetics | 69
useful products. Genetic engineering, also known of higher forms of life including human beings, and
as recombinant DNA (rDNA) technology, uses the their introduction into suitable microorganisms, in
techniques and tools developed by the bacterial which the genes would be functional, directing the
geneticists to purify, amplify, modify, and express production of the specific protein. Such cloning of
specific gene sequences. genes in microorganisms enables the preparation
of the desired protein in pure form, in large
Genetic Engineering Procedure (Fig. 10.16) quantities and at a reasonable cost.
This consists of isolation of the genes coding for any
desired protein from microorganisms or from cells Basic Tools of Genetic Engineering
1. Cloning vectors: Cloning vectors can be used
to deliver the DNA sequences into receptive
bacteria and amplify the desired sequence.
Many types of vectors are currently used
such as plasmid vectors, bacteriophages and
other viruses, cosmid vectors, and artificial
chromosomes.
2. Restriction endonucleases (restriction
enzymes): Restriction enzymes are microbial
enzymes which cleave double-stranded DNA at
specific oligonucleotide sequences. Restriction
enzyme recognizes and cuts, or digests, only
one particicular sequence of nucleotide bases
in DNA, and it cuts this sequence in the same
way each time. These enzymes are present in
many prokaryotes organisms, e.g. restriction
endonuclease Eco RI, Hind Ill, obtained from
E. coli, H. influenzae.
3. DNA ligase: The enzyme that links the fragment
Fig. 10.15: Structure of transposon to the cloning vector.
www.ebook3000.com
70 | Section 1: General Bacteriology
Applications of Genetic Engineering Hybridization: It is the process whereby two single
1. Production of vaccines: Foot and mouth disease, strands of nucleic acid come together to form a
hepatitis B, rabies viruses and DNA vaccine. stable double-stranded molecule.
2. Production of proteins of therapeutic interest: Detection of hybridization: Hybridization assays
Human growth hormones, human insulin, require that one nucleic acid strand (probe)
erythropoietin, blood-clotting factor VIII, originates from an organism of known identity
tissue plasminogen activator, interferons, and the other strand (the target) originates from
tumor necrosis factor, interleukin-1, 2, and unknown organisms to be detected or identified.
3, granulocyte colony stimulating factor, Identification of unknown organism is
epidermal growth factor, fibroblast growth established by positive hybridization (i.e. duplex
factor, somatostatin, growth hormones are formation) between a probe nucleic acid strand
proteins of therapeutic interest. (from unknown organisms) and a target nucleic
C l o n e d h u m a n i n s u l i n , i n t e r f e r o n s, acid strand from the organism to be identified.
somatostatin, growth hormones and many Failure to hybridize indicates lack of homology
other biologicals have already been marketed. between probe and target nucleic acid. Positive
3. Gene therapy. hybridization identifies the unknown organism
4. Others: It has also become essent ial to as being the same as the probe-source organisms.
laboratory diagnosis, forensic science, agricul With a negative hybridization test, the organism
ture, and many other disciplines. remains undetected or unidentified (Fig. 10.17).
www.ebook3000.com
72 | Section 1: General Bacteriology
www.ebook3000.com
74 | Section 1: General Bacteriology
4. Which of the following properties may be plasmid- 10. The transfer of genetic information through the
mediated? agency of free (naked) DNA is called:
a. Antibiotic resistance a. Transformation
b. Enterotoxin production b. Transduction
c. Bacteriocin production c. Conjugation
d. All of the above d. Lysogenic conversion
5. The mutation in which a purine is replaced by 11. The transfer of a portion of DNA from one bacterium
pyrimidine and vice-versa in base pairing, is named: to another by a bacteriophage is known as:
a. Transversion a. Transformation
b. Transition b. Transduction
c. Induced mutation c. Conjugation
d. None of the above d. Lysogenic conversion
6. The mutation in which one purine is replaced 12. Which of the following factors is responsible for
by other purine and a pyrimidine is replaced by transferable drug resistance in bacteria?
another pyrimidine, is named as: a. Resistance transfer factor
a. Transversion b. F factor
b. Transition c. Colicogenic factor
d. All of the above
c. Induced mutation
d. None of the above 13. Resistance transfer factor can be transferred from
7. Frame-shift mutation occurs: one bacterium to another by:
a. When one or more base pairs are added or a. Transformation
deleted in the DNA b. Transduction
c. Conjugation
b. When base substitution in DNA produces a
d. None of the above
terminal codon
c. When one base in the nucleotide sequence is 14. Which of the following statement is true for
inserted in place of another mutational drug resistance?
d. Transposons are integrated into the DNA a. Resistance to one drug at a time
b. Resistance to multiple drugs at one time
8. The DNA is transmitted from one bacterium to
c. High degree resistance
another by all the methods except:
d. Resistance is transferable to other organisms
a. Transformation
b. Transposition 15. Western blotting is done for:
a. Detecting DNA sequesing
c. Transduction
b. Analysis of RNA
d. Conjugation
c. Identification of proteins
9. The process of transfer of DNA from the donor d. All of the above
bacterium to the recipient bacterium during the
mating of two bacterial cells is known as: Answers (MCQs)
a. Transformation b. Transposition 1. d; 2. c; 3. d; 4. d; 5. a; 6. b; 7. a; 8. b; 9. d; 10. a; 11. b; 12.
c. Transduction d. Conjugation a; 13. c; 14. a; 15. c.
11
Chapter
Infection
Learning Objectives
After reading and studying this chapter, you should ∙∙ Name and define various types of carriers
be able to: ∙∙ Discuss modes of spread of infection giving suitable
∙∙ Define the terms saprophytes, parasite, commensal, examples
pathogen ∙∙ List the differences between exotoxins and
∙∙ Describe classification of infections endotoxins.
∙∙ Define and differentiate primary, secondary,
opportunistic and reinfections
www.ebook3000.com
76 | Section 1: General Bacteriology
2. Reinfections: Subsequent infections by the same 1. Convalescent carrier: An individual who has
parasite in the host are termed reinfections. recovered from the infectious disease but
3. Secondary infection: When a new parasite sets continues to harbor large numbers of pathogen.
up an infection in a host whose resistance is 2. Healthy carrier: A healthy carrier is an individual
lowered by a preexisting infectious disease, who harbors the pathogen but is not ill.
this is termed secondary infection. 3. Incubatory carrier: An incubatory carrier is an
4. Local infection: The term local infection individual who is incubating the pathogen in
(more appropriately local sepsis) indicates large numbers but is not yet ill.
a condition where, due to infection or sepsis
4. Temporary carriers: Convalescent, healthy, and
at localized sites such as appendix or tonsils,
incubatory carriers may harbor the pathogen
generalized effects are produced.
for only a brief period (hours, days, or weeks)
5. Cross-infection: When in a patient already suffer
and lasts less than six months.
ing from a disease, a new infection is set up from
another host or another external source, it is 5. Chronic carriers: They harbor the pathogen for
termed cross-infection. long periods (months, years, or life).
6. Nosocomial infections: Cross-infections 6. Contact carriers: The term contact carrier is
occurring in hospitals are called nosocomial applied to a person who acquires the pathogen
infections (from Greek nosocomion hospital). from a patient.
7. Iatrogenic infection: The term iatrogenic infect 7. Paradoxical carrier: This refers to a carrier who
ion refers to physician induced infections acquires the pathogens from another carrier.
resulting from investigative, therapeutic or
other procedures. B. Animals
8. Inapparent infection: Inapparent infection is
Reservoir hosts: Many pathogens are capable
one where clinical effects are not apparent.
of causing infections in both human beings and
9. Subclinical infection: The term subclinical
animals. Therefore, animals may act as a source of
infection is often used as a synonym to
infection of such organisms. These, animals serve to
inapparent infection.
maintain the parasite in nature and act as reservoir
10. Atypical infection: Atypical infection is one
and they are, therefore, called reservoir hosts.
in which the typical or characteristic clinical
manifestations of the particular infectious Zoonosis: The diseases and infections, which
disease are not present. are transmissible to man from animals are called
11. Latent infection: Some parasites, following zoonosis.
infection, may remain in the tissues in a latent
Examples of zoonotic diseases
or hidden form proliferating and producing
clinical disease when the host resistance is Bacterial: Anthrax, brucellosis, Q fever, leptospirosis,
lowered. This is termed latent infection. bovine tuberculosis, bubonic plague, Salmonella
food poisoning.
Sources of infection
Viral: Rabies, yellow fever, cowpox, monkeypox.
A. Human beings
B. Animals Protozoal: Leishmaniasis, toxoplasmosis, trypano
C. Insects somiasis, babesiosis.
D. Soil and water
Helminthic: Echinococcosis, taeniasis, trichinellosis.
E. Food.
Fungal: Microsporum canis, Trichophyton verru
A. Human Beings cosum.
The most common source of infection for human
beings is human beings themselves. The parasite
may originate from a patient or carrier.
C. Insects
Humans serving as the microbial reservoir: Arthropod-borne Diseases
i. Acquisition of “strep” throat through touching Blood-sucking insects, such as mosquitos, ticks,
ii. Hepatitis by blood transfusions mites, flies, and lice may transmit pathogens to
iii. Gonorrhea, syphilis, and AIDS by sexual contact human beings and diseases so caused are called
iv. Tuberculosis by coughing; and the common arthropod borne diseases.
cold through sneezing.
Carrier Vectors
A carrier is person who harbors the microorganisms Insects that transmit infections are called vectors.
without suffering from any ill effect` because of it. Vector-borne transmission can be of two types
There are several types of carriers: either mechanical (external) or biological (internal).
Chapter 11: Infection | 77
i. Mechanical vector: The disease agent is a. Direct contact: Diseases transmitted by direct
transmitted mechanically by the arthropod. contact include STD (sexually transmitted
Examples : Transmission of diarrhea, diseases), such as syphilis, gonorrhea, lympho
dysentery, typhoid, food poisoning and granuloma venereum, lymphogranuloma
trachoma by the housely. inguinale, trichomoniasis, herpes simplex type
ii. Biological vectors: Biological vectors are those 2, hepatitis B and acquired immunodeficiency
in whom the pathogens multiply sufficiently syndrome (AIDS).
or has undergone a developmental cycle. b. Indirect contact: Fomites: Indirect contact may
The interval between the time of entry of be through the agency of fomites, which are
the pathogen into the vector and the vector inanimate objects, such as clothing, pencils or
becoming infective is called the extrinsic toys which may be contaminated by a pathogen
incubation period. from one person and act as a vehicle for its
Examples: Aedes aegypti mosquito in yellow fever, transmission to another.
Anopheles mosquito in malaria.
2. Inhalation
Reservoir hosts: Besides acting as vectors, some
Droplet nuclei: Respiratory infections, such
insects may also act as reservoir hosts (for example,
as common cold, influenza, measles, mumps,
ticks in relapsing fever and spotted fever). Infection
tuberculosis and whooping cough are acquired
is maintained in such insects by transovarial or
by inhalation.
transstadial passage.
3. Ingestion
D. Soil and Water
Intestinal infections are generally acquired by the
i. Soil ingestion of food or drink contaminated by path
Some pathogens can survive in the soil for long ogens. Infection transmitted by ingestion may be
periods. waterborne (cholera), food borne (food poisoning)
Examples or handborne (dysentery). Diseases transmitted
a. Spores of tetanus and gas gangrene: Spores of by water and food include chiefly infections of
tetanus and gas gangrene remain viable in the the alimentary tract, e.g. acute diarrheas, typhoid
soil for several decades and serve as source of fever, cholera, polio, hepatitis A, food poisoning
infection. and intestinal parasites.
b. Fungi and parasites: Fungi (causing mycetoma,
4. Inoculation
sporotrichosis, histoplasmosis) and parasites
The disease agent may be inoculated directly in
such as roundworms and hookworms also
to the skin or mucosa, e.g. rabies virus depos
survive in the soil and cause human infection.
ited subcutaneously by dog bite, tetanus spores
implanted in deep wounds, and arboviruses
ii. Water injected by insect vectors.
Water may act as the source of infection Infection by inoculation may be iatrogenic
either due to contamination with pathogenic when unsterile syringes and surgical equipment
microorganisms (Shigella, Salmonella, Vibrio are employed. Hepatitis B and the human
cholerae, poliomyelitis virus, hepatitis virus) immunodeficiency virus (HIV).
or due the presence of aquatic vector (cyclops
containing larvae of guinea worm infection). 5. Insects
Vector-borne
E. Food Vector is defined as an arthropod or any living
Contaminated food may act as source of infection of carrier (e.g. snail) that transports an infectious
organisms causing food poisoning, gastroenteritits, agent to a susceptible individual. In some diseases,
diarrhea and dysentery. blood-sucking insects play an important role in the
spread of infection from one individual to another
Modes of Transmission of Infection (Refer to Chapter 85).
www.ebook3000.com
78 | Section 1: General Bacteriology
herpes virus), varicella virus, syphilis, hepatitis B, 3. Invasiveness
Coxsackie B and AIDS. Invasiveness signifies the ability of a pathogen to
spread in the host tissues after establishing infec
7. Iatrogenic and Laboratory Infections tion. Highly invasive pathogens characteristically
If meticulous care in asepsis is not taken, infections produce spreading or generalized lesions (e.g. strep
like AIDS and hepatitis B may sometimes be tococcal septicemia following wound infection),
transmitted during administration of injections, while less invasive pathogens cause more localized
lumber puncture and catheterization. These lesions (e.g. staphylococcal abscess).
are known as iatrogenic or physician-induced
infections. Modern methods of treatment, such 4. Toxigenicity
as exchange transfusion, dialysis, and heart and Some bacteria cause disease by producing toxins,
transplant surgery have increased the possibilities of which there are two general types: The exotoxins
for iatrogenic infections. and the endotoxins (Table 11.1).
Adhesins: The initial event in the pathogenesis is 9. Highly antigenic 9. Weakly antigenic
the attachment of the bacteria to body surfaces. 10. Action specifically neu 10. Neutralization by
This attachment is not a chance event but a specific tralized by antibody antibody ineffective
reaction between surface receptors on host cells 11. Usually do not produce 11. Usually produce
and adhesive structures (ligands) on the surface fever fever by release of
interleukin-1
of bacteria. These adhesive structures are called
adhesins. 12. Produced by both 12. Produced by gram-
gram-positive bacteria negative bacteria only
Adhesions may occur as organized structures, and gram-negative
such as fimbriae or fibrillae and pilli, or as bacteria
colonization factors. 13. Frequently controlled 13. Synthesized directly by
Adhesins as virulence factors: Adhesins are by extrachromosomal chromosomal genes
usually made of protein and are antigenic in nature. genes (e.g. plasmids)
Specific immunization with adhesins has been 14. Disease examples- 14. Gram-negative
attempted as a method of prophylaxis in some Botulism diphtheria infections,
tetanus meningococcemia
infections.
Chapter 11: Infection | 79
in minute amounts and include some of the most d. Antigenic Variation
poisonous substances known. Variation in surface antigen composition during
Treatment with formaldehyde converts the course of infection provides a mechanism of
exotoxin into toxoids, are thus useful in preparing avoidance of specific immune responses directed
vaccines. at those antigens.
They exhibit specific tissue affinity and Examples: (i) Pathogenic Neisseria; (ii) The borreliae
pharmacological activities. generate antigenic variation.
They are associated with specific diseases and
have specific mechanisms of action.
e. Serum Resistance
They are easily inactivated by formaldehyde,
iodine, and other chemicals to form immunogenic To survive in the blood, bacteria must be able to
toxoids. resist lysis.
Exotoxins are generally formed by gram-
positive bacteria but may also be produced by some f. Siderophore and Iron Acquisition
gram-negative organisms such as Shiga’s dysentery Siderophores can acquire iron from the host’s iron
bacillus, cholera vibrio and enterotoxigenic E. coli. binding proteins.
b. Endotoxins 6. Enzymes
These are heat-stable, lipopolysaccharide (LPS) Many species of bacteria produce tissue-degrading
components of the outer membranes of gram- enzymes that play important roles in the infection
negative. Their toxicity depends upon the the process.
component (lipid A). i. Coagulase: Coagulase is produced by
They are released into the host’s circulation Staphylococcus. aureus. This thrombin-like
following bacterial cell lysis. enzyme prevents phagocytosis by forming a
They are toxic only at high doses. fibrin barrier around the bacteria and walling
They cannot be toxoided. off the lesion.
They are poor antigens and weakly immunogenic.
ii. Lecithinase-C and collagenase: Clostridiun.
They do not exhibit specific pharmacological
perfringens produces lecithinase-C and
activities.
collagenase promoting spread of infection in
Intravenous injections of large doses of
tissue.
endotoxin and massive gram-negative septicemias
cause endotoxic shock marked by fever, leukopenia, iii. Hyaluronidases: Hyaluronidases split hyalu
thrombocytopenia, siignificant fall in blood ronic acid and thus facilitate the spread of
pressure, circulatory collapse and bloody diarrhea infection along tissue spaces, e.g. Streptococcus.
leading to death (Table 11.1). iv. Streptokinase (fibrinolysin): Many haemolytic
streptococci produce streptokinase (fibri
5. Avoidance of Host Defence nolysin) which promotes the spread of
Mechanisms infections.
v. Cytolysins: These include hemolysins capable
a. Capsules of destroying erythrocytes and leukocidins
Some bacteria such as Streptococcus pneumoniae, damage polymorphonuclear leukocytes.
Neisseria meningitidis, and Haemophilus influenzae vi. IgA 1 proteases: These enzymes specifically
can produce a slippery mucoid capsule that cleave immunoglobulin IgA which protects
prevents the phagocyte from effectively contacting at mucosal surfaces.
the bacterium.
www.ebook3000.com
80 | Section 1: General Bacteriology
9. Communicability Infection: Infections may be classified in various ways.
The ability of a microbe to spread from one host to Sources of infection: A. Human beings; B. Animals; C.
another is known as communicability. Insects; D. Soil and water; E. Food.
Modes of transmission of infection: 1. Contact; 2.
Inhalation; 3. Ingestion; 4. Inoculation; 5. Insects; 6.
10. Infecting Dose Congenital; 7. Iatrogenic and laboratory infections
Adequate number of bacteria is required for Determinants of virulence: 1. Transmissibility;
successful infections. The dosage may be estimated 2. Adhesion; 3. Invasiveness; 4. Toxigenicity; 5.
as the minimum infecting dose (MID) or minimum Avoidance of host defence mechanisms; Enzymes;
lethal dose (MLD). 7. Plasmids; 8. Bacteriophages; 9. Communicability;
10. Infecting dose; 11. Route of infection
These doses are more correctly estimated as
Types of infectious diseases
statistical expressions, ID50 and LD50 as the dose A. Localized infections may be superficial or deep
required to infect or kill 50 per cent of the animals seated
tested under standard conditions. B. Generalized infection: 1. Bacteremia; 2. Septicemia;
3. Pyemia.
11. Route of Infection
Certain bacteria are infective when introduced Important Questions
through optimal route, for example, cholera vibrios
1. Describe in detail the sources of infections to
can produce lesion only when administered by humans beings.
oral route, but unable to cause infection when
2. What are the various modes of spread of infection?
introduced subcutaneously. Describe each in brief giving suitable examples.
3. Describe the factors determining microbial
Types of infectious diseases pathogenicity and virulence.
Infectious diseases may be localized or generalized. 4. Distinguish between exotoxins and endotoxins in
a tabulated form.
A. Localized
Localized infections may be superficial or deep Multiple choice questions (MCQs)
seated. 1. The organisms can be transmitted vertically by all
the following ways except:
B. Generalized a. Sexual contact
1. Bacteremia: Circulation of bacteria in the blood b. Through the placenta
c. Within the birth canal
is known as bacteremia.
d. Through breast milk
2. Septicemia: It is the condition where bacteria
2. Which of the following may cause teratogenic
circulate and multiply in the blood, form toxic
infections?
products and cause high, swinging type of fever. a. Toxoplasma
3. Pyemia: It is a condition where pyogenic b. Rubella virus
bacteria produce septicemia with multiple c. Cytomegalovirus
abscesses in internal organs, such as the spleen, d. All of the above
liver and kidney. 3. Which of the following statement is not true for
exotoxins?
Epidemiological terminologies a. They are proteins
b. They are highly antigenic
1. Endemic: The disease which is constantly c. They are heat labile
present in a particular area, e.g. typhoid fever d. They are obtained by cell lysis
is endemic in most parts of India. 4. Which of the statement is not true for endotoxins:
2. Epidemic: The disease that spreads rapidly, a. These are lipopolysacaride components of
involving many persons in a particular area at outer membrane of gram negative bacteria
b. These are heat stable
the same time, is called epidemic disease, e.g.
c. They cannot be toxoided
meningococcal meningitis. d. They are highly antigenic
3. Pandemic: It is an epidemic that spreads through 5. The disease that spreads rapidly, involving many
many areas of the world involving very large persons in a particular area at the same time is
number of persons within a short period, e.g. known as:
cholera, influenza and enteroviral conjunctivitis. a. Sporadic
b. Endemic
Key Points c. Epidemic
Parasites are microbes that can establish themselves d. Pandemic
and multiply in the hosts. Parasite microbes may be
Answers (MCQs)
either pathogens or commensals
1. a; 2. d; 3. d; 4. d; 5. c.
Section 2: Immunology
12
Chapter
Immunity
Learning Objectives
After reading and studying this chapter, you should ∙∙ Differentiate between active and passive immunity.
be able to:
∙∙ Describe innate immunity, artificial active immunity,
natural passive immunity, herd immunity
www.ebook3000.com
82 | Section 2: Immunology
Factors influencing the level of immunity saturated and unsaturated fatty acids that kill many
1. Age bacteria and fungi.
i. Fetus in utero: The two extremes of life
B. Mucous Membrane
carry higher susceptibility to infectious
General protective mechanisms : A major
diseases as compared with adults. The
protective component of mucous membranes is
higher susceptibility of the young appears
the mucus itself.
to associate with immaturity of immune
system. Specific protective characteristics: Besides the
ii. Newborn animals: Newborn animals are general protective properties of mucosal cells,
more susceptible to experimental infection the lining of the different body tracts has other
than adult animals, e.g. coxsackievirus characteristics specific to each anatomic site.
causes fatal infection in suckling mice but
not in adult mice. a. Mouth or Oral Cavity
iii. In the elderly, besides a general waning The mouth or oral cavity is protected by the flow
of the activities of the immune system, of saliva that physically carries microorganisms
physical abnormalities (e.g. prostatic away from the cell surfaces and also contains the
enlargement leading to stasis of urine) lysozyme, which destroys bacterial cell walls and
or long-term exposure to environmental antibodies.
factors (e.g. smoking) are common causes
of increased susceptibility to infection. b. Gastrointestinal Tract
2. Hormonal Influences and Sex i. Stomach: The low pH and proteolytic enzymes
i. Endocrine disorders: There is an increased of the stomach help keep the number of
susceptibility to infection in endocrine micro-organisms low. The pH becomes
disorders, such as diabetes mellitus, progressively alkaline from the duodenum to
hypothyroidism and adrenal dysfunction the ileum.
(increased corticoids secretion).
ii. Small intestine: In the small intestine,
ii. Sex: In general, incidence and death rate protection is provided by the presence of bile
from infectious diseases are greater in males salts.
than in females.
iii. Ileum: The ileum contains a rich and varied
3. Nutrition: In general, both humoral and cell
flora and in the large intestine, the bulk of the
mediated immune processes are reduced
contents is composed of bacteria. Abundant
in malnutrition. Experimental evidence in
resident microflora in the large bowel also
animals has shown that inadequate diet may
contributes significantly to protection.
be correlated with increased susceptibility of
a variety of bacterial diseases, associated with
c. Upper Respiratory Tract
decreased phagocytic activity and leukopenia.
4. Stress: A growing body of evidence has i. Architecture of the nose: In the upper
demonstrated an inverse relation between respiratory tract, nasal hairs keep out large
stress and immune function. The end result is airborne particles that may contain micro-
an increased susceptibility to infection. organisms.
ii. Sticky mucus: The sticky mucus covering the
Mechanisms of Innate Immunity respiratory tract acts as a trapping mechanism
for inhaled particles.
1. Mechanical Barriers and Surface iii. Ciliary motion: Ciliary motion transports the
Secretions trapped organisms back up the respiratory
These surfaces include as follows: tract to the external openings.
A. Skin iv. Cough reflex: Cough reflex is an important
B. Mucous membranes. defence mechanism of the respiratory tract.
A. Skin v. Mucopolysaccharide: Nasal and respiratory
The intact skin and the mucous membranes provide secretions contain mucopolysaccharide
mechanical barriers that prevent the entrance of capable of combining with influenza and
most microbial species. Even though the structure certain other viruses.
of the skin itself undoubtedly gives a great deal of
protection, considerably more important are the d. Genitourinary Tract
fatty acids secreted by the sebaceous glands and i. Normal flow of urine: The normal flow of urine
the propionic acid by the normal flora of the skin. flushes the urinary system, carrying micro-
Secretions from the sebaceous glands contain both organisms away from the body.
Chapter 12: Immunity | 83
ii. Spermine and zinc: Spermine and zinc present Phagocytosis: Phagocytosis is apart of the innate
in the semen carry out antibacterial activity. immune response, during which microorganisms,
iii. Acidity of the adult vagina: The low pH (acidity) foreign particles, and cellular debris are engulfed
of the adult vagina, due to fermentation of by phagocytic cells, such as neutrophils and
glycogen in the epithelial cells by the resident monocytes in the circulation, and macrophages
aciduric bacilli, provides an inhospitable and neutrophils in interstitial spaces. Phagocytosis
environment for colonization by pathogens. consists of three stages:
A thick mucus plug in the cervical opening is 1. Attachment of the microorganisms or foreign
a substantial barrier. material to the phagocytic cell.
2. Ingestion by the cell and drawn into the cell by
e. Conjunctiva endocytosis.
3. Degradation of the foreign material or micro-
Lachrymal fluid: Conjunctiva is kept moist by the
organism within the phagocytic cells.
continuous flushing action of tears (lachrymal
fluid). Tears contain large amounts of lysozyme,
lactoferrin, and sIgA and thus provide protection.
5. Inflammation
If the surface chemical and physiologic def
ences of the body are breached by a pathogen,
2. Antibacterial Substances in Blood and inflammation can result, which is an
Tissues important, nonspecific defence mechanism.
Many microbial substances are present in the Sequences of events in acute inflammation in
tissue and body fluids. These are non-specific. The response to an injury will be: (i) vasodilation,
complement system possesses bactericidal activity (ii) increased vascular permeability; (iii) emigration
and plays an important role in the destruction of of leucocytes; (iv) chemotaxis; (v) phagocytosis.
pathogenic bacteria that invade the blood and
tissues. 6. Fever
Other Substances (i) beta lysin; (ii) basic Following infection, a rise of temperature is a
polypeptides, such as leukins extracted from natural defense mechanism. It not merely helps
leucocytes and plakins from platelets; (iii) acidic to accelerate the physiological processes, but
substances, such as lactic acid found in muscle may, in some cases, actually destroy the infecting
tissue and in the inflammatory zones; and pathogens. Fever aids recovery from viral infections
(iv) lactoperoxidase in milk. The production of by stimulating the production of interferon.
interferon is a method of defence against viral
infections. 7. Acute Phase Proteins
A sudden increase in the plasma concentration
3. Microbial Antagonisms of certain proteins, collectively termed as ‘acute
The skin and mucous surfaces have resident phase proteins’ occurs as a result of infection or
bacterial flora which prevent colonization by tissue injury. These include C-reactive protein
pathogens. Invasion by extraneous microbes (CRP), mannose-binding protein, alpha-I-acid
may be due to alteration of normal resident flora, glycoprotein, serum amyloid P component
causing serious diseases, such as staphylococcal and many others. The alternative pathway of
or clostridial enterocolitis or candidiasis following complement is activated by CRP and some other
oral antibiotics. acute phase proteins. They are believed to enhance
host resistance, prevent tissue injury and promote
repair of inflammatory lesions.
4. Cellular Factors in Innate Immunity
Natural defense against the invasion of blood ii. Acquired Immunity
and tissues by microorganisms and other foreign
Acquired immunity refers to the resistance that an
particles is mediated to a large extent by phagocytic
individual acquires during his lifetime. Acquired
cells which ingest and destroy them. Phagocytic
immunity is of two- types: active immunity and
cells, originally discovered by Metchnikoff
passive immunity. (Fig. 12.1 and Table 12.1).
(1883), were classified by him into microphages
(polymorphonuclear leucocytes) and macrophages.
a. Active Immunity
Macrophages: Macrophages consist of histiocytes Active immunity is induced after contact with
which are the wandering ameboid cells seen foreign antigens. It is also known as adaptive
in tissues, fixed ret iculoendothelial cells and immunity. This involves the active functioning
monocytes of blood. of the host’s immune apparatus leading to the
www.ebook3000.com
84 | Section 2: Immunology
In some infections like syphilis and malaria,
A. Active immunity
the immunity to reinfection lasts only as long as
1. Natural—Clinical
the original infection remains active. Once the
– Subclinical Infection
disease is cured, the patient becomes susceptible to
2. Artificial
the infection again. This special type of immunity
Vaccination—Live
is known as ‘premunition’ or infection-immunity.
– Killed
B. Passive immunity 2. Artificial Active Immunity
1. Natural—Through placenta Artificial active immunity is the resistance induced
– Through breast milk by vaccines. Vaccines are preparations of live or
2. Artificial—Immune serum killed microorganisms or their products used for
– Immune cells immunization. Vaccines are made with either:
Fig. 12.1: Types of immunity
(i) live attenuated microorganisms; (ii) killed
microorganisms; (iii) microbial extract; (iv) vaccine
conjugates; or (v) inactivated toxoids.
synthesis of antibodies and/or the production of
immunologically active cells. Examples of Vaccines
Immune Response I. Bacterial vaccines
a. Live (BCG vaccine for tuberculosis)
A. Primary Response b. Killed (cholera vaccine)
Active immunity sets in only after a latent period. c. Subunit (typhoid Vi antigen)
There is often a negative phase. Once developed, d. Bacterial products (tetanus toxoid)
the active immunity is long-lasting. 2. Viral vaccines
a. Live
B. Secondary Response –– Oral polio vaccine-Sabin
If an individual, who has been actively immunized –– 17D vaccine for yellow fever
against an antigen, experiences the same antigen –– MMR vaccine for measles, mumps, rubella.
subsequently, the immune response occurs more b. Killed
quickly and abundantly than during the first –– Injectable polio vaccine-Salk
encounter. This is known as secondary response. –– Neural and non-neural vaccines for rabies
–– Hepatitis B vaccine
Types of Active Immunity c. Subunit: Hepatitis B vaccine
1. Natural Live Vaccines
2. Artificial In general, live vaccines are more potent immunizing
agents than killed vaccines The immunity lasts for
1. Natural Active Immunity
several years but booster doses may be necessary.
Natural active immunity results from either a
Live vaccines may be administered orally or
clinical or an inapparent infection by a microbe.
parenterally.
Such immunity is usually long-lasting but the
duration varies with the type of pathogen. The Killed Vaccines
immunity is life-long following many viral diseases, Killed vaccines are usually safe and generally less
such as chickenpox or measles. immunogenic than live vaccines, and protection
The immunity appears to be shortlived in some lasts only for a short period. They have, therefore,
viral diseases, such as influenza or common cold. to be administered repeatedly, generally at least
www.ebook3000.com
86 | Section 2: Immunology
Local immunity Combined immunization is a combination of active
and passive methods of immunization which is
Local immunity is conferred by secretory sometimes employed
immunoglobulin A (secretory IgA) produced Local immunity is conferred by secretor y
locally by plasma cells present on mucosal surfaces immunoglobulin A (secretory IgA)
or in secretory glands. Herd immunity is the level of resistance of a
community or a group of people to a particular
disease and is relevant in the control of epidemic
Examples diseases.
1. Poliomyelitis immunization: In poliomyelitis,
active immunization provides systemic Important questions
immunity with the killed vaccine. Natural
1. Define and classify immunity. Discuss mechanisms
infection or immunization with the live oral of innate immunity.
vaccine provides local intestinal immunity. 2. Tabulate the differences between active immunity
2. Influenza, immunization: Natural infection and passive immunity.
or the live influenza vaccine administered 3. Write short notes on:
intranasally provides local immunity. a. Innate immunity
b. Artificial active immunity
c. Natural passive immunity
Herd immunity d. Artificial passive immunity
It is the level of resistance of a community or e. Herd immunity.
a group of people to a particular disease and
is relevant in the control of epidemic diseases. Multiple choice questions (MCQs)
Epidemics are likely to occur on the introduction 1. All are acute phase proteins except:
of a suitable pathogen, when herd immunity is low. a. C-reactive protein
b. Mannose-binding protein
Eradication of communicable diseases depends on
c. Serum amyloid P component
the development of a high level of herd immunity d. Antibody
rather than on the development of a high level of 2. Clinical or inapparent infection leads to:
immunity in individuals. a. Natural active immunity
b. Artificial active immunity
Key Points c. Natural passive immunity
d. Artificial passive immunity
Immunity refers to the resistance exhibited by the 3. Vaccine induces:
host towards injury caused by microorganisms and a. Active natural immunity
their products b. Active artificial immunity
Innate or natural immunity is the resistance to c. Passive natural immunity
infections, which an individual possesses by virtue d. Passive artificial immunity
of his genetic or constitutional make-up. It may be 4. All the following statements are true for artificial
nonspecific, or specific. Innate immunity may be passive immunity except:
considered at the level of species, race or individual. a. Artificial passive immunity is the resistance
Factors influencing the level of immunity are age, passively transferred to a recipient by the ad-
hormonal influences and sex, nutrition and stress ministration of antibodies.
Mechanisms of innate immunity are: 1. Mechanical b. It is shortlived and lasts only a few weeks to a
barriers and surface secretions; 2. Antibacterial few months
substances in blood and tissues; 3. Microbial c. This type of immunity is immediate
antagonisms; 4. Cellular factors in innate immunity; d. This immunity may be induced by maternal anti-
5. Inflammation; 6. Fever; 7. Acute phase proteins bodies.
Acquired immunity refers to the resistance that an 5. All the following are live vaccines except:
individual acquires during his lifetime. b-Acquired a. BCG b. Sabin vaccine
immunity is of two types: active immunity and c. MMR d. TAB vaccine
passive immunity 6. All the following are killer vaccines except:
Natural active immunity results from either a clinical a. Salk vaccine
or an inapparent infection by a microbe b. Non-neural vaccines for rabies
Artificial active immunity is the resistance induced c. Hepatitis B vaccine
by vaccines d. BCG
The immunity that is transferred to a recipient in
a ‘readymade’ form is known as passive immunity
Answers (MCQs)
1. d; 2. a; 3. b; 4. d; 5. d; 6. d
13
Chapter
Antigens
Learning Objectives
Antigens: Antigens (antibody generators) are the 2. Complex haptens: Complex haptens can
substances that can stimulate an immune response precipitate with specific antibodies. Complex
and, given the opportunity, react specifically by haptens are polyvalent.
binding with the effector molecules (antibodies)
and effector cells (lymphocytes). Antigenic determinant or epitome
The smallest unit of antigenicity is known as the
Types of antigens antigenic determinant or epitope. Each antigen
can have several antigenic determinant sites or
Based on the ability of antigens to carry out these two
epitopes. The combining area on the antibody
functions, they may be classified into different types:
molecule, corresponding to the epitope, is called
the paratope.
A. Complete Antigen
Complete antigen is able to induce antibody Valence: The number of antigenic determinant
formation and produce a specific and observable sites on the surface of an antigen is its valence. The
reaction with the antibody so produced. antigen is monovalent or multivalent.
www.ebook3000.com
88 | Section 2: Immunology
3. Foreignness associated with plasma membrane of tissue
Only antigens which are ‘foreign’ to the individual cells. These antigens are encoded by genes
(nonself ) induce an immune response because known as histocompatibility genes which
host distinguishes self from nonself and normally collectively constitute major histocompatibility
does not respond to self. In general, the greater the complex (MHC). MHC products present on the
difference between the antigen (Ag) and similar surface of leucocytes are known as human
molecules in the host’s body, the greater the leukocyte-associated (HLA) antigens.
immune response that is generated.
8. Autospecificity
4. Susceptibility to Tissue Enzymes Sequestrated Antigens
Only those substances which can be metabolized Autologous or self-antigens are ordinarily
and susceptibility to the tissue enzymes behave as nonantigenic but there are exceptions. Certain
antigens. Substances unsusceptible to the tissue self-antigens are present in closed system and are
enzymes are not antigenic. Substances that are not accessible to the immune apparatus and these
insoluble in body fluid and not metabolized are not are known as sequestrated antigens.
antigenic. Substances very rapidly broken down by
tissue enzymes are also not antigenic. Examples of Sequestrated Antigens
5. Antigenic Specificity i. Lens protein: Sequestrated antigens that are
not normally found free in circulation or tissue
Chemical Groupings fluids (such as lens protein normally confined
Foreignness of a substance to an animal can within its capsule) are not recognized as
depend on the presence of chemical groupings self-antigens. When the antigen leaks out
that are not normally found in the animal’s body. following penetrating injury, it may induce
Antigenic specificity varies with the position of an immune response causing damage to the
antigenic determinant, i.e. whether it is in ortho, lens of the other eye.
meta or para position. ii. Sperm: Similarly, antigens that are absent
In many cases, an antibody specific for one during embryonic life and develop later (such as
antigen may display significant cross-reactivity for sperm), are also not recognized as self-antigens.
an apparently unrelated antigen. When the sperm antigen enters the circulation,
it is immunogenic and is believed to be the
6. Species Specificities pathogenesis of orchitis following mumps.
Tissues of all individuals in a species possess
9. Organ Specificity
species-specific antigens. However, some degree
of cross-reactivity is seen between antigens from Some organs, such as brain, kidney and lens protein
related species. of different species share the same antigens. These
antigens are known as organ-specific antigens,
7. Isospecificities characteristic of an organ or tissue and found in
Isoantigens or alloantigens are antigens found different species. Injection of heterologous organ-
in some but not all members of a species. On the specific antigens may induce an immune response,
basis of isoantigens, a species may be divided into damaging the particular organs or tissue in the host.
different groups.
Example
Examples of Isoantigens Neuroparalytic complications: The neuroparalytic
i. Human erythrocyte antigens: The best complications following antirabic vaccination
example of isoantigens is human erythrocyte using sheep brain vaccines are a consequence of
antigens, on the basis of which all humans can brain-specific antigens shared by sheep and man.
be divided into different blood groups: A, B, The sheep brain antigens induce an immunological
AB and O. Each of these groups may be further response in the vaccines, damaging their nervous
divided into Rh-positive or Rh-negative. tissue due to the cross-reaction between human
and sheep brain antigens.
ii. Histocompatibility antigens: Histocompatibility
antigens are those cellular determinants
specific to each individual of a species. 10. Heterogenetic (Heterophile) Specificity
Histocompatibility typing is essential in organ/ Same or closely related antigens occurring in
tissue transplantation from one individual to different biological species, classes and kingdoms
another within a species. These antigens are are known as heterogenetic or heterophile antigens.
Chapter 13: Antigens | 89
Examples of Heterophile Antigens 2. Association with diseases: Superantigens should
i. Forssman antigen: It is a lipid carbohydrate be considered possible chronic associates in
complex widely distributed in man, animals, such diseases as rheumatic fever, arthritis,
birds, plants and bacteria. Kawasaki syndrome, atopic dermatitis, and one
ii. Weil-Felix reaction: It is an agglutination test type of psoriasis.
in which patient sera are tested for agglutinins
to the O antigens of certain nonmotile Proteus Key Points
strains OXl9, OX2, and OXK. Cross-reaction
between O antigen of these strains of Proteus Antigens are the substances that can stimulate an
immune response and, given the opportunity, react
and certain rickettsial antigens is the basis of
specifically by binding with the effector molecules
this test. (antibodies) and effector cells (lymphocytes)
iii. Paul-Bunnell test: During infectious-mono Types of antigen are: A. Complete antigen; B.
nucleosis, heterophile antibodies appear in Haptens (incomplete immunogen)
the serum of the patient. These antibodies Determinants of antigenicity are: 1. Size; 2.
agglutinate sheep erythrocytes. This test is Chemical nature; 3. Foreignness; 4. Susceptibility to
known as Paul-Bunnell test. tissueenzymes; 5. Antigenic specificity; 6. Species;
iv. Cold agglutinin test: Agglutination of human O specificities; Isospecifities; 8. Autospecicity; 9. Organ
specificity; 10. Heterogenetic (heterophile) specificity.
group erythrocytes at 4°C by the sera of patients
suffering from primary atypical pneumonia.
v. Agglutination of Streptococcus MG: Agglutin
Important questions
ation of Streptococcus MG by the sera of the
patients of primary atypical pneumonia. 1. What is an antigen? Discuss briefly various deter
minants of antigenicity.
Superantigens 2. Write short notes on:
a. Haptens
Superantigens are bacterial proteins which can
b. Heterophile antigens
interact with antigen-presenting cells (APCs) and
T cells in nonspecific manner. This activity does
not involve the endocytic processing required for Multiple choice questions (MCQs)
typical antigen presentation but instead occurs
1. The substances that are least immunogenic are
by concurrent association with MHC class II
a. Proteins
molecules of the APCs and the Vβ domain of the
b. Polysaccharides
T cell receptor. This interaction activates a large
c. Nucleic acids
number of T cells (10%) than conventional anti
d. None of the above
gens (about 1%), explaining the massive cytokine
expression and immunomodulation. The antigens 2. The test that does not use heterophilic antigen is
which provoke such a drastic immune response are a. TPHA test
termed as superantigens. b. Weil-Felix reaction
c. Paul-Bunnell test
Examples d. Cold agglutination test
www.ebook3000.com
14
Chapter
Antibodies—Immunoglobulins
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe structure and functions of IgG, IgA and
be able to: IgM
∙∙ Define antibody and draw labeled diagram of ∙∙ Discuss properties of IgM, IgG, IgA, IgD and IgE
immunoglobulin ∙∙ Draw labeled diagram of IgG, IgM and IgA.
ANTIBODY STRUCTURE
Porter, Edelman, Nisonoff and their colleagues Fig. 14.1: Basic structure of an immunoglobulin molecule and
poineered studies involving the cleavage of the the fragments obtained by the cleavage by papain and pepsin
Chapter 14: Antibodies—Immunoglobulins | 91
A B
Fig. 14.2: The four-peptide chain structure of the IgG molecule composed of two identical heavy (H) and two identical light
(L) chains linked by interchain disulfide bonds. Loops formed by intrachain disulfide bonds are domains (shown stippled).
Each chain has one domain in the variable region (VH and VL). Each light chain has one domain in the constant region (CL)
while each heavy chain has three domains in the constant region (CH1, CH2 and CH3). Between CH1 and CH2 is the hinge region
www.ebook3000.com
92 | Section 2: Immunology
the domain next to the variable domain. It is the
portion of H-chains present in Fab fragment.
H-chains carry a carbohydrate moiety, which is
distinct for each class of immunoglobulins.
4. Fc Fragment
The Fc fragment is composed of the carboxyterminal
portion of the H-chains. It can be crystallized, and
is, therefore, called Fc (fragment-crystallizable).
Functions of Fc
Fig. 14.3: Secretory IgA. 1. Heavy chain; 2. Light chain; 3. J
Binds complement leading to complement fix chain; 4. Secretory component; 5. Disulfide bond
ation.
∙∙ Binds to cell receptors (FcRs) fluid, and it is distributed nearly equally
∙∙ Determines passage of IgG across the placental between extra- and intravascular spaces.
barrier 7. Four subclasses of IgG (Ig1, Ig2, Ig3, Ig4) have
∙∙ Determines skin fixation and catabolic rate. been recognized.
8. Catabolism of IgG is unique in that it varies with
5. Immunoglobulin Domain its serum concentration. When its level is raised,
Each immunoglobulin peptide chain has internal as in chronic malaria, kala-azar or myeloma,
disulfide links in addition to interchain disulfide the IgG synthesized against a particular antigen
bonds which bridge the H- and L-chains. These will be catabolized rapidly and may result in
interchain disulfide bonds form loops in the peptide the particular antibody deficiency. Conversely,
chain, and each of the loops is compactly folded in hypogammaglobulinemia, the IgG given for
to form a globular domain, each domain having treatment will be catabolized only slowly.
a separate function. The variable region domains, Functions of IgG: IgG is a very versatile molecule.
VL and VH, are responsible for the formation of a It may be considered a general-purpose antibody,
specific antigen-binding site. The CH2 region in IgG protective against those infectious agents, which
binds C1q in the classical complement sequence, are active in the blood and tissues.
and the CH3 domain mediates adherence to the
monocyte surface. The areas of the H-chain in the Examples of its Functions
C-region between the first and second C-region i. Transfer from mother to fetus: IgG is the only
domains (CH1 and CH2) is the hinge region. It is class of Igs that can cross the placenta and is
more flexible and is more exposed to enzymes and responsible for the protection of the infant
chemicals. Papain acts here to produce one Fc and during the first few months of life.
two Fab fragments (Figs 14.3). ii. Opsonization: IgG binds to microorganisms
and enhances their phagocytosis.
IMMUNOGLOBULIN CLASSES iii. Fixing to guinea pig skin.
Human serum contains five classes of immuno iv. Immunological reactions: IgG participates
globulins—IgG, IgA, IgM, IgD and IgE in the in complement fixation, precipitation and
desending order of the concentration. Table14.2 neutralization of toxins and viruses.
shows their differentiating features. v. Immobilize bacteria.
vi. Suppresses the homologous antibody synthesis:
1. Immunoglobulin G (IgG) ( Fig. 14.3) Passively administered IgG suppresses the
1. This is the major immunoglobulin in human homologous antibody synthesis by a feedback
serum, accounting for about 80% of the total process.
immunoglobulin pool.
2. It has a sedimentation coeffient of 7S and a 2. Immunoglobulin A (IgA)
molecular weight of 150,000. 1. It is the second most abundant class, cons
3. It contains less carbohydrate than other tituting about 10–13 per cent of serum immuno
immunoglobulins. globulins.
4. The normal serum concentration of IgG is 2. The normal serum level is 0.6–4.2 mg per mL.
about 8-16 mg per mL. 3. It has a half life of 6–8 days.
5. It has a half-life of 23 days—the longest of all of 4. IgA is the primary immunoglobulin found in
the immunoglobulin isotypes. external secretions, such as mucus, tears, saliva,
6. IgG is the predominant immunoglobulin in gastric fluid, colostrum and sweat. It exists in
blood, lymph, peritoneal fluid and cerebrospinal different forms in these various solutions.
Chapter 14: Antibodies—Immunoglobulins | 93
Table14.2 Physical, physiologic, and biologic properties of human serum immunoglobulins
Property lgG IgA* lgM lgD IgE
A. Physical properties
1. Sedimentation coefficient(s) 7 7 19 7 8
2. Molecular weight in 150,000 160,000 900,000, 180,000 190,000
kilodaltons
3. Carbohydrate (%) 3 8 12 13 12
4. Number of four-chain units 1 1–3 5–6 1 1
per molecule
B. Physiologic properties
1. Normal adult serum 12 2 1.2 0.03 0.00004
concentration (mg/mL)
2. Half-life (in days) 23 6 5 2–8 1–5
4. Daily production (mg/kg) 34 24 3.3 0.4 0.0023
5. Intravascular distribution (%) 45 42 80 75 50
C. Biologic properties
1. Complement fixation
Classical ++ – +++ – –
Alternative – + – – –
2. Placental transport to fetus + – – – –
3. Present in milk + + – – –
4. Selective selection by – + – – –
submucus glands
5. Anaphylactic – – – – ++++
hypersensitivity
6. Heat stability + + + + –
D. Major characteristics Most Protects Very efficienct Mainly Initiates
abundant Ig; mucosal against lymphocyte inflammation;
Longest half surfaces bacteremia receptor; raised in helminth
life: Crosses major surface infection; causes
placenta components of allergy symptoms
opsonizes B cells
antigen
*
IgA may occur in 7S, 9S and 11S forms
5. IgA occurs in two forms. Serum IgA and The secretory piece is believed to protect IgA
secretory IgA (SIgA). from denaturation by bacterial proteases in sites
Serum IgA: Serum IgA is monomeric (one four- such as the intestinal mucosa which have a rich
chain unit) 7S molecule (MW about 160,000). and varied bacterial flora.
Subclasses: There are two subclasses of IgA in
Secretory IgA (SIgA): In contrast, IgA found on
humans: IgA1 and IgA2.
mucosal surfaces and in secretions is a dimer
formed by two monomer units joined together at Functions of IgA
their carboxy terminals by a glycopeptide termed
i. Local immunity: Secretory IgA (SIgA) is
as the J chain (J for joining). This dimeric form
believed to play an important role in local
is more important form, known as secretory IgA
immunity against respiratory and intestinal
(SIgA). Dimeric SIgA is synthesized by plasma
pathogens.
cells situated near the mucosal or glandular
epithelium. J chains are also found in other ii. Prevention of organisms’ entry into body tissues.
polymeric immunoglobulins such as IgM. iii. Newborn protection: IgA present in breast milk
provides the newborn with protection against
Secretory component: Secretory IgA (SIgA)
infection.
contains another glycine-rich polypeptide called
the secretory component or secretory piece. This is iv. Agglutination.
not produced by lymphoid cells but by mucosal or v. Alternative pathways activation.
glandular epithelial cells. vi. Phagocytosis and intracellular killing.
www.ebook3000.com
94 | Section 2: Immunology
3. Immunoglobulin M (IgM) 4. Immunoglobulin D (IgD)
1. About 10% of normal serum Igs consists of this 1. IgD has a monomer structure similar to IgG.
class. 2. Its molecular weight is 180,000 daltons.
2. It is a heavy molecule (19S; MW 900,000 to 4. IgD is an immunoglobulin found in trace
1,000,000 daltons, hence called ‘millionaire amounts in the blood serum (0.03 mg/mL).
molecule’). 5. Half-life is about 3 days.
3. The normal serum level of IgM is 1.2 mg/mL. 6. IgD antibodies are abundant in combination
4. It has a half-life of about 5 days. with IgM on the surface of B cells and bind
5. IgM is the first immunoglobulin to appear antigens, thus signaling the B cell to start
after exposure to an antigen. antibody production.
6. In the circulation, IgM exists as a pentamer 7. Two subclasses of IgD (IgD1 and IgD2) are known.
of five four-chain units. The five identical IgM
monomers are connected to each other by 5. Immunoglobulin E (IgE)
a polypeptide joining (J) chain (Fig. 14.4). 1. It resembles IgG structurally and also known
Polymerization of the subunits depends upon as reagin antibody.
the presence of the J chain as with IgA (Fig. 14.3). 2. IgE is an 8S molecule (MW 19,000) and half-
7. Most of IgM (80%) is intravascular in life of two days.
distribution. 3. It is present in extremely low amounts in serum.
8. IgM is the first class of antibody produced 4. It exhibits unique properties such as heat
during the primary immune response. It is also lability (inactivated at 56°C in one hour).
the earliest to be synthesized by fetus beginning 5. It is susceptible to mercaptoethanol.
by about 20 weeks of age. As it cannot cross 6. It does not pass the placental barrier.
the placental barrier, the presence of IgM in 7. IgE does not activate complement nor
the fetus or newborn indicates intrauterine agglutinate antigens.
infection. 8. Allergic reactions: IgE may be elevated in allergic
9. They are relatively shortlived, hence their (atopic) individuals, and is responsible for
demonstration in the serum indicates recent many of the symptoms of allergies, bronchial
infection. asthma, and even systemic anaphylaxis.
10. Treatment of serum with 0.12 M 2-mercaptoe Allergy mediated by IgE is termed as type I
thanol selectively destroys IgM without hypersensitivity response.
affecting IgG antibodies. 9. Immunity against helminthic parasites:
11. Isohemagglutinins (anti-A and anti-B) and anti Children living in insanitary conditions, with
bodies to S. Typhi O antigen and Wassermann a high load of intestinal parasites, have high
reaction antibodies in syphilis are usually IgM. serum levels of IgE.
12. IgM agglutinates bacteria activates complement 10. Extravascular: It is mostly found extra
by the classical pathway, and enhances the vascularly in the lining of the respiratory and
ingestion of pathogens by phagocytic cells. IgM intestinal tracts.
is normally restricted to the intravascular space 11. Protection against pathogens: The physiological
because of its high molecular weight. role of IgE appears to be protection against
pathogens by mast cell degranulation and
release of inflammatory mediators.
Abnormal Immunoglobulins
Other structurally similar proteins are seen in
serum in many pathological processes, and
sometimes even in healthy persons apart from
antibodies in the following pathological conditions:
A. Multiple myeloma
Fig. 14.4: Pentameric IgM molecule, composed of five B. Heavy chain disease
identical monomers C. Cryoglobulinemia
Chapter 14: Antibodies—Immunoglobulins | 95
A. Multiple Myeloma An antibody molecule consists of two identical
The abnormal plasma cells are myeloma cells light chains and two identical heavy chains, which
which also collect in the solid part of the bone. are linked by disulfide bonds. Each heavy chain has
The disease is called “multiple myeloma”. Myeloma an amino-terminal variable region followed by a
constant region
is a plasma cell dyscrasia in which there is
Within the amino-terminal variable domain of each
unchecked proliferation of one clone of plasma heavy and light chain are three complementarity-
cells, resulting in the excessive production of the determining regions (CDRs). These polypeptide
particular immunoglobulin synthesized by the regions contribute the antigen-binding site of an
clone. Such immunoglobulins are, therefore, called antibody, determining its specificity
Abnormal immunoglobulins are multiple myeloma,
monoclonal.
heavy chain disease C and cryoglobulinemia.
Waldenstrom’s macroglobulinemia: Multiple
myeloma may affect plasma cells synthesizing
IgG, IgA, IgD or IgE. Myeloma involving IgM- Important questions
producing cells (lympho-plasmacytoid cells) is
known as Waldenstrom’s macroglobulinemia. In this 1. What is an antibody? Draw a labeled diagram of IgG.
condition, there occurs excessive production of the 2. Name various classes of immunoglobulins and
respective myeloma proteins (M proteins) and of describe structure and functions of IgG, IgA and
their light chains (Bence-Jones proteins). IgM.
3. Write shot notes on:
Bence-Jones proteins: In most patients, the
a. Immunoglobulin G (IgG)
myeloma cells also secrete excessive amounts
b. Immunoglobulin M (IgM)
of light chains. These excess light chains were
first discovered in the urine of myeloma patients c. Immunoglobulin A (IgA)
and were named Bence-Jones proteins, for their
discoverer Bence Jones (1847). Bence Jones proteins Multiple choice questions (MCQs)
can be identified in urine by its characteristic 1. The immunoglobulin that crosses the placenta is:
property of coagulation when heated to 50°C but a. IgG b. IgM
redissolving at 70°C. These proteins are the light c. IgA d. IgE
chains of immunoglobulins and so may occur as 2. The J-chain is present in the immunoglobulin:
the kappa or lambda forms. But in any one patient, a. IgG b. IgM
the chain is either kappa or lambda only, and never c. IgA d. IgE
both, being uniform in all other respects also. 3. All the following immunoglobulins are heat-stable
except:
B. Heavy Chain Disease a. IgG b. IgM
It is a lymphoid neoplasia characterized by the c. IgA d. IgE
overproduction of the Fc parts of the immuno 4. Which is the earliest immunoglobulin to be
globulin heavy chains. synthesized by the fetus?
a. IgG b. IgM
C. Cryoglobulinemia c. IgA d. IgE
It is a condition in which there is the formation 5. The immunoglobulin that mediates type I hyper
of a gel or a precipitate on cooling the serum, sensitivity reaction is:
which redissolves on warming. It may not always a. IgG b. IgM
be associated with disease but is often found in c. IgA d. IgE
myelomas, macroglobulinemias and autoimmune 6. Antibodies that are bound to mast cells and involve
conditions such as systemic lupus erythematosus. in allergic reactions are:
Most cryoglobulins consist of either IgG, IgM or a. IgG b. IgM
mixture of the two. c. IgA d. IgE
7. The first antibodies synthesized, especially against
Key Points microorganisms:
An antibody or immunoglobulin (lg) is a glycoprotein a. IgG b. IgM
that is made in response to an antigen, and can c. IgA d. IgE
recognize and bind to the antigen that caused its
production Answers (MCQs)
1. a; 2. b; 3. d; 4. b; 5. d; 6. d; 7. b.
www.ebook3000.com
15
Chapter
Complement System
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss biological effects of complement
be able to: ∙∙ Describe complement deficiencies and associated
∙∙ Describe the sequence of events when the diseases.
classical pathway and the alternative pathway of
the complement system is activated
Complement: The term ‘complement’ (C) refers to 5. Complement fixation, binding or consumption:
a system of factors which occur in normal serum Complement (C) ordinarily does not bind to
and are activated characteristically by antigen- free antigen or antibody but only to antibody
antibody interaction and subsequently mediate a which has combined with its antigen.
number of biologically significant consequences. 6. Site of complement binding: The site of
complement binding is located on the Fc piece
Complement system of the Ig molecule.
The complement system belongs to the group
of biological effector mechanisms (called Complement Activation
triggered enzyme cascades), which also includes There are nine components of complement
coagulation, fibrinolytic and kinin systems. called C1 to C9. The fraction C1 occurs in serum
Pfeiffer phenomenon: It was discovered by as a calcium ion-dependent complex, which on
Pfeiffer (1894) that cholera vibrios were lysed chelation with EDT A yields three protein subunits
when injected intraperitoneally into specifically called C1q, r and s. Thus, C is made up of a total
immunized guinea pigs (bactereolysis in vivo or of 11 different proteins. C fractions are named C1
Pfeiffer phenomenon). to C9 in the sequence of the cascading reaction,
except that C4 comes after C1, before C2. The
General Properties of Complement components of the classical pathway are numbered
1. Present in the sera of all mammals and of most from C1 to C9, although they do not function in
lower animals: numerical order (C4 comes after C1, before C2).
2. Nonspecific serological reagent: Complement Activation of the complement system can be
from one species can react with antibodies from initiated either by antigen–antibody complexes or
other species. by a variety of nonimmunologic molecules.
3. Serum molecules: The complement system Complement components react in a specific
consists of approximately 30 serum molecules sequence as a cascade either through the classical or
constituting nearly 10% of the total serum alternative pathway.
proteins and forming one of the major defence
systems of the body. Principle pathways of complement
4. Heat labile: Complement as a whole is heat activation
labile, and being destroyed in 30 minutes at
56°C. A serum deprived of its complement Three principle pathways are involved in
activity by heating at 56°C for 30 minutes is then complement activation (Classical pathway, alternate
said to be ‘inactivated’. or properdin pathway, and Lectin pathway).
Chapter 15: Complement System | 97
A. Classical Complement Pathway instance of amplification. Activated Cl cleaves
The chain of events in which C components react in C4 into two pieces C4a and C4b (C4® C4a + C4b).
a specific sequence following activation of C1 and C4a is an anaphylotoxin and C4b which binds
typically culminate in immune cytolysis is known to cell membrane along with C1.
as the classical pathway (Fig. 15.1). It consists of C14b in the presence of magnesium ions
the following steps: cleaves C2 into two pieces (C2® C2a + C2b).
1. Antigen–antibody binding: The first step is the C2a remains linked to cell-bound C4b, and C2b
binding of C1 to the antigen–antibody (AB) which is released into fluid phase. The pieces
complex. The recognition unit of C1 is Clq, recombine, forming C4b2a has enzymatic
which reacts with the Fc piece of bound IgM activity and is referred to as the classical
of IgG. Clq binding in the presence of calcium pathway C3 convertase.
ions leads to sequential activation of Clr and s. 3. Production of C5 convertase: C3 convertase
In the presence of calcium ions, a trimolecular cleaves C3 into two fragments (C3® C3a + C3b).
complex (CI qrs-Ag-Ab) that has esterase C3a is soluble, and is an anahylotoxin, and
activity is rapidly formed. C3b which remains cell-bound along with
2. Production of C3 convertase: Activated Cls is C4b2a to form a trimolecular complex C4b2a3b,
an esterase (C1s esterase), one molecule of which has enzymatic activity and is called C5
which can cleave several molecules of C4—an convertase of the classic pathway.
www.ebook3000.com
98 | Section 2: Immunology
4. Formation of the membrane attack complex and a C5 convertase. These are factor B, factor D
(MAC): The terminal stage of the classic pathway and properdin (P).
involves creation of membrane attack complex The binding of C3b to an activator is the first
(MAC), which is also called the lytic unit. step in the alternative pathway. Although C3b
Initiation of membrane attack complex (MAC) is present in the circulation but in the free state
assembly begins with cleavage of C5 by C5 convertase it is rapidly inactivated by the serum protein
into C5a and C5b fragments (C5®C5a+C5b). The C5a factors H and I. However, bound C3b is protected
is the most potent anaphylatoxin in the body and from such inactivation. The bound C3b, in the
C5b continues with the cascade. C6 and C7 then presence of Mg++, interacts with plasma protein
join together. A heatstable trimolecular complex factor B forming C3bB which is also known as ‘C3
C567 is formed part of which binds to the cell proactivator convertase’) to form a magnesium-
membrane and prepares it for lysis by C8 and C9 dependent complex ‘C3bB’. Factor B in the complex
which join the reaction subsequently. This complex is cleaved by serum factor D (also called ‘C3
(C5b67) inserts itself into the plasma membrane proactivator convertase’) into two fragments—Ba
of the target cell. Most of C567 escape and serve to and Bb. Fragment Ba is released into the medium.
amplify the reaction by adsorbing onto unsensitized Fragment Bb remains bound to C3b producing
‘bystander cells’ and rendering them susceptible to C3bBb. C3bBb acts as the alternate pathway C3
lysis by C8 and C9. convertase, capable of producing more C3b. This
The unbound C567 has chemotactic activity, enzyme C3bBb is extremely labile. The function of
though the effect is transient due to its rapid properdin (also called Factor P), a serum protein
inactivation. C8 and C9 then bind, forming the is to stabilize the C3 convertase, which hydrolyzes
membrane attack complex (C5b6789) that creates a C3, leading to further steps in the cascade, as in the
pore in the plasma membrane of the target cell. The classical pathway (Fig. 15.1).
mechanism of complement-mediated cytolysis is
the production of ‘holes’, approximately 100° A in 2. Production of Alternative Pathway C5
diameter on the cell membrane. This disrupts the Convertase and MAC
osmotic integrity of the membrane, leading to the C3b produced by C3 convertase binds to C3bBb,
release of the cell contents. producing the alternative C5 convertase (C3bBb3b).
C5 convertase may or may not still have factor P
B. Alternative Complement Pathway attached. Properdin enters the reaction sequence
In the complement cascade, the central process is and binds both C3 and C5 convertase to protect
the activation of C3, which is the major component the complex from the action of factor I (a normal
of C. In the classical pathway, activation of C3 is component of serum that is capable of inactivating
achieved by C42 (classical C3 convertase). The C3b). Properdin interaction, therefore, is the
activation of C3 without prior participation of C142 terminal event in the assembly of the activation
is known as the ‘alternative pathway’ (Fig. 15.2). unit for this pathway.
Once the C5 convertase is formed, C5 is
1. Production of Alternative Pathway C3 cleaved to form C5a and C5b, and the spontaneous
formation of the attack complex (C5b-9) quickly
Convertase
follows. C5b is necessary for formation of the mem
This pathway bypasses both the recognition unit brane attack complex and cell lysis. The formation
and the assembly of the activation unit as described of this attack complex proceeds in the same
for the classic pathway. Instead, there are at least manner as it does in the classic pathway (Fig. 15.1).
three normal serum proteins that, when activated
together with C3, form a functional C3 convertase Biological Effects of Complement (C)
1. Bacteriolysis and cytolysis: Complement
mediates immunological membrane damage.
This results in bacteriolysis and cytolysis.
2. Virus neutralization: Neutralization of certain
viruses requires the participation of C.
3. Anaphylotoxins: C2 kinins are vasoactive amines
and increase capillary permeability. The cleavage
products of both pathways of complement
activation, C3a and C5a are anaphylatoxic
(histamine releasing) and chemotactic. C4a also
has anaphylotoxin activity but is less potent even
than C3a. C567 is chemotactic and also brings
Fig. 15.2: Complement cascade—the alternative pathway about reactive lysis.
Chapter 15: Complement System | 99
4. Immune adherence and opsonization : 2. Factor H: It has a strong affinity for C3b, and,
Opsonization is the process of making a after binding C3b, it exerts its control.
particulate antigen more easily identified 3. Anaphylatoxin inactivator: Anaphylatoxin
by a phagocyte. If immune complexes have inactivator is an alphaglobulin that
activated the complement system, the C3b enzymatically degrades C3a, C4a and C5a
bound to them stimulate phagocytosis and which are anaphylatoxins released during
removal of immune complexes. This facilitated the C cascade.
phagocytosis is referred to as opsonization. 4. C4 binding protein: It is a normal human
5. Chemotaxis: Factors Ba (the split product serum protein that binds tightly to activated
from the alternate pathway) and C5a are both C4 and enhances C4b degradation.
chemotactic for polymorphonuclears (PMNs)
and macrophages, thus contributing to local Quantitation of complement (c)
inflammation. C5b67, the partially formed and its components
attack complex, has also been implicated as a Measurement of the complement levels in the serum
chemotactic agent. can be accomplished by estimating the highest
6. Hypersensitivity reaction: Complement partici dilution of the serum lysing sheep erythrocytes
pates in: sensitized with antierythrocytic antibody. The
i. Type II hypersensitivity (cytotoxic) reactions: hemolytic unit of C (CH50) may be defined as that
ii. Type III (immune complex) hypersensitivity amount of complement that lyses 50% of sensitized
reactions: erythrocytes under defined conditions.
7. Autoimmune diseases: Serum C (complement)
components are decreased in many auto Biosynthesis of complement
immune diseases such as systemic lupus Various complement components are synthesized
erythromatosus and rheumatoid arthritis. in different parts of the body, e.g. intestinal
8. Endotoxic shock: Endotoxins can efficiently epithelium (C1), macrophages (C2, C4), spleen
activate the alternative pathway of the comple (C5, C8) and liver (C3, C6, C9). The site of synthesis
ment cascade. There is massive C3 fixation and of C7 is not known. The control mechanism
platelets adherence in endotoxic shock. Large that controls the synthesis of the complement
scale platelet lysis and release of large amounts of component is not known.
platelet factor lead to disseminated intravascular
coagulation (DIC) and throbocytopenia. In Complement deficiencies
endotoxic shock with gram-negative septicemia
Complement deficiencies have been associated
or dengue, hemorrhagic fever may have a similar
with recurrent bacterial and fungal infections
pathogenesis. Depletion of C protects against the
as well as with collagen-vascular inflammatory
Schwartzman reaction.
diseases. Human genetic deficiencies of
Regulation of the Complement System complement components and associated diseases
Several inbuilt control mechanisms regulate the are listed in Table 15.1.
complement cascade at different steps. These are
mainly of two kinds: Table 15.1 Human genetic deficiencies of comple
A. Inhibitors ment components and associated diseases
B. Inactivators
A. Inhibitors: They bind to complement compo Complement Association with disease
nents and halt their further functioning. deficiency
1. C1 esterase: Normal serum contains an C1 inhibitor Hereditary angioneurotic edema
inhibitor of C1 esterase (ClsINH). This heat C1r Systemic lupus erythematosus-
like disease, frequently fatal from
labile alpha neuraminoglycoprotein also overwhelming infection
inhibits many other esterases found in
C2 Increased susceptibility to infections
blood, such as plasmin, kininogen and the
C3 Recurrent bacterial infections
Hageman factor.
C4 Systemic lupus erythematosus-like
2. Vitronectin: It is also known as the S protein. disease
The S protein present in normal serum C5 Recurrent infections—lupus like disease
binds to C567 and modulates the cytolytic C6, C7, C8 Recurrent infections—Disseminated
action of the membrane attack complex. gonococcal infections
B. Inactivators: They are enzymes that destroy C9 Not more susceptible to disease
complement proteins. than other individuals in the general
1. Factor 1: Normal serum contains an endo population`
peptidase, called Factor 1 which cleaves C3b Factor 1 Low C3 levels with recurrent bacterial
and possibly C4b. infections
www.ebook3000.com
100 | Section 2: Immunology
Antigen–Antibody Reactions
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe applications of agglutination reactions
be able to: and their uses
∙∙ Differentiate between precipitation and agglutination ∙∙ Discuss principle and applications of agglutination
∙∙ Describe prozone phenomenon reactions
∙∙ Discuss mechanism and applications of precipit ∙∙ Describe principle of complement fixation test
ation reactions giving suitable examples
∙∙ Discuss principle and clinical applications of
∙∙ Describe types of precipitation reactions
immunofluorescence technique
∙∙ Describe principle and applications of Immuno
elec trophoresis, radial immunodiffusion, ∙∙ Discuss principle, various types and clinical
counteri mmunoelectrophoresis (CIE)), rocket applications of ELISA technique.
electrophoresis
www.ebook3000.com
Chap-16.indd 101 15-03-2016 11:12:02
102 | Section 2: Immunology
Table 16.1 Efficacy of the different Immuno Sensitivity
globulin classes in various serological reactions Sensitivity is defined as the ability of a test to
Serological Reaction IgM IgG IgA identify correctly all those who have the disease,
i.e. “true positives”.
1. Precipitation Weak Strong Variable
2. Agglutination Strong Weak Moderate
Specificity
3. Complement Weak Strong Negative
fixation Specificity has been defined as the ability of a test
to identify correctly those who do not have the
disease, i.e. “true negatives”.
immunity against infectious diseaseas well
as clinical allergy and other immunological SEROLOGICAL REACTIONS
diseases. The study of antigen–antibody reactions in vitro
is called serology [serum and ology]. Antigen–
GENERAL CHARACTERISTICS OF antibody reactions in vitro are known as serological
ANTIGEN–ANTIBODY REACTIONS reactions.
www.ebook3000.com
Chap-16.indd 103 15-03-2016 11:12:03
104 | Section 2: Immunology
Advantages ii. For screening sera for antibodies to influenza
1. The reaction is visible as a distinct band of viruses.
precipitation, which is stable and can be iii. For the laboratory diagnosis of multiple
stained for preservation, if necessary. myeloma or agammaglobulinemia.
2. These immunodiffusion reactions can be used to 4. Double diffusion in two dimensions (Ouch
determine relative concentrations of antibodies terlony Technique
or antigens, to compare antigens, or to determine In the Ouchterlony method, both antigen and
the relative purity of an antigen preparation. antibody diffuse (hence, double diffusion)
3. It also indicates identity, cross-reaction and radially form wells toward each other, thereby
nonidentity between different antigens establishing a concentration gradient. When
soluble antigen and soluble antibody are placed
Types of Immunodiffusion Tests in separate small wells punched into agar that
has solidified on a slide or glass plate, the
1. Single diffusion in one dimension (Oudin
antigen and the antibody will diffuse through
procedure)
the agar. The holes are located only a few
Antibody is incorporated in agar gel in a test
millimeters apart, and antibody and antigen
tube. Antigen solution is then layered over
will interact to form a line of precipitate in the
it. The antigen diffuses downward through
area in which they are in optimal proportions.
the agar gel and wherever it reaches in
In specimens containing several soluble
optimum concentration with antibody, a line
antigens, multiple precipitin lines are observed,
of precipitation is formed (Fig. 16.3). The
each occurring between the wells (Fig. 16.5).
number of bands indicates the number of
The visible line of precipitation permits a
different antigens present.
comparison of antigens for identity (same
2. Double diffusion in one dimension (Oakley-
antigenic determinants), partial identity
Fulthorpe procedure)
(cross-reactivity), or nonidentity against a given
Here the antibody is incorporated in gel in a test
selected antibody.
tube, above which is placed a column of plain
agar and antigen is layered on surface of this.
Antigen and antibody diffuse (double diffusion) Example
towards each other (in one dimension) through Elek test for toxigenicity in diphtheria bacilli:
the intervening column of plain agar and A special variety of double diffusion in two
forms a band of precipitate where they meet at dimensions is the Elek test for toxigenicity in
optimum proportion (Fig. 16.3). diphtheria bacilli.
3. Single diffusion in two dimensions (Radial
immunodiffusion—Mancini method) 5. Immunoelectrophoresis
The assay is carried out by incorporating Immunoelectrophoresis is a procedure that
monospecific antiserum into melted agar and combines electrophoresis and double diffusion
allowing the agar to solidify on a glass plate in a for the separation of antigen–antibody reactions
thin layer. Wells then are punched into the agar, in gels.
and different dilutions of the antigen are placed
into the various wells. As the antigen diffuses
into the agar, a ring of precipitation, a precipitin
ring, forms around the well (Fig. 16.4). The
area of the precipitin ring is proportional to the
concentration of antigen.
Uses
i. To quantitate serum immunoglobulins, Fig. 16.4: Single diffusion in two dimensions (Radial
immunodiffusion—Mancini method)
complement proteins, other substances.
i. Counterimmunoelectrophoresis (CIE) or
Countercurrent eletrophoresis (CIEP)
Counterimmunoelectrophoresis can be used only
for antigens and antibody that migrate in opposite
directions in the electric field. The test is set up
similarly to that for double diffusion in agar. Two
wells are punched about 1 cm apart in an agar
slab on a glass plate. The antigen and antibody
Fig. 16.6: Immunoelectrophoresis solutions are placed in these wells in such a way
that when the electric fleld is applied across the
plate, the antigen will migrate toward the antibody,
In this procedure, antigens are separated by and the antibody will migrate toward the antigen.
electrophoresis in an agar gel. A small drop of A precipitin line will form when the antigen and
solution containing the antigens (usually proteins) antibody meet in optimal proportions resulting in
is placed into a small well punched out of solidified precipitation at a point between them (Fig 16.7).
agar on a small glass plate. The plate then is placed Advantages of Counterimmunoelectrophoresis
in an electric field to allow for the electrophoretic (CIE)
migration of the antigens. A trough is then cut 1. More sensitive: It is 10 times more sensitive than
next to the wells and filled with antibody and simple diffusion in agar.
diffusion allowed to proceed for 18–24 hours.The
2. More rapid assay: It is a much more rapid assay
antibody and the antigens diffuse toward each
(as little as 30 minutes for some antigens) than
other, resulting in the formation of precipitin bands
is double diffusion in gel.
or arcs wherever they are in optimal proportions
(Fig. 16.6).
Clinical Applications
Uses i. For detection of various antigens: such as
i. To separate many antigens as well as to hepatitis B surface antigen (HBS antigen) and
indicate the potential purity of an antigen alpha-fetoprotein in serum and meningococcal
ii. To separate the major blood proteins in serum and cryptococcal antigens in CSF.
for certain diagnostic tests. ii. To detect the presence of anti-DNA antibody in
iii. For testing for normal and abnormal proteins the serum of patients with several autoimmune
in serum and urine. disorders.
www.ebook3000.com
Chap-16.indd 105 15-03-2016 11:12:05
106 | Section 2: Immunology
appropriate position. The precipitate formed antibody causes a clumping or agglutination of the
between antigen and antibody has the shape of cells. Antibodies that produce such reactions are
a rocket. (Fig. 16.8). The height (distance from called aggglutinin. Agglutination occurs optimally
the antigen well to the top of the precipitin band) when antigen and antibodies react in equivalent
of which is proportional to the concentration proportion.
of antigen in the well. is so named because the Agglutination reactions are more sensitive than
precipitin bands resemble rockets.Thus, this precipitin reactions because of the direct nature of
method is primarily a quantitative technique. the antigen/carrier/antibody interaction.
Prozone phenomenon: The prozone phenomenon
Main Application may be seen when either an antibody or an antigen
i. Quantitative estimation of antigens: For is in excess. Just an excess of antibody inhibits
quantitative estimation of antigens and does precipitation reactions, such excess can also inhibit
permit measurement of antigen levels. reactions. This inhibition is called the prozone
phenomenon. Several mechanisms can cause the
Laurell’s Two-dimensional prozone effect.
Electrophoresis Blocking antibodies: Occasionally antibodies
A variant of rocket electrophoresis is Laurell’s two- are formed that will react with the antigenic
dimensional electrophoresis. In this technique, determinants on a cell but will not cause
the antigen mixture is first electrophoretically agglutination (e.g. anti-Rh and anti-Brucella).
separated in a direction perpendicular to that of These antibodies are called blocking antibodies
the final rocket stage. By this method, one can because they inhibit agglutination by the complete
quantitate each of several antigens in a mixture antibody added subsequently.
(Fig 16.9).
APPLICATIONS OF AGGLUTINATION
B. Agglutination Reactions REACTION
When a particulate antigen is mixed with its
antibody in the presence of electrolytes at a 1. Slide Agglutination
suitable temperature and pH, the particles are A drop of sterile saline is placed on one of the
clumped or agglutinated. When antigen is present divisions of the slide or plate and is emulsified in
on the surface of a cell or particle, the addition of it bacterial culture. The diagnostic serum is taken
and is then mixed into the latter. The mixing is
completed by tilting the slide to and fro. Distinct
clumping within 60 seconds is a positive result.
On another area of the slide or plate, in parallel
with the test, a control test is performed, adding
only saline, instead of serum, to the bacterial
suspension. If clumping takes place, the bacterial
suspension is autoagglutinable and the reaction
with the serum must be disregarded.
Uses
Fig. 16.8: Rocket electrophoresis i. For the identification of unknown bacterial
cultures.
ii. Very rapid: It requires only small quantities of
culture and serum.
iii. Also the method for blood grouping and cross-
matching.
2. Tube Agglutination
Fig. 16.9: Laurell’s variant of rocket electrophoresis (two- Serum from a patient thought to be infected with
dimensional electrophoresis) a given bacterium is serially diluted in a series
1. First run—Antigens are separated by electrophoretic of tubes to which the bacteria is added. The last
mobility.
tube showing visible agglutination will reflect the
2. The second run is done at right angles to the first which
drives the antigens into the antiserum containing gel serum antibody titer of the patient. The reciprocal
to form precipitation peaks. The area of the peak is of the greatest serum dilution that elicits a positive
proportional to the concentration of the antigen agglutination is known as the agglutinin titer.
www.ebook3000.com
Chap-16.indd 107 15-03-2016 11:12:07
108 | Section 2: Immunology
highest dilution of the serum that lyses one unit
volume of washed sheep RBCs in the presence of
excess complement within a fixed time (usually
30–60 minutes at 37°C).
Principle
A known antigen is mixed with test serum lacking
complement. When immune complexes have had
time to form, complement is added to the mixture.
If the patient’s serum contains antibody to the
Fig. 16.11: Coagglutination antigen, the resulting antigen–antibody complexes
will bind all of the complement added. In most of
Staph. aureus cell wall binds the Fc portion of the cases, fixation of complement with antigen-
the immunoglobulin molecule, leaving the Fab antibody complex causes in itself no visible effect.
portion free to bind antigen. Visible agglutination (Antigen + Antibody + Complement).
of the staphylococcal cells serves as a positive test This test consists of two separate systems and
to indicate antigen-antibody binding (Fig.16.11). these two systems are tested in sequence (Fig.
16.12). Appropriate controls should be used,
Uses including the following: antigen and serum controls
to ensure that they are not anticomplementary,
i. Coagglutination can be used for detecting the
complement control to ensure that the desired
presence of antigens in serum, urine and CSF.
amount of complement is added, and cell control
ii. Several commercial suppliers have prepared
to see that sensitized erythrocytes do not undergo
coagglutination reagents for identification
lysis in the absence of complement.
of antigens of various streptococcal groups,
including Lancefield groups A, B, C, D, F, G, and N;
Procedure
Streptococcus pneumoniae; Neisseria meningitidis;
N. gonorrhoeae; and Haemophilus influenzae types A. Test system: It consists of (i) Antigen- suspected
A to F grown in culture. of causing the patient’s disease; (ii) Patient’s
serum (antibody); (iii) Complement.
C. Complement Fixation Test (CFT) B. Indicator system: It consists of sheep red cells
(antigen) coated with anti-sheep-red cell
When complement binds to an antigen–antibody
antibody and Complement—An exogenous
complex. it bec omes “fixed” and “used up.”
source of complement (usually guinea pig
Complement fixation tests are very sensitive and
serum). Afterward, sensitized indicator cells,
can be used to detect extremely small amounts of
usually sheep red blood cells previously coated
an antibody for a suspect micro-orgamism in an
with complement-fixing antibodies, are added
individual’s serum. This can detect as little as 0.04
to the mixture. Complement lyses antibody-
µg of antibody nitrogen and 0.01 µg of antigen.
coated red cells.
Standardization of Complement and
Interpretation—Positive CF Test—Absence
Amboceptor
of Lysis
Guinea pig serum is first titrated for complement
activity. One unit or minimum haemolytic dose The specific antibodies are present in the test serum
(MHD) of complement is the highest dilution of and complement is consumed by the immune
guinea pig serum that lyses one unit volume of complexes. Insufficient amount of complement
washed sheep RBCs in the presence of excess of will be available to lyse the indicator cells.
haemolysin (amboceptor) within a fixed time
(usually 30–60 minutes) at a fixed temperature Negative CF Test Lysis of The Indicator Cells
(37°C). Similarly, amboceptor should also be Lysis of the indicator cells indicates lack of antibody
titrated. One MHD of hemolysin is defined as and a negative CF test. Lysis of the indicator cells
www.ebook3000.com
Chap-16.indd 109 15-03-2016 11:12:08
110 | Section 2: Immunology
Therefore, hemolysis indicates a positive result ii. Nagler’s reaction: Cl. perfringens produces
in the indirect test. α-toxin and produces opalescence in serum
or egg yolk media. This reaction is specifically
D. Neutralization Tests neutralized by the antitoxin.
These are of two types:
1. Viral neutralization tests E. Opsonization
2. Toxin neutralization tests. Opsonization is the process in which micro-
organisms or other particles are coated by antibody
1. Viral Neutralization and/or complement, and thus prepared for
Neutralization of viruses by their antibodies “recognition” and ingestion by phagocytic cells.
can be demonstrated in various systems. This Immune opsonization: Phagocytes, such as
antibody-mediated viral inactivation is called viral macrophages, monocytes and neutrophils possess
neutralization. surface receptors (CRI) for C3b and Fc receptors for
Indicator systems: Laboratory animals or tissue antibody. If immune complexes have activated the
culture cells are used as “ indicator systems” in complement system, then Fc and CRI receptors,
these tests. The test serum is diluted serially, present on the phagocyte, bind Fc region of
incubated with a known amount of virus and antibody and C3b bound on immune complexes
the mixture is then added to indicator cultures: respectively, thus facilitating their phagocytosis.
animals, embryonated hen’s egg and tissue culture. This facilitated phagocytosis by antibody and
complement is known as immune opsonization.
2. Toxin Neutralization Nonimmune opsonization: On the contrary,
Bacterial exotoxins are good antigens and nonimmune opsonization requires only C3b
their activity may be completely neutralized by (opsonin) for opsonization. C3b binds to CR1
appropriate concentrations of specific antibody. receptors present on the phagocytes thus
Antibody to bacterial exotoxin is usually referred facilitating their phagocytosis (Fig 16.13).
to as antitoxin which is important clinically, in
protection against and recovery from diseases such F. Immunofluorescence
as diphtheria and tetanus. Toxin neutralization can Immunofluorescence is a process in which dyes
be tested in vivo or in vitro. called fluorochromes are exposed to UV, violet or
A. Toxin neutralization in vivo blue light to make them fluoresce or emit visible light.
i. Toxigenicity test: The neutralizing capa Fluorescent molecules absorb light of one wavelength
city of an antitoxin can be assayed by (excitation) and emit light of another wavelength
neutralization test in which mixture of toxin (emission). Albert Coons and his colleagues (1942)
and antitoxin is injected into a susceptible showed that fluorescent dyes can be conjugated to
animal and the least amount of antitoxin antibodies and that such ‘labelled’ antibodies can
that prevents death or disease in the animal be used and identify antigens in tissues. Antibody
is estimated. molecules bound to antigens in cells or tissue sections
ii. Schick test: With the diphtheria toxin, which can similarly be visualized. The emitted light can be
in small doses causes cutaneous reaction, viewed with a fluorescence microscope, which is
neutralization test can be carried out on the equipped with a UV light source.
human skin. The Schick test is based on the Fluorescent dyes: Rhodamine B and fluorescein
ability of circulating antitoxin to neutralize isothiocyanate (FITC) are the most commonly
the diphtheria toxin given intradermally.
Neutralization (no reaction) indicates
immunity and redness and erythema
indicates susceptibility to diphtheria.
B. Toxin neutralization in vitro: If a toxin has a
demonstrable in vitro effect, this effect can be
neutralized by specific antitoxin.
Examples
i. Antistreptolysin O (ASO) test: Antistreptolysin
O (ASO)-antitoxin, present in the serum of
the patient suffering from Strep. pyogenes
infection, neutralizes the haemolytic activity
of the streptococcal O hemolysin (toxin). Fig. 16.13: Opsonization
Sandwich Technique
The dyes can be conjugated to the Fc region of an
antibody molecule without affecting the specificity
of the antibody. For detection of antibodies by
immunofluorescence, the sandwich technique
can be employed. The antibody is first allowed to
B
react with unlabeled antigen, which is then treated
with fluorescent labeled antibody. A sandwich, is Fig. 16.14: Direct and indirect immunofluorescence.
thus formed, the antigen being in the middle and fluorochrome-labeled reagent. Indirect immuno
labeled and unlabeled antibodies on either side. fluorescence is used to detect the presence of
antibodies in serum following an individual’s expo
Types of Immunofluorescence sure to microorganisms.
There are two main kinds of fluorescent antibody In this technique, a known antigen is fixed
assays: direct and indirect. onto a slide. The test antiserum is then added, and
if the specific antibody is present, it reacts with
1. Direct Immunofluorescence (Fig. 16.14A) antigen to form a complex. When fluorescein-
Principle: In direct staining, the specific antibody labeled anti-immunoglobulin is added, it reacts
(the primary antibody) is directly conjugated with the fixed antibody. The slide is examined with
with fluorescein. It involves fixing the specimen the fluorescence microscope. The occurrence of
containing the antigen of interest onto a slide. fluorescence shows that antibody specific to the
Fluorescein-labeled antibodies are then added test antigen is present in the serum.
to the slide and examined with the fluorescence
microscope for a yellow-green fluorescence. The Uses
pattern of fluorescence reveals the antigen’s location. Diagnosis of syphilis: It is used to identify the
presence of Treponema pallidum antibodies in
Uses the diagnosis of syphilis (fluorescent treponemal
i. It is used to identify antigens, such as those antibody absorption (FTA-ABS).
found on the surface of group A streptococci. Advantages
ii. To diagnose enteropathogenic Escherichia i. The primary antibody does not need to be
coli, Neisseria meningitidis, Salmonella Typhi, conjugated with a fluorochrome.
Shigella sonnei, Listeria monocytogenes, ii. Increase the sensitivity of staining.
Haemophilus influenzae type b.
iii. To diagnose rabies virus. G. Radioimmunoassay (RIA)
One of the most sensitive techniques for detecting
Disadvantage antigen or antibody is radioimmunoassay
Separate fluorescent conjugates hence to be (RIA). The technique was first developed by two
prepared against each entigen to be tested. endocrinologists, SA Berson and Rosalyn Yalow, in
1960. The technique soon proved its own value for
measuring hormones, serum proteins, drugs, and
2. Indirect Immunofluorescence
vitamins at concentrations of 0.001 micrograms per
(Fig. 16.14B) milliliter or less. The significance of the technique
In indirect staining, the primary antibody is was acknowledged by the award of a Nobel Prize
unlabeled and is detected with an additional to Yalow in 1977, some years after Berson’s death.
www.ebook3000.com
Chap-16.indd 111 15-03-2016 11:12:09
112 | Section 2: Immunology
Principle of RIA its extreme sensitivity (measuring picograms
The principle of RIA is based on competitive of antigen per milliliter).
binding of radiolabeled antigen (e.g. 125I and ii. To determine the presence of the hepatitis B
unlabeled antigen to a high-affinity antibody. The antigen.
labeled and unlabeled (test) antigens compete
for the limited binding sites on the antibody. This H. Enzyme-linked Immunosorbent Assay
competition is determined by the level of the (ELISA) (Fig. 16.15)
unlabeled (test) antigen present in the reacting Enzyme-linked Immunosorbent Assay, commonly
system. known as ELISA or EIA), is similar in principle to
RIA but the radioactive tag used in RIA techniques
Procedure can be replaced with an enzyme. When this enzyme
1. Measured quantities of labeled antigen is linked to an antibody and used to detect and
(radiolabeled) antigen (of the same kind being measure other antibodies or antigens, the assay is
tested) and antibody (specific to the antigen called the enzyme-linked immunosorbent assay
being tested) are mixed and incubated, one (ELISA). An enzyme conjugated with antibody
mixture with and one without added test reacts with a colorless substrate to generate a
sample. colored reaction product. Such a substrate is called
The labeled antigen is mixed with antibody a chromogenic substrate.
at a concentration that saturates the antigen- Enzyme-linked immunosorbent assay is
binding sites of the antibody molecule. highly sensitive, highly specific and less expensive
technique used in serology to detect antigens or
2. Then increasing amounts of the test sample
antibodies.
(unlabeled antigen) of unknown concentration
are added. The antibody does not distinguish
labeled from unlabeled antigen, and so the two Principle
kinds of antigens compete for available binding ELISA is based on two principles:
sites on the antibody. 1. Solid phase immunoassays are more widely
3. With increasing concentrations of test antigen used. It refers to the binding of either antigen or
(unlabeled antigen), more labeled antigen will antibody to a variety of solid materials, such as
be displaced from the binding sites. polyvinyl or polycarbonate wells or membranes
4. The antigen–antibody complex is washed to (discs) of polyacrylamide, paper or plastic or
remove unbound radiolabeled antigen from the metal beads or some other solid matrix.
mixture. The antigen is separated into ‘free’ and 2. Antigens and antibodies can be covalently
‘bound’ fractions after the reaction and their attached to an active enzyme (such as alkaline
radioactive counts measured. phosphatase or horseradish peroxidase and
5. The radioactivity associated with the antibody β-galactosidase), with the resulting complexes
is then detected by means of radioisotope still fully functional, both immunologically
analyzers and autoradiography. A little amount and enzymatically. Enzyme activity is used to
of bound radioactivity indicates that there is measure the quantity of antigen or antibody
large amount of antigen; and a large amount of present in the test sample. After all the
bound radioactivity indicates that there is little unreacted material is washed away, the
antigen in the sample. substrate for the enzyme is added (usually
6. The standard dose response or reference one that will yield a colored product, such
curve has to be prepared first for any reacting as p-nitrophenol phosphate for alkaline
system. This is plotted on graph by taking the phosphatase), and the conversion of the
ratio of bound radiolabeled antigen to free substrate from colorless to color is a measure of
radiolabeled antigen against varying known antigen-antibody interaction. The test is usually
amounts of unlabeled antigen and the antigen done using microtiter plates (96 w ell) suitable
concentration of the test sample can be read for automation.
from this curve. This curve will allow the
measurement of antigen present by merely Types of ELISA (Fig 16.15)
counting the radioactivity present in the test 1. Indirect ELISA
antigen-antibody precipitated complexes. 2. Sandwich ELISA
3. Competitive ELISA
Uses 1. Indirect ELISA: The indirect immunosorbent
To measure the concentration of certain
i. assay detects antibodies rather than antigens.
hormones: such as insulin, testosterone, Serum or other sample containing primary
growth hormone, and glucagon because of antiboby is added to antigen-coated microtiter
well. Any free antibody is washed away and positive. If the antigen is not recognized by the
the presence of antibody bound to the antigen absorbed antibody, the ELISA test is negative
is detected by adding an enzyme-conjugated because the unatt ached antigen has been
secondary anti-isotype antibody (antibody 2), washed away, and no antibody-enzyme is
which binds to the primary antibody. Any free bound.
antibody 2 then is washed away, and a substrate 3. Competitive ELISA: In this technique, antibody
for the enzyme is added. The amount of colored is first incubated in solution with a sample
reaction product that forms is measured by containing antigen. The antigen–antibody
specialized spectrophotometric plate readers, mixture is then added to an antigen coated
which can measure the absorbance of all of microtiter well. The more antigen present
the wells of a 96-well plate in less than a few in the sampIe, the less free antibody will be
seconds. available to bind to the antigen-coated well.
2. Sandwich ELISA: The most frequently used Addition of an enzyme-conjugated, secondary
ELISA for detecting microbial antigen is the antibody specific for the isotype of the primary
sandwich solid-phase ELISA. In this technique, antibody can be used to determine the amount
the antibody (rather than the antigen) is of primary antibody bound to the well as in
immobilized on a microtiter well. The test an indirect ELISA. In the competitive assay,
sample is then exposed to the solid-phase however, the higher the concentration of
antibody, to which the antigen, if present, antigen in the original sample, the lower the
will bind. After the well is washed, a second absorbance.
enzyme-linked antibody specific for test
antigen is added and allowed to react with the USES OF ELISA
bound antigen. The conjugated antibody will
react with the antigen held to the solid-phase by ELISA has been used to detect antigens and
the first antibody, forming an antibody-antigen- antibodies of various microorgnisms.
antibody sandwich on the solid phase. After any
free second antibody is removed by washing, Examples
substrate is added, and the colored reaction 1. Parasites
product is measured.
If the antigen has reacted with the absorbed ∙∙ Entamoeba histolytica antigens in feces.
antibodies in the first step, the ELISA test is ∙∙ Toxoplasma antigens in the patient serum.
www.ebook3000.com
Chap-16.indd 113 15-03-2016 11:12:10
114 | Section 2: Immunology
2. Bacteria and Northern blotting which detects mRNAs. It is a
∙∙ Haemophilus influenzae antigens in spinal variation of an ELISA.
fluid. A specific antibody in a mixture can also be
∙∙ β-hemolytic streptococcal antigen in spinal identified by Western blotting. Known antigens of
fluid. well-defined molecular weight are separated by
SDS-polyacrylamide gel (SDS-PAGE), and blotted
∙∙ Labile enterotoxin of E. coli in stools.
onto nitrocellulose in this case. The separated
To detect antibody specific for Mycoplasmas, bands of known antigens are then probed with
Chlamydiae, Borrelia burgdorferi. the sample (test sample) suspected of containing
antibody specific for one or more of these antigens.
3. Viruses Reaction of an antibody with a band is detected
To detect antibody specific for hepatitis virus; by using either radiolabeled or enzyme-linked
∙∙ Herpes simplex viruses 1 and 2 secondary antibody that is specific for the species
of the antibodies in the test sample. The enzyme
∙∙ Respiratory syncytial virus (RSV)
substrate is subsequently added, which indicates
∙∙ Cytomegalovirus positive test. The substrate changes color in the
∙∙ Human immunodeficiency virus (HIV) presence of enzyme and permanently stains the
∙∙ Rubella virus. nitrocellulose paper. The position of the band on
the paper indicates the antigen with which the
Cassette-based Membrane-bound ELISA antibody has reacted.
Assays
Cassette-based membrane-bound ELISA assays,
Application
designed for testing a single serum, can be i. Confirmatory testing for human immuno
performed rapidly (often within 10 minutes) as deficiency virus.
compared with the 2–4 hours taken for microplate ii. Antibodies against microbes with numerous
ELISA. There is no need for microplate washers or cross-reacting antibodies.
readers. The result is read visually. Inbuilt positive
and negative controls are usually provided for J. Immunochromatographic Tests
validation of the test procedure.
A one-step qualitative immunochromatographic
Examples of Cassette ELISA techn ique has found wide application in
serodiagnosis due to its simplicity, economy
Used for the detection of HIV type 1 and 2 antibodies.
and reliability. A description of its use for Hbs
Antibody-capture ELISAs: Antibody-capture antigen detection illustrates the method. The test
ELISAs are particularly valuable for detecting IgM system is a small cassette containing a membrane
in the presence of IgG. Toxoplasmosis, rubella, impregnated with anti-Hbs antigen antibody
and other infections are diagnosed using this colloidal gold dye conjugate. The membrane is
technology. exposed at three windows on the cassette. The
test serum is dropped into the first window. As
Slot-blot and Dot-blot Assays the serum travels upstream by capillary action, a
Slot-blot and dot-blot assays force the target colored band appears at the second window (test
antigen through a membrane filter, causing it to site) if the serum contains Hbs antigen, due the
become affixed in the shape of the hole (a dot or formation of an Hbs antigen–antibody conjugate
a slot). When test (patient) serum is layered onto complex. This is the positive reaction. Absence of
the membrane, specific antibodies, if present, will a colored band at the test site indicates a negative
bind to the corresponding dot or slot of antigen. reaction. Simultaneously, a colored band should
Addition of a labeled second antibody and appear in every case at the third window, which
subsequent development of the label allows visual forms an inbuilt control, in the absence of which
detection of the presence of antibodies based on the test is invalid. The test is claimed to be nearly
the pattern of antigen sites. as sensitive and specific as EIA tests.
www.ebook3000.com
Chap-16.indd 115 15-03-2016 11:12:10
116 | Section 2: Immunology
8. Tube agglutination test is used for serological 12. Direct immunofluorescence test may be used for
diagnosis of: detection of:
a. Enteric fever a. Rabies virus antigens
b. Infectious mononucleosis b. Antibodies in syphilis
c. Typhus fever c. Both the above
d. All the above d. None of the above
9. Which of the following is/are example/s of 13. Indirect immunofluorescence test may be used for
heterophile agglutination test? detection of:
a. Weil-Felix reaction a. Rabies virus antigens
b. Paul-Bunnel test b. Antibodies in syphilis
c. Streptococcus MG agglutination test c. Both the above
d. All the above d. None of the above
10. Which of the following is/are example/s of passive 14. ELISA can be used for detection of antibodies in:
agglutination test? a. HIV b. Rubella virus
c. Hepatitis B virus d. All the above
a. Latex agglutination test
b. Haemagglutination test 15. The technique of immunoblotting to analyse RNA
c. Coagglutination is named:
d. All the above a. Southern blot
b. Northern blot
11. Which of the following is/are example/s of c. Western blot
neutralization test? d. None of the above
a. Schick test
b. Antistreptolysin ‘0’ test ANSWERS (MCQs)
c. Nagler reaction 1. a; 2. b; 3. a; 4. c; 5. b; 6. d; 7. d; 8. d; 9. d; 10. d; 11. d; 12. a;
d. All the above 13. b; 14. d; 15. b
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe cluster of differentiation (CD)
be able to: ∙∙ Discuss the following; Natural killer cells or NK cells;
∙∙ Differentiate between T and B cells in a tabulated killer cells or K cells or ADCC cells; human leukocyte
form antigen (HLA).
www.ebook3000.com
118 | Section 2: Immunology
Thymus (T)-dependent lymphocytes : B-cells and secrete various cytokines that are
Lymphocytes produced in the thymus are called required for development. A selection process
‘thymus (T) dependent lymphocytes’ or ‘T-cells’. within the bone marrow eliminates B-cells with
Lymphocyte proliferation in the thymus is not self-reactive antibody receptors like thymic
dependent on antigenic stimulation,unlike selection during T-cell maturation. Bone
in the peripheral organs. Differentiation marrow is not the site of B-cell development
and maturation are under the influence of in all species.
hormones such as thymopoietin and thymosin,
produced by the epithelial elements in the B. Peripheral (Secondary) Lymphoid Organs
thymus. 1. Lymph nodes: They are encapsulated bean-
Thymus-dependent antigens: T-lymphocytes shaped structures containing a reticular network
are selectively seeded into certain sites in the packed with lymphocytes, macrophages, and
peripheral lymphatic tissues, being found in dendritic cells. Clustered at junctions of the
the white pulp of the spleen, around the central lymphatic vessels, lymph nodes are the first
arterioles, and in the paracortical areas of organized lymphoid structure to encounter
lymph nodes. These regions have been termed antigens that enter the tissue spaces.
‘thymus-dependent’. While thymectomy affects
CMI primarily, it also diminishes antibody Structure of lymph node: Lymph nodes have an
response to many types of antigens (thymus- indentation called the hilus where blood vessels
dependent antigens) such as sheep erythrocytes enter and leave the node. A typical lymph node
and bovine serum albumin. is surrounded by a fibrous capsule from which
Thymus and immune function: The importance trabeculae penetrate into the nodes.
of thymus in lymphocyte proliferation and Morphology: Morphologically, a lymph node
development of CMI is evident from the can be divided into three roughly concentric
lymphopenia, deficient graft rejection and regions:,), each of which supports a distinct
the so called ‘runt disease’ seen in neonatal microenvironment.
thymectomized mice. i. Cortex: The outermost layer, the cortex (B-cell
A congenital birth defect in humans (DiGeorge’s area),, contains lymphocytes (mostly B-cells),
syndrome) and in certain mice (nude mice) in macrophages, and follicular dendritic cells
which the thymus fails to develop are other arranged in primary follicles. After antigenic
evidence of the importance of the thymus. In challenge, the primary follicles enlarge into
both the cases, there is an absence of circulating secondary follicles, each containing a germinal
T-cells and of cell-mediated immunity and an center.
increase in infectious disease. ii. Paracortex (T-cell area): Beneath the cortex
2. Bursa of Fabricius: In birds, undifferentiated is the paracortex, which is populated
lymphocytes move from the bone marraw to largely by T-lymphocytes and also contains
the Bursa of Fabricius where B-cells mature. The interdigitating dendritic cells. The paracortex
bursa is also a site of lymphocytic proliferation is sometimes referred to as a thymus-
and differentiation. Stem cells from the yolk sac, dependent area in contrast to the cort ex,
fetal liver and bone marrow enter the bursa, which is a thymus-independent area.
proliferate and develop into immunocompetent iii. Medulla: The innermost layer of a lymph
‘bursal dependent’ or B-cells (to designate their node, the medulla (consisting of cellular cords
origin in the bursa). These migrate and seed containing T-cells, B-cells, abundant plasma
selective areas in the peripheral lymphoid cells and macrophages) In the medulla, the
organs—the mantle, germinal follicles and lymphocytes, plasma cells and macrophages
perifollicular regions of the spleen, and the are arranged as elongated branching bands
far cortical areas and medullary cords of (medullary cords). The cortical follicles and
lymphocytes. These are called ‘bursa-dependent’ medullary cords contain B-lymphocytes
or ‘thymus-independent areas’. B-lymphocytes and constitute the bursa dependent areas.
transform into plasma cells and secretes Paracortical area contains T-lymphocytes and
antibodies following antigenic stimulation. constitutes the thymus dependent area.
3. Bone marrow: In humans and other mammals,
Functions of lymph node
the bone marrow acts as the bursa equivalent
and is the site of B-cell origin and development. i. Lymph nodes act as a filter for lymph.
Arising from lymphoid progenitors, immature ii. They phagocytose foreign materials, including
B-cells proliferate and differentiate within microorganisms.
the bone marrow, and stromal cells within iii. They help in the proliferation and circulation
the bone marrow interact directly with the of T- and B-cells.
Chapter 17: Structures and Functions of the Immune System | 119
iv. They enlarge following local antigenic Lymphocytes: Lymphocytes constitute 20%–40%
stimulation. of the body’s white blood cells and 99% of the cells
in the lymph.Many mature lymphoid cells are long-
2. Spleen
lived, and persist as memory cells for many years.
The spleen is the large secondary lymphoid organ
The lymphocytes can be broadly subdivided
located in the abdominal cavity.
into three populations—B-cells, T-cells, and
Structure of spleen: It has a capsule from that natural killer cells-on the basis of function and
extends a number of projections (trabeculae) into cell-membrane components. According to their
the interior to form a compartmentalized structure. size, lymphocytes can be classified into small (5–8
The compartments are of two types, white pulp and μm ), medium (8–12 μm) and large (12–15 μm)
the red pulp. lymphocytes.
White pulp: The splenic white pulp surrounds Lymphatic recirculation: Lymphopoiesis takes
the branches of the splenic artery, forming a place mainly in the central lymphoid organs where
periarteriolar lymphoid sheath (PALS) populated they differentiate and mature before entering the
mainly by T-lymphocytes. Primary lymphoid circulation and then the peripheral lymphoid organs
follicles are attached to the PALS. These follicles and tissues. These populations of lymphocytes do
are rich in B-cells and some of them contain not remain distinct but mix together in a process
germinal centers which develop following antigenic known as ‘lymphocyte recirculation’. There is
stimulation. The lymphatic sheath surrounding the a constant traffic of lymphocytes through the
central arterioles is the thymus dependent area blood, lymph, lymphatic organs and tissues. This
of the spleen. The perifollicular region, germinal recirculation ensures that following introduction of
centre and mantle layer form the bursa dependent antigen into any part of the body, lymphocytes of
(thymus-independent) areas. appropriate specificity would reach the site during
their ceaseless wandering and mount an immune
Red pulp: The splenic red pulp consists of a response. Recirculating lymphocytes are mainly
network of sinusoids. T-cells. B-cells tend to be more sessile. Chronic
Functions of spleen thoracic duct drainage will therefore result in
selective T-cell depletion.
i. Functions as the graveyard for blood cells.
ii. Mounting immune responses to antigens in the
blood stream. Differences between T- and B-Cells
Many tests help in differentiation of T- and B-cells
3. Mucosa-associated lymphoid tissue (MALT) (Table 17.1). These include:
The mucosa lining the alimentary, respiratory,
1. Thymus-specific antigens: Which are absent on
genitourinary and other lumina and surfaces are
B-cells.
constantly exposed to numerous antigens. These
vulnerable membrane surfaces are defended by 2. T-cell receptor (TCR): Which resembles but
a group of organized lymphoid tissues known differs from antibody, and CD2- and CD3-
collectively as mucosal-associated lymphoid tissue associated proteins on their surface.
(MALT). 3. Surface immunoglobulins: B-cells have
immunoglobulin on their surface.
Gut-associated lymphoid tissue (GALT): GALT 4. SRBC rosette: T-cells bind to sheep erythrocytes
includes the tonsils, adenoids, and Peyer’s patches forming rosettes (SRBC) or E rosette, through
in the intestine. CD2 molecule. B-cells do not bind.
Bronchial associated lymphoid tissue (BALT): 5. EAC rosettes: B-cells bind to sheep erythrocytes
Also occurs in the respiratory system. coated with antibody and complement,forming
EAC rosettes, due to the presence of a C3
Mucosal or secretory immune system: Mucous receptor (CR2) on the B-cell surface.
membranes are an effective barrier to the
6. Microvilli: Viewed under the scanning
entrance of most pathogens, which contributes to
microscope, T cells are generally free of
nonspecific immunity. This indicates the existence
cytoplasmic surface projections, while B-cells
of a common mucosal or secretory immune system.
have an extensively filamentous surface, with
Cells of the lymphoreticular system numerous microvilli
7. Blast transformation: T-cells undergo blast
Lymphoid Cells: Cells of the Immune System transformation, evidenced by enhanced DNA
The cells responsible for both nonspecific and synthesis, on treatment with mitogens, such as
specific immunity are the white blood cells called phytohemagglutinin (PHA) or Concanavalin
leukocytes. A (Con A), while B-cells undergo similar
www.ebook3000.com
120 | Section 2: Immunology
Table 17.1 Comparison of T-cells and B-cells differentiation and functional properties of the
cells. Thus a cell displaying CD1 is identified by
Property T cell B cell
the binding of antibodies against CD1. Each class
A. Origin Bone marrow Bone marrow of leucocyte displays a diagnostic pattern of CDs.
B. Maturation Thymus Bursal equivalent: Over 200 CD markers have been identified so far.
bone marrow,
Examples
Payer’s patches
∙∙ CD3 is expressed only by T-cells.
C. Location
∙∙ CD 19 is expressed only by B-cells.
1. Peripheral blood 65–85% 15–25%
∙∙ CD64 is expressed only by monocytes.
2. Lymph node 60–75% 30–35% ∙∙ CD66 is expressed only by granulocytes.
3. Spleen 25–45% 55–60% ∙∙ CD68 is expressed only by macrophages.
4. Thoracic duct 80–90% 10–20% ∙∙ On the other hand, CD18 and CD45 are expr
5. Thymus 96% Negligible essed by a variety of leucocyte types.
D. Thymus specific + – 2. Antigen recognition receptors
antigens These include membrane-bound (surface)
E. CD3 receptor + – immunoglobulins (mlgs or slgs) in B-cells and
F. Surface – + T-cell receptors (TCRs) in T-cells. In contrast to
immunoglobulins CDs, which can serve as diagnostic feature for
G. Receptor for Fc – +
all leukocytes, antigen recogn ition receptors
piece of IgG are limited to B- and T-lymphocytes only. These
H. SRBC rosette (E + –
receptors are required for B- and T-cells to be
rosette) antigen reactive. Reaction of antigens with mlgs
and TCRs activates B-cells and T-cells respectively,
I. EAC rosette (C3 – +
receptor leading to proliferation and differentiation.
J. Numerous – + b. On the Basis of Functions
microvilli, on
A. Regulatory T-cells
surface
1. Helper /inducer cell(TH): Helper/inducer
K. Blast transform + –
cell (TH), with CD 4 surface marker, MHC
ation with:
a. anti-CD 3 – + class II restriction; generally stimulating
b. anti-Ig + – and promoting the growth of T-cells and
c. PHA + – macrophages. They help B-cells make antibody
d. Concanavalin A – + in response to antigenic challenge; stimulate
e. Endotoxins
cell mediated immunity. Based on the different
profiles of cytokines produced, two subsets are
transformation with bacterial endotoxins, identified. Th1 and Th2.
Staphylococcus aureus (Cowan 1 strain) or EB Th1 cells: Th1 cells produce mainly the cytokines
virus. interferon gamma (IFN-g) and interleukin-2
(IL2) which activate macrophages and T-cells
T-lymphocytes promoting CMI, destruction of target cells and
Types of T-cells killing of intracellular microbes, such as tubercle
and lepra bacilli.
Based on their surface markers, MHC restriction,
TH2 cells: TH2 cells produce mainly the
target cells and function, the following T-cells sub
cytokines IL4, 5 and 6 which stimulate B-cells
types are recognised:
to form antibodies.
T-cells may be broadly classified into:
2. Suppressor T-cells (TS cells): These have CD8
surface marker and MHC class I restriction.
a. On the Basis of Surface Markers They can suppress B-cell and T-cell response.
Classification of lymphocytes on the basis of B. Effector cells
surface markers makes use of two important char
1. Delayed type-hypersensitivity T-cells (T-cells):
acteristics:
They are involved in delayed hypersensitivity
1. Cluster of differentiation or cluster determinant and cell-mediated immune response.
(CD) 2. Cytotoxic T-cells(TC-cells): They are also called
The term cluster of differentiation (CD) refers CD8+ cells with CD8 surface marker and MHC
families of surface glycoprotein antigens that can class I restriction. They can kill and lyse target
be recognized by specific antibodies produed cells carrying new or foreign antigens, including
against them. These markers reflect the stage of tumour, allograft and virus infected cells.
Chapter 17: Structures and Functions of the Immune System | 121
ii. B-cells and Plasma Cells i. They are not inducible by antigen.
ii. They lack the classic antigen-specificity of
B-cell Maturation
T-cells and B-cells.
The B-lymphocyte derived its letter designation iii. They are not restricted by MHC-encoded
from its site of maturation, in the bursa of Fabricius proteins.
in birds; and the bone marrow of mammals, iv. NK activity is ‘natural’ or ‘nonimmune’ as
including humans and mice. B-cell differentiation it does not require sensitisation by prior
also takes place in the fetal liver and fetal spleen. antigenic contact.
About 5–15% of the circulating lymphoid pool v. They belong to a different lineage from T- and
are B cells defined by the presence of surface B-cells.
immunoglobulin.
Mature B-cell undergoes clonal proliferation on Functions: They are considered to be important
contact with its appropriate antigen. Interaction i. Immune surveillance
between antigen and the membrane-bound ii. Natural defence against virus infected and
antibody on a mature naive B-cell, as well as maliganant mutant cells.
interactions with T-cells and macrophages, iii. NK-cells are capable of nonspecific killing
selectively induces the activation and differentiation of virus-transformed target cells and are
of B-cell clones of corresponding specificity. In involved in allograft and tumour rejection.
this process, the B-cell divides repeatedly and
differentiates, generating a population of plasma b. Antibody-dependent Cell-mediated
cells and memory cells. Plasma cells, synthesize
Cytotoxicity (ADCC)
and secrete antibody. Memory cells circulate until
activated by specific antigen. A subpopulation of LGLs possesses surface
receptors for the Fc part of Ig. They are capable
Plasma cell: They are fully differentiated
of lysing or killing target cells sensitised with
antibody-secreting effector cells of the B-cell
IgG antibodies. This is an example of a process
lineage. Antigenically stimulated B-cells undego
known as antibody-dependent cell-mediated
blast transformation, becoming successively
cytotoxicity (ADCC). This antibody dependent
plasmoblasts, intermediate transitional cells and
cellular cytotoxicity is distinct from the action of
plasma cells. Plasma cells are factories of antibody
cytotoxic T-cells, which is independent of antibody.
production. A plasma cell makes an antibody of a
ADCC-cells were formerly called killer (K)-cells but
single specificity, of a single immunoglobulin class
are now classified with NK-cells.
and allotype and a single light chain type only. An
exception is found in primary antibody response,
when a plasma cell producing IgM initiallyand later
c. Lymphokine-activated Killer (LAK)
it may switch to IgG production. Lymphocytes, Cells
lymphoblasts and transitional cells may also Lymphokine activated killer (LAK) cells are
synthesize Ig to some extent, while plasma cell is NK-lymphocytes treated with interleukin-2 (lL-2),
the best antibody producing cell. which are cytotoxic to a wide range of tumour cells
without affecting normal cells. IL-2 also acts as a
iii. Null Cells growth factor for NK-cells.
A small proportion (5-10%) of lymphocytes
that lack distinguishing phenotypic markers Phagocytic Cells
characteristic of T- or B-lymphocytes are called Phagocytic cells are the mononuclear macrophages
null cells. Because of their morphology, they are (of blood and tissues) and the polymorphonuclear
also known as large granular lymphocytes (LGL). microphages.
The member of this group is the
a. Natural killer (NK)-cells Mononuclear cells: The mononuclear phagocytic
b. Antibody dependent cellular cytotoxic (ADCC)- system consists of monocytes circulating in the
cells blood and macrophages in the tissues Both types
c. Lymphokine-activated killer (LAK)-cells. are highly phagocytic and make up the monocyte-
The term NK-cell is sometimes used as a macrophage system.
common name for all null cells. Macrophages: Monocytes leave the circulation and
reach various tissues to become transformed into
a. Natural Killer (NK) Cells macrophages, with morphological and functional
Natural killer (NK) cells are derived from large features characteristic of the tissues. Macrophages
granular lymphocytes (LGL). They differ from spread throughout the animal body and take up
K-cells in being independent of antibody. NK-cells residence in specific tissues where they are given
differ Tc cells in other properties as well: special names, e.g. alveolar macrophages in the
www.ebook3000.com
122 | Section 2: Immunology
lung, histiocytes in connective tissues, Kupffer cells Major Histocompatibility complex
in the liver, Mesangial cells in the kidney, microglial
cells in the brain and osteoclasts in the bone. The major histocompatibility complex (MHC) is
a remarkable cluster of genes that control T-cell
Functions of Macrophages recognition of self and nonself. MHC proteins
play a pivotal role in “presenting” antigens to
1. Phagoc ytosis: The primary function of T-cells. In fact, T-cells do not respond to foreign
macrophages is phagocytosis. peptides unless the antigen peptides are properly
2. Antigen presentation to T-cells to initiate “presented” (that is, offered in combination with
immune immune responses. a MHC molecule). In humans, the MHC is called
3. Secretion of cytokines to activate and promote the human leukoc yte antigen (HLA) complex.
innate immune response. These proteins were first detected by their effect on
Macrophages secrete interleukin-1, interleukin-6, transplant rejection (that is, tissue incompatibility).
tumor necrosis factor, and interleukin-12 In 1980, Snell, Dausset and Benacerrof were
in response to bacterial interaction, which awarded the Nobel prize for their work on MHC
stimulate immune and inflammatory responses, and the genetic central of immune response.
including fever. One of the most potent activators
of macrophages is interferon gamma (IFN-g) HLA Complex
secreted by activated TH cells.
The major antigens determining histocompatibility
Polymorphonuclear Microphages in human beings are alloantigens, characteristically
found on the surface of leukocytes. Human MHC
Microphages are the polymorphonuclear antigens are therefore synonymous with human
leucocytes of the blood.Because of the irregular- leucocyte antigens (HLA), and the MHC complex
shaped nuclei, granulocytes are also called of genes with the HLA complex.
polymorphonuclear leukocytes or PMNs. Three The HLA complex of genes is located on the
types of granulocytes exist: basophils, eosinophils, short arm of chromosome 6 (Fig. 17.1). It consists
and neutrophils. of three separate clusters of genes:
a. Neutrophils: They are actively phagocytic 1. HLA Class I comprising A, B and C loci: HLA
and form the predominant cell type in acute class I comprising A, Band C loci. Class 1
inflammation. The phagocytic property of proteins are encoded by the HLA-A, HLA-B and
neutrophils is nonspecific, except for its HLA-C genes in humans. HLA Class I antigens
augmentation by opsonins. (A, B and C) are found on the surface of virtually
b. Eosinophils: They possess phagocytic activity all nucleated cells and, in some species (i.e.
but only to a limited degree. They are found mice, but not humans), on red blood cells as
in large numbers in allergic inflammation, well.
parasitic infections and around antigen- They are the principal antigens involved in
antibody complexes. graft rejection and cell mediated cytolysis. Class
c. Basophils: Basophil leukocytes are found in the I molecules may function as components of
blood and tissues (mast cells). Their cytoplasm hormone receptors.
has large numbers of prominent basophilic 2. Class II or the D region consisting of DR, DQ
granules containing heparin, histamine, and DP loci: Class II proteins are encoded
serotonin and other hydrolytic enzymes. by the HLA-D region. There are three main
Degranulation of mast cells, with release of sets: the DP, DQ and DR-encoded molecules.
pharmacologically active agents, constitutes the HLA Class II antigens are more restricted in
effector mechanism in anaphylactic and atopic distribution, being found only on cells of the
allergy. immune system—macrophages, dendritic cells,
Dendritic cells: While macrophages are the activated T-cells, and particularly on B-cells.
major antigen presenting cells, dendritic cell also 3. Class III or the complement region: Class III or
performs this function. Dendritic cells are bone the complement region containing genes for
marrow derived cells of a lineage different from complement components C2 and C4 of the
the macrophages and T- or B-lymphocytes. They
possess MHC class II antigens.
They are more potent antigen-presenting cells
than macrophages and B-cells, both of which need
to be activated before they can function as antigen-
presenting cells (APCs). Dendritic cells are specially
involved in the presentation of antigens to T-cells
during the primary immune response. Fig. 17.1: HLA complex loci on chromosome
Chapter 17: Structures and Functions of the Immune System | 123
classical pathway, as well as properdin factor B 4. An association between HLA types and diseases:
of the alternative pathway, heat shock proteins For example, strong association has been found
and tumor necrosis factors alpha and beta. between ankylosing spondylitis and HLA-B27,
HLA loci are multiallelic, that is, the gene rheumatoid arthritis and HLA-DR4, and many
occupying the locus can be anyone of several autoimmune conditions and HLA-DR3.
alternative forms (alleles). As each allele
determines a distinct product (antigen), the MHC RESTRICTION
HLA system is very pleomorphic. For example,
at least 24 distinct alleles have been identified Both CD4+ and CD8+ T-cells can recognize antigen
at HLA locus A and 50 at B. only when it is presented by a self-MHC molecule,
an attribute called self-MHC restriction. Both class
I and class II antigens operate in this phenomenon.
HLA Molecules
Cytotoxic T-lymphocytes from immunised mice
HL A antigens are two-chain glycoprotein are able to kill and lyse virus infected target
molecules anchored on the surface membrane cells only when the T-cells and target cells are of
of cells. Class I and class II MHC are membrane- the same MHC type. Helper T-cells can accept
bound glycoproteins that are closely related in both antigen presented by macrophages only when
structure and function. the macrophages bear the same class II MHC
molecules on the surface. For T-cells participating
Role of MHC Diversity in delayed type hypersensitivity the antigen has to
1. Transplantation : The MHC system was be presented along with class II MHC
originally identified in the context of
transplantation, which is an artificial event. Key Points
2. For protecting the species from the broadest
The immune system is organized into several special
possible number of pathogens: The primary aim
tissues which are collectively termed as lymphoid or
of the MHC may be defence against microbes immune tissues
and not against the graft. The lymphoid organs, based on their functions,
3. Nonimmunological phenomena: Such as are classified into primary (central) and secondary
individual odour, body weight in mice and egg (peripheral) lymphoid organs
laving in chickens. There are three types of lymphocytes: B-cells, T-cells,
and natural killer cells (NK-cells)
HLA Typing T-cells perform two important functions: cytotoxicity
and delayed hypersensitivity
Antisera for HLA typing were obtained principally T-cells play a key role in regulating antibody
from multiparous women as they tend to production and CMI, and in suppression of certain
have antibodies to the HLA antigens of their immune responses
husbands, due to sensitisation during pregnancy. Bone marrow-derived lymphocytes are known as
Monoclonal antibodies to HLA antigens have B-lymphocytes or B-cells and perform two important
been developed. functions: First, they differentiate themselves into
Typing is done serologically by microcytotoxicity, plasma cells and produce antibodies. Second, they
can present antigen to helper T-cells
which tests for complement mediated lysis of
NK-cells have the ability to kill certain virally infected
peripheral blood lymphocytes with a standard set
cells and tumor cells without prior sensitization
of typing sera. However, serological typing is not
The MHC in humans is known as HLA complex. In
possible for HLA-DR antigens, which are detected humans, the HLA complex of genes is located on
by the mixed leucocyte reaction (MLR) and primed short arm of chromosome 6 containing several genes
lymphocyte typing (PLT), respectively. Genetic that are critical to immune function
methods are being used increasingly for HLA-typing The genes encode MHC proteins that are classified
in advanced centres. These employ restriction into three groups or classes known as the class I, class
fragment length polymorphism (RFLP) and gene II, and class III molecules
sequence specific oligonucleotide probe typing HLA typing or tissue typing are usually performed
for 1. Tissue transplantation; 2. Disputed paternity;
3. Anthropological studies; 4. An association between
Uses of HLA Typing HLA types and diseases.
1. Tissue transplantation: HLA typing is used
primarily for testing compatibility between
Important questions
recipients and potential donors before tissue
transplantation. 1. Differentiate between T- and B-cells in a tabulated
2. Disputed paternity. form.
3. Anthropological studies. 2. Give an account of lymphocytes.
www.ebook3000.com
124 | Section 2: Immunology
3. Write short notes on: 4. All the following statements are true for helper
a. Subsets of T-lymphocytes T-cells except that they:
b. B lymphocytes a. Carry CD4 marker
c. Null cells (or) Large granular lymphocytes (LGL) b. Help or induce immune responses
d. Natural killer cells or NK-cells c. Kill intracellular microorganisms by secreting
cytokines
e. Killer cells or K-cells or ADCC-cells
d. Recognize antigen in association with class I
4. Write briefly on:
MHC
a. Major histocompatibilty complex (MHC)
5. Class II MHC antigens are present on:
b. Human leukocyte antigen (HLA)
a. Macrophages
c. HLA typing b Monocytes
d. MHC restriction c. Activated T lymphocytes (CD4)
d. All the above
Multiple choice questions (MCQs) 6. Class I proteins are encoded by:
a. HLA-A, -B, and -C loci
1. All the following are examples of secondary- b. HLA-DR, HLA-DQ, and HLA-DP loci
lymphoid organs except: c. Complement loci that encode C2 and C4
a. Lymph nodes d. Complement loci that encode factor B
b. Thymus 7. Which of the following HLA types is associated
c. Spleen with ankylosing spondylitis?
d. Mucosa-associated lymphoid tissues a. HLA-B27 b. HLA-DR4
2. The CD4+ T-cells that recruit and activate phagocytic c. HLA-DP d. None of the
cells acting against intracellular microbes are called: above
a. Th-0 cells b. Th-1 cells 8. Which of the following HLA types is associated
c. Th-2 cells d. Antigen-present- with rheumatoid arthritis?
ing cells a. HLA-B27 b. HLA-DR4
3. The molecule expressed on surface of the mature c. HLA-Al d. None of the
T-cells is: above
a. CD 19 b. CD 8 Answers (MCQs)
c. CD 3 d. CD 1 1. b; 2. c; 3. c; 4. d; 5. d; 6. a; 7. b; 8. b
18
Chapter
Immune Response
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss monoclonal antibodies—principle, technique
be able to: and applications
∙∙ Differentiate between primary and secondary ∙∙ Describe the following: cytokines; immunological
humoral immune responses tolerance.
www.ebook3000.com
126 | Section 2: Immunology
produced is 10 or more times greater than during
the primary response.
Priming dose and booster doses: A single
injection of an antigen helps more in sensitising or
priming the immunocompetent cells producing the
particular antibody than in the actual elaboration
of high levels of antibody. Only by subsequent
injections of the antigen effective levels of antibody
are usually induced. The first injection is known as
the ‘priming’ dose and subsequent injections as
‘booster’ doses. With live vaccines, a single dose is
Fig. 18.1: Primary immune response. An antigenic stimulus
sufficient as multiplication of the organism in the
1. Latent period; 2. Long phase (rise in titer of serum antibody);
3. Steady stage of antibody titer; 4. Decline of antibody titer body provides a continuing antigenic stimulus that
acts as both the priming and booster dose.
on whether the humoral response results from Negative phase: If the same animal is subsequently
activation of naive lymphocytes (primary response) exposed to the same antigen already carrying the
or memory lymphocytes (secondary response). specific antibody in circulation, a temporary fall in
In both cases, activation leads to production of the level of circulating antibody occurs due to the
secreted antibodies of various isotypes, which combination of the antigen with the preexisting
differ in their ability to mediate specific effector antibody. This is known as the ‘negative phase’. It is
functions. followed by an increase in the titre of the antibody
exceeding the initial level.
a. Primary Humoral Response
The first contact of an exogenous antigen with an
Fate of antigen in tissues
individual generates a primary humoral response, The manner in which an antigen is dealt with in
characterized by the production of antibody- the body depends on factors such as the physical
secreting plasma cells and memory B-cells. and chemical nature of the antigen, its dose and
When first introduced the antigen selects the route of entry, and whether the antigenic stimulus
cells that can react with it. In all cases, however, a is primary or secondary.
primary response to antigen is characterized by a
Physical and chemical nature of the antigen:
lag phase, subsequent clonal expansion, and dif
Particulate antigens are removed from circulation
ferentiation into memory cells or plasma cells. The
in two phases. The first is the nonimmune phase
duration of the lag phase varies with the nature of
during which the antigen is engulfed by the
the antigen.
phagocytic cells, broken down and eliminated.
During a primary humoral response, IgM is
The phase of immune elimination begins with the
secreted initially, often followed by a switch to an
appearance of the specific antibody, during which
increasing proportion of IgG. Depending on the
persistence of the antigen, a primary response
can last for various periods, from only a few days
to several weeks.
www.ebook3000.com
128 | Section 2: Immunology
produced by a single clone of plasma cells directed 1. Selection of antigen: Monoclonal antibodies can
against a single antigenic determinant and hence be produced against any substance recognized
the antiboies are homogeneous and monoclonal. as an antigen by the immune system of the
animal being injected.
Hybridoma technology: The discovery of the
2. Animal immunization: Animal (usually mouse)
hybridoma technology for the production of
is immunised with a pure selective antigen ,it
unlimited quantities of identical monoclonal
is killed and B lymphocytes are harvested from
antibodies of the same Ig class, possessing uniform
the spleen or lymph node. A suspension of
specificity, affinity and other properties, created
spleen cells (B cells) is prepared.
a revolution in immunology by opening up
numerous diagnostic, therapeutic and research 3. Fusion of splenic lymphocytes and myeloma
applications. Monoclonal antibodies against cells: A suspension of splenic cells is then fused
several antigens are now available commercially. with a myeloma cell-line by incubating in the
presence of polyethylene glycol.
By laboratory manipulation, Kohler and
Because cells cannot remain viable in cell cul
Millstein (1975) prepared a hybrid cell line
ture for very long, they must be fused together
(hybridoma) by fusion of a mouse myeloma cell
with cells that are able to survive and multiply
with an antibody producing lymphocyte from
in tissue culture, that is, the continuously
spleen or lymph node of the-same inbred strain
propagating, or immortal cells, of multiple
of mouse. In recognition of the great importance
myeloma (a malignant tumor of antibody-
of this hybridoma technology, the Nobel Prize for
producing plasma cells).
Medicine was awarded to them in 1984.
4. Selection of hybrid lymphocyte-myeloma
cells: Lymphocytes from the spleen of mice
Procedure (Fig. 18.3) immunised with the desired antigen are fused
The production of monoclonal antibodies involves with mouse myeloma cells grown in culture
the following steps listed below: which do not form immunoglobulins and
www.ebook3000.com
130 | Section 2: Immunology
b. Bacterial: Freund’s complete adjuvant is the lymphocytes and has no antimitotic activity.
Freund’s incomplete adjuvant along with a It selectively inhibits helper T-cell activity.
suspension of killed tubercle bacilli. vi. Antilymphocyte serum (ALS): Antilymphocyte
c. Chemical: Silica particles, beryllium sulfate and serum (ALS) is a heterologous antiserum raised
endotoxins activate macrophages. against lymphocytes. Antibody prep ared
Action of Adjuvants against thymus cells is called antithymocyte
a. Sustained release of antigen from depot serum (ATS). The corresponding globulin
b. Liberation of lymphocytes activating factor preparations are called ALG and ATG. They
c. Lymphocytes stimulation-B-cell, T-cell or both. were used to prevent graft rejection.
d. Stimulate CMI. ALS acts primarily against T-lymphocytes
and therefore specifically on cell-mediated
Freund’s Complete Adjuvant
immunity. Humoral antibody response to thymus-
The most potent adjuvant is Freund’s complete
independent antigens is unaffected and may even
adjuvant, which is the incomplete adjuvant
be enhanced. ALS acts only against lymphocytes
along with a suspension of killed tubercle bacilli.
in circulation and not cells in lymphoid organs.
Besides increasing the humoral immune response,
As ALS is a foreign protein, its effect is decreased
it induces a high degree of cellular immunity
on repeated administration, which may also lead
(delayed hypersensitivity) as well. It is unsuitable
to serum sickness and other hypersensit ivity
for human use as it produces a local granuloma.
reactions.
7. Immunosuppressive Agents
8. Effect of Antibody
These inhibit the immune response. They are
Passive administration of the homologous antibody
useful in certain situations like transplantation,
will suppress specifically the humoral immune
when it becomes necessary to prevent graft
response to an antigen. The action appears to be
rejection.
by a feedback mechanism.
Examples: X-irradiation, radiomimetic drugs,
corticosteroids, antimetabolites and other cytotoxic 9. Superantigens
chemicals, and antilymphocytic serum
i. X-irradiation: Antibody response is suppressed Superantigens are certain protein molecules that
by sublethal whole body irradiation. activate very large numbers of T-cells irrespective of
ii. Radiomimetic drugs: They belong in general their antigenic specificities such as staphylococcal
to the class of alkylating agents (for example, enterotoxins.
cyclophosphamide, nitrogen mustard). In
human beings, cyclop hosphamide given 10. Mitogens
for three days after the antigen completely Mitogens are certain substances that induce
suppresses the antibody response. It is much division of lymphocytes and other cells.
less effective when given before the antigen.
iii. Corticosteroids: Corticosteroids cause depletion CELL-MEDIATED IMMUNE RESPONSES
of lymphocytes from the blood and lymphoid
organs. Therapeutic doses have little effect on The term ‘cell-mediated immunity’ (CMI) refers
the antibody formation in human beings. They to the specific immune responses which involves
inhibit the induction and manifestations of T-Iymphocyte-mediated functions that do not
delayed hypersensitivity in human beings. involve antibodies. This form of immunity can
iv. Antimetabolites: They include folic acid be transferred from donor to recipient with intact
antagonists (methotrexate), alkylating agents lymphocytes, but not with antisera, hence it is
(cyclophosphamide) and analogues of called cell-mediated immune reaction.
purine (6-mercaptopurine, azathioprine),
cytosine (cytosine arabinoside) and uracil Scope of CMI
(5-fluorouracil). CMI participates in the following immunological
Antimetabolites are substances that interfere functions:
with the synthesis of DNA, RNA or both and 1. Delayed hypersensitivity (type IV hyper
thus inhibit cell division and differentiation sensitivity).
necessary for humoral and cellular immune 2. Immunity in infectious diseases caused by
responses. Many antimetabolites find clinical obligate and facultative intracellular parasites.
application in the prevention of graft rejection. 3. Transplantation immunity and graft-versus-
v. Cyclosporine: The drug most widely used host reaction.
now for immunosuppression is cyclosporine, 4. Immunological surveillance and immunity
a cyclic polypeptide. It is not cytotoxic for against cancer.
Chapter 18: Immune Response | 131
5. Pathogenesis of certain autoimmune diseases Interferons, growth factors and others were
(for example, thyroiditis, encephalomyelitis). found to have similar effects. Therefore, all of them
have been grouped under the term cytokines.
Induction of CMI Recently cytokines have been grouped into
Antigen-specific cell-mediated immunity is the following categories or families: chemokines,
mediated by T-lymphocytes. A second, smaller hematopoietins, interleukins, and members of
population of cells includes natural killer (NK) and the tumor necrosis factor (TNF) family. Some
killer (K) cells. Cell-mediated immune response examples of these cytokine families are provided
can also be divided into primary and secondary in Table 18.1.
cell-mediated immune responses.
Features of Important Cytokines
Primary Cell-mediated Immune Response Various cytokines are shown in Table 18.1.
Foreign antigen is presented by antigenpresenting
celIs (APCs) to T-cells leading to their activation. A. Interleukins
Each T-cell bears on its surface a specific receptor Interleukin-1
(TCR) for one epitope and combines only with It is a stable polypeptide retaining its activity up
antigens carrying that epitope. On contact to 56°C and between pH 3 and pH 11. IL-l occurs
with the appropriate antigen, T-cells undergo in two molecular forms, IL-l alpha and beta.
blast transformation, clonal proliferation and IL-l is principally secreted by macrophages and
differentiation into memory cells and effector cells monocytes but can be produced by most other
providing CMI. T-cells recognise antigens only nucleated cells also. Its production is stimulated
when presented with MHC molecules. by antigens, toxins, injury and inflammatory
The T-cell group is composed of: processes and inhibited by cyc1osporin A,
1. Helper T (Th)-cells which react with antigens corticosteroids and prostaglandins.
presented on the surface of macrophages
or other cells, complexed with MHC class Immunological Effects of IL-1
II molecules. They then release biological i. Stimulation of T-cells for the production of IL2
mediators (lymphokines) which activate and other lymphokines
macrophages, enabling them to kill intracellular ii. B-cell proliferation and antibody synthesis
parasites; and
iii. Neutrophil chemotaxis and phagocytosis
2. T-cytotoxic cells (Tc-cells), which recognize
iv. It mediates a wide range of metabolic, physio
antigen on the surface of cells, in association
logical, inflammatory and hematological
with MHC class I molecules, secrete lympho
effects
kines and destroy the target cells.
Together with the tumor necrosis factor (TNF),
Secondary Cell-mediated it is responsible for many of the hematological
changes in septic shock
Immune Response
If the same host is subsequently exposed to the Interleukin-2
same antigen, then the secondary cell-mediated It is the major activator of T- and B-cells and
immune response is usually more pronounced and stimulates cytotoxic T-cells and NK-cells. It
occurs more rapidly. converts some null cells (LGL) into lymphokine-
activated killer (LAK) cells which can destroy NK
CYTOKINES resistant tumor cells. This property has been used
in the treatment of certain types of cancer.
Cytokines (Greek cyto, cell and kinesis, movement)
are biologically active substances produced by Interleukin-3
cells that influence other cells. Biologically active IL-3 is a growth factor for bone marrow stem cells.
substances released by activated T-lymphocytes It is also known as the multicolony-stimulating
were called lymphokines. When released from factor (multi-CSF).
mononuclear phagocytes, these proteins are called Interleukin-4
monokines. IL-4 activates resting B-cells and acts as a B-cell
The term interleukin was introduced for those differentiating factor. It also acts as a growth factor
products of leucocytes which exert a regulatory for T-cells and mast cells. It enhances the action
influence on other cells; and if their effect is of cytotoxic T-cells. Formerly, it was known as
to stimulate the growth and differentiation of the B-cell growth factor-l (BCGF-1). It may have a
immature leukocytes in the bone marrow, they are role in atopic hypersensitivity as it augments IgE
called colony-stimulating factors (CSFs). synthesis.
www.ebook3000.com
132 | Section 2: Immunology
Table 18.1 Cytokines: Hormones of the immune system-EDIT
Cytokine Main sources Major functions
A. Interleukins
IL-l (Alpha and b) Macrophages and other cell types Proliferation and differentiation ofT, B and other cells;
pyrogenic; induce acute phase proteins; bone marrow cell
proliferation
IL-2 T-cells Promote growth and differentiation of T- and B-cells,
cytotoxicity ofT and NK cells, secretion of other lymphokines
IL-3 T-cells Multi-CSF
IL-4 TH-cells Proliferation of B- and cytotoxic T-cells; increase IgGI and IgE
production; enhance MHC class II and IgE receptors
IL-5 TH-cells Proliferation of eosinophils, stimulate IgA and IgM production
IL-6 TH-cells Promote B-cell differentiation; IgG production, acute phase
proteins
IL-7 Bone marrow, spleen, stromal cells B- and T-cell growth factor
IL-8 Macrophages, others Neutrophil chemotactic factor
IL-9 T-cell T-cell growth and proliferation
IL-I0 T-, B-cells, macrophages Inhibit IFN production and mononuclear cell functions
IL-11 Bone marrow stromal cells Induce acute phase proteins
IL-12 T-cells Activate NK-cells
IL-13 T-cells Inhibit mononuclear cell functions
B. Colony-stimulating Factors
GM-CSF T-cells, macrophages, fibroblasts T-cell and macrophage growth stimulation
G-CSF Fibroblasts, endothelium Granulocyte growth stimulation
M-CSF roblasts, Fibroblasts, endothelium Macrophages growth stimulation
endothelium
C. Tumor Necrosis Factors
TNF-α Macrophages, monocytes Tumor cytotoxicity, lipolysis, wasting, acute phase proteins,
phagocytic cell activation, antiviral and antiparasitic effects,
endotoxic shock
TNF-β T-cells Induce other cytokines
D. Interferons
IFN-α Leukocytes
IFN-β Fibroblasts Antiviral activity
IFN-γ T-cells Antiviral, macrophage activation; MHC class I and II expression
on cells
E. Others
TGFb T- and B-cells Inhibit T- and B-cell proliferation and hematopoiesis; promote
wound healing
LIF T-cells Proliferation of stem cells; eosinophil chemotaxis
www.ebook3000.com
134 | Section 2: Immunology
rheumatoid arthritis) and diseases of unknown 2. Dose of Antigen
etiology (sarcoidosis, multiple sclerosis). The induction of tolerance is dose-dependent.
With certain antigens, tolerance can be induced by
Immunological tolerance two types of doses, one high and the other low, with
intermediate doses producing immunity instead of
Immunologic tolerance is defined as the absence tolerance. These are known as ‘high zone’ and ‘low
of a specific immune response resulting from a zone’ tolerance respectively. A special type of high
previous exposure to the inducing antigen. The zone tolerance is Felton’s immunological paralysis.
most notable example is immunologic tolerance
to self. Any antigen that comes into contact with 3. Route of Tolerogen Administration
the immunological system during embryonic life
would be recognized as a self-antigen and would Certain haptens that are immunogenic in guinea
not induce any immune response. pigs by the intradermal route are tolerogenic orally
Burnet and Fenner (1949) suggested that or intravenously.
the unresponsiveness of individuals to self-
antigens was due to the contact of the immature 4. Nature of Tolerogen
immunological system with self-antigens during Soluble antigens and haptens are more tolerogenic
embryonic life. Any antigen that comes into contact than particulate antigens.
with the immunological system during embryonic
life would be recognized as a self-antigen and would 5. Various Treatments
not induce any immune response. Medawar and
his colleagues (1953) proved this experimentally When human gammaglobulin is heat aggregated,
using two strains of syngenetic mice. When a it is highly immunogenic in mice but is tolerogenic
skin graft from one inbred strain of mice (CBA) when deaggregated.
is applied on a mouse of another strain (A), it
is rejected. If CBA cells are injected into fetal or 6. Genetic Background
newborn strain A mice, however, the latter when Rabbits and mice can be rendered tolerant more
they grow up will freely accept skin grafts from rapidly than guinea pigs and chickens.
CBA mice. The content of the self antigen appears
to have been enlarged by contact with a foreign Mechanism of Tolerance
antigen during embryonic life. This phenomenon
is called ‘specific immunological tolerance’. Tolerance can arise through three possible
mechanisms:
1. Clonal deletion
Types of Immunological Tolerance
2. Clonal anergy
Two forms of tolerance are known: 3. Suppression.
1. Natural tolerance: The body develops immune
tolerance towards self-molecules due to natural 1. Clonal Deletion
tolerance during growth of fetus. Autoimmune
disease wi11 develop in the event of breakdown In embryonic life clones of B- and T-cells
of tolerance to self-tissues. possessing receptors that recognize self-antigens
2. Acquired tolerance: Tolerance in later life may are selectively deleted or eliminated and, therefore,
occur under certain special circumstances, e.g. no longer available to respond upon subsequent
ingestion of bovine myelin in high concentrations exposure to that antigen. This is known as clonal
can tolerise an individual to myelin. deletion.
2. Clonal Anergy
Parameters that Affect the Induction of
Tolerance Clones of B- and T-cells expressing receptors that
recognize self-antigen might remain but they
These include age, dose of antigen, route of tolerogen cannot be activated. This is known as clonal anergy.
administration, physical nature of the antigen, and
various treatments that reduce activation of positive 3. Suppression
regulatory cells of the immune response.
Clones of B- and T-cells expressing receptors that
recognize self-antigens are preserved. Antigen
1. Age
recognition might be capable of causing activation,
The younger the recipient, the easier it is to induce however, expression of immune response might be
tolerance, and, of course, it is easiest in utero. inhibited or blocked through active suppression.
Chapter 18: Immune Response | 135
Theories of Antibody formation development in later life by somatic mutation
could lead to autoimmune processes. Each
Theories of immunity fall into two categories: immunocompetent cell was capable of reacting
instructive and selective. with one antigen (or a small number of
antigens) which could recognize and combine
A. Instructive Theories with antigens introduced into the body. The
1. Direct template theories: According to these, result of the contact with the specific antigen
the antigen or the antigenic determinant enters was cellular proliferation to form clones
the antibody-forming cell and serves as a synthesizing the antibody. This theory is more
template against which antibody molecules are widely accepted than other theories.
synthesized so that they have combining sites
Key Points
complementary to the antigenic determinants.
These are therefore known as ‘direct template’ The immune response is the specific reactivity
theories. induced in a host by an antigenic stimulus and can
be divided into two types—the humoral (antibody-
2. Indirect template theory: This theory was mediated) and the cellular (cell-mediated) types
proposed by Burnet and Fenner (1949). The production of antibodies consists of three
According to this theory, the entry of the steps: 1. Lag phase; 2. Log phase; 3. A plateau or
antigenic determinant into the antibody steady state
producing cell induced in it a heritable change. The humoral response results from activation of
naive lymphocytes (primary response) or memory
A ‘genocopy’ of the antigenic determinant
lymphocytes (secondary response)
was thus incorporated in its genome and Monoclonal antibodies: Such antibodies produced by
transmitted to the progeny cells (indirect a single clone and directed against a single antigenic
template). determinant are called monoclonal antibodies, e.g.
plasma cell tumor (myeloma)
Cell-mediated immunity’ (CMI): The term ‘cell-
B. Selective Theories mediated immunity’ (CMI) refers to the specific
1. Side chain theory: According to side chain immune responses which involves T-Iymphocyte-
theory, immunocompetent cells (ICCs) have mediated functions
Cytokines: Cytokines are biologically active
surface receptors capable of reacting with
substances produced by cells that influence other
antigens which have complementary side cells. Interferons, growth factors and others were
chains. When foreign antigens are introduced found to have similar effects. Therefore, all of them
into the body, they combine with those cell have been grouped under the term ‘cytokines’
receptors which have a complementary Transfer factor: Transfer of CMI in man by injection of
fit. This inactivates the receptors. There extract from the leukocytes from immunized individual
Immunologic tolerance is defined as the absence of a
is an overproduction of the same type of specific immune response resulting from a previous
receptors which circulate as antibodies as a exposure to the inducing antigen. Two forms of
compensatory mechanism. tolerance are known. 1. Natural; 2. Acquired
2. Natural selection theory: This theory was Tolerance can arise through three possible
mechanisms: 1. Clonal deletion; 2. Clonal anergy;
proposed by Jerne (1955) which postulates that
3. Suppression
about a million globulin (antibody) molecules Theories of immunity fall into two categories:
were formed in embryonic life, which covered instructive and selective:
the full range of antigenic specificities. These A. Instructive theories: 1. Direct template theories;
globulins were the ‘natural antibodies’. When 2. Indirect template theory
an antigen was introduced, it combined B. Selective theories: 1. Side chain theory; 2. Natural
selection theory; 3. Clonal selection theory.
selectively with the globulin that had the
nearest complementary ‘fit’. The globulin,
with the combined antigen, homed in on the Important questions
antibody-forming cells and stimulated them 1. Discuss the primary and secondary humoral
to synthesize the same kind of antibody. immune responses.
3. Clonal selection theory: This theory was 2. Discuss briefly about:
proposed by Burnet (1957). This theory states Monoclonal antibodies—production and
that during immunological development, cells applications.
Adjuvants
capable of reacting with different antigens
Cytokines
were formed by a process of somatic mutation.
Theories of antibody production
Clones of cells that had immunological 3. Write short notes on:
reactivity with self-antigens were eliminated a. Transfer factor
during embryonic life. Such clones are b. Burnet’s clonal selection theory
called forbidden clones. Their persistence or c. Immunological tolerance
www.ebook3000.com
136 | Section 2: Immunology
Multiple choice questions (MCQs) 4. Development of cell mediated immunity can be
detected by:
1. The synthesis and production of antibodies a. Skin test for delayed hypersensitivity
typically is dependent on complex interaction of b. Lymphocyte transformation test
all the following cells except: c. Migration inhibiting factor test
a. Macrophages d. All the above
b. Helper T cells 5. Transfer factor shows all the following features
c. Cytotoxic T cells except:
d. B cells a. It is an extract from the leukocytes from the
2. Which animal is used for for monoclonal immnized host
antibodies production: b. Transfers humoral immunity
a. Guinea pig c. Transfers CMI
b. Mouse d. Transferred immunity is systemic
c. Rabbit 6. Clonal selection theory was postulated by:
d. None of the above a. Breinl and Haurowitz
3. Interleukin-l is a protein produced mainly by: b. Burnet and Fenner
c. Ehrlich
a. Macrophages and monocytes
d. Burnet
b. Polymorphonuclear leukocytes
c. Lymphocytes Answers (MCQs)
d. Stem cells 1. c; 2. b; 3. a; 4. d; 5. b; 6. d
19
Chapter
Immunodeficiency Diseases
Learning Objectives
After reading and studying this chapter, you should ∙∙ List primary and secondary immunodeficiency
be able to: syndromes.
∙∙ Classify and enumerate immunodeficiency diseases
www.ebook3000.com
138 | Section 2: Immunology
Table 19.1 Classification of primary immuno proper signals from the T-cells. B-cells fail to mature
deficiency syndromes into plasma cells and secrete immunoglobulins.
www.ebook3000.com
140 | Section 2: Immunology
usually dies within the first year of life from Bacterial infection: The bacteria involved in
recurrent, intractable infections. the recurrent infections are catalase-positive
iv. Recombinase activating gene (RAG 1/2) pyogenic pathogens such as staphylococci
deficiency. and coliforms. Catalase-negative patho
v. Interleukin receptor γ chain (γc) deficiency gens such as streptococci and pneumococci
vi. Janus-associated kinase 3 (JAK3) deficiency are handled normally. Leukocytes from the
patients are unable to kill catalase positive
B. Disorders of complement bacteria following phagocytosis. The bacteria
multiply in the cells and, being protected from
a. Complement Component Deficiencies antibodies and antibiotics by their intracellular
Genetic deficiencies have been detected for almost position, set up chronic suppurative infection.
all the complement components in human beings. Bactericidal defect: The diminished bactericidal
The defects are transmitted as autosomal recessive capacity of the phagocytic cells is associated
traits. Hemolytic and other functional activities with a decrease of some metabolic processes like
are completely restored by supplying the deficient oxygen consumption, hexose monophosphate
factor. Deficiency of C1 and C4 is associated pathway activity and production of hydrogen
with systemic lupus erythematosus. Recurrent peroxide. The diminished H2O2 production
pyogenic infections were found associated with C3 appears to be the major reason for the bacteri
deficiency and neisserial infections with deficiency cidal defect.
of C6, C7 and C8. So far, there is no known disease Leukocytes from the patients fail to reduce nitro
with deficiency of C9. blue tetrazolium (NBT) during phagocytosis.
This property has been used as a screening
b. Complement Inhibitor Deficiencies method (NBT test) for the diagnosis of chronic
i. C1 inhibitor: Hereditary angioneurotic edema granulomatous disease.
(HAE) is due to a genetic deficiency of C1 b. Myeloperoxidase (MPO) deficiency: In this
inhibitor and is transmitted as an autosomal- rare disease, leukocytes are deficient in
dominant condition. It manifests clinically as myeloperoxidase (MPO). Patients are liable to
localized edema of the tissue, often following develop recurrent Candida albicans infection.
trauma, but sometimes with no known cause. c. Chediak-Higashi syndrome: The leukocytes
Management: Androgens, aminocaproic acid possess diminished phagocytic activity. The
and its analog tranexamic acid have been found individuals with this syndrome suffer from
useful in the management of this condition. Plasma recurrent infections similar to those seen in
infusions, once recommended for treatment, have persons with CGD
been given up as they were found to worsen the d. Leukocyte G-6-PD deficiency: Leucocytes are
condition in some cases. deficient in glucose-6-phosphate dehydrogenase.
ii. Deficiency of C3b inactivator: The rare defici These patients show diminished bactericidal
ency of C3b inactivator has been associated activity after phagocytosis leading to repeated
with chronic recurrent pyogenic lesions. bacterial infections.
e. Job’s syndrome: It is probably a primary defect
in phagocytic function.
C. Disorders of Phagocyte
f. Tuftsin deficiency: A leucokinin capable of
Phagocytosis may be impaired by either intrinsic or stimulating phagocytosis, discovered at Tufts
extrinsic defects. Intrinsic disorders may be due to University, Boston, has been designated ‘tuftsin’.
defects within the phagocytic cell, such as enzyme Chemically, it is a small tetrapeptide (Thr-Lys-
deficiencies. Extrinsic disorders may be due to a Pro-Arg) and patients have been reported to be
deficiency of opsonic antibody, complement or prone to local and systemic bacterial infections.
other factors promoting phagocytosis. Phagocytic g. Lazy leukocyte syndrome: The basic defect here
dysfunction leads to increased susceptibility is in chemotaxis and neutrophil mobility.
to infection, ranging from mild recurrent skin h. Hyper-IgE syndrome: Serum IgE levels are
infections to overwhelming systemic infection. usually more than ten times the normal level.
a. Chronic granulomatous disease (CGD): Chronic i. Actin-binding protein deficiency.
granulomatous disease (CGD) is a group of j. Shwachman’s disease.
disorders, most of which are X-linked recessive, k. Leucocyte adhesion deficiency (LAD).
with some that are autosomal recessive.
Individuals with CGD possess polymorpho
Secondary immunodeficiencies
nuclear leucocytes that phagocytize invading
bacteria normally but are unable to kill many Secondary or acquired deficiencies of immuno
of the ingested microorganisms. logical mechan isms can occur secondarily to
Chapter 19: Immunodeficiency Diseases | 141
a number of disease states, such as metabolic Important questions
disorders, malnutrition, malignancy and infections
1. What are immunodeficiency diseases? Classify
or after exposure to drugs and chemicals. AIDS
and enumerate immunodeficiency diseases.
is a secondary immunodeficiency secondary
2. Write short notes on:
immunodeficiency is far more common than
a. DiGeorge’s syndrome
primary immunodeficiency.
b. B-cell defect
c. T-cell defect
Deficiencies of Humoral and Cellular d. Disorders of complement
Immune Response
Deficiencies of humoral and cellular immune Multiple choice questions (MCQs)
response may occur secondarily during the course
of many disease processes. 1. Recurrent infections with certain viruses, protozoa,
and fungi indicate a:
A Humoral immunity depression: Humoral a. T-cell deficiency
deficiency results when B cells are depleted as b. B-cell deficiency
in lymphoid malignancy, particularly in chronic c. Combined T- and B-cell deficiency
lymphatic leukemia; when immunoglobulin d. Complement deficiency
catabolism is increased as in the nephrotic 2. X-linked agammaglobulinemia is:
syndrome; when excessive loss of serum protein a. Prototype of ‘pure’ T-cell deficiency
occurs as in exfoliative skin disease and in b. Prototype of ‘pure’ B-cell deficiency
protein-losing enteropathies; and when excessive c. Prototype of ‘pure’ combined T- and B-cell
production of abnormal immunoglobulins occurs deficiency
as in multiple myeloma. d. Prototype of ‘pure’ complement deficiency
3. All the following are the examples of T-cell
B. Cell-mediated immunity depression: Cell- deficiencies except:
mediated immunity is depressed in lymphoreticular a. DiGeorge’s syndrome
malignancies like Hodgkin’s lymphoma, obstruction b. Chronic mucocutaneous candidiasis
to lymph circulation and infiltration of the thymus- c. Chronic granulomatous disease
dependent area of lymph nodes with non lymphoid d. Purine nucleoside phosphorylase deficiency
cells as in leplromatous leprosy; and, transiently, 4. Which of the following defects occur in Nezelof’s
following certain viral infections, such as measles. syndrome?
a. T cell defects b. B cell defects
C. Humoral and Cell-mediated immunity
c. Both of the above d. None of the
depression: Both types of immune responses
5. The most common severe form of severe combined
are adversely affected in nutritional deprivation.
immunodeficiency is:
Aging also causes waning in the efficiency of
a. Patients with a defect in adenosine deaminase
acquired immunity. Immunodeficiency follows enzyme
the intentional or unintentional administration of b. Patients with a defect in purine nucleoside
immunosuppressive agents. phosphorylase enzyme
Key Points c. Chediak-Higashi syndrome
d. Swiss type agammaglobulinemia
Immunodeficiency disorders can occur in T-cells,
6. All are disorders of phagocytosis except:
B-cells, complement and phagocytes
These can be classified as primary or secondary
a. Chronic granulomatous disease
immunodeficiencies b. Myeloperxidase deficiency
A primary immunodeficiency may affect either c. Chediak-Higashi syndrome
adaptive or innate immune functions d. Digeorge’s syndrome
Secondary immunodeficiencies occur secondary to
numerous diseases or conditions. Answers (MCQs)
1. a; 2. b; 3. c; 4. c; 5. c; 6. d
www.ebook3000.com
20
Chapter
Hypersensitivity Reactions
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss type I, type II, type III, type IV hypersensitivity
be able to: reactions: Mechanism and examples.
∙∙ Compare major types of hypersensitivity reactions.
∙∙ Differentiate between immediate and delayed
hypersensitivity.
www.ebook3000.com
144 | Section 2: Immunology
Because of the reactions the individual can die with an antigen, the individual produces IgE
within a few minutes from reduced venous return, antibodies. IgE are bound to surface receptors on
asphyxiation, reduced blood pressure, and circula mast cells and basophils. IgE molecules attach
tory shock. to these receptors by their Fc end, leaving two
Antigens: A wide range of antigens have been antigen-binding sites free. Mast cells and basophils
shown to trigger this reaction in susceptible coated by IgE are said to be sensitized, making the
humans, including the venom from bee, wasp, individual allergic to the allergen.
hornet, and ant stings; drugs, such as penicillin, Following exposure to the shocking dose,
insulin, and antitoxins; and seafood and nuts. If the antigen molecules combine with the cell
not treated quickly, these reactions can be fatal. bound IgE, bridging the gap between adjacent
antibody molecules. This cross-linking increases
Treatment: Epinephrine (adrenaline) is the drug
the permeability of the cells to calcium ions and
of choice for systemic anaphylactic reactions, a
leads to degranulation, with release of biologically
drug that constricts blood vessels and raises the
active substances contained in the granules. The
blood pressure.
pharmacologically active mediators released
from the granules act on the surrounding tissues.
i. Cutaneous Anaphylaxis The manifestations of anaphylaxis are due
Small amounts of potential allergens are introduced to pharmacological mediators, which can be
at specific skin sites either by intradermal injection classified as two types, either primary or sec
or by superficial scratching. If a person is allergic ondary. These mediators trigger smooth muscle
to the allergen, there is a wheal and flare (local contractions, vasodilation, increased vascular
anaphylaxis) within 30 minutes. permeability, and mucous secretion (Fig. 20.1)
Use: Cutaneous anaphylaxis is useful in testing A.Primary mediators of anaphylaxis
for hypersensitivity and in identifying the allergen
responsible in atopic diseases. A. Primary Mediators of Anaphylaxis
These are produced before degranulation and are
ii. Passive Cutaneous Anaphylaxis (PCA) stored in the granules. The most significant primary
This test is an extremely sensitive in vivo method mediators are histamine, proteases, eosinophil
for detection of antibodies developed by Zoltan chemotactic factor, neutrophil chemotactic factor,
Ovary (1952). In this procedure, a small volume of and heparin.
the antibody is injected intradermally into a normal 1. Histamine: Histamine, which is formed by
animal. The corresponding antigen is injected decarboxylation of the amino acid histidine
intravenously 4–24 hours afterwards along with and is localized in the granules of mast cells
a dye, such as Evans blue that is strongly bound and basophils and in the platelets of some
to serum albumin. There will be an immediate species. Histamine induces smooth muscle
blueing at the site of intradermal injection due to contraction in diverse tissues and organs,
vasodilatation and increased capillary permeability including vasculature, intestines, uterus and
(wheal-and-flare reaction). especially the bronchioles. It also stimulates
secretions (secretagogue effect).
Use: PCA can be used to detect human IgG 2. Heparin: It contributes to anaphylaxis in dogs,
antibody which is heterocytotropic (capable of but apparently not in human beings.
fixing to cells of other species) but not IgE which 3. Serotonin (5-hydroxytryptamine): It is found
is homocytotropic (capable of fixing to cells of in the intestinal mucosa, brain tissue and
homologous species only). platelets. It induces contraction of smooth
Mechanism of Anaphylaxis
The immunologic basis for hypersensitivity is
cytotropic IgE antibody. After an initial contact Fig. 20.1: Antigen-induced mediator release from mast cell
Chapter 20: Hypersensitivity Reactions | 145
muscle, increased vascular permeability, and Anaphylactoid Reaction
capillary dilatation. Intravenous injection of peptone, trypsin and
4. Chemotactic Factors certain other substances provokes a clinical reaction
i. Eosinophil chemotactic factor (ECF- resembling anaphylactic shock. This is termed
A): Eosinophil chemotactic factors of ‘anaphylactoid reaction’. The clinical resemblance
anaphylaxis (ECF-A) are acidic tetrapeptides is due to the same chemical mediators participating
released from mast cell granules which are in both reactions.
strongly chemotactic for eosinophils. These Anaphylactoid shock has no immunological
probably contribute to the eosinophilia basis and is a nonspecific mechanism involving
accompanying many hypersensitivity states. the activation of complement and the release of
ii. Neutrophil chemotactic factors (NCF): anaphylatoxins which is the only difference.
A high molecular weight chemotactic
factor has been identified, which attracts B. Localized Anaphylaxis (Atopy)
neutrophils (NCF).
The term ‘atopy’ (literally meaning out of place
5. Proteases: Enzymatic mediators, such as or strangeness) refers to naturally occurring
proteases and hydrolases are also released familial hypersensitivities of human beings. It was
from mast cell granules. These enzymes most introduced by Coca (1923) and typified by hay fever
probably are involved in the digestion of blood and asthma. The antigens commonly involved in
vessel basement membranes, resulting in atopy are inhalants (for example, plant pollen,
increased permeability to a variety of cell types. fungal spores, animal dander, and house dust mites
or other types of fine particles suspended in air) or
B. Secondary Mediators of Anaphylaxis ingestants (for example, milk, milk products, eggs,
1. Platelet-activating factor (PAF): Platelet- meat, fish or cereal). Some of them are contact
activating factor (PAF) is a low molecu lar allergens, to which the skin and conjunctiva may
weight lipid released from basophils which be exposed. The symptoms depend primarily
causes aggregation of platelets and release of on the route by which the antigen enters the
their vasoactive amines. body. These atopens are generally not good
2. Leukotrienes and prostaglandin: They antigens when injected parenterally but induce IgE
are derived by two different pathways from antibodies, formerly termed as ‘reagin’ antibodies.
arachidonic acid, which is formed from Atopic sensitization is developed spontaneously
disrupted cell membranes of mast cells and following natural contact with atopens. It is difficult
other leukocytes. A substance originally to induce atopy artificially.
demonstrated in lungs, producing slow, Predisposition to atopy is genetically deter
sustained contraction of smooth muscles, and, mined, probably linked to MHC genotypes. Atopy,
therefore, termed as slow reacting substance of therefore, runs in families. What is inherited is not
anaphylaxis (SRS-A) has since been identified sensitivity to a particular antigen, or a particular
a family of leukotrienes (LTB4, C4, D4, lM). atopic syndrome but the tendency to produce
In humans, the leukotrienes are thought to IgE antibodies in unusually large quantities. All
contribute to the prolonged bronchospasm individuals are capable of forming IgE antibodies
and buildup of muc us seen in asthmatics. in small amounts but in atopics IgE response is
Prostaglandin F2-α and thromboxane A2 are preponderant. About 10% of persons have this
powerful, but transient, bronchoconstrictors. tendency to overproduce IgE. It has been reported
3. Cytokines: Human mast cells secrete IL-4, that bottlefed infants tend to develop atopy in later
IL-5, IL-6, and TNF-α. These cytokines alter the life more often than breastfed babies.
local microenvironment, eventually leading to
the recruitment of inflammatory cells such as Mechanism of Atopy
neutrophils and eosinophils. The mechanism of development of atopy is essen
tially the same as that of systemic anaphylaxis. The
Other Mediators of Anaphylaxis symptoms of atopy are caused by the release of
Besides the products of mast cells and other pharmacologically active substances following the
leucocytes, several other biologically substances combination of the antigen and the cell fixed IgE.
are known to induce mast cell degranulation and Atopic sensitivity is due to an overproduction
have been implicated in anaphylaxis. These include of IgE antibodies. This is often associated with
split products from complement activation, C3a a deficiency of IgA. When IgA is deficient, the
and C5a and bradykinin and other kinins formed antigens cause massive stimulation of IgE forming
from plasma kininogens. cells, leading to overproduction of IgE.
www.ebook3000.com
146 | Section 2: Immunology
Clinical Expression of Atopic Reactions
The clinical expression of atopic reactions is
usually determined by the portal of entry of the
antigen-conjunctivitis, rhinitis, gastrointestinal
symptoms and dermatitis following exposure
through the eyes, respiratory tract, intestine or
skin, respectively. Sometimes the effects may be at
sites remote from the portal of entry, for example,
urticaria following ingestion of the allergen.
Specific desensitisation (hyposensitisation) is
often practised in the treatment of atopy. Atopic
allergies, which afflict at least 20% of the population
in developed countries, include a wide range of Fig. 20.2: Type II hypersensitivity
IgE-mediated disorders, including allergic rhinitis
(hay fever), asthma, food allergies and atopic membrane of a foreign cell, or it can mediate
dermatitis (eczema). cell destruction by antibody-dependent cell-
mediated cytotoxicity (ADCC). Type II hyper
Methods to Detect Type I sensitivity is generally called cytolytic or cytotoxic
Hypersensitivity Reactions reactions because it results in the destruction of
Formerly, measurement of reaginic antibody could host cells, either by lysis or toxic mediators. Type
be done only by an in vivo assay or by skin testing. II hypersensitive reactions involve antibody-
1. Prausnitz–Kustner (PK) reaction mediated destruction of cells (Fig. 20.2).
Prausnitz and Kustner in 1921 demonstrated
transmission of IgE-mediated type I hyper Examples
sensitivity by injecting serum containing IgE 1. Transfusion reactions: A transfusion reaction
antibodies from allergic person into the skin can occur if a patient receives erythrocytes
of a normal or nonallergic person. Serum differing antigenically from his or her own
from Kustner, who was hypersensitive to during blood transfusion.
certain species of cooked fish, was injected 2. Hemolytic disease of the newborn: If the child
intracutaneously in Prausnitz (normal) followed is Rh+, Rh– mother can become sensitized to
24 hours later by an intracutaneous injection of this antigen during birth causing the mother’s
cooked fish, to which Kustner was sensitive, into body to produce anti-Rh antibodies of the IgG
the same site in Prausnitz. This led to wheal and type. If the fetus in a subsequent pregnancy
flare reaction within 20 minutes. As IgE antibody is Rh+, her anti-Rh antibodies will cross the
is homocytotropic, the test has to be carried placenta and destroy fetal RBCs. The fetal
out on human skin. Therefore, there is risk of body responds to this immune attack by
transmission of hepatitis B virus and human producing large numbers of immature RBCs
immunodeficiency virus. call erythroblasts.
2. Skin testing: It is done by injecting small 3. Drug-induced cytotoxic reactions: Some
amounts of allergen into the skin of an allergic persons develop antibodies against their blood
individual and looking for a wheal and flare elements, resulting in autoimmune hemolytic
reaction. anemia, agranulocytosis or thrombocytopenia.
3. Radioimmunosorbent test (RIST): This Blood platelets (thrombocytes) that are
highly sensitive techn ique, based on the destroyed by drug-induced cytotoxic reactions
radioimmunoassay, can detect nanomolar in the disease called thrombocytopenic
levels of total IgE. purpura (quinine is a familiar example).
4. Radioallergosorbent test (RAST): It detects Drugs may bind similarly to white or red blood
the serum level of IgE specific for a given cells (RBCs), causing local hemorrhaging and
allergen. yielding symptoms described as “blueberry
muffin” skin mottling. Immune-caused
Type II Hypersensitivity: cytolytic destruction of granulocytic white cells is called
agranulocytosis, and it affects the body’s
and cytotoxic
phagocytic defenses. When RBCs are destroyed
These reactions involve a combination of IgG (or in the same manner, the condition is termed
IgM)antibodies with an antigenic determinant hemolytic anemia.
on the surface of cells. Antibody can activate 4. Anemia due to infectious diseases: A variety
the complement system, creating pores in the of infectious diseases due to Salmonella
Chapter 20: Hypersensitivity Reactions | 147
organisms and mycobacteria are associated of the site with neutrophils. Leukocyte-platelet
with hemolytic anemia. thrombi are formed that reduce the blood supply
and lead to tissue necrosis. The Arthus reaction
Type III Hypersensitivity: immune can be passively transferred with sera containing
complex-mediated precipitating antibodies (IgG, IgM) in high titers.
Clinical syndrome: Arthus reaction forms
Type III reactions involve antibodies against
a pathogenic component of many clinical
soluble antigens circulating in the serum. The
syndromes. Examples:
antigen-antibody complexes are dep osited in
i. Farmer’s lung.
organs and cause inflammatory damage. The
ii. “Pigeon fancier’s disease”
tissue damage that results from the deposition of
immune complexes is caused by the activation of
B. Serum Sickness (Systemic Immune
complement, platelets and phagocytes; in essence,
an acute inflammatory response (Fig. 20.3). Complex Disease)
This is a systemic form of type III hypersensitivity.
Models of immune complex-mediated disease:
This appears 7–12 days following a single injection
Two basic models of immune complex-mediated
of a high concentration of foreign serum, such as the
disease have been well characterized: the Arthus
diphtheria antitoxin. The clinical syndrome consists
reaction and serum sickness. These differ
of fever, weakness, lymphadenopathy, splenomegaly,
somewhat in both their mode of development and
arthritis, glomerulonephritis, endocarditis, vasculitis,
their clinical manifestations.
urticarial rashes, abdominal pain, nausea and
vomiting.
A. Arthus Reaction (Local Immune Pathogenesis: The pathogenesis is the formation
Complex Disease) of immune complexes (consisting of the foreign
Arthus (1903) observed that when rabbits were serum and antibody to it that reaches high
repeatedly injected subcutaneously with normal enough titers by 7-12 days) and the circulating
horse serum, the initial injections were without immune complexes deposit in the blood vessel
any local effect, but with later injections, there walls and tissues, leading to increased vascular
occurred intense local reaction consisting of permeability and thus to inflammatory diseases
oedema, induration and haemorrhagic necrosis. such as glomerulonephritis and arthritis. Antigen-
This is known as Arthus reaction and is a local antibody aggregates can fix complement leading
manifestation of generalized hypersensitivity. to inflammation and tissue damage. The plasma
The tissue damage is due to formation of local concentration of complement falls due to massive
precipitating immune complexes which are complement activation and fixation by antigen-
deposited on the endothelial lining of the blood antibody complexes. The disease is self-limited.
vessels. Antigen-antibody complexes can then The latent period of 7–12 days is required only
trigg er and activate complement leading to for serum sickness following a single injection.
release of inflammatory molecules. This leads to Serum sickness differs from other types of
increased vascular permeability and infiltration hypersensitivity reaction in that a single injection
Fig. 20.3: Immune complex-mediated hypersensitivity. (1) Immune complexes on the basement membrane of the wall of
a blood vessel, where they: (2) activate complement and attract inflammatory cells such as neutrophils to the site; (3) The
neutrophilis discharge enzymes as they react with the immune complexes, resulting in damage to tissue cells
www.ebook3000.com
148 | Section 2: Immunology
can serve as both as sensitizing dose and a shocking tuberculin (infection) type and the contact
dose. dermatitis type.
Diseases associated with immune complexes:
Complexes of antibody with various bacterial, 1. Tuberculin (Infection) Type
viral and parasitic antigens have been shown to This form of hypersensitivity was originally
induce a variety of type III hypersensitive reactions. described by Koch. In tuberculin hypersensitivity
These include systemic lupus erythematosus, tuberculin or purified protein derivative (PPD) is
poststreptococcal glomerulonephritis, endo injected into the skin of the forearm. intradermally
carditis, dengue haemorrhagic fever, hepatitis B, in an individual sensitized to tuberculoprotein by
malaria, etc. prior infection or immunization. An indurated
(firm and hard) inflammatory reaction, 10 mm or
Type IV Hypersensitivity—delayed more in diameter, develops at the site of injection
hypersensitivity within 48–72 hours. It is characterized by erythema
Type IV hypersensitivity reactions (delayed due to increased blood flow to the damaged
hypersensitivity) constitute one aspect of cell- area and the infiltration with a large number of
mediated immune response and are caused mainly mononuclear cells, mainly T-lymphocytes and
by T-cells. It is named delayed hypersensitivity about 10–20% macrophages into the injection site
because it appears in 24–48 hours after the are responsible for the induration. In unsensitized
presensitized host encounters the antigen, while individuals, the tuberculin injection provokes no
immediate hypersensitivity reactions develop in response.
1/2 to 12 hours. The tuberculin test therefore provides useful
indication of the state of delayed hypersensitivity
Causes of Type IV Reactions (cell-mediated immunity) to the bacilli. The
Type IV reactions occur when antigens, especially tuberculin test differs from the skin test for Type I
those binding to tissue cells, are phagocytosed hypersensitivity not only in the longer interval for
by macrophages and then presented to receptors appearance but also in its morphology and histology.
on the THI cell surface in the context of class I Tuberculin type hypersensitivity develops in
MHC. Contact between the antigen and TH I cell many infections with bacteria, fungi, viruses and
causes the cell to proliferate and release cytokines. parasites, especially when the infection is subacute
Cytokines attract lymphocytes, macrophages, and or chronic and the pathogen intracellular. A similar
basophils to the affected tissue. Extensive tissue hypersensitivity is developed in allograft reaction
damage may result (Fig. 20.4). and in many autoimmune diseases.
Delayed hypersensitivity cannot be passively
transferred by serum but can be transferred by 2. Contact Dermatitis Type
Iymphocytes or the transfer factor. Allergic contact dermatitis is caused by haptens
Types of delayed hypersensitivity: Three types that combine with proteins in the skin to form
of delayed hypersensitivity are recognized—the the allergen that elicits the immune response.
The substances involved are in thems elves
not antigenic but may acquire antigenicity on
combination with skin proteins. Subs equent
contact with allergen in a sensitized individual
leads to contact dermatitis. As most of the sub
stances involved are fat soluble, passage along
sebaceous glands may be the method of entry of
the allergens.
Examples: Examples of these haptens include
cosmetics, plant materials (catechol molecules
from poison ivy and poison oak), topical chemo
therapeutic agents, metals (nickel and chromium),
and chemicals like dyes, picryl chloride and
dinitrochlorobenzene, drugs such as penicillin and
toiletries and jewelry (especially jewelry containing
nickel).
Mechanism of action: Most of these substances are
Fig. 20.4: Type IV (delayed or cell-mediated) small molecules that can complex with skin proteins.
hypersensitivity This complex is internalized by antigen-presenting
Chapter 20: Hypersensitivity Reactions | 149
cells in the skin (e.g. Langerhans’ cells), then along with hypersensitivity reactions because of
processed and presented together with class II superficial resemblance.
MHC molecules, causing activation of sensitized Shwartzman (1928) observed that when a
TH cells. The sensitized T cells travel to the skin culture filtrate of S. Typhi (endotoxin) is injected
site, where on contacting the antigen they release intradermally in a rabbit, followed by the same
various lymphokines. Approximately 48–72 hours filtrate (endotoxin) intravenously 24 hours later, a
after the second exposure, the secreted cytokines hemorrhagic necrotic lesion develops at the site of
cause macrophages to accumulate at the site. Tissue the intradermal injection.
damage results from lytic enzymes released from
activated macrophages. Contact with the allergen in a Mechanism
sensitised individual leads to ‘contact dermatitis’. The
The first dose is called preparatory dose. The
lesions varying from macules and papules to vesicles
second dose is called provocat ive dose. The
that break down, leaving behind raw weeping areas
preparatory injection causes accumulation of
typical of acute eczematous dermatitis.
leukocytes which condition the site by release
Detection by ‘patch test’: Hypersensitivity is of lysosomal enzymes. These enzymes damage
detected by the ‘patch test’. The allergen is applied capillary walls. Following provocative dose, there
to the skin under an adherent dressing. Sensitivity occurs intravascular clotting, the thrombi leading
is indicated by itching appearing in 4–5 hours, and to necrosis of vessel walls and hemorrhage. This is
local reaction which may vary from erythema to called local form of Shwartzman reaction.
vesicle or blister formation, after 24–28 hours. When both injections are given intravenously,
the animal dies 12–24 hours after the second dose.
3. Granulomatous Hypersensitivity The animal develops disseminated intravascular
Granulomatous hypersensitivity reactions develop coagulation (DIC). Autopsy reveals bilateral
over a period of 21–28 days; the granulomas are cortical necrosis of kidneys and patchy necrosis of
formed by the aggregation and proliferation of the liver, spleen and other organs. Sanarelli (1924)
macrophages, and may persist for weeks. In terms described an essentially similar phenomenon in
of its clinical consequences, this is by far the most experimental cholera. The reaction is, therefore,
serious type of Type 1V hypersensitivity response. called the Sanarelli–Shwartzman reaction or the
Examples are leprosy, tuberculosis, leish generalized Shwartzman reaction.
maniasis, candidiasis and herpes simplex lesions.
Clinical Conditions
Type V: Hypersensitivity 1. Waterhouse–Friderichsen syndrome: Pururic
(Stimulatory Type) Jones–Mote rashes of meningococcal septicemia and the
acute hemorrhagic adrenal necrosis found
Reaction (or) Cutaneous Basophil in overwhelming infections (Waterhouse–
Hypersensitivity Friderichsen syndrome) mechanisms similar
This is an antibody-mediated hypersensitivity and to the Shwartzman reaction may operate.
is a modification of type II hypersensitivity reaction. 2. Septic shock syndrome.
Antibodies interact with antigens on cell surface
which leads to cell proliferation and differentiation Key Points
instead of inhibition or killing. Antigen-antibody
reaction enhances the activity of affected cell. Hypersensitivity is an exaggerated immune
response that results in tissue damage and is
Example: Graves’ disease: Thyroid hormones are manifested in the individual on second or subsequent
produced in excess quantity in Graves’ disease. contact with an antigen. Hypersensitivity reactions
are of 5 types: Types I, II, III, IV, and V
Long-acting thyroid stimulating (LATS) antibody
Type I hypersensitive reaction is mediated by IgE
is an autoantibody to thyroid membrane antigen. antibodies
It is presumed that LATS combines with a TSH Clinical manifestations of type I reactions include
receptor on thyroid cell surface and brings about generalized or systemic anaphylaxis or localized
the the same effect as TSH resulting in excessive anaphylactic
secretion of thyroid hormone. Type II hypersensitive reactions, or cytotoxic
reactions are caused by antibodies that can destroy
normal cells by complement lysis or by antibody-
Schwartzman reaction dependent cellular cytotoxicity (ADCC). Transfusion
reactions and hemolytic disease of the newborn are
This is not an immune reaction but rather a type II reactions
perturbation in factors affecting intravascular Type III hypersensitivity reactions are mediated
coagulation. It is, however, traditionally described by small antigen–antibody complexes that activate
www.ebook3000.com
150 | Section 2: Immunology
2. All are primary mediators of anaphylaxis except:
complement and other inflammatory systems,
a. Histamine
attract neutrophils, and contribute to inflammation.
Deposition of immune complexes near the site
b. Proteases
of antigen entry can induce an Arthus reaction, c. Eosinophil chemotactic factors of anaphylaxis
(localized reaction) and serum sickness (systems d. Platelet-activating factor
form) 3. Schultz-Dale phenomenon is an example of:
Type IV hypersensitive reactions involve the cell- a. Type I hypersensitivity reaction
mediated branch of the immune system. b. Type II hypersensitivity reaction
Three types of delayed hypersensitivity are: c. Type III hypersensitivity reaction
i. Tuberculin skin test; ii. Contact d. Type IV hypersensitivity reaction
Hypersensitivities; iii. Granulomatous. 4. All the following statements are true for Arthus
reaction except:
a. It is a local manifestation of generalized hyper-
Important questions sensitivity.
b. The tissue damage is due to formation of local
1. What is hypersensitivity? How do you classify precipitating immune complexes.
various types of hypersensitivity reactions? Des c. This is systemic form of type III hypersensitivity.
cribe type I hypersensitivity reactions. d. It manifests after a single injection of a high
concentration of foreign serum
2. Write short notes on:
a. Anaphylaxis 5. All the following statements are true for serum
b. Atopy sickness except:
a. It manifests after a single injection of a high
c. Type III hypersensitivity or immune complex
concentration of foreign serum
diseases
b. Disease is self-limited and clears without
d. Arthus reaction
sequelae
e. Serum sickness
c. This is systemic form of type III hypersensitivity.
f. Type IV hypersensitivity or delayed hypersensi- d. It is a localized inflammatory reaction due to
tivity. deposition of immune complexes.
6. Delayed hypersensitivity reaction is mediated by:
Multiple choice questions (MCQs) a. T-lymphocytes b. B-lymphocytes
c. Macrophages d. Basophils
1. All the following statements are true for type I 7. Shwartzman reaction is an example of:
hypersensitivity reaction except: a. Type I hypersensitivity reaction
a. It is called immediate hypersensitivity reaction b. Type II hypersensitivity reaction
b. It always involves IgE-mediated degranulation c. Type III hypersensitivity reaction
of basophils or mast cells d. None of the above.
c. This reaction is always rapid Answers (MCQs)
d. Atopy is one of the manifestations 1. b; 2. d; 3. a; 4. d; 5. d; 6. a; 7. d
21
Chapter
Autoimmunity
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Classify autoimmune diseases
be able to: ∙∙ List autoimmune diseases.
∙∙ Describe the mechanisms of autoimmunity
www.ebook3000.com
Chap-21.indd 151 15-03-2016 11:12:48
152 | Section 2: Immunology
4. Polyclonal B-cell Activation the development of autoimmunity in certain
circumstances.
While an antigen generally activates only its
corresponding B-cell, certain stimuli nonspecifically
turn on multiple B clones. Such stimuli include 8. Genetic Factors
chemicals (for example, 2-mercaptoethanol), Their actual role in autoimmunity, if any, has not
bacterial products (PPD, lipopolysaccharides), been established.
enzymes (trypsin), antibiotics (nystatin) and
infection with some bacteria (mycoplasma), viruses CLASSIFICATION OF AUTOIMMUNE
(EB virus, CM virus) and parasites (malaria).
DISEASES
5. Activity of Helper and Suppressor Based on the site of involvement and nature of
lesions, autoimmune diseases may be classified
T-cells as (Table 21.1):
Enhanced helper T-cell and decreased suppressor A. Localized (or organ-specific)
T-cells functions have been suggested as causes of B. Systemic (or nonorgan-specific)
autoimmunity.
A. Localized (Organ-specific)
6. Sequestered Antigens Autoimmune Diseases (Table 21.1)
Certain self-antigens are present in closed systems The immune response is directed to a target
and are not accessible to the immune apparatus. antigen unique to a single organ or gland in an
These are known as sequestered antigens. organ-specific autoimmune disease, so that the
manifestations are largely limited to that organ.
Examples
B. Systemic (or Nonorgan-specific)
i. Lens antigen of the eye: The lens protein is
enclosed in its capsule and does not circulate Autoimmune Disease (Table 21.1)
in the blood. Hence, immunological tolerance In systemic autoimmune diseases, the response is
against this antigen is not established during directed toward a broad range of target antigens
fetal life. The release of lens protein after eye and meshces a number of organs and tissues.
damage has been shown to lead on occasion
to the formation of autoantibodies. When Pathogenesis of Autoimmune Disease
the antigen leaks out, following penetrating Many diseases are considered to be of autoimmune
injury, it may induce an immune response origin, based on their association with cellular or
causing damage to the lens of the other eye. humoral immune responses against self-antigens.
ii. Sperm antigens: Sperms arise late in develop
ment and sequestered from the circulation. As Humoral and cellular immune processes: The
spermatozoa develop only with puberty, the relative importance of humoral and cellular
antigen cannot induce tolerance during fetal immune processes in the etiology of autoimmune
life. However, after a vasectomy, some sperm diseases is not known. Antibodies may cause dam
antigens are released into the circulation age by the cytolytic or cytotoxic (type 2) and toxic
and can induce auto-antibody formation in complex (type 3) reactions. They are obviously
some men. This is also believed to be the important in hemocytolytic autoimmune diseases.
pathogenesis of orchitis following mumps. A third mechanism of autoimmune tissue
The virus damages the basement membrane damage is by sensitized T-lymphocytes (type 4
of seminiferous tubules leading to the leakage reaction). It is likely that humoral and ceIlular
of sperms and initiation of an immune immune responses may act synergistically in the
response resulting in orchitis. production of some autoimmune diseases. For
example, experimental orchitis can be induced
only when both types of immune responses are
7. Defects in the Idiotype–Anti-idiotype operative.
Network Autoimmune disease are usually treated with
It is possible that abnormalities in the generation immunosuppressants, or drugs that interfere
of appropriate anti-idiotype antibodies, either with T-cell signaling, steroids and other anti-
at the B- or T-cell level, are responsible for inflammatory drugs.
www.ebook3000.com
Chap-21.indd 153 15-03-2016 11:12:48
22
Chapter
Immunology of
Transplantation and Malignancy
Learning Objectives
After reading and studying this chapter, you should ∙∙ Histocompatibility antigens
be able to: ∙∙ Graft versus host (GVH) reaction
Discuss the following: ∙∙ Tumor antigens
∙∙ Types of transplants ∙∙ Immunological surveillance.
www.ebook3000.com
156 | Section 2: Immunology
Fetus as An allograft whole-body irradiation) that he cannot reject
a graft (allograft or xenograft).
Fetus is always a mixture of maternal and paternal
GVHD is a major complication of bone-
genes; therefore, fetal MHC antigens are always
marrow-transplantation and affects 50–70% of
different from those of the mother. The fetus can be
bone-marrow-transplant patients. It develops as
considered an intrauterine allograft, as it contains
donor T-cells recognize alloantigens on the host
antigens which are foreign to the mother. In spite
cells. The activation and proliferation of these
of this, fetus is not treated as foreign transplanted
T-cells and the subsequent production of cytokines
tissue and rejected. The fetus does not reject the
generate inflammatory reactions in the skin,
mother which may be due to the fact that the
gastrointestinal tract and liver.
developing immune system of the fetus cannot
The major clinical features of the GVH in
respond. The reason why the fetus is exempt from
animals are retardation of growth, emaciation,
rejection is not clear, though many explanations
diarrhea, hepatosplenomegaly, lymphoid atrophy
have been offered.
and anemia, terminating fatally. The syndrome has
1. Immunological barrier: The placenta acts
been called runt disease.
as an immunological barrier by generating a
hormone which is locally immunosuppressive.
2. Mucoproteins: They are produced by cells of Immunology of malignancy
the placenta which coat fetal cells, thus masking When a cell undergoes malignant transformation,
histoc ompatibility antigens and prevent it acquires new surface antigens. It may lose
recognition. some normal antigens and this makes a tumor
3. Soluble inhibitory factor: It can be produced antigenically different from the normal tissues of
by placental giant cells which suppresses T-cell the host. A tumor can, therefore, be considered an
proliferation and antibody production, and allograft and be expected to induce an immune
induces a population of T-cells. Hormones response.
(human chorionic gonadotropin, produced
by placenta, and the high levels of maternal
Tumor Antigens
progesterone produced during pregnancy) also
cause Immunosuppression. Tumor antigens are antigens that are present in
4. Blocking antibodies: The mother produces malignant cells but absent in the corresponding
specific blocking antibodies to fetal antigens of normal cell of the host. Two types of tumor antigens
the fetal cells thus blocking immune recognition have been identified on tumor cells:
and immune attack by maternal Tc-cells. 1. Tumor-specific transplantation antigens
5. Major histocompatibility complex (MHC) (TSTAs): Tumor-specific antigens are unique
antigens: There antigens are present only in to tumor cells and do not occur on normal
low density on trophoblastic cells and the cell cells in the body. Different tumors possess
membranes are relatively resistant to attack by different TSTA, even though induced by the
T- or K-cells. same carcinogen. In contrast, TSTA of virus
6. Alphafetoprotein: The high concentration of induced tumors is virus specific in that all
alphafetoprotein in fetal blood also may be a tumors produced by one virus will possess
factor, as it has immunosuppressive properties. the same antigen, even if the tumor occur in
different animal strains or species.
Graft-versus-host reaction 2. Tumor-associated transplantation antigens
(TATAs): These are present on tumor cells
Graft rejection is due to the reaction of the host to
and also on some normal cells. Since they are
the grafted tissue (host-versus-graft response). The
also present on some normal cells, therefore,
contrary situation, in which the grafted tissue may
they do not evoke an immune response and
react to and reject the host, is known as the graft-
are of little significance in tumor rejection.
versus-host (GVH) reaction.
The more common tumor-associated antigens
The GVH reaction occurs when the following
are oncofetal antigens and increased levels of
conditions are present:
normal oncogene products.TATAs fall into three
1. The graft (bone marrow, lymphoid tissue,
categories:
splenic tissue, etc.) contains immunocompetent
T-cells. i. Tumor associated carbohydrate antigens
2. The recipient possesses transplantation anti (TACAs), such as mucin-associated antigen
gens that are absent in the graft. detected in pancreatic and breast cancers.
3. The recipient must not reject the graft. The ii. Oncofetal tumor antigens: Oncofetal
host’s immunological responsiveness must tumor antigens, as the name implies,
be either destroyed or so impaired (following are found not only on cancerous cells but
Chapter 22: Immunology of Transplantation and Malignancy | 157
also on normal fetal cells. These antigens It is evident that tumor cells must develop
appear early in embryonic development, mechanisms to escape or evade the immune
before the immune system acquires system in immunocompetent hosts. Several such
immunocompetece. If these antigens mechanisms may be operative:
appear later on cancer cells, they are 1. Weak immunogenicity: Some tumors are
recognized as nonself and induce an weakly immunogenic, so in small numbers they
immunologic response. Two well-studied do not elicit an immune response. But when
oncofetal antigens are alpha-fetoprotein their numbers increase enough to provoke
(AFP) in hepatoma and cacinoembryonic immune response the tumor load may be too
antigen (CEA) found in colonic cancer. great for the host’s immune system to mount
an effective response.
iii. Differentiation antigens: They are peculiar
2. Modulation of surface antigens: Certain
to the differentiation state at which cancer
tumor-specific antigens disappear from the
cell are arrested. For example, CD10, an
surface of tumor cells in the presence of serum
antigen expressed in early B lymphocytes, antibody and then to appear after the antibody
is present in B-cell leukaemias. Similarly, is no longer present.
prostate specific antigen (PSA) is expreseed 3. Masking tumor antigens: Certain cancers
on the normal as well as cancerous produce copious amounts of a mucoprotein
prostatic epithelium. Both serve as useful called sialomucin. It binds to the surface of the
differentiation markers in the diagnosisi of tumor cells. Immune system does not recognize
lymphoid and prostatic cancer. these tumor cells as foreign since sialomucin is
a normal component.
Immune response in malignancy 4. Induction of immune tolerance: Some tumor
cells can synthesize various immunosup
Tumors can induce potent immune responses. In pressants.
experimental animals, tumor antigens can induce 5. Production of blocking antibodies: Antitumor
both humoral and cell-mediated immune responses antibody itself acts as a blocking factor.
that result in the destruction of the tumor cells. In 6. Low levels of HLA class I molecules: This
general, the cell-mediated response appears to play impairs presentation of antigenic peptides to
the major role. The immune response to tumor cytotoxic T-cells.
includes cytotoxic T lymphocytes (CTL) mediated
lysis, natural killer (NK)-cell activity, macrophage-
Immunotherapy of cancer
mediated tumor destruction, and destruction
mediated by antibody-dependent cell mediated Different approaches have been attempted in
cytotoxicity (ADCC). Several cytotoxic factors, the immunotherapy of cancer-active or passive,
including, TNF-α and TNF-b, help to mediate specific or nonspecific.
tumor-cell killing. A. Active
∙∙ Nonspecific
Immunological surveillance ∙∙ Specific
B. Passive
The immune surveillance theory was first
∙∙ Nonspecific
conceptualized in the early 1900s (1906) by Paul
∙∙ Specific
Ehrlich. He suggested that cancer cells frequently
arise in the body but are recognized as foreign ∙∙ Combined
and eliminated by the immune system. Some 50
years later, Lewis Thomas revived it in 1950s and A. Active Immunotherapy
was developed by Burnet. It postulates that the 1. Nonspecific active immunotherapy: Biological
primary function of cell mediated immunity is response modifiers (BRMs) are used to enhance
to ‘seek and destroy’ malignant cells that arise by immune responses to tumors and fall into four
somatic mutation. Such malignant mutations are major groups—(i) Bacterial products; (ii) Synthetic
believed to occur frequently and would develop molecules; (iii) Cytokines; (iv) Hormones.
into tumors but for the constant vigilance of the i. Bacterial products: Broadly speaking,
immune system. Inefficiency of the surveillance bacterial products such as BCG and nonliving
mechanism, either as a result of ageing or in Corynebacterium parvum have adjuvant
congenital or acquired immunodeficiencies, leads effects on macrophages. Intralesional BCG
to an increased incidence of cancer. can cause regression of melanoma and
The development of tumors represents a lapse nonspecific local immunization with BCG is
in surveillance. effective against bladder tumors.
www.ebook3000.com
158 | Section 2: Immunology
ii. Synthetic molecules: Dinitrochlorobenzene Graft-versus-host (GVH) reaction to occur requires
has been tried in the treatment of squamous three important components:
and basal cell carcinoma of the skin. Glucan, a The donor graft must contain immunocompetent
pyran polymer derived from micro-organisms, T-cells. The host must be immunocompromised.
and levamisole, originally introduced as an The recipient should express antigens which will be
antihelmintic, have been tried for stimulating identified as foreign to the donor
Tumor antigens can be classified as tumor-
cell mediated immunity and macrophage specific antigens (TSAs) and tumor-associated
functions. transplantation antigens (TATAs). The TSAs are
iii. Cytokines: Immunotherapy with cytokine can unique to tumors and not found on other cells of
cause tumor regression. the body
Cancer should not occur if immunological
iv. Hormones: Thymic hormones can be used to surveillance is effective.
enhance T-cell function.
2. Specific active immunotherapy: This immuno Important questions
therapy includes therapeutic vaccines of tumor 1. What is a transplant or graft? Define various types
cells, cell extracts, purified or recombinant of grafts.Describe allograft reaction.
antigens, peptides, heat shock proteins or DNA 2. What are histocompatibility antigens? Describe
antigen-pulsed dendritic cells. Specific active various procedures for histocompatibility testing.
immunotherapy by the injection of tumor cell 3. Write short notes on:
‘vaccines’ was tried early in this century but was a. Mechanism of allograft rejection
given up unprofitable. b. Graft versus host (GVH) reaction
c. Tumor antigens
d. Immunological surveillance
B. Passive Immunotherapy
i. Nonspecific (lymphokine-activated killer Multiple choice questions (mcqs)
(LAK) cells)
ii. Specific (antibodies alone or coupled to 1. Tissue transferred between two genetically different
drugs, pro drugs, toxins or radioisotope, members of the same species are known as:
a. Autografts b. Isografts
bi-specific antibodies, T-cells)
c. Allografts d. Xenografts
iii. Combined (LAK cells and bispecific antibody). 2. Acute rejection shows all the following features
except:
Key Points a. It takes days or weeks after transplantation.
b. It is due to the primary activation of T-cells
Transplantation can be defined as the transfer of c. Acute rejection is the typical first-set rejection
cells, tissues, or organs from one site in an individual d. Vascular endothelial injury is the most common
to another, or between two individuals feature
There are four different basic types of transplants: 3. The following cell may provide the first line of
autograft, isograft, allograft and xenograft defence against many tumors:
The immune response to tissue antigens encoded a. Eosinophils
within the major histocompatibility complex is the b. Natural killer cells
strongest force in rejection c. Monocytes
The match between a recipient and potential graft d. None of the above
donors is assessed by typing MHC class I and class
II tissue antigens Answers (MCQs)
1. c; 2. d; 3. b
23
Chapter
Immunohematology
Learning Objectives
After reading and studying this chapter, you should ∙∙ List various infectious agents transmitted via blood
be able to: transfusion.
∙∙ Describ Rh blood groups
∙∙ Discuss complications of blood transfusion
History H Antigen
The ABO system is the most important of all Red cells of all ABO groups possess a common
the blood group systems and its discovery made antigen, the H antigen or H substance which is the
blood transfusion possible. No other blood group precursor for the formation of A and B antigens. H
antigens were discovered for the next 25 years. antigen is not ordinarily important in grouping or
Subsequently, other blood groups (MN, P, Rh, blood transfusion due to its universal distribution.
Lutheran, Lewis, Kell, Duffy, Kidd, Diego, Yt, Kg, However, Bhende et al. (1952) from Bombay
Dombroc and Colton) were reported. reported a very rare instance in which A and B
antigens as well as H antigens were absent from the
red cells. This is known as Bombay or OH blood
ABO Blood Group System group. Sera of these individuals have anti-A, anti-B
The ABO blood group system was originally and anti-H antibodies. Therefore, they can accept
described by Landsteiner (1900) and now contains the blood only from the same rare blood group.
four blood groups. The blood group is determined A, B and H antigens are glycoproteins. They
by the presence or absence of two distinct antigens are also present in almost all the tissues and fluids
A and B on the surface of the erythrocytes. It is of the body in addition to erythrocytes. They are
these antigens (also called agglunotigens) that found in secretions (saliva, gastric juice, sweat) of
cause blood transfusion reactions. Red cells of only about 75% of all persons while these antigens
group A carry antigen A, cells of group B antigen B are always present in tissues. Such persons are
and cells of group AB have both A and B antigens, called ‘secretors’ and those who lack blood group
while group O cells have neither A nor B antigen. antigens in secretions are called ‘nonsecretors’.
The serum contains the isoantibodies specific
for the antigen that is absent on the red cell. The Table 23.1 Distribution of ABO antigens on the
serum of a group A individual has anti-B antibody, red blood cells and antibodies in the serum
group B has anti-A and group O both anti-A and Blood Antigens on Antibodies Occurrence
anti-B, while in group AB, both anti-A and anti-B group red blood cells in serum (%) in India
are absent (Table 23.1). A A Anti-B 22
The frequency of ABO distribution antigens B B Anti-A 33
differs in different people. Group O is the most
AB A and B None 5
common group and AB the rarest. In India, the
distribution is approximately O–40%, A—22%, O None Anti-A and 40
Anti-B
B—33% AB—5%.
www.ebook3000.com
160 | Section 2: Immunology
Rh (Rhesus) Blood Group System possessed neither A nor B antigen. Hence
It was discovered by Landsteiner and Weiner in the O group was designated as the ‘universal
1940. Their experiment was to produce an antibody donor’. The anti-A and anti-B antibodies in
to the red cells of the Rhesus monkey in rabbits the transfused O blood group do not ordinarily
and guinea pigs, but they discovered that not only cause any damage to the red cells of the A or B
did the antibody in the rodents’ serum agglutinate group recipients because they will be rendered
the Rhesus monkey red cells, it also agglutinated ineffective by dilution in the recipient’s plasma.
the red cells of 85% of the human population. If an But some O group plasma may contain
individual’s red cells were clumped together by this isoantibodies in high titers (1:200 or above)
antiserum, they were said to have the Rhesus factor so that damage to recipient cells may result.
on their red cells (i.e. Rh positive). If an individual’s This is known as the ‘dangerous O group’. The
cells were not agglutinated by the antiserum, they AB group persons were designated ‘universal
were said to lack the Rhesus factor (i.e. Rh negative). recipients’ due to the absence of isoantibodies
Rh antigens—“Rh-positive” and “Rh-negative” in plasma.
people. 2. Rh compatibility: An Rh positive person may
There are six common types of Rh antigens, safely receive either Rh positive or negative
each of which is called an Rh factor. These types are blood. But an Rh-negative individual receiving
designated as C, D, E, c, d, and e. The designations Rh-positive blood may form antibodies against
employed by the two system for the different Rh the Rh-antigen. A subsequent transfusion with
types are as follows: Rh positive blood may then cause a rapid,
serious hemolytic reaction.
The type D antigen (Rho) is widely present in the
population and considerably more antigenic than
the other Rh antigens. Rh-positive or Rh-negative Complications of blood
blood depends on the presence or absence of transfusion
D-antigen on the surface of red cells respectively. The complications of blood transfusion may be divi
It can be accomplished by testing with anti-D ded into immunological and nonimmunological.
(anti-Rh) serum. About 15% of the population have
A. Immunological complications: Immunological
no RhD antigens and thus are “Rh negative”. Among
complications may be caused by red cell,
Indians, approximately 93% are Rh positive and
leukocyte or platelet incompatibility or allergic
about 7% negative. The Rh factor can be detected
reaction to plasma components. Red cell
by testing the blood with anti-D (anti-Rh) serum.
incompatibility leads to acute intravascular
hemolysis or the red cells may be coated
Other blood group systems by antibodies and engulfed by phagocytes,
Blood group systems other than ABO and Rh are removed from the circulation and subjected to
of little clinical importance as they do not usually extravascular lysis. Hemolysis may also be due
cause transfusion reactions or hemolytic disease. to transfusion of group O whole blood or plasma
They have applications in genetics, anthropology, to group A or group B or group AB recipients.
tissue typing and forensic medicine. As blood group B. Nonimmunological complications: Non-
antigens are inherited from the parents, they are immunological complications of blood trans
often useful in settling cases of disputed paternity. fusion include transmission of infectious
agents and circulatory overload. Infectious
Lewis blood group system: It differs from other
agents which may be transmitted during
blood group systems in that the antigens are
blood transfusion may be viruses, bacteria and
present primarily in the plasma and saliva and
protozoa (Table 23.2). Massive transfusion may
antigens do not form an integral part of the red
lead to circulatory overload.
cell membrane.
MN system: The antigens are M, N, S and s. Hemolytic disease of the newborn
This system has expanded to include at least 28
antigens. When an Rh– woman and an Rh+ man produce
a child, there is a 50% chance that the child will
Medical applications of blood be Rh+. If the child is Rh +, the Rh– mother can
become sensitized to this antigen during birth,
groups when the placental membranes tear and the fetal
1. Blood transfusion: Ideally, the donor and Rh+ RBCs enter the maternal circulation, causing
recipient should belong to the same ABO group. the mother’s body to produce anti-Rh antibodies
It used to be held that O group cells could be of the IgG type. During subsequent pregnancy,
transfused to recipients of any group as they Rh antibodies of the IgG class pass from the
Chapter 23: Immunohematology | 161
Table 23.2 Nonimmunological complications of Detection of Rh-antibodies
blood transfusion Most Rh-antibodies are of the IgG class, and they
A. Transmission of Infectious Agents do not agglutinate Rh-positive cells in saline being
Viruses ‘incomplete antibodies’. IgG anti-D antibodies
• Hepatitis B virus* may be detected by the following techniques:
• Hepatitis C virus** 1. Using a colloid medium such as 20% bovine
• Human immunodeficiency virus 1 and 2*
• Human T-cell Iymphotrophic virus 1 and 2
serum albumin.
• Cytomegalovirus 2. Using red cells treated with enzymes such as
Bacteria trypsin, pepsin, ficin or bromelin, and
• Treponema pallidum* 3. By the indirect Coombs test: This is the most
• Leptospira interrogans sensitive method.
• Borrelia burgdorferi
Parasites
Plasmodium spp.* Prevention of Rh-isoimmunization
• Babesia spp.
• Trypanosoma cruzi HDNB is usually prevented today by passive immu
• Leishmania donovanii nization of the Rh– mother at the time of delivery
• Toxoplasma gondii (within 24–48 hours) of any Rh+ infant with anti-
B. Circulatory Overload Rh-antibodies, which are available commercially
* Mandatory tests in India
(Rhogam). To be effective, this should be employed
** Mandatory tests in India since June 2001 from the first delivery onwards.
www.ebook3000.com
162 | Section 2: Immunology
It has been shown that duodenal ulcer is more Multiple Choice Questions (Mcqs)
frequent in persons of blood group O than in
1. The ABO blood group system was described by:
others. An association has also been established
a. William Harvey
between group A and cancer of the stomach.
b. Ehrlich and Morgenroth
Key Points c. Philip Levine
Immunohematology is the study of blood group d. Karl Landsteiner
antigens and antibodies and their interactions in 2. All the following statements are true for H antigen
health and disease except:
ABO blood group system: The ABO blood group a. It is a glycoprotein
substances are glycopeptides
b. It is structurally an L-sucrose
Rh antigens: There are six common types of Rh
antigens designated as C, D, E, c, d, and e. Of all the c. It is a precursor for the production of A and B
Rh antigens, antigen D (Rho) is most important antigens
Transmission of infectious agents especially HIV I and d. It is present on red blood cells of all ABO groups
II, HBV, and HCV through blood is the most important 3. Hemolytic disease in newborn may occur when:
complication a. An Rh negative mother carries an Rh positive
Hemolytic disease of the newborn. Rh antibodies fetus
of the IgG class pass from the mother to the fetus
through the placenta and damage its erythrocytes. b. An Rh positive mother carries an Rh negative
This disease usually occurs even in the second or fetus
successive child. c. Both of the above
d. All of the above
Important questions 4. All the following infectious agents may be trans
mitted by blood transfusion except:
1. Name various blood group systems and discuss a. Human immunodeficiency viruses I and II
Rh blood group system. Add a note on hazards of b. Hepatitis A virus
incompatible blood transfusion.
c. Hepatitis B virus
2. Write short notes on: d. Hepatitis C virus
a. Complications of blood transfusion.
b. Hemolytic disease of the newborn (or) erythro Answers (MCQs)
blastosis fetalis. 1. d; 2. b; 3. a; 4. b
Section 3: Systemic Bacteriology
24
Chapter
Staphylococcus
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe staphylococcal diseases
be able to: ∙∙ Discuss laboratory diagnosis of infections caused
∙∙ Describe species of Staphylococcus by Staphylococcus aureus
∙∙ Describe morphology and culture characteristics ∙∙ Explain methicillin-resistant staphylococci and its
of Staphylococcus aureus clinical problem
∙∙ List characteristics of Staph. aureus strains ∙∙ D escribe the following: Coagulase-negative
∙∙ Explain coagulase test staphylococci (CNS); micrococci
∙∙ List and describe toxins and enzymes of Staphylo ∙∙ Distinguish characteristics of Staph. aureus, Staph.
coccus aureus epidemidis and Staph. saprophyticus.
www.ebook3000.com
Chap-24.indd 163 15-03-2016 11:33:06 AM
164 | Section 3: Systemic Bacteriology
They stain readily with aniline dyes and are food and dust, likely to contain a predominance
uniformly gram-positive. of other kinds of bacteria. Therefore, 7–10% of
sodium chloride may be added to nutrient agar
Cultural Characteristics (Salt agar) or milk agar (salt milk agar); mannitol
They are aerobes and facultative anaerobes. Opti salt agar containing 1% mannitol, 7.5% NaCl,
mum temperature for growth is 37°C ( range being and phenol red in nutrient agar; and Ludlam’s
12–44°C). Optimum pH is 7.5. They can grow medium containing lithium chloride and tellurite;
readily on ordinary media. and salt cooked meat broth (10% NaCl.).
1. Nutrient agar: After aerobic incubation for 24
hours at 37°C, colonies are 1–3 mm in diameter Biochemical Reactions
and have a smooth glistening surface, an 1. Sugar fermentation: S. aureus ferments a range
entire edge, a soft butyrous consistency and an of sugars producing acid but no gas. Sugar
opaque, pigmented appearance. Most strains fermentation is of no diagnostic value except
produce golden-yellow (aureus) pigment. These for mannitol, which is usually fermented
white-colonied strains of S. aureus are fully anaerobically by Staph. aureus but not by other
virulent. Pigmentation is characteristic of this species.
species when grown aerobically. 2. Catalase: Catalase positive (unlike streptococci).
Pigmentation is enhanced on fatty media, such 3. Lipolytic: When grown on media containing
as tween agar, by prolonged incubation, and by egg-yolk, produce a dense opacity because
leaving plates at room temperature. The pigment most strains are lipolytic.
is believed to be lipoprotein allied to carotene. 4. Phosphatase test: This is a useful screening
2. Blood agar: The colonies have the same procedure for differentiating Staph. aureus
appearances as on nutrient agar, but may from Staph. epidermidis in mixed cultures, as
be surrounded by a zone of β-hemolysis the former gives prompt phosphatase reaction,
(Fig. 24.2). Hemolysis is more likely to be while the latter is usually negative or only
present if sheep, human or rabbit blood is used. weakly positive. All strains of S. aureus produce
3. MacConkey agar: Colonies are smaller and are phosphatase which liberates phenolphthalein
pink due to lactose fermentation. from sodium phenolphthalein diphosphate
4. Milk agar: On this medium, after overnight The culture plate, with the culture, is inverted
incubation, the colonies of S. aureus are larger over the ammonia for a minute or so. Colonies
than those on nutrient agar and pigmentation of S. aureus become bright pink because
is well-developed and easily recognized against phenolphthalein is pink in alkaline pH. Most
the opaque white background. other staphylococci form colonies that remain
5. Phenolphthalein phosphate agar: This is an uncolored.
indicator medium and assists in the iden 5. Deoxyribonulease (DNAase) test: It produces a
tification of S. aureus in mixed cultures. deoxyribonulease (DNAase), and a heat-stable
Appearance of the colonies of S. aureus on this nuclease (thermonuclease, TNAase).
medium is similar to those on nutrient agar. 6. Other biochemical tests: Indole negative, MR
Colonies of S. aureus become bright pink when positive, VP positive, urease positive, hydrolyzes
culture plate is inverted over the ammonia for gelatin and reduces nitrates to nitrites.
a minute or so. Table 24.1 shows the characteristics of
6. Selective salt media: Selective medium may Staphylococcus aureus.
be useful for the isolation and enumeration of
staphylococci from materials, such as feces,
Table 24.1 Characteristics of Staph. aureus strains
Staph. aureus strains usually exhibit the following
characteristics:
1. Beta hemolysis—produce clear hemolysis on blood
agar
2. Golden yellow pigment
3. Coagulase positive
4. Greater biochemical activity, ferment mannite
5. Liquefy gelatin
6. Produce phosphatase
7. Black colonies on potassium tellurite blood agar—
produce black colonies in a medium containing
potassium tellurite by reducing tellurite to metallic
tellurium
8. Produce thermostable nucleases which can be
demonstrated by the ability of boiled cultures to
Fig. 24.2: Structure of staphylococcal cell wall degrade DNA in an agar diffusion test
www.ebook3000.com
Chap-24.indd 165 15-03-2016 11:33:07 AM
166 | Section 3: Systemic Bacteriology
iv. Delta (d) hemolysin: It lyses red blood cells of syndrome’ (SSSS). Two distinct forms of exfoliative
sheep, rabbit, horse and man. toxin (ETA and ETB) have been identified, and
v. Leukocidin: Leukocidin (called the Panton- either can produce disease. ETA is heat-stable and
Valentine toxin after its discoverers) is also a the gene is chromosomal, whereas ETB is heat-
two component toxin, like the gamma lysin, labile and plasmid-mediated. SSSS is seen mostly
S (slow) and F (Fast). These components act in young children and only rarely in older children
synergistically to damage polymorphonuclear and adults.
leukocytes and macrophages and to produce
dermonecrosis. B. Extracellular Enzymes
2. Enterotoxins Staph. aureus produces a number of enzymes such
This toxin is responsible for the manifestations as coagulase catalase, hyaluronidase, fibrinolysin,
of staphylococcal food poisoning n ausea, lipases, nucleases and penicillinase.
vomiting and diarrhea 2–6 hours after consuming Coagulase: S. aureus produces an extracellular
contaminated food containing preformed toxin. enzyme called coagulase which brings about
Enterotoxins are commonly produced by about clotting of human or rabbit plasma. It acts along
two-thirds of Staph. aureus strains, growing in with a ‘coagulase reacting factor’ (CRF) present
carbohydrate and protein foods. The toxin is in plasma, binding to prothrombin and converting
relatively heat stable, resisting 100°C for 10–40 fibrinogen to fibrin. Coagulase does not clot plasma
minutes depending on the concentration of the of guinea pigs because they lack CRF. Coagulase
toxin and nature of the medium. test is the standard criterion for the identification
Eight serologically distinct staphylococcal of Staph. aureus isolates.
enterotoxins (A-E, G-I) and three subtypes of Eight antigenic types (A-H) have been des
enterotoxin C have been identified. Enterotoxin cribed. Most human strains form coagulase type A.
A is most commonly associated with disease. Coagulase Fibrin (Clotting)
Fibrinogen →
Enterotoxins C and D are found in contaminated CRF
milk products, and enterotoxin B causes staphylo Coagulase test: Coagulase test is done by two
coccal pseudomembranous enterocolitis. methods—slide and tube coagulase test. The slide
The precise mechanism of toxin activity is not or tube coagulase test is performed to distinguish
understood. The toxin is believed to act directly on Staph. aureus from coagulase-negative species.
the autonomic nervous system to cause the illness, a. Slide coagulase test: The slide test detecting
rather than on the gastrointestinal mucosa. The bound coagulase is much simpler. It can be
toxin is antigenic and neutralized by the specific detected by emulsifying a few colonies of the
antitoxin. Type A toxin is responsible for most cases. bacteria in a drop of normal saline on a clean
For detection of the toxin sensitive serological tests, glass slide and mixing it with a drop of rabbit
such as latex agglutination and ELISA are available. plasma. Prompt clumping of the organisms
These toxins are supera ntigens, however, indicates the presence of clumping factor
capable of inducing nonspecific activation of T (bound coagulase). Positive and negative
cells and cytokine release. The toxin is potent, controls also are set up.
microgram amounts being capable of causing the Almost all the clumping factor producing
illness. strains of S. aureus produce coagulase.
b. Tube coagulase test: The tube coagulase test
3. Toxic Shock Syndrome Toxin-I (TSTT-1) detects free coagulase. 0.1 ml of an overnight
S. aureus is associated with toxic shock syndrome broth culture or broth suspension from an agar
(TSS), a severe and often fatal disorder characterized plate culture made up to the same density is
by multiple organ dysfunction. TSST-I (formerly mixed with 0.5 mL of a 1 in 10 dilution of human
called pyrogenic exotoxin C and enterotoxin F) or rabbit plasma. The mixture is incubated in
is heat and proteolysis resistant, chromosomally a water bath at 37°C for three to six hours. If
mediated exotoxin. It is antigenic and most persons positive, the plasma clots and does not flow when
over 30 years of age have circulating antibodies. the tube is inverted. If clot does not appear it is left
The enterotoxins and TSST-1 belong to a class overnight at room temperature and re-examined.
of polypeptides known as superantigens which On continued incubation, the clot may be
are potent activators of T lymphocytes leading to lysed by fibrinolysin produced by some strains.
the release of cytokines such as interleukins and Controls with plasma alone, known coagulase-
tumour necrosis factor. This results in clinical positive and coagulase-negative cultures must
condition of TSS. be set up with each batch of tests.
4. Epidermolytic Toxins (Exfoliative Toxins) False-positive reaction: Citrated plasma should not
This toxin, also known as ET or ‘exfoliatin’ is be used because contaminating gram-negative
responsible for the ‘staphylococcal scalded skin bacilli (e.g. Pseudomonas) may utilize the citrate
www.ebook3000.com
Chap-24.indd 167 15-03-2016 11:33:07 AM
168 | Section 3: Systemic Bacteriology
performed appropriate to the clinical situation.
This is important as staphyloc occi readily
develop resistance to drugs.
6. Bacteriophage typing: Bacteriophage typing
may be done if the informat ion is desired
for epidemiological purposes. Other typing
methods include antibiogram pattern, plasmid
profile, DNA fingerprinting, ribotyping and
PCR-based analysis for genetic pleomorphism.
7. Serological tests : Serological tests may
sometimes be of help in the diagnosis of
hidden deep infections. Antistaphylolysin
(anti-alphalysin) titers of more than two units
per mL, especially when the titer is rising, may
be of value in the diagnosis of deep seated
Fig. 24.3: Bacteriophage typing of staphylococci
infections, such as bone abscesses.
Microbiology, Maulana Azad Medical College,
New Delhi. Treatment
Benzyl penicillin is the most effective antibiotic,
Laboratory Diagnosis if the strain is sensitive but most clinical isolates
1. Specimens: The specimens to be collected of S.aureus are resistant to benzyl penicillin due to
depend on the type of lesion, for example: production of beta lactamase. Cloxacillin, oxacillin,
pus from suppurative lesions; sputum from flucloxacillin and methicillin are penicillinase-
respiratory infect ions; food remains and resistant penicillins.
vomit from cases of food poisoning; nasal and Methicillin-resistant Staphylococcus aureus
perineal swabs from suspected carriers. (MRSA) are also resistant to other penicillins
Swabs of the perineum, pieces of hair and and cephalosporins. Glycopeptides (vancomycin
umbilical stump—may be necessary in special or teicoplanin) are the agents of choice in the
situations. treatment of systemic infection, but these agents
2. Direct microscopy: Direct microscopy with are expensive and may be toxic.
Gram stained smears is useful in the case of For mild superficial lesions, topical applications
pus, where cocci in clusters may be seen. This of drugs as bacitracin, chlorhexidine or mupirocin
is of no value for specimens like sputum where may be sufficient.
mixed bacterial flora are normally present. The treatment of carriers is by local application
3. Culture: The specimens are cultured on a blood of antibiotics such as bacitracin and antiseptics
agar plate. Staphylococcal colonies appear such as chlorhexidine. In resistant cases, rifampicin
after overnight incubation specimens, where along with another oral antibiotic may be effective.
staphylococci are expected to be outnumbered
by other bacteria (e.g. wound swab and Control
feces), are inoculated on selective media like 1. The focus of infection (e.g. abscess) must be
Ludlam’s or salt-milk agar or Robertson’s cooked identified and drained.
meat medium containing 10% sodium. The 2. Treatment is symptomatic for patients with
inoculated media is incubated at 37°C for 18–24 food poisoning.
hours. On blood agar plate, look for hemolysis 3. Proper cleansing of wounds and use of
around the colonies. The plates are inspected disinfectant.
for golden-yellow or white colonies. Smears are 4. Thorough hand washing and covering of
examined from the culture and coagulase test exposed skin helps medical personnel prevent
done when stahylococci are isolated. infection or spread to other patients.
4. Identification: Relatively simple biochemical
5. Asymptomatic nasopharyngeal carriage—
tests (e.g. positive reactions for coagulase
the use of chemoprophylaxis consisting of
(clumping factor), heat-stable nuclease, alkaline
vancomycin and rifampin to prevent spread of
phosphatase, and mannitol fermentation) can
oxacillin-resistant organisms
be used to differentiate S. aureus and the other
staphylococci. Other Coagulate-positive
Coagulase test: Coagulase test is done by two
staphylococci
methods-slide and tube coagulase test.
5. Antibiotic sensitivity tests: As a guide to Other staphylocoagulase producing (coagulase
treatment antibiotic sensitivity tests should be p ositive) staphylococci are S. intermedius,
S. delphini, S. lutrae, and some strains of S. hyicus. to novobiocin and by its failure to ferment glucose
These are often animal-associated species and are anaerobically. It is nonhemolytic and does not
infrequently isolated from human samples. contain protein A. Table 24.3 lists the features
useful for distinguishing the major species of
Coagulase-negative staphylococci.
Staphylococci
Other Coagulase-negative Staphylococci
Coagulase-negative staphylococci (CONS) are
commonly found on the surface of healthy persons. S. haemolyticus has been reported in wounds,
They are opportunistic pathogens that cause bacteremia, endocarditis, and UTIs. Other species
infection in debilitated or compromised patients. include S. lugdunensis, S. warneri, S. capitis,
Various species are Staph. epidermidis, Staph. S. simulans, and S. schleiferi.
saprophyticus, Staph. hemolyticus, Staph. hominis,
and Staph. capitis. Sensitivity to Antibiotics
Before the introduction of penicillin, most of the
Staphylococcus epidermidis strains of S. aureus were sensitive to this antibiotic.
Staph. epidermidis is invariably present on normal Staphylococci quickly developed drug resistance
human skin. It is nonpathogenic ordinarily but after penicillin was introduced.
can cause disease when the host defences are Penicillin resistance is of three types:
breached. S. epidermidis has a distinct predilection 1. Production of beta lactamase (penicillinase):
for foreign bodies, such as artificial heart valves, Production of beta lactamase (penicillinase)
indwelling intravascular catheters, central nervous which inactivates penicillin by splitting the beta
system shunts, and hip prostheses. Their etiological lactam ring. Staphylococci produce four types
role is proved by repeated isolation. of penicillinases, A to D. Penicillinase is an
inducible enzyme and its production is usually
controlled by plasmids which are transmitted
Clinical Infection
by transduction or conjugation.
∙∙ Stitch abscesses; endocarditis of native Penicillinase plasmids are transmitted to
and prosthetic valves; intravenous catheter the sensitive staphylococci by transduction and
infections; CSF shunt infections; bacteremia; also possibly by conjugation.
osteomyelitis; wound infections, vascular graft 2. Changes in bacterial surface receptors:
infections, prosthetic joint infection, etc. Changes in bacterial surface receptors, reduc
ing binding of beta-lactam antibiotics to cells.
Staphylococcus saprophyticus This change is normally chromosomal in nature
S. saprophyticus is a common cause of urinary tract and is expressed more at 30°C than at 37°C. This
infections in sexually active young women. It may resistance also extends to cover beta lactamase-
also cause urethritis in men and women, catheter- resistant penicillins, such as methicillin and
associated urinary tract infections, prostatitis in cloxacillins. Some of these strains may show
elderly men, and rarely bacteremia, sepsis and resistance to other antibiotics and heavy metals
endocarditis. also and cause outbreaks of hospital infection.
This coagulase-negative Staphylococcus can be These strains have been called ‘epidemic
distinguished from S. epidermidis by its resistance methicillin-resistant Staphylococcus aureus’
www.ebook3000.com
Chap-24.indd 169 15-03-2016 11:33:08 AM
170 | Section 3: Systemic Bacteriology
Table 24.4 Differentiation between staphylococci and micrococci
Property Staphylococcus Micrococcus
Gram staining Gram-positive Gram-positive, darkly stained
Grape-like clusters In groups of four (tetrad) or eight
Uniform staining Often staining is not uniform
Colony characters Colonies are golden Colonies are white in color generally
yellow Size 1 mm Size Larger than staphylococcus
Anaerobic acid production from glucose + –
Aerobic acid production form glycerol + –
in the presence of erythromycin
Modified oxidase – +
Bacitracin sensitivity (0.04 unit disk) Resistant Sensitive
Lysostaphin sensitivity Sensitive Resistance
Furazolidone susceptibility (100 µg) of furazolidone disk Sensitive Resistance
or EMRM. (as methicillin is an unstable drug, samples. A high salt concentration (5.5% NaCl)
cloxacillin is used for sensitivity testing instead). and polymyxin B make the medium selective
3. Development of tolerance: Development of for staphylococci.
tolerance to penicillin, by which the bacterium 2. The gold standard for MRSA detection is the
is only inhibited but not killed. detection of the mecA gene by using nucleic
acid probes or polymerase chain reaction (PCR)
Methicillin-resistant Staphylococcus amplification.
aureus (MRSA) Control of MRSA: Control of MRSA requires strict
Methicillin was the first compound developed adherence to infection-control practices such as
to combat resistance due to penicillinase (beta barrier.
lactamase) production by staphylococci. Due
to the limitations in clinical use of methicillin, Micrococci
cloxacillins are used instead against penicillinase-
producing strains. But methicillin resistant strains Micrococci are catalase-positive, gram-positive,
of Staph. aureus (MRSA) became common, which coagulase-negative and usually oxidase-positive.
were resistant not merely to penicillin, but also They may ocassionally colonize the skin or mucous
to all other beta lactam antibiotics and many membrane of humans, but they are only rarely
others besides. Isolates that are resistant have associated with infections.
been traditionally termed methicillin-resistant Only two species, Micrococcus luteus and
staphylococci, with S. aureus being called MRSA Micrococcus lylae, remain in the genus. Micrococci,
and S. epidermidis referred to as MRSE. When especially M. luteus, have a tendency to produce
any staphylococcus isolated is identified as being a yellow pigmented colony. Nine species of genus
resistant to methicillin, this implies that it is Micrococcus have been described.
also resistant to nafcillin and oxacillin and to all Table 24.4 gives some differentiating features
β-lactam antibiotics, including the cephalosporins. of Staphylococcus and Micrococcus.
MRSA is also becoming more common in the
community, especially in long-stay institutions. Key Points
Staphylococcus
Treatment-glycopeptides (vancomycin or
Staphylococcus is gram-positive cocci arranged in
teicoplanin) are the agents of choice in the
clusters
treatment of systemic infection, but these agents
Species of staphylococci are classified by the
are expensive and may be toxic. Concerns with coagulase test into two groups: the coagulase-
rising resistance to glycopept ides call for the positive (Staphylococcus aureus) and coagulase-
restrictive use of these drugs. negative staphylococci. S. epidermidis and S.
saprophyticus
Laboratory Diagnosis Staphylococcus aureus
Staph. aureus strains usually exhibit following
1. For laboratory purposes, oxacillin is generally characteristics: (1) Beta hemolysis; (2) Golden
used for detection of methicillin resistance. yellow pigment; (3) Coagulase positive; (4) Greater
The use of an oxacillin-salt agar plate, such as biochemical activity, ferment mannite; (5) Liquefy
the oxacillin resistance screening agar can be gelatin; (6) Produce phosphatase; (7) Black colonies
used as a screening test for MRSA in clinical on potassium tellurite blood
S. aureus produces many virulence factors including: f. Epidermolytic toxins of Staphylococcus aureus
(1) Cytolytic or membrane-damaging toxins (alpha, g. Drug resistance in staphylococci.
beta, delta, gamma, and Panton-Valentine [P-V] h. Methicillin-resistant Staphylococcus aureus
leukocidin); (2) Exfoliative toxins: (3) Enterotoxins (MRSA)
(A-E, G-I); (4) Toxic shock syndrome toxin-1 (TSST-1) i. Coagulase-negative staphylococci (CNS)
Diseases: S. aureus causes cutaneous infections j. Micrococci.
such as folliculitis, boils, carbuncles, impetigo, and
purulent abscesses. These cutaneous infections
can progress to deeper abscesses involving other
Multiple choice questions (MCQs)
organ systems and progress to septicemia and 1. Staphylococcus aureus shows the following
bacteremia. Toxin-induced diseases, such as food characters except
poisoning, scalded skin syndrome (SSS), and toxic
a. It produces golden brown pigment on the blood
shock syndrome (TSS), are also associated with this
agar
organism
Other systemic diseases (frequently associated with b. It ferments mannitol
bacteremia) include pneumonia, empyema, septic c. It is novobiocin-sensitive
arthritis, osteomyelitis, acute endocarditis, and d. It has protein A
catheter-related bacteremia 2. Protein A is a cell wall components of
Diagnosis: It is done by microscopy, culture, antibiotic a. Staphylococcus aureus
sensitivity tests and serological tests b. Staphylococcus epidermidis
Bacteriophage typing may be done if the information
c. All of the above
is desired for epidemiological purposes
Treatment: The antibiotics of choice are oxacillin
d. None of the above
(or other penicillinase-resistant penicillin) or 3. The enzyme coagulase shows all the following
vancomycin for oxacillin-resistant strains features except
Methicillin-resistant strains (MRSA). Glycopeptides a. It has eight serotypes
(vancomycin or teicoplanin) are the agents of choice b. It is extracellular
in the treatment of systemic infection c. It is detected by tube coagulase test
Coagulase-negative staphylococci: Coagulase- d. Undiluted serum is used in the test
negative staphylococci are opportunistic pathogens
4. Scalded skin syndrome is caused by the
that cause infection in debilitated or compromised
following toxin of Staphylococcus aureus
patients. Staph. epidermidis accounts for about 75%
of all clinical isolates. Other species include Staph. a. Enterotoxins
haemolyticus, Staph. hominis, Staph. capitis and b. Toxin shock syndrome
Staph. saprophyticus c. Exfoliative toxin
Staph. saprophyticus can be distinguished from S. d. Leukocidin
epidermidis by its resistance to novobiocin and by 5. Which of the of the following Staphylococcus is
its failure to ferment glucose anaerobically novobiocin resistant?
Micrococci may occasionally colonize the skin or a. Staphylococcus aureus
mucous membrane of humans, but they are only
b. Staph. epidermidis
rarely associated with infections.
c. Staph. saprophyticus
d. None of the above
Important Questions 6. The most common cause of cystitis in a young
1. Describe the morphoplogy, cultural characteristics healthy sexually active women is:
and antigenic structure of Staphylococcus aureus. a. Staphylococcus aureus
2. Name various virulence factors of Staphylococcus b. Staphylococcus epidermidis
aureus. c. Staphylococcus saprophyticus
3. Classify staphylococci. Discuss pathogenicity and d. Staphylococcus saccharolyticus
laboratory diagnosis of Staph. aureus. 7. All are coagulase-negative staphylococci except:
4. Write short notes on: a. Staphylococcus epidermidis
a. Coagulase or staphylocoagulase b. Staphylococcus saprophyticus
b. Clumping factor c. Staphylococcus haemolyticus
c. Toxins and enzymes of produced by Staphylo d. Staphylococcus aureus
coccus aureus
d. Staphylococcal food poisoning. Answers (MCQs)
e. Toxic shock syndrome 1. a; 2. a; 3. d; 4. c; 5. c; 6. c; 7. d
www.ebook3000.com
Chap-24.indd 171 15-03-2016 11:33:08 AM
25
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ List and describe toxins and enzymes of Strepto
be able to: coccus pyogenes
∙∙ Classify streptococci ∙∙ Describe non-suppurative complications of Str.
∙∙ Describe antigenic structure of Str. pyogenes pyogenes infections
∙∙ List and describe toxins and enzymes of Strepto ∙∙ Discuss laboratory diagnosis of streptococcal
coccus pyogenes infections
∙∙ Discuss pathogenicity of streptococci ∙∙ Discuss group B streptococci, Group D streptococci
and viridans group.
Introduction Classification
The genus Streptococcus comprises a large and Streptococci are first divided into obligate anaerobe
biologically diverse group of gram-positive cocci and facultative anaerobes. Obligate anaerobe are
that grow in pairs or chains (Fig. 25.1). They are designated as peptostreptococci.
normal flora of humans and animals. They inhabit Three different schemes are used to classify the
various sites, notably the upper respiratory tract, organism, as shown in Figure 25.2.
and live harmlessly as commensels.
Streptococci were first described by Billroth
(1874) in exudates from erysipelas and wound
infections, who called them streptococci (streptos,
meaning twisted or coiled; coccus, a grain or
berry). Pasteur (1879) found similar organisms
in the blood of a patient with puerperal sepsis.
Ogston (1881) isolated them in acute abscesses,
distinguished them staphylococci and by animal
inoculation established their pathogenicity.
www.ebook3000.com
174 | Section 3: Systemic Bacteriology
Table 25.1 Characteristics and clinical significance of important streptococci and enterococci
Species Lancefield Hemolysis Natural habitat Associated diseases Laboratory tests
group
Str. pyogenes A Beta Throat, skin Pharyngitis, scarlet fever, Bacitracin sensitive;
pyoderma, erysipelas, PYR test positive;
cellulitis, necrotizing fasciitis, Ribose not
streptococcal toxic shock fermented
syndrome, bacteremia,
rheumatic fever, glomerulo
nephritis
Str. agalactiae B Beta Female genital Neonatal sepsis, meningitis, Hippurate
tract, rectum puerperal fever, pyogenic hydrolysis, CAMP
infections test,
S. equisimilis C Beta Throat Pharyngitis, endocarditis Ribose and
trehalose
fermentation
Enterococcus sp. Group D Variable Gastrointestinal Urinary tract infections, Growth in 6.5%
(Enterococcus hemolysis tract, oral cavity, endocarditis, bacteremia, NaCI; PYR positive
fecalis and other gallbladder, abdominal infections
enterococci) urethra, and
vagina
Nonenterococcal Group D Alpha- Gastrointestinal Neonatal meningitis No growth in 6.5%
Group D species hemolytic or tract NaCI
(Streptococcus nonhemolytic
bovis)
Str. anginosus A, C, F, G, Beta (alpha, Throat, colon, Pyogenic infections Group A strains
group untypable gamma) female genital bacitracin-resistant
tract PYR negative
colony variants of
other groups
Viridans Not typed Alpha Mouth, throat, Dental caries; endocarditis Optochin
streptococci (Str. (gamma) colon, female resistant, species
mitis, Str. mutans, genital tract classification
Str. salivarious, on biochemical
and many other properties
species)
www.ebook3000.com
176 | Section 3: Systemic Bacteriology
exotoxin (SPE). This toxin was originally called 5. Serum opacity factor (SOF)
‘erythrogenic’ toxins because its intradermal The exact biological significance is not known it is
injection into susceptible individuals produced an produced mainly by strains causing skin infections.
erythematous reaction (Dick test, 1924). Antitoxin
6. Nicotinamide adenine dinucleotidase (NADase)
injected into the skin of a patient with scarlet fever
It is believed to be leukotoxic.
causes localized blanching as a result of neutral
ization of erythrogenic toxin (Schultz-Charlton 7. Other enzymes
reaction). Many strains of streptococci also produce ATPase,
Three immunologically distinct types heat- phosphatase, esterases, amylase, N-acetylglucos
labile toxins (SPE A, B, and C) have been described in aminidase, neuraminidase. and other toxins or
S. pyogenes. Type A and C are encoded by enzymes.
lysogenic phages; the gene for B is located on the
bacterial chromosome. SPEs act as ‘superantigens’ Epidemiology
interacting with both macrophages and helper T Group A streptococci commonly colonize the
cells with the release of inflammatery cytokines. oropharynx of healthy children and young adults.
These cytokines cause important effects, including Person-to-person spread is by respiratory droplets
the fever, shock, the organ failure and the rash (pharyngitis) or through breaks in skin after direct
observed in patients with scarlet fever. contact with infected person, fomite, or arthropod
vector. Although the organism is ubiquitous, there
B. Enzymes are seasonal incidences of specific diseases.
Pharyngitis due to S. pyogenes is primarily a
1. Deoxyribonucleases (streptodornase DNAase) disease of childhood between the age of 5 and 15
These enzymes are capable of depolymerizing the years, but. Crowding, such as in classrooms and
highly viscous DNA which accummlates in thick pus daycare facilities, increase the opportunity for the
as a result of disintegration of polymorphonuclear organism to spread. No seasonal distribution has
leukocytes. Streptodornase helps to liquefy the been identified in the tropics. Immunity is type
thick pus and may be responsible for the thin serous specific and appears to be associated with antibody
character of streptococcal exudates. This property to the M protein. Reinfections occur because of the
has been applied therapeutically for breaking down multiplicity of the serotypes.
blood clots, thick pus and fibrinous exudates in
closed spaces such as joints or pleural cavity. Pathogenesis
There are four antigenically distinct nucleases
Acute diseases associated with Streptococcus
(A, B, C and D), of which B is the most antigenic in
pyogenes occur chiefly in the respiratory tract,
human beings. Antibody titers to DNase B are of
bloodstream, or the skin. Two post streptococcal
great value in the serodiagnosis of pharyngeal or
sequelae (rheumatic fever following respiratory
skin infection, especially the latter, where the ASO
infection and glomerulonephritis following
titer may be low.
respiratory or skin infection), occur in 1–3% of
2. Streptokinase (Fibrinolysin) untreated infections.
Streptokinase, also known as fibrinolysin, is
another spreading factor. This acts on plasminogen, A. Suppurative Streptococcal Disease
a factor present in normal plasma, which is 1. Respiratory Infections
converted into plasmin, an active proteolytic i. Sore throat is the most common of
enzyme that lyses fibrin. Fibrinolysin appears to streptococcal disease. It may be localized as
play a biological role in streptococcal infections by tonsillitis or may involve the pharynx more
breaking down the fibrin barrier around the lesions diffusely (pharyngitis). Tonsillitis is more
and facilitates the spread of infection. common in older children and adults than in
An antigenic protein and neutralizing anti younger children.
bodies appear in convalescent sera which provide ii. Scarlet fever : It is a complication of
retrospective evidence of streptococcal infection. streptococcal pharyngitis that occurs when
the infecting strain is lys ogenized by a
3. Hyaluronidase
temperate bacteriophage that stimulates
Hyaluronidase splits hyaluronic acid, and might
production of a pyrogenic exotoxin.
favor the spread of streptococcal infection along
iii. Suppurative complications: The infection
the intercellular spaces.
may spread to the surrounding tissues which
4. Proteinase may cause suppurative complications of
It destroys several proteins formed by the Strepto streptococcal pharyngitis, such as periton
coccus itself. sillar or retropharyngeal abscess, otitis
Chapter 25: Streptococcus and Enterococcus | 177
media, mastoitidis, quinsy, Ludwig’s angina, Table 25.2 Distinguishing features of rheumatic
suppurative adenitis and disseminated fever and glomerulonephritis
infections to brain, heart, bone, and joints.
Feature Acute Acute
2. Skin and Soft Tissue Infections rheumatic glomerulonephritis
Str. pyogenes causes a variety of suppurative fever
infections of the skin, including infection of Primary site of Throat Throat or skin
wounds or burns, with a predilection to produce infection
lymphangitis and cellulitis. Infection of minor Latent period Longer Shorter (1–3 weeks)
abrasions may at times lead to fatal septicemia. (2–5 weeks)
The two typical streptococcal skin infections are Prior Essential Not necessary
erysipelas and impetigo. sensitization
i. Erysipelas: Erysipelas is an acute infection of Repeated Common Absent
the skin. It is a diffuse infection involving the attacks
superficial lymphatics. Erysipelas occurs most Genetic Present Not known
commonly in young children or older adults. susceptibility
ii. Pyoderma (impetigo): Pyoderma (impetigo) Serotype of Any Pyodermal types 49,
is seen primarily in young children. Group Str. pyogenes 53–55, 59–61 and
A streptococci causing impetigo are pharyngitis strains 1
frequently nephritogenic, that leads to acute and 12
glomerulonephritis. Immune Marked Moderate
For retrospective diagnosis of pyoderma response
antecendent to acute glomerulonephritis, Complement Unaffected Lowered
antibody to DNAase B and hyaluronidase are level
more useful. Course Progressive or Not indicated
iii. Cellulitis: Typically involves the skin and static
deeper subcutaneous tissues. Prognosis Variable Good
iv. Necrotizing fascitis (Streptococcal gangrene). Penicillin Essential Not indicated
v. Streptococcal Toxic Shock Syndrome. prophylaxis
www.ebook3000.com
178 | Section 3: Systemic Bacteriology
5. Circulating immune complexes have been D. Identification
found in the serum of patients with acute Colonies of S. pyogenes on SBA are small,
poststreptococcal glomerulonephritis. transparent, and smooth with a well-defined area
of β-hemolysis. A Gram stain will reveal gram-
Laboratory diagnosis positive cocci with some short chains. Hemolytic
streptococci are grouped by the Lancefield
In acute infections, diagnosis is established by
technique using serologic methods, or biochemical
the isolation and identification of β-streptococci
tests can be performed. The fluorescent antibody
from the patient, while in non-suppurative
technique has been employed for the rapid
complications, diagnosis is based mainly on the
identification of group A streptococci. A key test
examination of the patient’s serum for a rising titer
that should be done is bacitracin susceptibility
of antibody to one or more streptococcal antigens.
or PYR hydrolysis.
Bacitracin susceptibility: S. pyogenes is more
1. Acute Suppurative Infections
sensitive to bacitracin than most other streptococci,
A. Specimens A disk containing 0.04 units of bacitracin is applied
Throat and nose swabs, high vaginal swabs, pus on the surface of an inoculated blood culture plate.
or pus swabs are the usual specimens collected. S. pyogenes should show a large inhibition zone
Serum is obtained for antibody demonstration. (e.g. >15 mm diameter) and most other streptococci
should show little or no inhibition. S. pyogenes is
susceptible to bacitracin and hydrolyzes PYR,
B. Microscopy
whereas the other β-hemolytic groups are resistant
Presumptive information may be obtained by an to bacitracin and are PYR negative.
examination of Gram stained films from pus and Typing of Str. pyogenes is required for epidemio
CSF. The observation of typical gram-positive logical in specialized reference laboratories.
cocci in chains may indicate the likelihood of the
presence of streptococcal infection. In contrast, E. Antigen Detection
smears are of no value in infections of throat or
genitalia, where streptococci may form part of part Detection of Str. pyogenes is done directly in throat
of the resident flora and has a poor predictive value. swabs without cultivation. Enzyme immunoassay
(ElA) or agglutination tests are used to demonstrate
the presence of the antigen.
C. Culture
For culture, swabs should be collected under vision 2. Non-suppurative Complications
from the affected site and either plated immediately Serological Tests
or if there is likely to be delay, swab should be sent
to the laboratory in Pike’s medium (blood agar Detection of antibodies against antigens of Str.
containing 1 in 1,000,000 crystal violet and in 1 in pyogenes is an important means of establishing
16,000 sodium azide). The specimen is plated on the diagnosis of poststreptococcal rheumatic
blood agar and incubated at 37°C anaerobically or fever and glomerulonephritis. Some immunologic
under 5–10% CO2, as hemolysis develops better tests used to detect past infection with S. pyogenes
und er these conditions. Sheep blood agar is include ASO, anti-DNase, antistreptokinase, and
recommended for primary isolation because it is antihyaluronidase titers.
inhibitory for Haemophilus haemolyticus, colonies Antistreptolysin O (ASO) test is used most
of which may be confused with those of hemolytic frequently. ASO titers higher than 200 Todd units/
streptococci but their hemolysis is stronger on mL are indicative of prior streptococcal infection.
aerobic than on anaerobic plates. High levels are usually found in acute rheumatic
Crystal violet blood agar and PNF medium fever but in glomerulonephritis, titers are often low.
are selective media that inhibit many throat Antideoxyribonuclease B (antiD Nase B)
commensal bacteria and may facilitate the estimation is also commonly employed. Titers
detection of small numbers of S. pyogenes in throat higher than 300 or 350 are taken as significant.
swabs.They are rarely used for routine culture. S. AntiDNase B and antihyaluronidase (ASH) tests
pyogenes does not grow on MacConkey’s bile-salt are very useful for the retrospective diagnosis of
medium. streptococcal pyoderma, for which ASO is of much
For isolating group A streptococci from throat less value.
swabs the most common medium is blood agar Commercial products are available for detection
supplemented with antibiotics trimethoprim/ of antistreptococcal antibodies. Streptozyme
sulfamethoxazole to suppress the groth of normal test detects a mixture of antibodies.It is a passive
flora. slide hemagglutination test using erythrocytes
Chapter 25: Streptococcus and Enterococcus | 179
sensitized with a crude preparation of extracellular Other group B infections in neonates: Osteomy
antigens of streptococci, is a convenient, sensitive elitis, conjunctivitis, sinusitis, otitis media,
and specific screening test. It becomes positive endocarditis and peritonitis may also occur.
after nearly all types of streptococcal infections,
2. Infections in the adult: Puerperal sepsis,
whether of the throat or the skin.
pneumonia endometritis, urinary tract infection,
wound infection, and bacteremia.
Treatment
S. pyogenes is very sensitive to penicillin and Identification
penicillin resistance has not yet been observed in Pre sumptive identification method is based on
S. pyogenes. Erythromycin or an oral cephalosporin their ability to hydrolyze hippurate. They may be
can be used in patients with a history of penicillin identified by the CAMP reaction (Christie, Atkins
allergy. Adequate treatment during acute infection and Munch-Peterson), which can be demonstrated
prevents the complications of acute rhematic fever. as an accentuated zone of hemolysis (arrowhead-
shaped area of enhanced hemolysis) when Str.
agalactiae is inoculated perpendicular to a streak
Prophylaxis of Staph. aureus grown on blood agar (Fig. 25.4). S.
Patients with a history of rheumatic fever require agalactiae produces a CAMP factor that enhances
long-term antibiotic prophylaxis to prevent the lysis of sheep red cells by staphylococcal β-lysin.
recurrence of the disease. Those persons who have Group B streptococci are identified definitively by the
recovered from ARF are given oral penicillin for many demonstration of the group-specific carbohydrate
years to prevent recurrence. Antimicrobial drugs or the use of commercially prepared molecular
have no effect on established cases of AGN and ARF. probes. Occasional strains are bacitracin sensitive.
Clinical Diseases
1. Infection in the neonate
A. Early-onset disease : It develops during the
first week of life and disease is characterized by
bacteremia, pneumonia, or meninigitis and is
often fatal.
B. Late-onset disease: It develops between
second and twelfth weeks of life. The predominant
manifestation is bacteremia with meningitis, but
septic arthritis. Fig. 25.4: CAMP reaction
www.ebook3000.com
180 | Section 3: Systemic Bacteriology
throat, pneumonia, septicemia, endocarditis, and media and on MacConkey agar, on which it forms
bone, joint, skin and wound infections. small (0.5–1 mm), usually magenta-colored
colonies.
Group D Streptococci
Distinctive Features of Enterococci
Until the mid-1980s, the group D streptococci were
The enterococci possess several distinctive
divided into the two groups:
features separting them from sreptocooci: The
1. Enterococcus group (enterococci or fecal
enterococci grow in the presence of 6.5% NaCl,
streptococci) which have been reclassified as
40% bile, at ph 9.6, at 45°C and in 0.1% methelyne
a separate genus called Enterococcus.
blue. It survives heating at 60°C for 30 min, a
2. Nonenterococcal group, for example, Str. bovis,
feature distinguishing it from streptococci, and
Str. equinus.
also grows within a wider range of temperatures
(10–45°C). On MacConkey medium, they produce
Enterococcus deep pink colonies. Enterococci are PYRase test
The enterococci (“enteric cocci”) were previously positive. They do not hydrolyze hippurate.
classified as group D streptococci (Table 25.3). The
enterococci were reclassified into the new genus Identification
Enterococcus, and there are currently 16 species The identification of Enterococcus species is made
in this genus. on biochemical characteristics. E. aecalis can
be identified by its ability to ferment mannitol,
Species sucrose, sorbitol and aesculin, and to grow on
Enterococcus faecalis (“pertaining to feces”) is tellurite blood agar producing black colonies with
the Enterococcus most often isolated from human gas production. It is VP positive.
sources.
Enterococcus faecium (“of feces”). Clinical Infections
Other species: E. durans, E. avium, E. casseliflavus, The enterococci inhabit the gasterointestinal
E. gallinarum, and E. raffinosus are observed tract and the genitourinary tract in humans and
occasionally. other animals. Enterococci are frequent causes of
nosocomial infections and may cause urinary tract
Characteristics of Enterococci infection, bacteremia, infective endocarditis,
The enterococci are gram-positive cocci typically biliary tract infection, intra-abdominal abscess
arranged in pairs and short chains, and is nonmotile complicating diverticulitis, peritonitis and
and noncapsulate. The cocci are facultatively wound infection.
anaerobic and grow optimally at 35°C, although
most isolates can grow in the temperature range Treatment
10°C to 45°C. They grow readily on blood agar Most strains of enterococci are resistant to
media, with large, white colonies appearing penicillin. They are also resistant to sulfonamides.
after 24 hours of incubation; the colonies are Recently they have developed resistance to newer
typically nonhemolytic but can be α-hemolytic or penicillins and cephalosporins, streptomycin and
β-hemolytic. It grows readily on ordinary nutrient gentamicin.
www.ebook3000.com
182 | Section 3: Systemic Bacteriology
Multiple choice questions (MCQs) 4. Which of the following is selective medium for
Streptococcus pyogenes?
1. Which type of hemolysis is produced by Strepto a. Blood agar
coccus pyogenes on blood agar?
b. Crystal violet blood agar
a. Alpha hemolysis b. Beta hemolysis
c. Gamma hemolysis d. None of the above c. Potassium tellurite blood agar
2. Group-specific antigen extraction of Streptococcus d. Chocolate agar
pyogenes by treating with hydrochloric acid 5. Susceptibility to bacitracin can be used to identify:
method is known as
a. Streptococcus pyogenes
a. Lancefield’s method b. Fuller’s method
b. Streptococcus viridans
c. Maxted’s method d. Randall’s method
3. Erythrogenic toxin is responsible for: c. Streptococcus mitis
a. Pyoderma d. Streptococcus agalactiae
b. Schultz-Charlton reaction
c. Necrotising facititis Answers (MCQs)
d. All of the above 1. b; 2. a; 3. b; 4. b; 5; a
26
Chapter
Pneumococcus (Diplococcus pneumoniae:
Streptococcus pneumoniae)
Learning Objectives
After reading and studying this chapter, you should ∙∙ Explain C-reactive protein
be able to: ∙∙ Discuss laboratory diagnosis of pneumococcal
∙∙ Describe morphology and cultural characters of infections
pneumococci ∙∙ Differentiate between Str. pneumoniae and Str.
∙∙ Describe Quellung reaction viridans.
Pneumococcus
(Diplococcus Pneumoniae,
Streptococcus Pneumoniae)
Morphology
Pneumococci are gram-positive cocci in pairs Fig. 26.1: Str. pneumoniae in pus
(diplococci). The cocci are about 1 μm, slightly
elongated cocci, with one end broad or rounded
and the other pointed, presenting a flame-shaped
or lanceolate appearance (Fig. 26.1). They are
nonmotile and nonsporing.
All freshly isolated strains are capsulate. The
capsule encloses each pair. The capsule may
be demonstrated as a clear halo in Indian ink
preparations (Fig. 26.2) or may be stained directly
by special techniques or by use of homologous
type-specific antibody in the Quellung reaction.
Cultural Characteristics
They are aerobes and facultative anaerobes. It Fig. 26.2: Pneumococci. Indian ink preparation to show
grows best in air or hydrogen with 5–10% CO2 , capsules
www.ebook3000.com
184 | Section 3: Systemic Bacteriology
in enriched media. It grows on ordinary media, but Optochin Sensitivity: Pneumococci are highly
better on media with serum, blood or heated blood. sensitive to killing by optochin (ethyl hydrocuprein
On blood agar, after incubation for 18 hours, hydrochloride), and is useful in distinguishing
the colonies are small (0.5–1 mm), dome-shaped on an area of a blood agar plate inoculated with
and glistening, with an area of green discoloration pneumococcus-like colonies from the primary
(alpha hemolysis) around them. On further diagnostic plate. A growth of Pneumococcus will
incubation, the colonies become flat with raised be inhibited in a zone extending radially for at least
edges and depressed centrally, so that concentric 5 mm from the margin of the disc on incubation.
rings are seen on the surface when viewed from Viridans streptococci will grow right up to the disc.
above (draughtsman or carrom coin appearance)
which is due to autolysis of bacteria within the flat Antigenic Structure
pneumococcal colonies.
1. Capsular Antigens
Under anaerobic conditions, however, a zone of
beta hemolysis is produced around the colony by The most important antigen of the Pneumococcus
an oxygenlabile pneumolysin O. In liquid media is the type specific capsular polysaccharide. It is
such as glucose broth, growth occurs as uniform also called the ‘specific soluble substance’ (SSS)
turbidity. The cocci readily undergo autolysis in as this polysaccharide diffuses into the culture
cultures due to the activity of intracellular enzymes. medium or infective exudates and tissues. These
Autolysis is enhanced by bile salts, sodium lauryl polysaccharides are antigenic and form the basis
sulfate and other surface active agents. for the separation of pneumococci into different
serotypes. A total of 90 different capsular serotypes
have been identified. The serotypes are designated
Biochemical Reactions
by numbers, and those that are structurally related
1. Inulin fermentation: Pneumococci ferment are grouped together (I, 2, 3. 4, 5, 6A, 68, etc.).
several sugars with the production of acid and The tests may be done by :
no gas. Fermentation of inulin by pneumococci 1. Agglutination of washed capsulate cocci.
is a useful test for differentiating them from 2. Precipitation of SSS from culture supernates.
streptococci as the latter do not ferment it. 3. Quellung reaction or capsule swelling reaction
Fermentation is tested in Hiss’s serum water It was described by Neufeld (1902). In the
or serum agar slopes. capsule swelling or ‘quellung’ reaction (quellung
2. Bile solubility test: = swelling), a suspension of pneumococci is mixed
Basis: Bile solubility test for identifying on a slide with a drop of the type specific antiserum
pneum ococci is based on the presence in and a loopful of methylene blue solution and then
pneumococci, of an autolytic amidase that examined using the oil-immersion objective. The
cleaves the bond between alanine and muramic capsule becomes apparently swollen, sharply
acid in the peptidog lycan. The amidase is delineated and refractile in the presence of the
activated by surface-active agents such as bile homologous antiserum. This reaction can be used
or bile salts, resulting in lysis of the organisms. to identify the organism directly from ‘sputum, CSF,
Procedure: When sodium deoxyc holate and other sources. It used to be a routine bedside
solution is added to broth culture, the culture procedure in the past.
clears due to lysis of cocci. Pneumococci are
soluble in bile; viridans and other streptococci 2. Somatic Antigen
are not.
a. C polysaccharide: The cell wall of S. pneumoniae
Alternatively, touch a suspected pneumo
contains a species specific carbohydrate
coccal colony with a loopful of 2% sodium
antigen, referred to as C substance. C-reactive
deoxycholate solution, it disappears, leaving
protein (CRP) is an abnormal protein (beta
an area of α-hemolysis on the blood agar.
globulin) that precipitates with the somatic ‘C’
3. Pneumococci are catalase and oxidase
antigen of pneumococci. It appears in acute
negative.
phase sera of cases of pneumonia but dis
appears during convalescence. It is known as
Resistance C-reactive protein because it precipitates with
Pneumococci are delicate organisms and are killed C antigen of pneumococci. CRP is present in
by moist heat at 55°C in 10 min, and readily by most low concentrations in healthy people but in
disinfectants. Most strains are highly sensitive to elevated concentrations in patients with acute
benzylpenicillin, other penicillins. A drug resistant inflammatory diseases.
Strep. pneumoniae (DRSP) strain originating in It is an ‘acute phase’ substance, produced
Spain has spread to most parts of the world posing in hepatocytes. Its production is stimulated by
problems in treament. bacterial infections, inflammation, malignancies
Chapter 26: Pneumococcus (Diplococcus pneumoniae: Streptococcus Pneumoniae) | 185
and tissue destruction. It disappears when the pneumococcal pneumonia. In children, types
inflammatory reactions subside. It is used as 6, 14, 19 and 23 are frequent causes.
an index of response to treatment in rheumatic ii. Bronchopneumonia: It is almost always a
fever and certain other conditions. secondary infection. This may be caused by
CRP testing, by passive agglutination using any serotype of Pneumococcus. Other causative
latex particles coated with anti-CRP antibody agents responsible for bronchopneumonia
is a routine diagnostic procedure. include Staph. aureus, K. pneumoniae, Str.
b. F antigen: The lipid bound teichoic acid in the pyogenes, H. influenzae, Fusobacterium species
bacterial cytoplasmic membrane is called the and Bacteroides.
F or Forssman antigen because it can cross-
react with the Forssman surface antigens on 2. Acute Exacerbations in Chronic Bronchitis
mammalian cells. Pneumococci are commonly associated with
c. M protein: Type-specific protein antigens the acute exacerbations in chronic bronchitis.
analogous to the M protein of Streptococcus Another bacterium commonly associated with this
pyogenes. condition is Haemophilus influenzae.
www.ebook3000.com
186 | Section 3: Systemic Bacteriology
3. Microscopy and Antigen Detection Table 26.1 Differential characters of pneumococci
A centrifuged deposit of the CSF should be and viridans streptococci
examined immediately in a Gram film in case of Character Pneumococcus Viridans
meningitis and presumptive diagnosis may be streptococci
made by finding gram-positive diplococci both Morphology Ovoid or Short or long
inside the polymorphs and extracellularly. lanceolate chains of
Pneumococcal antigen is often detectable by diplococci; some rounded cocci
short chains
coagglutination (COA), latex agglutination (LA) or
counterimmunoelectrophoresis (CIE) and ELISA. Capsule Present Usually absent
In addition to CSF, capsular polysaccharide Colonies Become Convex
can be demonstrated in the blood and urine by flattened
or draughtsman
counterimmunoelectrophoresis
Effect on Narrow zones of Wide or narrow
blood agar a-hemolysis zone of
4. Capsule Swelling Tests a-hemolysis
If typing sera are available, the most simple, rapid, Optochin Sensitive Resistant
and accurate method for the identification of sensitivity
pneumococci by direct examination is the quellung Bile solubility + –
reaction. Inulin + –
fermentation
5. Culture Virulence in mice + –
Specimen is inoculated on plates of blood agar and
heated-blood agar incubated in air with 5–10%
CO2 for 18–24 hours. Typical colonies develop Prophylaxis
with α-hemolysis. The colonies are small (0.5–1 Immunity is type-specific and associated
mm), dome-shaped and glistening, with an area with antibody to the capsular polysaccharide.
of green discoloration (alpha hemolysis) around The existence of some 90 serotypes makes a
them. On further incubation, the colonies have complete polyvalent vaccine impracticable. The
draughtsman or carrom coin appearance. current vaccine contains 23 different capsular
polysaccharides which is stated to give 80–90%
6. Identification protection. The vaccine is immunogenic in normal
Procedures commonly used to distinguish S. adults, and the immunity is long-lived. It is not
pneumoniae from the viridans streptococci are meant for only in persons at enhanced risk of
optochin susceptibility, bile solubility, and the pneumococcal infection, such as patients with
quellung reaction. S. pneumoniae is susceptible to absent or dysfunctional spleen, sickle cell disease,
optochin, whereas other α-hemolytic species are celiac disease, chronic renal, lung, heart and liver
resistant (Table 26.1). diseases, diabetes mellitus and immunodeficien
cies, including human immunodeficiency virus
7. Intraperitoneal Injection into Mice (HIV) infection and renal transplant.
Vaccination is contraindicated in young
Isolation may be obtained by intrap eritoneal
children and elderly with lymphoreticular malig
inoculation in mice from specimens where
nancies and immunosuppressive therapy.
pneumococci are expected to be scanty. Inoculated
mice die in 1–3 days, and pneumococci may be
demonstrated in the peritoneal exudate and heart Treatment
blood. Penicillin is the drug of choice for susceptible
strains, alt hough resistance is increasingly
8. Blood Culture common. Cephalosporins, er ythromycin,
chloramphenicol, or vancomycin are used for
In the acute stage of pneumonia, the organism may patients allergic to penicillin or for treatment of
be obtained from blood culture in glucose broth. penicillin-resistant strains.
The finding of pneumococci in the blood is much
better evidence of their pathogenic role in the Key Points
lung than is their finding in sputum. Isolation of Streptococcus pneumoniae are elongated or
pneumococci from blood indicates bad prognosis. “lancet-shaped,” gram-positive cocci arranged in
pairs (diplococci). They are capsulated
The Pneumococcus has complex nutritional
9. Antibiotic Sensitivity Test
requirements. On blood agar, the colonies are small
It is especially useful in strains which are resistant.
Chapter 26: Pneumococcus (Diplococcus pneumoniae: Streptococcus Pneumoniae) | 187
www.ebook3000.com
27
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe morphology, culture characteristics,
be able to: biochemical reactions of Neisseria gonorrhoeae
∙∙ Describe morphology, culture characteristics, ∙∙ Discuss pathogenicity of N. gonorrhoeae
biochemical reactions and antigenic structure of ∙∙ Discuss laboratory diagnosis of gonorrhoea
Neisseria meningitidis ∙∙ Explain nongonococcal urethritis (or) nonspecific
∙∙ Discuss pathogenicity and lab diagnosis of urethritis
meningococcal meningitis ∙∙ Describe Morexella (Branhamella) catarrhalis.
Neisseria meningitidis
(Meningococcus; Diplococcus
intracellularis meningitidis)
Morphology
Meningococci are gram-negative oval or spherical
cocci (0.6–0.8 μm in size), typically arranged
in pairs, with the adjacent sides flattened or
concave opposing edges and the long axes parallel.
They are typically seen in large numbers inside
polymorphonuclear leukocytes (Fig. 27.1).
Most fresh isolates are capsulated. They are Fig. 27.1: Neisseria meningitidis in cerebrospinal fluid.
nonsporing and nonmotile. Inset—enlarged view showing flat adjacent side of cocci
Chapter 27: Neisseria and Moraxella | 189
culturing meningococci. Modified Thayer -Martin Stages of Meningococcal Infections
(with vancomycin, colistin and nystatin) is a useful There are three stages.
selective medium.
On blood agar after 24 hours incubation, First Stage—Nasopharyngeal Infection
colonies are 1–2 mm in diameter, round, convex, The organisms appear in nasopharynx leading
gray, translucent and nonhemolytic. Heated blood to nasopharyngeal infection, which is usually
(chocolate) agar-colonies are slightly larger on asymptomatic.
heated blood (chocolate) agar than on ordinary
blood agar. Growth is poor in liquid media, producing Second Stage—Meningococcal Septicemia
a granular turbidity with little or no surface growth. In a small percentage of cases, the meningococci enter
the bloodstream from the posterior nasopharynx.
This stage is called meningococcemia. The patient
Biochemical Reactions
develops fever, malaise and petechial skin lesions.
1. They are catalase and oxidase positive. The organisms may also cause lesions in the
Oxidase test: When 1% solution of oxidase joints and lungs and rarely cause massive bilateral
reagent (tetramethylparaphenylene-diamine- hemorrhages in the adrenals (Waterhouse–
dihydrochloride) is poured on culture media, Friderichsen syndrome). It is an overwhelming
Neisseria colonies quickly turn deep-purple. and usually fatal condition, characterized by shock,
This prompt oxidase reaction helps in the disseminated intravascular coagulation (DIC)
identification of meningococci and gonococci and multisystem failure. Meningococcal disease is
in mixed cultures. favored by deficiency of the terminal complement
The test may also be performed by rubbing components (C5–C9).
a little of the growth with a loop on a strip of
filter paper moistened with the oxidase reagent Third Stage—Meningitis
(Kovacs’ method). A deep purple color appears In the third stage of meningococcal infection, the
immediately. organisms can cross the blood–brain barrier and
2. Sugar fermentation: Meningococci ferment infect the meninges. The route of spread from the
glucose and maltose with the production of nasopharynx to the meninges is controversial. The
acid but no gas, but not lactose or sucrose spread may be directly along the perineural sheath
(gonococci ferment glucose but not maltose). of the olfactory nerve, through the cribriform plate
to the subarachnoid space, or more probably,
3. Indole and hydrogen sulfide are not produced
through the bloodstream. In certain cases, the site of
and nitrates are not reduced.
entry of the Meningococcus may be the conjunctiva.
On reaching the central nervous system, a
Antigenic Classification suppurative lesion of the meninges is set up. Case
fatality is variable but in untreated cases may be as
Based on their capsular polysaccharide antigens,
high as 80%. Survivors may have sequelae such as
meningococci are classified into at least 13
blindness and deafness.
serogroups: A, B, C, X, Y, Z, Zl (29E) and W135.
The pathogenic agent in meningococcal disease
Further serogroups H, I, K and L have also been
appears to be the endotoxin (LPS) released by
described. A group D was described but no
autolysis. The vascular endothelium is particularly
capsular polysaccharide specific for this group has
sensitive to the endotoxin.
yet been demonstrated. Groups A, B and C are the
most important. Serogroups are further classified Epidemiology
into serotypes and subtypes.
Humans are the only natural carriers for N. menin
gitidis. The oral and nasopharyngeal carriage rates are
Resistance highest for school-aged children and young adults,
are higher in lower socioeconomic populations.
Meningococci are very delicate organisms, being
The carrier rate is higher in the members of the
highly susceptible to heat, dessication, alterations
household of a patient with meningococcal disease.
in pH and to disinfectants. They die within a few
Natural infection is limited to human beings.
days at room temperature. They are killed by
heating at 55°C in 5 minutes. Laboratory Diagnosis
1. Specimens
Pathogenicity
i. Cerebrospinal fluid (CSF),
Meningococci are strict human parasites ii. Blood for culture (which may come from a
inhabiting the nasopharynx. Infection is usually patient with meningitis, a hemorrhagic rash
asymptomatic. or pyrexia of uncertain origin)
www.ebook3000.com
190 | Section 3: Systemic Bacteriology
iii. Aspirate from skin lesions or pus from an The oxidase test is performed on colonies on
infected joint, solid medium.
vi. Throat or nasopharyngeal swabs from vi. Antibiotic sensitivity tests
suspected cases. Swabs should be transported Set up antibiotic sensitivity tests.
in Stuart’s transport medium. All speci vii. Serogrouping is performed by slide agglutina
mens where meningococcal infection is tion with hyperimmune sera that provide
suspected must be submitted to the laboratory important epidemiological information.
immediately.
3. Blood Cultures
2. Examination of CSF Blood culture is often positive in meningococcemia
If meningitis is suspected, a lumbar puncture and in early cases of meningitis. Cultures should
should be performed as soon as possible unless be incubated for 4–7 days, with daily subcultures.
there are signs of raised intracranial pressure. Subculture to blood agar and heated blood agar.
i. Perform a cell count. The exudate in mening Incubate cultures in 5–10% CO 2 for 24 hours
ococcal meningitis is typically polymor and examine oxidase-positive colonies of gram-
phonuclear. negative diplococci as above.
ii. Centrifuge the remaining CSF. Make a
smear of the centrifuged deposit and stain 4. Pus, Aspirates and Swabs
with Gram-stain. CSF from a typical case of Gram-stained films are examined. In addition
meningococcal meningitis will show gram- to blood agar and heated blood agar, Thayer–
negative diplococci inside a limited proportion Martin selective medium is used for the culture of
of the pus cells; many are extracellular. materials expected to yield a mixture of organisms
Stain a second film with methylene blue to such as pus, aspirates, and throat, nasopharyngeal
determine the cell type; occasionally, diplococci and genital swabs.
may be seen more easily with this stain.
If fluorescein isothiocyanate coupled
5. Petechial Lesions
antiserum is available, a smear of the deposit
may be examined for the direct identification Meningococci may sometimes be demonstrated in
of the meningococcal serogroup responsible petechial lesions by microscopy and culture.
for infection.
iii. Divide the supernatant CSF into two aliquots: 6. Serological Diagnosis
One to be kept if necessary for biochemical Paired sera may be tested for the presence of
examination, the other to be examined for the complement-fixing antibodies.
presence of meningococcal polysaccharide Specific antibodies to capsular polysaccharide
antigen by counterimmunoelectrophoresis, may be demonstrated by a (i) hemagglutination
latex agglutination or coagglutination using test (ii) ELISA tests.
meningococcal antisera. Similar tests are also
available for pneumococcus, H. influenzae 7. Polymerase Chain Reaction (PCR)
type b and group B Streptococcus antigens. Group specific diagnosis of infection can be made
Antigen detection is particularly useful in by detection of meningococcal DNA sequence in
partially treated patients in whom smear and CSF or blood by PCR amplification.
culture tests may be negative.
iv. Cuture: Plate out the centrifuged deposit on Treatment
both blood and heated blood agar (chocolate
agar) and incubate at 37°C in 5–10% CO2. Penicillin is currently the antibiotic of choice.
Colonies appear after 18–24 hours which Chloramphenicol is effective, but risks of blood
may be identified by morphology and dyscrasia have limited its use. Either chloramphenicol
biochemical reactions. or a third-generation cephalosporin, such as
Subculture: Add Robertson’s cooked meat cefotaxime or ceftriaxone is used in persons allergic
broth to the remaining deposit, incubate to penicillin allergy.
overnight and subculture in the same way. In At the end of a course of therapy with penicillin,
the absence of visible meningococci, glucose it is important to give eradicative treatment with
broth may be added to the remaining sed rifampicin or ciprofloxacin.
iment of the centrifuged deposit to facilitate
the isolation. Prophylaxis
v. Biochemical reactions 1. Chemoprophylaxis: Minocycline and rifampin
Sugar utilization tests or commercial kits are have been used effectively for antibiotic-
used to identify any gram-negative diplococci. mediated chemoprophylaxis. Ciprofloxacin is
Chapter 27: Neisseria and Moraxella | 191
widely used as a prophylactic for adolescents round, translucent, convex or slightly umbonate,
and adults as a single, oral dose. with finely granular surface and lobate margins
2. Immunoprophylaxis: A polyvalent vaccine after incubation for 24 hours..They are soft and
effective against serogroups A, C, Y, and W135, easily emulsifiable. After 48 hours, the colonies are
which can be administered to children older larger (1.5–2.5 mm), sometimes with a crenated
than 2 years, has been developed. The vaccine margin and an opaque raised center.
cannot be administered to children in younger Types of gonococci: Kellogg divided gonococci
age groups because they do not respond to into four types (T1–T4) on the basis of colonial
polysaccharide antigens. The immunity is appearance, auto-agglutinability and virulence.
group specific. There is no Group B vaccine
available at present. Types 1 and 2 form small brown colonies and bear
numerous fimbriae (piliated types P1 and P2). They
are auto-agglutinable and virulent.
Neisseria gonorrhoeae
(gonococcus) Types T3 and T4 are nonpiliated (P–), form smooth
suspensions and are avirulent.
N. gonorrhoeae causes the venereal disease Tl and T2 types are also known as P+ and P++,
gonorrhea. Gonococci resemble meningococci respectively, while T3 and T4 are known as P–.
very closely in many properties.
Biochemical Reactions
Morphology
Gonococcus is oxidase positive and resembles
Morphology and staining of N. gonorrhoeae are mening ococci except in the fermentation of
identical to those of N. meningitidis. In smears maltose. Gonococci ferment only glucose and
from the urethral discharge in acute gonorrhea, not maltose and neither species ferments lactose
the organism appears as a Diplococcus with the or sucrose. This can be remembered by G for
adjacent sides concave (Fig. 27.2), being typically Gonococcus and M+G for Meningococcus.
kidney-shaped. It is found predominantly within
the polymorphs, some cells containing as many
Antigenic Structure
as a hundred cocci.
The structure of N. gonorrhoeae is typical of gram-
Cultural Characterstics negative bacteria. The surface structures include
Gonococci are more diffic ult to grow than the following:
meningococci They are aerobic but may grow 1. Pili: Pili are hair-like appendages that extend
anaerobically also. Growth occurs best at pH 7.0– up to several micrometers from the gonococcal
7.4 and at a temperature of 35–36°C. It is essential surface. They act as virulence factors by
to provide 5–10% CO2. promoting attachment to host cells and
They grow well on chocolate agar and Mueller– inhibiting phagocytosis. The pili are composed
Hinton agar. A popular selective medium is of repeating protein subunits (pilins). Pili
the Thayer–Martin medium (chocolate agar undergo antigenic and phase variation.
containing vancomycin, colistin and nystatin) 2. Por proteins (protein I): The Por proteins
which inhibits most contaminants, including (formerly protein I) are porin proteins that form
nonpathogenic Neisseria. Colonies are small, pores or channels in the outer membrane. Two
classes of Por proteins (PorA and PorB), have
been identified. Anyone strain carries only
either IA or IB but not both.
3. Opa protein (protein II): These proteins facili
tate bacterial adherence to each other and to
eukaryotic cells and also for the clumping of
cocci seen in urethral exudate smears.
4. Rmp (protein III): These proteins stimulate
antibodies that block serum bactericidal
activity against N. gonorrhoeae.
5. Lipooligosaccharide (LOS): This antigen
possesses endotoxic activity.
6. Other proteins: Other important gonococcal
proteins are IgA1 protease, β-lactamase,
Fig. 27.2: N. gonorrhoeae in urethral pus. Inset— enlarged which degrades penicillin and Fbp (iron-
view showing diplococci with adjacent surfaces concave binding protein).
www.ebook3000.com
192 | Section 3: Systemic Bacteriology
Resistance Once ver y common, this has been
Gonococcus is a very delicate organism, readily controlled by the practice of instilling 1%
killed by drying, soap and water, and many other silver nitrate solution into the eyes of all
cleansing or antiseptic agents at their correct use- newborn babies (Crede’s method).
dilution. Organisms may remain viable for a day or ii. Vulvovaginitis: In prepubertal girls,
so in pus contaminating linen or other fabrics. In vulvovaginitis may be caused by gonococci.
cultures, the coccus dies in 3–4 days at room tem This occurs either in conditions of poor
perature. Freeze-drying is the most reliable method hygiene or by sexual abuse.
for long-term storage of gonococci but storage at
–70°C or in liquid nitrogen may be more convenient Epidemiology
for intermediate storage. Gonorrhea occurs only in humans. It has no other
known reservoir. N. gonorrhoeae is transmitted
Pathogenesis primarily by sexual contact. A higher incidence of
Gonorrhea gonorrhea has been observed in persons belonging
to blood group B. The basis for this is not known.
Gonorrhea is a venereal disease. The disease is
acquired by sexual contact. The incubation period
is 2–8 days. Laboratory Diagnosis
A. Disease in men: The most common clinical Diagnosis can be established readily in the acute
presentation is acute urethritis in the male. stage but chronic cases sometimes present great
Dysuria and a purulent penile discharge difficulties.
make most sufferers seek treatment rapidly.
The infection extends along the urethra to the 1. Specimens
prostate, seminal vesicles and epididymis. A. Specimens in Men
Chronic urethritis may lead to stricture 1. Urethra: In men, urethral samples usually suffice
formation. The infection may spread to the (with rectal cultures in homosexual males).
periurethral tissues, causing abscesses and In acute gonorrhea, the urethral discharge
multiple discharging sinuses (‘watercan contains gonococci in large numb ers. The
perineum’). meatus is cleaned with a gauze soaked in saline
B. Disease in women: Asymptomatic carriage and a sample of the discharge collected with a
in women is common, espec ially in the platinum loop for culture, or directly on slide for
endocervical canal. The infection may extend smears. Purulent discharge may be expressed at
to Bartholin’s glands, endometrium and the anterior urethra and collected with a swab.
fallopian tubes to give rise to acute salpingitis, In chronic infections, there may not be any
which may be followed by pelvic inflammatory uret hral discharge. The morning drop of
disease and a high probability of sterility. secretion may be examined or some exudate
Peritoneal spread occasionally occurs and may may be obtained after prostatic massage. It may
produce a perihepatic inflammation (Fitz– also be possible to demonstrate gonococci in
Hugh–Curtis syndrome). the centrifuged deposits of urine in cases where
Proctitis occurs in both sexes. Gonococcal no urethral discharge is available.
pharyngitis may follow orogenital contact in 2. Anal canal: In homosexual males.
either sex. Conjunctivitis may occur usually by
B. Specimens in Women
autoinoculation with fingers.
1. Endocervical swab: In women, uret hral,
C. Disseminated gonococcal disease: Blood cervical and rectal specimens should always be
invasion may occur from the primary site of examined. A single well taken endocervical swab
infection and may lead to metastatic lesions will detect approximately 90% of gonococcal
such as arthritis, ulcerative endocarditis and infections in women. A high vaginal swab is
very rarely meningitis. not suitable. Throat infection also occurs and
D. Disease in childern should be sought where appropriate.
i. Ophthalmic neonatorum: A nonvenereal 2. Urethral
infection is ophthalmia neonatorum in 3. Anal canal and
the newborn, in which the eyes are coated 4. Throat.
with gonococci as the baby passes down
the birth canal. A severe purulent eye C. Blood, Swabs of skin lesions, or pus aspirated
from a joint.
discharge with periorbital edema occurs
within a few days of birth. If untreated, D. Conjunctival Swab: Particularly in neonatal
ophthalmia leads rapidly to blindness. ophthalmia.
Chapter 27: Neisseria and Moraxella | 193
E. Urine Specimen: Any urine specimen showing 6. Genetic Probes
gram-negative diplococci in a Gram stain should Probes specific for the nucleic acids of N. gonorr
be cultured on an appropriate selective medium. hoeae have been developed for the direct detection
of bacteria in clinical specimens.
2. Transport
For culture, specimens should be inoculated on 7. Serological Diagnosis
prewarmed plates, immediately on collection. If No serological test has been found useful for
this is not possible, specimens should be collected routine diagnostic purposes.
with charcoal impregnated swabs and sent to the
laboratory in Stuart’s transport medium. Treatment
Penicillin is no longer the antibiotic of choice for
3. Direct Microscopy treatment of gonorrhea since the development and
Do the Gram staining shows characteristic kidney- widespread use of penicillin, gonococcal resistance
shaped gram-negative diplococci lying within to penicillin has gradually risen, owing to the selec-
polymorphonuclear leukocytes with a few extra tion of chromosomal mutants, so that many strains
cellular. Approximately 95% of infected men will now require high concentrations of penicillin G for
yield a positive smear. It has to be emphasized that inhibition (MIC 2 µg/mL).
diagnosis of gonorrhea by smear examination is Penicillinase p roducing gonococci (PPNG):
unreliable in women as some of the normal genital In 1976, gonococci producing β-lactamase
flora have an essentially similar morphology. (penicillinase) have appeared, rendering penicillin
Immunologic methods employing monoclonal treatment ineffective. Penicillinase production, in
antibodies may be used for the identification of gonococci, is plasmid-mediated.
N. g onorrho eae. These methods include
Chromosomally-mediated resistance (CMRNG):
coagglutination and fluorescent antibody testing.
This chromosomally-mediated resistance
(CMRNG) is not limited only to penicillin but
4. Culture extends to tetracyclines, erythromycin, and
In acute gonorrhea, cultures can be obtained aminoglycosides.
readily on chocolate agar or Mueller–Hinton agar Empirical therapy: Currently, the Centers for Dis
incubated at 35–36°C under 5–10% CO2. In chronic ease Control and Prevention (CDC) recommends
cases, where mixed infection is usual and in the that ceftriaxone, cefixime, ciprofloxacin, or
examination of lesions, such as proctitis; however, ofloxacin be used as the initial therapy for cases
it is better to use a selective medium such as the of uncomplicated gonorrhea. Doxycycline or
Thayer–Martin medium. Examine plates after 24 azithromycin should be added for infections
hours incubation and the growth is identified by complicated by dual infections with Chlamydia.
morphology and biochemical reactions. Incubation
of primary isolation plates is continued for 48 hours. Prophylaxis
Colonies are small, round, translucent, convex
Control of gonorrhea consists of early detection of
or slightly umbonate, with finely granular surface
cases, contact tracing, health education and other
and lobate margins. They are soft and easily
general measures.
emulsifiable. After 48 hours, the colonies are larger
As even clinical disease does not confer any
(1.5–2.5 mm), sometimes with a crenated margin
immunity, vaccination has no place in prophylaxis.
and an opaque raised center.
Smear is made from the colony and Gram Nongonococcal (nonspecific)
staining is done. Gonococci are gram-negative
cocci arranged in pairs (diplococci) with adjacent
urethritis
sides concave (pear or bean shaped). Nongonococcal urethritis (NGU), also known
as nonspecific urethritis (NSU), refers to chronic
5. Identification urethritis where gonococci cannot be demonstrated.
N. gonorrhoeae is identified preliminarily on the
basis of the isolation of oxidase-positive, gram-
Causative Agents
negative diplococci that grow on chocolate blood The most important causative agents are as follows:
agar or on media that are selective for pathogenic
Neisseria species.N gonorrhoeae is oxidase positive. A. Bacterial
It ferments glucose with acid only. It does not ∙∙ Chlamydia trachomatis
ferment maltose unlike meningococci. ∙∙ Ureaplasma urealyticum
www.ebook3000.com
194 | Section 3: Systemic Bacteriology
∙∙ Mycoplasma hominis N. flavescens, N. catarrhalis) have been reported
∙∙ Gardnerella vaginalis occasionally as having caused meningitis. N.
∙∙ Acinetobacter wolfii sicca, N. subflava, N.cinera, N. mucosa, and N.
∙∙ Acinetobacter calcoaceticus. flavescens are also members of the normal flora of
the respiratory tract, because of its carbohydrate
B. Viral fermentation pattern.
∙∙ Herpes virus
∙∙ Cytomegalovirus. Moraxella
C. Fungi The genus Moraxella is a member of the family
Neisseriaceae.
∙∙ Candida albicans.
Species: The Moraxella spp. of medical importance
D. Protozoa are M. lacunata, M. catarrhalis, M. osloensis, M.
∙∙ Trichomonas vaginalis. phenylpyruvica, M. atlantae and M. nonliquefaciens.
www.ebook3000.com
196 | Section 3: Systemic Bacteriology
Important questions 4. Neisseria meningitidis shows all the following
characteristics except:
1. Describe the morphology, pathogenicity and a. Oxidase test positive
laboratory diagnosis of meningococcal meningitis. b. Catalase test positive
2. Discuss laboratory diagnosis of gonorrhea. c. Ferments maltose with production of acid
3. Write short notes on: d. Ferments sucrose with production of acid
a. Antigenic structure of Neisseria gonorrhoeae. 5. Waterhouse–Friderichsen syndrome is caused by:
b. Nongonococcal urethritis (or) nonspecific ure- a. Naieisseria meningitidis
thritis b. Leptospira
c. Moraxella (Branhamella) catarrhalis. c. Streptococcus pyogenes
d. Neisseria gonorrhoeae
Multiple choice questions (MCQs) 6. Causative agent of nongonococcal urethritis is
caused by:
1. All of the following bacteria are oxidase positive a. Chlamydia trachomatis
except: b. Ureaplasma urealytium
a. Neisseria gonorrhoeae c. Mycoplasma hominis
b. Neisseria meningitidis d. All of the above
c. Vibrio cholerae 7. Following statements are true for quadrivalent
d. Enterobacter meningococcal polysaccharide vaccine (MPSV4)
2. The most common infective cause of vaginal except:
discharge in a sexually promiscuous female is: a. The vaccine is given intramuscularly
a. Trichomonas vaginalis b. Prevents disease caused by A, C, Y, and W135
b. Gardnerella vaginalis serogroups of meningococci
c. Neisseria gonorrhoeae c. Indicated for at risk population during out-
break of meningococcal infection
d. Candida albicans
d. Produce good antibody response in children
3. The specimen of choice for isolation of gonococci
below 2 years of age
from women with gonorrhea is:
a. Vaginal swab b. Cervical swab Answers (MCQs)
c. Urethral swab d. Urine 1. d; 2. c; 3. a; 4. d; 5. a; 6. d; 7. d
28
Chapter
Corynebacterium
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss diphtheria toxin
be able to: ∙∙ Discuss laboratory diagnosis of diphtheria
∙∙ Describe morphology, cultural characteristics, ∙∙ Toxigenicity tests/virulence tests of C. diphtheriae.
biochemical reactions, toxin production and ∙∙ Describe the following: Schick test; DPT vaccine or
pathogenesis of diphtheria triple vaccine; diphtheroids
∙∙ Differentiate three biotypes: Gravis, intermedius ∙∙ Differentiate between C. diphtheriae and diphthe
and mitis of C. diphtheriae roids.
Corynebacterium diphtheriae
Morphology
They are, thin, slender gram-positive bacilli but
are decolorized easily, particularly in old cultures,
measuring approximately 3–6 μm × 0.6–0.8 μm.
They have a tendency to clubbing at one or both
ends. They are highly pleomorphic. They are non-
motile, non-spore forming, and nonacid fast.
The bacilli are arranged in a characteristic
fashion in smears. They are usually seen in pairs, Fig. 28.1: Corynebacterium diphtheriae showing
palisades (resembling stakes of a fence) or small metachromatic granules and Chinese letter arrangement
www.ebook3000.com
198 | Section 3: Systemic Bacteriology
Albert’s stain, the granules stain bluish black and of cystine to a tellurite containing medium
the protoplasm green. The granules represent (Tinsdale’s medium) has greatly helped the
accumulation of polymerized polyphosphates. isolation of diphtheria bacilli. The growth
The granules formation is best seen on Loeffler’s of diphtheria bacilli may be delayed on the
serum slope. tellurite medium and colonies may take two
days to appear.
Cultural Characteristics Based on colonial morphology on the tellurite
medium and other properties, Mc Leod and
C. diphtheriae is an aerobe and facultative anaerobe;
Anderson described three different biotypes:
the optimum temperature for growth is 37°C (range
Gravis, intermedius and mitis (Table 28.1). The
15–40°C) and optimum pH 7.2. It can grow on
names were originally proposed to relate to the
ordinary nutrient agar, but its growth is improved
clinical severity of the disease produced by the
by the presence of animal proteins such as blood
three types—gravis, causing the most serious, and
or serum. Two media are useful for this purpose:
mitis the mildest variety, with intermedius being
1. Loeffler’s serum slope: Diphtheria bacilli
responsible for disease of intermediate severity.
grow on LoeffIer’s serum slope very rapidly
and colonies can be seen in 6–8 hours, long
before other bacteria grow. Colonies are at first Biochemical Reactions
small, circular white opaque disks but enlarge C. diphtheriae ferments glucose and maltose
on continued incubation and may acquire a with the production of acid (but no gas) but not
distinct yellow tint. lactose, mannitol, trehalose or sucrose. Starch and
2. Tellurite blood agar: The addition of potassium glycogen are used for biochemical differentiation
tellurite (0.03–0.04%) makes the medium of three biotypes of C. diphtheriae (Table 28.1).
selective for Corynebacteria by inhibiting most Gravis strains utilize glycogen and starch, while
other pathogenic and commensal bacteria. On mitis and intermedius do not. Fermentation of
this medium, C. diphtheriae give grey/black, sugars are usually done in Hiss’s serum peptone
shiny or dull black colonies. The addition water medium.
Table 28.1 Differentiating features of Corynebacterium diphtheriae
Gravis Intermedius Mitis
1. Morphology i. Usually short rods, with i. Long barred forms with i. Long, curved, rods
uniform staining clubbed ends; ii. Prominent granules
ii. Few or no granules. ii. Poor granulation iii. Pleomorphic
iii. P leomorphism (some iii. Very pleomorphic
degree), with irregularly
barred, snow-shoe and
teardrop forms
2. Colony on tellurite i. In 18 hours: Colony is i. 18 hour: Colony small, I i. Size variable, shiny black.
blood agar 1–2 mm in size, greyish mm in size, misty.
black centre, paler,
semitranslucent periphery
and commencing ii. i n 48 hours Does not ii. In 2–3 days: Colonies
crenation of edge. enlarge, dull granular become flat, with
ii. In 2–3 days: 3–5 mm in centre with smoother, more a central elevation
size, flat colony with raised glistening periphery and a ‘poached egg’ colony
dark centre and crenated lighter ring near the edge -
edge with radial striation - ‘frog’s egg’ colony
‘daisy head’ colony
3. Consistency of colonies i. Brittle, moves as a whole Intermediate between (i) Soft, buttery, (ii) Easily
on the plate like ‘cold gravis and mitis emulsifiable
margarine’.
ii. Not easily picked out or
emulsifiable
4. Hemolysis Variable Nonhemolytic Usually hemolytic
5. Growth in broth Surface pellicle. Deposit Turbidity in 24 hours, Turbidity diffuse with soft
granular. clearing in 48 hours, with pellicle later
Turbidity little or no fine granular sediment
6. Glycogen and starch Positive Negative Negative
fermentation
7. Toxigenic strains Almost 100% 95–99% 80–85%
8. Virulence Severe Moderate Mild
9. Predominant strains Epidemic areas Epidemic areas Endemic areas
Chapter 28: Corynebacterium | 199
C. diphtheriae is H2S positive and reduces and mediates the entry of fragment A into the
nitrate to nitrite. It does not liquefy gelatin or cytoplasm. The antibody to fragment B prevents the
hydrolyze urea or form phosphatase. binding of toxin to cells and is thus protective. The
Pyrazinamidase (PYZ) test: In pyrazinamidase toxin is heat labile. It is extremely potent (0.0001 mg
(PYZ) test, pyrazinamide is converted into pyrazinoic kills a guinea pig of 250 g weight). It is converted into
acid by the organisms which produce pyrazinamidase toxoid by heat (at 37°C for 4–6 weeks), treatment
(PYZ). This test is helpful to distinguish ‘C. diphtheriae’ with 0.2–0.4% formalin or by acidic pH. The toxoid
(PYZ-negative) from other Corynebacterium species is a toxin that has lost its toxicity but has retained the
(mostly PYZ-positive). antigenicity. It is capable of producing antitoxin. It
has a special affinity for certain tissues such as the
myocardium, adrenals and nerve endings.
Toxin
Toxigenic strains of C. diphtheriae produce a a Mode of Action
very powerful exotoxin. The toxicity observed
The diphtheria toxin acts by inhibiting protein
in diphtheria is directly attributed to the toxin
synthesis. It inhibits polypeptide chain elongation
secreted by the bacteria at the site of infection.
in the presence of nicotinamide adenosine
dinucleotide (NAD) by inactivating elongation
Synthesis factor 2 (EF-2), an enzyme required for elongation
Almost all strains of gravis, 95-99% of intermedius of polypeptide chains on ribosomes. Inhibition of
and 80-85% of mitis produce this toxin. Strains of protein synthesis is probably responsible for both
all three types are invariably virulent when isolated the necrotic and neurotoxic effects of the toxin.
from acute cases. Avirulent strains are common NAD+ + EF–2 = ADPR–EF–2 + nicotinamide + H+
among convalescents, contacts and carriers. The Active Inactive
strain almost universally used for toxin production
is the ‘Park Williams 8’ strain, which has been Resistance
variously described as a mitis (Topley and Wilson)
and an intermedius strain (Cruickshank). They are readily killed, however, by a 1-minute
exposure to100°C or a 10 minute exposure to 58°C.
They are susceptible to most of the routinely used
Lysogeny and toxin Production
disinfectants. It remains alive for weeks in dust and
The toxigenicity of the diphtheria bacillus depends on fomites when dry and protected from sunlight.
on the presence in it of corynephages (tox +), It is susceptible to penicillin, erythromycin and
which act as the genetic determinant controlling broad spectrum antibiotics.
toxin production. Nontoxigenic strains can be
converted to tox+ by infection with the appropriate Antigenic Structure
bacteriophage. This is known as lysogenic or phage
conversion. The bacillus loses the toxigenicity Diphtheria bacilli possess three distinct antigens:
when it is cured of its phage, as by growing it in the 1. A deep-seated antigen found in all Coryne
presence of antiphage serum. bacterial species
2. A heat-labile protein (K-antigen)
3. A heat-stable polysaccharide (O-antigen).
Iron for toxin Production
Toxin production is also influenced by the Serotypes: On the basis of agglutination reaction,
concentration of iron in the medium. The optimum biotypes gravis, intermedius and mitis have been
level of iron for toxin production is 0.1 mg per liter, divided into 13, 4 and 40 serotypes, respectively.
while a concentration of 0.5 mg per liter inhibits
the formation of toxin. The toxin is released in Bacteriophage Typing
significant amounts only when the available iron The susceptibility of C. diphtheriae to bacterio
in the culture medium is exhausted. phage strains has been most comprehensively
studied; 22 phages were used to type the gravis
Properties of toxin strains into 14 types, the intermedius into 3 and the
Diphtheria toxin is an iron-free, crystalline, heat mitis into 4. An additional set of 33 phages has also
labile protein. The diphtheria toxin is a protein and been used. The only other corynebacteria reported
has a molecular weight of about 62000. It consists as susceptible to the diphtheria typing phages are
of two fragments A (active) and B (binding) of C. ulcerans and C. pseudotuberculosis.
molecular weights 24000 and 38000 respectively.
Both fragments are required for toxicity. Fragment Pathogenesis
A has all the enzymatic activity whereas fragment The organism is carried in the upper respiratory tract
B is responsible for binding the toxin to the cells and spread by droplet infection or hand-to-mouth
www.ebook3000.com
200 | Section 3: Systemic Bacteriology
contact. The incubation period of diphtheria is 2–5 systemic complications are less common than from
days, with a range of 1–10 days. Diphtheria, which upper respiratory infections with C. diphtheriae.
occurs in two forms (respiratory and cutaneous),
is found worldwide. Laboratory Diagnosis
Diagnostic laboratory tests serve to confirm the
A. Respiratory Diphtheria clinical impression and are of epidemiologic
The illness begins gradually and is characterized significance but not for the treatment of individual
by low-grade fever, malaise, and a mild sore throat. cases. Specific treatment should be instituted
The most common site of infection is the tonsils immediately on suspicion of diphtheria without
or pharynx. The organisms rapidly multiply on waiting for laboratory tests. Any delay may be
the epithelial cells, and the toxigenic strains of C. fatal. Laboratory diagnosis consists of isolation
diphtheriae produce toxin locally, causing tissue of the diphtheria bacillus and demonstration of
necrosis and exudate formation triggering an its toxicity.
inflammatory reaction. This combination of cell 1. Specimens
necrosis and exudate forms a tough gray to white
Swabs from the nose, throat, or other suspected
pseudomembrane, which attaches to the tissues-
lesions must be obtained before antimicrobial
commonly over the tonsils, pharynx, or larynx. Any
drugs are administered. In suspected cases,
attempt to remove the pseudomembrane results
whether of faucial or nasal dipht heria, swabs
in bleeding. In nasopharyngeal infection, the
should be taken both from the throat and from the
pseudomembrane may involve nasal mucosa, the
nose, and preferably two swabs from the site most
pharyngeal wall and the soft palate. In this form,
affected. Swabs should also be taken from skin
oedema involving the cervical lymph glands may
lesions and wounds where diphtheritic infection is
occur in the anterior tissues of the neck, a condition
suspected, and both throat and nose swabs should
known as bullneck diphtheria. Laryngeal
be taken from suspected carriers.
involvement leads to obstruction of the larynx and
lower airways. 2. Microscopy
Systemic effects Direct microscopy of a smear is unreliable since
The toxin also is absorbed and can produce a vari C. diphtheriae is morphologically similar to
ety of systemic effects involving the kidneys, heart, other coryneforms. Smears stained with alkaline
and nervous system, although all tissues possess methylene blue or Gram’s stain show beaded rods
the receptor for the toxin and may be affected. in typical arrangement. Hence smear examination
Intoxication takes the form of myocarditis and alone is not sufficient for diagnosing diphtheria
peripheral neuritis, and may be associated with but is important in identifying Vincent’s angina.
thrombocytopenia. Visual disturbance, difficulty in For this, a Gram or Leishman stained smear is
swallowing and paralysis of the arms and legs also examined for Vincent’s spirochetes and fusiform
occur but usually resolve spontaneously. Complete bacilli. Toxigenic diphtheria bacilli may be
heart block may result from myocarditis. Death is identified in smears by immunofluorescence.
most commonly due to congestive heart failure and
cardiac arrhythmias. 3. Culture
The swab should be inoculated on Loffler’s serum
Complications slope, tellurite blood agar, and blood agar. The
The common complications are as follows: cultures should be incubated aerobically at 37°C.
1. Asphyxia due to mechanical obstruction of the Unless the swab can be inoculated promptly, it
respiratory passage by the pseudomembrane should be kept moistened with sterile horse serum
for which an emergency tracheostomy may so the bacilli will remain viable.
become necessary. i. Loeffler’s serum slope: After incubation for 6
2. Acute circulatorv failure, which may be hours or overnight, make a smear of growth from
peripheral or cardiac. all parts of the slope mixed in the condensation
3. Postdiphtheritic paralysis, which typically water, stain by the Albert-Laybourn method and
occurs in the third or fourth week of the disease; look for the presence of slender green-stained
palatine and ciliary but not pupillary paralysis bacilli containing the purple-black granules
is characteristic, and spontaneous recovery is characteristic of C. diphtheriae.
the rule. ii. Tellurite blood agar: Blood tellurite agar is
4. Septic, such as pneumonia and otitis media. examined after 24 hours and after 48 hours,
as growth may sometimes be delayed.
B. Cutaneous Diphtheria iii. Blood agar: It is used for differentiating
In cutaneous diphtheria, which is prevalent in the streptococcal or staphylococcal pharyngitis,
tropics, the toxin also is absorbed systemically, but which may simulate diphtheria.
Chapter 28: Corynebacterium | 201
4. Identification Tests Advantages of the intracutaneous test:
Identification is based on carbohydrate ferment a. The animals do not die
ation reactions and enzymatic activities. C. b. As many as ten strains can be tested at a time
diphtheriae ferments glucose and maltose, on a rabbit.
producing acid but not gas, and is catalase positive. B. In vitro Test
It reduces nitrate to nitrite and is nonmotile. i. Elek’s gel precipitation test: The in vitro
Commercial kits such as the API Coryne strip diphtheria toxin detection procedure is an
provide a reliable identification. immunodiffusion test first described by Elek.
Procedure: A rectangular strip of filter paper
5. Virulence Tests impregnated with diphtheria antitoxin (1000 units/
Such tests are really tests for toxigenicity of an ml) is placed on the surface of a 20% normal horse
isolated diphtheria-like organism. Diagnosis of serum agar in a Petri dish while the medium is still
diphtheria depends on showing that the isolate fluid. When the agar has set, the surface is dried.
produces diphtheria toxin. Virulence testing may The plate should be streaked with the test strain as
be by in vivo or in vitro methods. well as the control positive and negative strains at
A. In vivo tests right angles to the strip in a single straight line and
i. Subcutaneous test parallel to each other. The plate is incubated at 37°C
and examined after 24 and 48 hours.
ii. Intracutaneous test
B. In vitro test Interpretation: Toxins produced by the bacterial
growth will diffuse in the agar and where it meets the
i. Precipitation test
antitoxin at optimum concentration will produce a
ii. Tissue culture test line of precipitation (Fig. 28.2). A negative control
iii. Enzyme-linked immunosorbent assays should be free of any line. No precipitate will form
iv. Polymerase chain reaction (PCR). in the case of nontoxigenic strains.
A. In vivo Tests ii. Tissue culture test : The toxigenicity of
i. Subcutaneous test: The growth from an diphtheria bacilli can be demonstrated by
overnight culture on Loeffler’s slope is incorporating the strains in the agar overlay of
emulsified in 2–4 mL broth and 1 mL of the cell culture monolayers. The toxin produced
emulsion injected subcutaneously into diffuses into the cells below and kills them.
two guinea pigs or rabbits, one of which has iii. Enzyme-linked immunosorb ent assays
been protected with the diphtheria antitoxin (ELISA): Rapid, enzyme-linked immunosor
(500–1000 units) 18–24 hours previously and bent assays and immunochromatographic
was used as control. If the strain is virulent, strip assays are also available for the detection
the unprotected animal will die within of diphtheria toxin.
four days. Postmortem examination would iv. Polymerase chain reaction (PCR): In addition,
show hemorrhage at the site of injection procedures for detecting the C. diphtheriae tox
and injected blood vessels, with typically gene by the polymerase chain reaction (PCR)
hemorrhagic adrenal necrosis. have been developed. The PCR assay can also
Simple and reliable subcutaneous be applied directly to clinical specimens.
toxigenicity tests in rabbits or larger guinea- Schick Test
pigs were used in the past, at a time when
laboratories had many isolates each day. Schick (1913) introduced an intradermal test
The method is not usually employed as it is (Schick test) for distinguishing between susceptible
wasteful of animals. and immune persons.
ii. Intracutaneous (Intradermal) test: The
broth emulsion of the culture is inoculated
intracutaneously into two guinea pigs (or
rabbits) so that each receives 0.1 mL in two
different sites. One animal acts as the control
and should receive antitoxin (500 units)
the previous day. After four hours the skin
test, the other is given 50 units of antitoxin
intraperitoneally in order to prevent death.
In the test animal, toxigenicity is indicated by
inflammatory reaction at the site of injection,
progressing to necrosis in 48–72 hours and in
the control animal no change. Fig. 28.2: Elek’s test
www.ebook3000.com
202 | Section 3: Systemic Bacteriology
Principle DPT Vaccine: Diphtheria toxoid is usually given
This test depends upon the principle of toxin- in children as a trivalent preparation containing
antitoxin neutralization, in vivo, and the test is tetanus toxoid and pertussis vaccine also as the
carried out by injecting one Schick test dose of DPT or triple vaccine.
diphtheria toxin (0.2 mL containing 1/50 MLD)
intradermally on the anterior surface of the left Schedule of primary immunization: The schedule
forearm and a control injection in the right forearm of primary immunization of infants and children
contains a heat-inactivated dose (70°C for 30 consists of DPT given at the age of 6 weeks, 10
minutes) or preferably, purified diphtheria toxoid. weeks, 14 weeks and 16–24 months followed by
booster dose DT at the age of 5–6 years (school
Results entry).
Readings are taken after 1, 4 and 7 days. Four types
of reactions may occur:
B. Passive Immunization
1. Negative reaction: There is no reaction of any
kind in either arm. This indicates that the toxin This is an emergency measure to be employed
has been neutralized by the circulating antitoxin where susceptibles (nonimmunized) are exposed
and the person is immune to diphtheria and to infection, as when a case of diphtheria is
does not need immunization. admitted to general pediatric wards. It consists of
2. Positive reaction: In the test arm, there appears the subcutaneous administration of 500–1000 units
erythema and swelling at the site of inoculation in of antitoxin (antidiphtheritic serum, ADS). As this is
24–36 hours, reaching its maximum (1–5 cm) by a horse serum, precaution against hypersensitivity
the 4th to 7th days and then fading with superficial should be observed.
scaling and persistent brownish pigmentation. On
the control arm, there is no reaction. C. Combined Immunization
A positive Schick test indicates that the This consists of administration of the first dose
individual is susceptible to diphtheria and of adsorbed toxoid on, while ADS is given on the
little or no antitoxin (less than 0.01 unit/mL) is other arm, to be continued by the full course of
present. The subject is not immune and should active immunization since protection conferred
be immunized. by passive immunization is of short duration.
3. Pseudoreaction: There is erythema occurring Ideally, all cases that receive ADS prophylactically
within 6–24 hours and disappearing within four should receive combined immunization.
days. The reaction is the same on both arms.
This indicates that the individual is immune to
Treatment
diphtheria and also that he is hypersensitive to
one or more antigens in the toxin preparation. Specific treatment of diphtheria consists of
This individual does not need immunization. antitoxic and antibiotic therapy. Antitoxin
4. Combined reaction: Here the initial picture is should be given immediately as soon as clinical
that of pseudoreaction, but while the erythema diagnosis is made to neutralize the toxin being
in the control arm fades, within four days, it produced. The dosage recommended is 20,000
progresses in the test arm to a typical positive units intramuscularly for moderate cases and
reaction. This indicates that the individual is 50,000 to 100,000 units for serious cases, half the
susceptible to diphtheria and is sensitive to dose being given intravenously.
one or more antigens in the toxin preparation C. diphtheriae is sensitive to most antibiotics,
making immunization necessary but likely to including penicillin and erythromycin for the
induce reaction. treatment of patients as well as carriers. The
antibiotics do not neutralize circulating toxin.
Prophylaxis Penicillin-sensitive individuals can be given
The methods of immunization available are active, erythromycin. Erythromycin is more active than
passive or combined. penicillin in the treatment of carriers.
www.ebook3000.com
204 | Section 3: Systemic Bacteriology
Table 28.3 Differences between C. diphtheriae and diphtheroids
Feature C. diphtheriae Diphtheroids
1. Morphology i. Weakly gram-positive and thin bacilli Strongly gram-positive, short and thick bacilli
ii. Metachromatic granules present Few or absent
iii. Arranged in Chinese letter pattern Pallisade arrangement
iv. Pleomorphism present Very little pleomorphism present
2. Culture Grow on enriched media Can grow on ordinary media
3. Biochemical tests Ferments glucose only and does not Ferments both glucose and sucrose
ferment sucrose
4. Toxin production Toxic Nontoxic
5. Virulence tests Positve Negative
f. Nondiphtheria corynebacteria
Key Points g. Diphtheroids.
Corynebacterium is gram-positive bacilli with an
irregular shape, tendency to clubbing at one or both,
highly pleomorphic with Chinese letter or cuneiform
Multiple choice questions (MCQs)
arrangement. The granules in the cell are known as 1. Corynebacterium diphtheriae is classified into three
metachromatic granules volutin granules or Babes distinct biotypes (mitis, intermedius, and gravis)
Ernst granules. With Albert’s stain, the granules stain based on the morphologies of the colonies on:
bluish black and the protoplasm green a. Tellurite blood agar
Two media are useful. 1. Loeffler’s serum slope- b. Loeffler’s serum slope
tellurite blood agar c. Blood agar
Fermentations of sugars are usually done in Hiss’s
d. All the above media
serum peptone water medium
2. Diphtheria toxin shows following features except:
C. diphtheriae is H2S positive and reduces nitrate
a. It is a protein with a molecular weight of 58,300
to nitrite
Toxin: Toxigenic strains of C. diphtheriae produce
Da
a very powerful exotoxin. The toxigenicity of the b. It consists of two functionally distinct polypep-
diphtheria bacillus depends on the presence in it of tide chain fragments, A and B protein
corynephages (tox+). Diphtheria toxin is, heat labile c. It inhibits synthesis of fatty acids
protein, and consists of two fragments. Inhibition of d. It is a very potent toxin
protein synthesis is probably responsible for both 3. Following statements are true for diphtheria
the necrotic and neurotoxic effects of the toxin antitoxin except:
Clinical diseases: Diphtheria occurs in two forms a. It is the mainstay of therapy in diphtheria
(respiratory and cutaneous) b. It is of immense value in cutaneous diphtheria
Laboratory diagnosis: Depends upon micros c. It is of no value in treatment of asymptomatic
copy,culture and virulence tests. Virulence testing carriers
may be by in vivo or in vitro methods. In vivo tests d. It is ineffective after toxin has entered into the cell
are i. Subcutaneous test; ii. Intracutaneous test. 4. Diphtheria toxin has a special affinity for which of
In vitro test include i) Precipitation test; ii) Tissue the following tissue/s?
culture test; iii) Enzyme-linked immunosorbent a. Heart muscles b. Nerve endings
assays (ELISA); iv) Polymerase chain reaction (PCR) c. Adrenal glands d. All of the above
Prophylaxis: DPT given at the age of 6 weeks, 10 5. Which of the following sites is most commonly
weeks, 14 weeks and 16–24 months followed by
affected by diphtheria bacilli?
booster dose DT at the age of 5–6 years (school entry)
a. Upper respiratory tract b. Skin
Diphtheroids: Cor ynebacteria resembling
c. Cornea d. Conjunctiva
C. diphtheriae occur as normal commensals in the
throat, skin and other areas. These may be mistaken
6. Diphtheroids show following features except:
for diphtheria bacilli and are known as diphtheroids. a. They possess few or no metachromatic granules
The common diphtheroids are C. pseudodiphth b. They are usually arranged in parallel rows
eriticum and C. xerosis. c. Most of them do not produce toxins
d. They do not ferment sucrose
7. A positive Schick test implies that the person is:
Important questions a. Immune and nonhypersensitive.
b. Susceptible and nonhypersensitive
1. Discuss morphology, cultural characteristics c. Immune and hypersensitive
and biochemical characters of Corynebacterium d. Non-immune and hypersensitive
diphtheriae. 8. Which of the following bacteria can cause infection
2. Name different species of genus Corynebacterium. in immunocompetent patients?
Discuss in detail laboratory diagnosis of diphtheria. a. Corynebacterium ulcerans
3. Write short notes on: b. Corynebacterium haemolyticum
a. Diphtheria toxin c. Corynebacterium pseudotuberculosis
b. Pathogenicity of C. diphtheriae d. All of the above
c. Toxigenicity tests/Virulence tests of C. diphtheriae
d. Schick test Answers (MCQs)
e. Prophylaxis of diphtheria 1. a; 2. c; 3. b; 4. d; 5. a; 6. d; 7. b; 8. d
29
Chapter
Bacillus
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of anthrax
be able to: ∙∙ Descrbethe following: Anthracoid bacilli; Bacillus
∙∙ Describe the morphology, cultural characteristics cereus food poisoning.
and pathogenicity of Bacillus anthracis
www.ebook3000.com
206 | Section 3: Systemic Bacteriology
Fig. 29.1: Anthrax bacilli Fig. 29.2: Medusa head appearance of anthrax bacilli
B. Human infection
Resistance
Based on the mode of infection, human Anthrax
The spores are resistant to chemical disinfectants
presents in one of three ways: (1) cutaneous (2)
and heat. With moist heat, the vegetative bacilli are
pulmonary, or (3) intestinal. All types leading to
killed at 60°C in 30 minutes and the spores at 100°C
fatal septicemia or meningitis.
in 10 minutes. With dry heat the spores are killed at
150°C in 60 minutes. The spores are also killed by 1. Cutaneous Anthrax
4% formaldehyde or 4% potassium permanganate Cutaneous anthrax used to be caused by shaving
in a few minutes. brushes made with animal hair. It begins 2–5 days
The bacilli are sensitive to benzylpenicillin, after infection as a small papule that develops
streptomycin, tetracyclines, chloramphenicol, within a few days into a vesicle filled with dark
ciprofloxacin, the cephalosporins and sulfonamides. bluish black fluid. Rupture of the vesicle reveals
a black eschar at the base, with a very prominent
Antigenic Structure inflammatory ring of reaction around the eschar.
(The name anthrax, which means coal, comes
Three main antigens have been characterized: from the black color of the esc har). This is
1. Capsular polypeptide: It is polypeptide con sometimes referred to as a malignant pustule.
sisting exclusively of D-glutamic acid. The lesion is classically found on the hands,
2. Somatic polysaccharide: It is a component of forearms, or head and is painless. The disease used
the cell wall. to be common in dock workers carrying loads of
3. Complex protein toxin (anthrax toxin): hides and skins on their bare backs and hence was
Anthrax toxin is a complex exotoxin consisting of known as the ‘hide porter’s disease.’ Cutaneous
three protein components: Protective antigen anthrax generally resolves spontaneously, but
(PA), lethal factor (LF), and edema factor (EF). 10–20% of untreated patients may develop fatal
Each of the separate components is serologically septicemia or meningitis.
active and distinct and is also immunogenic.
2. Pulmonary Anthrax
Pulmonary anthrax, known as ‘wool-sorter’s
Virulence Factors disease, because it used to be common in workers
The pathogenesis of B. anthracis depends on two in wool factories, due to inhalation of dust from
important virulence factors: A poly (D-glutamic infected wool. It occurs in patients who handle raw
acid) capsule and a three-component protein wool, hides, or horsehair and acquire the disease
exotoxin. Fully virulent organisms have two large by the inhalation of spores. This is a hemorrhagic
plasmids that code for these products. pneumonia with a high fatality rate. Hemorrhagic
1. Capsule: The capsule interferes with phagocytosis. meningitis may occur as a complication.
2. Anthrax toxin: Anthrax toxin consists of three 3. Intestinal Anthrax
proteins called protective antigen (PA), edema Intestinal anthrax, is rare and occurs mainly in
factor (EF), and lethal factor (LF), each of primitive communities who eat the carcasses of
which individually is nontoxic but together act animals dying of anthrax. An individual may suffer
synergistically to produce damaging effects. after a day or so from hemorrhagic diarrhea, and
The three factors have been characterized and dies rapidly from septicemia.
cloned.
Laboratory Diagnosis of Human Anthrax
Pathegenesis
In laboratories unfamiliar with the disease,
Cattle, sheep, goats and other herbivores are
additional precautions for staff safety need to be
naturally infected.
organized. All procedures connected with the
handling of B. anthracis should be carried out with
A. Animal Infection greatest care in a safety cabinet.
Anthrax is a zoonosis. Animals are infected by the 1. Specimens: Material from a malignant pustule,
ingestion of the spores present in the soil. Direct sputum from pulmonary anthrax, gastric
spread from animal to animal is rare. The disease aspirates, feces or food in intestinal anthrax and
is generally a fatal septicemia but may sometimes in the blood in the septicemic stage of all forms
www.ebook3000.com
208 | Section 3: Systemic Bacteriology
of the infection. Specimens should be taken antigen, a ring of precipitation will appear at the
before antibiotic therapy has been instituted. junction of the two liquids within 5 minutes at
2. Microscopy: Prepare smears of each specimen room temperature.
and stain with Gram’s method and McFadyean’s With the availability of purified anthrax toxin
method or with Giemsa stain. Gram’s stain antigen, Ascoli’s test has been replaced by highly
may show typical large gram-positive bacilli. sensitive and specific immunoassays. EIA can also
Capsule appears as a clear halo around the detect antibody in the serum of animals surviving
bacterium by India-ink staining. anthrax infection.
Direct fluorescent antibody test (DFA) for
capsule specific staining and for polysaccharide 6. Polymerase Chain Reaction (PCR)
(cell wall) antigen confirms the identification. A sensitive and specific PCR technique has been
3. Culture: Culture the exudate on nutrient agar, developed for the detection of anthrax contami
blood agar, PLET medium and nutrient broth. nation of animal and agricultural products.
Incubate at 37°C for 18 hours. Examine plates
for the medusa-head colonies characteristic Treatment
of B. anthracis, nonhemolytic on the blood Ciprofloxacin is the drug of choice. Penicillin,
agar plate. Prepare a smear, stain it by Gram’s doxycycline, erythromycin, or chloramphenicol
method and look for tangled chains of large can be used (if susceptible).
gram-positive bacilli some of which have
central, oval, non-bulging spores. In nutrient Prophylaxis
broth, look for a pellicle and a deposit.
Control of human anthrax ultimately depends on
4. Confirmatory tests
control of the disease in animals. Animals with
i. Biochemical and physiological reactions:
known or suspected anthrax should be handled
Demonstration of non-motility, gelatin
with care and carcasses of animals suspected to have
liquefaction, growth in straight chains and died of anthrax are incinerated or buried deep in
enhanced growth aerobically, as seen in the quicklime. Wool, horsehair, and hides coming from
characteristic inverted fir tree appearance areas where epidemic anthrax is present should be
in a gelatin stab, will generally identify B. gas sterilized. A vaccine is available for control of
anthracis completely. outbreaks of human anthrax in an ‘industrial setting’.
Toxin production can be demonstrated by
immunological or gene probe methods in Immunization
reference laboratories. Live-attenuated bacilli were first used by Louis
ii. Animal inoculation: Inoculate intraperi Pasteur in May 1881. Pasteur’s vaccine was the
toneally in mice a 24 hours broth culture. anthrax bacillus attenuated by growth at 42–43°C.
The animal dies in 48–72 hours. Make The original Pasteur’s anthrax vaccine is of great
smears from heart blood and spleen, stain historical importance.
by Gram’s and McFadyean’s methods, and
Sterne strain of live spore vaccine: Subsequently,
look for typical anthrax bacilli. the Sterne strain of live spore vaccine has been
The anthrax bacillus can often be isolated used for animal immunization. The Sterne vaccine
from contaminated tissues by applying contained spores of a noncapsulated avirulent
them over the shaven skin of a guinea mutant strain. Live bacterial vaccines are not
pig. It is able to penetrate through minute considered safe for man.
abrasions and produce fatal infection.
Alum-precipitated toxoid prepared from the
5. Serological diagnosis: Serological diagnosis by
protective antigen has been shown to be a safe
enzyme-linked immunosorbent assay (ELlSA)
and effective vaccine for human use. It has been
is seldom used diagnostically.
used in persons occupationally exposed to
Ascoli’s thermoprecipitin test: If the sample anthrax infection. Three doses intramuscularly at
received is putrid so that viable bacilli are intervals of six weeks between first and second,
unlikely, diagnosis may be established by Ascoli’s and six months between second and third doses
thermoprecipitin test by demonstration of the induce good immunity, which can be reinforced if
anthrax antigen in tissue extracts.The original necessary with annual booster injections. Frequent
thermoprecipitin test devised by Ascoli (191l) booster doses are necessary.
was a ring precipitation by letting the boiled
tissue extract in a test tube react with the anthrax
ANTHRACOID BACILLI
antiserum. The tissue is ground up in saline, boiled
for 5 minutes, filtered and layered over anti anthrax Nonpathogenic aerobic spore bearing bacilli having
serum in a narrow tube. If tissue contains anthrax a general resemblance to anthrax bacilli have been
Chapter 29: Bacillus | 209
collectively called pseudoanthrax or anthracoid There is a longer incubation period occurring
bacilli. The important species include B. cereus, B. 8–24 hours after ingestion, during which the
subtilis, B. stearothermophilus B. licheniformis, B. organism multiplies in the patient’s intestinal
pumilus. B. cereus has been recognized as a frequent tract and produces the heat-labile enterotoxin.
cause of foodborne gastroenteritis. Table 29.1 lists Then the diarrhea, nausea, and abdominal
the main differentiating features between Anthracoid cramps develop.
bacilli and B. anthracis. The diarrheal disease is mostly caused
by serotypes 2, 6, 8, 9, 10 or 12 of B. cereus
Bacillus cereus strains. Isolates from the diarrheal type of
B. cereus has recently assumed importance as a disease produce enterotoxin which causes
cause of food poisoning. It is widely distributed in fluid accumulation in ligated rabbit ileal loop,
nature, may be readily isolated from soil, vegetables resembling the heat labile enterotoxin of
and a wide variety of foods including milk, cereals, Escherichia coli and Vibrio cholerae.
spices, meat and poultary. 3. Ocular infection: Common cause of post-
traumatic ophthalmitis.
4. Other opportunistic infections: Intravenous
Pathogenesis catheter and central nervous system shunt
1. Emetic form (the short incubation type): The infections and endocarditis as well as
emetic form results from the consumption of pneumonitis, bacteremia, and meningitis in
contaminated rice. The heat-stable enterotoxin severely immunosuppressed patients.
that is released is not destroyed when the rice
is reheated. After ingestion of the enterotoxin Laboratory Diagnosis
and a 1–6-hour incubation period, a disease of
If food is available for testing, confirmation is easy.
short duration (less than 24 hours) develops.
High numbers of B. cereus, in the absence of other
Symptoms consist of vomiting, nausea, and
food poisoning bacteria are sufficient to make the
abdominal cramps. Fever and diarrhea are
diagnosis.
generally absent.
Large facultatively anaerobic Gram-positive
Two mechanisms of action have been
bacilIi that produce anthracoid colonies on blood
described for the enterotoxin of B. cereus, one
agar after overnight incubation at 37°C are almost
involving stimulation of CAMP system and the
certain to be B. cereus.
other independent of it.
2. Diarrheal form: The diarrheal form of B. cereus Treatment : Both the emetic and diarrheal
food poisoning results from the consumption syndromes are shortlived and no specific treatment
of contaminated meat, vegetables or sauces. is needed.
www.ebook3000.com
210 | Section 3: Systemic Bacteriology
Prevention Important questions
Prevention is best accomplished by the prompt 1. Discuss laboratory diagnosis of anthrax.
refrigeration of boiled rice and other foods because 2. Write short notes on Bacillus cereus food poisoning.
it is nearly impossible to eliminate B. cereus spores
from food. Multiple choice questions (Mcqs)
Key Points 1. McFadyean’ s reaction is employed for the presump
The genus Bacillus consists of Gram positive, aerobic tive diagnosis of:
bacilli forming heat resistant spores a. Anthrax b. Tetanus
Bacillus has two important species—1. Bacillus c. Diphtheria d. Typhoid
anthracis—the organism responsible for anthrax 2. ‘Medusa head’ appearance of the colonies is
and 2. Bacillus cereuus—can cause food poisoning.
characteristic of:
Bacillus anthracis
a. Proteus mirabilis
Spore-forming gram-positive, capsulated bacilli.
B anthracis is a M’Fadyean’s reaction positive. It b. Clostridium tetani
grows as ‘Medusa head appearance’ on nutrient c. Bacillus anthracis
agar medium d. Pseudomonas aeruginosa
Diseases: Cutaneous anthrax, inhalation anthrax 3. Malignant pustule is characteristic of:
and gastrointestinal anthrax a. Cutaneous anthrax
Diagnosis: Isolation of the organism from clinical b. Pulmonary anthrax
specimens (e.g. papule or ulcer, blood)
c. Intestinal anthrax
Animal vaccination is effective, but human vaccines
d. All of the above
have limited usefulness
Aerobic spore bearing bacilli having a general resem 4. Ascoli’s thermoprecipitin test helps in confirming
blance to anthrax bacilli which have been collectively the laboratory diagnosis of:
called pseudoanthrax or anthracoid bacilli a. Anthrax b. Tetanus
Bacillus cereus Infections c. Typhoid d. Cholera
People at risk include those who consume food 5. Which of the following foods is most often
contaminated with the bacterium (e.g. rice, meat, associated with emetic type of food poisoning
vegetables, sauces), those with penetrating injuries
caused by Bacillus cereus?
(e.g. to eye), and those who receive intravenous
a. Meat b. Milk
injections
Diseases: Emetic (vomiting) and diarrheal forms c. Eggs d. Rice
of gastroenteritis; ocular infection; and other
opportunistic infections Answers (MCQs)
1. a; 2. c; 3. a; 4. a; 5. d
30
Chapter
Clostridium
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss morphology, cultural characteristics of
be able to: C. tetani
∙∙ Describe classification of clostridia and diseases ∙∙ Describe toxins produced by C. tetani
produced by different clostridia ∙∙ Describe the following: Pathogenesis of tetanus;
∙∙ Discuss morphology, cultural characteristics of prophylaxis of tetanus
C. welchii ∙∙ Discuss morphology and cultural characteristics
∙∙ Discuss Nagler reaction of C. botulinum
∙∙ Discuss lab. diagnosis and prophylaxis of gas ∙∙ Discuss lab. diagnosis of botulinum
gangrene ∙∙ Describe the following: gas gangrene; botulinum
toxin; Clostridium difficile.
www.ebook3000.com
212 | Section 3: Systemic Bacteriology
in dried earth or dust. All species are killed by Table 30.1 Clostridia as human pathogens
autoclaving at 121°C within 20 minutes. Among
A. The gas gangrene group
hospital disinfectants the greatest sporicidal
1. Established • C. perfringens
activity is shown by alcoholic hypochlorite and pathogens • C. septicum
glutaraldehyde. • C. novyi
In general, clostridia are susceptible to 2. Less pathogenic • C. histolyticum
metronidazole, penicillin chloramphenicol • C. fallax
3. Doubtful pathogens • C. bifermentans
and erythromycin; less so to tetracyclines, and
• C. sporogenes
resistant to aminoglycosides and quinolones.
5. Diseases produced: Clostridia are more B. Tetanus C. tetani
commonly associated with skin and soft tissue C. Food poisoning
infections, food poisoning, and antibiotic- 1. Gastroenteritis C. perfringens (Type A)
associated diarrhea and colitis (Table 30.1). 2. Necrotizing enteritis C. perfringens (Type C)
3. Botulism C. botulinum
D. Acute colitis C. difficile
Classification
The traditional method for classifying an isolate in
the genus Clostridium was based on a combination
of diagnostic tests, including the demonstration of
spores, optimal growth in anaerobic conditions, a
complex pattern of biochemical reactivity such as
saccharolytic and proteolytic capacities and the
findings yielded by gas chromatography analysis
of the metabolic byproducts. With these methods,
more than 130 species have been defined.
Fortunately, most of the clinically important
isolates fall within a few species. Fig. 30.1: Reverse CAMP test
Toxins
C. perfringens is one of the most prolific of toxin-
producing bacteria, forming at least 12 distinct
soluble substances or toxins, all of which are of
protein in nature and antigenic. Major lethal toxins
include: α (alpha), b (beta), e (epsilon) and i (iota)
and minor lethal toxins include: g (gamma), d
(delta), k (kappa), l (lambda), m (mu), n (nu), q
(theta) and h (eta). The four 'major toxins', alpha,
Fig. 30.2: Nagler’s reaction. C perfringens colonies on the
beta, epsilon and iota, are predominantly respon right half of the plate are surrounded by haloes, while colonies
sible for pathogenicity. on the left half (containing antiserum to alpha toxin) have no
haloes around them
Classification Interpretation: On the section containing no
C. perfringens can be divided into five types, A to antitoxin, C. perfringens colonies show surrounding
E on the basis of four major toxins. Strains of C. zone of opalescence, i.e. Nagler reaction. There
perfringens type A that produce enterotoxin are will be no opacity around the colonies on the half
associated with a mild form of food poisoning. of the plate with the antitoxin, due to the specific
neutralization of the alpha toxin (Fig. 30.2).
Alpha Toxin This reaction, however, is not totally specific
The alpha (a) toxin is produced by all types of for C. perfringens since the opalescence in the egg
C. perfringens and most abundantly by type A yolk media may be produced by other lecithinase
strains, is a lecithinase (phospholipase C) that lyses forming bacteria (C. novyi, C. bifermentans, some
erythrocytes, platelets, leukocytes, and endothelial vibrios, some aerobic spore bearers). The reaction
cells. It is lethal, dermonecrotic and hemolytic for produced by C. perfringens is specifically neutralized
the red cells of most species, except horse and by C. perfringens antitoxin, but serologically related
goat. The lysis is of the hot-cold variety, being best phospholipases of C. bifermentans and ‘C. sordellii
seen after incubation at 37°C followed by chilling and some other phospholipases are also inhibited.
at 4°C. This toxin increases vascular permeability, These organisms can be separated by other tests.
resulting in massive hemolysis and bleeding,
tissue destruction, hepatic toxicity, and myocardial Other Major Toxins
dysfunction. It is relatively heat stable. Beta (β), Epsilon (e) and iota (i) toxins have lethal
and necrotizing properties.
Nagler’s Reaction
Minor Toxins
Basis: The alpha (a) toxin is lecithinase C (or
phospholipidase C) splits lecithin into phosphoryl Gamma and eta toxins have minor lethal toxins.
choline and diglyceride, in the presence of Ca++ and Delta toxin has a lethal effect and is hemolytic for
Mg++ ions because the toxin is activated by Ca++ and the red cells of even- goat, pigs and cattle. Theta
Mg++ ions. This reaction is seen as opalescence in toxin is oxygen-labile hemolysin antigenically
serum or egg yolk media and is specifically neutral related to streptolysin O. It is also lethal and a
ized by the antitoxin. This is the basis of Nagler general cytolytic toxin.
reaction.
Enterotoxin
Procedure: For rapid detection of C. perfringens, a
C. perfringens type A strains produce a potent
culture plate containing 6% agar, 5% peptic digest
enterotoxin which causes diarrhaea and other
of sheep blood and 20% human serum or 5% egg-
symptoms of food poisoning.
yolk is prepared. The incorporation of neomycin
sulphate in the medium makes it more selective,
Pathogenesis
inhibiting coliforms and aerobic spore bearers.
On one half of the plate, 2–3 drops of C. 1. Soft Tissue Infections
perfringens antitoxin are spread and allowed to dry. Soft tissue infections caused by C. perfringens
The plate is then inoculated with the test organisms are subdivided into (1) cellulitis, (2) fasciitis or
or the exudate under investigation and incubated suppurative myositis, and (3) myonecrosis or gas
anaerobically at 37°C for 18 hours. gangrene.
www.ebook3000.com
214 | Section 3: Systemic Bacteriology
Clostridial myonecrosis or gas gangrene: The 5. C. perfringens Colitis
disease is characterized by rapidly spreading edema, A sporadic diarrheal syndrome, usually occurring in
myositis, necrosis of tissues, gas production and elderly patients during treatment with antibiotics,
profound toxemia occurring as a complication of has been described.
wound infection. The disease has been referred to in
the past as ‘malignant edema’. Other descriptive terms 6. Clostridial Endometritis
that have been used are ‘anaerobic (clostridial)
This condition is a grave complication of incom
myositis’ and ‘clostridial myonecrosis.
plete abortion, or the use of inadequately sterilized
Etiology: Amongst the pathogenic clostridia, instruments.
C. perfringens is the most frequently encountered
(app rox imately 60%), and C . n ov y i and Laboratory Diagnosis
C. septicum being the next common (20–40%), and
Gas gangrene is a medical emergency. The diagnosis
C. histolyticum less often. Other clostridia usually
of gas gangrene must be made primarily on clinical
found are C. sporogenes, C. fallax, C. bifermentans,
grounds, and the function of the laboratory is only
C. sordelli, C. aerofoetidum and C. tertium.
to provide confirmation of the clinical diagnosis
Mechanism of infection: Clostridial spores are as well as identification and enumeration of the
introduced into tissue, e.g. by contamination with infecting organisms.
dirt, or by endogenous transfer from the intestinal
tract. After injury there is incubation period may A. Specimens
be as short as seven hours or as long as six weeks,
(1) Edge of the affected muscles; (2) Exudates from
usually of 12–48 hours.
the wound; and (3) Necrotic tissue and muscle
When there is considerable cell injury or
fragments.
compromise of circulation, germination and
outgrowth of clostria spores occurs. Alpha toxin and
B. Microscopy
other exotoxins are secreted and extensive cell killing
ensues. The production of enzymes that break down Gram stained films give presumptive information
ground substance facilitates the spread of infection. about the species of clostridia present and their
Fermentation of tissue carbohydrates yields gas, and relat ive numbers. If gas gangrene is present,
an accumulation of gas bubbles in the subcutaneous Gram-positive rods may predominate. Thick,
spaces produces a crinkling sensation on palpation stubby, Gram-positive rods suggest C. perfringens
(crepitation), hence the name gas gangrene. or C. sordellii, ‘citron bodies’, boat- or leafshaped
pleomorphic bacilli with irregular staining, may
2. Septicemia indicate C. septicum; slender rods with round
terminal spores suggest C. tetani and large rods with
Invasion of the bloodstream may occur in associ oval sub terminal spores indicate C. novyi.
ation with malignancy and may involve a localized
myonecrosis in addition to a fulminating clostridial C. Culture
septicemia.
Fresh and heated blood agar are used for aerobic
and anaerobic cultures. To prevent swarming
3. Food Poisoning by some species of clostridia, the use of plates
The organisms usually involved are strains of type containing increased agar (5-6%) are considered. A
A. Meat, chicken, fish and their by-products are the plate of serum or egg yolk agar, with C. perfringens
most common vehicles for clostrial food poisoning. antitoxin spread on one half is used for the ‘Nagler
Clostridial food poisoning, is characterized by (i) a reaction’.
short incubation period (8–24 hours), (ii) a clinical Four tubes of cooked meat broth are inoculated
presentation that includes abdominal cramps and and heated at 100°C for 5, 10, 15 and 20 minutes,
watery diarrhea but no fever, nausea, or vomiting, incubated and subcultured on blood agar plates
and (iii) a clinical course lasting less than 24 hours. after 24–48 hours, to differentiate the organisms
The illness is self-limited and recovery occurs in with heat resistant spores. Blood cultures are
24–48 hours. often positive, especially in C. perfringens and
C. septicum infections. However, C. perfringens
4. Enteritis Necroticans (Necrotizing bacteremia may occur without gas gangrene.
Jejunitis, Necrotic Enteritis)
This is severe and often fatal enteritis known by D. Identification
different names in different countries: β-toxin- Examine plates for typical colonies. The isolates are
producing C. perfringens type C is responsible for identified based on their morphological, cultural,
this disease. biochemical and toxigenic characters.
Chapter 30: Clostridium | 215
Animal Pathogenicity
0.1 mL 24 hours growth in cooked meat broth is
injected into a healthy guinea pig by intramuscular
route. The animal dies within 24 hours. A control
animal protected with antiserum prior to test is also
included. On autopsy, bacteria can be recovered
from heart and spleen of the test animal.
www.ebook3000.com
216 | Section 3: Systemic Bacteriology
Somatic (O) antigen: There is a single somatic tetanus’, are demonstrable in experimental animals,
agglutination group for all strains that permits but the stages tend to merge in their clinical present
identification of the organism. ation in man.
www.ebook3000.com
218 | Section 3: Systemic Bacteriology
Clostridium botulinum The toxin is relatively stable being inactivated
at 80°C for 30–40 minutes and at 100°C for 10
C. botulinum (from the Latin botulus, “sausage”) minutes. Food suspected to be contaminated with
causes botulism. Botulism is a severe, often fatal, botulinum toxin can be rendered completely safe
form of food poisoning characterized by pronounced by pressure cooking or boiling for 20 minutes. It can
neurotoxic effects. The disease has been caused be toxoided. It is a good antigen and is specifically
by a wide range of foods, the disease can be a pure neutralized by the antitoxin.
intoxication. Botulinum toxin gains access to the peripheral
nervous system, where it acts preferentially on
Morphology cholinergic nerve endings to block the release of
C. botulinum is a strictly anaerobic gram-positive the neurotransmitter, acetylcholine, from the nerve
bacillus (about 5 × 1 mm). It is non-capsulated, terminals of neuromuscular junctions. A symmetric
motile with peritrichous flagella and produces descending paralysis is the characteristic pattern,
spores which are oval, subterminal and bulging. ending in death bv respiratory paralysis.
www.ebook3000.com
220 | Section 3: Systemic Bacteriology
Nonsporing Anaerobes
Learning Objectives
After reading and studying this chapter, you should ∙∙ List infections caused by nonsporing anaerobes
be able to: ∙∙ Discuss laboratory diagnosis of infections caused
∙∙ Describe classification of nonsporing anaerobes by nonsporing anaerobes.
www.ebook3000.com
222 | Section 3: Systemic Bacteriology
They produce black colonies on blood agar on They are found as part of the normal flora of
prolonged incubation due to the production of the mouth, stomach, intestines, and genitourinary
H2S. They occur as normal flora of skin, intestine tract. Lactobacilli are normally present in the mouth
and genitourinary tract. They may cause pyogenic and have been incriminated in the pathogenesis
infections of wounds, puerperal sepsis and urinary of dental caries. It is believed that lactobacilli
tract infections. ferment sucrose to produce lactic acid, which
dissolves the mineral components of enamel and
Peptostreptococcus dentine causing dental caries.
Several species of lactobacilli are present in the
They are cocci of small size (0.2–2.5 µm). Many of
intestine, the most common being L. acidophilus.
them are aerotolerant and grow well under 10%
Intestinal lactobacilli are beneficial in synthesizing
CO2 in an aerobic atmosphere.
vitamins, such as biotin, vitamin B12 and vitamin K,
Peptostreptococcus anaerobius is most often
which may be absorbed by the host.
responsible for puerperal sepsis and Pst. magnus
for abscesses. Lactobacillus species are major members of
the normal flora of the vagina and, typically, in
the adult vagina and these are collectively known
Gram-negative anAerobic cocci as Doderlein’s bacilli. They ferment the glycogen
Veillonella deposited in the vaginal epithelial cell and form
lactic acid, which accounts for the highly acidic
Veillonellae are gram-negative cocci of varying
pH of vaginal mucus epithelia. They protect
sizes, and measure 0.3–2.5 μm in diameter.
adult vagina from infections. In prepubertal and
Occurring as diplococci, short chains or groups.
postmenopausal vagina, lactobacilli are scanty.
They are normal inhabitants of the mouth,
It is generally nonpathogenic though L. cat
intestinal and genital tracts. Veillonella parvula
enaforme has been associated with bronchopulmo
has been reported from clinical specimens but its
nary infections. Lactobacilli are acidophilic
pathogenic role is uncertain.
and grow best at acidic pH. Some species have
strict growth requirements and are used for the
Anaerobic, nonspore-forming, microbiological assay of growth factors (vitamins).
gram-positive bacilli
This group contains many genera, of which medically 4. Mobiluncus
relevant are Eubacterium, Propionibacterium, Members of the genus Mobiluncus are obligate
Lactobacillus, Mobiluncus, Bifidobacterium and anaerobic, gram-variable or gram-negative, curved
Actinomyces. bacilli with tapered ends. Two species, Mobiluncus
curtisii and Mobiluncus mulieris, have been identified
1. Eubacterium in humans.
Mobiluncus curtisii and Mobiluncus mulieris
Members of the genus Eubacterium are strictly
have been isolated from the vagina in bacterial
anaerobic and grow very slowly. They are members
vaginosis, along with Gardnerella vaginalis. The
of the normal mouth and intestinal flora. Some
organisms colonize the genital tract in low numbers
species (E. brachy, E. timidum and E. nodatum)
but are abund ant in women with bacterial
are commonly seen in periodontitis. E. lentum is
vaginosis. Their microscopic appearance is a useful
commonly isolated from nonoral clinical specimens.
marker for this disease, but the precise role of these
organisms in the pathogenesis of bacterial vaginosis
2. Bifidobacterium is unclear.
Bifidobacterium is gram-positive bacilli nonmotile,
nonsporing and pleomorphic, showing true and 5. Propionibacterium
false branching. They occur as normal flora of Propionibacterium species are members of the
mouth, gastrointestinal tract (GI) tract and genito normal flora of the skin and cause disease when
urinary tract. They are usually nonpathogenic. they infect plastic shunts and appliances. Their
metabolic products include propionic acid, from
3. Lactobacillus which the genus name derives.
Lactobacilli are gram-positive bacilli, straight or Species: The two most commonly isolated species
slightly curved which frequently show bipolar and are Propionibacterium acnes and Propioni
barred staining. They are nonsporing and most bacterium propionicus.
strains are nonmotile. They form considerable P. acnes are responsible for acne and
amount of lactic acid from carbohydrates and grow oportunistic infections in patients with prosthetic
best at pH of 5 or less. devices or intravascular lines.
Chapter 31: Nonsporing Anaerobes | 223
Propionibacterium propionicus mouth and upper alimentary and respiratory tracts.
Causes lacrimal canaliculitis and abscesses. Its cell wall contains a strong endotoxin. Prevotella
infections are usually associated with the upper
6. Actinomyces—See Chapter 35. respiratory tract, causing, for example, dental
and sinus infections, pulmonary infections and
Anaerobic gram-negative bacilli abscesses, brain abscesses, and infections caused
Gram-negative, anaerobic,nonsporeforming, by a human bite.
non motile rods were previously classified in the Laboratory identification is similar to that of B.
the family Bacteroidaceae within three genera, tragilis. In addition, P. melaninogenica forms black
Bacteroides, Fusobacterium and Leptotrichia. colonies on blood agar—a characteristic from which
its name was derived. The color is not due to the
1. Bacteroides melanin pigment as was once thought. Cultures of
The genus Bacteroides can be divided. P. melaninogenica and even dressings from wounds
infected with the bacillus give a characteristic red
a. Bile-tolerant fluorescence when exposed to ultraviolet light. P.
i. Members of the B. fragilis group—B. fragilis, B. melaninogenica exhibits less drug resistance than
ovatus, B. distasonis, B. vulgatus, B. thetaiotao do the bacteroides, and are usually susceptible to
micron, and others. penicillin.
ii. Two other species—Bacteroides eggerthii and
Bacteroides splanchnicus. 2. Porphyromonas
b. Bile Sensitive species Porphyromonas species can be cultured from
i. Pigmented: Have been reclassified into—the gingival and periapical tooth infections and, more
genera Porphyromonas, Prevotella and commonly, breast, axillary, perianal, and male
porphyromonas. genital infections.
ii. Nonpigmented species: Many nonpigmented
species of Bacteroides also have been transferred ii. Nonpigmented Species
to the genus Prevotella. Many nonpigmented species of bacteraies have
been transferred to the genus prevotella.
2. Fusobacterium (Bacilli with pointed ends) Nonpigmented Prevotella spp. includes
Fusobacterium necrophorum, Fusobacterium Prevotella bivia, Prevotella buccae, etc.
nucleatum, Fusobacterium necrogenes.
www.ebook3000.com
224 | Section 3: Systemic Bacteriology
ulcerative gingivitis (Vincent’s gingivitis) or A. Specimen Collection and Transport
Vincent’s angina, together with Treponema, Specimens should be collected in such a manner as
Porphyromonas and Fusobacterium species. It is to avoid resident flora. For example, from a suspected
seen in patients with malnutrition, debility and case of lung abscess the sputum is unsatisfactory for
poor oral hygiene. It is characterized by pain, culture and only material collected by aspiration
hemorrhage, foul odor, destruction of interdental. would be acceptable. In general, material for
Vincent’s angina may resemble diphtheria, anaerobic culture is best obtained by tissue biopsy or
with the inflamed pharyngeal mucosa showing a by aspiration using a needle and syringe. Aspirated or
grayish membrane which peels easily. l. buccalis tissue specimens are preferable to swabs whenever
and T. vincentii are present in the exudate and feasible. Swabs are generally unsatisfactory but
pseudomembrane, and diagnosis is made by direct where they are to be used, they should be sent in
microscopy. Stained smears show large fusiform Stuart’s transport medium.
and spiral bacilli. Ideally, specimens should be placed in an
anaerobic transport device that consists of a
Anaerobic infections tube or vial containing an anaerobic gas mixture
substituted for air. Specimens should be delivered
Most of the anaerobic bacteria that cause infection
immediately (within 20 minutes) for culture.
are members of our normal indigenous flora.
Recapping a syringe and transporting the needle
Anaerobic infections are usually endogenous and
and syringe to the laboratory is no longer acceptable
are caused by tissue invasion by bacteria normally
because of safety concerns involving needle stick
resident on the respective body surfaces. Anaerobic
injuries. Therefore, even aspirates must be injected
bacteria are normally present on the skin, mouth,
into some type of oxygen-free transport tube or vial.
nasopharynx and upper respiratory tract, intestines
In some laboratories, gas-liquid chromatography
and vagina (Table 31.2).
is carried out directly on pus and other clinical
Anaerobic infections generally follow some specimens in order to detect metabolic products,
precipitating factor, such as trauma, tissue necrosis, such as butyric and propionic acids, that are
impaired circulation, hematoma formation or the characteristic of certain anaerobes.
presence of foreign bodies. Diabetes, malnutrition,
malignancy or prolonged treatment with aminoglyco B. Direct Microscopy
side antibiotics, corticosteroids and cytotoxic agents
Examination of a gram stained smear is very useful.
may act as predisposing factors. Anaerobic infections
Pus in anaerobic infection usually shows a large
are typically polymicrobial.
variety of different organisms and numerous pus
Table 31.3 lists the common sites and type of cells.
anaerobic infections and the bacteria responsible. Examination of the specimen under ultraviolet
light may show the bright red fluorescence of P.
Laboratory Diagnosis melaninogenica.
As anaerobes form part of the normal flora of the
skin and mucous surfaces, their isolation from C. Culture
specimens has to be interpreted cautiously. The Several special media have been described for
mere presence of an anaerobe does not prove its anaerobes but for routine diagnostic work, freshly
causal role. prepared blood agar with neomycin, yeast extract,
hemin and vitamin K is adequate. Plates are incu 3. An indication of antimicrobial agents likely to
bated at 37°C in an anaerobic jar, with 10% CO2. The be used.
Gas-Pak system provides a convenient method of
routine anaerobic cultures. Plates are examined after E. Antibiotic Sensitivity Tests
24 or 48 hours. Some anaerobes, such as fusobacte Antibiotic sensitivity tests can be done by disc
ria, require longer periods of incubation. Since many diffusion or dilution methods.
anaerobes are relatively slow-growing, it is essential
that cultures are incubated for several days before F. Other Anaerobic Techniques
being discarded. In mixed infections, fast-growing Gloved anaerobic chambers with continuous gas
aerobic or facultatively anaerobic organisms are flow may be used for culture of specimens. Pre-
often detected within 24 hours, whereas some reduced anaerobically sterilised media (PRAS) can
anaerobes may require incubation for 7–10 days also be employed but are not essential for rountine
before their colonies can be recognized. diagnostic procedures.
Other anaerobic media, such as cooked meat
broth (CMB) and thioglycollate broth, may also Treatment of Anaerobic Infections
be used for incoculating the specimens. Parallel Treatment of mixed anaerobic infections is by
aerobic cultures (such as Pseudomonas aeruginosa surgical drainage (under most circumstances) plus
should always be set up. This is necessary as a antimicrobial therapy.
control for the growth on anaerobic plates and The most active drugs for treatment of anaerobic
also because in most anaerobic infections aerobic infections are clindamycin and metronidazole.
bacteria are also involved. Clindamycin is preferred for infections above the
D. Identification diaphragm. Penicillin G remains the drug of choice
Colony morphology, pigmentation, and fluorescence for treatment of anaerobic infections that do not
are helpful in identifying anaerobes. Biochemical involve β-lactamase-producing Bacteroides and
activities and production of short-chain fatty acids Prevotella species.
as measured by gas-liquid chromatography are used
Key Points
for laboratory confirmation. It takes time and is
difficult, but it is possible to report on the following: Many anaerobic bacteria are pathogenic for human
1. Whether the infection is solely aerobic, anaerobic beings, and they outnumber aerobic bacteria in
or mixed. many habitats
Anaerobic gram-positive cocci: Most of important
2. The identification of the more common anaero anaerobic cocci belong to the genus Peptostreptococcus
bes, particularly of B. fragilis.
www.ebook3000.com
226 | Section 3: Systemic Bacteriology
b. Lactobacillus
Anaerobic gram-negative cocci: Veillonella parvula c. Bacteroides
is the species frequently reported from clinical d. Fusobacterium
specimens e. Anaerobic gram-positive bacilli.
Anaerobic nonspore-forming gram-positive
bacilli: Include Eubacterium, Propionibacterium,
Lactobacillus, Bifidobacterium and Mobiluncus Multiple choice questions (MCQs)
Anaerobic gram-negative bacilli: Medically
important anaerobic gram-negative bacilli belong 1. The following is an example of gram-negative
to the family Bacteroidaceae and are classified into anaerobic cocci
the genera Bacteroides, Prevotella, Porphyromonas, a. Veillonella
Fusobacterium and Leptotrichia b. Peptococcus
Laboratory diagnosis: Specimens should be placed c. Peptostreptococcus
in an anaerobic transport device. Examination of d. Bacteroides
a Gram stained smear is very useful. The Gas-Pak 2. Lactobacilli constitute the normal flora of:
system provides a convenient method of routine a. Adult vagina
anaerobic cultures. Other anaerobic media, such b. Prepubertal vagina
as cooked meat broth (CMB) and thioglycollate c. Post-menopausal vagina
broth, may also be used. Colony morphology, d. None of the above
pigmentation, and fluorescence are helpful in
3. All the following anaerobic bacteria cause head
identifying anaerobes. Biochemical activities and
production of short-chain fatty acids as measured
and neckinfections except.
by gas-liquid chromatography are used for labora a. Bacteroides thetaiotaomicron
tory confirmation. b. Fusobacterium nucleatum
c. Fusobacterium necrophorum
d. Porphyromonas asaccharolytica
Important questions 4. Which of the following bacterial colonies fluoresce
brick-red in UV light?
1. Classify nonsporing anaerobes. Discuss the a. Bacteroides fragilis
laboratory diagnosis of infections caused by b. Bacteroides melaninogenicus
nonsporing anaerobes. c. Bacteroides gingiva/is
2. Give an account of infections caused by nonsporing d. Bacteroides levii.
anaerobes.
3. Write short notes on: Answers (MCQs)
a. Anaerobic cocci 1. (a); 2. (a); 3. (a); 4. (b)
32
Chapter
Mycobacterium tuberculosis
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Differentiate between Mycobacterium tuberculosis
be able to: and M. bovis
∙∙ Classify mycobacteria ∙∙ Describe pathogenesis of Mycobacterium tuberculosis
∙∙ Discuss morphology and culture characteristics ∙∙ Discuss the laboratory diagnosis of pulmonary
and biochemical characteristics of Mycobacterium tuberculosis
tuberculosis ∙∙ Describe the following: Koch’s phenomenon;
Tuberculin test; BCG vaccine.
www.ebook3000.com
Chap-32.indd 227 15-03-2016 11:13:50
228 | Section 3: Systemic Bacteriology
tuberculosis and Hansen’s disease. A large group
of mycobacteria, excluding the M. tuberculosis
complex and M. leprae, normally inhabit the
environment and can cause disease that often
resembles tuberculosis in humans. These organ
isms are sometimes referred to as atypical
mycobacteria or mycobacteria other than the
tubercle bacillus (MOTI). Classification of mycobac
teria are shown in Table 32.1.
MYCOBACTERIUM TUBERCULOSIS
Morphology
M. tuberculosis is a slender, straight, or slightly
curved rod with rounded ends, about 3 µm × 0.3 µm, Fig. 32.1: Mycobacterium tuberculosis in Ziehl–Neelsen
in pairs or as small clumps. The bacilli are nonmotile, stained smear
nonsporing, noncapsulated and acid-fast.
When stained with carbol fuchsin by the Ziehl–
Neelsen method or by fluorescent dyes (auramine
O, rhodamine), they resist decolorization by 20%
sulphuric acid and absolute alcohol for 10 minutes
(acid and alcohol fast). With Ziehl–Neelsen acid-
fast stain, the tubercle bacilli stain bright red, while
the tissue cells and other organisms are stained
blue (Fig. 32.1).
Acid fastness has been ascribed variously to
the presence in the bacillus of an unsaponifiable
wax (mycoloic acid) or to a semi permeable
membrane around the cell. It is related to the
integrity of the cell and appears to be a property
of the lipid-rich waxy cell wall. Staining may be
uniform or granular. In M. tuberculosis beaded or
barred forms are frequently seen, but M. bovis stains
more uniformly. M. bovis appear straighter, stouter Figs 32.2: L–J media without growth (A) and with growth (B)
and shorter with uniform stainin (Table 32.2).
other bacteria and to provide a contrasting color
Cultural Characteristics against which colonies of mycobacteria are easily
seen. In this medium egg acts as a solidifying agent.
M. tuberculosis is an obligate aerobe while M. The addition of 0.5% glycerol improves the growth
bovis is microaerophilic on primary isolation, of M. tuberculosis, but has no effect on or may even
becoming aerobic on subculture. The optimal impair the growth of M. bovis. Sodium pyruvate
growth temperature of tubercle bacilli is 35–37°C. helps the growth of both types (Figs 32.2A and B).
Optimum pH is 6.4–7.0. The bacilli grow slowly, Human tubercle bacilli produce visible growth
the generation time in vitro being 14–15 hours. on LJ medium in about 2 weeks, although on
Colonies appear in about two weeks and may primary isolation from clinical material colonies
sometimes take up to eight weeks. may take up to 8 weeks to appear. On solid media,
M. tuberculosis forms dry, rough, raised, irregular
Solid Medium colonies with a wrinkled surface. They are creamy
Tubercle bacilli are able to grow on a wide range of white, becoming yellowish or buff colored on
enriched culture media and do not have exacting further incubation. They are tenacious and not
growth requirements. The solid media contain egg easily emulsified. Mycobacterium tuberculosis has
(Lowenstein Jensen, Petragnini, Dorset), blood a luxuriant growth (eugenic growth) as compared
(Tarshis), serum (Loeffler) or potato (Pawlowsky). to Mycobacterium bovis which grows poorly on LJ
The solid medium most widely employed glycerol medium (dysgonic growth) and colonies,
for routine culture is Lowenstein–Jensen (L–J) in comparison are flat, smooth, moist, white and
medium. This consists of coagulated hens’ egg, break up easily when touched. The growth of M.
mineral salt solution, asparagine and malachite bovis is much better on LJ pyruvate medium (media
green, the last acting as a selective agent inhibiting containing sodium pyruvate in place of glycerol).
www.ebook3000.com
Chap-32.indd 229 15-03-2016 11:13:51
230 | Section 3: Systemic Bacteriology
However, not all srains produce a positive
reaction after the culture is heated to 68° C for
20 minutes.
4. Tween 80 hydrolysis: Some mycobacteria
possess a lipase that can split detergent Tween
80 into oleic acid and polyoxyethylated sorbitol
which modifies the optical characteristics of
the test solution from a straw yellow to pink
whereas pathogenic species do not. This test is
useful in distinguishing scotochromogenic and
Fig. 32.3: Cell wall of Mycobacterium tuberculosis
nonchromogenic mycobacteria. M. tuberculosis
gives variable result.
5. Arylsulfatase test: The enzyme is present in of the cell wall. The agglutinogen antigens
most mycobacteria. This test is positive only have been identified as the sugar moieties on
with atypical mycobacteria. mycosides.
6. Neutral red test: M. tuberculosis, M. bovis, M. The cell wall antigens include arabinomanan,
avium and M. ulcerans give positive tests. arabinogalactan and lipoarabinomannan.
7. Amidase tests: Atypical mycobacteria can be
differentiated by their ability to split amides. 2. Cytoplasmic Antigens
A useful pattern is provided by testing five Cytoplasmic antigens are protein antigens which
amides, viz. acetamide, benzamide, carbamide, are employed to type the mycobacteria. These
nicotinamide and pyrazinamide. include antigen 5, antigen 6, antigen 14, antigen
M. tuberculosis produces nicotinamidase and 19, antigen 32, antigen 38 and antigen 60. All are
pyrazinamidase which splits nicotinamide and protein in nature except antigen 60 which is a
pyrazinamide. lipopolysaccharide protein complex.
8. Susceptibility to pyrazinamide: M. tuber
culosis is sensitive to pyrazinamide, while M.
bovis and other mycobacteria are resistant. Mycobacteriophages
9. Growth inhibition by thiophen-2-carboxylic Numerous phages with activities on many
acid hydrazide (TCH): This test is used to mycobacterial species, including M. tuberculosis, have
distinguish M. bovis from M. tuberculosis, been isolated from both clinical and environmental
because only M. bovis is usually susceptible sources. Many mycobacteriophages have been
while M. tuberculosis is usually not susceptible isolated from soil, water and other environmen
to this chemical. tal sources as well as from lysogenic strains. Many
mycobacteria infected with phage are not truly lyso
Antigenic Structure genic. Instead of being integrated with the bacterial
Mycobacteria possess two types of antigens, chromosome, the phage genome appears free, like
cell wall (insoluble) and cytoplasmic (soluble) a plasmid. This is called pseudolysogeny.
antigens. M. tuberculosis has been classified into four
phage types - A,B,C, and I (a type intermediate
1. Cell Wall Antigens between A and B). Phage type A is the most common
and is present worldwide. Type B is common in
The cell wall consists of lipids, proteins and
Europe, the Middle East and North America. Type
polysaccharides. The lipid content accounts for
C is seen rarely. Type I is common in India and
60% of the cell wall weight. The cell wall is made
neighboring countries. Phage 33D can lyse M.
up of four distinct layers (Fig. 32.3).
tuberculosis, M. bovis, M. africanum and M. microti.
i. Peptidoglycan (murein) layer: It is the
innermost layer which maintains the shape
and rigidity of the cell. Pathogenesis
ii. Arabinogalactan layer: It lies external to the The source of infection is usually an open case of
peptidoglycan layer. pulmonary tuberculosis. The mode of infection is
iii. Mycolic acid layer: It is the principal constituent by direct inhalation of aerosolized bacilli contained
of cell wall and is a dense band on long chain in droplet nuclei of expectorated sputum tubercle
a-alkyl and β-hydroxy fatty acids attached by bacilli are acquired from persons with active
ester bonds to the terminal arabinose units of disease who are excreting viable bacilli by means
arabinogalactan. of coughing, sneezing, or talking. Infection also
iv. Mycosides (peptidoglycolipids or phenolic occurs infrequently by ingestion, for example,
glycolipids): These form the outermost layer through infected milk, and rarely by inoculation.
www.ebook3000.com
Chap-32.indd 231 15-03-2016 11:13:52
232 | Section 3: Systemic Bacteriology
2. Microscopy acid fast bacilli provides only presumptive evidence
Direct or concentration smears of sputum are of tuberculous infection, as even saprophytic
examined. Smears should be prepared from the mycobacteria may present a similar appearance.
thick purulent part of the sputum. Smears are
dried, heat fixed and stained by the Ziehl–Neelsen 3. Concentration Methods
technique (hot stain procedure). Several methods have been described for the
In the Ziehl–Neelsen (Z–N) staining technique, homogenization and concentration of sputum
heat-fixed smears of the specimens are flooded and other specimens. Concentration methods
with a solution of carbol fuchsin (a mixture of basic that do not kill the bacilli and so can be used for
fuchsin and phenol) and heated until steam rises culture and animal inoculation. Several methods
but without boiling. Allow the preparation to stain are in use:
for 5 minutes, applying heat at intervals to keep
i. Petroff’s method: This simple method is widely
the stain hot. The slide is then washed with water
used. Equal volumes of sputum and 4% sodium
and decolorized with 20% sulphuric acid till no
hydroxide are mixed and incubated at 37°C with
more stain comes off and then with 95% alcohol
frequent shaking till it gets liquefied and beco
(ethanol) for two minutes. Decolorization may be
mes clear. It is then centrifuged at 3,000 r.p.m.
carried out as a single step with acid alcohol (3%
for 30 minutes. The supernatant fluid is pipetted
HCl in 95% ethanol). After washing, the smear is
off and the deposit is neutralized by adding 8%
counterstained with Loeffler’s methylene blue, I%
hydrochloric acid in the presence of a drop of
picric acid or 0.2% malachite green for one minute.
phenol red indicator and used for smear, culture
Under the oil immersion objective, acid fast
and animal inoculation.
bacilli are seen as bright red rods while the
background is blue, yellow or green depending on ii. Other methods: Instead of alkali, homogenization
the counterstain used. At least 10,000 acid fast bacilli can be achieved by treatment with dilute acids (6%
should be present per mL of sputum for them to be sulphuric acid, 3% hydrochloric acid or 5% oxalic
readily demonstrable in direct smears. acid), N acetyl cysteine with NaOH, pancreatin,
Smears should be examined carefully by desogen, zephiran and cetrimide.
scanning at least 300 oil immersion fields before
reporting a smear as negative (Table 32.3). 4. Culture
The classic carbolfuchsin (Ziehl–Neelsen) stain It is a very sensitive diagnostic technique for tubercle
requires heating the slide for better penetration of bacilli, detecting as few as 10–100 bacilli per mL. The
stain into the mycobacterial cell wall. Hence it is concentrated material is inoculated onto at least
also known as the hot stain procedure. Kinyoun two bottles of L–J medium. A direct drug sensitivity
acid-fast stain is similar to the Ziehl–Neelsen stain test may also be set-up if the specimen is positive
but without heat ; hence the term cold stain. by microscopy.
It is more convenient to use fluorescent
Inoculated media are incubated at 35–37°C.
microscopy. Smears are stained with auramine
Growth of most strains of M tuberculosis may
phenol or auramine rhodamine fluorescent
appear in 2–8 weeks.
dyes. Screening of specimens with rhodamine or
Cultures are examined for growth after
rhodamine-auramine will result in a higher yield
incubation at 37°C for four days (for rapid growing
of positive smears and will substantially reduce
mycobacteria, fungi and contaminant bacteria)
the amount of time needed for examining smears.
and at least twice weekly thereafter. Any bacterial
In general, specificity of acid-fast smear exami
growth is stained by the ZN method and, if acid-fast,
nation is very high. Microscopic demonstration of
it is subcultured for further identification. The first
Table 32.3 Methods for reporting numbers of acid step in identification is to determine whether an
fast bacilli observed in stained smears isolate is a member of the M. tuberculosis complex
(Fig. 32.4).
No. of bacilli Observed in (oil Report
immersion field)
For routine purposes, a slow growing, nonpig
mented, niacin positive acid fast bacillus is taken
0 300 fields Negative
as M. tuberculosis. Confirmation is by detailed
1–2 100 fields ± biochemical studies. When the isolate is niacin
1–9 100 fields 1+ negative, a battery of tests may be needed for
1–9 10 fields 2+ identification, including growth at 25°C and
1–9 1 field 3+
45°C, animal pathogenicity and biochemical tests
(Tables 32.2 and 32.4).
More than 9 1 field 4+
In recent years, isolation of mycobacteria from
Fields—Oil immersion field clinical samples has been improved by newer
Table 32.4 Some differential characteristics of tubercle bacilli causing human disease
Species Oxygen Glycerol Niacin Nitrate TCH Pyrazinamide Pathogenicity
preference enhanced reduction
www.ebook3000.com
Chap-32.indd 233 15-03-2016 11:13:52
234 | Section 3: Systemic Bacteriology
iii. The spleen is enlarged with irregular necrotic methods, such as column chromatography and
areas. Tubercles are seen in the peritoneum thin-layer chromatography, have been replaced
and sometimes in the lung, but the kidneys by gas–liquid chromatography and, most recently,
are unaffected. high-pressure liquid chromatography (HPLC).
The autopsy lesions have to be confirmed as Chromatography is rapid and highly reproducible,
tuberculous by acid fast staining of smears, to but the initial cost of equipment is high.
exclude Y. pseudotuberculosis, Brucella, Salmonella
and several fungi which may resemble the lesions of Extrapulmonary Tuberculosis
tuberculosis but will be smear negative. M. tubercu For diagnosis of extrapulmonary tuberculosis,
losis is highly pathogenic for guinea-pigs and virtu extrapulmonary tuberculosis microscopy, culture
ally nonpathogenic for rabbits, while M. bovis is and occasionally animal inoculation are also used,
highly pathogenic for both guinea-pigs and rabbits. though it is difficult to get conclusive results as the
Guinea pig inoculation, is now seldom resorted to bacilli are present in far fewer numbers in these
because it is cumbersome, costly and less sensitive lesions than in pulmonary disease.
than culture, particularly with catalase negative, CSF from tuberculous meningitis often develops
INH resistant strains isolated from south India. a spider web clot on standing, examination of
The animal inoculated with strains of low virulence which may be more successful than of the fluid.
may have to be observed for 12 weeks or longer and The use of PCR and DNA probes may help detect
sometimes the only lesion demonstrable may be an the bacilli speedily and more often.
enlarged lymph node. Bone marrow and liver biopsy specimens from
Guinea-pig inoculation is very rarely used miliary tuberculosis and blood from those with
nowadays. HIV coinfection are useful for culture. Pus from
6. Immunodiagnosis tuberculous abscesses often yields positive results
in smear and culture.
i. Serology: Various serological tests like enzy
Pleural effusion and other exudates may be
me-linked immunosorbent assay (ELISA),
collected with citrate to prevent coagulation. If free
radioimmunoassay (RIA) and latex agglutination
from other bacteria, they may be used for culture
have been tried for the serodiagnosis of
after centrifugation. If other bacteria are present,
tuberculosis. ELISA is considered to be the
prior concentration is necessary.
most sensitive and specific. ELISA test has been
Urinary excretion of bacilli in renal tuberculosis is
attempted using several antigenic materials
intermittent. Hence it is advisable to test 3–6 morning
such as antigen 5, antigen 6, antigen 60, purified
samples of urine. Each sample is centrifuged for 3000
mycobacterial glycolipids, unheated sterile
rpm for 30 minutes and the sediment used for culture
culture filtrate of M. tuberculosis and purified
after concentration.
protein derivative derived from M. tuberculosis.
ii. Tuberculin testing: Demonstration of hyper
sensitivity to tuberculoprotein (tuberculin Sensitivity Testing
testing) is a standard procedure. Its scope and Drug-resistant mutants continuously arise at a low
limitations are discussed below. rate in any mycobacterial population. The purpose
of sensitivity testing is to determine whether the
7. Hybridization and Nucleic Acid Technology great majority of the bacilli in the culture are
i. Nucleic acid probes: are available for the sensitive to the antitubercular drugs currently in
identification of the M. tuberculosis complex use. Several methods have been described:
and specifically, M. tuberculosis. 1. Absolute concentration method: Resistance is
ii. Polymerase chain reaction (PCR) and ligase expressed as the lowest concentration of drug
chain reaction (LCR) are used as diagnostic that inhibits all or almost all of the growth,
techniques. that is, the minimum inhibitor concentration
iii. Transcription mediated amplification, (MIC). This method is inferior to resistance
targeting ribosomal RNA has been introduced ratio method because known sensitive strain
as an improvement on PCR based DNA is not tested for MIC.
amplification. 2. Resistance ratio method: The resistance-ratio
iv. DNA ‘fingerprinting: Methods for epidemio method in which test strains and susceptible
logical purposes. controls, i.e. a known sensitive strain (H37Rv)
of M. tuberculosis are inoculated on sets of LJ
8. Chromatography medium containing doubling dilutions of drug.
The cell walls of Mycobacterium organisms contain The results are expressed as the ratio of the drug
long chain fatty acids called mycolic acids, which concentration inhibiting the test strain to that
may be detected chromographically. Earlier inhibiting the control strains.
www.ebook3000.com
Chap-32.indd 235 15-03-2016 11:13:53
236 | Section 3: Systemic Bacteriology
‘old tuberculin’ (OT). Initially Koch employed and other exanthematous reactions; 5. Occasionally
OT in the treatment of tuberculosis but it was after chemotherapy and removal of lung lesion; 6.
soon given up. Advanced age; 7. Immunosuppressive therapy
ii. Purified Protein Derivative (PPD): OT was and defective cell mediated immunity (CMI); 8.
first used for allergic (tuberculin) testing by von Lymphoreticular malignancy; 9. Sarcoidosis; 10.
Pirquet (1906). It was replaced by a partially Severe malnutrition; 11. False-negative results
purified protein antigen introduced by Seibert may also be due to inactive PPD preparations and
as OT was a crude product. This is known as the improper injection technique.
purified protein derivative (PPD).
Dose of PPD: Purified protein derivative (PPD) Uses of Tuberculin Test
is the skin test reagent that is primarily used to 1. To diagnose active infection in infants and
detect hypersensitivity in these persons. One large young children.
batch of PPD made by Seibert (PPD-S) in 1939 was 2. To measure prevalence of infection in an area .
recognized as the international standard PPD- 3. To select susceptibles for BCG vaccination.
tuberculin and arbitrarily designated to contain of 4. Indication of successful BCG vaccination.
OT or 0.00002 mg of PPD-S. In recent years in vitro interferon-γ release
Another PPD is RT-23 with tween 80. In India, assay (IGRA) have been introduced sensitive and
PPDRT-23 of strength 1TU and 2TU are available more specific to tuberculin test. This test is used in
1TU of PPD RT-23 is equivalent to 5TU of PPD-S. blood specimens which contains lymphocytes. It
uses ELISA for measuring interferon-γ production
Method by sensitized T lymphocytes.
1. Mantoux test: In the Mantoux test, 0.1 mL of
PPD containing 5 TU is injected intradermally Prophylaxis
on the flexo r aspect of the forearm with a In the prevention of tuberculosis general measures
tuberculin syringe. such as adequate nutrition, good housing and
A PPD dose of 1 TU is used when extreme health education are as important as specific anti
hypersensitivity is suspected; and doses of 10 bacterial measures. The latter consists of early
or 100 when 5 TU test is negative. detection and treatment of cases, BCG vaccination
Interpretation: Tuberculin tests should be and by chemoprophylaxis.
read 48–72 hours after injection. Induration of
diameter 10 mm or more is considered positive, BCG Vaccination
5 mm or less negative and 6–9 mm is of doubtful
Immunoprophylaxis is by intradermal injection of
significance because it may be due to other
the live attenuated vaccine developed by Calmette
mycobacterial infections.
and Guerin (1921), the Bacille Calmette Guerin
2. Heaf test: Multiple puncture testing as Heaf test
or BCG. This is a strain of M. bovis attenuated by
is done with a spring-loaded gun which fires
239 serial subcultures in a glycerine-bile-potato
six prongs into the skin through a drop of PPD
medium over a period of 13 years between the
(Heaf method). Single-test disposable devices
years 1908 and 1920 which was avirulent for man
with PPD dried onto prongs (tine tests) are also
while retaining its capacity to induce an immune
available for individual testing.
response. The foller widely used BCG strains
include pasteur 1173 P2, Tokyo-172, copenhagen
Result 1331 and Glaxo-1077.
I. Positive Test
Aim: The aim of BCG vaccination is to induce
A positive tuberculin test indicates hypersensitivity a benign, artificial primary infection which will
to tuberculoprotein denoting infection with tuber stimulate an acquired resistance to possible
cle bacillus or BCG immunization, recent or past, subsequent infection with virulent tubercle bacilli.
with or without clinical disease. The test becomes
positive 4–6 weeks after infection or immunization. Dose and administration: BCG vaccine is available
in liquid form and freeze-dried (lyophilized) form.
Freeze-dried (lyophilized) form is more stable
II. False-positive reactions
preparation and commonly used. The lyophilized
False–positive reactions may be seen in infections vaccine is reconstituted by sterile physiological
with related mycobacteria (‘atypical’ mycobacteria). saline to make a final concentration of 0.1 mg
(moist weight) in 0.1 mL of the vaccine and it is
III. False-negative Tests (Tuberculin Anergy) supplied by BCG vaccine laboratory,Chennai.
1. Early tuberculosis; 2. Advanced tuberculosis; Vaccine should be utilized within 3–6 hours once
3. Miliary tuberculosis; 4. In patients with measles reconstituted. The organisms grow to a limited
www.ebook3000.com
Chap-32.indd 237 15-03-2016 11:13:53
238 | Section 3: Systemic Bacteriology
Risk factors for drug resistance may include Liquid media are Dubos’, Middlebrook’s, Proskauer
previous treatment for TB, residence in an area and Beck’s, Sula’s and Sauton’s media are the more
endemic for drug resistance, or close contact with common
an individual who is infected with MDR-TB. Drug They are weakly catalase positive, neutral-red positive,
resistance is usually acquired by spontaneous amidase positive, nitrate reduction test positive, niacin
mutations as a result of the inappropriate use of test negative, and arylsulfatase negative
antimicrobial agents to treat M. tuberculosis and Pathogenesis and immunity: The source of infection
is usually an open case of pulmonary tuberculosis.
the lack of patient compliance. Another serious
The initial infection with M. tuberculosis is referred
condition extensively drug resistant-tuberculosis to as a primary infection. Subsequent disease in a
(XDR-TB) has emerged recently. XDR-TB is due to previously sensitized person, known as postprimary
M. tuberculosis strains which are resistant to any (secondary or reinfection) tuberculosis
fluoroquinolone and at least one of three injectable Diseases: M. tuberculosis causes primarily pulmonary
second line drugs (capreomycin, kanamycin and tuberculosis
amikacin) in addition to isoniazid and rifampicin. Diagnosis: Bacteriological diagnosis of tuberculosis
Between 50 and 100 million people worldwide can be established by direct microscopy, culture
examination or by animal inoculation test
are thought to be infected with strains of drug
1. Sputum is the specimen of choice for pulmonary
resistant tuberculosis. MDR-TB requires an
tuberculosis
extended treatment period compared with drug-
2. Microscopy: Z–N staining and auramine-
susceptible isolates. For cases of resistance to rhodamine staining for demonstration of AFB in
isoniazid or rifampin, second-line. antituberculosis stained smears is most
drugs may include aminoglycosides (kanamycin, 3. Culture is the definite method to detect and
amikacin, capreomycin), and fluoroquinolones. identify M. tuberculosis and is sensitive and specific
With the numbers of cases of multidrug-resistant 4. Serology is of limited value in the diagnosis of
M. tuberculosis increasing, newer agents are pulmonary tuberculosis
being tested in vitro to determine their efficacy. If 5. D irect detection by molecular probes is
compliance is an issue, a single daily dose of all first relatively insensitive
line antitubercular drugs is preferred, and ‘directly 6. The recent rapid and automated methods include
observed treatment strategy’ (DOTS) has been automated radiometric culture methods (e.g.
BACTEC) , SEPTICHEK, MGITs, etc.
recommended by WHO. Otherwise resistance may
Resistance ratio method, absolute concentration
be assumed and tested for in vitro. method and proportion method are used to
determine the sensitivity testing of M. tuberculosis.
Key Points Other methods for sensitivity testing include BACTEC
radiometric method, MGIT, chemiluminescence and
Mycobacteria are aerobic, nonmotile, noncapsulated
Epsilometer test (E-test)
and nonsporing. Growth is generally slow
Tuberculin test: This test is delayed (Type IV) hyper
Mycobacteria are, known as acid-fast bacilli (AFB).
sensitivity reaction. Many methods such as 1. Mantoux
Acid fastness has been ascribed variously to the
test and 2. Heaf test had been described for tuberculin
presence in the bacillus of mycolic acid or to a sem
testing
ipermeable membrane around the cell
Treatment, Prevention, and Control
Mycobacterium tuberculosis
Chemotherapy forms the mainstay of treatment of
Mycobacterium tuberculosis is weakly gram-positive,
tuberculosis . Drug resistance in M. tuberculosis is due
strongly acid-fast, aerobic bacilli
to mutation. The emergence of multidrug resistance-
Ziehl-Neelsen acid-fast stain is useful in staining tuberculosis (MDR-TB) is a very serious problem. DOT
organisms. With this stain, the tubercle bacilli stain is a method now being widely followed for treatment
bright red. Tubercle bacilli may also be stained with of cases of tuberculosis. Another serious condition
the fluorescent dyes (auramine 0, rhodamine) and extensively drug resistant-tuberculosis (XDR- TB) has
appear yellow luminous bacilli under the fluorescent emerged recently
microscope
Preventive measures against tuberculosis include
They are aerobes, slow growers, produce luxuriant chemoprophylaxis, vaccination, and general health
eugonic growth after 2–8 weeks measures. Immunoprophylaxis with BCG in endemic
The solid medium most widely employed for routine countries.
culture is Lowenstein–Jensen (L–J) medium without
starch
On L–J media, M. tuberculosis forms dry, rough, raised, IMPORTANT QUESTIONS
irregular colonies with a wrinkled surface. They are
creamy white, becoming yellowish or buff colored 1. Describe the morphology, cultural characteristics
on further incubation. They are tenacious and not and pathogenicity of M. tuberculosis.
easily emulsified. Mycobacterium tuberculosis has 2. Classify mycobacteria. Describe the laboratory
a luxuriant growth (eugenic growth) as compared diagnosis of pulmonary tuberculosis.
to Mycobacterium bovis, which grows poorly on LJ
3. Differentiate between Mycobacterium tuberculosis
glycerol medium (dysgonic growth)
and Mycobacterium bovis in a tabulated form.
www.ebook3000.com
Chap-32.indd 239 15-03-2016 11:13:53
33
Chapter
Mycobacterium leprae
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following:
be able to: – Lepromatous leprosy; tuberculoid leprosy;
∙∙ Describe morphology of M. leprae lepromin test
∙∙ Discuss cultivation of lepra bacilli – Differentiate between lepromatous and tuber
∙∙ Explain animal models in leprosy culoid leprosy
∙∙ Describe pathogenesis of leprosy – Discuss laboratory diagnosis of leprosy.
www.ebook3000.com
242 | Section 3: Systemic Bacteriology
the immune status of the host. It is therefore not 2. Tuberculoid (TT) Leprosy
permanent and varies with chemotherapy and The skin lesions are few and sharply demarcated,
alterations in host resistance. TT and LL are stable. consisting of macular anesthetic patches. In
The others are unstable, especially BT, which in tuberculoid leprosy, skin biopsies show mature
the absence of treatment can regress to BB or BL. granuloma formation in the dermis consisting of
epithelioid cells, giant cells, and rather extensive
Pathogenesis infiltration of lymphocytes.
Leprosy (Hansen’s disease) is a chronic granulo There are very few acid-fast bacilli (AFB) so
matous disease of humans primarily involving that they are generally not seen microscopically
the skin, peripheral nerves and nasal mucosa but (paucibacillary disease) and infectivity is minimal.
capable of affecting any tissue or organ. The organisms invade the nerves and selectively
M. leprae is an obligate intracellular parasite colonize the Schwann cells. The local nerves are
that multiplies very slowly within the mononuclear involved in the early stage and gradually the infec
phagocytes, especially the histiocytes of the skin tion extends into the bigger nerve trunks which are
and Schwann cells of the nerves. M. leprae has an thickened, hard and tender. This leads to deformi
especially strong predilection for nerves and is the ties of hand and feet.
only known human pathogen that preferentially
attacks the peripheral nerves. The two extreme or 3. Borderline (BT) Leprosy
polar forms of the disease are the lepromatous and
Borderline leprosy (BB), sometimes called
tuberculoid types TT) types (Table 33.2).
dimorphous or intermediate leprosy, has features
of both tuberculoid and lepromatous forms. This is
1. Lepromatous Leprosy an unstable form of the disease and may shift to the
Patient develops numerous nodular skin lesions lepromatous or tuberculoid part of the spectrum
(lepromata) on face, ear lobes, hands, feet and depending on chemotherapy or alterations in host
less commonly trunk. Skin lesions contain many resistance.
macrophages, often seen as large foamy cells
packed with AFB. Skin biopsy specimens may 4. Indeterminate Type
contain up to 109 bacilli per gram of tissue. This is
There is an early unstable tissue reaction with mild
known as ‘multibacillary disease’. Nodular skin
transient tissue lesion, often resembling maculo-
lesions ulcerate due to repeated trauma as a result
anesthetic patches which is not characteristic of
of loss of sensation. The ulcerated nodules become
either the lepromatous or the tuberculoid type. In
secondarily infected that leads to distortion and
many persons, the indeterminate lesions undergo
mutilation of extremities.
healing spontaneously. In others, the lesions may
Bacilli invade the mucosa of the nose, mouth
progress to the tuberculoid or lepromatous types.
and upper respiratory tract and are shed in large
numbers in nasal and oral secretions. Bacillemia
is common. Eyes, testes, kidneys and bones are Epidemiology
also involved. Leprosy is an exclusively human disease and the
Lepromatous leprosy is more infectious than only source of infection is the patient. Disease is
other types and has a poor prognosis. Cell-mediated spread by person-to-person contact. The mode
immunity is deficient and the lepromin test is of entry may be either through the respiratory
negative in lepromatous leprosy. Humoral antibodies tract or through the skin. Asymptomatic infection
against mycobacterial antigens are produced in high appears to be quite common in endemic areas.
concentrations which play no protective role. Most Bacilli may also be transmitted via breast milk
cases show biological false positive reaction in from lepromatous mothers, by insect vectors, or
standard serological tests for syphilis. by tattooing needles.
www.ebook3000.com
244 | Section 3: Systemic Bacteriology
The nodule may even ulcerate and heal with dries, it is fixed by passing the slide twice or thrice
scarring if the antigen is crude. It takes several over a flame with the surface carrying the smear
weeks to heal. Histologically, there is infiltration uppermost. About 5–6 different areas of the skin
with lymphocytes, epithelioid cells and giant should be sampled, including the skin over the but
cells. tocks, forehead, chin, cheek and ears.
www.ebook3000.com
246 | Section 3: Systemic Bacteriology
Nontuberculous Mycobacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Name the diseases caused by nontuberculous
be able to: mycobacteria
∙∙ Classify nontuberculous mycobacteria and ∙∙ Discuss the clinical significance of nontuberculous
examples of different groups of nontuberculous mycobacteria.
mycobacteria
www.ebook3000.com
Chap-34.indd 247 15-03-2016 11:14:38
248 | Section 3: Systemic Bacteriology
4. Staining: They are acid fast as well as alcohol is principally isolated from cases of pulmonary
fast. They may differ or resemble in their disease. Natural reservoir is tap water. Pulmonary
morphology from those of tubercle bacilli. disease is the most common clinical form of M.
5. Biochemical reactions: They are arylsulfatase kansasii infection. It occurs primarily in middle-
test positive, but are niacin and neutral red aged or elderly white men, most of whom have
reactions negative. some pre-existing form of lung disease.
6. Animal pathogenicity: They are nonpathogenic M. kansasii may also occasionally cause
for guinea pigs but pathogenic for mouse. infections of the cervical lymph nodes, penetrating
7. Treatment: They are usually resistant to anti wound infections, and granulomatous synovitis. It
tubercular drugs such as streptomycin, INH, can produce generalized infection in HIV-positive
and para-aminosalicylic acid. patients.
8. Transmission: Soil or water Mycobacterium marinum (M. marinum): M.
9. Classification: They are classified by Runyon marinum (previously termed the fish tubercle
into four groups on the basis of their rate of bacillus) the cause of a warty skin infection known
growth and their ability to produce pigments as swimming pool granuloma. Microscopically, it
in the presence or absence of light. resembles M. kansasii. but can be differentiated by
its poor growth at 37°C, negative nitratase, positive
CLASSIFICATION pyrazinamide hydrolase and L-fucosidase activities.
Runyon(1959) classified NTM into four groups Mycobacterium simiae (M. simiae): M. simiae,
(Table 34.2) based on phenotypic characteristics of which, like M. kansasii, grows at 37°C and is
the various species, most notably pigment (yellow occasionally involved in pulmonary disease. Several
or orange) production and rate of growth. These photochromogenic mycobacteria were isolated
include: from monkeys exported from India. They have
∙∙ Group I: Photochromogens subsequently been associated with pulmonary
∙∙ Group II: Scotochromogens disease in human beings.
∙∙ Group III: Nonphotochromogens
∙∙ Group IV: Rapid growers. Group II: Scotochromogens
Species identification depends on several
additional characteristics (Table 34.2). The scotochromogens are slow-growing NTM
whose colonies are pigmented (yellow-orange-red)
when grown in the dark or the light. They are widely
Group I: Photochromogens distributed in the environment and sometimes
Photochromogens develop a bright yellow or contaminate cultures of tubercle bacilli.
orange coloration if young cultures are exposed to a i. Mycobacterium scrofulaceum (M. scrofulaceum):
light source. They are slow growing, though growth M. scrofulaceum is principally associated with
is faster than that of tubercle bacilli. The important scrofula or cervical lymphadenitis in children,
species in this group are M. kansasii, M. marinum but also causes pulmonary disease.
and M. simiae.
ii. Mycobacterium gordonae (M. gordonae):
Mycobacterium kansasii (M. kansasii): Myco M. gordonae (formerly M. aquae), frequently
bacterium kansasii, which grows well at 37°C and found in water and a common contaminant of
clinical material, is a rare cause of pulmonary
Table 34.2 Runyon classification scheme of non- disease.
tuberculous mycobacteria iii. Mycobacterium szulgai (M. szulgai): M.
szulgai, an uncommon cause of pulmonary
Runyon group Name Species
disease and bursitis.It is a scotochromogen
I Photochromogens M. kansasii when incubated at 37°C but a photochromogen
M. marinum
M. simiae at 25°C.
II Scotochromogens M. scrofulaceum
M. gordonae
Group III: Nonphotochromogens
M. szulgai The nonphotochromogens are slow-growing NTM
III Non- M. auium whose colonies produce no pigment whether they
photochromogens M. intracellulare are grown in the dark or the light. Colonies may
M. xenopi resemble those of tubercle bacilli.
M. ulcerans Of the organisms classified in this group, those
M. malmoense
belonging to nonpathogenic for humans are M.
IV Rapid growers M. chelonei terrae complex and M. gastri. Medically important
M. fortuitum
species are M. avium. M. intracellulare, M. xenopi
www.ebook3000.com
Chap-34.indd 249 15-03-2016 11:14:38
250 | Section 3: Systemic Bacteriology
A. Lymphadenitis: Most patients are children aged forming indolent ulcers which slowly extend under
less than 5 years. The M. avium complex is the the skin. Initially, smears from the edge of the ulcer
predominant cause worldwide. Some reports claim show large clumps of bacilli which are acid fast and
a high incidence of M. scrofulaceum. alcohol fast. Later, the immunoreactive phase sets in
and the bacilli disappear. The ulcers then heal with
B. Skin lesions: Three main types have been
disfiguring scars.
described:
C. Pulmonary disease: These infections resemble
1. Postinjection (and posttraumatic) abscesses
tuberculosis. In most but not all cases, there is some
2. Swimming pool granuloma
predisposoing lung disese. This is most frequently
3. Buruli ulcer.
seen in middle-aged or elderly men with lung
1. Postinjection abscesses: These are usually
damage. The disease may be caused by many
caused by the rapidly growing pathogens M.
species, but the most frequent are the M. avium
chelonae and M. fortuitum. Abscesses occur
complex and M. kansasii.
when batches of injectable materials are con
Organism must be isolated repeatedly from the
taminated by these bacteria. Abscesses are
sputum to diffrentiate true disease from transient
painful and last for many months.
colonization.
2. Swimming pool granuloma: It is caused by
D. Disseminated disease: Up to a half of all
M. marinum and is also known as fish tank
persons dying of AIDS in the USA had disseminated
granuloma and fish fancier’s finger. M. marinum
mycobacterial disease in the 1980s and early 1990s,
is a natural pathogen of cold-blooded animals,
almost always due to the M. avium complex.
causing tuberculosis in fish and amphibia.
Human infection originates from contaminated
swimm ing pools or fish tanks. The lesion, Laboratory Diagnosis
beginning as a papule and breaking down to A. Specimen: Sputum, pus or exudates.
form an indolent ulear, usually follows abrasions. B. Microscopy: Ziehl–Neelsen staining of smear
It was first described from Sweden under shows acid fast bacilli. Reapeted smear
the name ‘swimming pool granuloma’, examination is necessary.
and the bacillus was named M. balnei (from C. Culture: They grow well on LJ medium.
balneum, meaning bath). The disease is usually Several LJ media should be inoculated with
self-limiting although chemothera py with the specimen. These are incubated in the dark
minocycline, co-trimoxazole or rifampicin with and in the light at different temperatures for
ethambutol hastens its resolution. distinguishing the species.
3. Buruli ulcer: This disease, caused by M. D. Identification: There is no universally recognized
ulcerans, was first described from human skin identification scheme, although reliance is
lesions in Australia (1948). The name Buruli is usually placed on cultural characteristics (rate
derived from the Buruli district of Uganda where and temperature of growth and pigmentation),
a large outbreak was extensively investigated. various biochemical reactions and resistance
Ulcers are usually seen on the legs or arms. to antimicrobial agents (Table 34.4). The
Indurated nodules appear, which break down most discriminative methods are based on
Table34.4 Differentiation between tubercle bacilli and some species of atypical mycobacteria
Test M. M. M. M M M. M. avium M. M. M. M.
tubercu bovis microti kansasii marinum scrofula intra fortuitum chelonei phlei smegmatis
losis ceum cellulare
complex
Growth in – – – – – – – + + + +
7 days
Growth at – – – + + + ± + + + +
25°C
Growth at + + + + ± + + + + + +
37ºC
Growth at – – – – – ± – – – + +
45°C
Pigment – – – + + + – – – + –
in light
Pigment – – – – – + – – – + –
in dark
Niacin + – ± – – – – – – – –
Nitrate + – – + – – – + – + +
reduction
Urease + + + – + + – + + + +
www.ebook3000.com
Chap-34.indd 251 15-03-2016 11:14:38
35
Chapter
Actinomycetes
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of actinomycosis
be able to: ∙∙ Differentiate between the genera Actinomyces and
∙∙ Describe morphology of Actinomyces Nocardia
∙∙ Explain clinical forms of Actinomyces ∙∙ Describe the following: nocardiosis, mycetoma.
Introduction Morphology
Actinomyces are Gram-positive, nonmotile, non
Actinomycetes are traditionally considered to be
sporing, nonacid-fast. They often grow in mycelial
transitional forms between bacteria and fungi.
forms and break-up into coccoid and bacillary
They form a mycelial network of branching
forms. Most show true branching.
filaments like fungi but, like bacteria, they are
thin, possess cell walls containing muramic acid,
have prokaryotic nuclei and are susceptible to
Cultural Characteristics
antibacterial antibiotics. They are therefore true They are facultative anaerobes. They grow best
bacteria, bearing a superficial resemblance to under anaerobic or micro-aerophilic conditions
fungi. Actinomycetes are related to mycobacteria with the addition of 5–10% CO2. The optimum tem
and corynebacteria. perature for growth is 35–37°C. They can be grown
They are gram-positive, nonmotile, nonsporing, on brain heart infusion agar, heart infusion agar
noncapsulated filaments that break up into supplemented with 5% defibrinated horse, rabbit
bacillary and coccoid elements. Although mostly or sheep blood. Suitable liquid media include brain
soil saprophytes, occas ionally cause chronic heart infusion broth and thioglycollate broth which
granulomatous infections in animals and man. may be supplemented with 0.1–0.2% sterile rabbit
serum. Most species show good growth after 3–4
Important genera: The family Actinomycetes days incubation however, A. israelii may take 7–14
contains three major medically important genera, days.
Actinomyces, Nocardia and Actinomadura. Another
genus, Streptomyces rarely causes disease in man.
Pathogenesis
Actinomyces is anaerobic or microaerophilic
and nonacid-fast, while Nocardia is acid-fast and Actinomycosis: The Actinomyces causes the
aerobic. Streptomyces is nonacid-fast and aerobic. disease known as actinomycosis. Actinomycosis is a
chronic disease characterized by multiple abscesses
and granulomata, tissue destruction, extensive
Actinomyces
fibrosis and the formation of sinuses. Within
A mould-like organism in the lesion of ‘lumpy jaw’ diseased tissues the actinomycetes form large
(actinomycosis) in cattle was found by Bollinger masses of mycelia embedded in an amorphous
(1877).The name Actinomyces was coined by protein–polysaccharide matrix and surrounded by
Harz to refer to the raylike appearance of the a zone of gram-negative, weakly acid-fast, club-like
organism in the granules that characterize the structures (Fig. 35.1). The mycelial masses may
lesions (Actinomyces, meaning ray fungus). This be visible to the naked eye and are called sulfur
was named Actinomyces israelii. It causes human granules, as they are often light yellow in color. The
actinomycosis. sulfur granules may be dark brown and very hard in
Chapter 35: Actinomycetes | 253
using 1% sulfuric acid for decolorization. Gram
staining shows a dense network of thin gram-positive
filaments, surrounded by a peripheral zone of
swollen radiating club shaped structures, presenting
a sun-ray appearance (Fig. 35.1). The ‘clubs’ are
believed to be antigen-antibody complexes.Acid
fast staining shows central part as non-acidfast
surrounded by acid-fast ‘clubs’. In absence of sulfur
granules, Gram’s staining of pus shows gram-positive
branching filaments.
Sulfur granules and mycelia in tissue sections
Fig. 35.1: Sulfur granule. Section of tissue showing an
actinomycotic clolony, the clubs at the periphery giving a can also be identified by direct fluorescence
‘sun-ray’ appearance microscopy.
older lesions because of the deposition of calcium
3. Culture
phosphate in the matrix.
In man, actinomycosis.is usually caused by Sulfur granules or pus containing actinomycetes are
Actinomyces israelii. Less common causes include washed and inoculated into thioglycollate liquid
A. gerencseriae, A. naeslundii, A. odontolyticus, A. medium or streaked on brain h eart infusion agar
viscosus, A. meyeri, Propionibacterium propionicum (BHI agar) blood agar and incubated anaerobically
and members of the genus Bifidobacterium. at 37°C. On solid media, A. israelii may form
so-called spider colonies that resemble molar teeth
in 48–72 hours that become heaped up, white and
Human Actinomycosis
irregular or smooth, large colonies in 10 days. Other
Human actinomycosis may take several forms: species have different types of colonies.
1. Cervicofacial: This is the most common type
and it occur mainly in cheek and submaxillary 4. Identification
regions. The disease is endogenous in origin. The identity may be confirmed by direct fluo
Dental caries is a predisposing factor, and rescence microscopy and biochemical tests or
infection may follow tooth extractions or other by gas chromatography of metabolic products of
dental procedures. carbohydrate fermentation.
2. Thoracic: Thoracic actinomycosis commences
in the lung, probably as a result of aspiration of 5. Biopsy
actinomyces from the mouth. In hematoxylin and eosin stained sections, the
3. Abdominal: The lesion is usually around the sulfur granules are deeply stained with hematoxy
cecum, with the involvement of the neighboring lin except in the periphery which is stained by
tissues and the abdominal wall. eosin, which shows short, radiate, club-like
4. Pelvic: Pelvic actinomycosis occasionally structures. On Gram staining, the filaments are
occurs in women fitted with plastic intra- gram-positive and periphery gram-negative.
uterine contraceptive devices.
5. Punch actinomycosis: It is a rare infection of Epidemiology
the hand acquired by injury of the knuckles on
Actinomycosis is an endogenous infection.
an adversary’s teeth.
Actinomycosis is more common in rural areas and
Laboratory Diagnosis in agricultural workers. Young male persons (10–30
years old) are most commonly affected. About
1. Specimens 60% of the cases are cervicofacial and some 20%
Pus, sinus discharge, bronchial secretions, sputum abdominal. Pelvic actinomyces is seen mainly in
or infected tissues are collected aseptically. women using intrauterine devices.
2. Microscopy Treatment
‘Sulfur granules’ may be demonstrated in pus Treatment for actinomycosis involves the combin
by shaking it up in a test tube with some saline. ation of surgical debridement of the involved
On standing, the granules sediment may be tissues and prolonged treatment with penicillin or
withdrawn with a capillary pipette. Granules may tetracycline.
also be obtained by applying gauze pads over the
discharging sinuses. Nocardia
Granules are crushed between two slides and Nocardia resemble Actinomycetes morphologically
stained with Gram and Ziehl–Neelsen staining but are aerobic. Most species (such as N. asetroides
www.ebook3000.com
254 | Section 3: Systemic Bacteriology
Table 35.1 Differences between the genera Cutaneous infection: Primary or secondary
Actinomyces and Nocardia cutaneous infection may lead to mycetoma, lymph
ocutaneous infections, cellulites, subcutaneous
Actinomyces spp. Nocardia spp.
abscesses.
• Facultative anaerobes • Strict aerobes
• Grow at 35–37°C • W
ide temperature Laboratory Diagnosis
range of growth
Diagnosis is by demonstration of branching fila
• Oral commensals • E nvironmental
ments microscopically and by isolation in culture.
saprophytes
• Nonacid-fast mycelia • Usually weakly acid-fast
1. Specimens
• E ndogenous cause of • Exogenous cause of
disease disease
Pus or purulent sputum.
www.ebook3000.com
36
Chapter
Enterobacteriaceae: Escherichia,
Klebsiella, Proteus and Other Genera
LEARNING OBJECTIVES
After reading and studying this chapter, you should – Discuss various groups of E. coli producing diarrhea
be able to: – Differentiate between heat labile toxin (LT) and
∙∙ Describe general characters of the family Enter heat stable toxin (ST) of E. coli
obacteriaceae – Discuss laboratory diagnosis of urinary tract
– Classify the family Enterobacteriaceae infections caused by E. coli
– Describe morphology, culture characteristics and – Discuss morphology, culture characteristics and
biochemical reactions of E. coli biochemical reactions of Klebsiella sp
– Discuss pathogenicity of E. coli – Differentiate between E. coli and Klebsiella sp.
INTRODUCTION CLASSIFICATION OF
The family Enterobacteriaceae is the largest, most ENTEROBACTERIACEAE
heterogeneous collection of medically important
Gram-negative bacilli. Because of their normal
Lactose Fermentation
habitat in humans, these organisms are referred The oldest method was to classify these bacteria
to as the “enteric bacilli” or “enterics” into three groups based on their action on
lactose. Lactose fermenters (LF), Late lactose
CHARACTERISTICS OF THE FAMILY fermenter and nonlactose-fermenting (NLF).
ENTEROBACTERIACEAE The specimen is plated on a medium containing
lactose and neutral red indicator. (MacConkey
Members of the family Enterobacteriaceae have agar). The organisms fermenting the lactose form
the following characteristics: acid and in acidic pH, neutral red is red in colour,
i. They are gram-negative bacilli. therefore, the colonies of lactose-fermenting
ii. They are aerobes or and facultative anaerobes bacteria are red or pink and those of nonlactose-
and grow readily on ordinary laboratory media fermenting (NLF) bacteria are pale. All lactose-
including MacConkey’s lactose bile-salt agar. ferm enting enterobacteria, e.g. Escherichia,
iii. All species ferment glucose with the production Klebsiell a, Enterobacter and Citrobacter are
of acid or acid and gas. popularly known as ‘coliform bacilli’ as the
iv. They are either non-motile or motile with most common member of this group is the colon
peritrichous flagella. bacillus or Escherichia coli. The major intestinal
pathogens, Salmonella and Shigella are non-
v. They are catalase positive (except for Shigella
Iactose-fermenters (NLF). There remained a
dysenteriae type 1 which is catalase-negative)
small group which showed delayed fermentation
vi. They are oxidase-negative. of lactose (2–8 days) and with the exception of
vii. They reduce nitrate to nitrites Shigella sonnei, they were all commensals. This
viii. They are typically intestinal parasites of heterogenous group of late lactose fermenters was
humans and animals. called paracolon bacilli.
www.ebook3000.com
Chap-36.indd 257 15-03-2016 11:15:18
Chap-36.indd 258
Table 36.2 Important distinguishing features of the different genera of Enterobacteriaceae
Test Escherichia Shigella1 Edwardsiella Klebsiella Enteobacter Serratia Hafnia Citrobacter Salmonella2 Proteus Morganella Providencia
Motility + – + – + + + + + + + +
Gas from glucose + – + + + d + + + d + +
Acid from lactose + – – + + – – + – – – –
Acid from sucrose d – – + + + – d – d – d
Growth in KCN – – – + + + + + d + + +
Indole + d + – – – – d – d + +
MR + + + – – – – + + + + +
258 | Section 3: Systemic Bacteriology
VP – – – + + + + – – – – –
Citrate – – – + + + + + + d d d
H2S – – + + – – – + + + – –
Urease – – – + d – – – – + + d
Phenylalanine – – – – – – – – – + + +
deaminase (PPA)
Arginine d – – – d – – d + – – –
dehydrolase
Lysine + – + d d + + – + – – –
decarboxylase
Ornithine d d + – + + + d + d + –
decarboxylase
(d = results different in different species or strains)
Important exceptions:
1. Sh. sonnei ferments lactose and sucrose late
2. S. Typhi does not produce gas from sugars
15-03-2016 11:15:18
Chapter 36: Enterobacteriaceae: Escherichia, Klebsiella, Proteus and Other Genera | 259
Table 36.3 K antigens (group I and group II) of colonization factor antigens (CFAs) (CFAI,
E. coli CFAII, CFA/III) expressed by enterotoxigenic E.
coli (ETEC) causing diarrheal disease in humans.
Properties Group I Group II
1. Molecular weight >100 000 <50 000 B. Toxins
2. O groups 08.09 Many 1. Exotoxins: E. coli produces two kinds of
3. Acidic component Hexuronic acid Glucuronic acid, exotoxins hemolysins and enterotoxins.
phosphate, a. Hemolysins: Hemolysins do not appear to
KDO, NeuNAc be relevant in pathogenesis.
4. Electrophoretic Low High b. Enterotoxins: Three distinct types of E. coli
mobility enterotoxins have been identified:
5. Expressed at Yes No i. Heat-labile toxin (LT): Mechanism
17-20°C of action of LT: E. coli LT is heat labile
6. Chromosome site His SerA protein and is closely related to the
KDO, ketodeoxyoctonate; NeuNAc, N-acetylneuraminic toxin produced by strains of Vibrio
acid cholerae. There are two main forms,
termed LT-I and LT-II. Different forms
of LT-1 have been described. Similarly,
the agglutinability, antigenicity and antibody two forms of LT-II (LT-IIa and LT- lIb)
binding power of bacterial strains that express have been detected.
them. Later it was shown that the B antigen was LT is a complex of polypeptide subunits-
not a separate entity. K antigens are therefore each unit of the toxin consisting of
currently classified into two groups, I and II, one subunit A (A for active) and five
(Table 36.3). subunits B (B for binding). The toxin
4. Fimbrial antigen (F antigen): These are binds to the Gm1 ganglioside receptor
thermolabile proteins. Heating the organisms on intestinal epithelial cells by means of
at 100°C leads to detachment of fimbriae. The subunit B, following which the subunit
F antigen has no role in antigenic classification A is activated to yield two fragments—A1
of E. coli. and A2. The A1 fragment activates adenyl
cyclase in the enterocyte to form cyclic
Virulence Factors
adenosine 5’ monophosphate (cAMP),
Two types of virulence factors have been recognized leading to increased outflow of water
in E. coli—surface antigens and toxins. and electrolytes into the gut lumen, with
consequent diarrhea.
A. Surface Antigens LT is a powerful antigen and can
1. Somatic antigen (O antigen): The somatic therefore, be detected by a number of
lipopolysaccharide surface O antigen, besides serological as well as biological tests.
exerting endotoxic activity, also protects the ii. Heat-stable toxin (ST): The heat stable
bacillus from phagocytosis and the bactericidal toxins of E. coli (ST) have a low molecular
effects of complement. weight which is probably responsible for
2. K antigen: Most strains of E. coli responsible their heat stability and poor antigenicity.
for neonatal meningitis and septicemia carry There are two major classes, designated
the KI envelope antigen which is a virulence ST-I (or STa) and ST-II (or STb). STa is
factor resembling the group B antigen of associated with human disease.
meningococcci. a. ST-I (or STa): STa is a small, monomeric
3. Fimbriae: Like many other members of the toxin that binds to guanylate cyclase,
Enterobacteriaceae, strains of E. coli exhibit leading to an increase in the level of
common fimbriae which are chromosomally cyclic guanosine monophosphate and
determined, present in large numbers and subsequent hypersecretion of fluids. ST-I
causing mannose sensitive hemagglutination is methanol soluble, is plasmid encoded.
and probably not relevant in pathogenesis. b. ST-II (STb)—ST-II (STb) is distinguished
Filamentous protein structures resembling from ST-I (STa) by its biological activity
fimbriae cause mannose-resistant hemagglutin and by its insolubility in methanol. It
ation and play an important part in the stimulates fluid accumulation in ligated
pathogenesis of diarrheal disease and in intestinal loops of young piglets (upto
urinary tract infection. They include the nine weeks) but not in the infant mouse
K88 antigen found in strains causing enteritis test. The mechanism of action is not
of pigs, the K99 antigen found in strains known but it appears not to act via cAMP
causing enteritis of calves and lambs, and the or cGMP.
www.ebook3000.com
Chap-36.indd 259 15-03-2016 11:15:18
260 | Section 3: Systemic Bacteriology
Table. 36.4 Differentiating proper ties of age groups in the local population. In developing
verocytotoxins by E. coli countries. ETEC are a major cause of mortality in
children under the age of 5 years.Persons from
VT1 VT2 VT2v1
developed countries visiting endemic areas often
• Synonyms SLT1 SLT2 SLT2v suffer from ETEC diarrhea—a condition known as
• Cytotoxicity ‘traveller’s diarrhea’.
Vero cells + + + ETEC produce a heat-stable toxin (ST) or a
HeLa cells + + – heat-labile toxin (LT) or both (See under virulence
factors above). The organism must initially be able
Genes phage-encoded + + –
to adhere to the mucosal surface of the epithelial
1
Human and porcine variants cells of the small intestine. This adhesion is usually
mediated by fimbriae that bind to specific receptors
iii. Verocytotoxin or Verotoxin (VT): The in the intestinal cell membrane. These adhesins
biological properties, physical char have been termed colonization factors antigens
acteristics and antigenicity of VT are (CFAs) of which a number of have been identified
very similar to those of Shiga toxin (Stx), (CFAI, II, III,IV). Plasmids that simultaneously
produced by strains of Sh. dysenteriae carry genes for both CFAs and enterotoxin
type 1 so it is also known as ‘Shiga-like production have been described.
toxin’ (SLT). Though plasmids with enterotoxin genes may
2. VT1 and VT2 form: Serological tests have be present in any strain of E. coli, in practice only a
revealed two antigenically distinct forms, small number of serotypes become enterotoxigenic
termed VTl and VT2. Antibodies prepared to (for example, 06, 08, 015, 025, 027, 0167).
VT1 neutralize Shiga toxin, while antibodies
specific for VT2 do not. Variant forms of VT2 C. Enteroinvasive E. coli (EIEC)
(VT2v) have been described in strains of human These are closely related by phenotypic and
and porcine origin (Table 36.4). pathogenic properties to Shigella. Many of these,
VT1 and VT2, like Shiga toxin, are cytotoxic for strains are nonmotile, do not ferment lactose or
Vero and HeLa cells, although VT2 variant toxins ferment it late with acid, but without producing any
do not bind to HeLa cells. Enterotoxicity in ligated gas and do not form lysine decarboxylase. Many of
rabbit gut loops and mouse paraliytic lethality can these show 0 antigen cross- reaction with shigellae.
also be used to detect VTs. These ‘atypical’ E. coli strains had earlier been
grouped under the Alkalescens-Dispar Group
Clinical Infections and given names such as ‘Shigella alkalescens’
Four main types of clinical syndromes are caused (resembling Sh. flexneri except in fermenting
by E. coli. dulcitol and forming alkali in litmus milk) and ‘Sh.
dispar’ (late lactose fermenter like Sh. sonnei but
1. Diarrhea indole positive). Besides these atypical strains
At least five different types of diarrheagenic E. many typical E. coli strains can also cause clinical
coli are now recognized, each associated with illness resembling shigellosis. These have been
specific serotypes and with different pathogenic termed enteroinvasive E. coli because they have
mechanisms. the capacity to invade interstitial epithelial cells
in vivo and penetrate HeLa cells in tissue culture.
A. Enteropathogenic E. coli (EPEC) EIEC strains usually belong to serogroups 028ac,
These have been associated mainly with diarrhea 0112ac, 0124, 0136, 0143, 0114, 0152, 0154. The
in infants and child ren usually occurring as most common serogroup is 0124.
institutional outbreaks. Certain strains belonging to
characteristically EPEC serogroups, such as O26 and Pathogenesis
O111, were later shown to express verocytotoxins. EIEC, like those of Shigella species, can penetrate
Pathogenesis of EPEC diarrhea: EPEC neither the epithelial cells of the large intestine and
ordinarily produce enterot oxins, nor are they multiply intracellularly, giving rise to blood and
invasive. In infantile enteritis, the bacilli are seen mucus in the stool. Infection is by ingestion.
to be adherent to the mucosa of the upper small Clinically, EIEC infection resembles shigellosis,
intestine, intimately attached to cup-like projections ranging from mild diarrhea to frank dysentery, and
(pedestals) of the enterocyte membrane, causing occurs, in children as well as adults.
disruption of the brush border microvilli.
D. E. coli Verocytotoxin or Verotoxin (VT)
B. Enterotoxigenic E. coli (ETEC) E. coli verocytotoxin or verotoxin (VT) causes
Diarrhea caused by ETEC is endemic in the hemorrhagic colitis and hemolytic uremic
developing countries in the tropics, among all syndrome. Outbreaks were first recognized in
www.ebook3000.com
Chap-36.indd 261 15-03-2016 11:15:19
262 | Section 3: Systemic Bacteriology
Table 36.5: Methods for detection of ETEC enterotoxins
Assay LT ST
In vivo tests
Ligated rabbit ileal loop
Read at 6 hours ± +
Read at 18 hours + −
Infant rabbit bowel + +
Infant mouse intragastric (4 hours) − +
Adult rabbit skin (vascular permeability factor) + −
In vitro tests
i. Tissue culture tests
ii. Rounding of Y1 mouse adrenal cells
iii. Elongation of Chinese hamster ovary (CHO) cells + −
iv. Serological tests
ELISA + (ST-ELISA with monocolonial antibody)
Passive agglutination tests, passive immune hemolysis, + −
precipitin (Biken’s) test
v. Genetic tests
DNA probes + +
www.ebook3000.com
Chap-36.indd 263 15-03-2016 11:15:19
264 | Section 3: Systemic Bacteriology
KLEBSIELLA Table 36.6 Distinguishing reactions of Citrobacter
Introduction Species Test/substratea
ind mal H2S KCN adon arbtl mel
Members of the genus Klebsiella are gram-negative,
nonsporing, non-motile bacilli that grow well on C. freundii − − ± + − − ±
ordinary media, produce pink mucoid colonies C. koseri + + − − + + −
on MacConkey’s agar. They are usually found in C. + − − + − − −
the intestinal tract of humans and animals or free- amalonaticus
living in soil, water, and on plants. a
Ind, indole production; mal, utilization of malonate; H2S,
H2S produced in TSI agar; KCN, growth in KCN medium;
Classification adon, arbtl, mel, fermentation of adonitol, D-arabitol,
melibiose.
Their classification has undergone various
modifications. The name K. pneumoniae is used
for the species as a whole. It is further divided into 1. Capsular (K) antigen: On the basis of capsular (K)
4 subspecies. The most frequently encountered, antigens, the klebsiellae have been differentiated,
biochemically typical form of it is known as K. into 80 serotypes. Members of capsular types 1–6
pneumoniae subsp. aerogenes, K. pneumoniae occur most frequently in the human respiratory
subsp. ozaenae, K. pneumoniae subsp. pneumoniae tract.
K. pneumoniae subsp. rhinoscleromatis (Table Capsular antigens are usually detected by means
36.6). Indole-producing strains that resemble of the capsular ‘swelling’ reaction, countercurrent
K. pneumoniae subsp. aerogenes biochemically are immunoelectrophoresis and enzyme-linked
classified in a separate species, K. oxytoca. immunosorbent-assay (ELISA).
2. Somatic (O) antigen: Five different somatic or O
Morphology antigens (01–05) occur in various combinations
They are short,plump, gram-negative, non-sporing, with the capsular antigens. Four of the five
capsulated, non-motile bacilli, 1–2 µm long and Klebsiella O antigens are identical to or related
0.5–0.8 µm wide with parallel or bulging sides and to E. coli O antigens.
slightly pointed or rounded ends.
Typing Methods
Cultural Characteristics 1. Bacteriocin (Klebocin or pneumocin) typing;
Klebsiellae grow well on ordinary media at a 2. Phage typing; 3. Biotyping; 4. Resistotyping; 5.
temperatures between 12°C and 43°C (optimum, Molecular typing methods: Plasmid analysis,
37°C) in 18–24 hours. On MacConkey agar, the DNA profiling by random amplified polymorphic
colonies typically appear large, mucoid and red in DNA (RAPD) and pulsed-field gel electrophoresis.
colour. Mucoid nature of colonies is due to capsular
Pathogenicity: Klebsiella pneumoniae can cause
material produced by the organism.
a primary community-acquired pnuemonia,
nosocomial infections, urinary tract infections,
Biochemical Reactions
wound infections, bacteremia and meningitis and
They ferment sugars (glucose, lactose, sucrose, rarely diarrhea. In some hospital, K. pneumoniae
mannitol) with production of acid and gas. They has replaced E. coli as the leading blood culture
are urease positive, indole negative, MR negative, isolate. As most strains are resistant to antibiotics,
VP positive and citrate positive (IMViC – – ++). treatment poses serious problems.
These reactions are typical of K. pneumoniae subsp.
aerogenes. Pneumonia: Klebsiella pneumonia is a serious
disease with high case fatality. The typical patient
Glucose Lactose Sucrose Mannitol is a middle- or older-aged man who have medical
+ (AG) + + +
problems. Positive blood cultures can be obtained
in about 25% of the cases.
Urease Indole MR VP Citrate
+ – – + + Diarrhea: Some strains of K. pneumoniae isolated
from cases of diarrhea have been shown to produce
Biochemical reactions of different subspecies of K. an enterotoxin very similar to the heat stable toxin
pneumoniae and K. oxytoca are given in Table 36.6. of E. coli.
K. ozaenae is a bacillus associated with ozena,
Antigenic Structure an uncommon, chronic disease in which there is
Klebsiella possess capsular (K) and somatic(O) atrophy of the nasal mucosa characterized by foul
antigens. smelling nasal discharge.
K. rhinoscleromatis causes rhinoscleroma, a urine, pus and other pathological materials. They
chronic granulomatous hypertrophy of the nose. may cause urinary tract infections and hospital
K. oxytoca may be rarely isolated from clinical infections. They are occasionally associated with
specimens (Table 36.7). meningitis and septicemia.
Treatment: Aminoglycosides are often effective in
Laboratory Diagnosis the treatment of Enterobacter infections.
Diagnosis is made by culturing appropriate spec
imens on blood agar and Mac Conkey agar and HAFNIA
identifying the isolate by biochemical reactions.
Antibiotic sensitivity should invariably be done. These organisms are probably best regarded
Many strains carry plasmids determining multiple as non-lactose fermenter (NLF) member of
drug resistance. genus Enterobacter. This is a motile, nonlactose-
fermenting bacillus which is indole and MR
Treatment negative and VP and citrate positive. Biochemical
They are normally susceptible to cephaloporins, reactions are evident best at 22°C but at 37°C they
especially β-lactamase stable derivatives may be negative or irregular. Hafnia alvei is the
such as cefuroxime and cefotaxime, and to only species.
fluoroquinolones. They are often sensitive to Strains are isolated from feces of man and
gentamicin and other aminoglycosides., but other animals and are also found in sewage, soil,
transferable enzymic resistance to aminoglycosides water and dairy products. They are occasionally
and other antimicrobial agents has become encountered as opportunistic pathogens that has
common in strains found in some hospitals. been recovered from infected wounds, abscesses,
Klebsiella infection of the urine often responds sputum urine, blood and other sites.
to trimethoprim, nitrofurantoin, co-amoxiclav
or oral cephalosporins. Pneumonia and other SERRATIA
serious infections require vigorous treatment S. marcescens is the one most commonly
with aminoglycoside or a cephalosporin, such as encountered species in clinical specimens. It is
cefotaxime. small, motile, gram-negative bacillus. This differs
from Hafnia in forming a pink, red or magenta,
ENTEROBACTER nondiffusible pigment called prodigiosin which
Enterobacter is a motile, capsulated, lactose is formed optimally at room temperature.
fermenting bacillus which is indole and MR negative
and VP and citrate positive (IMViC – - + +). These Pathogenesis
characteristics are similar to those of Klebsiella but It is a saprophyte found in water, soil and food.
can be differentiated from Klebsiella because it is It may grow in sputum after collection and may
motile and ornithine positive. Two clinically relevant suggest hemoptysis because of the pigment formed
species are E. cloacae and E. aerogenes. (pseudohemoptysis).Nosocomial infections due to
Clinical Infections: They are normally found S. marcescens are being reported with increasing
in feces, sewage, soil and water and rarely in frequency. The bacillus has been associated with
www.ebook3000.com
Chap-36.indd 265 15-03-2016 11:15:19
266 | Section 3: Systemic Bacteriology
infections of the urinary and respiratory tracts, 5. Write short notes on:
meningitis, wound infections, septicemia and a. Antigenic structure of Escherichia coli
endocarditis. Multiple drug resistance is common b. Enterotoxins of Escherichia coli
in hospital strains. c. Verotoxin (VT) or Shiga-like toxin (SLT)
6. Write briefly about:
a. Citrobacter
Key Points
b. Klebsiella pneumoniae
Escherichia coli c. What are the properties which differentiate E.
Gram-negative bacilli aerobe and a facultat ive coli from Klebsiella
anaerobe. On MacConkey’s medium, colonies are d. Enterobacter
red or pink due (lactose fermenter). Selective media e. Serratia
such as DCA or SS agar growth
B. Toxins: 1. Exotoxins: Hemolysins and enterotoxins.
Enterotoxins: (e.g. heat-stabile and heat-labile MULTIPLE CHOICE QUESTIONS (MCQs)
enterotoxins, and Verotoxin (VT) also known as
1. Which of the following properties is/are shown
Shiga-like toxin (SLT)-
by the organisms belonging to the family Enter
Clinical infections: 1. Urinary tract infection; 2.
Diarrhea, 3. Pyogenic infections, 4. Bacteremia obacteriaceae?
Diagnosis: Organisms grow rapidly on most culture a. They are catalase positive
media b. They are oxidase negative
Edwardsiella: Edwardsiella tarda mainly causes c. They ferment glucose
wound infection, but meningitis and septicemia d. All of the above
have also been reported 2. Which of the following bacteria is/are known as
Citrobacter: It may cause infections of the urinary coliform bacilli?
tract, gallbladder, middle ear and meninges. C. koseri a. Escherichia b. Klebsiella
occasionally causes neonatal meningitis
c. Enterobacter d. All of the above
Klebsiella: Members of the genus Klebsiella are
gram-negative, nonsporing, nonmotile bacilli that 3. Vero cytotoxin of Escherichia coli is similar to:
grow well on ordinary media, produce pink mucoid a. Shiga toxin
colonies on MacConkey’s agar b. Cholera toxin
The name K. pneumoniae is used for the species as a c. Heat-labile enterotoxin of Escherichia coli
whole. It is further divided into 4 subspecies d. Heat-stable enterotoxin of Escherichia coli
Klebsiella pneumoniae can cause a primary 4. Traveler’s diarrhea is caused by
communiy-acquired pnuemonia, nosocomial a. Enteropathogenic Escherichia coli:
infections, urinary tract infections, wound infections,
b. Enterotoxigenic Escherichia coli
bacteremia and meningitis and rarely diarrhea
K. ozaenae- is associated with ozena
c. Enteroinvasive Escherichia coli
K. rhinoscleromatis causes rhinoscleroma, a chronic d. Verotoxigenic Escherichia coli
granulomatous hypertrophy of the nose 5. Haemolytic uraemic syndrome is caused by:
K. oxytoca may be rarely isolated from clinical a. Enteropathogenic E. coli
specimens b. Enterotoxigenic E. coli
Enterobacter: They may cause urinary tract c. Enteroinvasive E. coli
infections and hospital infections, occasionally d. Vero cytotoxin producing E. coli:
associated with meningitis and septicemia
6. Sereny test is used for detection of
Hafnia: They are has been recovered from infected
wounds, abscesses, sputum urine, blood and other
a. Enteropathogenic Escherichia coli
sites b. Enterotoxigenic Escherichia coli
Serratia: S. marcescens is associated with infections c. Enteroinvasive Escherichia coli
of the urinary and respiratory tracts, meningitis, d. Verotoxigenic Escherichia coli
wound infections, septicemia and endocarditis. 7. Prodigiosin (red pigment) is produced by members
of genus:
a. Serratia b. Enterobacter
IMPORTANT QUESTIONS c. Hafnia d. Citrobacter
8. Which of the following species/subspecies of
1. Discuss various mechanisms by which Escherichia Klebsiella produces indole?
coli produces diarrhea. Describe the laboratory a. K. pneumoniae subspecies aerogenes
diagnosis of bacterial diarrheas. b. K. oxytoca
2. Discuss the pathogenicity of Escherichia coli. c. K. pneumoniae subspecies pneumoniae
3. Discuss the laboratory diagnosis of various d. K. pneumoniae subspecies rhinoscleromatis
infections caused by Escherichia coli.
4. Discuss the pathogenesis and laboratory diagnosis ANSWERS (MCQs)
of urinary tract infections caused by Escherichia coli. 1. d; 2. d; 3. a; 4. b; 5. d; 6. c; 7. a; 8. b
Learning Objectives
After reading and studying this chapter, you should ∙∙ List various differences among Proteus, Morganella
be able to: and Providentia.
∙∙ Describe morphology, culture characteristics and
biochemical reactions of Proteus sp
www.ebook3000.com
268 | Section 3: Systemic Bacteriology
are usually first recognized by their characteristic interest. Weil and Felix (1916) studying Proteus
putrefactive odor described as ‘fishy’ or ‘seminal’ bacilli observed that flagellated strains growing
and swarming appearance on noninhibitory on agar formed a thin surface film resembling the
solid media, such as nutrient agar and blood agar. mist produced by breathing on glass and named
Swarming is a striking feature of Pr. mirabilis and Pr. this variety the ‘Hauch’ form (from Hauch, meaning
vulgaris. Swarming of Proteus appears to be due to film of breath). Nonflagellated variants grew as
vigorous motility of the organisim although the exact isolated colonies without the surface film and
cause is not yet established. were called ‘Ohne Hauch’ (meaning without film
Swarming growth is a problem in the laboratory of breath). These names came to be abbreviated
when mixed growth is obtained in which Pro as the Hand 0 forms. Subsequently, the H and O
teus bacilli are present with other bacteria. A were extended to refer to the flagellar and somatic
number of methods have been devised to inhibit antigens of other bacilli as well.
swarming. Swarming of Proteus can be inhibited Weil and Felix also observed that certain
by (i) increasing concentration of agar (6%) and nonmotile strains of Pr. vulgaris, called the ‘X
by (ii) incorporation of chloral hydrale (1 : 500), strains’, were agglutinated by sera from typhus fever
sodium azide (1:500), alcohol (5–6%), sulfonamide, patients. This heterophilic agglutination due to the
surface. active agents or boric acid (1:1000). sharing of an alkali stable carbohydrate antigen
Swarming does not occur on MacConkey’s by certain strains of Proteus (OX2, OX19 and OXK)
medium, on which smooth colorless (NLF) formed. and rickettsiae forms the basis of the Weil-Felix
Proteus produces uniform turbidity with a slight reaction for the diagnosis of some rickettsial
powdery deposit and an ammonical odor in liquid infections. Three nonmotile Proteus strains OX2,
medium (peptone water). OX19 and OXK are used in the agglutination test.
Dienes phenomenon: When two identical strains OX19, OX2 are the strains of P. vulgaris serotype 01
of Proteus are inoculated at different places of the and serotype 02 and OXK is the strain of P. mirabilis
same culture plate, the resulting swarms of growth serotype 03.
coalesce and no line is formed between swarming Typing methods: Phage typing, bacteriocin
culture of the same strain. When, however, two (proticin) typing and serotyping schemes have been
different strains of Proteus species are inoculated, developed for Proteus and Providencia species.
the spreading films of growth fail to coalesce and Swarming Proteus strains exhibit the Dienes
remain separated by a narrow but easily visib1e phenomenon and this forms the basis for a precise
furrow. This is known as Dienes phenomenon. method of differentiation among such strains.
It has been used to determine the identity or
non-identity of various strains of Proteus. Pathogenesis
Proteus bacilli are widely distributed in nature as
Biochemical Reactions saprophytes, being found in decomposing animal
The distinctive characters of this genus are: matter, in sewage, in manured soil and in human and
i. PPA test—Deamination of phenyl alanine animal feces. They are frequently present on the moist
to phenyl pyruvic acid (PPA test) is always areas of the skin. They are opportunistic pathogens,
positive. commonly responsible for urinary and septic infec
ii. Urea hydrolysis—Urea hydrolysis by enzyme tions, often nosocomial.
urease is another characteristic of Proteus, but P. mirabilis accounts for the majority of human
is negative in some Providencia strains. infections seen with this group of organisms. All
iii. All species of Proteus produce acid from members of the tribe can cause urinary tract
glucose. infections (UTI), wound infections, pneumonia,
iv. Lactose is not fermented. infection of the ear, respiratory tract infection,
v. They are malonate utilization negative. septicemia and nosocomial infections. Strains of
vi. Indole is formed by Pr. vulgaris but is negative Pr. mirabilis are a prominent cause of urinary tract
in Pr. mirabilis. infection in children and in domiciliary practice.
vii. They are MR positive and VP negative. UTI caused by Proteus tends to be more serious
viii. H2S is produced by Pr. vulgaris and Pr. mirabilis. than that caused by E. coli and other coliforms.
ix. Nitrate reduction positive. It produces urease which splits urea into carbon
Other biochemical characters of species of dioxide and ammonia. Ammonia inactivates
Proteus are given in Table 37.1. complement, damages renal epithelium and
makes the urine alkaline.This increase in pH causes
Antigenic Structure precipitation of calcium and magnesium salts from
Proteus bacilli possess somatic O and flagellar the urine and results in the formation of urinary
H antigens, which are of considerable historical calculi.
Chapter 37: Tribe Proteeae: Proteus, Morganella and Providencia | 269
Labortory Diagnosis isolated from respiratory and urinary infections in
Culture: Laboratory diagnosis of the infections predisposed or hospitalized patients.
caused by species Proteus can be carried out by
culture of the specimen on MacConkey agar or Key Points
DCA. The tribe Proteeae is classified into three genera
Proteus, Morganella and Providencia
Identification: The isolate is identified by its
Genus Proteus has four species: P. mirabilis, P. vulgaris,
morphological, biochemical and agglutination P. myxofaciens and P. penneri
reactions. Cultural characteristics: Proteus organisms are
usually first recognized by their characteristic pu
Treatment trefactive odor described as ‘fishy’ or ‘seminal’ and
swarming appearance on noninhibitory solid media
Proteus bacilli are resistant to many of the common such as nutrient agar and blood agar
antibiotics. An exception is P. mirabilis which is Pathogenesis: All members of the tribe can cause
sensitive to ampicillin and cephalosporins. urinary tract infections (UTI), wound infections,
pneumonia
Morganella morganii causes urinary infection infre
Morganella quently. Nosocomial wound infections also occur
Five Providencia species may be recovered from feces
Morganella morganii is the only species in the
Erwinia: E. herbicola has occasionally been
genus Morganella. It is motile and lactose-non- isolated from respiratory and urinary infections in
fermenting, and do not swarm on solid media. predisposed or hospitalized patients.
M. morganii is commonly found in human
and animal feces and causes urinary infection
infrequently. Nosocomial wound infections also Important question
occur. Write short notes on:
a. Classification of tribe Proteeae
Providencia b. Pathogenicity of Proteus species
c. Swarming growth
Five Providencia species include P. alcalifaciens, d. Dienes phenomenon
P. rettgeri, P. rustigianii and P. stuartii and P. e. Weil-Felix rection
heimbachae.All species may be recovered from f. Genus Morganella
feces. However, only P. alcalifaciens may be g. Genus Providencia.
associated with diarrhea. P. rettgeri and P. stuartii
have been associated with hospital-acquired Multiple choice questions (MCQs)
urinary tract, wound and other infections, 1. The bacterium that shows swarming on blood agar
particularly in immunocompromised patients. is:
P. stuartii may occasionally cause hospital- a. Proteus stuartii
associated urinary tract infection, infection of b. Proteus mirabilis
c. Providencia rettgeri
burns and pneumonia and septicemia in elderly
d. Morganella morganii
and immunodeficient patients. 2. Phenylalanine deaminase test is positive in:
P. rettgeri is part of the normal fecal flora of a. Proteus b. Morganella
reptiles and amphibians, and sometimes causes c. Providencia d. All of the above
nosocomial infection of the urinary tract, wounds, 3. Diene’s phenomenon is used to detect identity of
burns and blood. strains of:
a. Klebsiella b. Salmonella
c. Shigella d. Proteus
Laboratory Diagnosis
4. Swarming of Proteus strains on solid media can be
Culture: Laboratory diagnosis of the infections inhibited by:
caused by species Proteus, Morganella and a. Increasing the concentration of agar in the me-
Providencia can be carried out by culture of the dium
specimen on MacConkey agar or DCA. b. Incorporation of chloral hydrate in the medium
c. Incorporation of sodium azide in the medium
Identification: The isolate is identified by its d. All of the above methods
morphological, biochemical and agglutination 5. Which of the following Proteus strains is/are used
reactions. in Weil-Felix reaction?
a. Proteus vulgaris OX2
b. Proteus vulgaris OX19
Erwinia c. Proteus mirabilis OXK
These are anaerogenic bacilli forming a yellowish d. All of the above
pigment, usually found in soil and causing plant Answers (MCQs)
infections. E. herbicola has occasionally been 1. b; 2. d; 3. d; 4. d; 5. d.
www.ebook3000.com
38
Chapter
Shigella
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss the antigenic structure and toxins of
be able to: Shigella
∙∙ Describe morphology and various culture media ∙∙ Discuss laboratory diagnosis of bacillary dysentery.
for the isolation of Shigella
www.ebook3000.com
Chap-38.indd 271 15-03-2016 11:16:01
272 | Section 3: Systemic Bacteriology
Table 38.2 Biotypes of Sh. flexneri type 6 illness may also be caused by members of S. flexneri
and S. boydii groups. In contrast dysentery associated
Fermentation of
with S. sonnei (Sonne dysentery) in an otherwise
Glucose Mannitol healthy person may be confined to the passage of a
Boyd 88 A A few loose stools with vague abdonimal discomfort
Manchester AG AG and the patient often continues at school or work.
Newcastle A or AG − Complications: Complications are most often seen
A, acid; AG, acid and gas in patients with S. dysenteriae serotype 1 infection.
These include arthritis, toxic neuritis, conjunctivitis,
parot itis, and, in children, intussusception.
S. sonnei causes the mildest form of bacillary Hemolytic uremic syndrome (HUS) may occur as
dysentery. In many cases the disease may only be a a complication in severe cases.
mild diarrhea. However, S. sonnei infection persists The severity of the disease may vary from acute
as the most common shigellosis in the advanced fulminating dysentery to mild diarrhea. As the term
countries. For epidemiological purposes, S. sonnei bacillary dysentery refers only to the more severe
has been classified into 26 colicin types. cases, the term ‘shigellosis’ has been employed
to include the whole spectrum of disease caused
Pathogenic Mechanisms by shigellae.
1. Surface properties: Lipopolysaccharide (LPS)
has been implicated to entering intestinal cells. Epidemiology
2. Invasiveness: Invasive property is related to Bacillary dysentery has a global distribution. It is
the presence in the bacillus of large plasmids mostly associated with the overcrowding and bad
coding for the outer membrane protein hygienic conditions. Humans are the only known
responsible for cell penetration. These proteins reservoir of Shigella organisms. Transmission may
are called ‘virulence marker antigens’ (VMA). occur by direct person-to person contact, and spread
3. Toxins: S. dysenteriae type 1 forms a produces may take place via the fecal. oral route, with carriers
a powerful exotoxin (Shiga toxin) (details see as the source. From the carries the organisms can
under heading group A (S. dysenteriae). be spread by flies, fingers, food and feces. Young
children in daycare centers, people living in crowded
Pathogenicity and less-than-adequate housing, and young male
Shigellae cause bacillary dysentery. Humans are homosexuals as part of the gay bowel syndrome who
the only known reservoir of Shigella organisms participate in anal-oral sex are most likely affected.
infection occurs by ingestion. The minimum Sh. sonnei is the predominant infecting agent.
infective dose is low, as few as 10–100 bacilli being S. sonnei is the main type in the north in the USA,
capable of initiating the disease. while S. flexneri is more common in the south. In
Shigella cause disease by invading and India, S. flexneri has been the predominant species,
replicating in cells lining the colonic mucosa. After having formed 50–85% of isolates in different series.
reaching the large intestine, the shigellae multiply S. dysenteriae (8–25%) and S. sonnei (2–24%) are the
in the gut lumen. The shigellae multiply within the next common species. S. boydii (0–8%) has been
epithelial cells and spread laterally into adjacent isolated least frequently.
cells, where cell-to-cell passage occurs, and deep
into the lamina propria. The infected epithelial Laboratory Diagnosis
cells are killed and the lamina propria and Diagnosis depends on isolating the bacillus from
submucosa develop an inflammatory reaction with feces.
capillary thrombosis. Patches of nerotic epithelium
are sloughed and ulcer form. The cellular response A. Specimens
is mainly by polymorphonuclear leukocytes.
i. Feces: A specimen of feces is always preferable
The ulcers of the bacillary dysentery are much
to a rectal swab
shallower than amoebic ulcers. Bacteremia may
ii. Rectal swabs: A direct swab may be taken from
occur in severe infections.
the ulcer by sigmoidoscopic examination.
Bacillary dysentery has a short incubation
period (1–7 days, usually 48 hours). The main clinical
features are frequent passage of loose, scanty feces B. Transport
containing blood and mucus, along with abdominal Fresh feces should be inoculated without delay or
cramps and tenesmus. Fever and vomiting may be transported in a suitable medium such as Sachs’
present. Infection is usually self-limited. buffered glycerol saline. pH 7.0–7.4, which prevents
In dysentery caused by S. dysenteria type 1, the dysentery bacilli from being destroyed by the
patients experience more severe symptoms. Severe acid produced during the growth of other organisms.
www.ebook3000.com
Chap-38.indd 273 15-03-2016 11:16:01
39
Chapter
LEARNING OBJECTIVES
After reading and studying this chapter, you should Salmonella; antigenic variation of Salmonellae;
be able to: Kauffmann-White scheme
∙∙ Describe morphology, culture charactrestics and ∙∙ Discuss laboratory diagnosis of enteric fever
biochemical reactions of Salmonella sp ∙∙ Describe vaccination against enteric fever
∙∙ Describe the following: Antigenic structure of ∙∙ Discuss Salmonella gastroenteritis.
Biochemical Reactions
1. Salmonellae ferment glucose, mannitol, arabi
nose, maltose, dulcitol and sorbitol, forming
acid and gas except S. Typhi, Gallinarum and
rare anaerogenic variants in other serotypes form
only acid and no gas.
2. Lactose, sucrose, salicin or adonitol are not
fermented.
3. They are, indole positive, MR positive, VP
negative and citrate positive (IMViC – + – +) Fig. 39.1: Antigenic structure of salmonellae
except by S. Typhi and S. Paratyphi A which are Several strains carry fimbriae. Fimbrial antigens
citrate negative as they need tryptophan as the are not important in identification but may cause
growth factor. confusion due to their nonspecific nature and
4. Hydrogen sulfide is produced except by S. widespread sharing among enterobacteria.
Paratyphi A, S. Choleraesuis, S. Typhisuis and 1. H antigen: These antigens represent deter
S. Sendai. minant groups on the flagellar protein. They
5. Urease is not hydrolyzed. are heat-labile and alcohol-labile, but are well
6. Salmonellae decarboxylate the amino acids preserved in 0.04–0.2% formaldehyde. In most
lysine, ornithine and arginine. salmonellae, H antigen exists in two alternative
The enteric fever group may be separated phases called phase 1, and phase 2.
biochemically (Table 39.1). When mixed with antisera, H suspensions
agglutinate rapidly, producing large, loose, fluffy
Resistance clumps. The H antigen is strongly immunogenic
Salmonellae are readily killed by moist heat, at 55°C and induces antibody formation rapidly and in
in 1 hour or at 60°C in 15 minutes and most strong high titers following infection or immunization.
disinfectants. Boiling or chlorination of water and 2. O antigens: The somatic O antigen is a
pasteurization of milk destroy the bacilli. They are phospholipidprotein-polysaccharide complex
killed within five minutes by mercuric chloride (1: which forms an integral part of the cell wall.
500) or 5% phenol. The O antigen is unaffected by boiling (heat-
stable), alcohol (alcohol-stable) or weak acids.
Antigenic Structure The O antigen is less immunogenic than the H
antigen and the titer of the O antibody induced
Antigenic structure of salmonellae is shown in after infection or immunization is generally lower
Figure 39.1. Salmonellae possess the following than that of the H antibody. O agglutination takes
antigens based on which they are classified and place more slowly and at a higher temperature
identified: optimum (50–55°C) than H agglutination (37°C).
1. Flagellar antigen H. 3. Vi antigen: Almost all recently isolated strains of
2. Somatic antigen O. S. Typhi form Vi antigen as a covering layer
3. Surface antigen Vi, found in some species. outside their cell wall. This antigen is an acidic
Table 39.1 Biochemical characters of typhoid and polysaccharide.When fully developed it renders
paratyphoid bacilli the bacteria agglutinable by Vi antibody and
inagglutinable by O antibody. Felix and Pitt,
Glucose Xylose d-Tartrate Mucate who first described this antigen, believed that
S. Typhi A d A d it was related to virulence and gave it the name
S. Paratyphi A AG − − − ‘Vi antigen’ (Vi for virulence). It is analogous to
S. Paratyphi B AG AG − AG
the K antigens of coliforms.
The Vi antigen is heat-labile. It can be removed
S. Paratyphi C AG AG AG −
from the bacteria by heating a suspension for 1
A = Acid; AG = Acid and Gas; d = Delayed hour at 100°C and centrifuging the bacteria from
www.ebook3000.com
Chap-39.indd 275 15-03-2016 11:16:23
276 | Section 3: Systemic Bacteriology
the Vi-containing fluid. It is also destroyed by N at both ends, in such a way that it projects above
HCl and 0.5 N NaOH. It is unaffected by alcohol the agar. The strain is inoculated carefully into
or 0.2% formol. the inner tube. After shortest period (8–16 hours)
Originally observed in S. Typhi, the Vi antigen incubation, subcultures are withdrawn from
with similar antigenic specificity is present in S. the top of the agar outside the central tube. This
Paratyphi C and S. Dublin, as well as in certain yields a population of motile cells (Fig. 39.2).
strains of Citrobacter (the Ballerup-Bethesda A U-tube of soft agar may be employed
group). The Vi antigen tends to be lost on serial instead of Craigie’s tube. Inoculation is made into
subculture. The Vi polysaccharide acts as a one limb and subculture taken from the other.
virulence factor by inhibiting phagocytosis, 2. Phase variation: The flagellar antigens of most
resisting complement activation and bacterial salmonellae occur in one of two phases. Phase
lysis by the alternative pathway and peroxi 1 antigens are either specific for a species or
dase mediated killing. Strains possessing the shared by a few species only. Hence phase I is
Vi antigen were found to cause clinical disease called the ‘specific’ phase. Phase 2 antigens are
more consistently than those lacking it in human widely shared and hence phase 2 is called the
volunteer experiments. ‘nonspecific’ or ‘group’ phase. The presump
The Vi antigen is poorly immunogenic and tive identification of serotypes, therefore, mainly
following infection only low titers of antibody are depends on the identification of the H antigens
produced. Vi antigen is not employed in the Widal in phase I, which are relatively ‘specific’.
test because detection of the Vi antibody is not Phase 1 antigens are designated a, b, c, d,
helpful for the diagnosis of cases. The total absence etc., and after z, as z 1, z2, etc. Phase 2 antigens
of the Vi antibody in a proven case of typhoid fever are far fewer and are termed I, 2, etc. In some
indicates poor prognosis. The antibody disappears species, antigens belonging to phase 1 may
early in convalescence. Its persistence indicates the occur as the phase 2 antigens (for example, e, n,
development of the carrier state. The Vi antigen x, z 15). The strains that possess both phases are
affords a method of epidemiological typing of the S. known as ‘diphasic’ and the strains that possess
Typhi strains based on specific Vi bacteriophages. only one phase are known as monophasic (e.g.
Phenolized vaccine induces no Vi antibody S. Typhi, S. Paratyphi A, S. Enteritidis, etc.).
though low titers are produced by the alcoholized A culture will contain cells with the flagellar
vaccine. The protective efficacy of the Vi antigen antigens of both phases but generally one or
is demonstrated by the success of the purified Vi the other phase will predominate so that the
vaccine for typhoid now in routine use. culture is agglutinated only by one of the phase
antisera. It may be necessary to identify the
Antigenic Variations flagellar antigens of both phases for serotyping
The antigens of salmonellae undergo phenotypic of salmonella isolates. A culture in phase 1 can
and genotypic variations. be converted to phase 2 by passing it through
1. H–O variation: This variation is associated a Craigie’s tube containing specific phase 1
with the loss of flagella. When salmonellae antiserum, and the reverse conversion achieved
are grown on agar containing phenol (1 : 800), by using phase 2 antiserum (Fig. 39.2).
flagella are inhibited. This change is phenotypic 3. V–W variation: Almost all recently isolated
and temporary. Flagella reapp ear when strains of S. Typhi form Vi antigen as a covering
the strain is subcultured on media without
phenol. Salmonellae may rarely lose flagella by
mutation resulting in the development of stable
or irreversible mutant. For example, a stable
nonmotile mutant of S. Typhi is the 901-O strain
which is widely employed for the preparation
of O-agglutinable bacterial suspensions.
Craigie’s tube: Generally, the loss of flagella is
not total and there occurs only a diminution
in the number of flagella and the quantity of
the H antigen. Flagellated cells are found in
small numbers in such cultures. To obtain a
population of motile cells, rich in H antigen,
from such cultures, selection may be carried out
by using Craigie’s tube. This consists of a wide
tube containing soft agar (0.2%) at the center of
which is embedded a short, narrow tube open Fig. 39.2: Craigie’s tube
www.ebook3000.com
Chap-39.indd 277 15-03-2016 11:16:23
278 | Section 3: Systemic Bacteriology
Table 39.3 Kauffmann–White classification–Antigenic formulae of some representative serotypes of
Salmonella
Serogroup Oantigen Serotype name O antigens and Vi H antigens
group
Phase 1 Phase 2
2 A Paratyphi A 1, 2, 12 a [1, 5]
4 B Paratyphi B 1, 4, [5], 12 b 1, 2
Stanley 1.4, [5], 12, 27 d 1, 2
Typhimurium 1.4, [5], 12 i 1, 2
Heidelberg 1.4, [5], 12 r 1, 2
7 C1 Choleraesuis 6, 7 c 1, 5
Paratyphi C 6, 7 [Vi] c 1, 5
Typhisuis 6, 7 c 1, 5
Virchow 6, 7 r 1, 2
8 C2-C3 Muenchen 6, 8 d 1, 2
Newport 6, 8, 20 e, h 1, 2
Hadar 6, 8 z10 e, n, x
Miami 1, 9, 12 a 1, 5
- Sendai 1, 9, 12 a 1, 5
9 D1 Typhi 9, 12 [Vi] d –
Enteritidis 1, 9, 12 g, m [1, 7]
Dublin 1, 9, 12, [Vi] g, p –
Panama 1, 9, 12 I, v 1, 5
Gallinarum 1, 9, 12 – –
3,10 E1 Anatum 3, 10, [12], [1Q,; H) e, h 1, 6
not found in other O groups. The O groups first more than 2400 serotypes of salmonellae, giving
defined were designated by capital letters A to Z individual names is not realistic.
and those discovered later by the number (51–67)
of the’ characteristic O antigen. It would be more Differentiation of Antigenically Similar
logical to name the serogroups according to their Strains
characteristic O antigen factor number rather than
Serotypes that share the same antigenic formula
by letters, i.e. to abandon the letters A–Z used to
may be distinguished from one another by bio
designate early O groups. Hence, O groups become:
chemical tests, e.g. Paratyphi C has the same O and
O2 (A), O4 (B), O7 (Cl), O8 (C2–C3), 09, 12 (Dl),
H antigens as Choleraesuis, and Typhisuis. Thus,
09, 46 (D2), 03, 10 (EI) etc. Some serogroups were
Paratyphi C ferments d-tartrate and trehalose,
subdivideed into subserogroups (C1–C4; E1–E4).
but not Stern’s glycerol. Choleraesuis ferments
Serogroups A–Z and 51–67 represented O antigens
d-tartrate, but not trehalose or Stern’s glycerol, and
2–50 and 51–67 respectively. Groups 02 to 03, 10
Typhisuis only trehalose.
(A–EI) contain nearly all the salmonellae that are
important pathogens in man and animals.
Within each O group the different serotypes are Bacteriophage Typing
distinguished by their particular H antigen or com Strains within a particular serotype may be differen
bination of H antigens (Table 39.3). Kauffmann– tiated into a number of phage types by their patterns
White classification gave species status to each of susceptibility to lysis by members of a series of
serotype. The species were named according to phages with different specificities. Intraspecies
the disease caused (S. Typhi), the animal source (S. classification of S. Typhi for epidemiolocal
Gallinarium), the discoverer (S. Schottmulleri), the purpose was made possible by bacteriophage
name of the patient from whom the first strain was typing, first developed by Craige and Yen (1937).
isolated (S. Thompson), or the place of isolation They observed that a bacteriophage acting on the
(S. Poona). This was satisfactory so long as the Vi antigen of the typhoid bacillus (Vi phage II) is
serotypes were not too many but now with some highly adaptable. The parent phage is called phage
www.ebook3000.com
Chap-39.indd 279 15-03-2016 11:16:24
280 | Section 3: Systemic Bacteriology
S. Paratyphi C may also cause paratyphoid fever A. Isolation and Identification of the Bacilli
but more often it leads to a frank septicemia with It may be done by culture of specimens such
suppurative complications. as patient’s blood, feces, urine, bone marrow,
duodenal drainage rose spots etc. For the
2. Bacteremia with Focal Lesion leborotatory diagnosis of enteric fever, selection
This is associated with S. Cholerae-suis but may of relevant specimens depends upon duration
be caused by any Salmonella serotype. Infection of illness, e.g. blood for culture must be taken
occurs by oral route and there is early invasion of repeatedly. Urine culture may be positive after
the bloodstream with possible focal lesions and second week and stool second or third week.
in particular may cause septicemic disease (with
focal lesions, in lungs, bones, meninges, etc.) but Blood Culture
intestinal manifectations are often absent. Blood
Blood cultures are positive in approximately 90%
culture are positive.
cases in the first week of fever, in approximately
75% of cases in the second week, 60% in the third
3. Gasteroenteritis or Food Poisoning
week and 25% thereafter till the subsidence of
This is the most common manifestation of pyrexia (Fig. 39.3, Table 39.4).
Salmonella infection. In the USA, Salmonella With all aseptic precautions, about 5–10 mL of
Typhimurium and Salmonella enteritis are blood is collected by venipuncture and inoculated
prominent (For detail see under the heading, into a culture bottle containing 50–100 mL of 0.5%
“Salmonella gastroenteritis”.) bile broth. Large quantity (5–10 mL) of blood is
required because the number of organisms are not
4. Asymptomatic Carrier State numerous and may be quite small. The blood is
Most people infected with salmonella continue
to excrete the organism in their stools for days
or weeks after complete clinical recovery, but
eventual clearance of the bacteria from the body
is usual. A few patients continue to excrete the
salmonellae for prolonged periods.
Epidemiology
The typhoid and paratyphoid bacilli are essentially
human parasites. Man is the reservoir host and
most infections can be traced to a human source,
or at least to a source of human sewage. All other
salmonellae have animal hosts.
S. Paratyphi A is prevalent in India and other
Asian countries, Eastern Europe and South
America, S. Paratyphi B in Western Europe, Britain
and North America; and S. Paratyphi C in Eastern Fig. 39.3: Laboratory diagnosis of typhoid fever. The appro
Europe and Guyana. Enteric fever is endemic in all ximate percentages of tests found positive during different
parts of India. Paratyphoid B is rare and C very rare. stages of the disease (from 1st to 5th week). A. Widal aggluti
nation. B. Feces culture. C. Blood culture
The disease occurs at all ages but is probably most
common in the 5–20 year age group.
The source of infection is a patient, or far more
Table 39.4 Positivity of various specimens at
different phases of enteric fever
frequently, a carrier. Food handlers or cooks who
become carriers are particularly dangerous. The Duration Specimen % positivity
best known of such typhoid carriers was Mary 1st week Blood culture 90
Mallon (‘Typhoid Mary’), a New York cook who Feces culture –
caused at least seven outbreaks affecting over 200
Widal test –
persons over a period of 15 years.
2nd week Blood culture 75
Laboratory Diagnosis Feces culture 50
Bactriological dignosis of enteric fever consists of: Widal test Low titer
A. Isolation and identification of the bacilli. 3rd week Blood culture 60
B. Demonstration of circulating antigen.
Feces culture 80
C. Demonstration of antibodies in patient’s serum.
Widal test 80–100
D. Other laboratory tests.
www.ebook3000.com
Chap-39.indd 281 15-03-2016 11:16:24
282 | Section 3: Systemic Bacteriology
(S. Paratyphi A, B and C) will form acid and gas i. Widal Test
from sugars. Testing the patient’s serum for Salmonella antibodies
Identification: Identification of the isolate is by is useful only in the diagnosis of enteric fever (Widal
slide agglutination. reaction). Tests for the presence of Salmonella
antibodies in the patient’s serum may be of value
Slide agglutination test: A loopful of the growth in the diagnosis of enteric fever. This is a test for
from an agar slope is emulsified in two drops of measurement of H and O agglutinins for typhoid
saline on a slide. One emulsion acts as a control to and paratyphoid bacilli in the patient’s serum. The
show that the strain is not autoagglutinable. If S. patient’s serum is tested by tube agglutination for its
Typhi is suspected (that is, when no gas is formed titers of antibodies against H, O and Vi suspensions
from glucose), a loopful of typhoid O antiserum of the enteric fever bacteria likely to be encountered,
(factor 9/group D) is added to one drop of e.g. S. Typhi and S. Paratyphi A in India. Two types
bacterial emulsion on the slide, and agglutination of tubes are generally used for the test:
looked for after rocking the slide gently. Prompt 1. Dreyer’s agglutination tube (narrow tubes with
agglutination indicates that the isolate belongs a conical bottom) for the H agglutination.
to Salmonella group D. Its identity as S. Typhi is 2. Felix tube (a short round bottomed tube) for
established by agglutination with the flagellar the O agglutination.
antiserum (anti-d serum). Quite often, fresh Equal volumes (0.4 mL) of serial dilutions of
isolates of S. Typhi are in the V form and do not the serum (from 1/10 to 1/640) and the H antigens
agglutinate with the O antiserum. Such strains may of S. Typhi (TH) and S. Paratyphi A (AH) and O
be tested for agglutination against anti-Vi serum. antigen of S. Typhi (TO) are mixed in Dreyer’s and
Alternatively, the growth is scraped off in a small Felix’s agglutination tubes respectively. Incubate H
amount of saline, boiled for 20 minutes and tested agglutinations for 2–4 hours in water bath at 37°C
for agglutination with the O antiserum. and read after standing on the bench for half an
Where the isolate is a nontyphoid Salmonella hour. Incubate O agglutinations for 4–6 hours at
(producing gas from sugars), it is tested for 37°C and read after overnight refrigeration at 4°C.
agglutination with O and H antisera for groups A, Controls tubes containing the antigen and normal
B and C. For identification of unusual serotypes, saline are set to check for autoagglutination. The
the help of the ‘National Salmonella Reference agglutination titers of the serum are read.
Center’ should be sought. The National Salmonella H agglutination leads to the formation of loose,
Reference Center in India is located at the Central cotton woolly clumps, while O agglutination is
Research Institute, Kasauli. The reference center seen as a disc like pattern at the bottom of the
for salmonellae of animal origin is at the Indian tube. Control (Felix) tube shows a compact
Veterinary Research Institute, Izatnagar. deposit. In both, the supernantant fluid is rendered
clear. The highest dilution of the serum showing
B. Demonstration of Circulating Antigen agglutination indicates the titer of the antibody.
In the early phase of the disease, typhoid bacillus The antigens used in the test are the H and O
antigens are consistently present in the blood antigens of S. Typhi and S. Paratyphi A and B. The
and also in the urine of patients. The antigen can paratyphoid O antigens are not employed as they
be demonstrated by sensitized staphylococcal cross react with the typhoid O antigen due to their
coagglutination. Staphylococcus aureus containing sharing of factor 12. Although suspensions may be
protein A (Cowan 1 strain) is stabilized with prepared from suitable stock laboratory cultures,
formaldehyde and then coated with S. Typhi anibody. there is little need for that nowadays because
When a 1% suspension of such sensitized cells is ready made Widal kits of stained antigens available
mixed on a slide with serum from patients in the first commertially are now widely used.
week of typhoid fever, the typhoid antigen present in
the serum combines specifically with the antibody
Interpretation of Widal Test
attached to the staphylococcal cells producing visible 1. Stage of the disease: The agglutinins titer will
agglutination within two minutes. The test is rapid, depend on the stage of the disease. Usually
sensitive and specific but is not positive after first week agglutinins appear by the end of first week
of the disease. ELISA test has also been employed to (seventh to tenth day) of the illness, so that
detect typhoid antigen in blood and urine. blood taken earlier may give a negative result
and is inconclusive. The titer then increases
C. Demonstration of Antibodies in steadily till the third or the fourth week, after
which it declines gradually.
Patient’s Serum 2. Rising titer: Demonstration of a rise in titer of
i. Widal test. antibodies by testing two or more samples is
ii. Other serological tests. more meaningful than a single test.
www.ebook3000.com
Chap-39.indd 283 15-03-2016 11:16:24
284 | Section 3: Systemic Bacteriology
Note: In India a divalent vaccine containing S. Typhi chloramphenicol did not pose any problem
and S. Paratyphi A are now in use instead of the TAB in typhoid fever till 1972, when resistant
vaccine, eliminating S. paratyphi B which is rare in strains emerged in Mexico and Kerala in India.
the country or the monovalent typhoid vaccine is Chloramphenicol resistant typhoid fever appeared
preferred. In Europe and USA, monovalent vaccine in epidemic form first in Calicut (Kerala) in early
containing S. Typhi is employed as paratyphoid A 1972. It became endemic and was confined to
and B infections are rare in these countries. Kerala till 1978. Subsequently such strains carrying
drug resistance plasmids appeared in many
Oral Vaccine–Live Oral (Ty21) Typhoid Vaccine other parts of India. Resistance was originally
The live oral vaccine (typhoral) is a stable mutant of confined to phage type D1-N, but later to types
S. Typhi strain (Ty21a), lacking the enzyme UDP- C5, A and O. This multidrug resistance was due
galactose-4-epimerase (Gal E mutant). On ingestion, to a transmissible plasmid carrying resistance
it initiates infection but ‘self destructs’ after four or determinants to chloramphenicol, streptomycin,
five cell divisions, and therefore, it cannot induce sulfadiazine and tetracycline (CSSuT).
any illness. This oral vaccine is available in enteric A ciprofloxacin-resistant S. Typhi isolate was
coated capsule containing 109 viable lyophilized first reported from the United Kingdom in 1992. At
mutant bacilli. present, the drugs useful in treatment of such multire
sistant typhoid cases are the later fluoroquinolones
Dose schedule: Three doses of the vaccine are (such as ciprofloxacin, pefloxacin, ofloxacin) and
given on alternate days to children. The course the third generation celphalosporins (such as
consists of one capsule orally, taken an hour before ceftazidime, ceftrioxone, cefotaxime).
food, with a glass of water or milk, on days 1, 3, and
5. No antibiotic should be taken during this period. SALMONELLA GASTROENTERITIS
Protection: It is safe and confers 65–96% protection Salmonella gastroenteritis (more appropriately
for 3–5 years. enterocolitis) or food poisoning is generally a
zoonotic disease. The source of infection being ani
Vaccine of Purified Vi Antigen (Typhim-Vi) mal products. It may be caused by any salmonella
This injectable vaccine (typhim-Vi) contains except S. Typhi. In most parts of the world, S.
purified Vi polysaccharide antigen (25 µg per dose) Typhimurium is the commonest (30–40%) species.
from S. Typhi strain Ty2. Some other common species have been S. Enteri
It is given as a single subcutaneous or intra tidis, S. Haldar, S. Heidelberg, S. Agona, S. Virchow,
muscular injection. This vaccine is not recom S. Seftenberg, S. Indiana, S. Newport and S. Anatum
mended for children younger than 2 years. S. Dublin.
In the trials conducted the protection conferred
Source of Infection
by the vaccine was 64% and 72%.
The most frequent sources of Salmonella food
Treatment poisoning are poultry, meat, milk and milk products.
Specific antibacterial therapy for enteric fever Of great concern are eggs and egg products.
became available only in 1948 with the introduction Food contamination may also result from the
of chloramphenicol, which continued as the sheet droppings of rats, lizards or other small animals.
anchor against the disease till the 1970s when Human carriers do occur but their role is minimal.
resistance became common. Ampicillin, amoxicillin,
and trimethoprim-sulfamethoxazole have been used Clinical Features
successfully. Clinically, the disease develops after a short
In the past decade, third-generation cephalo incubation period of 24 hours or less, with diarrhea,
sporins, particularly ceftriaxone and cefoperazone, vomiting, abdominal pain and fever. It usually
and fluoroquinolones including norfloxacin, subsides in 2–4 days but in some cases a more
ciprofloxacin, ofloxacin, and pefloxacin all are at prolonged enteritis develops.
least as effective as chloramphenicol in treating
typhoid fever. Ciprofloxacin has emerged as the Laboratory Diagnosis
drug of choice for the treatment of adult typhoid,
The laboratory diagnosis depends on the isolation
and is proving equally effective and free from side-
of the causal organism from samples of feces or
effects in children.
suspected foodstuffs.
www.ebook3000.com
Chap-39.indd 285 15-03-2016 11:16:25
286 | Section 3: Systemic Bacteriology
5. Which of the following serotypes of Salmonella is/ 9. Most important complications of enteric fever is/
are anerogenic? are:
a. S. Typhi b. S. Paratyphi A a. Intestinal perforation b. Hemorrhage
c. S. Enteritidis d. All of the above c. Circulatory collapse d. All of the above
6. Citrate is not used by the following Salmonella: 10. Most important specimen for detection of carriers
a. Salmonella Typhi in enteric fever is:
b. Salmonella Paratyphi A
a. Feces b. Urine
c. Both of the above
d. None of the above c. Blood d. Sputum
7. Which of the following Salmonella is/are primarily 11. All the following are the examples of killed vaccines
human pathogens? used against typhoid fever except:
a. S. Typhi b. S. Paratyphi A a. TAB vaccine
c. S. Paratyphi B d. All of the above b. Vi capsular polysaccharide antigen vaccine
8. The most important specimen for isolation of c. Acetone-inactivated parenteral vaccine
Salmonella Typhi culture in first week of enteric d. Ty21a vaccine
fever?
a. Blood b. Urine ANSWERS (MCQs)
c. Feces d. CSF 1. a; 2. a; 3. d; 4. c; 5. a; 6. c; 7. d; 8. a; 9. d; 10. a; 11. d
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Differentiate between classical and El Tor vibrios
be able to: ∙∙ Discuss laboratory diagnosis of cholera
∙∙ Describe morphology, cuture characteristics and ∙∙ Describe the following: cholera vaccine; nonagglut
biochemical reactions of Vibrio cholerae inating vibrios; halophilic vibrios
∙∙ Discuss antigenic structure of Vibrio cholerae
∙∙ Describe the following: pathogenesis of cholera;
mechanism of action of cholera toxin
VIBRIO CHOLERAE
Morphology
These are gram-negative, short, curved, cylindrical
rods, about 1.5 μm × 0.2–0.4 μm in size. The cell
is typically comma-shaped. S-shaped or spiral
forms may be seen due to two or more cells lying
end-to-end. In old cultures, they are frequently
highly pleomorphic. The vibrios are seen arranged
in parallel rows, described by Koch as the ‘fish in
stream’ appearance in stained films of mucous
flakes from acute cholera cases. Fig. 40.1: Cholera vibrios
www.ebook3000.com
Chap-40.indd 287 15-03-2016 11:17:33
288 | Section 3: Systemic Bacteriology
A. Ordinary Media C. Plating Media
i. Nutrient agar: After overnight growth, 1. Alkaline bile salt agar (BSA); pH 8.2
colonies are moist, translucent, round disks, This is modified nutrient agar medium containing
about 1–2 mm in diameter, with a bluish tinge 0.5% sodium taurocholate. The colonies on BSA are
in transmitted light. The growth has a dis similar to those on nutrient agar medium.
tinctive odor. 2. Monsur’s gelatin taurocholate trypticase
ii. MacConkey agar: The colonies are colorless, tellurite agar (GTTA) medium; pH 8.5
but become reddish on prolonged incubation After 24 hours incubation, vibrios produce small
due to the late fermentation of lactose. (1–2 mm) translucent colonies with grayishblack
iii. Blood agar: Colonies are initially surrounded center and a turbid halo, due to hydrolysis and
by a zone of greening, which later becomes denaturation of gelatin. After 48 hours incubation,
clear due to hemodigestion. colonies increase in size to 3–4 mm.
iv. Gelatin stab culture: At first a white line 3. Thiosulfate-citrate-bile-sucrose (TCBS) agar—
of growth appears along the track of the pH 8.6
inoculating wire. Liqu efaction of gelatin This is most used selective plating medium for
begins at the top, which spreads downwards in vibrios. Constituents of this medium are sodium
infundibuliform (funnel shaped) or napiform thiosulphate, sodium citrate, ox bile, sucrose, yeast
(turnip-shaped) in 3 days at 22°C. extract, peptone, sodium chloride, ferric citrate,
v. Peptone water: When incubated at 37°C in thymol blue, bromothymol blue (indicator) and
liquid media, such as peptone water, it forms water. On this differential medium, the colonies
a fine surface pellicle because of its affinity for of sucrose-fermenting vibrios, e.g. V. cholerae, are
oxygen. yellow, those of sucrose-non-fermenting vibrios,
e.g. V. parahaemolyticus, are green.
B. Special Media Biochemical Reactions
a. Holding or Transport Media 1. Sugar fermentation: It ferments glucose,
1. Venkatraman–Ramakrishnan (VR) medium mannitol, maltose, mannose and sucrose and
A simple modified form of this medium is prepared ferments lactose only after several days (late
by dissolving 20 g crude sea salt and 5 g peptone lactose-fermenter).
in one liter of distilled water and adjusting the pH 2. Cholera red reaction: V. cholerae is strongly
to 8.6–8.8. It is dispensed in screw capped bottles indole positive and reduces nitrates to nitrites.
in 10–15 mL amounts. About 1–3 mL stool is to be These two properties contribute to the ‘cholera
added to each bottle. Vibrios do not multiply in red reaction’ which is tested by adding a few
this medium but remain viable for several weeks. drops of concentrated sulfuric acid to a 24-hour
peptone water culture at 37°C. A reddish pink
2. Cary–Blair medium color is developed due to the formation of
This is a buffered solution of disodium hydrogen nitroso-indole with cholera vibrios.
phosphate, sodium thioglycollate, sodium chloride 3. It is catalase and oxidase-positive, methyl red
and agar to distilled water and pH 8.4. It is a suitable and urease negative.
transport medium for Salmonella and Shigella as 4. It decarboxylates lysine and ornithine but does
well as for vibrios. not utilize arginine.
3. Autoclaved sea water: Autoclaved sea water also 5. Gelatin is liquefied.
serves as a holding medium. 6. Voges-Proskauer reaction and hemolysis of
sheep RBCs are positive in El Tor biotype and
b. Enrichment Media both these reactions are negative in classical
biotype.
The rapid growth and tolerance of vibrios for alkaline 7. String test: Vibrio colonies may be identified
conditions is exploited in the formulation of media by the ‘string test’. A loopful of the growth is
used for their isolation. mixed with a drop of 0.5% sodium deoxycholate
1. Alkaline peptone water: Alkaline peptone water in saline on a slide. If the test is positive, the
at pH 8.6 is useful for preliminary enrichment suspension loses its turbidity, becomes mucoid
of vibrios from feces or other contaminated and forms a ‘string’ when the loop is drawn
materials. slowly away from the suspension.
2. Monsur’s taurocholate tellurite peptone
water at pH 9.2. RESISTANCE
Both these are good transport as well as enrich Cholera vibrios are susceptible to heat, drying and
ment media. acids, but resist high alkalinity. Vibrios are killed
www.ebook3000.com
Chap-40.indd 289 15-03-2016 11:17:33
290 | Section 3: Systemic Bacteriology
Table 40.2 Heiberg grouping of vibrios Table 40.4 Serotypes of cholera vibrios
Group Fermentation Sucrose Arabinose Serotype O antigens
of mannose Ogawa AB
I A A – Inaba AC
II – A – Hikojima ABC
III A A A
IV – A A 3. Sensitivity to cholera phage IV
V A – – All strains of classical cholera vibrio are lysed
VI – – – by Mukherjee’s group IV phage routine test
dilution (RTD), while all EI Tor strains are
VII A – A
not lysed. This is considered to be the most
VIII – – A dependable test for differentiating between EI
Tor and classical strains.
V. cholerae 0139
Table 40.3 Differeces between classical cholera In 1992 cases of cholera indistinguishable from that
and El Tor vibrios caused by V. cholerae 01 were reported in Madras
Test Classical cholera El. Tor (Chennai), India. By mid-January 1993 similar
isolates were found in neighboring Bangladesh,
Hemolysis − +*
and these rapidly spread north, following the
Voges-Proskauer − +* course of the major rivers and raising fears of a new
Chick erythrocyte − + pandemic.
agglutination V. cholerae 0139 may have evolved from V.
Polymyxin B sensitivity† + − cholerae 01, but with modified lipopolysaccharide
Group IV phage + − structure. V. cholerae 0139 makes a polysaccharide
susceptibility capsule like other non-O 1 V. cholerae strains, while
El Tor phage 5 − + V. cholerae 01 does not make a capsule. In the
susceptibility affected areas, this strain replaced the El Tor vibrios
* Strain isolated after 1961 give variable results. as the epidemic and environmental serovar. It also
†
50 i. u. disk. showed a tendency to be more invasive, causing
bacteremic illness in some. Both O-1 El Tor and
2. Sensitivity to polymyxin B: The organism is O-139 strains began to coexist in endemic areas.
tested by the disc diffusion method using discs
containing 50 units of polymyxin B. All strains Phage Typing
of classical cholera vibrio are sensitive and all Phage typing schemes have been standardized
strains of EI Tor vibrio are resistant. for classical and El Tor biotypes. New molecular
www.ebook3000.com
Chap-40.indd 291 15-03-2016 11:17:34
292 | Section 3: Systemic Bacteriology
since 1817, resulting in thousands of deaths and by 1994 the El Tor strain regained its dominance
major socioeconomic changes. and the threat of an 0139 pandemic diminished.
Cholera is an exclusively human disease.
Infection is generally spread by contaminated Laboratory Diagnosis
water or foods. The source of the contamination A. Specimen
is usually the feces of carriers or patients with
∙∙ Watery stool
cholera.
∙∙ Rectal swabs.
All pandemics of cholera caused by serotype 01,
although 0139 can cause similar diseases and may B. Collection of Specimen
cause a pandemic.
a. Stool: A fresh specimen of stool should be
India, more specifically the large deltaic area
collected for laboratory examination. Sample
of the Ganges and Brahmaputra in Bengal, is its
should be collected before the person is treated
homeland, where it has been known from very
with antibiotics. Collection may be made
ancient times. Till early in the nineteenth century,
generally in one of the following ways:
cholera was virtually confined to India, periodically
i. Rubber catheter: Fecal specimens from early
causing large epidemics in different parts of the
acute cases should be collected into a sterile
country. From 1817 to 1923, cholera vibrios had
container, preferably through a soft sterile
spread from Bengal, in six separate pandemic waves,
rubber catheter inserted into the rectum.
involving most parts of the world. After the end of the
ii. Rectal swab: Rectal swabs are useful in
6th pandemic in 1923, till 1961 the disease remained
collecting specimens from convalescents.
confined to its endemic areas, except for an isolated
Collection from a bedpan should be avoided
epidemic in Egypt in 1947. It was largely due to the
because of the risk of contamination or the
threat of pandemic cholera that international health
presence of disinfectant.
organizations came into being.
The seventh pandemic occurred in 1961 and was C. Transportation
first to be caused by the El Tor biotype. It originated
If possible, specimens should be processed without
from Sulawesi (Celebes), Indonesia. V. cholerae 0 I
delay but, if there is to be a delay of more than 6
biotype El Tor was first isolated by Gotschlich at the
hours in their reaching the laboratory, feces or
El Tor Quarantine Station in Egypt. After spreading
rectal swabs should be placed in a liquid alkaline
to Hongkong and the Philippines, it spread steadily
transport medium. Stool samples may be preserved
westwards, invading India in 1964. By 1966, it had
in VR fluid or CaryBlair medium for long periods.
spread throughout the Indian subcontinent and
If the specimen can reach the laboratory in a
West Asia. In the 1970s the pandemic extended to
few hours, it may be transported in enrichment
Africa and parts of Southern Europe.
media, such as alkaline peptone water or Monsur’s
Wherever the El Tor vibrio has caused long
medium.
standing infection, in all those areas it has
Strips of blotting paper may be soaked in the
displaced the classical vibrio. It supplanted the
watery stool and sent to the laboratory packed
classical biotype in India during the 1960s and
in plastic envelopes if transport media are not
spread into other parts of the country previously
available.
free from this infection. Similarly, in Bangladesh
Whenever possible, specimens should be
it entirely displaced classical biotype by 1973, but
plated at the bedside and the inoculated plates sent
the latter staged a comeback in 1982 and replaced
to the laboratory.
El Tor vibrio in many areas.
In January, 1991, the pandemic reached Peru, D. Microscopy
thus encircling the globe in 30-year time. By 1994
most parts of Central and South America had been Direct microscopic examination of feces is not rec
involved and rendered endemic. ommended. For rapid diagnosis, the characteristic
In 1992, cases of cholera indistinguishable motility of the vibrio and its inhibition by antiserum
from that caused by V. cholerae 01 were reported can be demonstrated under the dark field or phase
in Madras (Chennai), India. By mid-January contrast microscope.
1993 similar isolates were found in neighboring
Bangladesh, and these rapidly spread north, E. Culture
following the course of the major rivers and raising The specimens sent in enrichment media should
fears of a new pandemic. The new strain was be incubated for 6–8 hours, including transit time.
assigned to a new serogroup, 0139 Bengal. The new The specimens sent in holding media should be
strain continued spreading, eastwards to the South inoculated into enrichment media, to be incubated
East Asian countries, and westwards to Pakistan, for 6–8 hours before being streaked on a selective
China and some parts of Europe. But surprisingly, and a nonselective medium.
www.ebook3000.com
Chap-40.indd 293 15-03-2016 11:17:34
294 | Section 3: Systemic Bacteriology
Immunity ii. Live oral vaccine
Gastric acid provides some protection against Recombinant DNA vaccine with expression of V.
cholera vibrios. In cholera, the vibrios remain cholerae 01 in attenuated strain of S. Typhi Ty21 as
confined to the intestine, where they multiply and a carrier bacterium has been developed. The live
elaborate the enterotoxin which is responsible salmonellae colonize the Peyer’s patches of the
for the disease. Immunity, therefore, may be small intestine and induce IgA response by local
directed against the bacterium or against the toxin- immune system of the gut.
antibacterial or antitoxic. Natural infection confers
some amount of immunity but it does not seem to HALOPHILIC VIBRIOS
last for more than 6-12 months and reinfections are Vibrios that have a high requirement of sodium
known after this period. chloride are known as halophilic vibrios. Their
An attack of cholera is followed by immunity natural habitat is sea water and marine life. Some
to reinfection, but the duration and degree of halophilic vibrios have been shown to cause human
immunity are not known. The presence of antitoxin disease—V. parahaemolyticus, V. alginolyticus and
antibodies has not been associated with protection. V. vulnificus.
Immunity may be local, in the intestine, or
systemic. The appearance of local antibodies in the Vibrio parahaemolyticus
intestine has been known for a long time. These are Morphologically, it resembles V. cholerae except
known as ‘coproantibodies as they appear in the that it is capsulated, shows bipolar staining and
faeces. They consist of IgG, IgM and IgA. has a tendency to pleomorphism, especially when
grown on 3% salt agar and in old cultures. However,
Prophylaxis being a halophilic species, it grows well in peptone
1. General Measures water with 8% (but not 10%) sodium chloride.
It grows well on blood agar. On MacConkey agar
Most important are the provision of safe drinking
it forms pale, nonIactosefermenting colonies and
water supplies and the proper disposal of human
on sheep blood agar it produces β-hemolysis. On
feces. Control rests on education and on improvement
TCBS agar, the colonies are green (non-sucrose-
of sanitation, particularly of food and water.
fermenting). Unlike other vibrios, it produces
peritrichous flagella when grown on solid media.
2. Specific Measures—Vaccines
Polar flagella are formed in liquid cultures.
a. Killed whole organism vaccine
It is killed suspension containing 8000 million V. Biochemical Reactions
cholerae per mL, composed of equal numbers of It is oxidase, catalase, indole and citrate positive.
Ogawa and Inaba serotypes. The concentration of It reduces nitrate to nitrite. It ferments glucose,
the vaccine has been increased to 12,000 million maltose, mannitol, mannose and arabinose with
per mL, in order to improve the antigenic stimulus. the production of acid only. Lactose, sucrose
Primary immunization consists of 2 equal doses, salicin, dulcitol or inositol are not fermented.
injected subcutaneously, at an interval of 4–6 weeks. It is VP positive and decarboxylates lysine and
This vaccine offers about 60% protection for 3–6 ornithine but not arginine.
months. A single dose of vaccine is ineffective in
children below five years of age while two doses at Pathogenesis
1–4 week intervals are protective. However, it confers Not all strains of V. parahaemolyticus are patho
protection in adults due to its action as a booster genic for human beings. Strains isolated from
because of prior natural infection. environmental sources (such as water, fish,
Cell free somatic antigen preparations are as crabs or oysters) are nearly always nonhemolytic
effective as whole cell vaccine. when grown on a special high salt blood agar
(Wagatsuma’s agar), while strains from human
b. Oral vaccine: Two types of oral cholera vaccines
patients are alm ost always hemolytic. This
are available in some countries.
hemolysis is known as the Kanagawa phenomenon
i. Nonliving oral B subunit-whole cell (BS-WC) and is due to a heat stable hemolysin.
vaccine: Kanagawa positive strains being considered
This vaccine consists of killed whole-cell V. cholerae pathogenic for human beings and negative strains
01 in combination with a recombinant B-subunit nonpathogenic.
of cholera toxin (WC/rBS). Vibrio parahaemolyticus causes acute gastro
It is given orally in two dose schedule, 10–14 enteritis following ingestion of contam inated
days apart. On an average, the vaccine confers seafood, such as raw fish or shellfish. After an
50–60% protection for at least 3 years. incubation period of 12–24 hours, nausea and
www.ebook3000.com
Chap-40.indd 295 15-03-2016 11:17:34
296 | Section 3: Systemic Bacteriology
of mammals, including dogs, cats, goats, sheep h. Kanagawa phenomenon
and monkey. It is rare1y recovered from human i. Aeromonas
feces. It commonly infects various cold-blooded j. Plesiomonas.
animals like frogs, snakes, turtles and lizards. Man
is infected primarily by ingesting contaminated MULTIPLE CHOICE QUESTIONS (MCQs)
water or food. It causes: 1. All the following Vibrio species require sodium
1. Gastroenteritis, which manifests as a mild, chloride as a growth factor except:
watery diarrhea in which stools are free of blood a. Vibrio cholerae
and mucin. b. Vibrio parahaemolyticus
2. Extraintestinal lesions, including septicemia, c. Vibrio vulnificus
endophthalamitis, septic arthritis, meningitis, d. Vibrio alginolyticus
cellulitis, and acute cholecystitis. 2. Which of the following media can serve a transport
medium for Vibrio cholerae?
Key Points a. Selenite-F broth
b. Tetrathionate broth
Vibrio and Aeromonas are classified in the families
Vibrionaceae and Aeromonadaceae, respectively. c. Venkatraman–Ramakrishnan medium
Plesiomonas are closely related to Proteus and have d. Nutrient broth
now been placed in the Enterobacteriaceae family 3. Classical and El Tor biotypes of Vibrio cholerae can
Vibrio cholerae is short, typically comma-shaped, show be differentiated by which of the following tests?
typical darting type motility. It is strongly aerobic a. Sensitivity to polymyxin B
It grows well on a wide variety of media including b. Agglutination of fowl RBCs
ordinary and special media c. Sensitivity to Mukerjee group IV phage
Transport media such as VR medium and Cary–Blair d. All of the above
medium, o Enrichment media such as alkaline
4. The DNA expressing for production of cholera
peptone water and Monsur’s taurocholate tellurite
peptone, Selective media such as TCBS medium, toxin of Vibrio cholerae is located in:
Monsur’s GTTA medium, and alkaline BSA a. Chromosome b. Plasmid
Two biotypes of V. cholerae 01 strains-EI Tor and c. Transposon d. Phage
classical 5. Cholera toxin resembles which of the following
Classical and El Tor biotype vibrios can be differentiated toxins?
by acetoin in the Voges-Proskauer test, agglutination a. Labile toxin of E. coli
of fowl erythrocytes, lysis of sheep erythrocytes, b. Stable toxin of E. coli.
polymyxin B senisitivity, susceptibility to Mukerjee’s c. Diphtheria toxin
group IV phage and sensitivity to the group V phage
d. Tetanus toxin
Disease: Cholera. Spread is by consumption of
contaminated food or water 6. Stools from suspected cholera cases can be trans
Laboratory diagnosis: Fresh stool specimen is used for ported to the laboratory in:
dark-field microscopy and direct immunofluorescence. a. Venkatraman–Ramakrishnan medium
The specimen inoculated on a selective and b. Cary–Blair medium
nonselective media. Suspected V. cholerae are tested c. Thick blotting paper
by slide agglutination using specific V. cholerae 01 d. All of the above
antisera. If the colony is identified as V. cholerae 01 7. Which of the following vaccines is/are available for
then it is tested by various tests to determine whether prophylaxis against cholera?
isolated V. cholerae 01 is classical or El tor a. Killed parenteral vaccine
Halophilic vibrios
b. Killed parenteral vaccine
Vibrio parahaemolyticus, Vibrio alginolyticus, and
Vibrio vulnificus are three important halophilic vibrios c. Live oral vaccine
species known to cause infection in humans. V. d. All of the above
parahaemolyticus in humans causes gastroenteritis 8. Which of the following bacteria is/are associated
Aeromonas: Aeromonas species in humans cause with food poisoning due to consumption of sea fish?
gastroenteritis and wound infections a. Vibrio alginolyticus
Plesiomonas: P. shigelloides (the only species) in b. Vibrio parahaemolyticus
humans cause gastroenteritis, celullitis, septic c. Vibrio vulnificus
arthritis, septicemia, neonatal meningitis, etc. d. All of the above
9. Acute diarrhea resembling cholera can be caused by:
IMPORTANT QUESTIONS a. Vibrio parahaemolyticus
b. Vibrio vulnificus
1. Discuss laboratory diagnosis of cholera. c. Vibrio alginolyticus
2. Write short notes on: d. Aeromonas hydrophila
a. Classification of vibrios 10. Which of the following conditions can be caused
b. Noncholera vibrios by Plesiomonas?
c. Differences between classical and El Tor vibrios a. Gastroenteritis b. Septicemia
d. Pathogenesis of cholera
c. Cellulitis d. All of the above
e. Cholera toxin
f. Prophylaxis against cholera ANSWERS (MCQs)
g. Halophilic vibrios 1. a; 2. c; 3. d; 4. b; 5. a; 6. d; 7. d; 8. b; 9. d; 10. d
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss morphology, culture characteristics and
be able to: biochemical reactions of Helicobacter
∙∙ Describe morphology, culture characteristics and ∙∙ Discuss laboratory diagnosis of Helicobacter pylori
biochemical reactions of Campylobacter infections.
www.ebook3000.com
298 | Section 3: Systemic Bacteriology
are zoonotic, with a variety of animals serving lost fluids and electrolytes. Antimicrobial treatment
as reservoirs. Hum ans acquire the infections should be reserved for patients with severe or
with C. jejuni and C. coli after consumption of complicated infections. Severe gastroenteritis and
contaminated food, milk, or water. septicemia are treated with erythromycin (drug of
choice), tetracyclines and quinolones.
Laboratory Diagnosis Control: Gastroenteritis is prevented by proper
Laboratory diagnosis depends on isolation of the preparation of food (particularly poultry), and
Campylobacter from feces. consumption of pasteurized milk; prevention of
contaminated water supplies also controls infection.
A. Specimens
Diarrheal stool is the usual specimen. Other Campylobacters
Campylobacter species other than C jejuni are
B. Microscopy encountered infrequently.
Gram-stained smears of stool may show the
typical “gull wing”—shaped rods. Dark-field or 1. Campylobacter fetus
phase contrast microscopy may show the typical Campylobacter fetus subspecies fetus is a very
darting or tumbling motility of the spiral rods. important veterinary pathogen. It causes infective
abortion in cattle and sheep.It is an opportunistic
C. Culture pathogen that causes systemic infections in
Feces or rectal swabs are plated on selective media. man in immunocompromized patients. It may
A transport medium has to be employed in case of occasionally cause diarrhea.
delay in culturing. Campylobacters survive for 1–2
weeks at 4°C in Cary–Blair transport medium but 2. C. concisus
glycerol-saline is not satisfactory. Selective media It has been isolated from cases of gingivitis and
for isolation of C. jejuni are Butzler’s selective periodontal disease. It has also been isolated from
medium, Skirrow’s Campylobacter selective faeces.
medium, Preston Campylobacter selective medium C. fetus subsp. venerealis: It causes enzootic
and Blaser’s medium (Campy-BAP). Skirrow’s sterility (infectious infertility) of cattle but has not
medium contains vancomycin, polymyxin B, and been associated with human infection.
trimethoprim to inhibit growth of other bacteria.
Inoculated plates are incubated at 42°C to favor 3. C. jejuni subsp. doylei
growth of the thermophilic Campylobacters (C. This organism can be distinguished from other
jejuni, C. coli, C. lari and C. hyointestinalis) over that campylobacters because it does not reduce nitrate to
of other faecal bacteria. If, however, the presence of nitrite and hydrolyzes hippurate. The pathogenicity
C. fetus (nonthermophile) is suspected, additional of this organism is unknown. It has been isolated
plates should be incubated at 37°C to allow growth from human gastric epithelium biopsy and from
of this nonthermophile. Incubation must be done faeces of children with diarrhea.
in an atmosphere of 5% O2 10% CO2 and 85% N2.
Plates are incubated for 48 hours. 4. C. coli
Colonies are typically flat and effuse, with a It causes an infection clinically indistinguishable from
tendency to spread on moist agar. They are nonhe that of C. jejuni. It is commonly found in healthy pigs.
molytic, gray or colorless, moist, and flat or convex.
5. C. lari
D. Identification C. lari (formerly known as e. laridis). It causes
Suggestive colonies are screened by Gram staining, enteritis simulating C. jejuni infections in humans.
motility and oxidase tests. Confirmation is by
further biochemical tests, including positive 6. C. sputorum subsp. sputorum
catalase and nitrate reduction tests. It constitutes a part of normal flora of respiratory
tract and gingival crevices of man. It has also been
E. Serology isolated from feces of 2% of healthy people. It may
Complement fixation test and enzyme-linked occasionally cause diarrhea, abscess and septicemia.
immunosorbent assay (ELISA) are group-specific
tests that can detect recent infection with C. jejuni Helicobacter
or C. coli.
These are strict microaerophiles with a spiral or
Treatment helical morphology. They possess sheathed flagella.
Campylobacter gastroenteritis is typically a self- Species: Various species included in this genus
limited infection managed by the replacement of are: H. pylori, H. cinaedi, H. fennelliae, H. canis,
Chapter 41: Campylobacter and Helicobacter | 299
H pullorum H.rappininae and H. canadensis. Of vicinity of the organism, thus favoring bacterial
these, first three are medically important. multiplicatfun.
3. The bacterial antigens cross-react with antran
Helicobacter pylori gastric antigens stimulating an autoimmune
Warren and Marshall in Australia in 1983 observed response.
spiral, Campylobacter-like bacteria in close 4. Protease produced by the organism degrades
apposition to the gastric mucosa in several cases of gastric mucosa.
gastritis and peptic ulcer. They were originally named 5. H. pylori infected patients show hypergast
Campylobacter pyloridis then C. pylori and now rinemia that upsets gastrin-HCI homeostasis.
redesignated as Helicobacter pylori. Features that Pathogenesis
distinguish this organism from Campylobacters are
its multiple sheathed flagella, its strong hydrolysis of H. pylori colonize the surface of the gastric
urea and its unique fatty acid profile. mucosa, especially of the antrum but any part of
the stomach may be colonized. Colonization often
extends into gastric glands, but the mucosa is not
Morphology
invaded by the bacteria.
H. pylori is a gram-negative spirally shaped Although gastric acid is potentially destructive
bacterium, 0.5–0.9 mm wide by 2–4 mm long. It is to H. pylori, protection is provided by its powerful
motile by means of a tuft of sheathed unipolar flagella, urease. H. pylori produce potent urease activity,
unlike the unsheathed flagella of campylobacters. It which yields production of ammonia and further
is non-sporing. buffering of acid and neutralizes acid around the
bacteria. Where they are numerous, the underlying
Cultural Characteristics mucosa usually shows a superficial gastritis of the
Like campylobacters, H. pylori is microaerophic. type known as chronic active or type B gastritis.
The optimum temperature for its growth is 35–37°C, H. pylori are associated with antral gastritis,
some grow poorly at 42°C but none grows at 25°C. It duodenal (peptic) ulcer disease, gastric ulcers.
can grow in an atmosphere of 5% O2, 10% CO2 and It is also recognized as a risk factor for gastric
85% N2. It does not grow anaerobically or in air. It malignancies, namely, ‘adenocarcinoma’ and
can be grown on moist freshly prepared chocolate ‘mucosa associated lymphoid tissue’ (MALT)
agar and Skirrow’s Campylobacter selective lymphomas.
medium. H. pylori produce circular, convex and
translucent colonies after incubation at 35–37°C in Laboratory Diagnosis
a microaerophilic atmosphere for 3–5 days. Diagnostic tests are of two kinds:
www.ebook3000.com
300 | Section 3: Systemic Bacteriology
1. Microscopy: The biopsy specimen can be
Helicobacter pylori: Curved gram-negative bacilli.
examined by microscopic examination of Urease production at very high levels is typical of
Gram’s staining, silver staining, hematoxylin gastric helicobacters (e.g., H. pylori)
and eosin (Hand E) staining, Giemsa staining or Diseases–H. pylori causes gastritis, peptic ulcers,
immunofluorescence for the presence of bacteria. gastric adenocarcinoma
Warthin-Starry silver stain is the most sensitive. Diagnosis: A. Noninvasive tests: 1. Serology 2. Urea
2. Culture: Culture is done on nonselective breath test. 3. Faecal antigen test 4. Polymerase chain
medium such as chocolate agar and a selective reaction (PCR) B. Invasive tests: 1. Microscopy 2.
medium Skirrow’s campylobacter selective Culture 3. Biopsy urease test.
medium. Plates are incubated for 2–7 days in a
moist, microaerophilic atmosphere at 35–37°C Important questions
in the presence of 5–10% CO2.High humidity
is essential. The organism is identified on the 1. Discuss pathogenesis and laboratory diagnosis of
basis of its colonial morphology, Gram staining, diarrhea caused by Campylobacter.
biochemical properties and positive urease tests. 2. Write short notes on:
3. Biopsy urease test a. Campylobaeter infections
The biopsy urease test can be performed by b. Helicobacter pylori
c. Helicobacter pylori infections.
crushing biopsy tissue in 0.5 mL urea solution with an
d. Laboratory diagnosis of Helicobacter pylori in-
indicator and incubated at 37°C. If H. pylori is present,
fections
the pH changes within a few minutes to 2 hours due e. Urea breath test.
to the production of ammonia. The abundance of
urease produced by the organism permits detection
of the alkaline byproduct in less than 2 hours.
Multiple choice questions
1. Most common Campylobacter species known to
Treatment cause diarrhea in humans is:
The standard treatment is a combination of a. Campylobacter lari
bismuth subsalicylate, tetracycline (or amoxicillin) b. Campylobacter fetus
c. Campylobacter jejuni
and metronidazole for two weeks. An alternative
d. Campylobacter coli
schedule employs a proton pump inhibitor like
2. All the following media are commonly used for
omeprazole and clarithromycin. cultivation of Campylobacter except:
Prevention and control: Prophylactic treatment a. Butzler medium
of colonized individuals has not been useful b. Skirrow’s medium
and potentially has adverse effects, such as c. Preston’s Campylobacter selective medium
predisposing patients to adenocarcinomas of d. TCBS medium
the lower esophagus. Human vaccines are not 3. Which of the following bacteria is/are microaerophilic?
currently available. a. Campylobacter jejuni
b. Helicobacter pylori
Helicobacter cinaedi c. Mycobacterium bovis
d. All of the above
H. cinaedi (formerly known as Campylobacter 4. Campylobacter and Helicobacter can be different
cinaedi) has been associated with proctitis in iated on the basis of:
homosexual men. a. Multiple sheathed flagella
b. Strong hydrolysis of urea
Helicobacter fennelliae c. Both of the above
H.fennelliae (formerly known as Campylobacter d. None of the above
fennelliae) like H. cinaedi has been associated with 5. Helicobacter pylori have been implicated in all of
proctitis in homosexual men. the following clinical conditions except
a. Chronic gastritis
b. Peptic ulcer disease
Key Points
c. Septicemia
Campylobacters are thin, curved gram-negative d. Idiopathic thrombocytopenic purpura
bacilli. They are are microa erobic and strongly 6. Which of the following tests is/are useful in
oxidase positive identification of Helicobacter pylori?
Campylobacter jejuni and Campylobacter coli have a. Warthin-starry silver staining
emerged as common human pathogens b. Biopsy urease test
Diseasess—Zoonotic infection; improperly prepared c. Urea breath test
poultry is a common source of human infections d. All of the above
Diagnosis—Microscopy is insensitive. Culture
Answers (MCQs)
requires use of specialized media incubated
1. c; 2. d; 3. d; 4. c; 5. c; 6. d
42
Chapter
Pseudomonas,
Stenotrophomonas, Burkholderia
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Pigments of Pseudomonas;
be able to: pathogenicity of Pseudomonas aeruginosa;
∙∙ Describe morphology, cultural characteristics, Burkholderia mallei.
biochemical reactions and laboratory diagnosis of
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Morphology
It is a slender gram-negative bacillus, 1.5–3 μm ×
0.5 μm, actively motile usually with a single polar
flagellum. Occasional strains have two or three
flagella. It is nonsporing, noncapsulated but many
strains have a mucoid slime layer.
Cultural Characteristics
It is a strict aerobe but can grow anaerobically if Fig. 42.1: Pseudomonas aeruginosa on nutrient
nitrate is available. Growth occurs at a wide range agar
www.ebook3000.com
302 | Section 3: Systemic Bacteriology
5. Cetrimide agar is selective medium for Ps. Epidemiological Typing Methods
aeruginosa 1. Serotyping: Identification of group-specific
heat-stable lipopolysaccharide antigens by
Pigment Production agglutination forms the basis of O serotyping.
P. aeruginosa produces at least 4 distinct pigments: Typically, nine serotypes account for over 90%
i. Pyocyanin: It is a bluish-green phenazine of isolates.
pigment soluble in chloroform and water. This 2. Bacteriocin (pyocin) typing: Three types
pigment is not produced by other species of of bacteriocins (pyocins) are produced by P.
this genus. Demonstration of the presence aeruginosa which are known as R, F and S.
of the blue phenazine pigment pyocyanin Depending upon the growth inhibition of these
is absolute confirmation of a strain as P. 13 indicator strains, 105 types are recognized.
aeruginosa. Pyocin typing is easy to perform, results are
ii. Pyoverdin (fluorescein): The yellow/green available by the third day and has a reasonable
pigment pyoverdin (fluorescein) is also reproducibility and good discrimination.
produced by most strains, giving the charac 3. Phage typing: In bacteriophage typing
teristic blue-green appearance of infected pus considerable difficulties have been encounterd.
or cultures. It is insoluble in chloroform but 4. Molecular methods: Restriction endonuclease
soluble in water. typing wih pulsed field gel electrophoresis
iii. Pyorubrin: It is a bright red water soluble (PFGE) is the most reliable typing method
pigment and is insoluble in chloroform. and discriminatory of the present DNA-based
iv. Pyomelanin: It is a brown to black pigment typing methods and is considered to be the gold
and its production is uncommon. standard.
www.ebook3000.com
304 | Section 3: Systemic Bacteriology
gentamicin and tobramycin. Ciprofloxacin exhibits 3. Septicemia, particularly in patients with
good activity against P. aeruginosa and penetrates contaminated intravascular catheters.
well into most tissues. 4. Endocarditis, especially in drug addicts,
pneumonitis, osteomyelitis, dermatitis and
Control wound infections
Prevention of Ps. aeruginosa cross infection in
Treatment
hospitals requires constant vigilance and strict
attention to asepsis. The inappropriate use of B. cepacia is susceptible to trimethoprim-
broad-spectrum antibiotics should also be avoided. sulfamethoxazole.
Immunotherapy in human burns cases with
antiserum to Ps. aeruginosa may be useful. Vaccine Burkholderia mallei (formely
from appropriate types administered to high-risk Pseudomonas mallei)
patients provides protection against Pseudomonas
The bacillus was discovered by Loeffler and Schutz
sepsis.
(1882) from a horse dying of glanders. It is the
causative agent of glanders (malleus,in Latin),
Stenotrophomonas maltophilia a disease primarily of equine animals, such as
(Formerly Pseudomonas maltophilia) horses, mules and asses—but capable of being
It is responsible for infections in debilitated patients transmitted to other animals and to human beings.
with impaired host defense mechanisms. The
spectrum of nosocomial infections with S. maltophilia Morphology
includes bacteremia, pneumonia, meningitis, Ps. mallei is a slender, nonmotile, gram-negative
wound infections, and urinary tract infections. bacillus, 2–5 μm × 0.5 μm staining irregularly and
often giving a beaded appearance.
Burkholderia cepacia (formerly
Pseudomonas cepacia) Culture
Morphology It is an aerobe and facultative anaerobe, growing
on ordinary media. Colonies which are small and
B. cepacia is a slender, motile, gram-negative rod.
transluscent initially become yellowish and opaque
The bacillus accumulates poly-β-hydroxybutyrate
on aging. On potato, a characteristic amber, honey-
as granules, so stains irregularly.
like growth appears, becoming greenish yellow
resembling Ps. aeruginosa.
Culture
It can grow in many common disinfectants and can Biochemical Reactions
even use penicillin G as a sole source of carbon It is It is quite inactive biochemically, attacking only
aerobic and grows well on nutrient agar optimally glucose. It is the only nonmotile species in the
at 25–30°C. On prolonged incubation, colonies genus Pseudomonas.
become reddish-purple due to the formation of a
nondiffusible phenazine. Animal Pathogenicity
The natural disease in equines occurs in two
Biochemical Reactions
forms—glanders and farcy.
lt is weak oxidase-positive. It utilizes glucose, 1. Glanders: In glanders, respiratory system is
maltose, lactose, and mannitol and is lysine affected. The infected animal develops profuse
decarboxylase positive, ornithine decarboxylase catarrhal discharge from the nose and the nasal
positive, and arginine dihydrolase negative. Isolates septum shows nodule formation. Later the
are motile by means of polar tuft of flagella. nodules break down with the production of
irregular ulcers.
Pathogenicity 2. Farcy: This follows infection through skin with
Like P. aeruginosa, B. cepacia can colonize a variety involvement of superficial lymph vessels and
of moist environmental surfaces and is commonly lymph nodes. The lymph vessels are thickened
associated with nosocomial infections. and stand out as hard cords under the skin which
Infections caused by this organism include the are called ‘farcy pipes’
following: Strauss reaction: Guinea-pigs are susceptible
1. Respiratory tract infections in patients with and intraperitoneal injection into male guinea
cystic fibrosis or chronic granulomatous disease. pigs induces the Strauss reaction. This consists of
2. Urinary tract infections in catheterized patients. swelling of the testes, inflammation of tunica vaginal
Chapter 42: Pseudomonas, Stenotrophomonas, Burkholderia | 305
and ulceration of the scrotal skin. The Strauss Toxins
reaction is not diagnostic of glanders, as it may Two thermolabile exotoxins, one lethal and the
also be produced by inoculation of other bacteria other necrotizing have been identified in culture
such as Brucella species, Preisz–Nocard bacillus filtrates.
Actinobacillus lignieresii and Ps. pseudomallei.
www.ebook3000.com
306 | Section 3: Systemic Bacteriology
Table 42.1 Some characteristics of nonfermenters
Organisms Oxidase Habitat Pathogenicity
test
Acinetobacter spp. _ Saprophytes found in soil, water Opportunist pathogens, serious infections include
and sewage, and occasionally meningitis, pneumonia and septicemia
as commensals of moist areas of
human skin.
Alcaligenes spp. + Human feces UTI, wound infection
Achromobacter spp. + − CSOM, Postoperative meningitis
Flavobacterium spp. + Saprophyte of soil and moist Opportunistic nosocomial infections, particularly in
environments infants and associated with meningitis
Eikenella spp. + Commensal of mucosal surfaces Endocarditis, meningitis, pneumonia, and infect
ions of wounds and various soft tissues.
Legionella
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Legionella pneumophilia;
be able to: diseases caused by Legionella.
∙∙ Describe morphology, culture characteristics and
biochemical reactions of Legionella
Introduction Culture
In the summer of 1976, public attention was Legionellae are nutritionally fastidious. Their
focused on an outbreak of severe pneumonia that growth is enhanced with iron salts and depends
caused many deaths in members of the American on the supplementation of media the L-cysteine.
Legion convention in Philadelphia. The disease They have fastidious requirements and grow on
was characterized by fever, cough and chest pain, complex media such as buffered charcoal, yeast
leading on to pneumonia and often ending fatally. extract (BCYE) agar, with L-cysteine and antibiotic
The causative agent has been called Legionella supplements, with 5% CO2, at pH 6.9, 35°C and 90%
pneumophila. Subsequent studies found this humidity. Growth is slow and colonies take 3–6
organism to be the cause of multiple epidemic and days to appear.
sporadic infections. It is now recognized to be a
ubiquitous aquatic saprophyte. Biochemical Reactions
The organisms are nonfermentative. Most species
Species: Taxonomic studies have shown that are motile and catalase-positive, liquefy gelatin,
the family Legionellaceae consists of one genus, and do not reduce nitrate or hydrolyze urea.
LegionelIa, with 40 species and more than 60
serogroups. The original isolate in this genus is Epidemiology
designated L. pneumophila serogroup 1 (SG1),
The bacteria are commonly present in natural
which accounts for nearly all severe infections.
bodies of water, such as lakes and streams, as well
Examples of other species that cause human
as in air conditioning cooling towers and condens
infection less often are L. micdadei, L. bozemanii,
ers and in water systems (e.g. showers, hot tubs).
L. dumoffii and L. gormanii.
Legionellae survive and multiply inside free-living
amebae and other protozoa. They also multiply in
LEGIONELLA PNEUMOPHILA some artificial aquatic environments, which serve
Morphology as amplifiers.
Legionellae are thin, noncapsulated bacilli, 25 mm Human infection is typically by inhalation of
× 0.3–0.1 mm coccobacillary in clinical material aerosols produced by cooling towers, air conditioners
and assuming longer forms in culture. Most are and shower heads which act as disseminators. Man-
motile with polar or subpolar flagella. They are to-man transmission does not occur.
gram-negative but stain poorly, particularly in
smears from clinical specimens. They stain better Pathogenesis
by silver impregnation, but are best visualized by Respiratory tract disease caused by Legionella
direct fluorescent antibody (DFA) staining with species develops in susceptible people who inhale
monoclonal or polyclonal sera. infectious aerosols.
www.ebook3000.com
308 | Section 3: Systemic Bacteriology
Clinical Diseases Treatment
Asymptomatic Legionella infections are relatively For treatment, the newer macrolides,ciprofloxacin,
common. Symptomatic infections primarily affect and tetracyclines are effective. Rifampicin is
the lungs and present in one of two forms: employed in severe cases.
1. Pontiac fever: An influenza-like illness
2. Legionnaires’ disease: A severe form of Prevention
pneumonia Prevention of legionellosis requires identification of
1. Pontiac fever: Pontiac fever is a milder, nonfatal the environmental source of the organism and reduc
‘influenza-like’ illness with fever, chills, myalgia tion of the microbial burden. Hyperchlorination of
malaise, and headache but no clinical evidence the water supply and the maintenance of elevated
of pneumonia. Outbreaks with high attack rates water temperatures have proved moderately
may occur. successful. Howe ver, complete elimination of
2. Legionnaire’s disease: The incubation period Legionella organisms from a water supply is often
is 2–10 days. ‘The disease presents with fever, difficult or impossible. Because the organism
nonproductive cough and dyspnea, rapidly has a low potential for causing disease, reducing
progressing, if untreated, to pneumonia. Multi the number of organisms in the water supply is
organ disease involving the gastrointestinal frequently an adequate control measure. Hospitals
tract, central nervous system, liver and kidneys with patients at high risk for disease should monitor
is common. their water supply on a regular basis for the presence
Case fatality may be 15–20%. All age groups are of Legionella and their hospital population for
susceptible, though more cases have occurred in disease.
the elderly. Key Points
Legionella pneumophilia serogroup 1 is most
Laboratory Diagnosis common human pathogen
1. Microscopy L. pneumophilia are small, slender, pleomorphic,
Gram-negative bacilli
Legionellae in clinical specimens stain poorly Human infection is typically by inhalation of aerosols
with Gram stain. Nonspecific staining methods, produced by cooling towers, air conditioners and
such as those using Dieterle’s silver or Gimenez’s shower heads
stain, can be used to visualize the organisms. L. pneumophilia causes Legionnaire’s disease
The most sensitive way of detecting legionellae Laboratory diagnosis depends on direct fluorescent
antibody (DFA) test, culture, detection of antigen and
microscopically in clinical specimens is to use the demonstration of serum antibodies
direct fluorescent antibody (DFA) test, in which Hyperchlorination of the water and superheating of
fluorescein-labeled monoclonal or polyclonal water is of value in preventing the disease.
antibodies directed against Legionella species are
used.
Important questions
2. Culture 1. Discuss pathogenicity and laboratory diagnosis of
The medium most commonly used for the isolation Legionnaire’s disease.
2. Write short notes on:
of legionellae is buffered charcoal-yeast extract
a. Legionella pneumophilia
(BCYE) agar. Antibiotics can be added to suppress b. Legionnaire’s disease
the growth of rapidly growing contaminating c. Pontiac fever.
bacteria. Legionellae grow in air or 3% to 5% carbon
dioxide at 35°C after 3–5 days. Their small (1–3 mm) Multiple choice question (mCQs)
colonies have a groundglass appearance. 1. The causative agent of Pontiac fever is:
a. Legionella pneumophila
3. Antigen Detection b. Pseudomonas putida
c. Yersinia pseudotuberculosis
Enzyme-linked immunoassays, radioimmuno
d. Francisella tularensis
assays, the agglutination of antibody-coated latex 2. The natural habitat of Legionella pneumophila is:
particles, and nucleic acid analysis studies have a. Water b. Sputum
all been used to detect legionellae in respiratory c. Faeces d. Urine
specimens and urine. 3. The antibiotic of choice in Legionella infections is:
a. Erythromycin b. Cephalosporins
4. Serology c. Penicillins d. All of the above
Detection of serum antibody is done by ELISA or Answers (MCQs)
indirect immunofluorescent assay. 1. a; 2. a; 3. a
44
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe laboratory diagnosis of plague
be able to: ∙∙ Describe the following: prophylaxis against plague;
∙∙ Describe morphology, culture characteristics and Yersinia enterocolitica; Yersinia pseudotuberculosis;
biochemical reactions of Yersinia pestis Pasteurella multocida; Francisella tularensis.
www.ebook3000.com
310 | Section 3: Systemic Bacteriology
The bacillus is surrounded by a slime layer Biochemical Reactions
(envelope or capsule). It is nonmotile, nonsporing It ferments glucose, mannitol and maltose with the
and nonacid fast. production of acid but no gas. Lactose and sucrose
or rhamnose are not fermented.
Cultural Characteristics It is catalase positive, indole negative, MR
The plague bacillus is aerobe and facultative positive, VP and citrate negative (IMViC – + – –),
anaerobe. Growth occurs over a wide range of pH nitrate reduction positive, aesculin positive and
5–9.6 (optimum pH 7.2). Optimum temperature oxidase and urease negative. Gelatin is not liquefied.
for primary culture is 27°C (range 14–37°C). Several
phenotypic characteristics are best expressed at room Physiological varieties of Y. pestis: Devignat has
temperature but the envelope develops at 37o C. distinguished three physiological varieties of Y.
1. Nutrient agar: Colonies are small (as small as pestis based on the fermentation of glycerol and
0.1–0.2 mm), delicate, transparent disks and reduction of nitrate. These biotypes have been
become opaque on continued incubation. designated orientalis, medieval and antigua, and
2. Blood agar: Colonies are dark brown due to are characterized by differences in their geo
absorption of the haemin pigment. graphic distribution. This typing appears to be of
3. DCA and MacConkey agar: On DCA and epidemiological significance because of the different
MacConkey agar it grows poorly, producing pin- geographical distribution of the types (Table 44.1).
point, reddish colonies after 24 hours incubation.
4. In broth, a flocculent growth occurs at the Resistance
bottom and along the sides of the tube, with little
The plague bacillus is easily destroyed by exposure
or no turbidity. A delicate pellicle may form later.
to heat, sunlight, drying and chemical disin
5. Ghee broth: If grown in a flask of broth with
fectants. It is killed by moist heat at 55°C in 5
sterile oil or ghee (clarified butter) floated
min and by 0.5% phenol in 15 minutes. It is very
on top (ghee broth) a characteristic growth
susceptible to drying. It remains viable for long
occurs which hangs down into the broth from
periods in cold, moist environments.
the surface, resembling stalactites (stalactite
growth) (Fig. 44.2).
Antigens, Toxins and
Other Virulence Factors
At least 20 different antigens have been detected
in Y. pestis.
1. F-l antigen or envelope antigen: The heat-
labile Fraction I (FI) protein capsular antigen
is a soluble antigen contained within the
bacterial envelope. It helps the organism to
resist phagocytosis.
2. V and W antigens: These antigens have been
considered to be the virulence factors as they
inhibit phagocytosis. Production of V and W
antigens is plasmid mediated.
3. Pigment binding and iron-regulated surface
proteins: In yersiniae, avirulent and low-
pathogenicity strains require iron overload to
produce septicemia in humans or lethality in
Fig. 44.2: Y. pestis in ghee broth culture. Stalactite growth mice.
www.ebook3000.com
312 | Section 3: Systemic Bacteriology
(domestic plague, urban plague), and (iii) human Postmortem examination shows a marked local
plague, which may be acquired by contact with inflammatory condition at the site of inoculation
either of the former cycles and which may be with necrosis and edema. The regional lymph
transmitted by pneumonic spread or, rarely, by the nodes are enlarged, the spleen is enlarged and
bite of a human flea. congested and may show grayish-white patches
In the 1990s, there has been a re-emergence in the tissue. Prepare films from the local lesions,
of plague in countries where it had ceased to be lymph nodes, spleen pulp and heart blood; stain
noticed for many years. Y. pestis has been employed and examine for characteristic plague bacilli.
as a biological warfare agent.
5. Antigen Detection
Laboratory Diagnosis Demonstration of the FI capsular antigen by
Plague is confirmed by demonstrating the bacilli immunospecific staining and ELISA test will
in fluid from buboes or local skin lesions, in the confirm the presence of Y. pestis.
sputum and in blood films. Blood culture may be Dipstick test: FI glycoprotein can be detected by
intermittently positive in all forms of the disease. dipstick test using monoclonal antibodies. It is
Postmortem, the bacilli can usually be isolated rapid test and produces reliable result within 15
from a wide range of tissues, especially spleen, lung minutes.
and lymph nodes.
6. Serology
1. Specimens
The antibodies to F-I antigen can be demonstrated
i. Bubonic plague—pus or fluid aspirated. in patient’s serum by complement fixation test
ii. Pneumonic plague—sputum and blood. (CFT), hemagglutination test and enzyme linked
iii. Septicemic plague—blood. immunosorbent assay (ELISA).
iv. Meningeal plague—cerebrospinal fluid (CSF).
v. On postmortem—splenic tissue.
7. Polymerase Chain Reaction (PCR)
2. Microscopy A polymerase chain reaction (PCR), with primers
based on FI gene sequences, offers a rapid and less
Smears of exudate or sputum are stained with hazardous means of diagnosis than culture.
methylene blue or Giemsa stain. Characteristic
gram-negative coccobacilli and bacilli showing
Diagnosis of Plague in Wild Rats
bipolar staining with methylene blue suggest
plague bacilli. The smears are also stained by Before examining rats that may have died as a result
Gram’s method and observed for Gram negative, of plague, immerse them in disinfectant to kill any
ovoid coccobacilli with bipolar bodies. The infected fleas. Necropsy has the following features
fluorescent antibody technique may be of use in in plague:
identifying plaguqe bacilli. i. Enlargement of the lymphatic nodes with
periglandular inflammat ion and edema; ii.
3. Culture Pleural effusion; iii. Enlargement of the spleen; iv.
Liver congested and mottled; v. Congestion and
Culture the samples on blood agar plates,
hemorrhages under the skin and in internal organs.
MacConkey agar, nutrient agar and ghee broth
and incubated at 27°C. Colonies on blood agar Make films and cultures of heart blood, lymph
are dark brown. Colonies on MacConkey agar are nodes and spleen. Inoculate material from lesions
colorless. In ghee broth, a characteristic stalactite on to the nasal mucosa of guinea pigs or white
growth is produced. The growth is identified by rats as with sputum. Examine stained films and
biochemical tests and slide agglutination tests. pure cultures for characteristic morphology and
Demonstration of the FI capsular antigen by biochemical reactions of Y. pestis.
immunospecific staining will confirm the presence
of Y. pestis. Prophylaxis
Plague is one of the internationally quarantinable
4. Animal Inoculation diseases, and reporting of cases is mandatory.
If exudate is inoculated subcutaneously into Prophylaxis can be carried out by:
guinea-pigs or white rats, or on to their nasal
A. General Measures
mucosa, infection follows and the animals die
within 2–5 days. The bacilli may then be isolated 1. Control of fleas and rodents.
from the blood or from smears of spleen tissue 2. Spray insecticide (DDT) inside the rodent,
taken postmortem. burrows and houses to kill the fleas.
Chapter 44: Yersinia, Pasteurella, Francisella | 313
3. After the fleas have been killed, kill the rat with pseudotuberculosis and Y. enterocolitica. They are
rat-poison. found in the intestinal tract of a variety of animals,
in which they cause diseases, and are transmissible
B. Vaccination to humans, in which they produce a variety of
Two types of vaccine have been in use-killed and clinical syndromes. These are zoonotic diseases.
live attenuated vaccines. These are nonlactose fermenting gram-
negative rods that are urease-positive and oxidase-
1. Killed vaccine: The k illed vaccine used in India
negative. They resemble Y. pestis because they are
(prepared at the Haffkine Institute, Bombay) is
small, gram-negative rods with bipolar staining
a whole culture antigen. A virulent strain of the
and rodents, wild and domestic animals are
plague bacillus is grown in casein hydrolysate
reservoirs of infection. They differ from Y. pestis by
broth for 2–4 weeks at 32°C and killed by 0.05%
motility when grown at 22°C (nonmotile at 37°C),
formaldehyde and preserved with phenyl
noncapsulated, urease positive, oxidase-negative
mercuric nitrate (Sokhey’s modification of
and insusceptible to Y. pestis bacteriophage.
Haffkine’s vaccine). In Asia, Haffkine Institute,
Mumbai is the only vaccine producing center.
Yersinia pseudotuberculosis
Vaccine dose schedule: The vaccine is given
subcutaneously, two doses at an interval of 1–3 Y. pseudotuberculosis is a small, oval, Gram-
months, followed by a third six months later. negative,bipolar-stained bacillus. It is non-sporing,
Vaccination can confer significant protection non-capsulated and slightly acid-fast.
against bubonic but not pneumonic plague. The The organisms may be differentiated from
protection does not last for more than six months. Y. pestis by: by its relatively poor growth on
MacConkey agar.
Side effects: Fever, headache, malaise, lym ∙∙ Motility when grown at 22°C (but not at 37°C)
phadenopathy, and erythema and induration at ∙∙ Ability to produce urease
the site of inoculation. It may cause fetal damage ∙∙ Fermentation of rhamnose and melibiose
and abortion in pregnant women. Vaccination ∙∙ Failure to be lysed by the antiplague bac
is not very effective, therefore, even vaccinated teriophage at 22°C.
individuals should also be given chemoprophylaxis ∙∙ Lack of the FI antigen as shown by immunospecific
when exposed to plague. staining or PCR.
2. Live attenuated vaccines: Live vaccines are There are eight major O serotypes, several of
prepared from two avirulent strains of Y. pestis, which can be separated into subtypes. The majority
Otten’s Tjiwidej strain from Jawa and Girard’s of human cases of Y. pseudotuberculosis infection
EV 76 strain from Malagasey. Killed vaccine are due to serotype 1.
is recommended for general use because
live vaccines are difficult to prepare and may Pathogenesis
provoke unacceptable reactions. Live vaccines
Animal infection: It causes pseudotuberculosis
are not in use now.
which is a zoonotic diseses. In animals, the natural
mode of infection is by gastrointestinal tract. In
Chemoprophylaxis infected guinea pigs, the liver, spleen and lungs
A person exposed to definite risk of infection, show multiple nodules resembling tuberculosis
whether vaccinated or not, should be given lesions (hence the name pseudotuberculosis).
chemoprophylaxis-cotrimoxazole or tetracycline
Human infection: Human infection probably
orally for at least five days. Close contacts of
results from ingestion of materials contaminated
patients with plague should be given a course
with animal feces. Infection may be subclinical, but
of tetracycline (500 mg 6 hourly for one week).
occasionally results in a severe typhoid-like illness
with fever, purpura and enlargement of the liver
Treatment and spleen, which is usually fatal. More frequently
Streptomycin is the drug of choice. Chloramphenicol it causes mesenteric lymphadenitis and terminal
is recommended in patients with meningitic ileitis simulating acute or subacute appendicitis.
symptoms. Tetracycline may be adequate in It has also been reported to cause immunological
uncomplicated bubonic plague. Gentamicin and sequelae such as erythema nodosum or reactive
ciprofloxacin are also effective. arthritis in some patients.
www.ebook3000.com
314 | Section 3: Systemic Bacteriology
lesions or mesenteric nodes, or demonstration by demonstration of antbodies in the patient
of antibodies in patient serum during the acute serum. Isolation of the organisms can be done
phase of the illness by tube agglutination tests, from feces, blood or from mesenteric lymph
hemagglutination of red cells sensitized with nodes. Blood agar and MacConkey agar are used
LPS, or ELISA. for growing this organism. Selective medium for
recovery of Y. enterocolitica from faeces, food
Treatment soil etc. is cefsulodin-irgasan novobiocin (CIN)
Ileitis and mesenteric lymphadenitis are usually Yersinia selective agar. Colonies of Y. enterocolitica
self-limiting but septicem ia may be treated have a bull’s eye appearance, colored dark red
with parenteral ampicillin, chloramphenicol, and surrounded by a transparent border on CIN
gentamicin or tetracycline. agar. Identify the strain by biochemical tests and
serotype determination.
Yesinia enterocolitica
It is a gram-negative coccobacillus showing Pasteurella multocida (formerly
pleomorphism in older cultures. This bacillus Pasteurella septica)
resembles Y. pseudotuberculosis in being motile at
22°C but differs from it in fermenting sucrose and A group of related bacteria isolated from hemor
cellobiose and decarboxylating ornithine. It does rhagic septicemia in a variety of animals and birds
not ferment rhamnose or melibiose. Many strains had, in the past, been named according to their
are indole and VP positive. species of origin—Pasterella boviseptica. Pasterella
lepiseptica, Pasterella aviseptica, etc. Though they
Cultural Characters show some degree of host specificity, they are so
It is aerobe and facultative anaerobe. Optimum alike in other respects that they are now considered
temperature for growth is 22–29°C. On blood strains of a single species designated P. multocida.
agar, it forms nonhemolytic, smooth, translucent
colonies, 2–3 mm in diameter in 48 hours. On Morphology
MacConkey medium, it forms pinpoint non- They are gram-negative, nonmotile, nonsporing
lactose fermenting (NLF) colonies. coccobacilli which show bipolar staining. Some
strains are encapsulated.
Antigenic Structure
Based on O and H antigens, more than 60 different Cultural Characteristics
O antigens and 19 H factors have been identified. Pasteurellae are aerobes and facultative anaerobes.
Serotypes O3, O8 and O9 account for most human They grow on blood agar and chocolate agar.
infections.
Pathogenesis Pathogenesis
The bacillus is usually normal inhabitant of the
Y. enterocolitica has been isolated from a wide
upper respiratory tract of a variety of animals
range of domestic and wild animals. Human
such as dogs, cats, cattle and sheep. Pasteurella
disease usuaIly results from ingestion of contam
infections are considered zoonoses. The most
inated food or from contact with the environment.
common presentation is a history of animal bite.
Blood transfusion is a significant hazard.
Human infections usually present as:
Types of Disease in Humans 1. A local abscess at the site of a cat or dog bite;
2. Infections of the respiratory system.
1. Gastroenteritis or enterocolitis, which is self-
3. Meningitis or cerebral abscess (usually follow
limited and occurs in young children.
ing head injury), endocarditis, pericarditis or
2. Mesenteric lymphadenitis and terminal ileitis
septicemia.
in older children that may mimic appendicitis
3. Septicemia, which is often fatal. Animals and birds: P. multocida can be extremely
4. Pneumonia and meningitis are rare present virulent to many species of animals and birds,
ations. causing fowl cholera; hemorrhagic septicemia. It
5. Postinfectious complications include erythema respiratory infections and atrophic rhinitis in pigs.
nodosum, polyarthritis, Reiter’s syndrome and
thyroiditis. Laboratory Diagnosis
1. Culture: Swabs from bite wounds, from blood,
Laboratory Diagnosis from CSF in cases of meningitis and from
The laboratory diagnosis of Y. enterocolitica secretions in suppurative conditions of the
consists of isolation of the organism and indirectly respiratory tract are cultured on blood agar
Chapter 44: Yersinia, Pasteurella, Francisella | 315
plates incubated at 37°C for 24 hours. The Prophylaxis
organisms are identified by various cultural and A vaccine based on the live-attenuated LVS strain
biochemical tests. confers some protection. It can be administerd by
2. Serology: Serology is of no value in diagnosis. scarification to persons who are subject to high
3. Polymerasrs chain reaction (PCR): PCR is risk of infection.
potentially useful but rarely available. F. tularensis has been developed as a biological
warfare agent and has potential application in
Francisella tularensis bioterrorism.
(Pasteurella tularensis, Brucella Key Points
tularensis) Yersinia are gram-negative bacilli that are facultative
Francisella tularensis produces tularaemia in man anaerobes, positive by the catalase test and negative
by the oxidase test
and certain small mammals, notably rabbits, hares,
The genus Yersinia consists of 10 species, with Y. pestis,
beavers and various rodent species. Tularemia was Yersinia enterocolitica, and Yersinia pseudotuberculosis
originally described in Tulare county, California. It the well-known human pathogens
can be transmitted by direct contact, by biting flies, Y. pestis is a gram-negative, shows bipolar staining
mosquitoes and ticks, by contaminated water or (safety pin appearance), pleomorphic, nonmotile,
meat, or aerosols. and is capsulated
F-l antigen or envelope antigen has been considered
a virulence determinant
Morphology Plague is a zoonotic infection in humans. Disease is
It is a very small, nonmotile, non-sporing, capsulate, spread by flea bites or direct contact with infected
tissues or person-to-person by inhalation of infectious
Gram- negative coccobacillus, about 0.3–0.7 µm ×
aerosols from a patient with pulmonary disease
0.2 µm in size. Diseases: Yersinia pestis causes plague, which
manifests in one of three forms: Bubonic plague;
Cultural Characters pneumonic plague; septicemic plague
Diagnosis: For diagnosis, material is aspirated
F. tularensis is strictly aerobic. It will not grow on from the bubo, for demonstration of bipolar
ordinary nutrient media, but grows well on blood staining short rods (coccobacilli) using Wright’s or
agar containing 2.5% glucose and 0.1% cysteine Gram stain, for demonstration of the bacteria by
hydrochlor ide. Minute droplet-like colonies immunofluorescence. Culture of the bacteria on
blood agar. Serology can be used to detect antibody
develop in 72 hours. to capsular antigen
Yersinia pseudotuberculosis: It causes pseudotuber
Pathogenesis culosis, a zoonotic disease
Yesinia enterocolitica
The infection, which is a typical zoonosis, is mainly
It produces in humans-gastroenteritis or enterocol
spread by insects or ticks among lagomorphs and itis, mesenteric lymphadenitis and terminal ileitis
rodents. It is transmitted to man through handling in older children, septicemia, pneumonia and
of infected animals. meningitis, erythema nodosum, polyarthritis, Reiter’s
In human beings, tularemia may present as a syndrome and thyroiditis
local ulceration with lymphadenitis, a typhoid like Pasteurella multocida
fever with glandular enlargement or an influenza Pasteurella infections are considered zoonoses
like respiratory infection. Francisella tularensis
Francisella tularensis produces tularemia in man and
certain small mammals. The infection, is a typical
Laboratory Diagnosis zoonosis.
Diagnosis may be made by culture or by inoculation
into guinea pigs or mice. A PCR has been Important questions
described, but is not widely available. Serology is
most likely to be positive after 3 weeks. Rising titers 1. Describe the pathogenesis and laboratory diagnosis
of plague.
of agglutinins to F. tularensis or individual titers
2. Write short notes on:
of 160 are diagnostic. An intradermal delayed
a. Prophylaxis against plague
hypersensitivity test has been used in the past but
b. Yersinia pseudouberculosis
the antigen is not readily available.
c. Yersinia enterocolitica
d. Pasteurellosis
Treatment e. Pasteurella multocida
Streptomycin or gentamicin are the antibiotics of f. Francisella tularensis
choice in tularemia and are usually curative. g. Tularemia
www.ebook3000.com
316 | Section 3: Systemic Bacteriology
Multiple choice questions (Mcqs) 5. Which of the following vaccines is/are recommended
for immunization against plague?
1. Bipolar staining is characteristic of: a. Live b. Killed
a. Yersinia pestis c. Subunit d. All of the above
b. Yersinia enterocolitica 6. Yersinia enterocolitica shows all the following
c. Yersinia pseudotuberculosis characteristics except:
d. Proteus mirabilis a. They are motile at 22°C
2. Bubonic plague is transmitted by: b. They produce hemolytic colonies on blood agar
a. Inoculation b. Inhalation c. They ferment sucrose and cellobiose
c. Ingestion d. All of the above d. They decarboxylate ornithine
routes 7. Which of the following diseases is/are caused by Y.
3. Pneumonic plague is transmitted to humans by: enterocolitica in man?
a. Rat flea b. Droplet infection a. Gastroenteritis
c. Ingestion d. Inoculation b. Mesenteric lymphadenitis
c. Septicemia
4. The name black death is given to which of the d. All of the above
following diseases?
a. Tuberculosis b. Diphtheria Answers (MCQs)
c. Plague d. AIDS 1. a; 2. a; 3. b; 4. c; 5. b; 6. c; 7. d
45
Chapter
Haemophilus
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of infections caused
be able to: by Haemophilus influenzae
∙∙ Describe morphology and culture characteristics ∙∙ Discuss Haemophilus influenzae biogroup aegyptius
of Haemophilus influenzae ∙∙ Describe morphology and culture characteristics of
∙∙ Describe the following: X and V factors; Satellitism; Haemophilus ducreyi
Antigenic structure of Haemophilus influenzae ∙∙ Discuss Haemophilus ducreyi or chancroid or soft
∙∙ Describe pathogenicity of H. influenzae sore and its laboratory diagnosis.
www.ebook3000.com
318 | Section 3: Systemic Bacteriology
V factor: V factor is coenzyme, nicotinamide This is a routine test in clinical bacteriology for the
adenine dinucleotide (NAD), NAD phosphate identification of H. influenzae.
(NADP) which acts as a hydrogen acceptor in
the metabolism of the cell. It is heat labile being Biochemical Reactions
destroyed at 120°C in a few minutes. It is present in H. influenzae ferments glucose and galactose but
red blood cells and in many other animal and plant does not ferment sucrose, lactose and mannitol.
cells. It is synthesized by some fungi and bacteria Catalase and oxidase reactions are positive. It
such as Staph. aureus.The differential requirement reduces nitrates to nitrites. H. influenzae can
of X and V factors help to differentiate various be divided into eight biotypes on the basis of
species of haemophilus (Table 45.1). indole production, urease activity and ornithine
The growth is scanty on blood agar because V decarboxylase reactions (Table 45.2). Biotypes
factor is present inside the RBC. Chocolate agar I-III are the most common, and biotype I is most
(heated blood agar) is superior to plain blood frequently responsible for meningitis.
agar for the growth of H. influenzae, because V
factor is released from within the erythrocytes
and inactivates serum NADase. Clear transparent Antigenic Structure
media may be prepared by boiling and filtering a There are three major surface antigens:
mixture of blood and nutrient broth (Levinthal’s 1. Capsular antigens: The major antigenic
medium) or by adding a peptic digest of blood to determinant of capsulated strains is the capsular
nutrient agar (Fildes agar). polysaccharide based on which H. influenzae
Colonies: The colonies are small, translucent and strains have been classified by Pittman into six
nonhemolytic on blood agar. Capsulated strains capsular types—types a to f. Typing was originally
produce larger, distinctive iridescent colonies. done by agglutination but other methods such
Fildes agar is best for primary isolation of H. as Quellung reaction (swelling of the capsule)
influenzae and gives a copious growth. Capsulated with type-specific antisera, precipitation,
strains produce translucent colonies with a coagglutination, counterimmunoelectrophoresis
distinctive iridescence on Levinthal’s agar. (CIE) and enzyme-linked immunosorbent assay
(ELISA) may also be used. Diagnostic kits for the
Satellitism: The V factor is intracellular and
identification of H. influenzae type b (Hib) are
is present inside the RBC. It is synthesized by
commercially available.
some fungi and bacteria such as Staph. aureus
The type b capsular polysaccharide has
in excess of their requirements and released into
a unique chemical structure, containing the
the surrounding medium. When Staph. aureus
pentose sugars ribose and ribitol. The capsular
is streaked across a plate of blood agar on which
polyribosyl ribitol phosphate (PRP) antigen of
a specimen containing H. influenzae has been
Hib induces IgG, IgM and IgA antibodies which
inoculated, after overnight incubation, the colonies
are bactericidal, opsonic and protective. Hib
of H. influenzae will be large and well developed
PRP is, therefore, employed for immunization.
alongside the streak of Staphylococcus, and
H. influenzae strains lacking a capsule cannot
smaller farther away. This phenomenon is called
be typed and are called ‘nontypable strains’.
satellitism and demonstrates the dependence of H.
influenzae on the V factor, which is available in high 2. Somatic antigen: The cell envelope of H. influenzae
concentrations near the staphylococcal growth and consists of an outer and inner membrane
only in smaller quantities away from it (Fig. 45.1). containing protein and lipooligosaccharide
(LOS) antigens. Outer membrane protein (OMP)
antigens of Hib have been classified into at least
13 subtypes. OMP and LOS subtyping may be of
epidemiological value.
www.ebook3000.com
320 | Section 3: Systemic Bacteriology
iii. Antigen detection due to the production of β-lactamase. Amoxycillin-
Type b capsular antigen can be detected in patient clavulanate or clarithromycin is more effective.
serum, urine, CSF or pus by several methods.
These include latex agglutination, coagglutination, Prophylaxis
countercurrent immunoelectrophoresis radio
Active Immunization
immunoassay (RIA) enzyme linked immonosorbent
assay (ELISA). i. A purified type b capsular polysaccharide
vaccine
4. Culture It is used in children of 18–24 months. Vaccine
is administered in two doses at an interval of
i. CSF culture: CSF should be plated promptly
two months.
on blood agar or chocolate agar. A strain of S.
ii. Conjugate vaccines: Hib PRP vaccine in which
aureus should be streaked across the blood
the polysaccharide is covalently coupled
agar plate on which the specimen has already
to various proteins (e.g. tetanus toxoid,
been inoculated. Plate is then incubated
Neisseria meningit idis outer membrane
at 37°C with 5–10% CO2 and high humidity
protein, and diphtheria toxoid) produce a
overnight. It may also be cultured on Levinthal
lasting anamnestic response. Such Hib PRP
and Fildes blood-digest agar The growth
are available for use in young children.
is identified by colony morphology, Gram
staining, satellite phenomenon, biochemical iii. Household contacts of patients
reactions and serotyping. Rifampicin given for four days prev ents
secondary infection in contacts and also
ii. Blood culture: Blood cultures are often
eradicates carrier state.
positive in cases of epiglottitis and pneumonia.
iii. Sputum culture: Sputum should be homo
genized by treatment with pancreatin or by
Haemophili other than H. influenzae
shaking with sterile water and glass beads Haemophilus influenzae Biogroup Aegyptius
for 15–30 minutes. The rate of isolation is (Koch-Weeks Bacillus, Formerly H. Aegypticus)
increased by culturing several samples of
sputum from the patient. Koch (1883) observed a small bacillus in conjunc
tivitis cases in Egypt. It was first cultivated by Weeks
(1887) in New York.This organism, formerly known
5. Identification
as the Koch-Weeks bacillus and H. aegyptius, is
H. influenzae colonies have a characteristic seminal now thought to be a subgroup of H. influenzae.
odour. Confirmation of the identity depends on It is indistinguishable from H. influenzae
demonstrating a requirement for one or both of the biotype III in routine tests, but can be identified
growth factors, X and V H. influenzae requires both. by a PCR method.
www.ebook3000.com
322 | Section 3: Systemic Bacteriology
Bordetella
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss pathogenesis of pertussis
be able to: ∙∙ Discuss laboratory diagnosis of pertussis
∙∙ Describe various factors which determine the ∙∙ Describe the following: Cough plate method;
virulence of Bordetella pertussis vaccination against pertussis
∙∙ Describe culture media for Bordetella pertussis
www.ebook3000.com
324 | Section 3: Systemic Bacteriology
Table 46.1 Differentiatial characterstics of Bordetella species
Characteristics Bord. pertussis Bord. parapertussis Bord. bronchiseptica Bord. avium
1. Motility – – + +
2. Growth on:
Nutrient agar – + + +
Growth on Bordet- 3–6 1–2 1 1
Gengou medium (days)
MacConkey agar – + + +
3. Urease – – + +
4. Citrate utilization – V + +
5. Nitrate reduction – – + –
6. Toxins
Pertussis toxin + – – –
Adenylate cyclase toxin + + + –
Heat labile toxin + + + +
Tracheal cytotoxin + + + +
Lipopolysaccharide + + + +
7. Agglutinogens 1–7,13 7–10,14 7–13 Not known
www.ebook3000.com
326 | Section 3: Systemic Bacteriology
Bordetella parapertussis Important questions
This organism is readily distinguished from B. 1. Discuss various factors which determine the
pertussis by its ability to grow on nutrient agar, with virulence of Bordetella pertussis.
the production of a brown diffusible pigment after 2. Write short notes on:
a. Culture media for Bordetella pertussis
2 days (Table 46.1).
b. Pathogenesis of pertussis
This is an infrequent cause of whooping
c. Laboratory diagnosis of pertussis
cough (5% cases) and disease is mild. The
d. Cough plate method
pertussis vaccine does not protect against Bord.
e. Vaccination against pertussis
parapertussis infection. It usually causes less severe f. Acellular pertussis vaccine.
illness than B. pertussis.
Multiple choice questions (MCQs)
Bordetella bronchiseptica (Bord.
1. Which of the following species of Bordetella is the
bronchicanis) most important human pathogen?
It differs from the other species by also being a. Bordetella pertussis
motile by peritrichate flagella and by producing an b. Bordetella parapertussis
obvious alkaline reaction in the Hugh and Leifson c. Bordetella bronhiseptica
medium. d. Bordetella avium
2. Bordet–Gengou medium contains all the following
It can grow on nutrient agar and is antigenically ingredients except:
related to Bord. pertussis and Brucella abortus a. Potato b. Blood
(Table 46.1). It has been found to cause a very small c. Glycerine d. Agar
proportion (0.1%) of cases of whooping cough. 3. Pertussis toxin is produced by:
a. Bordetella pertussis
Bord. avium: It causes respiratory disease in
b. Bordetella parapertussis
Turkeys (rhinotracheitis).
c. Bordetella bronchiseptica
Key Points d. All of the above
4. The most infective stage in whooping cough is:
The genus Bordetella cons titutes a group of
minute, gram-negative, non-acid-fast, non-sporing, a. Catarrhal stage
coccobacilli b. Paroxysmal stage
Three important species of Bordetella include Bordetella c. Convalescent stage
pertussis, B. parapertussis and B. bronchiseptica d. None of the above
B. pertussis are extremely small, ovoid, gram-negative 5. Which of the following methods is most suitable
coccobacilli showing pleomorphism; nonmotile and for culture of Bordetella pertussis?
nonsporing a. Pernasal swab culture
Bordetella pertussis are strict aerobes and nutritionally
b. Postnasal swab culture
fastidious, grow on specialized media such as Bordet–
Gengou agar and charcoal agar with 10% blood c. Cough plate method
B. pertussis produces a number of factors that are d. Sputum culture
involved in the pathogenesis of disease, such as 6. The following Bordetella species cause infections
pertussis toxin (PT) in humans except:
Diseases: Pertussis and is characterized by three a. Bordetella pertussis
stages: catarrhal, paroxysmal, and convalescent b. Bordetella parapertussis
stages. Children younger than 1 year at greatest risk c. Bordetella bronchiseptica
for infection
d. Bordetella avium
Laboratory diagnosis: Depends on microscopy,
culture and polymerase chain reaction (PCR) 7. Vaccines available for prophylaxis of whooping
Treatment with macrolide (i.e. erythromycin, cough is/are:
azithromycin) is effective in. Erythromycin has been a. Whole cell pertussis vaccine
used for prophylaxis. Vaccination with whole-cell b. Acellular pertussis vaccine
vaccines is effective but associated with side effects. c. Both the of the above
Acellular vaccines are effective and associated with d. None of the above
fewer adverse effects 8. The most common clinical manifestation of
Bordetella parapertussis is responsible for only tularemia is:
about 5% of cases of whooping cough and relatively
a. Ulceroglandular tularemia
causes a mild form
Bordetella bronchiseptica: It is responsible to cause b. Glandular tularemia
in very small proportion (0.1%) of cases of whooping c. Oculoglandular tularemia
cough d. Oropharyngeal tularemia
Bord. avium: It causes respiratory disease in Turkeys
Answers (MCQs)
(rhinotracheitis).
1. a; 2. c; 3. a; 4. a; 5. a; 6. c; 7. c; 8. a
47
Chapter
Brucella
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss pathogenesis of brucellosis
be able to: ∙∙ Discuss laboratory diagnosis of brucellosis
∙∙ Discuss classification of Brucella ∙∙ Describe Castaneda method of blood culture.
∙∙ Describe culture characteristics and biochemical
reactions of Brucella sp.
www.ebook3000.com
328 | Section 3: Systemic Bacteriology
melitensis about 20 times M as A. B. suis has an ∙∙ CO2 requirement
intermediate antigenic pattern. Absorption of the ∙∙ H2S production
minor antigenic component from an antiserum ∙∙ Sensitivity to dyes (basic fuchsin and thionin).
will leave most of the major antibody component ∙∙ Agglutination with monospecific antisera.
and such absorbed A and M monospecific sera are ∙∙ Lysis by specific bacteriophage.
useful for species identification by the agglutination
test. Species and biotype identification depends on Biotypes
a variety of other factors besides antigenic structure The three major species are B. melitensis, B.
(Table 47.1). abortus, B suis infecting primarily goats or sheep,
Antigenic cross reactions exist between brucelIae cattle and swine, respectively. Many biotypes have
and V. cholerae, with E. coli 0: 116; 0: 157, SalmonelIa been recognized in these species (Table 47.1).
serotypes group N (0:30 antigen), Ps. maltophila, Y. i. B. melitensis: three biotypes.
enterocolitica and F. tularensis. ii. B. abortus: 7 biotypes (1-6 and 9, biotypes 7
and 8 have been discarded as invalid).
Brucella Bacteriophage iii. B. suis: 5 biotypes.
B. suis strains that produce H 2S are known
The Tblisi (Tb) phage has been designated as the as ‘American’ strains and those that do not as
reference phage and at routine test dose (RTD). It ‘Danish’ strains.
lyses B. abortus at both RTD and 10,000 TTD. B.
suis is lysed at 10,000 RTD, while B. melitensis is Pathogenesis
not lysed at all. They have been classified into six All three major species of brucellae are pathogenic
groups based on their host specificity. to human beings. B. melitensis is the most
pathogenic, B. abortus and B. suis of intermediate
Classification of Brucellae pathogenicity.
Brucellae may be categorized into species and Mode of infection: The modes of infection are
biovars by the following tests (Table 47.1): by ingestion, contact, inhalation or accidental
Thionin 1:50,000
CO2 requirement
H2S production
RTD x 104
Biotypes
Species
RTD
A M R
B. melitensis 1 − − − − + − + − + Sheep,
2 − − − − + − + + − − goats
3 − − − − + − + + + −
B. abortus 1 + + ± + + − − + − − Cattle
2 + + + + − − − + − −
3 + + ± + + + + + − −
4 + + ± + + − − − + −
5 + + − − + + + − + −
6 + + − − + + + + − −
9 + + ± ± + + + − + −
B. suis 1 − + − + − − + + − − Pigs
2 − + − − − − + + − − Pigs, hare
3 − + − − + + + + − − Pigs
4 − + − − + + + + + − Reindeer
B. neotoma'e − + − + − − − + − − Wood rat
B. ovis − − + − + + + − − + Sheep
B. canis − − − − − + + − − + Dogs
RTD: Routine test dilution
A—abortus; M—melitensis; R—rough
Chapter 47: Brucella | 329
inoculation. Person-to-person spread does not goat, sheep, cattle, buffaloes, and swine. Infection
ordinarily occur, but very rarely transmission has is transmitted among animals directly or through
been reported through the placenta, breast feeding blood-sucking arthropods, particularly ticks.
and sex. Brucellosis is a zoonosis of worldwide impor
i. Ingestion: The most important vehicle of tance, particularly in developing countries.
infection is raw milk. Brucellosis may also be potentially transmitted
ii. Contact: Contact infection is especially in association with biowarfare, bioterrorism, or
important as an occupational hazard in agri biocriminal activities.
cultural workers, veterinarians, butchers, Almost all human infections in various parts of
animal handlers, and others in occupations that India are due to B. melitensis acquired from goats
involve handling of animals or uncooked animal and sheep. Animal brucellosis is reported from
tissues are at higher risk for direct inoculation. practically every state in India.
iii. Inhalation: Infection is transmitted by inhal
ation of dried material of animal origin such Laboratory Diagnosis
as dust from wool. The clinical manifestations of human brucellosis are
Course of disease: Brucellosis is primarily an variable, so clinical diagnosis is almost impossible
intracellular pathogen affecting reticuloendothelial and laboratory aid is therefore essential. Laboratory
system. Brucellae have a special predilection for methods include culture of brucellae, serology,
intracellular growth and may be demonstrated polymerse chain reaction and hypersensitivity type
inside phagocytic cells. The brucellae spread skin tests.
from the initial site of infection through lymphatic
channels to the local lymph glands, in the cells of 1. Specimens
which they multiply. They then spill over into the
Blood culture is most important. Material from
bloodstream and are disseminated throughout the
bone marrow or liver biopsy, is also cultured, lymph
body. Once liberated into the bloodstream, they
nodes, cerebrospinal fluid, urine and abscesses, and
may infect a variety of organs but are most often
on occasion, also from sputum, breast milk, vaginal
localized in the reticuloendothelial system, where
discharges and seminal fluid.
they reside within phagocytic cells. Granulomas or
abscesses most often develop in the bone marrow,
2. Culture
liver, spleen, lymph nodes, or lungs. Other sites
of infection may include subcutaneous tissue, a. Blood culture
testes, epididymis, ovary, gallbladder, kidneys, and Blood culture is the most definitive method for
brain. Meningitis and endocarditis are commonly the diagnosis of brucellosis. When brucellosis is
reported complications. suspected, blood culture should be attempted
repeatedly, not only during the febrile phase.
Types of Human Infection Because the organisms may be scanty, at least
10 mL of blood should be withdrawn on each
The incubation period is usually about 10–30 days.
occasion, 5 mL being added to each of two blood
Human infection may be of three types:
culture bottles containing serum dextrose (SD)
1. Latent or subclinical infection
broth. One of these bottles should be incubated in
There is no clinical evidence of disease but is
an atmosphere containing 10% carbon dioxide at
detectable only by serological tests.
37°C. Subcultures are made on solid media (serum
2. Acute brucellosis: Acute brucellosis is mostly
dextrose agar) every 3–5 days, beginning on the
due to B. melitensis. It is associated with
fourth day. Growth may often be delayed and blood
prolonged bacteremia and irregular fever. It is
cultures should be retained for 6–8 weeks before
also known as undulant fever or Malta fever
being discarded as negative. Preliminary lysis and
because of the periodic noctural fever that may
centrifugation of the blood improves the isolation
occur over weeks, months or years particularly
rate. Automated blood culture systems may also
in untreated cases.
be used.
3. Chronic brucellosis: Chronic brucellosis is a low
grade infection with periodic exacerbations. It is b. Castaneda’s method of blood culture (Fig.
usually nonbacteremic. The illness lasts for years. 47.1)
Advantages: The Castaneda method of blood
Epidemiology culture has several advantages and is recommended.
Brucella organisms are distributed throughout i. A two-phase Castaneda culture system, in
the world. Human brucellosis is acquired from which the broth is periodically allowed to flow
animals, directly or indirectly. The animals that over agar contained within the blood culture
commonly act as sources of human infection are bottle, may be used. The blood is inoculated
www.ebook3000.com
330 | Section 3: Systemic Bacteriology
usually persist throughout the active phase of the
disease and in some cases long thereafter. As the
disease progresses, IgM antibodies decline, while
the IgG antibodies persist or increase in titer.
In chronic infections, IgM may often be absent
and only IgG can be demonstrated.
The agglutination test identifies mainly the
IgM antibody, while both IgM and IgG can fix
complement. However, as IgG and IgA antibodies
are formed during the course of the infection
some of them bind with antigen, thus preventing
its agglutination by larger IgM molecule. These
IgG and IgA antibodies are known as blocking
or non-agglutinating antibodies which may
prevent agglutination. It is thus evident that the
agglutination test is usually posit ive in acute
infection but may be only weakly positive or even
negative in chronic cases.
Fig. 47.1: Castaneda's method of blood culture
Brucella antibodies can be detected by a variety
into the broth and the bottle incubated in the of serological tests.
upright position. For subculture, it is sufficient i. Standard agglutination test (SAT): This is a
if a bottle is tilted so that the broth flows tube agglutination test in which equal volumes
over the surface of the agar slant. It is again of serial dilutions of the patient’s serum and the
incubated in an upright position. Colonies standardized antigen (a killed suspension of a
appear on the slant. standard strain of Br. abortus) are mixed and
ii. This method minimizes materials and incubated at 37°C for 24 hours or 50°C for 18
manipulation. hours. It detects antibodies against B. abortus,
iii. It reduces chances of contamination during B. suis, and B. melitensis but not B. canis. A titer
the period of incubation and risk of infection of 160 or more is considered significant.
to laboratory workers. Prozone phenomenon: Sera often contains
Blood cultures are positive only in about 30–50 ‘blocking’ or ‘nonagglutinating’ antibodies. A
per cent cases, even when repeated samples blocking factor may interfere with agglutination at
are tested. B. melitensis and B. suis are more low serum dilutions (the prozone phenomenon)
frequently isolated from blood than are B. although positive in higher dilutions.
abortus or B. canis. The blocking effect may sometimes be removed
c. Bone marrow or liver biopsy by prior heating of the serum at 55°C for 30 minutes
Isolation rates can be markedly improved if material or by using 4% saline as the diluent for the test. The
from bone marrow or liver biopsy is also cultured. most reliable method for obviating the blocking
Identification: Depends upon biochemical effect and detecting the ‘incomplete’ antibodies is
tests and may be classified into different species the antiglobulin (Coombs) test.
and biovars, based on CO 2 requirements, H 2S Cross-reactions: Cross-reactions may be observed
production, sensitivity to dyes (basic fuchsin and with antibodies directed against Francisella
thionin), agglutination with monospecific sera, tularensis, Vibrio cholerae, or Yersinia enterocolitica
lysis by specific phage and oxidative metabolic tests or immunization. Cholera induced agglutinins may
with amino acids and carbohydrates. be differentiated by the agglutinin absorption test
and also as they are removed by treatment with
3. Serological Tests 2-mercapto-ethanol. In order that results from,
In the absence of positive cultures, the diagnosis different laboratories are comparable; it is the
of brucellosis usually depends on serological tests, practice to express agglutinin titers in International
the results of which tend to vary with the stage of Units. This is done by using a standard reference
the infection. serum for comparison.
Antibodies appear within 7–10 days of onset ii. Mercaptoethanol test: The mercaptoethanol
of the disease. IgM antibodies appear first which test is carried out simultaneously and in
are rapidly followed and superseded by IgG and the same manner as the standard aggluti
to lesser extent IgA antibodies. These antibodies nation test except that the saline diluent
reach their maximum titers in the third or fourth contains 0.05 M 2-mercaptoethanol. The
week of disease and then slowly decline but they agglutinating ability of IgM, and sometimes
Chapter 47: Brucella | 331
IgA, is destroyed by 2-mercaptoethanol, and Result
therefore agglutination in this test is indicative Positive test: The bacilli are agglutinated and rise
of the continuing presence of IgG and the with the cream to form a blue ring at the top, leav
likelihood of persisting infection. ing the milk unstained.
iii. Complement fixation test: The complement
fixation test is more useful in chronic cases as Negative: No colored ring is formed and the milk
it detects IgG and IgM antibody also. remains uniformly blue.
iv. Enzyme-linked immunosorbentassay
(ELISA) and radio immunoassay (RIA): These ii. Whey Agglutination Test
tests are very sensitive useful for differentiation It is another useful method for detecting the
between the acute and chronic phases of antibodies in milk.
brucellosis. ELISA is sensitive, specific and can
detect IgM and IgG antibody separately. Prophylaxis
v. Rose Bengal plate test: This is a rapid slide
1. Prevention consists of checking brucellosis in
agglutination test. It is widely used as a
dairy animals.
screening test in farm animals.
2. Control of this disease in cattle has been
vi. Rapid dipstick assay: The rapid dipstick
achieved by serologic surveillance, vaccination
assay, a relatively simple screening procedure
(B. abortus strain 19), and elimination of
for Brucella specific IgM, shows promise as a
reactor cattle.
field test in areas without direct access to a
reference laboratory. 3. Pasteurization of infected milk or milk products.
4. Vaccines have been developed for use in animals.
Polymerase Chain Reaction (PCR) The live-attenuated B. abortus strain S19 vaccine
has been is now being replaced by the rough
Promising results have been obtained in clinical
strain B. abortus RB51, which gives comparable
studies.
protection, but does not induce interfering
Hypersensitivity Test (Brucellin Skin Test) antibodies and is less hazardous to man.
The live-attenuated smooth strain B. melitensis
Delayed hypersensitivity type skin tests with
Rev I is used to protect sheep and goats from B.
brucella antigens (‘brucellins’) are not useful in
melitensis infection. Strain 2 has been used in
diagnosing acute brucellosis. This test, similar to
China.
the tuberculin test, is no longer recommended.
They parallel the tuberculin test in indicating only 5. Human vaccination is not recommended.
prior sensitization with the antigens, and may
remain positive for years. Treatment
Brucella infections respond to a combination of
Detection of Animal Infection streptomycin or gentamicin and tetracycline or to
The methods used for the laboratory diagnosis rifampicin and doxycycline. Tetracycline alone is
of human brucellosis may also be employed for often adequate in mild cases. Treatment should be
the diagnosis of animal infections. In addition, continued for at least 6 weeks. Cotrimoxazole and
brucellae may be demonstrated microscopically rifampicin can be used in children.
in pathological specimens by suitable staining or
Key Points
by immunofluorescence.
Several rapid methods have been employed for The genus Brucella consists of very small, nonmotile,
the detection of brucellosis in herds of cattle. These aerobic, Gram-negative coccobacilli. They are
essentially pathogens of goats, sheep, cattle and pigs
include the rapid plate agglutination test and the
Six species are currently recognized and three major
Rose Bengal card test. For the detection of infected species of the genus Brucella are B. melitensis, B.
animals in dairies, pooled milk samples may be abortus, B. suis
tested for bacilli by culture and for antibodies by They grow best on media employed currently is
several techniques such as milk ring test. serum dextrose agar, serum potato infusion agar,
trypticase soy agar, or tryptose agar. They are
i. Milk Ring Test catalase and oxidase positive
Animal reservoirs are goats and sheep, cattle, swine,
1. In the milk ring test a sample of whole milk is and dogs. Individuals at greatest risk for disease are
mixed well with a drop of the stained brucella people who consume unpasteurized dairy products,
antigen (a concentrated suspension of killed Br. people in direct contact with infected animals, and
abortus stained with hematoxylin). laboratory workers
Human brucellosis is primarily a zoonotic bacterial
2. It is incubated in a water bath at 70°C for 40–50 infection
minutes.
www.ebook3000.com
332 | Section 3: Systemic Bacteriology
3. Brucellae are transmitted to humans by:
Laboratory diagnosis: Depends on the culture of
brucellae serology. Detection of Brucella species. Milk a. Direct contact with animal tissues
ring test is a screening test used for demonstration b. Ingestion of contaminated meats
of antibodies in the milk of animals c. Ingestion of raw infected milk
Human disease is controlled by eradication of the d. All of the above
disease in the animal reservoir; pasteurization of 4. The most common Brucella species causing human
dairy products; and use of proper safety techniques infection in India is:
in clinical laboratories working with this organism. a. Brucella melitensis b. Brucella abortus
c. Brucella suis d. Brucella canis
Important questions 5. The specimen of choice for isolation of Brucella is:
a. Blood b. Bone marrow
1. Discuss laboratory diagnosis of brucellosis. c. Urine d. Stool
2. Write short notes on: 6. Rose Bengal Card’ test is employed for which of the
a. Castaneda method of blood culture following infections?
b. Serodiagnosis of brucellosis a. Brucellosis
c. Diagnosis of brucellosis in animals. b. Salmonellosis
c. Tularensis
Multiple choice questions (Mcqs) d. None of the above
7. Milk ring test is used for diagnosis of:
1. All the following statements are true regarding
a. Bovine tuberculosis b. Brucellosis
cultural characteristics of Brucella except:
c. Salmonellosis d. Anthrax
a. They are strict aerobes
b. They require 5–10% of CO2 for better growth 8. Vaccination of cattle against brucellosis is done
c. They produce detectable colonies within 24–48 with:
hours a. Attenuated B. abortus vaccine
d. Erythritol has stimulatory effect on growth of the b. Attenuated B. melitensis vaccine
bacteria. c. Attenuated B. ovis vaccine
2. Humans acquire Brucella melitensis infection from: d. Attenuated B. neotome vaccine
a. Sheep b. Cow Answers (MCQs)
c. Pig d. Wood rat 1. c; 2. a; 3. d; 4. a; 5. a; 6. a; 7. b; 8. a
4848
Chapter
Spirochetes
LEARNING OBJECTIVES
∙∙ Describe diseses caused by different spirochetes ∙∙ Describe the following: fluorescent treponemal
∙∙ Name various pathogenic treponemes antibody-absorption (FTA-ABS) test; TPHA (or) T.
∙∙ Describe morphology of Treponema pallidum pallidum hemagglutination test
∙∙ Discuss BFP (Biological false-positive) reactions
∙∙ Discuss pathogenesis of syphilis
∙∙ Describe the following: i. Endemic syphilis (or)
∙∙ Describe diseses caused by Treponema pallidum
Bejel; ii. Yaws; iii. Treponema pertenue; iv. Pinta; v.
their laboratory diagnosis
Borrelia recurrentis; vi. Borrelia vincentii; vii. Borrelia
∙∙ Explain serological tests for syphilis burgdorferi or Lyme disease; viii. Leptospirosis; ix.
∙∙ Discuss standard tests for syphilis (STS) Weil’s disease.
www.ebook3000.com
Chap-48.indd 333 15-03-2016 11:18:34
334 | Section 3: Systemic Bacteriology
Morphology
It is a very delicate, spiral filament 6–14 µm (average
10 µm) by 0.2 µm, with 6–12 coils which are com
paratively small, sharp and regular. The length of
the coils is about 1 µm and the depth 1–1.5 µm. The
ends are pointed and tapering. Spirochaetes show
rotary corkscrew-like motility and also movements
of flexion; angulation, with the organism bending
almost to 90° near its centre, is highly characteristic
of T. pallidum. During motion, secondary curves
Fig. 48.1: Species designation of spirochetes based on
appear and disappear in succession but the
morphology primary spirals are unchanged (Fig. 48.2).
T. pallidum cannot be seen under the light
microscope in wet films but can be made out by
TREPONEMA negative staining with Indian ink. Its morphology
and motility can be seen under the dark ground
The name Treponena is derived from the Greek
or phase contrast microscope (Fig. 48.2). It does
words trepo :to turn and nema, meaning thread) are
not take ordinary bacterial stains but stains light
relatively short slender spirochetes with fine spirals
rose red with prolonged Giemsa staining. It can
and pointed or rounded ends. Some of them are
be stained by silver impregnation methods.
pathogenic, while others occur as commensals in
Fontana’s method is useful for staining films
the mouth, intestines, and genitalia.
and Lev aditi’s method for tissue sections.
The two treponemal species that cause human Immunofluorescence methods can now be used
disease are Treponema pallidum (with three to detect treponemes in tissues and body fluids.
subspecies) and Treponema carateum. The species Ultrastructurally, the cytoplasm of T. pallidum
T. pallidum is now considered to include three is surrounded by a trilaminar cytoplasmic
subspecies—subspecies pallidum, endemicum membrane, enclosed by a cell wall containing
and pertenue. peptidoglycan. External to this is the rigid rich outer
membrane layer. Usually three, but occasionally
Treponemes Cause the Following four, endoflagella lie inside the outer membrane
Diseases in Humans and are inserted at the tapering portion at each
1. T. pallidum subspecies pallidum causes end of the cell. In contrast to other motile bacteria,
Venereal syphilis these flagella do not protrude into the surrounding
2. T. pallidum, subspecies endemicum (T. endem medium but are enclosed within the bacterial outer
icum) causes endemic syphilis. membrane. A capsular or slime layer has been
observed occasionally on the surface of T. pallidum.
3. T. pallidum subspecies pertenue causes yaws
4. T. carateum causes pinta. Cultivation
It is possible to maintain T. pallidum in motile and
Treponema pallidum Subspecies pallidum virulent form for 10–12 days in complex media
Treponema pallidum is the causative agent of syphilis. under anaerobic conditions. Virulent T. pallidum
The name pallidum refers to its pale staining. strains have been maintained by serial testicular
Pathogenesis
Treponema pallidum subsp. pallidum causes
syphilis. Venereal syphilis is acquired by sexual
contact. Syphilis can also be acquired by nongeni
tal contact with a lesion (e.g. on the lip) or
transplacental transmission to a fetus, resulting in
congenital syphilis. T. pallidum enters tissues by
penetration of intact mucosae or through abraded
skin. Clinical disease sets in after an incubation
period of about a month (range 10–90 days). The
Fig. 48.2: Treponema pallidum-dark ground illumination natural course of syphilis can be divided into
primary, secondary, and tertiary stages based
passage in rabbits for many decades. One such
on the clinical manifestations.
strain (Nichol’s strain) was isolated in 1912 from a
patient of general paralysis of the insane. i. Primary Disease
Cultivable treponemes such as T. phagedenis
The bacteria multiply at the initial entry site and
(Reiter’s treponeme) and T. refringens (Noguchi
the primary lesion in syphilis is the chancre.
strain) are nonpathogenic. They can be grown
The chancre is a painless, relatively avascular,
under strict anaerobic conditions.
circumscribed, indurated, superficially ulcerated
lesion. It is covered by a thick, glairy exudate very
Resistance
rich in spirochetes. It is known as ‘hard chancre’.
T. pallidum is very delicate, being readily inactivated The chancre is most frequently on the external
by drying or by heat (41–42°C in one hour). It is genitalia.
inactivated by contact with oxygen, distilled water, The regional lymph nodes are swollen,
soap, arsenicals,mercurials, bismuth, common disc rete, rubbery and nontender. The chan
antiseptic agents and antibiotics. It is killed in 1–3 cre invariably heals in about 10–40 days, even
days at 0–4°C, so that transfusion syphilis can be without treatment, leaving a thin scar. Chancres
prevented by storing blood for at least four days in usually occur singly but multiple or persistent
the refrigerator before transfusion. Stored frozen chancres may develop in immunocompromised
at –70°C in 10% glycerol, or in liquid nitrogen individuals, such as those infected with the human
(–130°C), it remains viable for 10–15 years. immunodeficiency virus (HIV).
Antigenic Structure ii. Secondary Syphilis
The antigenic structure of T. pallidum is complex. Secondary syphilis sets in 1–3 months after healing
Treponemal infection induces at least three types of primary lesion. The secondary lesions are due
of antibodies. On the basis of these antibodies, to widespread multiplication of the spirochetes
the treponemal antigens may be divided into and their dissemination through the blood. In this
nonspecific and specific antigens. stage, patients typically experience a “flu-like”
syndrome, lymphadenopathy, and a generalized
A. Nonspecific Antigen mucocutaneous rash. Characteristic lesions
The first is the reagin antibody in which a hapten are roseolar or papular skin rashes, mucous
extracted from the beef heart is used as the antigen. patches in the oropharynx and condylomata at
This lipid hapten is known as cardioliom and is the mucocutaneous junctions. Condylomata lata
chemically a diphosphatidyl glycerol. This lipid occur around moist areas, such as the anus and
has been detected in T. pallidum but it is not vagina. The patient is most infectious as with the
known whether the reagin antibody is induced primary chancre. There may also be ophthalmic,
by cardiolipin that is present in the spirochete or osseous and meningeal involvement.
released from damaged host tissues.
iii. Latent Syphilis
B. Specific Antigens After the secondary lesions disappear there is a
1. Group-specific antigen: It is a protein anti period of quiescence known as ‘latent syphilis’. No
gen present in T. pallidum as well as in clinical manifestations are evident and diagnosis
www.ebook3000.com
Chap-48.indd 335 15-03-2016 11:18:35
336 | Section 3: Systemic Bacteriology
during this period is possible only by serological applying thin coverslips, examined under the dark
tests. In many cases, this is followed by natural cure ground microscope.
but in others, after several years, manifestations of
tertiary syphilis appear. A. Demonstration of Treponemes
a. Demonstration of Treponemes in the
iv. Tertiary Syphilis or Late Syphilis Exudate
Tertiary or late syphilis, which may develop decades 1. Dark Ground Microscopy
after the primary infection, is a slowly progressive, Dark field microscopy: Diagnosis by microscopy
destructive inflammatory disease that may affect is applicable in primary and secondary stages and
any organ. The three most common forms of late in cases of congenital syphilis with superficial
syphilis are cardiovascular syphilis, gummatous lesions. This bacterium is too thin to be visualized
syphilis and neurosyphilis. with a standard Gram stain so two techniques
a. Cardiovascular syphilis: These consist of to visualize it with a light microscope are dark
cardiovascular lesions including aneurysms, field microscopy and immunofluorescence. T.
chronic granulomata (gummata) and meningo pallidum is recognized by its slender structure,
vascular manifestations. regularity of its spirals and slightly pointed ends.
b. Gummatous syphilis: It is a rare granulomatous A treponemal concentration of 104 per mL in the
lesion of the skeleton, skin or mucocutaneous exudates is required for the test to be positive.
tissues. 2. Direct Fluorescent-antibody Staining for
c. Neurosyphilis: Neurological manifestations, Treponema pallidum (DFA- Tp)
such as tabes dorsalis or general paralysis of the For direct fluorescent-antibody staining for T.
insane develop several decades after the initial pallidum (DFA-TP) test whereby a smear of exudate
infection in a few cases. These are known as late is made on a slide, fixed in acetone, and DFA-TP test
tertiary or quaternary syphilis. is done using fluorescent tagged anti T. pallidum
antiserum. The use of specific monoclonal
Congenital Syphilis antibody has made the test more reliable. The
In conngenital syphilis infection is transmitted treponemes appear distinct, sharply outlined and
from mother to fetus transplacentally. The lesions exhibit an apple green fluorescence. It is a better
of congenital syphilis usually develop only after and safer method for microscopic examination.
the fourth month of gestation. Congenital syphilis
can be prevented if the mother is given adequate b. Demonstration of Treponemes in Tissues
treatment before the fourth month of pregnancy. Treponemes in the tissues can be demonstrated by
immunofluorescence staining or silver impregn
Syphilis Acquired Nonvenereally ation method of staining (Levaditis stain).
In syphilis acquired nonvenereally (as occupa c. Demonstration of Treponemal Antigen in
tionally in doctors or nurses), the natural evolution
the Lesion
is as in venereal syphilis except that the primary
chancre is extragenital, usually on the fingers. In T. pallidum antigen in the lesion can be detected
the rare instances where syphilis is transmitted by enzyme immunoassay and polymerase chain
by blood transfusion, the primary chancre does reaction (PCR).
not occur.
B. Serological Tests
Laboratory Diagnosis These tests form the mainstay of laboratory
diagnosis. (Table 48.2). Two major types of serologic
Laboratory diagnosis consists of demonstration tests exist: nontreponemal tests and treponemal
of the spirochetes under the microscope and of tests. In nontreponemal tests or standard tests
antibodies in serum or CSF. for syphilis (STS) cardiolipin or lipoidal antigen
is used, while in treponemal tests treponemes are
Specimen Collection and Handling used as the antigen.
Specimens should be collected with care as
the lesions are highly infectious. The lesion is a. Nontreponemal Tests or Standard Tests for
cleaned with a gauze soaked in warm saline and Syphilis
the margins gently scraped so that the superficial Reagin antibodies are detected by cardiolipin
epithelium is abraded. Gentle pressure is applied antigen in standard tests for syphilis (STS).
to the base of the lesion and the serum that exudes The antigen used in these tests is an alcoholic
is collected preventing admixture with blood. extract of beef heart tissue (cardiolipin) to which
Wet films are prepared with the exudate and after lecithin and cholesterol are added. The STS
includes Wassermann, Kahn, Venereal Diseases Serum is inactivate heated to it prior to the test,
Research Laboratory (VDRL) and the rapid plasma whereas CSF need not be heated.
reagin (RPR) tests. All these tests are flocculation 2. Inactivated patient serum (0.05 mL) is pipetted
tests except Wassermann reaction which is a into the paraffin ring on the glass slide. Each
complement fixation test (CFT). The Wassermann (0.05 mL) of positive and negative control sera
reaction is no longer in use. Similarly Kahn test is are pipetted into other paraffin rings.
rarely done these days. 3. One drop of working antigen suspension is
The two nontreponemal tests widely used today added to each of these paraffin rings from a
are the Venereal Disease Research Laboratory syringe delivering 60 drops in 1 mL.
(VDRL) and rapid plasma reagin (RPR) tests.
4. Mix with wooden sticks and rotate slide at 180
Veneral Disease Research Laboratory revolutions per minute for four minutes on a
(VDRL) Test mechanical VDRL or manually. Flocculation
occurs in a positive reaction and is observed
It is more rapid test which gives more quantitative
microscopically.
results (VDRL, for Veneral Disease Research
Laboratory, USPHS, New York, where the test was
developed). These tests are cheaper, more rapid Interpretation
and simpler to perform and control. The results of qualitative test are reported as
VDRL is the most widely used simple and rapid ‘reactive’, ‘weak reactive’ or ‘nonreactive’.
test which requires only a small quantity of serum. Reactive’ means positive (large clumps of antigen
The VDRL test uses a cardiolipin antigen that is with marked background clearing are obtained.)
mixed with the patient’s serum or CSF. Flocculation while ‘nonreactive’ is negative.The reciprocal of
occurs in a positive reaction and is observed the end point is given as the titer for reporting
microscopically. of quantitative test, e.g. reactive in 1:4 dilution is
reported as ‘reactive 4 dilution’ or R4.
Method Sometimes VDRL test may give false-negative
1. The test is done in a specially prepared slide, reaction due to high titers of antibody in patient’s
with depressions of 14 mm diameter each. serum (prozone phenomenon). The test is
www.ebook3000.com
Chap-48.indd 337 15-03-2016 11:18:35
338 | Section 3: Systemic Bacteriology
performed with diluted serum in such cases and damage and are typically seen in: 1. SLE and
it becomes positive. other collagen diseases; 2. Leprosy; 3. Malaria;
4. Relapsing fever; 5. Infectious mononucleosis;
Rapid Plasma Reagin Test 6. Hepatitis; 7. Tropical eosinophilia
The black carbon particles are bound to cardiolipin;
when mixed with a positive serum on a disposable b. Treponemal Tests (Table 48.2)
card, the particles clump together. Agglutination is Treponemal tests in which treponemes are
easily observed without a microscope. used as the antigen. These are of two types:
RPR test employs a stabilized VDRL carbon 1. Those using cultivable treponemes, such
antigen which make the result more clear cut and as Reiter treponemes (T. phagedenis) as the
is read macroscopically. antigen-Reiter protein complement fixation
(RPCF) test
Advantages of RPR Test 2. Species tests using pathogenic T. pallidum
(Nichol’s strain)
i. It enables the result to be read by eye instead
of microscopically. i. Using Live T. Pallidum
ii. Useful in field studies in developing countries. Treponema pallidum immobilisation (TPI)
test
iii. RPR test can be done with unheated serum or
plasma. ii. Using Killed T. Pallidum
iv. A fingerprick sample of blood is sufficient. a. Treponema pallidum immune
adherence (TPIA) test.
b. Treponema pallidum agglutination
Disadvantage
(TPA) test.
It is not suitable for testing cerebrospinal fluid c. Fluorescent treponemal antibody-
(CSF). absorption (FTA-Abs) test.
iii. Using an extract of T. pallidum
Toluidine Red Unheated Serum Test (TRUST)
a. Treponema pallidum
Instead of carbon particles it uses paint pigment hemagglutination assay (TPHA) test.
toner toludine red particles. b. Enzyme immunoassay (EIA).
Automated RPR test (ART): Automated RPR test
is available for large scale tests. 1. Group-specific Tests
Using Reiter Treponeme
VDRL–ELISA test: An automated VDRL–ELISA test
has been developed which can measure IgG and Reiter Protein Complement Fixation (RPCF)
IgM antibodies separately and is suitable for large Test
scale testing of sera. The principle of this test is the same as that of
Wassermann test. These employed the cutivable
Disadvantages of STS Reiter treponemes for prepation of antigen. Its
Since cardiolipin antigen tests detect antibodies sensitivity and specificity were lower than those
against a nonspecific antigen a positive result of tests using T. pallidum. RPCF and other Reiter
is sometimes obtained with sera from healthy treponeme tests are not now in general use.
individuals or patients without clinical evidence
of syphilis. These reactions are termed biological 2. Species Tests Using Pathogenic
false-positives (BFP) rections. These are not T. Pallidum (Nichol’s Strain)
caused by technical faults. They represent i. Tests Using Live T. pallidum
nontreponemal cardiolipin antibody responses. Treponema pallidum immobilization (TPI) test
BFP antibody is usually IgM, while reagin The test serum is incubated with complement and
antibody in syphilis is mainly IgG. Clinically, BFP T. pallidum maintained in a complex medium
reactions may be classified as acute or chronic. anaerobically. If ant ibodies are present, the
a. Acute BFP reactions: Acute BFP reactions last treponemes are immobilized, that is, rendered
only for a few weeks or months and are usually nonmotile, when examined under dark ground
associated with acute infections, injuries or illumination. The test is considered positive if the
inflammatory conditions. percentage of treponemes immobilized is 50 or
b. Chronic BFP reactions: Chronic BFP reactions more, negative if 20 or less, and inconclusive if in
persist for longer than six months. These between.
may occur in a wide variety of infectious and TPI was the most specific test available for
noninfectious conditions associated with tissue diagnosis of syphilis and was considered the gold
www.ebook3000.com
Chap-48.indd 339 15-03-2016 11:18:35
340 | Section 3: Systemic Bacteriology
IgG are available and are increasingly replacing 4. Diagnosis of Congenital Syphilis
the TPHA and VDRL tests for routine screening. In the diagnosis of congenital syphilis it
is necessary to differentiate between passive
Treponema pallidum Particle Agglutination
transplacental transfer of maternal antibody to the
(TP-PA) fetus and production by the fetus of endogenous
The TP-PA test has largely replaced the antitreponemal antibody. As IgM does not cross the
microhemagglutination assay for antibodies to placenta, its presence in neonatal serum confirms
T. pallidum (MHA-TP) test. The TP-PA test uses cong enital syphilis. If IgM-FTA-ABS test gives
gelatin particles sensitized with T. pallidum a reactive test result with infant blood then it is
antigens instead of the sensitized red blood cells strong evidence of active congenital disease. Serial
used in the MHA-TP assay. The TP-PA test is testing is also useful because the titer of passively
simpler to perform than the FTA-ABS test, does transferred antibody decreases rapidly, the VDRL
not require an absorption step or expensive UV test becoming negative by three months.
microscope, and is more specific than the MHA-TP
test. Table 48.3 shows the relative sensitivities of Syphilis Associated with Human
the serological tests in common use.
Immunodeficiency Virus (HIV) Infections
Interpretation of Various Serological Tests HIV-infected patients who acquire syphilis may fail
to produce antitreponemal antibodies. This has
1. Serological Screening
extremely serious implications for the diagnosis
Because of their simplicity and accuracy, the and control of syphilis.
cardiolipin antigen tests are used as screening or
first line procedures for both routine diagnosis
Epidemiology
and mass screening programs. When used together,
the VDRL and TPHA tests provide a highly efficient Syphilis is found worldwide in distribution.
screen for the detection or exclusion of treponemal Natural syphilis is exclusive to humans and has
infection. no other known natural hosts. The most common
route of spread is by direct sexual contact. The
2. Response to Treatment disease can also be acquired congenitally or by
Reagin tests usually become negative 6–18 months transfusion with contaminated blood. Syphilis is
after effective treatment of syphilis, depending not highly contagious. High-risk sexual behavior
on the stage at which treatment is given. Serial and coinfection with HIV continue to complicate
quantitative VDRL testing provides the best means syphilis control efforts.
of measuring response to treatment in most stages
of treponemal infection. Treatment in the primary Immunity
stage leads to seroreversal in about four months; in The immune mechanisms in syphilis are not
the secondary and early latent stages, it takes 12–18 adequately understood. Humoral immune
months; in later stages, it may take five years or response against the treponeme does not appear
more. In some cases low titer reactivity may persist to be effective but Cell-mediated immunity may
indefinitely, in spite of effective treatment. Specific be more relevant.
treponemal tests tend to remain positive in spite of In a person already having active infection
treatment so they are of little value as indicators of reinfections do not appear to occur. It was believed
clinical cure. TPHA titers may fall rapidly following that premunition or infection immunity, as seen
treatment in secondary syphilis but remain positive in some parasitic infections, holds good in syphilis
for life in low titers. also and that a patient becomes susceptible to
reinfection only when his original infection is cured.
3. Biological False-positive (BFP) Reactions
TPHA and FTA-ABS are helpful in excluding
Prophylaxis
or confirming the diagnosis of syphilis and for
identifying BFP reactions. 1. As transmission is by direct contact, it is possible
to protect against syphilis.
Table 48.3 Frequency of reactive serological tests 2. When this is not complied with, the use of
in untreated syphilis (percentage) in common use physical barriers (such as condoms), antiseptics
Stage VDR/RPR FTA-ABS TPHA (potassium permanganate) or antibiotics may
minimize the risk.
Primary 70–80 85–100 65–85
3. Educating people about sexually transmitted
Secondary 100 100 100
diseases.
Latent/late 60–70 95–100 95–100
4. Protective vaccines are not available.
www.ebook3000.com
Chap-48.indd 341 15-03-2016 11:18:35
342 | Section 3: Systemic Bacteriology
(formerly, T. cuniculi), which has a very similar the development of immunity to all the antigenic
appearance and causes natural venereal infection variants.
in rabbits, may pose problems. Agglutinating, complement fixing and lytic
antibodies develop during infection but their dem
BORRELIA onstration is not possible as a routine diagnostic
Species of Borrelia test due to the difficulty in preparing satisfactory
Borreliae of medical importance are: antigens.
1. B. recurrentis causing relapsing fever
2. B. vincenti sometimes causes fusospirochetosis Pathogenicity
3. B. burgdorferi—causative agent of Lyme Relapsing Fever
disease.
Relapsing (RF) is an arthropod-borne infection,
Morphology and two types of which occur louse-borne and
tick-borne. The borreliae causing them are
Borrelia are helical organisms 0.2–0.5 mm wide indistinguishable in morphology and many other
and 8–20 mm in length. They are gram-negative, features but differ in their arthropod hosts.
actively motile and possess 5–8 irregular spirals at
intervals of 2 mm with pointed ends (Fig. 48.3).
1. Epidemic or Louse-borne Relapsing Fever
Spirals are coarser and more irregular than those of
the treponemes or leptospires and usually can be The causative agent of louse-borne or epidemic RF
seen with light microscopy in preparations stained is B. recurrentis. It is an exclusive human pathogen,
with aniline dyes, such as Wright’s or Giemsa stains. being transmitted from person to person through
body lice (Pediculus humanus corporis). Humans
Cultural Characteristics are the only reservoir for B. recurrentis.
Borrelia are microaerophilic. Optimum temperature
for growth is 28–30°C. The organism can be grown 2. Endemic or Tick-borne Relapsing Fever
on Noguchi’s medium (ascitic fluid containing The second form of relapsing fever is endemic and
rabbit kidney), chorioallantoic membrane (CAM) of tick-borne. It is caused by as many as 15 species
chick embryos and in mice or rats intraperitoneally. of borreliae and cause RF (B. duttonni, B. hermsii,
B. parkeri B. turicatae, etc.) and is spread by
Antigenic Properties infected soft ticks of the genus Ornithodoros that
Antigenic variations: The most striking property vary according to the country where the infection
of relapsing fever is the capacity of Borrelia to occurs.
undergo several antigenically distinct variations
within a given host during the course of a single Pathogenicity
infection. This is believed to be the reason for the
After an incubation period of 2–10 days relapsing
occurrence of relapses in the disease. Ultimate
fever sets in as fever of sudden onset. During this
recovery after a number of relapses may be due to
period, borreliae are abundant in the patient’s
blood. The fever subsides in 3–5 days. Another bout
of fever sets in after an afebrile period of 4–10 days
during which borreliae are not demonstrable in
blood. The borreliae reappear in blood during the
relapses of fever. The disease ultimately subsides
after 3–10 relapses. Subsequent relapses are usually
milder and of shorter duration.
Epidemiology
The etiologic agent of louse-borne epidemic
relapsing fever is B. recurrentis. The vector is
the human bodylouse, and humans are the only
reservoir. The infection is transmitted not by the
bite of lice but by their being crushed and rubbed
into abraded skin.
Louse-borne relapsing fever tends to occur as
epidemics whenever poverty, overcrowding and
lack of personal hygiene encourage louse infesta
Fig. 48.3: Borrelia recurrentis in peripheral blood smear tion.
www.ebook3000.com
Chap-48.indd 343 15-03-2016 11:18:36
344 | Section 3: Systemic Bacteriology
and lymphadenopathy. Some develop meningeal Morphology
or cardiac involvement. Leptospires are delicate spirochetes about 6–20
Stage 3: The third stage of ‘persistent infection’ mm long and 0.1 mm thick. They have numerous
sets in months or years later with chronic closely wound primary coils, so closely set together
arthritis, polyneuropathy, encephalopathy and that they are difficult to demonstrate in stained
acrodermatitis. preparations although they are quite obvious in the
living state by darkfield microscopy or by electron
Laboratory Diagnosis microscopy. Their ends are hooked and resemble
A. Isolation of the borrelia: The borrelia has been umbrella handles (Fig. 48.4). They are actively
isolated from ticks as well as from skin lesions, motile, Gram-negative, but take up conventional
CSF and the blood of patients, but culture is too stains poorly. They can be visualized by Giemsa or
slow and difficult to be of use in diagnosis. silver deposition methods or by use of fluorescent
B. Serology: Serological tests such as ELISA and antibody.
immunofluorescence (IF) have been described
and immunob lotting recommended for Cultural Characteristics
confirmation. Antibodies take 1–2 months to They are aerobic and microaerophilic. Optimum
appear. temperature is 25–30°C and optimum pH 7.2–7.5.
False-positive syphilis serology may be seen, Leprospires can be grown in media enriched with
with FTA-ABS being positive and VDRL test rabbit serum.
negative. Liquid medium consists of Stuart’s or Korthof’s
medium. Semisynthetic media, such as EMJH
Treatment (Ellinghausen, McCullough, Johnson, Harris)
Penicillins, the newer macrolides, cephalosporins medium are now commonly used. A simple
and tetracyclines have all been used successfully in semisolid medium is Fletcher’s medium which
Lyme disease. consists of nutrient agar and rabbit serum. In
semisolid media, growth occurs characteristically
a few millimeters below the surface.
LEPTOSPIRA
Leptospires may be grown on the chorioallantoic
Introduction: Leptospires are actively motile, membrane (CAM) of chick embryos.
delicate spirochetes, possessing a large number of
closely wound spirals and characteristic hooked Resistance
ends. They are too thin to be seen under the light Leptospires are susceptible to heat; 10 min at
microscope (leptos, meaning fine or thin). They 50°C or 10 seconds at 60°C kills them. They are
may be visualized under dark ground illumination. also sensitive to acid and are destroyed by gastric
They do not stain readily. juice in 30 minutes. They are rapidly killed by bile
or trypsin.
Classification They are also readily destroyed by chlorine and
The family Leptospiraceae belongs to the order most other antiseptics and disinfectants. Their
Spirochetales and can be subdivided into three
morphologically indistinguishable genera:
Leptospira, Leptonema and Tumeria. Only
Leptospira spp. are considered to be pathogenic
for animals or man.
General characteristics: The genus Leptospira is
now classified into two species L. interrogans, and
L. biflexa. Pathogenic species are called Leptospira
interrogans, and most saprophytic leptospires are
called L. bifiexa.
1. Leptospira interrogans: It comprises the
parasitic and pathogenic leptospires. L.
interrogans is classified into 23 serogroups
(Icterohemorrhagiae, Canicola, Pyrogenes,
Autumnalis, Australis, Pomona, Hebdomadis,
Grippotyphosa, etc.). Within each serogroup
over 200 serovars are recognized. Fig. 48.4: Dark ground microscopy shows the appearance
2. L. bifiexa: It contains saprophytic leptospires. of living leptospires
survival in water or soil depends on tempera Duration of the illness varies from less than
ture, acidity, salinity and nature and amount of 1 week to 3 weeks. Late manifestations may be
pollution, dying rapidly in acid urine, nonaerated caused by the host immunologic response to the
sewage, saltish or brackish water. They can survive infection (Table 48.4).
for days in moist conditions at pH 6.8–8. Serious cases of leptospirosis are caused most
often by serotype icterohemorrhagiae, though
Antigenic Properties they m ay also be due to coenhageni and less
Leptospires exhibit considerable antigenic cross often batavitae, gripotyphosahosa, pyrogenes
reaction. A genus specific somatic antigen is and some others. Aseptic meningitis is common
present in all members of the genus. Classification in canicola infection and abdominal symptoms in
into serogroups and serotypes (now referred to grippotyphosa infections.
as sero-varieties or serovars) is based on surface
antigens. Laboratory Diagnosis
Diagnosis may be made by 1. Demonstration of
Pathogenesis the leptospires microscopically in blood or urine;
In natural reservoir hosts, leptospiral infection is 2. Isolation in culture; 3. Animal inoculation; 4.
asymptomatic. It is transmitted to humans when Serological tests.
the leptospires in water contaminated by the urine
of carrier animals enters the body through cuts or 1. Demonstration of Leptospiras in Blood
abrasions on the skin or through intact mucosa of
or Urine
mouth, nose or conjuctiva. During the acute phase
of the disease, leptospires are seen in the blood but Microscopy
can seldom be demonstrated after 8–10 days. They As leptospires disappear from the blood after the
persist in the internal organs, and most abundantly first week, blood examination is helpful only in
in the kidneys, so that they may be demonstrated in the early stages of the disease. Leptospires may be
the urine in the later stages of the disease. demonstrated by examination of the blood under the
Diseases: Mild virus-like syndrome. The incubation dark field microscope or by immunofluorescence
period is usually about 10 days (range 2–26 days). but this is of little practical value.
The onset of clinical illness is usually abrupt, Leptospires may be found in the urine during
with nonspecific, influenza-like constitutional the second week and intermittently for 4–6 weeks
symptoms such as fever, chills, headache, severe or even longer. Centrifuged deposit of the urine
myalgia, and malaise. may be examined under dark ground illumination.
Systemic leptospirosis with aseptic meningitis.
The patient may develop a more advanced disease- 2. Culture
including aseptic meningitis. Three or four drops of blood are inoculated into
Severe systemic disease (Well’s disease) each of several bijou bottles containing EMJH
includes renal failure, hepatic failure, and intra or similar medium. The bottles are incubated
vascular disease, and may result in death. at 37°C for two days and left thereafter at room
www.ebook3000.com
Chap-48.indd 345 15-03-2016 11:18:36
346 | Section 3: Systemic Bacteriology
temperature in the dark for two weeks. Samples Pathogenic leptospires survive for long periods in
from the cultures are examined every third day the convoluted tubules of the kidneys in natural
for the presence of leptospires under dark ground hosts, multiply and are shed in the urine. Humans:
illumination. accidental end-stage host. People at risk are those
Direct culture of urine is seldom successful but exposed to urine-contaminated streams, rivers,
isolation is usually possible by inoculation into and standing water; occupational exposure to
guinea pigs. infected animals for farmers, meat handlers,
veterinarians.
3. Animal Inoculation When human beings come into contact with
such water, the leptospires enter the body through
The blood or urine from the patient is inoculated
abraded skin or mucosa and initiate infection.
intraperitoneally into young guinea pigs. The
Several animals act as carriers. Rats are particu
animals develop fever and die within 8–12 days
larly important as they are ubiquitous and carry
with jaundice and hemorrhage into the lungs
the most pathogenic serotype icterohemorrhagiae
and serous cavities with virulent serotypes like
in the convoluted tubules of the kidney long-term,
icterohemorrhagiae.
resulting in chronic excretion of viable leptospires
in their urine. Field mice carry grippotyphosa, pigs
4. Serological Diagnosis pomona and dogs canicola serotypes.
Tests for detection of leptospiral antibodies in sera
are of two kinds: i.Genus-specific tests; ii. Sero- Prophylaxis
groupspecific tests General measures of prevention such as:
i. Rodent control
A. Genus-specific Tests ii. Disinfection of water
iii. Wearing of protective clothing.
The antigens for these tests are prepared from the
nonpathogenic L. biflexa Patoc 1 strain. The tests Vaccination: Vaccination has been attempted with
employed include sensitized erythrocyte lysis some success in dogs, cattle and pigs. Immunity
(SEL), complement fixation, agglutination and following vaccination or infection is serotype
indirect immunofluorescence. ELISA has been specific. Vaccination has also been tried in persons
used to detect IgM and IgG antibodies separately, at high risk such as agricultural workers.
in order to indicate the stage of infection. A simple Treatment
and rapid dip-stick assay has been developed for
Penicillin is given intravenously (IV), 1–2 million
the assay of leptospira-specific IgM antibody in
units 6 hourly for 7 days in serious cases. For milder
human sera.
infections a 7–10-day course of oral amoxicillin is
appropriate. Patients allergic to penicillins can be
B. Serogroup-specific Tests
treated with erythromycin. Doxycycline 200 mg
Serogroup-specific tests are reactive mainly with orally once a week is effective in prophylaxis.
strains of the same serogroup as the infecting
strain. They comprise agglutination tests that Key Points
are either macroscopic, or microscopic. In Spirochetes are slender unicellular, motile, helical or
macroscopic agglutination tests, formalinized spiral rods. They are motile because of endoflagella.
suspensions of prevalent leptospira serovars are The members of genera Treponema, Borrelia and
Leptospira are pathogenic to man
tested for macroscopic agglutination with serial
Treponema pallidum
dilutions of the test serum. Treponema pallidum is the causative agent of syphilis.
The micros copic agglutination test (MAT) T. pallidum is thin, coiled spirochete and is observed
generally uses live cultures of different serotypes by dark field or phase contrast microscopy
and agglutination is observed under the low power Diseases: Venereal syphilis. Syphilis is a sexually
transmitted disease
dark field microscope. This test is more specific and Lab diagnosis: Dark field microscopy is useful for
is generally accepted as “gold standard”. demonstration of treponemes in the clinical specimen.
Direct fluorescent antibody T. pallidum (DFA-TP) is a
Examination of Water for sensitive method for direct detection of treponemal
antigen in the exudates for diagnosis of syphilis
Pathogenic Leptospires Serological tests: Two major types of serologic tests
If a shaved and scarified area of the skin of a young exist: nontreponemal tests and treponemal tests.
guinea pig is immersed in water for an hour, In nontreponemal tests or Standard tests for
infection takes place through the abrasions. syphilis (STS) cardiolipin or lipoidal antigen is
used. Nontreponemal tests are Venereal Diseases
Epidemiology: Leptospirosis is most common Research Laboratory (VDRL) test and rapid plasma
zoonotic bacterial disease throughout the reagin (RPR). These are flocculation tests
world. Rodents are most important reservoirs.
www.ebook3000.com
Chap-48.indd 347 15-03-2016 11:18:36
4949
Chapter
LEARNING OBJECTIVES
After reading and studying this chapter, you should pneumoniae, Mycoplasma hominis, and Ureaplasma
be able to: urealyticum
∙∙ Describe the general characteristics of the ∙∙ Analyze the diagnostic methods appropriate for
Mycoplasma spp. and how they differ from other detection of mycoplasmal and ureaplasmal infections
bacterial species ∙∙ Discuss the use of serologic tests for diagnosing M.
∙∙ Describe morphology and characteristies of pneumoniae infections
Mycoplasma spp ∙∙ Compare mycoplasmas and L forms
∙∙ Compare the clinical diseases caused by Mycoplasma ∙∙ Describe Ureaplasma urealyticum.
Cultural Characteristics
Most mycoplasmas are facultatively anaerobic but,
since organisms from primary tissue specimens
frequently grow only under anaerobic conditions,
an atmosphere of 95% nitrogen and 5% carbon
dioxide is preferred for primary isolation. They
grow within a temperature range of 22–41°C, the
parasitic species growing optimally at 35–37 °C and
the saprophytes at lower temperatures.
Media for cultivating Mycoplasma are enriched
with 20% horse or human serum and yeast extract. Fig. 49.1: Typical large Mycoplasma colony showing ‘fried
The high concentration of serum is necessary egg” appearance
www.ebook3000.com
Chap-49.indd 349 15-03-2016 11:19:14
350 | Section 3: Systemic Bacteriology
Antigenic Structure children and young adults are especially susceptible
The surface antigens presented to the infected host to infection. Clinical disease is uncommon in very
are components of the cell membrane and consist young children and older adults. Other groups at
of glycolipids and proteins. risk include closed-in populations such as prisoners,
college students and military personnel. Epidemics
A. Glycolipid antigen: The glycolipid antigens of
are known to occur in these populations. Infection
M. pneumoniae are major membrane antigens
is not considered seasonal, but many cases occur
found in the lipid fraction. The purified
in autumn and early winter. Transmission is proba
glycolipids act as antigens in the complement-
bly through aerosol droplet spray produced while
fixation test and other in vitro antigen–antibody
coughing.
reactions.
Pneumonias not attributable to any of the
B. Protein antigens: During the course of
common bacterial causes were labeled historically
infection a number of protein antigens are
as primary atypical pneumonias. Eaton (944) was
recognized. Among them are two major surface
the first to isolate the causative agent of the disease
antigens, one of which is the PI protein that
and because it was filterable, it was considered
mediates attachment. Antibody to these surface
to be a virus (Eaton agent). The most important
proteins is found consistently in convalescent
species is Mycoplasma pneumoniae (also called
human sera and in respiratory secretions by
Eaton’s agent after the investigator. Who originally
radioimmunoprecipitation, gel electrophoresis,
isolated it.
and Western blot analysis.
The cold agglutinins that are induced in The disease has an incubation period of 1–3
patients with M. pneumoniae infection are directed weeks, and early symptoms are nonspecific,
against the I antigen determinant of erythrocytes, consisting of headache, low-grade fever, malaise,
which is also present on the surface of other blood and anorexia. Sore throat, dry cough, and earache
cells. are accompanying symptoms. However, patients
usually do not appear seriously ill and few warrant
Resistance: Mycoplasmas are killed by heating at admission to hospital.
56°C for 30 minutes. For long-term preservation, The disease is characterized by paucity of
lyophilization or freezing broth cultures at-70°C respiratory signs on physical examination but
(or in liquid nitrogen) are the preferred storage radiological evidence of consolidation, which is
methods. usually patchy involving one of the lower lobes,
M. pneumoniae can grow in the presence of starting at the hilum, and fanning out to the
0.002% methylene blue in agar, while many other periphery. About 20% of patients suffer bilateral
species are inhibited. Antiseptic solutions, such as pneumonia, but pleurisy and pleural effusions
chlorhexidine and cetrimide inhibit the growth of are unusual. The disease is usually self-limited,
mycoplasmas. recovery occurring in 1–2 weeks, but can be
They are resistant to penicillin and cepha prolonged.
losporin as well as to lysozymes that act on the Extrapulmonary complications, including
bacterial cell walls but are sensitive to tetracycline cardiovascular, central nervous system, dermato
and many other antibiotics that inhibit protein logic, and gastrointestinal problems, are rare
synthesis. occurrences. Secondary complications include otitis
media, erythema multiforme (Stevens–Johnson
Pathogenicity syndrome), hemolytic anemia, myocarditis, peri
Parasitic mycoplasmas exhibit host specificity. They carditis, and neurologic abnormalities.
generally produce surface infections by adhering
to the mucosa of the respiratory, gastrointestinal
Epidemiology
and genitourinary tracts. Mycoplasma causes
two types of diseases in humans—peumonia and M. pneumoniae is worldwide in its distribu
genital infections. tion and is found at all ages. Transmission is by
droplets of nasopharyngeal secretion. Disease is
A. Mycoplasma pneumoniae most common in school aged children and young
Infection with M. pneumoniae typically produces adults (5–15 years). Close contact is necessary for
mild upper respiratory tract disease. More transmission, and the infection usually occurs
severe disease with lower respir atory tract among classmates or family members. Spread is
symptoms occurs in less than 10% of patients. favored by close contact, as in families and most
Tracheobronchitis can occur. Pneumonia typically among military recruits. Mycoplasma may
(referred to as primary atypical pneumonia or remain in the throat for two or more months after
walking pneumonia) can also develop. School age recovery from the disease.
1. Specimens
M. pneumoniae may be recovered from throat
swabs, nasopharyngeal swabs, sputum, throat
washings, bronchoalveolar lavage, tracheal
aspirate and lung tissue specimens.
2. Culture
In the laboratory, if inoculation is not possible
immediately, then the specimen may be held up
to 24 hours at 4°C. If delay more than 24 hours is
expected, then the specimen should be frozen at
Fig. 49.2: Typical large Mycoplasma pneumoniae colony
– 70°C. A widely used isolation medium contains showing “mullberry shaped” appearance. (Courtesy
bovine heart infusion (PPLO broth) with fresh Dr Surinder Kumar, Director Professor, Department of
yeast extract and horse serum supplemented with Microbiology, Maulana Azad Medical College, New Delhi, India)
penicillin (to inhibit other bacteria), thallium
ii. Species identification: These colonies may
acetate, glucose and with phenol red as a pH
be identified by:
indicator because mycoplasmas do not produce
turbidity in broth media. For genital Mycoplasma, a. Hemadsorption test: The characteristic
polymymin B and amphotericin B and lincomycin of guinea pig red blood cells is adhering to
are also added to mycoplamal broth. colonies of M. pneumoniae.
b. Tetrazolium reduction test: Colonies of
1. Isolation and Identification M. pneumoniae appear red when these
In general, the growth of M. pneumoniae from are flooded with solution oftetrazolium
clinical specimens is detected by the ability of compound which is colourless. M.
these organisms to produce acid from glucose. pneumoniae reduces tetrazolium (colour
Most broth media are diphasic such as methylene less) to red coloured compound.
blue-glucose diphasic medium. Broth cultures are c. Serological techniques: Identification of
incubated at 35°C with the caps tightened. Tubes isolates can be confirmed by inhibition of
are inspected daily for color changes (from salmon their growth with specific antisera.
to yellow) in the medium. A slight, gradual shift in
the pH indicator over an 8–15 day period without 2. Polymerase Chain Reaction (PCR)
gross turbidity suggests a true-positive culture. Amplification
The broth must be subcultured to appropriate agar
M. pneumoniae in respiratory exudates or secretions
medium as soon as color changes in the medium
is detected by polymerase chain reaction (PCR)
are apparent. Inspection of the agar surface under
amplification of a chosen sequence in its genome.
the low power of the microscope will reveal small
colonies of the organisms. In the absence of
obvious color change in diphasic media, a blind 3. Antigen Detection Techniques
subculture to agar media should be performed after Detection of antigen in respiratory exudates by
1 and 3 weeks of incubation. direct immunofluorescence and counterimmuno
i. Colonies: M. pneumoniae may take 21 days or electrophoresis techniques, immunoblotting
more, while M. hominis colonies may appear with monoclonal antibodies and antigen capture
within 2–4 days. Colonies of M. pneumoniae are enzyme immunoassay (EIA).
small, betahemolytic and have a homogeneous
granular appearance (“mulberry shaped”), 4. DNA Probes
unlike the fried-egg morphology of other
DNA probes can be used to detect M. pneumoniae
mycoplasmas. Because the organisms do not
RNA in sputum specimens.
stain well with gram or acridine orange stains,
Mycoplasma like colonies are stained with the
Dienes or methylene blue stains. M. hominis 5. Serological Tests
colonies have a typical large “fried egg” Serologic tests are available only for M. pneumoniae
appearance (Fig. 49.2 ). Serological diagnosis may be made by:
www.ebook3000.com
Chap-49.indd 351 15-03-2016 11:19:14
352 | Section 3: Systemic Bacteriology
A. Specific tests using mycoplasmal antigens rubella, adenovirus infections, psittacosis, tropical
B. Nonspecific methods. eosinophilia, trypanosomiasis, cirrhosis of liver,
paroxysmal hemog lobinuria and haemolytic
A. Specific Tests Using Mycoplasmal Antigens anemia.
The development of antibody to M. pneumoniae by
infected subjects may be measured by a range of Treatment
techniques of widely differing sensitivity, viz. com Tetracyclines and erythromycin are drugs of choice
plement fixation, metabolic inhibition, inhibition for the treatment of Mycoplasma and Ureaplasma
of tetrazolium reduction, immunofluorescence on infections. Patients with NGU should receive one
sections of chick embryo lung, direct or antibody of the tetracyclines and ureaplasmas resistant to
capture enzyme- immunosorbent assays (EIA), or tetracyclines, patients should then be treated with
agglutination of antigen-coated erythrocytes, latex erythromycin, to which most tetracycline-resistant
or gelatin particles. ureaplasmas are sensitive.
i. Complement fixation test : Detection of
antibodies directed against M. pneumoniae by Infections in Immunocompromised Patients
complement fixation is a useful. Titers peak in 4 M. pneumoniae may cause severe pneumonia
weeks and persist for 6–12 months; diagnostic titer in immunodeficient patients and may persist
is ≥ 1: 32, or a fourfold increase. for many months in the respiratory tract of
hypogammaglobulinemia patients, despite
ii. Enzyme-linked immunosorbent assays: The
apparently adequate treatment.
tests are easier to perform and are highly sensitive
if both immunoglobulin M (IgM) and IgG are mea Mycoplasma and HIV Infection
sured.
Mycoplasmas tend to cause more severe and
prolonged infections in the human immuno
B. Nonspecific Serological Tests deficiency virus (HIV) infected and other
The nonspecific serological tests are Streptococcus immunodeficient subjects. A synergetic effect
MG and cold agglutination tests. had been proposed between HIV and Mycoplasma
i. Sterptococcus MG test (particularly M. fermentans).
It is done by mixing serial dilutions of the patient’s
unheated serum and a heat killed suspension of MYCOPLASMA AS CELL CULTURE
Streptococcus MG, and observing agglutination after CONTAMINANTS
overnight incubation at 37°C. A titer of 1 : 20 or over
is considered suggestive. Few primary cell cultures become infected with
mycoplasmas, but continuous cell lines do so
ii. Cold agglutination test
frequently. The contamination may originate from
The cold agglutination test is based on the appear
the worker or from animal sera or trypsin used in cell
ance in a high proportion of cases with primary
culture. The mycoplasmas most responsible are M.
atypical pneumonia, of macroglobulin antibodies
arginini. M. fermentans, M. hyorhinis, M. orale, M.
that agglutinate human group O cells at low
salivarium and A. laidlawii.
temperature. Cold agglutinins appear about one
week after infection with a peak at 4–5 weeks. Contamination generally does not produce
Thereafter, titer declines rapidly and the test beco cytopathic effects but may interfere with the
mes negative after about 5 months. growth of viruses in such cell cultures and may
This test is easily performed by mixing serial also produce misleading results in serological
dilutions of the patient’s serum with an equal volume tests. Eradication of mycoplasmas from infected
of a 0.2% washed human O group erythrocytes and cells is difficult.
clumping observed after incubating the mixture
overnight. The test is based on the development MYCOPLASMAS AND L FORMS OF
of hemagglutination, which can be reversed by
placing the tubes at 37oC. A titer of 1 : 32 or over
BACTERIA
is suggestive but demonstration of rise in titer in Kleineberger (1935) found pleuropneumonia-like
paired serum samples is more reliable. Because forms in a culture Streptobacillus moniliformis
paired sera are not always available, a single and termed them L forms, after Lister Institute,
antibody titer of 64–128 or more with either test, in London, where the observation was made. It was
a suggestive clinical setting, should be sufficient to subsequently shown that many bacteria, either
institute therapy. spontaneously or induced by certain substances
Cold agglutinins are occasionally induced in like penicillin, lost part or all of their cell wall
other diseases such as infectious mononucleosis, and develop into L forms. Such L forms may
www.ebook3000.com
Chap-49.indd 353 15-03-2016 11:19:14
354 | Section 3: Systemic Bacteriology
Miscellaneous Bacteria
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss rat-bite fever
be able to: ∙∙ Describe Klebsiella granulomatis and disease caused
∙∙ Describe morphology and culture characteristics by it
of Listeria ∙∙ Acinetobacter spp.
∙∙ Describe infections caused by Listeria monocytogenes
and their laboratory diagnosis
www.ebook3000.com
Chap-50.indd 355 15-03-2016 11:20:04
356 | Section 3: Systemic Bacteriology
Table 50.1 Distinguishing characters of Listeria spp
Species Hemolysis CAMP test with Acid production from Nitrate
reduction
S t a p h . Rhodococcus D-Mannitol L-Rhamnose D-Xylose a-Methyl
aureus equi D-mannoside
L. monocytogenes + + −a − + − + −
L. innocua − − − − +/− − + −
L. ivanovii ++ − + − − + − −
L. seeligeri ± + − − − + +/− −
L. welshimeri − − − − +/− + + −
L. grayi − − − + +/− − NK +/−
L. murayi − − − + V − NK +
L. denitrificans − − − − − + NK +
a
Regarded as ±; + = Positive reaction; – = Negative reaction; V = Variable; NK = Not known
www.ebook3000.com
Chap-50.indd 357 15-03-2016 11:20:04
358 | Section 3: Systemic Bacteriology
Culture Pathogenesis
It can be cultured readily in the egg-yolk medium The organisms survive well in the hospital environ
and on a modified Levinthal agar. ment, and are increasing in importance as opportunist
pathogens. Serious infections, including meningitis,
Pathogenicity pneumonia and septicemia, are most commonly
associated with Acinetobacter baumannii.
Donovanosis is a venereal disease, first described
by McLeod in India in 1882 and seen mainly in the Treatment
tropics. It can be transmitted after repeated exposure
through sexual intercourse or nonsexual trauma to Most strains are resistant to sulphonamides,
penicillins, erythromycin, the tetracyclines and
the genitalia. The incubation period ranges from
chloramphenicol. But empirical therapy for serious
1 to 12 weeks. It begins as a painless papule on
infections should consist of a b-Iactam antibiotic (e.g.,
the genitalia, which leads to a slowly progressive,
ceftazidime, imipenem) and an aminoglycoside.
autoinoculable ulcers. The disease runs a chronic
course.It causes a chronic granulomatous disease RAT-BITE FEVER (STREPTOBACILLUS
known as granuloma inguinale, granulomatous
MONILIFORMIS AND SPIRILLUM MINUS)
venereum or donovanosis.
Streptobacillus moniliformis and Spirillum minus
Laboratory Diagnosis are the causative agents of two distinct diseases
referred to collectively as rat-bite fever. Rat-bite fever
Laboratory confirmation of granuloma inguinale (RBF) is characterized by relapsing fever, rash and
is made by scraping the border of the lesion, by arthralgia occurring days or weeks after a rat-bite.
spreading the collected tissue on a slide, and Two different bacteria can cause this condition—
by staining it with Giemsa or Wright’s stain. Streptobacillus moniliformis and Spirillum minus,
Pathognomonic Donovan bodies are observed both of which are natural parasites of rodents.
within mononuclear phagocytes.
Streptobacillus moniliformis
Treatment Morphology
Cases of donovanosis respond well to tetracycline, S. moniliformis is a long, thin (0.1–0.5 × 1–5 µm),
chloramphenicol, erythromycin, clindamycin, gram- negative bacillus that tends to stain poorly
cotrimoxazole, streptomycin and other aminogly and to be more pleomorphic in older cultures.
cosides. Granules and bulb ous swellings resembling a
string of beads may be seen hence the species
ACINETOBACTER (MIMA POLYMORPHA; name ‘moniliformis’. It may lose its cell wall and
BACTERIUM ANITRATUM exists as L-form. In fact, L-forms were originally
discovered during the study of this bacillus.
The genus Acinetobacter contains strictly aerobic
short, stout, often capsulate, nonmotile gram- Culture
negative bacilli or coccobacilli that grow well on S. moniliformis is a nutritionally exacting aerobe and
simple media. They are oxidase negative. facultative anaerobe. Growth requires the presence
The genus contains only one species, Acinet of blood or other body fluids. It grows well on moist
obacter calcoaceticus, which embraces two variants: Loeffler’s serum slope or moist plate of nutrient
A. calcoaceticus var. anitratus produces acid agar containing 20% horse serum and in 20% serum
oxidatively from glucose whereas A. calcoaceticus broth. Colonies appear on the surface after 48 hours
var. lwoffii does not. incubation and are discrete granular and greyish
yellow. L phase variants show minute colonies
A. baumannii (0.1–0.2 mm diameter) with a fried egg appearance.
They consist mainly of small coccoid bodies.
These form pinkish colonies on MacConkey medium.
Acid without gas is formed in glucose, arabinose, Biochemical Reactions
xylose, and occasionally in rhamnose. A characteristic
It attacks sugars fermentatively and produces
reaction is the formation of acid in 10%, but not 1%
acid without gas from glucose, galactose, dextrin,
lactose. Several serotypes have been identified by
raffinose and starch. It is catalase, oxidase, indole
capsule swelling and immunofluorescence.
production, urease and nitrate reduction negative.
A. lowffi Pathogenesis
This forms yellow colonies on MacConkey medium In humans, it causes streptobacillary rat-bite fever.
and does not acidify sugars. Some strains are Another type of clinically indistinguishable, rat-bite
oxidase positive. fever is caused by Spirillum minus.
www.ebook3000.com
Chap-50.indd 359 15-03-2016 11:20:04
360 | Section 3: Systemic Bacteriology
human beings. It is an opportunistic pathogen that are hemolytic on human or rabbit blood agar. It is
causes infections a human bite wound or fist fight catalase, oxidase, indole and urease negative.
injury, endocarditis, sinusitis, meningitis, brain G. vaginalis is considered responsible for
abscesses, pneumonia, and lung abscesses. bacterial vaginosis, a mild but common condition
characterized by raised vaginal pH > 4.5, foul smelling
Treatment discharge and the presence of ‘clue cells’, which are
E. corrodens is susceptible to penicillin, ampicillin, vaginal epithelial cells with their surface studded
extended-spectrum cephalosporins, tetracyclines, with numerous small bacteria. Bacterial vaginosis is
and fluoroquinolone. also associated with anaerobic bacteria, particularly
Mobiluncus. Metronidazole is effective in treatment.
CARDIOBACTERIUM HOMINIS
Key Points
This gram-negative bacilli are nonmotile, char
Listeria monocytogenes is a small, coccoid, Gram-
acteristically small (1 × 1–2 mm) but sometimes
positive bacillus and exhibits a characteristic, slow,
pleomorphic. The organism grows slowly in culture. tumbling motility when grown at 25 °C but at 37 °C
On blood agar the colonies are small, 1–2 mm in is nonmotile
diameter, smooth, glistening, circular and opaque The disease chiefly affects pregnant women, unborn
after 48 hours incubation at 35°C. The organism or newly delivered infants, the immunosuppressed
does not grow on MacConkey agar or other selective and elderly. It is predominantly transmitted by the
media commonly used for gram-negative bacilli. consumption of contaminated food
The bacteria are fermentative, indole- and oxidase- Erysipelothrix rhusiopathiae: Disease is common in
swine but rare in humans. Three forms of human
positive, and catalase and nitrate negative.
infection-erysipeloid, generalized cutaneous form
C. hominis occurs commonly as a commensal and septicemia
in the human nose and throat may cause endo Alcaligenes faecalis: It causes urinary tract infection,
carditis, particularly in those with pre-existing infantile gastroenteritis, and typhoid-like fever in
cardiovascular disease. humans
It can be diagnosed by isolation of the causative Chromobacterium violaceum: It is associated
with intestinal and genitourinary infections and
agent in blood culture.
septicemic illnesses with pneumonia
C. hominis is susceptible to multiple antibiotics, Flavobacterium meningosepticum: causes oppor
and most infections are successfully treated with tunistic infections, neonatal meningitis in premature
penicillin or ampicillin for 2–6 weeks. infants and pneumonia in immunocompromised
hosts
CAPNOCYTOPHAGA Calymmatobacterium (Donovania) granulomatis
is the causative agent of granuloma inguinale, a
The Capnocytophaga species are gram-negative, sexually transmitted disease
slow-growing, capnophilic, fusiform or filamentous Acinetobacter species Acinetobacter species may
bacilli. They are fermentative and facultative cause nosocomial infections
anaerobes that require CO2 for aerobic growth. They Rat bite fever is caused by two different bacilli:
may show gliding motility which can be seen as Streptobacillus moniliformis and Spirillum minus
outgrowths of colonies. Eikenella corrodens: Causes a human bite wound
or fist fight injury, endocarditis, sinusitis, meningitis,
C. ochracea, C. gingivalis and C. sputigena are
brain abscesses, pneumonia, and lung abscesses
members of the normal oral flora of humans. They
Cardiobacterium hominis: C. hominis may cause
have been associated with severe periodontal disease endocarditis, particularly in those with preexisting
in juveniles. They occasionally cause bacteremia and cardiovascular disease
severe systemic disease in immunocompromised Capnocytophaga species have been associated
patients, especially granulocytopenic patients with with severe periodontal disease in juveniles,
oral ulcerations. bacteremia and severe systemic disease in immuno
Most Capnocytophaga infections can be compromised patients
treated with broad-spectrum cephalosporins, Gardnerella vaginalis is considered responsible for
bacterial vaginosis, a mild but common condition
fluoroquinolones, or penicillin. characterized by raised vaginal pH >4.5, foul
smelling discharge and the presence of clue cells’.
GARDNERELLA VAGINALIS Metronidazole is effective in treatment.
Gardnerella vaginalis is a small, gram-negative,
nonmotile, pleomorphic rod which shows IMPORTANT QUESTION
metachromatic granules. It was formerly known as Write short notes on:
Haemophilus vaginalis or Corynebacterium vaginale. a. Listeria monocytogenes
It grows on blood or chocolate agar aerobically under b. Elysipelothrix rhusiopathiae
5% CO2, minute colonies appear in 24–48 hours and c. Donovan bodies
www.ebook3000.com
Chap-50.indd 361 15-03-2016 11:20:04
51
Chapter
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss the laboratory diagnosis of rickettsial
be able to: infections
∙∙ Describe diseases caused by different rickettsiae ∙∙ Describe Weil–Felix reaction
∙∙ Describe Brill–Zinsser disease ∙∙ Discuss Q fever, Trench fever and Oroya fever.
www.ebook3000.com
Chap-51.indd 363 15-03-2016 11:20:55
364 | Section 3: Systemic Bacteriology
dead end. Man to man transmission does not fever group is widespread vasculitis involving the
occur. In Kashmir and China, lice have been known skin (with production of a rash) and internal organs
to transmit endemic typhus in human beings, (producing dysfunctions of the brain, heart, lungs,
producing smouldering outbreaks. Endemic and kidneys).
typhus is worldwide in prevalence.
Differences between R.typhi (R. mooseri) and Tick Typhus
R. prowazekii Rocky Mountain Spotted Fever
R.typhi and R. prowazekii are closely similar Rocky mountain spotted fever (RMSF) is a potent
but may be differentiated by biological and ially life-threatening infection. R. rickettsii is the
immunological tests. etiologic agent of Rocky Mountain spotted fever.
i. Neill–Mooser or the tunica reaction
When male guinea pigs are inoculated Vector: Ixodid (hard) ticks are the vectors. It is
intraperitoneally with blood from a case of transmitted by Dermacentor andersoni and related
endemic typhus or with a culture of R. typhi, species of ticks. The ecology and epidemiology of
they develop fever and a characteristic scrotal spotted fever are directly related to the life cycle of
inflammation. The scrotum becomes enlarged four species of ixodid (hard) ticks vectors. Humans
and the testes cannot be pushed back into the are only accidentally infected.
abdomen because of inflammatory adhesions Clinical diseases: The incubation period is about
between the layers of the tunica vaginalis. This one week. Clinical picture is similar to that of
is known as the Neill–Mooser or the tunica typhus fever but the rash appears earlier and is
reaction. The Neill-Mooser reaction is negative more pronounced. It is prevalent in many parts of
with R. prowazekii. North and South America.
ii. Other methods used include-IFA, ELISA and
PCR based DNA tests. Other Spotted Fever Rickettsiae (Table 51.2)
R. siberica causes the Siberian tick typhus which
B. Spotted Fever Group is mild rickettsial disease. Boutonneuse fever is
They are all transmitted by ticks, except R. akari, caused by R. conorii. R. conori strains are isolated
which is mite-borne. Rickettsiae of this group from the Mediterranean littoral; Kenya, South
possess a common soluble antigen and multiply Africa and India are indistinguishable. Australian
in the nucleus as well as in the cytoplasm of host tick typhus is caused by R. australis. All these
cells. The basic pathologic process in the spotted three rickettsiae are maintained in nature in ixodid
www.ebook3000.com
Chap-51.indd 365 15-03-2016 11:20:55
366 | Section 3: Systemic Bacteriology
i. Weil–Felix (WF) reaction Prophylaxis
The Weil–Felix reaction is an agglutination test
1. General measures: General measures, such
in which sera are tested for agglutinins to the
as control of vectors and animal reservoirs are
a-antigens of certain nonmotile Proteus strains OX
useful to prevent rickettsial diseases.
19, OX 2 and OX K.
2. Vaccination: Immunization is useful in special
Basis of the test: The basis of the test is the sharing situations. There is no safe, effective vaccine for
of an alkali-stable carbohydrate antigen by some any of the rickettsial diseases.
rickettsiae and by certain strains of Proteus, P.
i. Weigl’s vaccine: In earlier days, phenolized
vulgaris OX 19, and OX 2 and P. mirabilis OX K.
intestinal contents of lice infected per
Procedure: The test may be performed as a micro- rectum with R. prowazekii (Weigl’s vaccine)
agglutination reaction in microtiter plates with was developed. It was too complicated for
round bottomed wells with hematoxylin-stained mass production.
antigen or as a tube agglutination test, though rapid
ii. A live vaccine: Containing attenuated E.
slide agglutination methods have been employed
strain of R. prowazekii grown in yolk sac have
for screening.
been found to be effective but a proportion
of vaccines develop mild disease.
Interpretation
iii. Formalin inactivated R. rickettsii has
Sera from epidemic and endemic typhus aggluti
been used but does not prevent the disease
nate OX 19 and sometimes OX 2 also. The test is
completely.
negative or only weakly positive in Brill–Zinsser
disease. In tick-borne spotted fever both OX 19 and iv. Recombinant vaccines may be more
OX 2 are agglutinated. OX K agglutinins are found successful.
only in scrub typhus. (Table 51.3).
The Weil–Felix reaction is a simple and useful EHRLICHIA, ANAPLASMA AND
test for the diagnosis of some rickettsial diseases. NEORICKETTSIA
The antibody appears rapidly during the course of
the disease, reaches peak titers of up to 1 : 1000 or Ehrlichia and Anaplasma are small, obligate
1 : 5000 by the second week and declines rapidly intracellular, gram-negative bacteria. They infect
during convalescence. False-positive reaction may circulating leukocytes, erythrocytes, and platelets,
occur in some cases of urinary or other infections where they multiply within phagocytic vacuoles,
by Proteus and at times in typhoid fever and liver forming clusters with inclusion-like appearance.
diseases. Hence it is desirable to demonstrate a rise These clusters of ehrlichiae resemble mulberries
in titer of antibodies for the diagnosis of rickettsial and are called morulae (Latin morum = mulberry).
infection.
Species
ii. Specific tests using rickettsial antigens
Serological methods using rickettsial antigens are The ehrlichiae that cause disease in humans have
specific, which include complement fixation test, been classified in a limited number of species
latex agglutination test and enzyme immunoassay. which are as follows: (i) Ehrlichia chaffeensis,
causes human monocyte ehrlichiosis (HME); (ii)
Ehrlichia ewingii causes E ewingii ehrlichiosis;
Treatment (iii) Anaplasma phagocytophilum causes human
Rickettsial infections may be treated with tetracyc granulocyte anaplasmosis (HGE) (Table 51.4). The
lines or chloramphenicol. Both these drugs are same genera contain additional species that infect
rickettsiostatic. animals but apparently not humans. The human
pathogens in the group have animal reservoirs and
Table 51.3 Weil–Felix reaction in rickettsial diseases can cause disease in animals as well.
Disease Agglutination pattern with
1. Ehrlichia chaffeensis
OX 19 OX2 OXK
It is tick-borne ehrlichiosis. Deer and rodents are
1. Epidemic typhus +++ + –
believed to be reservoir hosts. Ehrlichia chaffeensis
2. Brill–Zinsser disease usually or weak causes human monocyte ehrlichiosis (HME). E
negative positive
chaffeensis frequently causes severe or fatal illness.
3. Endemic typhus +++ +/- –
4. Tickborne spotted ++ ++ – 2. Ehrlichia ewingii
fever
E. ewingii has a geographic distribution similar to
5. Scrub typhus – – +++
E. chaffeensis. Ehrlichia ewingii, causes E. ewingii
ehrlichiosis; It is transmitted by ticks. Leukopenia year or more at 4°C and in meat at least for one
and thrombocytopenia are seen in patients. month. It is not completely inactivated at 60°C or
by 1% phenol in one hour. In milk it may survive
3. Anaplasma phagocytophilum pasteurization by the holding method, but the
It infects human granulocytic cells. Anaplasma flash method is effective. It can survive in dust
phagocytophilum, causes human granulocyte and aerosols, therefore, can be transmitted as an
anaplasmosis (HGE). The human pathogens in airborne infection. It can be inactivated by 2%
the group have animal reservoirs and can cause formaldehyde, 5% hydrogen peroxide and 1% lysol.
disease in animals as well
Antigenic Variation
4. Neorickettsia sennestu An important characteristic of Coxiella infections
It was previously called Ehrlichia sennestu, an is the ability to undergo antigenic variation in
intraleukocytic bacterium. It causes sennestu expression of the cell wall LPS antigen. Cox. bumetii
ehrlichiosis, an illness resebling glandular fever shows phase variation. Fresh isolates are in Phase I.
(sennetsu being the Japanese name for glandular It becomes Phase II on repeated passage in yolk sac,
fever). No insect vector has yet been implicated in its but reverts to Phase I by passaging in guinea pigs.
transmission. It is associated with ingestion of raw fish
infested with infected flukes. Similar cases have been Pathogenesis
reported in Malaysia and Philippines. Coxiella burnetii causes Q fever which has a world
wide distribution, as a zoonosis solidly established
Laboratory Diagnosis in domestic livestock.
1. Identification of characteristic morulae: Typical Cycles of infection: In nature there are two cycles
morulae in white blood cells is diagnostic in of infection of C. burnetii. One involves arthropods
Giemsa stained peripheral blood smear. (especially ticks) and a variety of vertebrates. The
2. Specific antibodies can be demonstrated by other cycle is maintained among domestic animals
indirect immunofluorescence methods. (cattle, sheep and goats).
3. Polymerase chain reaction (PCR) for ehrlichial
DNA. Reservoirs of the disease: The primary reservoirs
of the disease are wild and domestic ungulates,
including cattle, sheep, goats, rabbits, cats and dogs.
GENUS COXIELLA: Q FEVER It is transmitted among them and to cattle, sheep
The name Q fever (Q for “query”) was first used when and poultry by ixodid ticks.
Derrick (1935) was investigating an outbreak of Animal infection: Domestic animals have
typhoid-like fever in abattoir workers in Australia. Q inapparent infections but may shed large quantities
fever is caused by Coxiella burnetit. Coxiella burnetii, of infectious organisms in their urine, milk, feces,
is an obligately intracellular prokaryote. and especially, their placental products.
www.ebook3000.com
Chap-51.indd 367 15-03-2016 11:20:56
368 | Section 3: Systemic Bacteriology
pneumonia, hepatitis, pericarditis, myocarditis, Pathogenesis
meningoencephalitis. Chronic diseases include Oroya fever: Bartonella bacilliformis is responsible
endocarditis, hepatitis, pulmonary disease, and for bartonellosis, an acute febrile illness consisting
infection of pregnant. of severe anemia (Oroya fever) followed by a
chronic cutaneous form (verruga). Oroya fever
Laboratory Diagnosis presents as fever and progressive anemia due to
1. Isolation: Isolation of C. burnetii from patient bacterial invasion of erythrocytes. Mortality is high
specimens is a specialized procedure and is in untreated cases.
not generally recommended because of the
Verruga peruana: Some of the survivors developed
extremely infectious nature of the organism.
nodular ulcerating skin lesions, called verruga
2. Serology: Using complement fixation test
peruana. It is a late sequel in survivors or in those
(CFT) or indirect immunofluorescence assay.
with asymptomatic infection.
Treatment: Tetracyclines are the drugs of choice
for acute infections. Rifampin combined with either Carrion’s disease: Oroya fever and verruga
doxycycline or trimethoprim-sulfamethoxazole is peruana are also known as Carrion’s disease.
used to treat chronic infections. Etiology: It is spread by the sandfly vector Phlebo
tomus.
Prophylaxis
Prevention of disease is by observing basic hygienic Laboratory Diagnosis
precautions while dealing with infected animals.
i. B. bacilliformis can be demonstrated in blood
Pasteurization of raw milk helps prevent milk-
smears stained by Giemsa. Organisms are
borne transmission. Vaccines have been developed
seen in the cytoplasm as well as adhering to
from formalin killed whole cells.
the cell surfaces.
BARTONELLA ii. Organisms may be isolated from the blood,
in semi-solid medium containing rabbit
Family Bartonellaceae contains two genera: serum and hemoglobin. Identification can be
Bartonella and Grahamella. achieved by PCR or by cultural and serological
Members of genus Bartonella are very small tests.
gram-negative bacilli. The genus Bartonella, which iii. Guinea pig inoculation leads to verruga
now consists of 11 species, including 5 that cause peruana but not Oroya fever.
human disease (Table 51.5). Members of the iv. Other tests
genus Grahamella preferentially grow within the a. Polymers chain reaction (PCR)
erythrocytes of vertebrates, but do not infect humans.
b. Serology: Indirect hemagg lutination,
indirect immunofluorescence and ELISA.
1. Bartonella bacilliformis
It is a pleomorphic gram-negative rod, which is Treatment: B. bacilliformis is susceptible to
motile by a tuft of polar flagella. It can be cultivated in penicillin, streptomycin, tetracycline and chloram
semisolid agar with rabbit or human blood. Growth phenicol.
is slow and becomes visible after 10 days incubation. Prophylaxis: Insecticides such as DDT should be
used to eliminate the sandfly inside and outside
Table 51.5 Bartonella species associated with the houses.
human infections
Species Diseases
2. Bartonella quintana
1. B. bacilliformis Oroya fever (bartonellosis, Carrion’s B. quintana is a small, gram-negative bacillus. It
disease) does not possess flagella, although it may exhibit
2. B. qintana Trench fever; cutaneous, subcutaneous,
twitching movement caused by fimbriae. It grows
and osseous manifestations of bacillary slowly on rabbit or sheep blood agar at 35°C in
angiomatosis; endocarditis 5% CO2 in air. Colonies appear after two weeks
3. B. henselae Cat-scratch disease; bacteremia; in primary culture and 3–5 days in subsequent
cutaneous, lymphatic, and passages.
hepatosplenic (peliosis hepatis)
manifestations of bacillary
angiomatosis; endocarditis
Pathogenesis
4. B. clarridgeiae Endocarditis, cat-scratch disease (rare)
Trench fever or five-day fever: B. quintana
causes trench fever or five-day fever occurred
5. B. elizabethae Endocarditis (rare)
among soldiers fighting in the trenches in Europe
www.ebook3000.com
Chap-51.indd 369 15-03-2016 11:20:56
370 | Section 3: Systemic Bacteriology
3. Human body louse is responsible for transmission
Ehrlichia of which of the following diseases?
These tick-borne bacteria cause three human a. Epidemic typhus b. Murine typhus
infections: E sennetsu causes a type of glandular fever; c. Rickettsial pox d. Q fever
E chaffeensis causes human monocytic ehrlichiosis,
4. Mites are responsible for transmission of diseases:
E phagocytophila causes human granulocytic
ehrlichiosis a. Epidemic typhus b. Murine typhus
c. Scrub typhus d. Trench fever
Coxiella burnetii
5. The rickettsial disease transmitted by ixodid ticks
It is small, pleomorphic coccobacillary bacterium is:
with a gram-negative cell wall, intracellular bacteria
a. Rickettsial pox:
Disease: Q fever is a worldwide zoonosis. Most
b. Rocky Mountain spotted fever
disease acquired through inhalation; possible
c. Trench fever
disease from consumption of contaminated milk
d. Scrub typhus
Bartonella 6. Weil-Felix reaction is positive in all the following
The genus Bartonella contains B. bacilliformis, diseases except:
B. quintana and B. henselae which cause Oroya a. Epidemic typhus b. Endemic typhus
fever, trench fever and cat scratch disease in man
c. Brill–Zinsser’s disease d. Q fever
respectively.
7. Coxiella differs from rickettsial pathogens as it:
a. Has typical Gram-negative cell walls
IMPORTANT QUESTIONS b. Contains both DNA and RNA
c. Is susceptible to antibiotics
1. Write short notes on: d. Is transmitted by inhalation or ingestion
a. Typhus fevers 8. Human infections due to Coxiella can be acquired
b. Brill-zinsser disease (Recrudescent typhus) by:
c. Rocky mountain spotted fever a. Consumption of infected milk.
d. Scrub typhus b. Handling of infected wool or hides.
e. Trench fever c. Soil contaminated with feces of infected ani-
f. Coxiella burnettii or Q fever mals
g. Weil-Felix reaction d. All of the above
h. Cat scratch disease 9. Oroya fever is caused by:
i. Neil–Mooser reaction or Tunica reaction a. Capnocytophages ochracea
j. Ehrlichia b. Legionella pneumophilia
k. Oroya fever c. Streptobacillus moniliformis
2. Discuss laboratory diagnosis of rickettsial infections. d. Bartonella bacilliformis
10. The causative agent of cat scratch disease is:
a. Bartonella henselae
MULTIPLE CHOICE QUESTIONS (MCQs) b. Bartonella quintana
1. Which of the following bacteria does not require c. Coxiella burnettii
an arthropod vector for its transmission? d. Rickettsia prowazekii
a. Coxiella burnetii 11. All the statements are true for Bartonella quintana
b. Rickettsia akari except:
c. Bartonella quintana a. It is nonmotile
d. Rickettsia prowazekii b. It requires 1–2 weeks to produce demonstrable
2. The causative agent of epidemic typhus is: colonies on rabbit blood agar
a. Rickettsia prowazekii c. It is transmitted by hard ticks
b. Rickettsia rickettsii d. It is the causative agent of trench fever
c. Coxiella burnettii ANSWERS (MCQs)
d. Rickettsia akari 1. a; 2. a; 3. a; 4. c; 5. b; 6. d; 7. d; 8. d; 9. d; 10. a; 11. c
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss morphology and the unique growth cycle
be able to: of Chlamydia, describing elementary body (EB) and
∙∙ Describe differences between chlamydiae and viruses reticulate body (RB) bodies
∙∙ Describe serotypes of various chlamydiae and ∙∙ Discuss laboratory diagnosis of chlamydial infections
diseases produced by them ∙∙ Describe the following: TRIC agents, inclusion
∙∙ Associate each of the major diseases with the three conjunctivitis lymphogranuloma venereum (LGV),
most important human species of Chlamydia and Frei’s test.
Chlamydophila
INTRODUCTION CLASSIFICATION
Chlamydiaceae is a family of obligate intracellular Genus Chlamydia is in the order Chlamydiales
bacterial parasites, small, nonmotile and gram- and the family Chlamydiaceae. The proposed new
negative with a tropism for columnar epithelial taxonomic classification for the family Chlamy
cells lining the mucous membranes. diaceae consists of two genera: (1) Chlamydia to
Based on the human diseases they were then include C. trachomatis and (2) Chlamydophila to
known to cause, they were called psittacosis- include C. pneumoniae, C. psittaci, and C. pecorum.
lymphogranuloma-trachoma (PLT) viruses, or Other species have been placed into the two genera,
noncommittally as ‘PLT agents’ or TRIC (trachoma- but they are uncommon human pathogens and are
inclusion conjunctivitis) organisms. not discussed in this chapter (Table 52.1).
Chlamydia Species
Differences between Chlamydiae and Chlamydia trachomatis: C. trachomatis is divi
Viruses ded into two biovars—those causing trachoma,
The Chlamydiaceae were once considered viruses. inclusion conjunctivitis (the so-called TRIC agents)
However, they differ from viruses in many respects and oculogenital infection.
and the organisms have the following properties
Chlamydophila Species
of bacteria:
1. Chlamydophila psittaci: It may lead to severe
1. They possess inner and outer membranes and sometimes fatal pneumonia in man (psittacosis
similar to those of gram-negative bacteria. or ornithosis).
2. They contain both DNA and RNA.
Table 52.1 Revised classification of the family
3. They possess prokaryotic ribosomes.
Chlamydiaceae
4. They synthesize their own proteins, nucleic
acids, and lipids. Genus Chlamydia Genus Chlamydophila
5. They multiply by binary fission. C. trachomatis C. pneumoniae
C. muridarum C. psittaci
6. They do not have ‘eclipse phase’ following C. suis C. pecorum
cellular infection. C. abortus
7. They are susceptible to numerous antibacterial C. caviae
antibiotics. C. felis
www.ebook3000.com
Chap-52.indd 371 15-03-2016 11:21:32
372 | Section 3: Systemic Bacteriology
2. Chlamydophila pneumoniae: It is a common Reticulate body: The reticulate body is the intra
cause of acute respiratory disease worldwide (Table cellular growing and replicative form, 500–1000 nm
52.2). in size (larger than the EB).
The chlamydiae are nonmotile, lacking flagella,
Morphology and nonpiliated.
www.ebook3000.com
Chap-52.indd 373 15-03-2016 11:21:33
374 | Section 3: Systemic Bacteriology
organisms are acquired from the mother during sole natural hosts of this infection caused by the
birth. The disease can be prevented by local LG V biovar of C. trachomatis, Ll, L2 and L3 most
application of antibiotics. commonly L2. (Table 52.2).
b. Inclusion conjunctivitis: Inclusion con
junctivitis (paratrachoma) is most prevalent in Clinical Manifestations
sexually active young people, being spread from The usual incubation period of LGV is 1–4 weeks.
genitalia to the eye and occurs worldwide. It
1. Primary lesion: The primary lesion is
was known as ‘swimming pool conjunctivitis’
painless, small, inconspicuous, and vesicular
as infection was associated with bathing in
and often escapes notice. Characteristically the
community swimming pools which presumably
presenting complaint concerns the enlarged
get contaminated with chlamydia from the
matted inguinal and femoral lymph nodes
genital secretions of bathers. Contamination of
which are moderately painful and firm and may
the eye with the patient’s own genital secretion
become fluctuant.
may be the cause more often. The disease is
much milder, is usually self-limiting and rarely 2. Secondary stage: The secondary stage results
causes visual loss, presumably because, unlike from lymphatic spread to the draining lymph
trachoma, repeated infection is less common. nodes. In men the inguinal lymph nodes
are involved most often and in women the
2. Genital Infections intrapelvic and pararectal nodes. Women and
homosexual men may develop hemorrhagic
C. trachomatis infections of the genital tract are of proctitis with regional lymphadenitis. The
two types and are sexually transmitted: nodes enlarge, suppurate, become adherent
1. Those caused by the oculogenital serotypes to the skin and breakdown to form sinuses
D through K collectively referred to as ‘genital discharging pus. Metastatic complications may
chlamydiasis’ sometimes occur, with involvement of joints,
2. LGV caused by serotypes L1, L2, L2a and L3. eyes, and meninges.
3. Tertiary stage: The tertiary stage is chronic,
I. Genital chlamydiasis lasting for several years, representing the
Ch. trachomatis D to K are called genital chlamy sequelae of scarring and lymphatic blockage.
diasis. Rectal stricture and rectal perforation are
recognized late sequelae to LGV proctitis. The
i. Infection in men: In men, they cause urethritis
course of the disease is variable. Late sequelae
(nongonococcal urethritis), epididymitis,
are more distressing in women and lymphatic
proctitis, conjunctivitis and Reiter’s syndrome.
obstruction in women can lead to elephantiasis
Reiter’s syndrome is a triad of recurrent
of the vulva, called esthiomene.
conjunctivitis, polyarthritis and urethritis
or cervicitis. Anal intercourse may cause
chlamydial proctitis in either sex. Associated Laboratory Diagnosis
symptoms include rectal pain and bleeding, The primary lesion usually goes unnoticed and
mucopurulent discharge and diarrhea. the disease is seen commonly first in the stage of
ii. Infection in women: Most genital tract inguinal adenitis (bubo).
infections in women are asymptomatic (as
many as 80%) but can nevertheless become A. Microscopy
symptomatic. The clinical manifestations
Smears of material aspirated from the bubos
include acute urethral syndrome, bartholinitis,
may show the elementary bodies (Miyagawa’s
mucopurulent cervicitis, endometritis,
granulocorpuscles). The sensitivity of microscopic
salpingitis, pelvic inflammatory disease (PID),
diagnosis is very low.
conjunctivitis, perihepatitis (Fitz–Hugh–
Curtis syndrome) and Reiter’s syndrome.
Genital chlamydiasis may cause infertility, B. Culture
ectopic pregnangy, premature deliveries, Isolation of the Chlamydia by intracerebral
perinatal morbidity and postpartum fever. inoculation into mice and into yolk sac of eggs has
been replaced by cell cultures.
II. Lymphogranuloma Venereum
Lymphogranuloma inguinale, climatic bubo, C. Serology
tropical bubo, and esthiomene are synonyms of LGV patients develop high titers of circulating
this sexually transmissible disease. It characterised antibodies, with titers of 1:64 or more in CF test
by suppurative inguinal adenitis. Humans are the and 1:512 or more in micro-IF.
www.ebook3000.com
Chap-52.indd 375 15-03-2016 11:21:33
376 | Section 3: Systemic Bacteriology
used in PCR assays to detect organisms and appear characteristic inclusions can be found in yolk sac
to be more sensitive than cell culture. material from infected 6–8 day embryos. However,
this procedure is impractical for use by clinical
Treatment laboratories since it is tedious, time-consuming,
Macrolides (erythromycin, azithromycin, clarithro and less sensitive than tissue culture.
mycin), tetracyclines (tetracycline, doxycycline), or b. Animal inoculation
levofloxacin administered for 10–14 days has been Chlamydiae can be propagated in experimental
used to treat infections. animals such as mice. C. psittaci strains infect
Control of exposure to C. pneumoniae is likely mice by intracerebral, intranasal, intraperitoneal
to be difficult because the bacterium is ubiquitous. and subcutaneous routes. Among C. trachomatis
strains, only the LGV serovars (Ll, L2, L3) infect
Laboratory Diagnosis of Chlamydia mice when injected intracerebrally, but other C.
Infections trachomalis strains (The TRIC serovars) will not
The laboratory diagnosis of Chlamydia infections infect mice by any route of infection.
can be acc omplished by various approaches: c. Tissue culture
(1) microscopic demonstration of inclusion or The most widely used method of cultivation is by
elementary bodies, (2) isolation of the organisms, the use of tissue cultures. Monkey kidney cell lines,
(3) demonstration of chlamydial antigen and McCoy and HeLa cell lines and, more recently, the
(4) detection of specific antibodies (5) Skin test- heteroploid HL cell line are commonly used.
Hypersensitivity against these bacteria.
Cell cultures used for isolation are pretreated
The specimens collected are conjunctival by irradiation or chemicals such as 5-iodo-2-
scrapings, sputum, throat swab, bubo pus, genital deoxyuridine, cytochalasin B, or cycloheximide to
swabs and blood. enhance chlamydial replication and to allow easier
1. Demonstration of Inclusion or Elementary recognition of inclusions. Pretreatment of the cells
with diethylaminoethyldextran (DEAE-dextran)
Bodies
or centrifugation of the chlamydiae onto the host
i. Light microscopy: Smears may be prepared cells increases contact between the infectious
from conjunctival scrapings or bubo pus and chlamydial particles and the host cell monolayer,
stained by Giemsa, Castaneda, Machiavello or with a subsequent increase in infectivity.
Giminez stains to demonstrate characteristic
LGV strains grow well, while TRIC strains are
inclusion bodies under light microscope.
less infective. C. psittaci can be isolated from
Chlamydia are gram-negative but are stained
infected material by methods similar to those used
better by, Castaneda, Machiavello or Giminea
for other Chlamydia.
stains. Chlamydial elementary bodies and
Propagation of C. pneumoniae in cell culture
inclusions are large enough to be seen under
has proven to be difficult. HeLa 229 cells and, more
the light microscope.
recently, the heteroploid HL cell line have been
Glycogen-containing inclusions of C. tracho
described as being more sensitive than McCoy cells
matis can be stained with Lugol’s iodine. The
for the isolation of this organism.
inclusion bodies of trachoma and inclusion
conjunctivitis are named Halberstaedter 3. Demonstration of Chlamydial Antigen
Prowazek or HP bodies whereas the elementary a. Immunofluorescence (IF): For diagnosis by
bodies of C. psittaci are called Levinthal Cole demonstration of chlamydial antigens, the
Lillie or LCL bodies. method commonly used is micro-IF. This test
ii. Immunofluorescence (IF): Immunofluore approaches cultures in sensitivity.
scence (IF) can identify not only inclusions b. ELISA : The ELISA method is preferred for
but also extracellular elementary bodies. screening as it enables rapid testing for LPS
Besides ocular infections, IF is useful also in antigen in large numbers of specimens.
examination of cervical or urethral specimens, c. Molecular methods: Molecular methods like
which may contain elementary bodies. DNA probes and amplification techniques
(polymerase chain reaction, ligase chain
2. Isolation of the Organisms reaction) have greatly increased the sensitivity
Traditionally the isolation of Chlamydia has been and specificity of antigen detection.
accomplished by the inoculation of infected material
into either embryonated eggs, experimental 4. Detection of Specific Antibodies or
animals, or selected tissue culture cell lines. Hypersensitivity Response
a. Yolk sac inoculation The diagnosis of chlamydial infections can
All known strains of Chlamydia will infect the be accomplished by either the group-specific
chick embryo, and group-specific antigen and complement-fixation (CF) test or type-specific
www.ebook3000.com
Chap-52.indd 377 15-03-2016 11:21:33
Section 4: Virology
5353
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ List various cell cultures
be able to: ∙∙ Discuss detection of virus growth in cell cultures
∙∙ Describe size, shape and symmetry of viruses ∙∙ List DNA and RNA viruses
∙∙ Describe cultivation of viruses ∙∙ Describe prions and viroids.
3. Complex Symmetry
Viruses (e.g. poxviruses) which do not show either
icosahedral or helical symmetry due to complexity
of their structure are referred to have complex
symmetry.
A B
C. Viral Envelope
Virions may be enveloped or nonenveloped
(naked). Enveloped Virus: The envelope or
outer covering of virus containing lipid is derived
from the plasma membrane of the host cell during
their release by budding from the cell surface. The
envelope is glycoprotein in nature. The lipid is largely
C D of host cell origin while the protein is virus-encoded.
Fig. 53.1: Schematic diagram illustrating the components of Peplomers: In mature virus particle, the glyco
the complete virus particle (the virion). (A) Naked icosahedral proteins often appear as projecting spikes on the
virus, consisting of an inner core of nucleic acid enclosed by a outer surface of the envelope. These are known as
capsid, which is made of capsomers; (B) Enveloped virus; (C)
Naked helical virus composed of capsomers wound round the
peplomers. A virus may have more than one type
nucleic acid to form a tubular structure; (D) Envelope helical of peplomers, e.g. the influenza virus carries two
virus in which the tubular capsid is pliable and is enclosed kinds of peplomers, the hemagglutinin which is
within an envelope a triangular spike and the neuraminidase which
www.ebook3000.com
Chap-53.indd 379 15-03-2016 11:33:19 AM
380 | Section 4: Virology
Fig. 53.2: Shapes and relative sizes of animal viruses of families that infect vertebrates
is a mushroom-shaped structure. Envelope confer information necessary for replication of the virus.
chemical, antigenic and biological properties on The genome may be single-stranded or double-
viruses. stranded, circular or linear, and segmented or
nonsegmented. The type of nucleic acid, its
Functions of Peplomers strandedness, and its size are major characteristics
i. Mediate attachment of the virus to the host- used for classifying viruses into families.
cell receptors.
ii. Attach to receptors on red blood cells.
Susceptibility to physical and
iii. Enzymatic activity chemical agents
iv. Major antigens: For protective immunity. 1. Heat and Cold
i. Heat-labile: With few exceptions, viruses are
D. Viral Nucleic Acids very heat-labile. They are inactivated within
Viruses contain a single kind of nucleic acid— seconds at 56°C, minutes at 37 °C and days at
either DNA or RNA—that encodes the genetic 4°C.
Viral hemagglutination
A large number of viruses contain hemagglutinin
spikes (peplomers) on the capsid or envelope
which can agglutinate red cells of different Fig. 53.3: Viral hemagglutination. Virus containing fluid is
diluted in doubling dilutions and 0.5% suspension of chick
species. Hemagglutinin of influenza virus is due
red cells added. There is diffuse widespread even pattern
to the presence of hemagglutinin spikes on the on the bottom of the wells in the plastic plate where virus is
surface of the virus. When RBCs are added to present. The cells settle down to a button like aggregate with
serial dilutions of viral suspension, the RBCs and sharp edges where no virus is present
www.ebook3000.com
Chap-53.indd 381 15-03-2016 11:33:21 AM
382 | Section 4: Virology
synthetic machinery of the host cell for replication. ii. Translation of the mRNA into ‘early
The viral multiplication cycle can be divided proteins’. These are enzymes which initiate
into six sequential phases, though the phases and maintain synthesis of virus components.
may sometimes be overlapping: (1) adsorption They may also induce shut down of host
or attachment, (2) penetration, (3) uncoating, protein and nucleic acid synthesis.
(4) biosynthesis, (5) maturation, and (6) release. iii. Replication of viral nucleic acid.
iv. Synthesis of ‘late’ or structural proteins,
1. Adsorption and Absorption which are the components of daughter virion
Virions come in contact with cells by random capsids.
collision but adsorption or attachment is specific Viruses have been categorized into six classes
and is mediated by the binding of virion surface by Baltimore (1970) based on thier replication
structures, known as ligands, to receptors on mechanisms (Table 53.2).
cell surface. In case of influenza virus, a surface
glycoprotein (the hemagglutinin) binds specifically 5. Maturation
to sialic acid residue of glycoprotein receptor sites Newly synthesized viral genomes and capsid
on the surface of respiratory epithelium. In case polypeptides assemble together to form progeny
of human immunodeficiency virus-1 (HIV1), viruses. Assembly of the various viral components
surface glycoprotein gp120 acts as a ligand. It binds into virions occurs shortly after the replication
to the CD4 60 kDa glycoprotein on the surface of of the viral nucleic acid and may take place in
mature T lymphocytes. either the nucleus (herpes and adenoviruses) or
cytoplasm (picorna and poxviruses). In case of
2. Penetration enveloped viruses, the envelopes are derived from
After binding, the virus particle is taken up inside Table 53.2 Baltimore classification of viruses
the cell.This is accomplished by receptor-medi based on replication
ated endocytosis (viropexis), with uptake of
the ingested virus particles within endosomes. Class
Enveloped viruses fuse their membranes with 1. Double stranded DNA viruses: In the case of fully
cellular membranes to deliver the nucleocapsid double stranded DNA viruses, the DNA enters the
or genome directly into the cytoplasm. host cell nucleus and uses the host cell enzymes
for transcription.
Examples: (i) Hepadnaviruses, (ii) Poxviruses
3. Uncoating 2. Single stranded DNA viruses: The DNA molecule
This is the process of stripping the virus of its moves into the host cell nucleus and is converted
outer layers and capsid so that the nucleic acid is into the duplex form. Transcription is achieved by
host enzymes, for example, parvovirus
released into the cell. With most viruses, uncoating
is affected by the action of lysosomal enzymes of 3. Double stranded RNA viruses: The double
the host cell. stranded RNA is transcribed to mRNA by viral
polymerases, e.g. reoviruses
4. Single stranded RNA viruses: are classified into
4. Biosynthesis two categories
This phase includes synthesis not merely of the (i) Positive strand (plus strand, positive sense):
viral nucleic acid and capsid protein but also of The viral RNA itself act as the mRNA. Viral RNA
is infectious by itself and is translated directly
enzymes necessary in the various stages of viral
into viral proteins in the host cell cytoplasm e.g.
synthesis, assembly and release. In addition, picorna-, togaviruses
certain ‘regulator proteins’ are also synthesized (ii) The negative strand (minus sense) RNA
which serve to shut down the normal cellular viruses: The RNA is ‘antisense’, with polarity
metabolism and direct the sequential production opposite to mRNA. They possess their own RNA
polymerases for, mRNA transcription. Extracted
of viral components. In general, most DNA viruses
nucleic acids from these viruses are not infectious
synthesize their nucleic acid in the host cell for example rhabdo-, orthomyxo-, paramyxoviridae
nucleus. The exceptions are the poxviruses. Most
5. Retroviridae exhibit a unique replicative strategy.
RNA viruses synthesize all their components in Their single stranded RNA genome is converted
the cytoplasm, except for orthomyxoviruses, some into an RNA: DNA hybrid by the viral reverse
paramyxoviruses and retroviruses. Viral protein is transcriptase (RNA directed DNA polymerase)
synthesized only in the cytoplasm. enzyme. Double stranded DNA is then synthesized
from the RNA: DNA hybrid. The double stranded
DNA form of the virus (provirus) is integrated into
Steps of Biosynthesis the host cell chromosome. This integration may
Transcription of messenger RNA (mRNA)
i. lead to transformation of the cell and development
of neoplasia
from the viral nucleic acid.
www.ebook3000.com
Chap-53.indd 383 15-03-2016 11:33:21 AM
384 | Section 4: Virology
and a buffering system generally consisting of
bicarbonate in equilibrium with atmosphere
containing about 5% carbon dioxide. This is
supplemented with upto 5% calf or fetal calf serum.
Antibiotics are added to prevent bacterial con
taminants and phenol red as indicator.
A B
C D
Figs 53.5A to D: A. Normal vero cell monolayer. B. Vero cell monolayer infected with Coxsackie B virus C. HeLa cell
monolayer. D. HeLa cell monolayer infected with Coxsackie virus B3, stained after 48 hours
www.ebook3000.com
Chap-53.indd 385 15-03-2016 11:33:22 AM
386 | Section 4: Virology
erythrocytes will adsorb onto the surface of cells 1. Total Virus Particles
if the viruses are multiplying in the culture. This is
A. Electron Microscopy
known as ‘hemadsorption.’ This reaction becomes
positive before cytopathic changes are visible and By simple negative staining, the virus particles in
in some cases occurs in the absence of cytopathic a suspension can be counted directly under the
effects. electron microscope. The virus suspension can
be mixed with a known latex particles. The ratio
4. Interference between particles under the electron microscope
gives an indication of the virus count.
The growth of a noncytopathogenic virus in cell
culture can be tested by the subsequent challenge
with a known cytopathogenic virus. The growth of B. Hemagglutination
the first will inhibit infection by the second virus A convenient method of quantitation is the
by interference. determination of hemagglutination titers with
Example: Rubella virus does not produce cytopathic hemagglutinating viruses. Hemagglutination is not
changes, although they multiply within the cell. a very sensitive indicator of the presence of small
A known cytopathogenic challenge virus is then amount of virus particles. Thus, approximately
introduced into the cells. No CPE will be seen in the 10 7 influenza virions are required to produce
cell culture as replication of challenge virus will be macroscopic agglutination.
prevented because of interference by rubella virus.
2. Infectious Virion Assay
5. Transformation Two types of infectivity assays can be carried out—
Tumor forming (oncogenic) viruses induce cell quantitative and quantal assays.
‘transformation’ and loss of contact inhibition,
so that growth appears in a piled-up fashion A. Quantal Assays
producing microtumors. Some herpes viruses,
adenoviruses, hepadnaviruses, papovaviruses and Quantal assays only indicate the prese nce or
retroviruses (human T cell lymphotropic virus type absence of infectious viruses. Quantal assays of
I) can transform cells. infectivity can be carried out in animals, eggs
or tissue culture for those viruses that do not
6. Immunofluorescence form plaques, and the effects of the virus can
be observed. Examples of endpoints used for
Cells from virus infected cultures can be stained by
infectivity titration are the death of the animal,
fluorescent conjugated antiserum and examined
production of hemagglutinin in allantoic fluid or
under the UV microscope for the presence of
the appearance of CPE in cell cultures. The titer is
virus antigen. This gives positive results earlier
usually expressed as the 50 percent infectious dose’
than other methods and, therefore, finds wide
(1D50) per mL, which indicates the highest dilution
application in diagnostic virology.
of the inoculum that would produce an effect in
50% of animals, eggs or cell cultures inoculated.
7. Detection of Virus-specific Nucleic Acid
Molecular-based assays, such as polymerase chain B. Quantitative Infectivity Assay
reaction, provide rapid, sensitive, and specific
Quantitative assays measure the actual number of
methods of detection.
infectious particles in the inoculum. Two methods
are available—plaque assay in monolayer cell
8. Detection of Enzymes culture and pock assay on chick embryo CAM.
The virus isolate can be identified by detection
of viral enzymes, such as reverse transcriptase in i. Plaque Assay
retroviruses, in the culture fluid. A viral suspension is added to a monolayer of
cultured cells in a bottle or Petri dish, and after
9. Electron Microscopy allowing time for absorption, the mediu m is
removed and replaced with a solid agar gel to
Viruses have distinctive appearances and can be
prevent virus spreading throughout the culture,
detected by electron microscopy of ultra thin sections
to ensure that the spread of progeny virions is
of infected cells.
confined to the immediate vicinity of infected cells.
In this system, each infectious viral particle gives
Viral assay
rise to a localized focus of infected cells that can
The virus content of a specimen can be assayed in be seen with the naked eye. Such foci are known as
two ways: Either with reference to the total virus ‘plaques’ and each plaque indicates an infectious
particles or with reference to infectious virion only. virus (Fig. 53.6).
B. Recombination
When two different, but related, viruses infect a
Fig. 53.6: Plaque formation in monkey kidney cells by cell simultaneously, genetic recombination may
poliovirus take place. The two viruses exchange segments
of nucleic acid between them so that a hybrid
ii. Pock Assay is formed possessing genes from both parents.
Certain viruses, e.g. herpes and vaccinia, form Recombinants may occur between (1) two active
pocks when inoculated onto the chorioallantoic (infectious) viruses, (2) one active and one inactive
membrane of an embryonated egg. Such viruses virus, and (3) two inactive viruses.
can be assayed by counting the number of pocks i. Recombinants between two active (infect
formed on CAM by appropriate inocula of virus. ious) viruses: When two inactive viruses
This is known as pock assay. markers (such as pock morphology or antigenic
properties) are grown together, recombinants
Viral genetics may be derived that possess the distinctive
Viruses obey the laws of genetics like all other ‘living properties of both parents. Thus, if a human
beings’. Several properties of viruses, such as virulence and an avian strain of influenza virus (whose
and antigenicity are under genetic control. Main hemagglutinin and neuraminidase antigens
mechanisms for genetic modification in viruses are: are different and easily identifiable) are grown
A. Mutation. together, a hybrid may be obtained with
B. Recombination. the hemagglutinin of one parent and the
C. Nonheritable variations: In addition, viruses neuraminidase of the other. This may be one
may exhibit many nonheritable variations due of the ways by which the pandemic strains of
to gene product interactions. the influenza virus originate in nature.
ii. Recombinants between one active and one
A. Mutation inactive virus: Cross-reactivation or marker
rescue
It is a random, undirected and heritable variation. When a cell is ‘infected’ with an active virus
The frequency of mutation in viruses is about 10-4 and a different but related inactivated virus,
to 10-8, approximately the same as in bacteria. progeny possessing one or more genetic traits
Mutations, therefore, occur during every viral of the inactivated virus may be produced. This
infection. Many mutations are lethal, because phenomenon is known as cross-reactivation
the mutated virus is unable to replicate. A mutant or marker rescue.
becomes evident only if the mutation confers some iii. Recombinants between two inactive viru
readily observable property or affords the mutant ses: Multiplicity reactivation: When a cell is
virus some selection or survival advantage. Mutation ‘infected’ with a large dose (high multiplicity)
may occur spontaneously or may be induced by of a single virus inactivated by UV irradiation,
mutagens, physical agents, such as UV light or live virus may be produced. The different
irradiation or chemical agents such as 5-fluorouracil. virions that cause multiple infection of a
cell may have suffered damage to different
Conditional lethal Mutant genes. Thus from the total genetic pool it may
Mutations in essential genes are termed lethal be possible to obtain a full complement of
mutations. A class of mutants that are of great undamaged genes. This phenomenon is called
www.ebook3000.com
Chap-53.indd 387 15-03-2016 11:33:22 AM
388 | Section 4: Virology
multiplicity reactivation. There is the potential Classification of viruses
danger of multiplicity reactivation taking place
following the administration of UV irradiated Main Criteria Used for the Classification
vaccines. of Viruses
Pseudovirion: As a general rule, virus capsids 1. Type of nucleic acid: Viruses are classified
enclose viral nucleic acids. Sometimes segments into two main divisions depending on the type
of host nucleic acid become encapsidated instead. of nucleic acid they possess: Riboviruses are
This is known as pseudovirion. For example, in those containing RNA and deoxyriboviruses
a papovavirus capsid, a linear piece of host DNA are those containing DNA.
roughly the same size as the papovavirus genome 2. Number of strands of nucleic acid: Single-or
may be found. double-stranded, linear, circular, circular with
breaks, segmented;
C. Nongenetic interactions 3. Polarity of the viral genome: RNA viruses in
1. Phenotypic Mixing which the viral genome can be used directly
as messenger RNA are by convention termed
When two different viruses infect the same cell, some
‘positive-stranded’ and those for which a
‘mix up’ may take place during assembly so that
transcript has first to be made are termed
progeny genome of one virus may be surrounded by
‘negative-stranded’
the capsid belonging entirely or partly to the other
4. The symmetry of the nucleocapsid
virus. This is known as phenotypic mixing. This is
not a stable variation. On subsequent passage, the 5. The presence or absence of a lipid envelope.
capsid will be found to be of the original type only. If
the genome is encased in a completely heterologous Short Descriptions of the Major Groups
protein coat, this extreme example of phenotypic of Viruses
mixing may be called “phenotypic masking” or DNA Viruses (Fig. 53.7)
“transcapsidation.” 1. Herpesviridae family: Human herpesviruses
include herpes simplex types 1 and 2 (oral
2. Genotypic Mixing or Heterozygosis
and genital lesions), varic ella-zoster virus
It results from the incorporation of more than (chickenpox and shingles), cytomegalovirus,
one complete genome into a single virus particle. Epstein-Barr virus (infectious mononucleosis
There is no recombination between the different and association with human neoplasms),
genomes so that the two kinds of viral progeny are human herpesviruses 6 and 7 (T Iymphotropic),
formed on passage. and human herpesvirus 8 (associated with
Kaposi’s sarcoma). Other herpesviruses occur
3. Complementation in many animals.
The basis for complementation is that one virus 2. Hepadnaviridae family: This consists of
provides a gene product in which the second is the human hepatitis type B virus and related
defective, allowing the second virus to grow. The viruses of animals and birds. (The name comes
genotypes of the two viruses remain unchanged. from hepa = liver, and dna for DNA core.)
If both mutants are defective in the same gene 3. Papovaviridae family: Two genera have been
product, they will not be able to complement each recognized. Papillomavirus and Polvomavirus.
other’s growth. Papillomavirus is also a former member of the
Papovaviridae family. Polyomaviruses were
4. Interference formerly a part of the Papovaviridae family
Infection of either cell cultures or whole animals before it was split into two families.
with two viruses often leads to an inhibition of 4. Adenoviridae family: Members have been
multiplication of one of the viruses, an effect called classified into two genera: Mastadenovirus
interference. (mammalian adenoviruses) and Aviadenovirus
Viral interference has been applied in the field in (adenoviruses of birds).
controlling poliomyelitis outbreaks by introducing 5. Parvoviridae family: Three genera have been
into the population, the live attenuated poliovirus described: Parvovirus, Adenosatellovirus and
vaccine. The vaccine virus interferes with the spread Densovirus.
of wild poliovirus and halts the outbreak. 6. Poxviridae family: The family is divided into
several genera. All poxviruses tend to produce
5. Enhancement skin lesions. Some are pathogenic for humans
Mixed infection of cells may sometimes lead to (smallpox, vaccinia, molluscum contagiosum);
increased virus yield or greater CPE. This is known others that are pathogenic for animals and can
as ‘enhancement’. infect humans (cowpox, monkeypox).
www.ebook3000.com
Chap-53.indd 389 15-03-2016 11:33:24 AM
390 | Section 4: Virology
iii. Pestivirus, consisting of the mucosal disease without a protein coat. They cause disease of plants.
virus, hog cholera virus and related viruses. Viroids are agents that do not fit the definition of
classic viruses.
8. Coronaviridae Family
Only one genus Coronavirus has been recognized. PRIONS
Toroviruses, which cause gastroenteritis, form a Prions (proteinaceous infectious particles) are
distinct genus. infectious particles composed solely of protein
with no detectable nucleic acid. Unlike viruses, the
9. Retroviridae Family: (Re = reverse, tr = agents are resistant to a wide range of chemical and
transcriptase) physical treatments. They are highly resistant to
These are RNA tumor viruses and related agents. inactivation by heat, formaldehyde, and ultraviolet
The characteristic biochemical feature is the light that inactivate viruses. The prion protein is
presence of RNA-dependent DNA polymerase encoded by a single cellular gene.
(reverse transcriptase) within the virus that Prion diseases: Prion diseases, called “transmissible
produces a DNA copy of the RNA genome. This spongiform encephalopathies,’’ include scrapie in
DNA becomes circularized and integrated into host sheep, mad cow disease in cattle, and kuru and
chromosomal DNA. The virus is then replicated Creutzfeldt–Jakob disease in humans. Prions do
from the integrated “provirus” DNA copy. Three not appear to be viruses. It has been suggested that
subfamilies are recognized: they may also be responsible for some other chronic
1. Oncovirinae, the RNA tumor virus group. neurological degenerative diseases of humans.
2. Spumivirinae, the foamy virus group (Spuma =
foam) Key Points
3. Lentivirinae, (Lenti = slow) visna and maedi
Viruses are the smallest known infective agents
viruses of sheep belonging to the slow virus The viruses are obligate intracellular parasites
group. They do not grow in inanimate media. They are
resistant to antibiotics
10. Flaviviridae Family The extracellular infectious virus particle is the virion
The viruses vary widely in their size: Range from 20
This group of arboviruses includes yellow fever nm virus (arboviruses) to 300 nm virus (poxvirus)
virus and dengue viruses. The virion consists of a nucleic acid core, the genome,
surrounded by a protein coat, the capsid
11. Caliciviridae Family Viruses may have icosahedral (cubical) symmetry,
helical symmetry, or complex symmetry
An important human pathogen is Norwalk virus, The capsid together with the enclosed nucleic acid
the cause of epidemic acute gastroenteritis. is known as the nucleocapsid. Some viruses are
surrounded by envelopes
12. Filoviridae Family Three methods are employed for cultivation of
viruses namely animal inoculation, embryonated egg
This contains the Marburg and Ebola viruses inoculation and tissue culture
causing human hemorrhagic fevers. Embryonated egg inoculation is done by one of the
several routes such as chorioallantoic membrane
13. Reoviridae Family (CAM), allantoic cavity, amniotic sac and yolk sac
Tissue culture are of three types: Organ culture;
Three genera have been recognized: explant culture and cell culture
1. Reovirus, containing reoviruses from humans, The viruses show variation in their genomic structure
other mammals and birds. by mutations and recombination
2. Orbivirus, containing several species of Depending on the type of nucleic acids viruses
possess, they are classified into two groups:
arboviruses such as blue tongue virus, African Deoxyriboviruses that contain DNA (DNA virus) and
horse sickness virus. riboviruses that contain RNA (RNA virus)
3. Coltivirus includes Colorado tick fever virus of Prions (proteinaceous infectious particles) are
humans. infectious particles composed solely of protein with
no detectable nucleic acid
4. Rotavirus including human rotaviruses, calf
Prion diseases, called “transmissible spongiform
diarrhea virus and related agents. Other genera encephalopathies,’’ include scrapie in sheep, mad
may have to be defined to include plant and cow disease in cattle, and kuru and CreutzfeldtJakob
insect viruses belonging to this family. disease in humans.
www.ebook3000.com
Chap-53.indd 391 15-03-2016 11:33:24 AM
5454
Chapter
Learning Objectives
www.ebook3000.com
394 | Section 4: Virology
is the most frequent means of viral entry into the the primary agents responsible for congenital
host. Some viruses, such as influenza A, B and C, defects in humans and produce maldevelopment
parainfluenza types 1–4, respiratory syncytial virus or severe neonatal disease.
(RSV), Rhinovirus, Coronavirus, adenovirus and
coxsackievirus are restricted to respiratory tract Spread of virus in the body
where they multiply and produce local disease.
Other viruses, multiply locally to initiate a silent
Generalized Infections
local infection which is followed by lymphatic or Sequence of events: The sequence of events,
hematogenous spread to other parts of the body summarized as follows:
where more extensive multiplication takes place 1. Entry: Virions enter through an epithelial
before producing generalized disease. surface, where they undergo limited replication.
2. Migration: They then migrate to the regional
2. Alimentary Tract lymph nodes where some are taken up by
The alimentary tract is the next most important macrophages and inactivated, but others enter
route of entry for viruses. The viruses that initiate the bloodstream.
infection of humans via the alimentary tract are 3. Primary viremia: Virions which enter the
many enteroviruses including poliovirus types 1–3, bloodstream. This is the primary viremia
hepatitis A virus (HAV), hepatitis E virus (HEV), 4. Secondary viremia: From the blood, the virus
adenoviruses, rotaviruses, Norwalk and related gains access to the large reticuloendothelial
viruses, Coronavirus, Astrovirus, coxsackie A and organs—liver, spleen, and bone marrow—in
B and echovirus. Polioviruses, HAV and HEV do which it again multiplies, and a large amount
not produce any intestinal symptoms, but cause of virus is produced which again spills over into
generalized infection. the bloodstream, causing a secondary viremia.
This heralds the onset of clinical symptoms (the
3. Skin prodromal phase in eruptive fevers).
Of the viruses that enter through the skin, only a few 5. Target organ: The virus reaches the target
produce local lesions. Some obtain entry through organ through the bloodstream. Multiplication
small abrasions of the skin (papillomaviruses, in the target sites produces the distinctive
molluscum contagiosum, cowpox, orf, milker’s lesions.
nodes viruses, herpes simplex and HBV), insect
bite (arboviruses), animal bite (rab ies virus, Significance of the
herpes B virus) or injection (HBV, HCV, HIV, HTLV,
incubation period
CMV, EBV and Ebola virus) (Table 54.1).
Viruses that are introduced deeper into the The incubation period represents the time taken
dermis have access to blood vessels, lymphatics, for the virus to spread from the site of entry to
dendritic cells, and macrophages and usually the organs of viral multiplication and hence to
spread and cause systemic infections. Rabies virus the target organs for the production of lesions. Its
travels along the nerves to the spinal cord or brain. duration is therefore influenced by the relation
between the sites of entry, multiplication and
4. Conjunctiva lesion. They are classifed into four main groups:
The conjuctiva also may act as a portal of entry for short, medium, long, and very long.
viruses. This may lead to local disease (adenovirus) 1. Short means less than a week and primarily
or to systemic spread (measles). applies to viruses causing localized infections
that spread rapidly on mucous surfaces.
5. Genital Tract 2. Medium incubation periods range from about
Papillomaviruses and herpes simplex viruses are 7–21 days.
sexually transmitted and produce lesions on the 3. Long refers to periods measured in weeks or
genitalia and perineum. HIV, HTLV, HBV and HCV months (e.g. 2–6 weeks for hepatitis A and 6–20
are also sexually transmitted but do not produce weeks for hepatitis B). Rabies may also have
local lesions. incubation periods extending for many months.
4. Very long incubation periods are measured
6. Congenital Viral Infections in years, which is why the agents involved were
Congenital infection may occur at any stage from originally termed ‘slow’ viruses. This group
the development of the ovum up to birth. If the comprises the prions and a few’conventional’
virus crosses the placenta and infection occurs in viruses, such as papovaviruses and measles,
utero, serious damage may be done to the fetus. which very occasionally cause delayed disease
Rubella virus and cytomegalovirus are presently of the central nervous system.
Chapter 54: Virus–Host Interactions: Viral Infections | 395
Host response to virus infections against viral infections. On the other hand,
macrophages phagocytose viruses and
The outcome of a virus infection is influenced are important in clearing viruses from the
by the virulence of the infecting strain and the bloodstream.
resistance offered by the host. Mechanisms of host
2. Fever: Fever may act as a natural defence
resistance may be immunological or nonspecific.
mechanism against viral infections as most
viruses are inhibited by temperatures above
A. Immunological Response 39°C.
Virions in general are good antigens and induce 3. Hormones: Corticosteroid administration
both antibody- and cell-mediated immunity (CMI). enhances most viral infections. Injudicious
For some diseases such as poxviruses, measles, use of steroids in the treatment of herpetic
herpes simplex virus and CMV infections, CMI keratoconjunctivitis may cause blindness.
appears to be the main immunospecific defence,
4. Malnutrition: Malnutrition can exacerbate viral
for others, such as enterovirus and arbovirus
infections, for example, measles produces a much
infections, antibody production appears to be the
higher case fatality in malnourished than in well-
main immunospecific defence. However, some
fed children.
viruses have evolved strategies to evade detection
and persist in their host. 5. Age: Most viral infections are commoner and
more dangerous at the two extremes of age.
1. Antibody-mediated Immunity 6. Interferon: Interferons are a family of host
coded proteins produced by cells on induction
In mediating humoral antiviral immunity, the
by viral or nonviral inducers. Isaacs and
important classes of antibodies are IgG, IgM
Lindenmann in 1957 discovered that virus
and IgA. IgG and IgM play a major role in blood
infected cells produce a soluble factor that
and tissue spaces, while secretory IgA antibody
protects other cells from infection and they gave
is important in protecting against infection by
the name interferon to this antiviral substance.
viruses through the respiratory or gastrointestinal
tracts. Humoral immunity protects the host against Mode of action of interferon: On exposure to
reinfection by the same virus. Mechanisms of interferon, cells produce a protein (translation
antibodies effecting virus neutralization inhibiting protein, TIP) which selectively inhibits
Antibodies effect virus neutralization by several translation of viral mRNA, without affecting cellular
mechanisms: mRNA. What has been called TIP is “actually
a mixture of at least three different enzymes (a
i. They may prevent adsorption of the virus to cell
protein kinase, an oligonucleotide synthetase and
receptors, cause enhanced virus degradation
an RNAase) which together block translation of
or prevent release of the progeny virus from
viral mRNA into viral proteins. It has also been
infected cells.
suggested that inhibition of viral transcription
ii. Opsonization of virions for phagocytosis and
may also be responsible for the antiviral activity
killing by macrophages.
of interferon.
iii. Complement acts in conjunction with
antibodies in causing surface damage to
enveloped virions and in producing cytolysis
Synthesis of Interferons
of virus infected cells. Interferons are produced by all vertebrate species.
Normal cells do not generally synthesize interferon
2. Cell-mediated Immunity until they are induced to do so. Viruses also vary
in their capacity to induce interferon, cytocidal
Cell-mediated immunity prevents infection of
and virulent viruses being poor inducers and
target organs and promotes recovery from disease
avirulent viruses being good inducers. Infection
by destroying virus and virus-infected cells by any
with viruses is a potent insult leading to induction;
of the following four different processes:
RNA viruses are stronger inducers of interferon
1. Cytolysis by cytotoxic T cells(Tc) cells. than DNA viruses.
2. Cytolysis by natural killer (NK) cells.
3. Antibody-complement-mediated cytotoxicity. Types of Interferon
4. Antibody-dependent cell-mediated cytotoxicity
There are multiple species of interferons that fall
(ADCC).
into three general groups, designated IFN-a, IFN-
b, and IFN-g.
B. Nonimmunological Responses 1. Alpha-interferon (IFN-a): It is produced by
1. Phagocytosis: Polymorphonuclear leukocytes leukocytes following induction by suitable
do not play any significant role in the defence viruses.
www.ebook3000.com
396 | Section 4: Virology
2. Beta-interferon (IFN-b): It is produced by fibro Interferons are a family of host coded proteins
blasts and epithelial cells following stimulation produced by cells on induction by viral or nonviral
by viruses or polynucleotides. It is a glycoprotein. inducers. Interferons fall into three general groups,
3. Gamma-interferon (IFN-g): It is produced by designated IFN-a, IFN-b, and IFN-g
T lymphocytes, on stimulation by antigens or
mitogens. Important Questions
1. Discuss various methods by which viruses can be
Main Biological Effects of Interferons transmitted to human beings.
1. Antiviral effects: Induction of resistance to 2. Write short notes on:
infections. a. Inclusion bodies b. Interferons
2. Antimicrobial effects: Resistance to intracellular
infections, for example, toxoplasma, chlamydia, Multiple choice questions (MCQs)
malaria. 1. Syncytium formation is the typical cytopathic
3. Cellular effects: Inhibition of cell growth and effect produced by:
proliferation; and of DNA and protein synthesis; a. Adenovirus b. Herpes virus
c. Measles virus d. SV40
increased expression of MHC antigens on cell
2. Intracytoplasmic inclusion bodies are found in
surfaces.
cells infected with:
4. Immunoregulatory effects: Enhanced cytotoxic a. Rabies virus
activity of NK, K and T cells; activation of b. Vaccinia virus
macrophage cytocidal activity; modulation of c. Molluscum contagiosum virus
antibody formation; activation of suppressor T d. All of the above
cells; suppression of DTH. 3. All of the following viruses are transmitted by the
respiratory route except:
a. Mumps virus b. Measles virus
Key Points c. Rubella virus d. Norwalk virus
4. All of the following viruses may be transmitted
Virus-host interactions may be considered at the
level of the cell, individual and community through genital tract except:
The cellular response to viral infection may range a. Papillomavirus
from no apparent effect to cytopathology with b. Herpes simplex virus
accompanying cell death to hyperplasia or cancer c. Hepatitis C virus
Inclusion bodies are structures with distinct size, d. Coronavirus
shape, location and staining properties that can be 5. Which type/s of interferon is/are produced by T
demonstrated in virus infected cells under the light lymphocytes?
microscope a. a b. b
Viruses enter the body through the respiratory tract, c. g d. g and b
the alimentary tract, the skin, conjunctiva and the 6. Which type/s of interferon are produced by virus
genital tract; many viruses (cytomegalovirus and infected cells?
rubella) can also be transmitted vertically from a. a
parent to progeny b. b
The immune response is the best and most c. Both of the above
important means of controlling virus infections. d. None of the above
Nonimmunological responses include age;
phagocytosis, fever, hormones, age and interferon Answers (MCQs)
1. c; 2. d; 3. d; 4. d; 5. c; 6. c.
55
Chapter
Laboratory Diagnosis, Prophylaxis and
Chemotherapy of Viral Diseases
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe immunoprophylaxis of viral diseases
be able to: ∙∙ Describe the following: Live viral vaccines; killed
∙∙ Describe laboratory diagnosis of viral infections viral vaccines.
www.ebook3000.com
398 | Section 4: Virology
through the finding of Negri bodies (rabies virus DNA molecule in the clinical specimens are
inclusions) in brain cells of animals. first separated, then, allowed to hybridize with
a labeled single-stranded DNA or RNA probe
B. Virus Isolation present in excess. Depending on the type
For virus isolation it is imperative that the specimen of label attached to the probe, hybridized-
be collected properly and transported with least labeled probe can be detected by radiography,
delay to the laboratory. The reasons are that many gamma-counting or a simple colorimetric
viruses are labile and that the samples are susceptible evaluation (dot-blot hybridization). The DNA
to bacterial and fungal overgrowth. Viruses are best probes are detected with autoradiography
transported and stored on ice and in special media. or with fluorescent or EIA-like methods. By
In general, the methods used for isolation use of nucleic acid probes cytomegalovirus,
consist of inoculation into animals, eggs or tissue papillomavirus and Epstein-Barr virus have
culture, after the specimen is processed to remove been identified.
bacterial contaminants. iii. Southern, Northern andd Dot blot analysis:
The isolates are identified by neutralization Viral genomes can also be detected in clinical
or other suitable serological procedures. Many samples with the use of dot blot or Southern
viruses (for example adenoviruses, enteroviruses) blot analysis. Electrophoretically separated
are frequently found in normal individuals. The viral RNA (Northern blot-RNA: DNA probe
results of isolation should always be interpreted hybridization) blotted onto a nitrocellulose
in the light of the clinical data. filter can be detected in a similar manner.
iv. Polymerase chain reaction: The polymerase
C. Detection of Viral Proteins chain reaction (PCR) for DNA, reverse
The viral proteins can be assayed by the following transcriptase polymerase chain reaction(RT-
methods: PCR) for RNA, and branched-chain DNA
i. Protein patterns (by electrophoresis)—HSV. (DNA, RNA) assays are becoming very
ii. Enzyme activities (e.g. reverse transcriptase)— important for viral detection.
Retrovirus. Use of the appropriate primers for PCR can
iii. Hemagglutination and hemadsorption— promote a million-fold amplification of a
Influenza virus. target sequence in a few hours. This technique
iv. Antigen detection: Viral antigens on the cell is especially useful for detecting latent and
surface or within the cell can be detected by integrated sequences of viruses, such as
immunofluorescence and enzyme immuno retroviruses, herpes viruses, papillomavi
assay (EIA). Virus or antigen released from ruses, and other papovaviruses as well as
infected cells can be detected by enzyme- the sequences of viruses present in low
linked immunosorbent assay (ELISA), radio concentrations.
immunoassay (RIA), and latex agglutination v. Reverse transcriptase polymerase chain
(LA). reaction (RT-PCR) for RNA: This approach
was very useful for identifying and disting
D. Detection of Viral Genetic Material uishing the Hantaviruses.
i. The electrophoretic patterns of RNA (influ vi. Branched-chain DNA assays: Quantitate
enza, reovirus) or restriction endonuclease viral DNA or RNA much like ELISA.
fragment lengths from DNA viral genomes
are like genetic fingerprints for these viruses. E. Serological Diagnosis
ii). Different strains of HSV-l and HSV-2 can The demonstration of a rise in titer of antibodies
be distinguished in this way by restriction to a virus during the course of a disease is strong
fragment length polymorphism. evidence that it is the etiological agent. For this,
ii. DNA probes: DNA probes with sequences it is essential to examine paired sera, the ‘acute’
complementary to spec ific regions of a sample collected early in the course of the disease
viral genome can be used, like antibodies, and the ‘convalescent’ sample collected 10–14
as sensitive and specific tools for detecting days later. Examination of a single sample of serum
a virus. Enzyme-labelled or radiolabeled for antibodies may not be meaningful except
nucleic acid (DNA or RNA) sequences when IgM specific tests are done which is present
complementary to unique regions in nucleic during the first 2 or 3 weeks of a primary infection,
acid sequences of most viruses are now generally indicates a recent primary infection.
manufactured commercially. These labeled The serological techniques employed would
complementary sequences are known as depend on the virus but those in general uses
nucleic acid probes. Two strands of the target are neutralization, complement fixation, ELISA,
Chapter 55: Laboratory Diagnosis, Prophylaxis and Chemotherapy of Viral Diseases | 399
hemagglutination inhibition tests, indirect fluore ∙∙ Recombinant live viral or bacterial vectors, e.g.
scent antibody test, latex agglutination test. influenza.
www.ebook3000.com
400 | Section 4: Virology
2. The immunity conferred is often brief and must Table 55.2 Major antiviral compounds used for
be boosted. treatment of viral infections
3. Parenteral administration of killed-virus
Drug Viral spectrum
vaccine, even when it stimulates circulating
Viral polymerase inhibitors
antibody (IgM, IgG) to satisfactory levels, has
sometimes given limited protection because Acyclovir Herpes simplex, varicella-
zoster
local resistance (IgA) is not induced adequately.
4. Cell-mediated response to inactivated vaccines Cidofovir Cytomegalovirus, herpes
simplex
is generally poor.
Foscarnet Herperviruses, HIV-1, HBV
5. Some killed-virus vaccines have induced hyper
sensitivity to subsequent infection. Ganciclovir Cytomegalovirus
Valacyclovir Herpesviruses
B. Passive Immunization Vidarabine Herpesviruses, vaccinia,
HBV
Passive immunization with human gammaglobulin,
convalescent serum or specific immune globulin Blocking of viral uncoating
gives temporary protection against many viral Amantadine Influenzavirus A
diseases such at. measles, mumps and infectious HIV protease inhibitors
hepatitis. These are indicated only when Indinavir HIV-1, HIV-2
nonimmune individuals who are as special risk
Ritonavir HIV-1, HIV-2
are exposed to infection. Combined active and
passive immunization is an established method Saquinavir HIV-1, HIV-2
for the prevention of rabies. Reverse transcriptase inhibitors
Lamivudine HIV-1, HIV-2, HBV
Chemoprophylaxis and (dideoxythiacytidine)
chemotherapy of virus diseases Nevirapine HIV-1
Stavudine HIV-1, HIV-2
Because viruses are obligate intracellular parasites, (didehydrodexoythymidine)
antiviral agents must be capable of selectively
Zidovudine HIV-1, HIV-2, HBV
inhibiting viral functions without damaging the
(azidothymidine, AZT)
host, making the development of such drugs very
Zatcitabine HIV-1, HIV-2, HBV
difficult. Viral infection may be checked at the
(dideoxycytidine, ddC)
level of attachment, transcription of viral nucleic
acid, translation of viral mRNA, replication of viral Didanosine HIV-1, HIV-2
(dideoxyinosine, ddl)
nucleic acid and’ assembly and release of viral
progeny. Blocking of capping of mRNA
www.ebook3000.com
56
Chapter
Bacteriophages
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss lytic and lysogenic cycle of bacteriophage
be able to: ∙∙ Describe phage typing.
∙∙ Describe morphology of bacteriophage
Morphology
Certain bacteriophages that infect E. coli, called the
T even phages (T2, T4, T6), have been studied in
great detail and traditionally serve as the prototypes
in describing the properties of bacteriophages.
Most phages are tadpole-shaped, possess a
hexagonal head and a cylindrical tail.
1. Head: The head consists of a tightly packed
core of nucleic acid (double stranded DNA) Fig. 56.1: Morphology of bacteriophage. A. hexagonal head,
surrounded by a protein coat or capsid. The B. DNA core, C. demarcation between head and tail, D. tail,
head of phage T4 has a diameter of 65 nm and E. base plate, F. tail fibers, G. prongs; right, process of injection
is 100 nm long. of phage DNA into host cell
Chapter 56: Bacteriophages | 403
phage DNA becomes integrated with the bacterial protein capsid outside. The process of penetration
genome, replicating synchronously with it, causing resembles injection through a syringe. Following
no harm to the host cell (Fig. 56.2). adsorption, six tail pins make contact with the host
cell surface and firmly attach the phage plate to it.
1. Lytic Cycle The contractile tail sheath then contracts forcing
Replication of a virulent phage can be considered the hollow interior tail tube into the bacterial cell
in the following stages adsorption, penetration wall. The phage DNA then passes through the
and synthesis of phage components, assembly, hollow interior tail tube. The empty head and tail
maturation and release of progeny phage particles. of the phage remain outside the bacterium as the
shell or ‘ghost’ after penetration.
When bacteria are mixed with phage particles at
i. Adsorption
high multiplicity (that is very large number of phages
By random collision, phage particles attach to per bacterial cell), multiple holes are produced on
virus-specific receptors on the host cell by its tail. the cell with the consequent leakage of cell contents.
Adsorption is a specific process and depends on Bacterial lysis occurs without viral multiplication.
the presence of complementary chemical groups This is known as ‘lysis from without’.
on the receptor sites on the bacterial surface and on
the terminal base plate of the phage. The infection iii. Synthesis of Phage Nucleic Acid and Proteins
of a bacterium by the naked phage nucleic acid is The synthesis of the phage components is initiated
known as transfection. immediately after penetration of the phage nucleic
acid. The first products to be synthesized (called
ii. Penetration early proteins) are the enzymes necessary for the
After adsorption, most phages inject their nucleic building of the complex molecules peculiar to the
acid into the bacterial cytoplasm and leave their phage. Subsequently, late proteins appear which
Fig. 56.2: Lytic and lysogenic life of bacteriophage. 1. Adsorption, 2. Injection of phage DNA, 3. Circularization of phage
DNA, 4. Replication of phage DNA, 5. Production of phage components, 6. Assenbly of phage, 7. Release of progeny phage,
8. Integration of phage DNA with host chromosome, 9. Binary fission of lysogenic bacterium, 10. Daughter bacteria carrying
prophage, 11. Excision of prophage, 12. Same stage as 3 (1 to 3 injection; 4 to 7 lytic; 8 to 10 lysogenic cycle; 11 and 12 induction)
www.ebook3000.com
404 | Section 4: Virology
include the protein subunits of the phage head and bacillus and nontoxigenic strains can be made
tail. During this period, the synthesis of bacterial toxigenic by lysogenization.
protein, DNA and RNA ceases and the cell is forced ii. Clostridium botulinum types C and D produce
to make viral constituents. toxin only if these are infected with phage CE
b and DE b, respectively.
iv. Assembly and Maturation iii. A wide variety of temperate phages of Salmo
Phage DNA, head protein and tail protein are nella can modify the antigenic properties of
synthesized separately in the bacterial cell. The somatic O antigen. When Salmonella is infected
DNA is condensed into a compact polyhedron by an epsilon phage, the structure of its outer
and ‘packaged’ into the head and, finally, the tail lipopolysaccharide layer may be modified.
structures are added. This assembly of the phage The prophage may become ‘excised’ from
components into the mat ure infective phage occasional cells during the multiplication of
particle is known as maturation. lysogenic bacteria. The excised prophage initiates
lytic replication and the daughter phage particles
v. Release are released, which infect other bacterial cells
Release of the mature progeny phages typically and render them lysogenic. This is known as
occurs by lysis of the bacterial cell. Phage enzymes act spontaneous induction of prophage. While this is
on the weakened cell wall causing it to burst or lyse a rare event, all lysogenic bacteria in a population
resulting in the release of mature daughter phages. can be induced to shift to the lytic cycle by
exposure to certain physical and chemical agents.
Such inducing agents include UV rays, hydrogen
Eclipse Phase
pero xide and nitrogen mustard. A lysogenic
The interval between the entry of the phage nucleic bacterium is resistant to reinfection by the same
acid into the bacterial cell and the appearance of or related phages. This is known as superinfection
the first infectious intracellular phage particle is immunity.
known as the eclipse phase. The interval between
the infection of a bacterial cell and the first release Significance of phages
of infectious phage particles is known as the latent
period (20–40 minutes). Immediately following the 1. Virulent Phage
latent period, the number of phage particles released i. Phage Assay
increases for a few minutes till the maximum number
of daughter phages is attained. This period, during When a phage is applied on the lawn culture of
which the number of infectious phages released a susceptible bacterium, areas of clearing occur
rises, is known as the rise period. The average yield after incubation. These zones of lysis are called
of progeny phages per infected bacterial cell is plaques. The size, shape and nature of plaques are
known as the burst size (100–300 phages). characteristic for different phages. Plaque assay
can be employed for titrating the number of viable
phages in a preparation.
2. Lysogenic Cycle
Unlike virulent phages which produce lysis of the
host cell, temperate phages enter into a symbiotic ii. Phage Typing
relationship with their host cells without destroying Bacteriophage (phage) typing is based on the
them. Following entry into the host cell, the specificity of phage surface receptors for cell surface
temperate phage nucleic acid becomes integrated receptors. The limited host range of many phages
with the bacterial chromosome. The integrated enables them to be used as an epidemiological
phage nucleic acid is known as the prophage. marker to discriminate between bacterial strains
The prophage behaves like a segment of the that are biochemically or serologically indisting
host chromosome and replicates synchronously uishable.
with it. This phenomenon is called lysogeny The strain to be typed is inoculated on a plate
and a bacterium that carries a prophage within of nutrient agar to produce a lawn culture. After
its genome is called a lysogenic bacterium or drying, the phages are applied over marked squares
lysogen. in a fixed dose (routine test dose). The highest
The prophage confers certain new properties dilution of the phage preparation that just produces
on the lysogenic bacterium. This is known as lyso confluent lysis known as the routine test dose
genic or phage conversion. (RTD). After overnight incubation, the culture will
i. Toxin production in Corynebacterium be observed to be lysed by some phages but not by
diphtheriae is determined by the presence in others. The phage type of the strain is expressed by
it of the prophage b. The elimination of this the designation of the phage/phages that lyse it.
prophage abolishes the toxigenicity of the Area of lysis caused by phage is known as plaque.
Chapter 56: Bacteriophages | 405
The most important application of phage typing
(virulent) cycle, intracellular multiplication of the
is for intraspecies typing of bacteria, as in the phage phage ends in lysis of the host bacterium and release
typing of Salmonella Typhi and staphylococi. of progeny virions
Phages are available that lyse all members of In the lysogenic (temperate) cycle, phage DNA
a bacterial genus (for example genus-specific becomes incorporated within the bacterial genome
bacteriophage for Salmonella), all members of and then replicates synchronous with it, causing no
harm to the host cell
a species (for example specific bacteriophage
Phage typing has been used for Staphylococcus
for B. anthracis), and all members of a biotype aureus, Vibrio cholerae, Salmonella, and many other
or subspecies (for example Mukerjee’s phage IV bacteria.
which lyses -all strains of classical V. cholerae but
not V. cholerae biotype EI Tor).
Important questions
1. Draw a labeled diagram of a T-even phage.
2. Temperate Phage 2. Discuss the life cycle of bacteriophages.
i. Transduction: Bacteriophages may act 3. Write short notes on:
as carriers of genes from one bacterium a. Lysogenic cycle of phages
to another. This is known as transduction. b. Lysogenic conversion
Two types of transduction are recognized: c. Phage typing
d. Significance of phages.
generalized transduction, in which any
portion of the donor DNA can be transferred,
and specialized transduction, in which only Multiple choice questions (Mcqs)
a specific set of genes can be carried to a 1. T-even bacteriophages possess
recipient cell. a. Single-stranded RNA
ii. Toxin production: Toxin production in b. Double-stranded RNA
Corynebacterium diphtheriae and Clostridium c. Single-stranded DNA
botulinum are determined by genes carried in d. Double-stranded DNA
prohage DNA. 2. All the following statements for stage of adsorption
iii. Antigenic property: A wide variety of in lytic cycle of bacteriophage are true accept
a. It is a specific process
temperate phages of Salmonella can modify
b. It depends upon the susceptibility of the bacte-
the antigenic properties of somatic O antigen. rium to the specific phages
Such acquisition of new properties by c. It occurs by random collision
bacterial cells following phage infection is d. It is a very slow process
called “phage conversion”. 3. All the following are the examples of phage
iv. Cloning vector: Bacteriophages have been used conversions except
as cloning vectors in genetic manipulations. a. Phage-mediated conversion of somatic anti-
gens of Salmonella spp.
Key Points b. Toxigenicity of Corynebacterium diphtheriae
Bacteriophages are bacterial viruses. They are c. Toxicity of Clostridium botulinum
associated with transmission of genetic material d. Toxigenicity of Vibrio cholerae
from one bacterium to another 4. Which of the following bacteria can be typed by
Morphology: Most phages are tadpole-shaped, phage typing method?
possess a hexagonal head and a cylindrical tail. Most a. Staph. aureus
of the phages usually consist of single, linear, and b. Salmonella typhi
doublestranded DNA molecule genome. The phages c. Vibrio cholerae
show high host specificity d. All of the above
Life cycle: Phages exhibit two different types of
life cycle: lytic cycle and lysogenic cycle. In the lytic Answers (MCQs)
1. d; 2. d; 3. d; 4. d.
www.ebook3000.com
57
Chapter
Poxviruses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe molluscum contagiosum.
be able to:
∙∙ Describe structure of vaccinia virus
A B
Fig. 57.1: Structure of vaccinia virus. The nucleic acid is Fig. 57.2: Variola and vaccinia pocks on cam. A. Variola,
contained within a dumb-bell shaped core. Fitting into the showing small, uniform pocks; B. Vaccinia, showing large,
concavities of the core are two lateral bodies. The virion is irregular pocks
enclosed within a protein shell which has an irregular surface
www.ebook3000.com
408 | Section 4: Virology
deliberate inoculation with smallpox material 7. Molluscum Contagiosum
failed to induce the disease. Molluscum contagiosum is a benign epidermal tumor
For thousands of years, smallpox raged as a that occurs only in humans. The causative agent is
scourge of mankind, causing death and disfigure molluscum contagiosum virus. It is a contangious
ment. The global eradication of smallpox, achieved disease. The lesions of this disease are small, pink,
after 10 years of concerted campaigns under the wart-like tumors on the face, arms, back, and buttocks
auspices of the WHO, has been a most impres are more frequent in children than in adults. It is
sive medical achievement. Naturally occurring spread by direct contact [e.g. sexual contact, wrestling
smallpox came to an end in 1977. On May 8, or fomites (e.g. towels)]. The disease is more common
1980, the WHO formally announced the global in children than adults but its incidence is increasing
eradication of smallpox. in sexually active individuals.
The diagnosis of molluscum contagiosum can
OTHER POXVIRUS DISEASES usually be made clinically. Sections of skin lesions
show large, eosinophilic intracytoplasmic inclusions,
1. Cowpox which displace the nuclei to the margin, known as
Cowpox is, a zoonosis. Cowpox lesions are seen on molluscum bodies under light microscope. It can
the udder and teats of cows and may be transmitted be detected under electron microscope and PCR
to humans during milking. The lesions in humans can detect viral DNA sequences. The virus cannot
usually appear on the hands or fingers. The natural be grown in animals or tissue cultures.
reservoir of cowpox seems to be a rodent.
Key Points
2. Monkeypox The family Poxviridae is a large family of ovoid or
brick-shaped viruses, and large enough to be just
This orthopoxvirus zoonosis causes an illness in visible under a light microscope
humans very similar to smallpox, squirrels seem to The genus Orthopoxvirus includes the viruses
be the main reservoir of infection, and the infection causing cowpox, vaccinia and variola. The variola
virus is an orthopoxvirus and the cause of smallpox
is seen mainly in children who may acquire it from
They can grow in chorioallantoic membrane CAM)
playing with captive animals. of chick embryos and tissue culture
Smallpox was the first viral disease to be eradicated
3. Buffalopox from the world
Molluscum contagiosum: The virus causing mollus
Buffalopox was identified in cattle in India in 1934 cum contagiosum is a poxvirus. The virus is spread by
and was considered an outbreak of vaccinia in close contact, often through sexual contact. It causes
them. Epizootics had occurred in buffaloes and small, pink, papular, pearl-like benign tumors of the
lesions had been observed on the hands of persons skin or mucous membranes.
in contact with infected animals.
Important question
4. Milker’s Node Write short notes on:
a. Variola virus. b. Vaccinia virus
Milker’s node (paravaccinia) is a trivial occupa c. Molluscum contagiosum
tional disease that humans get by milking infected
cows. The lesions are small ulcerating nodules. Multiple choice questions (mcqs)
1. All the statements are true for vaccinia virus except:
5. Orf (Contagious Pustular Dermatitis or a. It is an artificial virus
Sore Mouth) b. It is used for preparation of smallpox vaccine
c. It produces small, shiny, white, convex colonies
Orf is a disease of sheep and goats transmitted to on the chorioallantoic membrane
human beings by contact. It is an occupational d. It can be cultured in vitro on chorioallantoic
disease of sheep handlers. In humans, the disease membrane of ape
occurs as a single papulovesicular lesion with a 2. The last case of naturally occurring smallpox was
central ulcer usually on the hand, forearm, or face. detected in:
a. India in 1980
b. Bangladesh in 1979
6. Tanapox c. England in 1978
This virus takes its name from the Tana River in d. Somalia in 1977
Kenya, where it was first diagnosed. It is prevalent 3. What is/are the route/s of transmission of Molluscum
in monkeys and appears to be spread by insect contagiosum virus?
bites. Monkeys are the only animals susceptible. a. Direct contact. b. Sexual contact.
c. None of the above. d. Both of the above
There is usually only one vesicular lesion but its
appearance is preceded by fever and quite severe Answers (MCQs)
malaise. Recovery is uneventful. 1. c; 2. d; 3. d
58
Chapter
Herpesviruses
Learning Objectives
Classification
The family Herpesviridae is divided into three
subfamilies based on biological, physical and
genetic properties (Table 58.1).
Eight different types of herpesviruses are known
whose primary hosts are human (Table 58.1).
www.ebook3000.com
410 | Section 4: Virology
Table 58.1: Classification of human herpesviruses
Subfamily Virus Primary target cell Site of latency Mode of spread
Alphaherpesvirinae
Human herpesvirus 1 Herpes simplex type 1 Mucoepithelial cells Neuron Close contact
Human herpesvirus 2 Herpes simples type 2 Mucoepithelial cells Neuron Close contact (sexually
transmitted disease)
Human herpesvirus 3 Varicella-zoster virus Mucoepithelial cells Neuron Respiratory and close
contact
Gammaherpesvirinae
Human herpesvirus 4 Epstein-Barr virus B cells and epithelial cells B cell Saliva (kissing disease)
Human herpesvirus 8 Kaposi’s sarcoma- Lymphocyte and ? Close contatct (sexual),
related virus other cells saliva?
Betaherpesvirinae
Human herpesvirus 5 Cytomegalovirus Monocyte, lymphocyte, Monocyte, Close contact,
and epithelial cells lymphocyte, transfusions, tissue
and ? transplant, and
congenital
Human herpesvirus 6 Herpes lymphotropic T cells and ? T cells and ? Respiratory and close
virus contact?
Human herpesvirus 7 Human herpesvirus 7 T cells and ? T cells and ? ?
serologically. They can be differentiated by the Once HSV has established itself in a ganglion in
following features: vivo, reactivations are liable to take place at inter
1. Antigenic differences can be made out using vals of weeks or months thereafter triggered by a
type specific monoclonal antibodies. variety of stimuli. The virus follows axons back to
2. On chick embryo CAM type 2 strains form the peripheral site, and replication proceeds at the
larger pocks resembling variola. skin or mucous membranes.
3. Types 2 strains replicate well in chick embryo HSV-1 is usually associated with infections
fibroblast cells, while type 1 strains do so above the waist, and HSV-2 with infections below
poorly. the waist, consistent with the means of spread for
4. The infectivity of type 2 is more temperature these viruses.
sensitive than that of type 1.
5. Type 2 strains are more neurovirulent in labora Clinical Features
tory animals than type 1. 1. Cutaneous infections: (i) The most common
6. Type 2 strains are more resistant to antiviral site is the face—on the cheeks, chin, around
agents like IUDR and cytarabine in culture. the mouth or on the forepead; (ii) Napkin
7. Restriction endonuclease analysis of viral DNA rash; (iii) ‘Fever blister’ or herpis febrilis. In
enables differentiation between the two types some sensitive persons, very minor stimuli, like
as well as between strains within the same type. common cold, exposure to sun or even mental
strain or menses may bring on such reactivation;
Pathogenesis (iii) Herpetic whitlow- an infection of the
finger; (iv) Herpes gladiatorum an infection
The mechanisms involved in the pathogenesis of of the body; (v) Eczema herpeticum.
HSV-1 and HSV-2 are very similar. Both viruses 2. Oral infection: Acute gingivostomatitis,
initially infect and replicate in mucoepithelial cells herpetic stomatitis, pharyngitis, tonsillitis and
and then establish latent infection of the innervating localized lymphadenopathy may occur.
neurons. Skin and mucous membranes are the 3. Ophthalmic: Severe keratoconjunctivitis,
portals of entry in which the virus also multiplies, follicular conjunctivitis with vesicle formation
causing lysis of cells and formation of vesicles. on the lids, dendritic keratitis or corneal
Soon after replication is under way in the skin ulcers or as vesicles on the eyelids, corneal
or a mucous membrane, virions travel to the root scarring and impairment of vision.
ganglia via the sensory nerves supplying the area. 4. Nervous system: (i) HSV encephalitis; (ii)
Primary infections of the orofacial and genital Sporadic encephalitis; (iii) HSV meningitis;
areas involve respectively the trigeminal and (iv) Sacral autonomic dysfunction; (v) rarely
lumbosacral dorsal root ganglia. The virus then transverse myelitis or the Guillain–Barre
becomes latent in the ganglia. syndrome and Bell’s palsy.
Chapter 58: Herpesviruses | 411
5. Visceral: (i) HSV esophagitis; (ii) tracheo be used. Typical cytopathic changes may appear
bronchitis and pneumonitis; (iii) hepatitis; (iv) as early as in 24–48 hours but cultures should be
Erythema multiforme; (v) disseminated HSV observed for two weeks before being declared
infection. negative.
6. Genital infections: Genital disease is usually HSV isolates can be typed by biochemical,
caused by HSV-2. Genital herpetic ulcers are biologic, nucleic acid, or immunologic methods.
known to increase the risk of transmission of The restriction endonuclease cleavage patterns
infection with human immunodeiciecy virus of the DNA of HSV-1 and HSV-2 are unique allow
(HIV). unequivocal typing of the isolates. HSV type-
(i) In male patients: The lesions typically specific DNA probes, specific DNA primers for PCR
develop on the glans or shaft of the penis and and antibodies are also available for detecting and
occasionally in the urethra; (ii) In female differentiating HSV-1 and HSV-2.
patients: The lesions may be seen on the vulva,
vagina, cervix, perianal area, or inner thigh; C. Serology
(iii) Inguinal lymphadenopathy: In patients
of both sexes; (iv) Herpetic proctitis: in Rise in titer of antibodies may be demonstrated by
homosexuals; (v) Association between HSV-2 ELISA, neutralization or complement fixation tests.
and carcinoma of the cervix uteri: There have
been several reports of an association between D. Polymerase Chain Reaction
HSV -2 and carcinoma of the cervix uteri but a
causal relationship has not been established. Polymerase chain reaction is useful for detecting
viral DNA in cerebrospinal fluid when herpetic
7. Neonatal Herpes: Transplacental infection with
HSV1 or 2 can lead to congenital malformations. infection of the CNS is suspected.
8. Infections in immunocompromised hosts.
Treatment
Laboratory Diagnosis Idoxuridine used topically in eye and skin infections
was one of the first clinically successful antiviral
1. Specimens
agents. Acyclovir and vidarabine enabled the effective
2. Microscopy management of deep and systemic infections.
i. Tzanck smear: Characteristic cytopathologic Intravenous, oral, and topical preparations are
effects (CPEs) can be identified in a Tzanck available. Drug-resistant virus strains may emerge.
smear, Papanicolaou smear, or biopsy Early treatment with intravenous acyclovir has
specimen. CPEs include syncytia, “ballooning” improved the outcome of encephalitis.Valaciclovir
cytoplasm, and Cowdry type A intranuclear and famciclovir are more effective oral agents.
inclusions. When resistance to these drugs develop, drugs like
The Tzanck smear is a rapid, fairly sensitive foscarnet which are independent of viral thymidine
and inexpensive diagnostic method. Smears kinase action may be useful.
are prepared from the lesions, preferably
from the base of vesicles and stained with Herpes virus simiae: B virus
1% aqueous solution of toluidine blue ‘0’ for
15 seconds. Multinucleated giant cells with Herpes B virus of Old World monkeys is highly
faceted nuclei and homogeneously stained pathogenic for humans. This virus was isolated
‘ground glass’ chromatin (Tzanck cells) by Sabin and Wright (1934) from the brain of a
constitute a positive smears. laboratory worker who developed fatal ascending
myelitis after being bitten by an apparently healthy
ii. Electron microscopy: The virus particle may
monkey. It came to be known as the ‘B’ virus from
also be demonstrated under the electron
the initials of this patient.
microscope.
The virus is transmitted to humans by monkey
iii. Fluoroscent ant ibody technique: The
bites or saliva or even by tissues and cells widely
fluorescent antibody test on brain biopsy
used in virology laboratories.
specimens provides reliable and speedy
diagnosis in encephalitis. Virus isolation or serologic tests can be used to
establish the diagnosis of B-virus infections.
B. Virus Isolation
Varicella-zoster virus
Primary human embryonic kidney, human amnion
and many other cells are susceptible, but human Varicella-zoster virus (VZV) causes chickenpox
diploid fibroblasts are preferred. Specimen such (varicella) and, with recurrence, causes herpes
as vesicle fluid, spinal fluid, saliva and swab may zoster, or shingles. Varicella (chickenpox) and
www.ebook3000.com
412 | Section 4: Virology
herpes zoster are different manifestations of the residual immunity. It is believed that years after the
same virus infection. Thus, chickenpox is ‘caught’ initial infection, when the immunity has waned,
but not zoster. the virus may be reactivated, and triggered by
some precipitating stimulus, travel along the sen
Properties of the Virus sory nerve to produce zoster lesions on the area of
Varicella-zoster virus is morphologically identical the skin or mucosa supplied by it. It usually starts
to herpes simplex virus. It can be grown in cultures with severe pain in the area of skin or mucosa
of human fibroblasts, human amnion or HeLa cells. supplied by one or more groups of sensory nerves
Cytopathic changes are more focal and spread and ganglia. The most common sites are the areas
much more slowly than those induced by HSV. Only innervated by spinal cord segments D3 to L2 and
one antigenic type of VZV is known. the trigeminal nerve, particularly, its ophthalmic
branch. Within a few days after onset, a crop of
vesicles appears over the skin supplied by the
Varicella (Chickenpox) affected nerves.
Varicella (chickenpox) is one of the mildest highly
communicable and most common of childhood Complications
infections. 1. Postherpetic pain: In the affected area is
It is usually a mild disease of childhood and is frequent, particularly in the elderly.
normally symptomatic, although asymptomatic 2. Ophthalmic zoster.
infection may occur. The portal of entry of the 3. Generalized zoster
virus is the respiratory tract or conjunctiva. After an 4. Ramsay Hunt syndrome: It is a rare form of
incubation period of about two weeks (7–23 days) zoster affecting the facial nerve, with eruption
the lesions begin to appear. on the tympanic membrane and external
In children, there is little prodromal illness and auditory canal, and often a facial palsy.
the disease is first noticed when skin lesions appear.
Initially macular, the rash rapidly evolves through
papules to the characteristic clear vesicles. The
Immunity
rash is characteristically centripetal in distribution, Previous infection with varicella is believed to
affecting mainly the trunk, and is very superficial confer lifelong immunity to varicella.
without involving the deeper layers of the skin. The The development of varicella-zoster virus-
rash appears in successive crops, so that all stages specific cell-mediated immunity is important
of the eruption can be seen at the same time. It in recovery from both varicella and zoster.
matures very quickly, beginning to crust within 48 Appearance of local interferon may also contribute
hours.Recovery is usually uneventful. to recovery.
Complications: Primary infection is usually more
severe in adults than in children. The rash may Laboratory Diagnosis
become hemorrhagic. Varicella pneumonia is Diagnosis is usually clinical.
more common in adults. A variety of organs may 1. Microscopy: Multinucleated giant cells and type
be affected with complications like myocarditis, A intranuclear inclusion bodies may be seen
nephritis, acute cerebellar ataxia, meningitis in smears prepared by scraping the base of the
and encephalitis. Secondary bacterial infections, early vesicles (Tzanck smear) and stained with
usually due to staphylococci or streptococci, may toluidine blue, Giemsa or Papanicolou stain.
occur. Reyes’ syndrome may follow varicella in Direct examination by electron microscopy will
some cases with a history of administration of reveal herpes particles.
salicylates. 2. Virus isolation: Virus isolation can be
attempted by inoculating human amnion,
Herpes zoster (Shingles, Zona) human fibroblast, HeLa or Vero cells.
3. Virus antigen: The virus antigen can be
The name is derived from Heroein, meaning to detected in scrapings from skin lesions by
creep; Zoster, meaning girdle. immunofluorescence, and in vesicle fluid by
While varicella is typically a disease of child counterimmunoelectrophoresis with zoster
hood, herpes zoster is one of old age. immune serum. ELISA and PCR techniques
are also in use.
Pathogenesis 4. Serological diagnosis: A rise in specific
Herpes zoster usually occurs in persons who antibody titer can be detected in the patient’s
had chickenpox several year earlier. The virus serum by various tests, including fluorescent
remaining latent in the sensory ganglia, may leak antibody, latex agglutination, and enzyme
out at times but is usually held in check by the immunoassay.
Chapter 58: Herpesviruses | 413
Prophylaxis and Treatment and the reticuloendothelial system. Clinical
features—include intrauterine growth retar
Active Immunization
dation, jaundice, hepatosplenomegaly, throm
A live-attenuated varicella vaccine was developed bocytopenia, microcephaly, chorioretinitis and
by Takahashi in Japan in 1974 by attenuating a cerebral calcification resembling congenital
strain of varicella virus (Oka strain, so named after toxoplasmosis.
the patient) by serial passage in tissue culture.
Perinatal infection may be acquired from the
This vaccine, given by subcutaneous injection,
infected mother through genital secretions or
has been found to be immunogenic with good
breast milk.
antibody response but it was very labile and had
to be stored frozen. A modified lyophilized form of D. Postnatal infection: This can be acquired in
the vaccine is now available, which can be stored many ways-such as saliva, sexual transmission,
between 2°C and 8°C. It is recommended as a single blood transfusion and donated organs
subcutaneuos dose for childern 1–12 years old, and
for those older as 2 doses 6–10 weeks apart. It is safe Laboratory Diagnosis
and effective. It is not considered safe in pregnancy. A. Specimens: CMV can be isolated from the
urine, saliva, breast milk, semen, cervical
Passive Immunization secretions and blood leucocytes.
Varicella-zoster immunoglobulin (VZIG) prepared B. Demonsration of cytom egalic cell: The
from the patients convalescing from herpes zoster histologic hallmark of CMV infection is the
seems to be of some use in preventing or modifying cytomegalic cell, which is an enlarged cell
severe disease in immunodeficient patients. that contains a dense, central, “owl’s-eye,”
basophilic intranuclear inclusion body. The
Laboratory Diagnosis inclusions are readily seen with Papanicolaou
or hematoxylin-eosin staining
Diagnosis is easily made clinical. Laboratory
C. lsolation of virus: Human cytomegaloviruses
diagnosis is as for chickenpox.
can be grown in human fibroblast cultures.
Cultures have to be incubated for prolonged
Treatment periods as the cytopathic effects (swollen
It is as for chickenpox. refractile cells with cytoplasmic granules) are
slow in appearance.
Cytomegalovirus D. DNA probes: DNA probes are used to directly
detect the CMV antigens in tissues or fluids.
Cytomegaloviruses (CMV), formerly known as
salivary gland viruses, are a group of ubiquitous E. Polymerase chain reaction (PCR): To directly
herpesviruses of humans and animals. detect the genome in tissues or fluids.
F. Serology: IgM antibodies suggests a current
infection and can be detected in serum by
Pathogenesis
ELISA.
The congenital, oral, and sexual routes, blood
transfusion, and tissue transplantation are the major Treatment and Prevention
means by which CMV is transmitted. Congenitally
infected infants have viruria for upto 4–5 years. They Ganciclovir (dihydroxypropoxymethyl guanine)
are highly infectious in early infancy. and foscarnet (phosphonoformic acid) have been
approved for the treatment of CMV infections.
A. Normal hosts: Primary cytomegalovirus
infection of older children and adults is Prevention is indicated only in high risk
usually asymptomatic but occasionally causes cases. Screening of blood and organ donors and
a spontaneous infectious mononucleosis administration of CMV immunoglobulins have
syndrome. been employed in prevention.
B. Immunocompromised hosts: This occurs No vaccine is available.
in transplant recipients, cancer patients on
chemotherapy, and more particularly in the
Epstein–Barr virus
HIV infected. Epstein–Barr virus (EBV) is in some respects the
C. Congenital and perinatal infections: Intra most sinister herpesvirus, for its association with
uterine infection leads to fetal death or malignant disease is now well established. Only
cytomegalic inclusion disease of the newborn human and some subhuman primate B cells have
which is often fatal. Cytomegalic inclusion receptors for the virus. EBV infected B cells are
disease of newborns is characterized by transformed so that they become capable of con
involvement of the central nervous system tinuous growth in vitro.
www.ebook3000.com
414 | Section 4: Virology
Pathogenesis A. Infectious Mononudeosis (Glandular Fever)
Epstein–barr virus (EBV) is commonly transmitted This is an acute self-limited illness usually seen
by infected saliva and initiates infection in the in nonimmune young adults. The incubation
oropharynx. The virus enters the pharyngeal period is 4–8 weeks.Infectious mononucleosis is
epithelial cells through CR2 (or CD21) receptors. characterized by high fever, malaise, pharyngitis,
It multiplies locally, invades the bloodstream lymphadenopathy (swollen glands), and, often,
and infects B lymphocytes in which two types hepatosplenomegaly. A mild transient rash may
of changes are produced. In most cases, the virus be present. Some patients treated with ampicillin
becomes latent inside the lymphocytes, which may develop a maculopapular rash due to immune
become transformed or ‘immortalized so that complex reaction to the drug. In most patients
they become capable of indefinite growth in vitro. the spleen is palpable and there is some liver
Lytic infection is a second type of effect, shown by dysfunction, occasionally with frank jaundice. The
a few infected B cells with cell death and release of typical illness is self-limited and lasts 2–4 weeks.
mature progency virions. Complications are rare but some are serious:
The classic lymphocytosis associated with i. Acute airway obstruction.
infectious mononucleosis results mainly from the ii. Splenic rupture.
activation and proliferation of T cells. These appear iii. Neurological complications include meningitis,
as atypical lymphocytes (also called Downey encephalitis and the Guillain-Barre syndrome.
cells).
Intermittent reactivation of the latent EB virus B. Oral Hairy leukoplakia
leads to clonal proliferation of infected B cells. In This lesion is a wart-like growth that develops
immunocompetent subjects, this is kept in check on the tongue in some HIV-infected persons and
by activated T cells. In the immunodeficient, B transplant patients. It is an epithelial focus of EBV
cell clones may replicate unchecked, resulting in replication.
lymphomas. Hyperendemic malaria prevalent in
Africa is believed to be responsible for the immune C. Chronic Disease
impairment in children with Burkitt’ lymphoma. Epstein-barr virus (EBV) can cause cyclic recurrent
The frequency of lymphomas seen in many types of disease in some people. This disorder is different
immunodeficiencies, most typically in AIDS, may from chronic fatigue syndrome, which has another
have a similar pathogenesis. etiology.
Nasopharyngeal carcinoma seen in men of
Chinese origin. Genetic and environmental factors D. Burkitt’s lymphoma
are said to be important in the and EB virus DNA
Epstein–barr virus (EBV) is associated with the
is regularly found in the tumour cells.
development of Burkitt’s lymphoma (a tumor of
the jaw in African children and young adults).
Epidemiology Most African tumors (>90%) contain EBV DNA
Epstein–Barr (EB) virus is ubiquitous in all human and express EBNA1 antigen. Malaria, a recognized
populations. Infection with EBV is transmitted by cofactor.
saliva, and requires intimate oral contact.
The source of infection is usually the saliva E. Nasopharyngeal Carcinoma
of infected persons who shed the virus in This cancer of epithelial cells is common in males
oropharyngeal secretions. Saliva sharing between of Chinese origin. It mainly affects people aged
adolescents and young adults often occurs during 20–50 years, males preponderating. EBV DNA is
kissing; thus, the nickname “kissing disease” for regularly found in nasopharyngeal carcinoma
infectious mononucleosis. Children can acquire (NPC) cells. Genetic and environmental factors
the virus at an early age by sharing contaminated are believed to be important in the development
drinking glasses. Infection may also follow blood of nasopharyngeal carcinoma.
or marrow transfusion but these are rare events.
F. Lymphoproliferative Diseases in
Clinical conditions
Immunodeficient Hosts
1. Infectious mononucleosis.
Immunodeficient patients are susceptible to EBV
2. EBV associated malignancies:
induced lymphoproliferative diseases that may
a. Burkitt’s lymphoma. be fatal. AIDS patients are susceptible to EBV-
b. Lymphomas in immunodeficient persons. associated lymphomas and hairy oral leukoplakia
c. Nasopharyngeal carcinoma. of the tongue.
Chapter 58: Herpesviruses | 415
Laboratory Diagnosis IgG anti-VCA antibody indicates past or recent
infection and persists throughout life.
1. Differential White Cell Count
Early antigen (EA) antibodies are often found in
Blood examination during the initial phase may patients with Burkitt’s lymphoma or nasopharyngeal
show leucopenia. Later there is a prominent carcinoma.
leucocytosis with the appearance of abnormal Antibodies to the EB nuclear antigen (EBNA)
mononuclear cells. These atypical mononuclear reveal past infection with EBV, though detection
cells are lymphoblasts derived from T cells reactive of a rise in anti-EBNA antibody would suggest a
to the virus infection. The blood picture may primary infection.
sometimes resemble lymphocytic leukemia.
4. Antigen Detection
2. Paul–Bunnell Test
Epstein-barr virus (EBV) antigen can be detected
B cells transformed by EBV undergo polyclonal by immunofluorescence using monodonal
expansion. These antib odies include an IgM antibodies.
heterophile antibody. Agglutination of horse or
sheep red cells by serum absorbed to exclude a 5. Nucleic Acid Hybridization
natural antibody is the basis of this test. It is the most sensitive means of detecting EBV in
patient materials.
Procedure
Inactivated serum. 56°C for 30 minutes in doubling 6. Virus Isolation
dilutions is mixed with equal volumes of a 1% Epstein-barr virus (EBV) can be isolated from
suspension of sheep erythrocytes. After incubation saliva, peripheral blood, or lymphoid tissue by
at 37°C for four hours the tubes are examined for immortalization of normal human lymphocytes,
agglutination. An agglutination titer of 100 or above usually obtained from umbilical cord blood.
is suggestive of infectious mononucleosis.
7. Polymerase Chain Reaction.
Confirmation
For confirmation, differential absorpt ion of Human Herpesviruses 6 (HHV6)
agglutinins with guineapig kidney and ox red cells Human herpesviruses 6 was first isolated from the
is necessary. The Forssman antibody induced blood of patients with AIDS and grown in T-cell
by injection of horse serum is removed by cultures. HHV6 is lymphotropic and ubiquitous. It
treatment with guineapig kidney and ox red cells. is present in the saliva of most adults and is spread
Normally occurring agglutinins are removed by by oral secretions. Two variants are recognized,
guineapig kidney, but not by ox red cells. Infectious A and B. Variant B is the cause of the mild but
mononucleosis antibody is removed by ox red cells common childhood illness ‘exanthem subitum’
but not guinea pig kidney (Table 58.2). (roseola infantum or ‘sixth disease’). In older age
groups, it has been associated with infectious
3. Epstein–Barr Virus-specific Antibodies mononucleosis syndrome, focal encephalitis and,
Tests are also available for the demonstration of in the immunodeficient, with pneumonia and
specific EB virus antibodies. Immunofluorescence disseminated disease.
and ELISA are commonly employed. Laboratory dignosis: HHV6 can be isolated
from peripheral blood mononuclear cells in early
The IgM antibody to VCA (virus capsid antigen)
febrile stage of the illness by co-cultivation with
appears soon after primary infection and disappears
lymphocytes.
in 1–2 weeks and indicative of current infection. The
Virus antigen-can be detected by immuno
fluroscence using monoclonal antibodies. ELISA
Table 58.2: Differential absorption test for Paul- is used for detecting both antigen and antibidies
Bunnell antibody in patient serum.
Result of absorption Ox red cells
on guinea pig kidney Human Herpesvirus 7 (HHV7)
Normal serum Absorbed Absorbed
Like HHV6, HHV7 also appears to be widely distri
Antibody after Absorbed Not absorbed buted and transmitted through saliva. However,
serum therapy
HHV7 remains an orphan virus with no disease
Infectious Not absorbed Absorbed association. It shares with HIV the same CD4
mononucleosis
receptor on T cells.
www.ebook3000.com
416 | Section 4: Virology
It may cause an illness resembling infectious
Laboratory diagnosis—Atypical lymphocytes are
mononucleosis, some cases of exanthem subitum. probably the earliest detectable indication of an
EBV infection
Human Herpesvirus 8 (HHV8) Paul-Bunnell test is most frequently used test
to detect heterop hile antibodies in infectious
A new herpesvirus, also called Kaposi’s sarcoma- mononucleosis patients
associa ted herpesvirus (KSHV) is the cause Human Herpesvirus 8 (HHV8)
of Kaposi’s sarcomas, vascular tumors of mixed It is the cause of Kaposi’s sarcomas, vascular tumors
cellular composition, and is involved in the of mixed cellular composition, body cavity-based
lymphomas occurring in AIDS patients and of
pathogenesis of body cavity-based lymphomas multicentric Castleman’s disease.
occurring in AIDS patients and of multicentric
Castleman’s disease.
It appears to be sexually transmitted among Important questions
men who have sex with men. Infections are
common in Africa with infections acquired early in 1. Name various viruses of the family Herpesviridae.
life by nonsexual routes, possibly through contact Discuss the various infections caused by herpes
with oral secretions. simplex virus types 1 and 2.
Diagnosis depends mainly on detection of viral 2. Classify human herpesviruses. Discuss briefly
DNA by PCR. their pathogenesis and laboratory diagnosis.
3. Describe the lesions caused by herpes simplex
virus and their laboratory diagnosis.
Key Points
4. Write short notes on:
Herpesviruses are DNA viruses a. Varicella-zoster virus
Human herpesviruses include human herpesvirus b. Varicella or chickenpox
1 (HV1) to human herpesvirus 8 (HV-8). HHV 3, HHV c. Cytomegalovirus
4 and HHV 5 are varicella-zoster virus, Epstein-Barr d. Epstein-Barr virus (or) EB virus
(EB) and cytomegalovirus (CMV) respectively. Herpes e. Infectious mononucleosis
simplex virus type 1 and 2 are designated as HHV 1 f. Paul-Bunnel test
and HHV 2
g. Human herpesvirus 6 (HHV6)
Clinical syndromes
HSV-l is generally transmitted orally; HSV-2 is gener
ally transmitted sexually Multiple choice questions (mcqs)
HSV-1 causes acute herpetic gingivostomatitis, acute
herpetic pharyngotonsillitis, herpes labial is, herpes 1. Which of the following infection/s is/are caused by
encephalitis, eczema herpeticum, and herpetic herpes simplex virus type I?
whitlow. HSV-2 causes genital herpes, neonatal a. Acute gingivostomatitis
infection, and aseptic meningitis b. Keratoconjunctivitis
Laboratory diagnosis: (A) Direct microscopic examin c. Encephalitis
ation of cells from base of lesion; (B) Cell culture; d. All of the above
(C) Assay of tissue biopsy, smear, or vesicular fluid 2. All the following infections are caused by HSV-2
for HSV antigen; (D) HSV type distinction (HSV-l vs. except:
HSV-2) type a. Neonatal infection
Varicella Zoster Virus (VZV)—causes chickenpox b. Aseptic meningitis
(varicella) and herpes zoster or shingles, two distinct
c. Herpetic whitlow
clinical entities in humans
d. Genital herpes
Virus causes lifelong infection
3. All the following infections are caused by HSV-2
Cytomegalovirus
except:
Virus is transmitted orally and sexually, in blood
a. Neonatal infection
transfusions, in tissue transplants, in utero, at birth,
and by nursing b. Aseptic meningitis
Clinical syndromes—Congenital CMV infection,
c. Herpetic whitlow
acquired CMV infection, CMV infec t ion in d. Genital herpes
immunocompromised patients, and CMV infection 4. Ramsay–Hunt syndrome can be caused by:
in immunocompetent adult hosts. CMV generally a. Herpes simplex virus
causes subclinical infection b. Herpes-zoster virus
Epstein–Barr Virus c. Cytomegalovirus
EBV causes heterophile antibody-positive infectious d. Epstein–Barr virus
mononucleosis and has been causally associated
5. Shingles is caused by:
with Burkitt’s lymphoma, Hodgk in’s disease, and
a. Varicella–zoster virus
nasopharyngeal carcinoma
b. Cytomegalovirus
Transmission occurs via saliva, close oral contact
(“kissing disease”), or sharing of items c. Epstein–Barr virus
d. Herpes simplex virus type 1
Chapter 58: Herpesviruses | 417
6. Owl’s eye is the characteristic feature of the cell 8. Paul–Bunnell test is used for serodiagnosis of:
infected by: a. Infectious mononucleosis
a. Herpes simplex virus b. Genital herpes
b. Epstein-Barr virus c. Neonatal infection
d. Aseptic meningitis
c. Cytomegalovirus
9. Which of the following viruses is associated in
d. Human herpesvirus 8
causation of Kaposi’s sarcoma?
7. Which of the following malignancies is/are associated a. Herpes simplex virus
with Epstein–Barr virus? b. Herpesvirus simiae
a. Burkitt’s lymphoma c. Human herpesvirus 8
b. Nasopharyngeal carcinoma d. Human herpesvirus 6
c. B cell lymphoma Answers (MCQs)
d. All of the above 1. d; 2. c; 3. c; 4. b; 5. a; 6. c; 7. d; 8. a; 9. c.
www.ebook3000.com
59
Chapter
Adenoviruses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe diseases associated with adenovirus
be able to: ∙∙ Describe adenovirus-associated viruses (AAV).
∙∙ Describe morphology of adenovirus
C. Gastrointestinal Disease
Diarrhea: Some fastidious adenoviruses can cause
Fig. 59.1: Morphology of advenovirus diarrheal disease in children (for example, types
40 and 41).
A. Respiratory Diseases
1. Pharyngitis: Adenoviruses are the major cause D. Other Diseases
of nonbacterial pharyngitis and tonsillitis,
Mesenteric adenitis and intussusception in children,
presenting as febrile common cold. Types 1–7
pertussis-like illness, acute hemorrhagic cystitis with
are commonly responsible.
dysuria and hematuria in young boys, musculoskeletal
2. Pneumonia: Adenovirus types 3 and 7 are
disorders, genital and skin infections.
associated with pneumonia in adults resembling
primary atypical pneumonia. Adenoviruses,
E. Systemic Infection in Immunocompromised
particularly types 3, 7, and 21 are thought to be
responsible for about 10–20% of pneumonias Patients Include Pneumonia and Hepatitis
in childhood. In infants and young children
type 7 may lead to more serious and even fatal Laboratory Diagnosis
pneumonia.
3. Acute respiratory diseases: Adenoviruses are A. Specimens
the cause of an acute respiratory disease (ARD) Depending on the clinical disease, virus may be
syndrome among military recruits. Serotypes 4, recovered from stool or urine or from a throat,
7 and 21 are the agents commonly isolated. conjunctival, or rectal swab.
www.ebook3000.com
420 | Section 4: Virology
B. Direct Demonstration of Virus deaminase deficiency), cystic fibrosis, lysosomal
i. Electron microscopy: Virus particles may storage diseases, and even cancer.
be seen directly in stool extracts by electron
Adenovarus-associated Viruses
microscopy.
The adenovirus-associated viruses (AAVs) are
ii. Virus antigen: The presence of viral antigen
members of the parvoviridae. They are about 22
in the nasopharynx may be identified by
nm in diameter, appear to be more hexagonal
immunofluorescence with group-specific
than circular in outline and contain insufficient
antibodies (polyclonal or monoclonal) directly
single-stranded DNA to replicate on their own.
on aspirates. Alternatively, viral antigen may be
They form a genus, dependoviruses, indicating their
detected by enzyme immunoassays.
dependence on adenoviruses (or herpes simplex
iii. Viral DNA: It is also possible to detect viral
virus) to provide the missing functions.
DNA directly from feces by polyacrylamide gel
They can be detected by electron microscopy
electrophoresis.
and complement fixation or immunofluorescence
iv. Latex agglutination method: It uses latex
with specific antisera. Types 1, 2 and 3 are of human
particles coated with specific antibody to each
origin and cause natural infection, while type 4 is of
virus.
simian origin. Their pathogenic role is uncertain.
True AAV (also known as adenovirus satellite
C. Virus Isolation
virus) has not been implicated in clinical disease
The clinical specimens are inoculated in tissue and its pathogenic role is uncertain.
culture such as HeLa, Hep., KB and human embryo
kidney cells. The development of characteristic Key Points
cytopathic effects-rounding and clustering of Adenoviruses are nonenveloped, icosahedral, DNA
swollen cells—indicates the presence of adenovirus viruses. The virion has the appearance of a space
in inoculated cultures. vehicle
Isolates can be identified as adenoviruses by Adenoviruses infect and replicate in epithelial cells
of the respiratory tract, eye, gastrointestinal tract,
immunofluorescence tests using an antihexon urinary bladder, and liver.
antibody and infected cells hemagglutination Adenovirus-associated viruses (AAVs) are members of
inhibition and neutralization tests. the parvoviridae. They form a genus, dependoviruses,
Characterization of viral DNA by hybridization indicating their dependence on adenoviruses
or by restriction endonuclease digestion patterns (or herpes simplex virus) to provide the missing
can identify an isolate as an adenovirus and group it. functions.
Papovaviruses
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Describe the following: Papillomaviruses; Polyomaviruses.
www.ebook3000.com
422 | Section 4: Virology
and 18 are more commonly associated with lesions 4. BK Polyomaviruses
of greater severity and invasive cancer. The human polyomaviruses (BK and JC) have been
isolated from immunocompromised patients. It
Laboratory Diagnosis was named after the initials of the person from
A. Morphological identification: HPV infection whom it was first isolated. BK virus isolated from
may be readily diagnosed when there are the urine of a patient with kidney transplant.
typical clinical lesions.
Subclinical infection requires labo ratory Laboratory Diagnosis
confirmation using:
1. Electron Microscopy
1. Cytological and histological detection.
2. Immunocytochemical detection: HPV capsid Human polyomavirurses can be detected by
antigen in sections of tissues or in cell smears electron microscopy from brain tissue in a case
can be detected by immunoperoxidase test of PML (JC virus) and from the urine of a renal
using antiserum. It detects all the genital transplant case (BK virus).
HPV types.
3. Molecular methods: Amplification of HPV 2. Virus Isolation
DNA by polymerase chain reaction (PCR). Human fetal glial cell culture and human diploid
B. Serology: Are appropriate for epidemiological fibroblasts are used for the isolaton of JC polyomavirus
studies. BK virus respectively. Hemaggltination inhibition is
used to differentiate these two viruses.
Polyomaviruses
These are small viruses (diameter 45 nm) that 3. Viral Antigen Detection
possess a circular genome of double-stranded DNA The brain biopsy or autopsy material can be examined
enclosed within a nonenveloped capsid exhibiting directly for JCV antigen by immunofluorescence or
icosahedral symmetry. immunoperoxidase staining.
The name is derived from ‘poly’ (many) and
‘oma’ (tumor). The viruses are species-specific. 4. Viral nucleic Acid Detection
Recognized members of this group include:
Viral nucleic acid can be detected by nucleic acid
1. Mouse polyomavirus
hybridization and polymerase reaction (PCR).
2. Simian vacuolating virus 40 (SV 40)
3. JC virus (JCV) 5. Cytopathology
4. BK virus (BKV)
Exfoliated urinary epithelial cells show the
1. Mouse Polyomavirus presence of enlarged deeply stained basophilic
nuclei with a single inclusion.
It causes harmless infections in mice by natural
routes. However, it induces different types of
Key Points
malignant tumors when injected into infant rodents.
Papovaviruses are double-stranded DNA viruses and
has two genera, Papillomavirus and Polyomavirus
2. Simian Vacuolating Virus 40 (SV 40) Papillomaviruses are species-specific DNA viruses
The simian vacuolating virus (SV 40) was isolated that infect the squamous epithelia and mucous
from uninoculated rhesus and cynomolgus membranes of vertebrates, including man
monkey kidney tissue cultures. SV40 is oncogenic Over seventy types of human papillomaviruses
in newborn hamsters. Its only medical importance (HPVs) are now recognized
Papillomaviruses cause several different kinds of
is that because of its oncogenic potential, live viral
warts in humans, including cutaneous warts, genital
vaccines should be manufactured only in monkey warts, respiratory papillomatosis, oral papillomas
kidney tissue cultures tested and found free from and cancer
SV 40 infection. Polyomaviruses include: Mouse polyomavirus, simian
virus 40 (SV 40) of monkeys, JC virus (JCV) and BK
3. JC Virus (JCV) polyomavirus.
www.ebook3000.com
61
Chapter
Parvoviruses
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Describe Parvoviruses.
www.ebook3000.com
6262
Chapter
Picornaviruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Differentiate coxsackie A and coxsackie B viruses
be able to: ∙∙ Describe diseases caused by coxsackie viruses
∙∙ Describe enteroviruses ∙∙ D escribe the following: acute hemorrhagic
∙∙ Discuss prophylaxis against poliomyelitis conjunctivitis; Rhinoviruses.
∙∙ Differentiate live and killed polio vaccines
www.ebook3000.com
Chap-62.indd 427 15-03-2016 11:22:28
428 | Section 4: Virology
B. Culture 2. Oral polio vaccine (OPV) (Sabin vaccine)
Primary monkey kidney cells are usually employed, Oral polio vaccine (OPV) was described by Sabin
though any other human or simian cell culture may in 1957. It contains live attenuated virus (types
be used. The virus growth is indicated by typical 1, 2 and 3) grown in primary monkey kidney or
cytopathic effects in 2–3 days. An isolated virus is human diploid cell. cultures. The use of molar
identified and typed by neutralization with specific MgCl2 or sucrose stabilizes the vaccine against heat
antiserum. inactivation, particularly under tropical conditions.
It can be given to young infants, as the maternal
The mere isolation of poliovirus from feces does
antibody has little effect on intestinal infection.
not constitute a diagnosis of poliomyelitis. Virus
isolation must be interpreted along with clinical The shelf life of the vaccine at 4–8°C is four
and serological evidence. months and at –20°C is two years. Improper storage
conditions and ‘cold chain’ failure may be partly
responsible for the apparent failure of OPV to
C. Serological Tests
control poliomyelitis in the developing countries.
Serodiagnosis is less often employed. Antibody rise
can be demonstrated in paired sera by neutralisa Criteria of attenuated strains for live vaccine
tion or complement fixation tests. 1. They should not be neurovirulent.
2. They should be able to set up intestinal infect
Immunity ion following feeding and should induce an
Immunity is permanent to the type causing immune response.
the infection. Humoral immunity provided by 3. They should not acquire neurovirulence after
circulating and secretory antibody is responsible serial enteric passage.
for protection against poliomyelitis. 4. They should possess stable genetic character
The virus also induces cell mediated immunity, istics (markers) by which they can be differenti
but its importance appears to be uncertain. ated from the wild virulent strains.
Markers for differentiating the wild from the
Prophylaxis attenuated strains
Immunization 1. d marker: Wild strains will grow well in low
Both killed and live attenuated vaccines are levels of bicarbonate but avirulent strains will
available. Two types of vaccines are used throughout not.
the world; they are: 2. rct 40: Wild strains grow well at 40°C, while
1. Inactivated (Salk) polio vaccine (lPV) avirulent strains grow poorly.
2. Oral (Sabin) polio vaccine (OPV). 3. MS: Wild strains grow well in a stable cell line
of monkey kidney, while avirulent strains grow
1. Inactivated polio vaccine(IPV)—Salk’s killed poorly.
polio vaccine
4. McBride’s intratypic antigenic marker shown
By 1953, Salk had developed a killed vaccine.
by the rate of inactivation by specific antiserum.
Salk’s killed polio vaccine is a formalin inactivated
preparation of the three types of poliovirus grown in
monkey kidney tissue culture. Killed vaccine is given Immunization Schedule
by injection and is therefore called inactivated or The WHO Program on Immunization (EPI) and
injectable poliovaccine (lPV). The primary or initial the National Immunization Program in India
course of immunization consists of 4 inoculations. recommend a primary course of 3 doses of OPV
The first 3 doses are given at intervals of 1–2 months at one-month intervals, commencing the first
and 4th dose 6–12 months after the third dose. First dose when the infant is 6 weeks old. One booster
dose is usually given when the infant is 6 weeks old dose of OPV is recommended 12–18 months later.
to ensure that immune response is not impaired by It has been recommended that in the tropics, the
residual maternal antibodies. Additional doses are number of doses of vaccine be increased to five, in
recommended prior to school entry and then every. order to enhance seroconversion in the vaccinees.
5 years until the age of 18. It is very important to complete vaccination of all
IPV induces humoral antibodies (lgM, IgG infants before 6 months of age. This is because most
and IgA serum antibodies) but does not induce polio cases occur between the ages of 6 months
intestinal or local immunity. The circulating and 3 years.
antibodies protect the individual against paralytic There has been much controversy about the
polio, but do not prevent reinfection of the gut by relative merits of killed and live vaccines. The
wild viruses. In the case of an epidemic, IPV is differences between IPV and OPV are given in
unsuitable. Table 62.3.
www.ebook3000.com
Chap-62.indd 429 15-03-2016 11:22:28
430 | Section 4: Virology
Table 62.4: Features of coxsackieviruses A and B children and adults but are most threatening in
infection in the laboratory newborns.
Features Coxsackievirus A Coxsackievirus B 3. Aseptic meningitis with paralyisis.
1. Growth in + + 4. Juvenile diabetes
monkey Juvenile diabetes has been claimed to be associated
kidney with coxsackie B4 infection.
2. Effect in Generalized Patchy focal
suckling myositis myositis 5. Neonatal infections
mice Flaccid paralysis Spastic paralysis, Transplacental and neonatal transmission has
Death within a Necrosis of the been demonstrated with coxsackie B viruses,
week brown fat and, resulting in a serious disseminated disease that
often, pancreatitis,
may include hepatitis, meninoencephaIitis and
hepatitis,
myocarditis and adrenocortical involvement.
encephalitis
6. Chronic fatigue syndrome
3. Types 23 (1–24*) 6 (1–6) There is some evidence of a possible association
*Coxsackievirus A23 now classified as echovirus 9. between chronic fatigue syndrome and infection
with enteroviruses, particularly coxsackie B viruses.
by the fecal–oral route. Young infants are most
commonly affected. The incubation period of Laboratory Diagnosis
coxsackievirus infection ranges from 2 days to 9 A. Virus Isolation
days. The clinical manifestations of infection with
yarious coxsackieviruses are diverse and may The virus is isolated readily from throat washings
present as distinct disease entities (Table 62.4). conjunctival swabs, throat swabs and feces.
i. Inoculation into suckling mice: Specimens
Group A Viruses are inoculated into suckling mice. In suckling
mice, signs of illness appear usually within 3–8
1. Herpangina (vesicular pharyngitis)
days with group A strains and 5–14 days with
Herpangina is a severe febrile pharyngitis and is a
group B strains. Identification is by studying
common clinical manifestation of coxsackie group
the histopathology in infected mice and by
A infection in children.
neutralization tests.
2. Aseptic meningitis ii. Tissue culture: Specimens are inoculated into
Aseptic meningitis is caused by all types of tissue cultures. Of group A coxsackieviruses,
group B coxsackieviruses and by many group A only A7 and A9 grow in monkey kidney cells,
coxsackieviruses, most commonly A7 and A9. and coxsackievirus A21 can be grown in
3. Hand-foot-and-mouth disease HeLa, HEp2 or human embryonic kidney cell
Hand-foot-and-mouth disease is a vesicular exan cultures. All group B coxsackieviruses grow
them and is characterized by oral and pharyngeal readily in monkey kidney cell cultures.
ulcerations and a vesicular rash of the palms and
soles. This disease has been associated particularly B. Serology
with coxsackievirus A16, but A5 and Al0 have also Serologic tests are difficult to evaluate (because of
been implicated. the multiplicity of types).
4. Respiratory infections
A number of the enteroviruses have been asso C. Nucleic Acid Detection
ciated with common colds; among these are Reverse transcription–polymerase chain reaction
coxsackieviruses A21, A24, B1, and B3–5. tests can be broadly reactive.
Group B Viruses Prevention: Vaccination is not practicable as there
1. Epidemic myalgia or Bornholm disease are several serotypes and immunity is type-specific.
It is so called because it was first described on the
Danish island of Bornholm. It is an acute illness ECHOVIRUSES
in which patients have a sudden onset of fever Echoviruses (enteric cytopathogenic human
and unilateral low thoracic, pleuritic chest pain orphan viruses) are grouped together because
that may be excruciating. Coxsackie B virus is the they infect the human enteric tract. They were
causative agent. called orphans as they could not be associated
2. Myocardial and pericardial infections with any particular clinical disease then. There is
Myocardial and pericardial infections caused still no clear association of some types with specific
by coxsackie B virus occur sporadically in older diseases.
www.ebook3000.com
Chap-62.indd 431 15-03-2016 11:22:28
432 | Section 4: Virology
RHINOVIRUSES Treatment and Prophylaxis
Rhinoviruses are the common cold viruses and Antiviral drugs are thought to be a more likely
are the most important cause of the common cold control measure for rhinoviruses. Pleconaril is one
and upper respiratory tract infections. They are such drug showing activity against rhinoviruses
usually isolated from nasal secretions but may also and enteroviruses.
be found in throat and oral secretions. Hand washing and the disinfection of contamin
ated objects are the best means of preventing the
spread of the virus.
Properties of the Virus
Key Points
They differ from enteroviruses in being more
acid labile, but more heat stable. Inactivation of The Picornaviridae family has divided into six genera:
Enterovirus, Rhinovirus, Cardiovirus, Aphthovirus,
rhinoviruses occurs below pH 6.0 and is more rapid
Hepatovirus and Parechovirus
the lower the pH. They are relatively stable in the
Enteroviruses include the polioviruses, echoviruses,
range from 20 to 37°C (Table 62.2).
coxsackieviruses
By neutralization tests, they have been classified Polioviruses
into over 113 serotypes. Immunity is type-specific. Poliovirus is the causative agent of poliomyelitis
Prevention of poliomyelitis is accomplished by an
inactivated (killed) injectable vaccine (Salk vaccine)
Host Range and Growth or an attenuated live, orally administered vaccine
(Sabin vaccine), which confers immunity by raising
Rhinoviruses can be grown in tissue cultures of
neutralising antibody
human or simian origin with cytopathic changes.
Coxsackieviruses
Rhinoviruses were classified into three groups, H,
Based on the pathological changes produced in
M and O, depending upon growth in tissue culture,. suckling mice, they are classified into two group
H strains grew only in human cells, while M strains A and B
grew equally well in human and monkey cells. Zero The clinical manifestations of infection with yarious
strains could be grown only in nasal or tracheal coxsackieviruses are diverse and may present as
ciliated epithelium. distinct disease entities
Echoviruses
Echoviruses are found in the intestinal tract of the
Pathogenesis infected humans
Most echovirus infections are asymptomatic but
The virus enters via the upper respiratory tract. some have been associated with clinical syndromes
Local inflammation and cytokines may be Rhinoviruses
responsible for the symptoms of common cold. Rhinoviruses are the most important causative
agents of the common cold and upper respiratory
tract infections.
Clinical Syndromes
The incubation period is brief from 2 days to 4 IMPORTANT QUESTIONS
days. The acute illness usually lasts for 7 days.
Usual symptoms in adults include sneezing, nasal 1. Name the picornaviruses. Discuss pathogenesis
and laboratory diagnosis of poliomyelitis.
obstruction, nasal discharge, and sore throat; other
2. Write short notes on:
symptoms may include headache, mild cough, a. Prophylaxis against poliomyelitis
malaise and a chilly sensation. There is little or b. Coxsackieviruses
no fever. c. Echoviruses
d. Enterovirus 70
e. Acute hemorrhagic conjunctivitis
Laboratory Diagnosis f. Rhinoviruses.
The clinical syndrome of the common cold is
usually so characteristic that laboratory diagnosis MULTIPLE CHOICE QUESTIONS (MCQs)
is unnecessary.
1. The following statement is not true for polioviruses:
A. Specimens: Nose, throat swabs and nasophary a. Poliovirus was the first animal virus to be ob-
ngeal aspirates. tained in crystalline form
B. Culture: Cell cultures of human origin such as b. They are enveloped viruses
MRC5 or WI38 are preferred for the isolation c. They have single-stranded RNA genome
d. They have three serotypes
of rhinoviruses. Cultures are incubated at 33°C
and observed microscopically for a CPE. 2. The prototype strains for type 1 poliovirus are:
a. Brunhilde and Mahoney strains
C. Serology: Serology is not feasible. b. Lansing and MEFI strains
D. Nucleic acid detection: Likely to become a c. Leon and Saukett strains
significant tool in diagnosis. d. None of the above
www.ebook3000.com
Chap-62.indd 433 15-03-2016 11:22:29
63
Chapter
Orthomyxovirus
Learning Objectives
After reading and studying this chapter, you should ∙∙ D escrbe the following: Hemagglutinin (H) and
be able to: neuraminidase (NA); Antigenic variation in Influenza
∙∙ Differentiate between orthomyxoviruses and virus; Antigenic shift and antigenic drift
paramyxoviruses ∙∙ Discuss laboratory diagnosis of influenza
∙∙ Describe morphology of influenza virus ∙∙ D escribe the following: Influenza pandemics;
∙∙ Discuss types and subtypes of orthomyxoviruses prophylaxis against influenza; influenza vaccines.
Antigenic Variation
Influenza viruses are remarkable because of the
frequent ‘antigenic changes that occur in HA and
NA. This is of great importance in the epidemiology
of the disease. Antigenic variability is highest in
influenza virus type A and less in type B, while it
has not been demonstrated in type C.
The internal RNP antigen and M protein
Fig. 63.1: Diagrammatic representation of influenza virus antigen are stable but both the surface antigens,
www.ebook3000.com
436 | Section 4: Virology
hemagglutin in and neuraminidase, undergo hemagglutinin titers, but low infectivity. This has
independent antigenic variations, which may be of been called the von Magnus phenomenonand is
two types a
ntgenic drift (minor antigenic changes) due to the formation of incomplete virus particles
and antigenic drift (major antigenic changes) in HA lacking nucleic acid.
or NA result in the appearance of a new subtype.
Antigenic drift: Antigenic drift is the gradual
Pathogenesis
sequential change in antigenic structure occurring Influenza virus spreads from person to person
regu larly at frequent intervals. Here the new by airborne droplets. The viral neuraminidase
antigens, though different from the previous facilitates infection by reducing the viscosity
antigens, are yet related to them. Antigenic drift of the mucous film lining the respiratory tract
is due to mutation and selection. Antigenic drift and exposing the cell surface receptors for virus
accounts for the periodical epidemics of influenza. adsorption. The ciliated cells of the respiratory
tract are the main sites of viral infection. Within a
Antigenic shift: Antigenic shift is an abrupt, short time, many cells in the respiratory tract are
drastic, discontinuous variation in the antigenic infected and eventually killed. Influenza infections
structure, resulting in a novel virus strain unrelated cause cellular destruction and desquamation of
antigenically to predecessor strains. Such changes superficial mucosa of the respiratory tract. This
may involve hemagglutinin, neuraminidase or both. renders the respirator y tract highly vulnerable
The mechanism for shift is genetic reassortment to bacterial invasion, especially staphylococci,
between human and avian influenza viruses. streptococci, and Haemophilus influenzae. Viral
Antibodies to predecessor viruses do not neutralise pneumonia is seen only in more severe cases.
the new variants and can, therefore, spread widely
in the population causing major epidemics or Clinical Features
pandemic. Influenza B and C viruses do not exhibit
A. Uncomplicated Influenza
antigenic shift because few related viruses exist in
animals. The incubation period is 1–3 days. The disease
varies in severity from a mild coryza to fulminating
and rapidly fatal pneumonia. Most infections are
Antigenic Classification
subclinical.
Antigenic differences exhibited by two of the inter Symptoms of classic influenza usually appear
nal structural proteins, the nucleocapsid (NP) and abruptly and include chills, headache and dry
matrix (M) proteins, are used to divide influenza cough, followed closely by high fever, generalized
viruses into types A, B and C. These proteins muscular aches, malaise and anorexia. The fever
possess no cross reactivity among the three types. usually lasts 3–5 days, as do the systemic symptoms.
Antigenic variations in the surface glycoproteins,
HA and NA, are used to subtype the viruses. Only
Complications
type A has designated subtypes. Influenza virus
type A strains can be classified into subtypes based Complications of influenza include primary viral
on variations in their surface antigens. pneumonia, secondary bacterial pneumonia,
myositis and cardiac complicat ions, such as
congestive failure or myocarditis and, neurological
Host Range
involvement, such as Guillain–Barre syndrome,
1. Animals: Intranasal inoculation in ferrets encephalopathy, encephalitis and Reye’s syndrome
produces an acute respiratory disease. may occur.
2. Egg inoculation: The virus grows well in
the amniotic cavity of chick embryos. After Reye’s Syndrome
a few egg passages, the virus grows well in
Influenza, particularly infection with type B, has
the allantoic cavity also, except for the type
been associated with Reye’s syndrome. It is an
C virus which does not generally grow in the
acute encephalopathy of children and adolescents,
allantoic cavity. Virus growth is detected by the
usually between 2 and 16 years of age and is
appearance of hemagglutinin in the allantoic
characterized by acute degenerative changes in
and amniotic fluids.
the brain, liver and kidneys. Type B infections
3. Cell culture: The virus grows in primary may sometimes cause gastrointestinal symptoms
monkey kidney cell cultures, as well as in some (gastric flu).
continuous cell lines.
von Magnus phenonomenon: When passaged Laboratory Diagnosis
serially in eggs, using as inocula undiluted infected Diagnosis of influenza relies on isolation of the
allantoic fluid, the progeny virus will show high virus, identification of viral antigens or viral nucleic
Chapter 63: Orthomyxovirus | 437
acid in the patient’s cells, or demonstration of a and convalescent sera are necessary. A fourfold
specific immunologic response by the patient. or greater increase in titer must occur to indicate
influenza infection.
1. Demonstration of the Virus Antigen Neutralization tests are the most specific and
Rapid diagnosis of influenza may be made by the best predictor of susceptibility to infection.
demonstration of the virus antigen on the surface of ELISA test is more sensitive than other assays.
the nasopharyngeal cells by immunofluorescence. It is possible to estimate the neuraminidase
This test is rapid but is not as sensitive as viral antibody by enzyme neutralization tests.
isolation.
Detection of influenza RNA by reverse trans Immunity
criptase polymerase chain reaction may be more Immunity to influenza is long-lived and subtype-
sensitive than antigen detection but is not widely specific. Antibodies against HA and NA are
available in diagnostic laboratories. important in immunity to influenza, whereas
antibodies against the other virus-encoded proteins
2. Isolation of the Virus are not protective.
Nasal washings, gargles and throat swabs are the An attack of influenza confers protection
best specimens for viral isolation and should be effective for about one or two years. The apparent
obtained within 3 days after the onset of symptoms short duration of immunity is due to the antigenic
but less often in later stages. Throat garglings variation that the antigenic variation that the virus
are collected using broth saline or other suitable undergoes frequently. Following infection and
buffered salt solution. The sample should be held at immunization, circulating antibodies are formed
4°C until inoculation into cell culture, or if the delay against the various antigens of the virus. However,
is long, at –70°C. The specimen should be treated it the local concentration of anthaemagglutinin
with antibiotics to destroy bacteria. Isolation may and, to a smaller extent, of anineuraminadase
be made in eggs or in monkey kidney cell culture. antibodies (mainly IgA) in the respiratory tract that
The material is inoculated into the amniotic is more relevant in protection.
cavity of 11–13 day-old eggs, using at least six eggs When an individual experiences repeated
per specimen. After incubation at 35°C for three infections with different antigenic variants of
days, the eggs are chilled and the amniotic and influenza virus type A, he responds by forming
allantoic fluids harvested separately. The fluids antibodies not only against each infecting strain
are tested for hemagglutination using guinea pig but also against the strain that he first comes into
and fowl cells in parallel, at room temperature and contact with. The dominant antibody response
at 4°C. Some strains of the influenza virus type A will be against the strain that caused the earliest
agglutinate only guinea pig cells on initial isolation. infection. This phenomenon called the doctrine
The type B virus agglutinates both cells, while type of “original antigenic sin”.
C strains agglutinate only fowl cells at 4°C. Subtype Influenza virus infection induces cell-mediated
identification is made by hemagglutination immunity also but its role in protection has not
inhibition test. Some of the recent type A strains been clarified.
can be isolated by direct allantoic inoculation of the
clinical specimen into 9–11-day-old eggs. However, Epidemiology
type Band C viruses will be missed if only allantoic Influenza viruses occur worldwide and occurs
inoculation is used. sporadically, as epidemics or in pandemic form.
For primary isolation the most suitable cells are The source of infection is an infected individual.
primary monkey kidney or human embryo kidney Influenza infection is spread readily via small
cells, but most laboratories now use secondary airborne droplets expelled during talking,
baboon kidney cells or Madin–Darby canine breathing, and coughing. Influenza C is least
kidney cells. Incubation at 33°C in roller drum significant; it causes mild, sporadic respiratory
is recommended. The presence of virus may be disease but not epidemic influenza. Influenza B
detected by hemadsorption with human O group, sometimes causes epidemics, but influenza type A
fowl or guineapig red blood cells. Rapid results can sweep across continents and around the world
can be obtained by demonstrating virus antigen in massive epidemics called pandemics.
in infected cell cultures by immunofluoroscence. What makes influenza an important and
challenging disease is its propensity for causing
3. Serology pandemics. Domestic birds like ducks can get
Complement fixation tests (CFTs) and hemaggluti infected from wild birds and carry the infection to
nation inhibition (HI) tests are employed for the pigs.The genetic reassortment may take place in
serological diagnosis of influenza. Paired acute pigs that have receptors for both human and avian
www.ebook3000.com
438 | Section 4: Virology
and rimantadine which block the viral M2 protein
which functions as an ion channel. These act only
with type A virus and not with type B, which lacks
the M2 components.
B. Influenza Vaccines
Influenza vaccines have been in use for many
decades. The aim of immunization is to produce
hemagglutinat ion inhibiting or neutralizing
antibody in all vaccinees. However, cert ain
characteristics of influenza viruses make prevention
and control of the disease by immunization
especially difficult. Existing vaccines are continually
being rendered obsolete as the viruses undergo
antigenic drift and shift. Vaccines are of two types
as follow.
www.ebook3000.com
64
Chapter
Paramyxoviruses
Learning Objectives
Subfamily Paramyxoviridae
1. Respirovirus (para-influenza viruses 1, 3).
2. Rubulavirus (mumps virus, para-influenza
viruses 2, 4a, 4b).
3. Morbillivirus (Measles).
4. Henipavirus—Nipah virus and Hendra virus.
Subfamily Pneumovirinae
1. Pneumovirus (respiratory syncytial virus (RSV)
Fig. 64.1: Schematic diagram of a paramyxovirus showing 2. Metapneumovirus—Human metapneumo
major components virus.
Chapter 64: Paramyxoviruses | 441
Table 64.1: Characteristics of genera in the subfamilies of the family Paramyxoviridae
Paramyxovirinae Pneumovirinae
Property Respirovirus Rubulavirus Morbillivirus Henipavirus 1
Pneumovirus Metapneumovirus
Human Parainfluenza Mumps, Measles Hendra, Respiratory Human
viruses 1,3 parainfluenza Nipah syncytial metapneumovirus
2, 4a, 4b virus
Serotypes 1 each 1 each 1 ? 2 ?
Diameter of 18 18 18 ? 13 13
nucleocapsid
(nm)
Membrane + + + + + +
fusion
(F protein)
Hemolysin2 + + + ? 0 0
Hemaglutinin +3 +3 +4 0 0 0
Hemadsorption + + + 0 0 0
Neuraminidase +3 +3 0 0 0 0
Inclusions 5
C C N, C ? C ?
1
Zoonotic paramyxoviruses
2
Hemolysin activity carried by F glycoprotein
3
Hemagglutination and neuraminidase activities carried by HN glycoprotein
4
Hemagglutination of monkey erythrocytes only, by H glycoprotein that lacks neuraminidase activity
5
C, cytoplasm; N, nucleus
www.ebook3000.com
442 | Section 4: Virology
C. Serology variation. Immunity is permanent after a single
Serodiagnosis should be based on paired sera and infection.
can be tested by neutralization, enzyme linked
immunosorbent assay (ELISA) or Hemagglutination Laboratory Diagnosis
inhibition test (HI) or complement fixation test for Laboratory studies are not usually required to
rise in titre of antibodies. establish the diagnosis of typical cases.
The diagnosis may be established by virus
Genus Rubulavirus isolation and serological tests.
Mumps Virus A. Direct Demonstration
Mumps is an acute contagious disease commonly Direct demonstration by immunofluorescence on
affecting children characterized by nonsuppurative secretions is very rarely successful.
enlargement of one or both parotid glands.
Morphology: Mumps virus is a typical paramyxo B. Virus Isolation and Identification
virus; possess both hemagglutinin and neuramini Virus can be recovered from the saliva and CSF.
dase (HN) or a fusion (F) protein. The envelope Monkey kidney cells are preferred for viral isola
also contains a matrix (M) protein. It agglutinates tion. For rapid diagnosis, immunofluorescence
the erythrocytes of fowl, guinea pig, humans and using mumps-specific antiserum can detect
many other species. Hemagglutination is followed mumps virus antigens as early as 2–3 days after the
by hemolysis and elution at 37°C. inoculation of cell cultures in shell vials.
Isolation can also be made by inoculation into
Pathogenesis six to eight day old chick embryos by the amniotic
Transmission is from person to person by large route and testing the amniotic fluid after 5–6 days
droplets. Primary replication occurs in nasal or for hemagglutinins. The virus can be identified
upper respiratory tract epithelial cells. Virernia by hemagglutination inhibition using specific
then disseminates the virus to the salivary glands antisera.
and other major organ systems and infects the
parotid gland. The virus is spread by the viremia C. Serology
throughout the body to the testes, ovary, pancreas,
Enzyme linked immunosorbent assay (ELISA) or
thyroid, and other organs. Infection of the central
hemagglutination inhibition test is commonly used.
nervous system, especially the meninges, immunity
Mumps specific IgM antibodies can be detected in
is lifelong.
serum by enzyme linked immunosorbent assay
Clinical features: Infection is acquired by inhalation, (ELISA) for rapid diagnosis.
and probably also through the conjunctiva. The
incubation period may range from 7 days to 25 days Prophylaxis
but is typically about 16–18 days. The generalized
1. Vaccination
phase is the usual ‘flu-like’ illness with fever
and malaise, followed by developing pain in the An effective attenuated live Mumps virus vaccine
parotid glands, which then swell rapidly. Parotitis is based on the Jeryl Lynn or Urabe strains. Mumps
nonsuppurative and usually resolves within a week. vaccine is available in monovalent form (mumps
However, involvement of the extraparotid sites may only) or as combined vaccine, viz. combined
be more serious. mumps-rubella (MR) or into a triple vaccine
against measles, mumps and rubella (MMR) live-
Complications: Epididymo-orchitis, aseptic
virus vaccines. The vaccine is recommended for
meningitis, postinfection encephalitis, mumps
children over age 1 year.
meningitis. Other less common complications
are arthritis, oophoritis, nephritis, pancreatitis, The vaccine is given as a single dose (0.5 mL)
thyroiditis and myocarditis. intramuscularly. It provides effective protection for
at least ten years. Contraindications are pregnancy,
Epididymo-orchitis: Is a complication seen in immunodeficiency, severely ill and hypersensitivity
about a third of postpubertal male patients and to neomycin or egg protein.
rarely causes sterility.
2. Immunoglobulin
Immunity A specific immunoglobulin (mumps immuno
There is only one antigenic type of mumps virus, globulin) is available and is of no value either for
and it does not exhibit significant antigenic postexposure prophylaxis or treatment.
Chapter 64: Paramyxoviruses | 443
Complications
1. Complications may be due to the virus
(croup, bronchitis) or to secondary bacterial
infection (pneumonia, otitis media). Giant
cell pneumonia, particularly in children with
immunodeficiencie’s or severe malnutrition.
2. Post-measles encephalitis.
3. Subacute sclerosing panencephalitis (SSPE)
Is an extremely serious, very late neurologic
sequel of measles. It occurs in children or
early adolesents who have measles early in life,
usually less than 2 years of age. This disease
Fig. 64.2: Schematic diagram of measles virus occurs when a defective measles virus persists
Genus morbillivirus in the brain and acts as a slow virus. The virus
can replicate and spread directly from cell to
Measles (Rubeola) cell but is not released. Within infected cells
Measles is one of the five classic childhood exant is a defective form of measles virus, which,
hems, along with rubella, roseola, fifth disease, and because it is unable to induce the production
chickenpox. of a functional M protein, is not released as
complete virus from the cell.
Morphology 4. Protracted diarrhea in children in the poor
nations.
The virus has the general morphology of paramyxo
viruses (Fig. 64.2). It is a roughly spherical but often
Laboratory Diagnosis
pleomorphic particle, 120–250 nm in diameter. The
tighdy coiled helical nucleocapsid is surrounded Typical measles is reliably diagnosed on clinical
by the lipoprotein envelope carrying on its surface grounds, usually in the patient’s home. Laboratory
hemagglutinin (H) spikes. The envelope also diagnosis may be necessary in cases of modified or
has the F protein which mediates cell fusion and atypical measles.
hemolytic activities. The measles virus agglutinates
monkey erythrocytes but there is no elution as the A. Demonsration of Virus Antigen
virus does not possess neuraminidase activity. A simple diagnostic test, which can be used even
before the rash appears, is the demonstration of
Pathogenesis multinucleated giant cells in Giemsa stained smears
The virus gains access to the human body via the of nasal secretions. The measles virus antigen can
respiratory tract, or the conjunctiva where it multiplies be detected in these cells by immunofluorescence.
locally. The infection then spreads to the regional
lymphoid tissue, where further multiplication occurs. B. Virus Isolation
Primary viremia disseminates the virus, which then The virus can be isolated from the nose, throat,
replicates in the reticuloendothelial system. Finally, conjunctiva and blood during the prodromal phase
a secondary viremia seeds the epithelial surfaces and upto about two days after the appearance of the
of the body, including the skin, respiratory tract, and rash. The virus may be obtained from the urine for
conjunctiva, where focal replication occurs. a few more days. Primary human or monkey kidney
and amnion cells are most useful. Cytopathic
Clinical Features changes may take up to 7–10 days to develop
Measles is a serious febrile illness. Incubation but earlier diagnosis of viral growth is possible
period is 9–11 from the time of exposure to infection by immunofluorescence. Typical cytopathic
for the first signs of clinical disease to appear. After effects (multinucleated giant cellscontaining
2 days of illness, the typical mucous membrane both intranuclear and intracytoplasmic inclusion
lesions, known as Koplik’s spots, appear most bodies) suggests the presence of measles virus.
commonly on the buccal mucosa across from the
molars. Their appearance in the mouth establishes C. Serological Diagnosis
with certainty the diagnosis of measles. Demonstration of measles-specific IgM in a single
Before the rash there is a prodromal stage specimen of serum drawn between one and two
lasting 2 or 3 days, with high fever and CCC and weeks after the onset of the rash is confirmatory.
P-cough, coryza, and conjunctivitis, in addition to Enzyme linked immunosorbent assay (ELISA),
photophobia. hemagglutination inhibition (HI), and neutralization
www.ebook3000.com
444 | Section 4: Virology
(Nt) tests all may be used to measure measles resulting in at least one fatal infection in human
antibodies, though ELISA is the most practical in Australia.
method. A four fold rise in titer is diagnostic. The taxonomic position of these new viruses
High titer measles antibody in the CSF is has yet to be established, but they will most
diagnostic of SSPE. probably be put in a separate genus within the
Paramyxoviridae. Fruit bats appear to be the
Epidemiology natural reservoir of both viruses, with transmission
The key epidemiologic features of measles are as to mammals (including man) an exceptional event.
follows: the virus is highly contagious, there is a
single serotype, there is no animal reservoir, in Genus pneumovirus
apparent infections are rare, and infection confers Respiratory Syncytial Virus (RSV)
lifelong immunity. Transmission is person-to-
person, probably by respiratory droplets, but the Respiratory syncytial virus (RSV) first isolated from
associated conjunctivitis may also be a source. In a chimpanzee was named respiratory syncytial
general, epidemics recur regularly every 2–3 years. virus (RSV) because it caused cell fusion and the
formation of multinucleated syncytia in cell cultures.
Prophylaxis
Description
A. Passive immunization
RSV is pleomorphic and ranges in size from 150–
Passive protection with normal immunoglobulin 300 nm. The viral envelope has two glycoproteins—
(NHIG) given within six days of exposure can the G protein by which the virus attaches to
prevent or modify the disease, depending on the cell surfaces, and the fusion (F) protein which
dose. Passive immunization is recommended in brings about fusion between viral and host cell
children with immunodeficiency, pregnant women membranes. The F protein is also responsible for
and others at risk. cell-to-cell fusion, which leads to the characteristic
syncytial cytopathic changes in RSV infection.
B. Active Immunization
It is placed in a separate genus—Pneumovirus
A highly effective, safe, attenuated live measles
because of these minor physical differences (Table
virus vaccine is available. The original live vaccines
64.1).
used the Edmonston strain developed by multiple
RSV does not grow in eggs but can be propagated
passage through human kidney, amnion and chick
on heteroploid human cell cultures, such as HeLa
embryo cultures. The Schwartz and Moraten
and Hep-2. It is highly labile and is inactivated
strains so developed were safe but effective only
rapidly at room temperature. It is antigenically stable
in children older than 15 months. In the tropics,
and for most purposes there is only one serotype.
measles is common and serious in children below 12
months. Recent observations have suggested that the
Edmonston–Zagreb strain, attenuated by passage in Clinical Features
human diploid cells, may protect children from 4 to The spectrum of respiratory illness ranges from the
6 months of age. The recommended age for measles common cold in adults, through febrile bronchitis
vaccination in the developing countries is 9 months, in infants and older children and pneumonia in
while in the advanced countries it remains 15 months. infants, to bronchiolitis in very young babies.
The vaccine is given either by itself, or in 1. Common cold: In adults RSV infection may
combination as the MMR vaccine. A single present as a febrile common cold. It can cause
subcutaneous injection of the measles vaccine pneumonia in the elderly.
provides protection beginning in about 12 days 2. Febrile rhinitis and pharyngitis
and lasting for over 20 years, probably for life. 3. Bronchiolitis, pneumonia, or both: The most
Contraindications are pregnancy, acute illnesses, serious illness caused by RS virus are bronchi
immunodeficiency and untreated tuberculosis. olitis in young babies.
A live attenuated vaccine has been developed 4. Sudden infant death syndrome: A relation
which can be given by intranasal aerosol in young between RSV and the sudden death syndrome
babies and gives good protection irrespective of the in infants has been proposed but not proven.
presence of maternal antibodies.
Epidemiology
Nipah and Hendra viruses Respiratory syncytial virus is distributed worldwide
Nipah virus is distinct genetically from all the other and is recognized as the major pediatric respiratory
paramyxoviruses and is most closely related to tract pathogen. It is the most common cause of viral
Hendra, causing epidemic fatal respiratory disease pneumonia in children under age 5 years. RSV is
in horses and which can be transmitted to man, highly contagious.
Chapter 64: Paramyxoviruses | 445
RSV infections almost always occur in the Within the family Paramyxoviridae two subfamilies-
winter.It causes nosocomial infections in nurseries Parmyxoviridae and Pneumovirinae are recognized
and on pediatric hospital wards. Parainfluenza viruses 1, 2, and 3 may cause respiratory
tract syndromes ranging from a mild coldlike upper
Laboratory Diagnosis respiratory tract infection to bronchiolitis and
pneumonia. Parainfluenza virus type 4 does not
A. Demonstration of Virus Antigen cause serious disease, even on first infection
Immunofluorescence and enzyme immunoassay Mumps virus causes mumps
tests are available for direct detection of the viral Measles virus causes measles, atypical measles, and
subacute sclerosing panencephalitis (SSPE)
antigen in infected cells and nasal washings.
Measles vaccine is a live attenuated vaccine which
now uses Schwartz and Moraten attenuated strain
B. Virus Isolation of the original Edmonston B strain
Human heteroploid cell lines HeLa and HEp-2 are Measles vaccine along with mumps and rubella
the most sensitive for viral isolation. The presence of (MMR) vaccine is currently used for universal
immunization of the children
respiratory syncytial virus can usually be recognized
Respiratory syncytial virus (RSV) causes infection
by development of giant cells and syncytia in of the respiratory tract, ranging from the common
inoculated cultures but cytopathic effects may take cold in adults, through febrile bronchitis in infants
as long as 10 days for to appear. Definitive diagnosis and older children and pneumonia in infants, to
can be established by detecting viral antigen in bronchiolitis in very young babies.
infected cells using a defined antiserum and the
immunofluorescence test. Important questions
C. Serology 1. Classify and discuss the morphology of paramyxo
Serological diagnosis is by demonstration of viruses.
rising antibody titers in paired serum samples by 2. Write short notes on:
enzyme linked immunosorbent assay (ELISA), a. Parainfluenza viruses
b. Mumps virus
complement fixation (CF), neutralization or
c. Measles virus
immunofluorescence tests.
d. Respiratory syncytial virus (RSV)
e. Measles, mumps and rubella (MMR) vaccine.
Prophylaxis
f. Subacute sclerosing panencephalitis (SSPE)
No vaccine is currently available for RSV prophy g. Atypical measles.
laxis. The use of recombinant DNA technology for
making RSV vaccine is now being studied.
Multiple choice questions (mcqs)
Treatment 1. The paramyxovirus virus that possesses a fusion
In otherwise healthy infants, treatment is protein but lacks both hemagglutinin and neura
supportive care. Ribavirin is administered by minidase activities is:
inhalation (nebulization) and has been found a. Mumps virus
b. Measles virus
beneficial in hospitalized patients, decreasing the
c. Parainfluenza virus
duration of illness and of virus shedding. d. Respiratory syncytial virus
2. Which of the following conditions can be caused
Metapnemovirus by infection with mumps virus?
Human metapneumovirus is a respiratory a. Swelling of parotid glands
pathogen and is an important cause of respiratory b. Orchitis
tract infection in children. It can also cause disease c. Meningoencephalitis
in adults. It causes a disease similar to that of d. All of the above
human respiratory syncytial virus. 3. Koplik’s spots are characteristic of:
It is a single stranded RNA virus like other a. Mumps
paramyoviruses. Respiratory secretions are clinical b. Measles
specimen for test. The virus is difficult to grow. c. Herpes
Polymerase chain reaction (PCR) can be used d. Rubella
for diagnosis. No specific antiviral treatment or 4. MMR vaccine is:
vaccine is available. a. a live attenuated vaccine
b. a killed vaccine
Key Points c. a subunit vaccine
d. a synthetic peptide vaccine
Unlike the orthomyxoviruses, the paramyxoviruses
with their unsegmented genome do not undergo
Answers (MCQs)
genetic recombinations or antigenic variations
1. d; 2. d; 3. b; 4. a
www.ebook3000.com
65
Chapter
Arboviruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ List the arboviruses prevalent in India
be able to: ∙∙ Describe the following: Chikungunya; Japanese B
∙∙ Classify arboviruses encephalitis; Dengue fever (or) Break bone fever;
∙∙ Describe laboratory diagnosis of arboviruses Kyasanur forest disease (KFD).
∙∙ List mosquito-borne and tick-borne arboviruses
cultures of primary cells and in cultures of reverse transcriptase polymerase chain reaction
appropriate insect tissues. (RTPCR).
4. Many arboviruses multiply in continuous tissue
cultures of mosquito cells when incubated at D. Serology
34°C or lower temperatures. Diagnosis may also be made serologically by
5. Mosquito-borne arboviruses multiply after oral demonstrating rise in antibody titer in paired
feeding or intrathoracic injection of several serum samples by hemagglutination inhibition,
Aedes and Culex mosquito species. complement fixation or neutralization tests. Virus-
6. Tick-borne arboviruses multiply after oral specific IgM antibody may be detected within I day
feeding to larval or nymphal ixodes ticks. of onset of clinical symptoms using an IgM capture
7. In general, arboviruses are labile, being readily ELISA test.
inactivated at room temperature and by bile
salts, ether and other lipid solvents. PATHOGENESIS
The virus enters the body through the bite of the
LABORATORY DIAGNOSIS insect vector. After multiplication in the reticulo
A. Specimen endothelial system, viremia of varying duration
ensues and, in some cases, the virus is transported
As all arbovirus infect ions are viremic, blood
to the target organs, such as the central nervous
collected during the acute phase of the disease may
system in encephalitides, the liver in yellow fever
yield the virus. Isolation may also be made from
and the capillary endothelium in hemorrhagic
the CSF in some encephalitic cases but the best
fevers. Arboviruses cause the following clinical
specimen for virus isolation is the brain.
syndromes (Table 65.3).
∙∙ Fever with or without rash and arthralgia
B. Virus Isolation ∙∙ Encephalitis
i. Suckling Mice ∙∙ Hemorrhagic fever
Specimens are inoculated intracerebrally into suck ∙∙ The characteristic systemic disease, yellow
ling mice. The animals develop fatal encephalitis. This fever
is the most sensitive method for isolation of viruses. Arboviruses are maintained in natural trans
mission cycles involving reservoir hosts and
ii. Tissue Cultures arthropod vectors, typically: ticks, mosquitoes and
Some viruses may also be isolated in tiss ue other biting flies.
cultures or, less readily, in eggs. Specimens are
inoculated into Vero, BHK-21 and mosquito cello FAMILIES OF ARBOVIRUSES
lines. Isolates are identified by hemaglutination A. Family Togaviridae
inhibition, complement fixation, gel precipitation,
Togaviruses
immunofluorescence, immunochromatography,
ELISA or neutralization with appropriate antisera. Morphology
Togaviruses are spherical enveloped viruses with
iii. Virus Isolation from Insect Vectors and a diameter of 50–70 nm. The genome is single
Reservoir Animal stranded RNA. The name Togavirus is derived from
‘toga’, meaning the Roman mantle or cloak, and
C. Arbovirus-specific RNA Detection refers to the viral envelope.
Viral RNA is extracted from serum or suspensions
of tissues from patients, or from tissue culture cells Classification
or mosquito homogenates. This is amplified by The family Togaviridae contains two genera:
www.ebook3000.com
Chap-65.indd 447 15-03-2016 11:23:04
448 | Section 4: Virology
Table 65.3: Arboviruses associated with different clinical syndromes
Family Genus Virus Vector Geographic Reservoir
distribution
Fever with or without rash and arthralgia
Togaviridae Alphavirus Chikungunya Mosquito Africa, Asia Not known (? Monkeys)
Alphavirus Onyong-nyong Mosquito Africa Not known
Alphavirus Ross River Mosquito Australia Small animals
Alphavirus Sindbis Mosquito Africa, Asia Birds, mammals
Alphavirus Mayaro Mosquito South America Monkeys, marsupials
Flaviviridae Flavivirus Dengue, types 1–4 Mosquito Widespread, Not known
especially (? Monkeys)
Asia Pacific,
Caribbean
Flavivirus West Nile Mosquito Asia, Africa, USA Birds
Bunyaviridae Phlebovirus Sandfly fever Sandfly Mediterranean, Asia, not known
Tropical America (? Small mammals)
Phlebovirus Rift valley fever Mosquito Americas Sheep, cattle
Bunyavirus Oropouche Mosquito South America Not known
Reoviridae Orbivirus Colorado tick fever Tick USA Rodents
Encephalitis
Togaviridae Alphavirus Eastern equine Mosquito Americas Birds
encephalitis
Alphavirus Western equine Mosquito Americas Reptiles (? Birds)
encephalitis
Alphavirus Venezuelan equine Mosquito Americas Rodents
Flaviviridae Flavivirus St. Louis encephalitis Mosquito Americas Birds
Flavivirus West Nile Mosquito Africa, Europe, USA, Birds
West Asia
Flavivirus Japanese encephalitis Mosquito East and South East Birds
Asia
Flavivirus Murray valley Mosquito Australia Birds
encephalitis
Flavivirus RSSE complex Tick East Europe, USSR Rodents, other
mammals, birds, ticks
Flavivirus Louping ill Tick Britain Sheep
Flavivirus Powassan Tick North America Rodents
Bunyaviridae Bunyavirus California Mosquito North America Rodents
Hemorrhagic fever
Togaviridae Alphavirus Chikungunya Mosquito Africa, Asia Not known
(? Monkeys)
Flaviviridae Flavivirus Dengue types 1–4 Mosquito Tropics Not known
(? Monkeys)
Flavivirus Yellow fever Mosquito Africa, South America Monkeys, man
Flavivirus Kyasanur forest Tick Southwest India Rodents (? Ticks)
disease
Flavivirus Omsk hemorrhagic Tick USSR Small mammals
fever
Flavivirus Crimean Congo Tick USSR, Central Asia, Small mammals
hemorrhagic fever Africa
Alphavirus Rubivirus
The Alphavirus genus consists of about 32 viruses Rubivirus, which is not arthropod-borne and which
of which at least 13 are known to infect humans. causes rubella.
All of them are mosquitoborne.
www.ebook3000.com
Chap-65.indd 449 15-03-2016 11:23:04
450 | Section 4: Virology
1. St. Louis encephalitis virus (SLEV): This is of the disease in man may be divided into three
prevalent in North and Central America and is the stages:
most important mosquito-borne disease in the a. Prodromal stage: The onset of illness is usually
USA. acute and is heralded by fever, headache, and
Wild birds act as the reservoir and Culex tarsalis malaise.
as the vector. No vaccine is yet available. b. Acute encephalitic stage: The prominent
2. Ilheus virus: This occurs in South and Central features are fever, nuchal rigidity, focal central
America, maintained in forests by a cycle involving nervous system (CNS) signs, convulsions and
mosquitoes, wild birds and monkeys. Human altered sensorium progressing in many cases
infection is largely subclinical or leads to febrile to coma.
illness. Encephalitis is rare. c. Late stage and sequelae: Convalescence may
be prolonged and residual neurological deficits
3. West Nile virus (WNV): WNV was first isolated may not be uncommon. The case fatality rate
from a febrile human in the West Nile district of varies between 20 and 40%.
Uganda in 1937, has since been reported from many The large majority of infections are, however,
African countries, Israel, Cyprus, France and India. asymptomatic.
Vector—both mosquitoes and ticks have been
reported as vectors. The principal vectors are Epidemiology: Unlike the dengue viruses, JE virus
considered to be mosquitoes of the Culex genus. infects several extrahuman hosts, e.g. animals and
birds. The disease is transmitted to man by the bite
4. Murray valley encephalitis virus (MVEV): This of infected mosquitoes. Available evidence indicates
is confined to Australia and New Guinea. Vector- that the basic cycles of transmission are:
Natural cycles of transmission of MVEV involve Cx.
a. Pig → Mosquito → Pig
annulirostris as the principal mosquito vector and
water birds as reservoirs. b. The Ardeid bird → Mosquito → Ardeid bird
The disease has been recognised in Japan since Control of Japanese Encephalitis
1871 and was named Japanese ‘B’ encephalitis to
distinguish it from ‘encephalitis A (encephalitis Preventive measures include mosquito control and
lethargica, von Economo’s disease) which was locating piggeries away from human dwellings.
then prevalent. The virus was first isolated in Japan
during an epidemic in 1935. i. Formalin Inactivated Mouse Brain Vaccine
A formalin inactivated mouse brain vaccine
Problem in India using the Nakayama strain has been employed
Recognition of JE was first made in 1955 when the successfully for human immunization in Japan
virus was isolated from mosquitoes of the Culex and, in a small scale, in India also. For primary
vishnui complex from Vellore. From 1976, there have immunization, 2 doses of 1 ml each (0.5 mL for
been periodical outbreaks of the disease in various children under the age of 3 years) should be
parts of India—Dibrugarh (Assam), Gorakhpur (Uttar administered subcutaneously at an interval of
Pradesh) and Haryana and Goa and Maharashtra, 7–14 days. A booster injection of 1 mL should be
Kolar in Karnataka, Andhra Pradesh, in Tamil given after a few months (before one year) in order
Nadu, in Pondicherry and lately in Kerala. Japanese to develop full protection. Protective immunity
encephalitis has become a major public health develops in about a month’s time after the second
problem of national importance in India. dose. Revaccinations may be given after 3 years.
www.ebook3000.com
Chap-65.indd 451 15-03-2016 11:23:05
452 | Section 4: Virology
associated with dengue are similar to those of other suckling mice. Further identification is done by
Arboviruses from other families. using fluorescent antibody test. Live mosquito
inoculation is the most sensitive technique for
Clinical Findings isolation of virus.
Classic dengue usually affects older children and B. Polymerase chain reaction (PCR): PCR based
adults.After incubation period of 2–7 days, patient methods are available for rapid identification
develops fever of sudden onset and often biphasic and subtyping.
with severe headache, chilies, retrobulbar pain, C. Serology: Demonstration of circulating
conjunctivitis and severe pain in the back, muscles IgM antibody provides early diagnosis, as it
and joints (breakbone fever). A maculopapular appears within two to five days of the onset of
rash generally appears on the trunk in 3–5 days of illness and persists for one to three months.
illness and spreads later to the face and extremities. IgM ELISA test offers reliable diagnosis. A
Lymph nodes are frequently enlarged. Leukopenia strip immunochromatographic test for IgM is
with a relative lymphocytosis is a regular occurrence. available for rapid diagnosis.
Complications and death are rare. IgG antibody appears later than IgM antibody.
ELISA is used for detection of IgG antibody.
Complications of Dengue: Dengue may also Detection of four fold rise in IgG titer in paired
occur in more serious forms, with hemorrhagic sera taken at an interval of ten days or more is
manifestations (dengue hemorrhagic fever) or confirmatory.
with shock (dengue shock syndrome). They are
more common in previously healthy children in the Hematological diagnosis: Thrombocytopenia
indigenous populations of endemic areas. (100,000 cells or less per mm3); Hemoconcentration
(720% in hematocrit).
Pathogenesis: The pathogenesis of the severe
syndrome involves preexisting dengue antibody.
Epidemiology
It is postulated that virus–antibody complexes
are formed within a few days of the second Dengue virus is transmitted from person to person
dengue infection and that the non-neutralizing by Aedes aegypti mosquitoes. Most subtropical and
enhancing antibodies promote infection of tropical regions around the world where Aedes
higher numbers of mononuclear cells, followed vecrors exist are endemic areas. Dengue was
by the release of cytokines, vasoactive mediators, initially confined to the east coast of India and has
and procoagulants, leading to the disseminated caused epidemics. All four types of dengue virus
intravascular coagulation seen in the hemorrhagic are present in this country.
fever syndrome.
Control
Dengue hemorrhagic fever: Dengue hemorrhagic
Control depends upon antimosquito measures, as
fever (DHF) may occur in individuals (usually
no vaccine is currently available.
children) with passively acquired (as maternal
A live attenuated vaccine containing all four
antibody) or preexisting non-neut ralizing
dengue serotypes is under clinical trial in order to
heterologous dengue antibody due to a previous
avoid DHF/DSS in immunized persons.
infection with a different serotype of virus.
Dengue shock syndrome (DSS), a more severe
form of the disease characterized by shock and B. Tick-borne Group
hemoconcentration, may ensue. Circumstantial These viruses produce two clinical syndromes,
evidence suggests that secondary infection with encephalitis hemorrhagic fevers.
dengue type 2 following a type 1 infection is a
particular risk factor for severe disease. a. Tick-borne Encephalitis Viruses (TBEV)
Russian spring summer encephalitis (RSSE)
Laboratory Diagnosis
complex: A number of viruses belonging to the
(a) Specimens Russian spring summer encephalitis (RSSE)
For antibody detection—Serum complex cause encephalitis along a wide area of
For isolation of virus and PCR. Serum, plasma, the northern landmass from Scotland to Siberia.
whole blood (washed buffy coat), autopsy tissues The names given to the disease vary from one area
and, mosquitoes collected in nature. to another. Thus, in Scotland, it is called ‘louping ill’
A. Virus detection: Isolation of the virus is as the disease occurs primarily in sheep in which
difficult. Virus isolation can be done by it causes a curious ‘leaping’ gait. Human cases that
inoculating clinical specimen into mosquitoes, result from contact with these sheep are mild and
mosquitoes cell lines (C6/36 or AP-61 cells), or present as aseptic meningitis. It is called Central
www.ebook3000.com
Chap-65.indd 453 15-03-2016 11:23:05
454 | Section 4: Virology
California encephalitis virus, La Crosse virus, and (HFRS). Principal vertebrate reservoirs com
Chittor virus. California encephalitis virus and La prise Apodemus agrariusrodents in Asia and
Crosse virus are isolated from the United States Clethrionomys glareolus (bank vole) in Europe.
and Chittor virus from India. The clinical disease Species: The genus contains at least four’
caused is encephalitis, aseptic meningitis and fever. species:
All are mosquito-borne infections. (i) Hantaan virus; (ii) Seoul virus; (iii) Puumala
virus; (iv) Prospect hill virus. Hantavirus
B. Phlebovirus species are natural pathogens of rodents-field
a. Sandfly fever (Phlebotomus fever): Sandfly mice (Apodemus agrarius) being the major host
fever (Phlebotomus fever) also known as for Hantaan. Transmission to humans occurs by
Pappataci fever and three-day fever, is a inhaling Aerosols of rodent excreta. The disease
self, nonfatal fever transmitted by the bite of occurs in two forms—the milder epidemic
sandfly Phlebotomus papatasii. It occurs along nephritis (EN) common in Scandinavia and the
the mediterranean Coast and Central Asia, more serious epidemic hemorrhagic fever (EHF)
extendIng as far east as Pakistan and North in the far east. The clinical picture resembles
West India. Cases have also been reported from typhoid, leptospirosis and scrub typhus.
South and Central America. Domestic rats appear to be the source of
Natural vectors are Phlebotomus papatasi and infection in urban cases of HFRS. HFRS should
other phlebotomine sandflies. be considered a robovirus and not stricly an
Twenty antigenic types of the virus exist, of arbovirus infection in the absence of proved
which only five cause human disease—Naples, arthropod transmission.
Sicilian, Punta Toro, Chagres, Candiru. Its 2. Sin nombre virus (SNV): In 1993 an outbreak
occurrence in India was thought to be doubtful. of severe respiratory illness in the United States,
b. Rift valley fever: The agent of this disease, now designated the hantavirus pulmonary
a bunyavirus of the Phleb ovirus genus, is a syndrome (HPS), was found to be caused by a
mosquito-borne zoonotic virus pathogenic novel hantavirus.
primarily for sheep, cattle, and goats. Humans The disease is caused by a newly identified
are secondarily infected. hantavirus, the Sin nombre (meaning nameless)
virus, which is associated with the deer mouse and
C. Nairovirus other rodents.
Members of the Crimean Congo hemorrhagic Infection appears to be caused by inhalation
group are the major human pathogens in this of the virus aerosols in dried rodent feces. Person-
genus. to-person transmission of hantaviruses seldom
Crimean Congo Hemorrhagic Fever (CCHF) occurs.
virus: is distributed widely. Cattle, sheep, goats
and other domesticated animals act as natural Laboratory Diagnosis
reservoirs. It is transmitted by Hyalomma ticks. Laboratory diagnosis depends on detection
During the acute phase of the disease, the blood of viral nucleic acid by reverse transcription-
of the patients is highly infectious and direct polymerase chain reaction or detection of specific
transmission may occur through contact. antibodies using recombinant proteins of different
hantaviruses. Hantaviruses can be isolated in
Hazara virus: A related virus, Hazara, has been cultured cells.
isolated in Pakistan.
Nairobi sheep disease: It is an acute, hemorrhagic Reoviridae
gastroenteritis caused by a Nairovirus in sheep and Family Reoviridae contains four genera-Orbivirus,
goats in East Africa. It is transmitted by Rhipice Coltivirus, Orthoreovirus, and Rotavirus
phalus ticks. The genus Orbivirus contains arthropod
Ganjam virus: Ganjam virus, isolated from ticks borne viruses. Rotaviruses and reoviruses have no
collected from sheep and goats in Orissa, India, is arthropod vectors.
closely related to the Nairobi sheep disease virus. A. Genus Orbivirus: The genus Orbivirus contains
arthropod borne viruses which infect animals
D. Hantavirus and humans.
1. Hantaviruses: Hantaan viruses are classified i. African horse sickness virus: African horse
in the Hantavirus genus of the Bunyaviridae sickness virus, transmitted by Culicoides.
family. The viruses are found worldwide and It caused extensive disease among army
cause hemorrhagic fever with renal syndrome horses and mules in India.
www.ebook3000.com
Chap-65.indd 455 15-03-2016 11:23:05
456 | Section 4: Virology
MULTIPLE CHOICE QUESTIONS (MCQs) 5. All the following statements are true for dengue
hemorrhagic fever except:
1. Which of the following viral genera is/are not a. This occurs in children with passively acquired
arthropod-borne? maternal antibodies
a. Alphavirus b. Flavivirus b. It is a most severe manifestation of the disease
c. Rubivirus d. All of the above c. This condition shows a fatality rate as high as
2. All the following viruses are transmitted by 10%
arthropod vectors except: d. Immunization is carried out by, a heat-killed
a. Hantavirus vaccine
b. Chandipura virus
6. The vaccine is not available for:
c. Rift Valley fever virus
a. Dengue fever
d. Sandfly fever virus
b. Japanese encephalitis
3. Mosquitoes act as the vector in all of the following
c. Yellow fever
viruses except:
a. Chikungunya virus d. Russian spring-summer encephalitis
b. Dengue virus 7. Hantavirus pulmonary syndrome is caused by:
c. Semiliki Forest virus a. Nairobi sheep disease virus
d. Omsk hemorrhagic fever virus b. Chittor virus
4. Arbovirus transmitted by tick is: c. Sin Nombre virus
a. Western equine encephalitis d. West Nile virus
b. B. Kyasanur Forest disease
c. Russian spring-summer encephalitis ANSWERS (MCQs)
d. Omsk hemorrhagic fever 1. c; 2 a; 3. d; 4. a; 5. d; 6. a; 7. c
Rhabdoviruses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Discuss laboratory diagnosis of rabies
be able to: ∙∙ Discuss prophylaxis against rabies
∙∙ Describe morphology of rabies virus ∙∙ Describe neural and non-neural vaccines against
∙∙ Describe the following: Street virus vs. fixed virus; rabies.
pathogenesis and clinical features of rabies; Negri
bodies
Rabies virus
Morphology
1. Virion: The rabies virion consists of a helical
nucleocapsid contained in a bullet-shaped
lipoprotein envelope 180 × 75 nm, with one
end rounded or conical and the other plane or
concave (Fig. 66.1).
2. Proteins: Protruding from the lipid envelope
are approximately 200 glycoprotein (G) spikes
of the virus, hemagglutinin activity. Spikes do
not cover the planar end of the virion.
3. Membrane or matrix (M) protein: Beneath Fig. 66.1: Rabies virus: Bullet-shaped virion, showing tightly
the envelope is the membrane or matrix (M) wound helix of ribonucleoprotein in the core, and bilayered
protein and is the major structural protein membranous envelope carrying glycoprotein spikes
www.ebook3000.com
458 | Section 4: Virology
Resistance Street Virus
Rabies virus is sensitive to ethanol, iodine prepar The rabies virus isolated from natural human
ations, quaternary ammonium compounds, or animal infection is termed the street virus.
soap, detergents and lipid solvents such as ether, Following inoculation by any route, it can
chloroform and acetone. It is inactivated by phenol, cause fatal encephalitis in laboratory animals
formalin, beta propiolactone, ultraviolet irradiation, after a long and variable incubation period of
sunlight and heat at 50°C for 1 hour or 60°C for 5 about 1–12 weeks (usually 21–60 days in dogs).
minutes. Rabies virus survives storage at 4°C for Intracytoplasmic inclusion bodies (Negri bodies)
weeks and at –70°C for years or by lyophilization It can be demonstrated in the brain of animals dying
is inactivated by CO2 so on dry ice it must be stored of street virus infection.
in glass-sealed vials.
Fixed Virus
Antigenic Properties After several serial intracerebral passages in rabbits,
There is a single serotype of rabies virus. the virus undergoes certain changes and becomes
A. Glycoprotein G: The surface spikes are what is called the fixed virus that no longer multiplies
composed of glycoprotein G, which is important in extraneural tissues. The fixed (or murant) virus
in pathogenesis, virulence and immunity. is more neurotropic, though it is much less infective
Purified spikes containing the viral glycoprotein by other routes. After intracerebral inoculation, it
elicit neutralizing antibody in animals. The produces fatal encephalitis after a short and fixed
purified glycoprotein may therefore provide a incubation period of 6–7 days. Negri bodies are
safe and effective subunit vaccine. usually not demonstrable in the brain of animals
Hemagglutinating activity: Rabies virus dying of fixed virus infection. The fixed virus is used
possesses hemagglutinating activity, optimally for vaccine production.
seen with goose erythrocytes at 0–4 °C and
pH 6.2. Hemagglutination is a property of B. Chick Embryos
the glycoprotein spikes. The hemagglutinin The rabies virus can be grown in chick embryos.
antigen is species specific and distinct from the The usual mode of inoculation is into the yolk sac.
antigens on rabies related viruses. Serial propagation in chick embryos has led to the
B. Nucleocapsid protein: Complement fixing development of attenuated vaccine strains like
antibodies are induced by the nucleocapsid Flury and Kelev.
protein and are not protective. This antigen
is group specific and cross-reactions are seen C. Tissue Culture
with some rabies related viruses. Antiserum pre
The rabies virus can grow in chick embryo
pared against the purified nucleocapsid is used
fibroblast, porcine or hamster kidney cells human
in diagnostic immunofluorescence for rabies.
diploid cell and vero cell cultures.
C. Other antigens: Other antigens identified
include two membrane proteins, glycolipid
and RNA dependent RNA polymerase.
Pathogenesis
Rabies infection usually results from the bite of
Host Range and Growth Characteristics rabid dogs or other animals. The virus can also be
transmitted following nonbite exposures through
A. Animals the inhalation of aerosolized virus (as may be found
Rabies virus has a wide host range. All warm- in bat caves), in transplanted infected tissue (e.g.
blooded animals, including humans, can be cornea), and by inoculation through intact mucosal
infected. All mammals are susceptible to rabies membranes. The virus present in the saliva of the
infection, though differences in susceptibility animal is deposited in the wound.
exist between species. Susceptibility varies among The virus appears to multiply in the muscles,
mammalian species, ranging from very high connective tissue or nerves at the site of deposition.
(foxes, coyotes, wolves) to low (opossums); those The virus remains at the site for days to months
with intermediate susceptibility include skunks, (Fig. 66.2) before progressing to the central
raccoons, and bats. Humans and dogs occupy an nervous system (CNS). Rabies virus travels by
intermediate position. Pups are more susceptible retrograde axoplasmic transport to the dorsal root
than adult dogs. Experimental infection can be ganglia and to the spinal cord. Once the virus gains
produced in any laboratory animal but mice are access to the spinal cord, the brain becomes rapidly
the animals of choice. They can be infected by any infected. The virus then disseminates from the CNS
route. After intracerebral inoculation, they develop via afferent neurons to highly innervated sites, such
encephalitis and die within 5–30 days. as the skin of the head and neck, salivary glands,
Chapter 66: Rhabdoviruses | 459
b. Acute neurologic phase: During the acute
neurologic phase, which lasts 2–7 days, patients
show signs of nervous system dysfunction such
as nervousness, apprehension, hallucinations,
and bizarre behavior.
Hydrophobia: The pathognomonic feature
is difficulty in drinking, together with intense
thirst. Patients may be able to swallow dry solids
but not liquids. Attempts to drink bring on
such painful spasms of the pharynx and larynx
producing choking or gagging that patients
develop a dread of even the sight or sound of
water (hydrophobia). Generalized convulsions
follow.
c. Coma: Patients who survive the stage of acute
neurological involvement lapse into coma.
Death usually occurs within 1–6 days due to
respiratory arrest during convulsions.
B. Rabies in Dogs
Clinical Picture
Fig. 66.2: Pathogenesis of rabies virus infection Rabies in dogs may manifest itself in two forms—
Furious rabies and Dumb rabies.
retina, cornea, nasal mucosa, adrenal medulla, a. Furious rabies: This is the typical “mad dog
renal parenchyma, and pancreatic acinar cells. syndrome”, characterized by (i) A change in
The presence of the virus in the saliva and behavior; (ii) Running amuck; (iii) Change in
the irritability and aggression brought on by the voice; (iv) Excessive salivation and foaming at
encephalitis ensure the transmission and survival the angle of the mouth; (v) Paralytic stage—
of the virus in nature. The virus ultimately reaches Paralysis, convulsions and death follow.
virtually every tissue in the body, though the
b. Dumb rabies: In this type, the excitative or
centrifugal dissemination may be interrupted at
irritative stage is lacking. The dumb form is as
any stage by death. The virus is almost invariably
infectious as the furious type.
present in the cornea and the facial skin of patients
because of their proximity to the brain. The virus
may also be shed in milk and urine. Laboratory Diagnosis
With rare exception (three known cases), rabies 1. Diagnosis of Human Rabies
is fatal once clinical disease is apparent.
Tests are performed on samples of saliva, serum,
spinal fluid, and skin biopsies of hair follicles at the
Clinical Features nape of the neck.
A. Humans
Rabies is primarily a disease of lower animals and A. Rabies Antigens by Immunofluorescence
is spread to humans by bites of rabid animals or
The method most commonly used for diagnosis
by contact with saliva from rabid animals. The
is the demonstration of rabies virus antigens by
infection has also been acquired from aerosols in
immunofluorescence. The specimens tested are
bats’ caves.
corneal smears and skin biopsy (from face or neck)
The incubation period in humans is typically
or saliva antemortem, and brain postmorterm.
1–2 months but may be as short as 1 week or as long
Direct immunofluorescence is done using antirabies
as many years (up to 19 years). It is usually shorter
serum tagged with fluorescein isothiocyanate. The
in children than in adults.
use of monoclonal antibody instead of crude
antiserum makes the test more specific.
Phases of Clinical Spectrum
The clinical spectrum can be divided into three B. Virus Isolation
phases:
a. Prodromal phase: The prodrome, lasting a. Mouse Inoculation
2–10 days, may show any of the nonspecific Samples of brain tissue, saliva, CSF, or urine may
symptoms. be injected intracerebrally into newborn mice for
www.ebook3000.com
460 | Section 4: Virology
isolation of the virus. Infection in mice results in Italian physician who first discovered them. This is
encephalitis and death. The inoculated mice are still the method most commonly used as facilities
examined for signs of illness and their brains are for immunofluorescence and biological tests are
examined at death or at 28 days postinoculation not available in many laboratories.
for Negri bodies, or by immunofluorescence rabies Impression smears of the brain are stained by
antigen. Seller’s technique (basic fuchsin and methylene
blue in methanol), which has the advantage that
b. Isolation in Cell Culture fixation and staining are done simultaneously.
Negri bodies are seen as intracytoplasmic, round
A more rapid and sensitive method is isolation
or oval, purplish pink structures with characteristic
of the virus in tissue culture cell lines (WI38,
basophilic inner granules. Negri bodies vary in size
BHK21,CER). Virus isolations is identified by
from 3–27 µm. Other types of inclusion bodies may
immunofluorescence. A positive IF test can be
sometimes be seen in the brain in diseases such
obtained as early as 2–4 days after inoculation.
as canine distemper but the presence of inner
The identity of the isolate can be established by the
structures in the Negri bodies makes differentiation
neutralization test with specific antirabies antibody.
easy. Failure to find Negri bodies does not
exclude the diagnosis of rabies. The microscopic
C. Serology examination for Negri bodies identifies 75–90% of
Rabies antibodies can be detected in the serum and cases of rabies in dogs. Failure to find Negri bodies
CSF of the patient by ELISA. High titer antibodies does not exclude the diagnosis of rabies.
are present in the CSF in rabies but not after Negri bodies contain rabies virus antigens and
immunization. can be demonstrated by immunofluorescence.
Both Negri bodies and rabies antigen can usually
D. Detection of Nucleic Acid be found in animals or humans infected with
rabies, but they are rarely found in bats.
Reverse transcription-polymerase chain reaction
(RT-PCR) testing can be used to amplify parts If impression smears are negative, the tissue
of a rabies virus genome from fixed or unfixed should be sectioned and stained by Giemsa or
brain tissue for detection of rabies virus RNA. This Mann’s method.
technique can confirm dFA results and can detect 3. Isolation of the rabies virus (biological test)
rabies virus in saliva and skin biopsy samples. This is done as described above, for human rabies
diagnosis.
2. Animal Rabies 4. Corneal test
The head of the animal is cut off and sent to the Rabies virus antigen can be detected in live animals
nearest testing laboratory, duly packed in ice in an in corneal impressions or in frozen sections of skin
air-tight container. Alternatively, the brain may be biopsies by the flueroscent antibody test.
removed with antiseptic precautions and sent in
50% glycerol-saline for examination and the other Prophylaxis
in Zenker’s fixative, sent for biological test and
microscopy, respectively. The portion of brain sent This may be considered under:
should include the hippocampus and cerebellum
as Negri bodies are most abundant there. The A. Postexposure Prophylaxis
following tests are done in the laboratory: B. Preexposure Prophylaxis
1. Demonstration of rabies virus antigen by A. Post-exposure Prophylaxis
immunofluorescence This consists of: a. Local treatment , b. Antirabic
This is a highly reliable and the best single test vaccines, c. Hyperimmune serum.
currently available for the rapid diagnosis of rabies a. Local treatment of wound:
viral antigen in infected specimens. This test i. Cleansing: Immediate flushing and
can establish a highly specific diagnosis within washing the wound(s), scratches and the
a few hours. Examination of salivary glands by
adjoining areas with plenty of soap and
immunofluorescence is useful.
water, preferably under a running tap, for
2. Demonstration of inclusion bodies (Negri at least 5 minutes as soap inactivates virus
bodies) by destroying its envelope.
A definitive pathologic diagnosis of rabies can be ii. Chemical treatment: After cleansing
based on the finding of Negri bodies in the brain wound should be inactivated by irrigation
or the spinal cord. Negri bodies, named after the with virucidal agents - either alcohol
Chapter 66: Rhabdoviruses | 461
(400–700 ml/liter), tincture or 0.01% 3. Infant brain vaccines
aqueous solution of iodine or povidone The encephalitogenic factor in brain tissue is a
iodine. basic protein associated with myelin. It is scanty
iii. Suturing: Bite wounds should not be or absent in the nonmyelinated neural tissue of
immediately sutured. newborn animals. So vaccines were developed
iv. Antirabies Serum: The local application of using infant mouse, rat or rabbit brain.
antirabies serum or its infiltration around
the wound has been shown to be highly II. Non-neural Vaccines
effective in preventing rabies. 1. Egg vaccines
v. Antibiotics and antitetanus measure:
a. Duck embryo vaccine (DEV): It was disconti
When indicated should follow the local
nued because of its poor immunogenicity. (b)
treatment recommended above.
Live attenuated chick embryo vaccines—These
b. Antirabic vaccines: Antirabic vaccines fall into vaccines were used for vaccination of animals.
two main categories: neural and non-neural Two types of vaccines were developed with the
(Table 66.2). The former are associated with Flury strain.
serious risk of neurological complications and
i. Low Egg Passage (LEP) vaccine: at 40–50 egg
have been replaced by the latter.
passage level for immunization of dogs.
I. Neural Vaccines ii. High Egg Passage (HEP) vaccine: at 180
passage level for cattle and cats. These are not
These are suspensions of nervous tissues of
in use now.
animals infected with the fixed rabies virus.
2. Cell culture vaccines
1. Semple Vaccine
Semple (1911) developed this vaccine at the Central The cell culture vaccines are of two types:
Research Institute, Kasauli (India), had been the
i. Human origin
most widely used vaccine for over half a century.
It is a 5% suspension of sheep brain infected with Human diploid cell vaccine (HDCV)
fixed virus and inactivated with phenol at 37°C, The first cell culture vaccine was the human diploid
leaving no residual live virus. cell (HDC) vaccine developed by Koprowsky,
Wiktor and Plotkin. It is a purified and concentrated
2. Beta propiolactone (BPL) vaccine preparation of fixed rabies virus (Pitman–Moore
This is a modification of the Semple vaccine, strain) grown on human diploid cells (WI 38 or
in which beta propiolactone is used as the MRC 5) and inactivated with beta propiolactone
inactivating agent instead of phenol. It is prepared or tri-n-butyl phosphate. It is highly antigenic and
from fixed virus grown in the brains of adult sheep free from serious side effects. Its only disadvantage
(Semple type) or other animals. It is believed to be is its high cost. HDC vaccine is now licensed for use
more antigenic. in a number of countries including India for both
pre- and postexposure immunization.
www.ebook3000.com
462 | Section 4: Virology
Indications for Antirabies Treatment Dosage Schedules
Antirabies treatment should be started immediately: Two dosage schedules (one recommended by
i. If the animal shows signs of rabies or dies the Central Research Institute, Kasauli and the
within 10 days of observation. other recommended by the Pasteur Institute of
ii. If the biting animal cannot be traced or Southern India, Coonoor) are followed in India.
identified. A full schedule consists of 7–10 daily inoculations
iii. Unprovoked bites. followed by 1–2 boosters.
iv. Laboratory tests (e.g. fluorescent rabies anti
body test or test for Negri bodies) of the brain Site for Vaccination
of the biting animal are positive for rabies.
v. All bites by wild animals. The ideal site for vaccination is the anterior
abdominal wall, for this area offers enough space
to accommodate the large quantity of vaccine
Vaccination Schedules to be injected. The injections are given deep
Neural Vaccines subcutaneously.
The dosage of the vaccine depends on the degree
of risk to which the patient has been exposed. Adverse Reactions
Accordingly, patients are classified as follows.
1. General: Headache, insomnia, giddiness,
palpitation, diarrhea.
Classification of Exposures
2. Local: Itching irritation, pain, tenderness,
One of the factors determining the dose of anti-
redness and swelling at the site of injection.
rabies vaccine is the degree of risk of rabies to
which the person is exposed. Accordingly, patients 3. Allergic: Urticaria, syncope, angioneurotic
are classified as follows. edema, anaphylactic reaction.
4. Neuroparalysis: Post-vaccinial paralysis due to
Class I (Slight risk) sensitization.
a. Licks on healthy unbroken skin.
b. Licks on intact mucous membrane or conjunctiva.
Cell Culture Vaccines
c. Bites or scratches which have raised the
epidermis but have not drawn blood, on all All three cell culture vaccines available in India
parts of the body except the head, face, neck or (HDC, PCEC and PVC) have the same dosage
fingers. schedule, which is the same for both adults and
d. Consumption of unboiled milk or handling raw children.
flesh of rabid animals.
Class II (Moderate risk) i. Preexposure Prophylaxis
a. Licks on definitely remembered fresh cuts or This is indicated for persons at high risk of contact
abrasions on the fingers. with rabies virus such as laboratory workers
b. Scratches with oozing of blood. handling rabies virus or with rabid animals
c. All bites except those on head, neck, face, palms (veterinarians). Preexposure prophylaxis requires
and fingers. three doses of the vaccine injected on day 0, 7, 21 or
d. Minor wounds less than 5 in number. 0, 28 and 56. A booster dose is recommended after
one year and then one every five years.
Class III (Severe risk)
a. Licks on definitely remembered fresh cuts or
abrasions on head, face or neck. ii. Postexposure Prophylaxis
b. All bites or scratches on the head, face or neck. Postexposure prophylaxis requires five or six doses,
c. All bites or scratches on the fingers which are on days 0, 3, 7, 14, 30 and optionally 90. The vaccine
lacerated, more than half centimeter long or is to be given intra muscular (1M) or subcutaneous
have penetrated the true skin. (SC) in the deltoid region, or in children on the
d. All bites penetrating the true skin and drawing anterolateral aspect of the thigh. Gluteal injections
blood, when there are five teeth marks or more. are to be avoided as they are found to be less
e. All bites on any part of the body causing immunogenic. This course is expected to give
extensive laceration. protection for at least five years, during which
f. Bites from wild animals—All jackal and wolf period any further exposure may need only one or
bites. two booster doses (on days 0, 3) depending on the
g. Any Class II patient who has not received degree of risk. After five years, it is advisable to give
treatment within 14 days of exposure. a full five injection course if exposed to infection.
Chapter 66: Rhabdoviruses | 463
C. Passive Immunization Table 66.3: Lyssavirus sera/genotypes
Passive immunization is an important adjunct to Genotype/ Virus Isolated Disribution
vaccination and should be invariably employed Serotype from
whenever the exposure is considered of high risk. Virus
Two preparations of anti rabies serum are available 1. Rabies Warm Worldwide
for passive immunization: blooded with few
animals exceptions
i. Horse Antirabies Serum: It should be given on 2. Lagos bat/ Bat/cat Nigeria/
day 0 in a single dose of 40 IU/kg of body weight Natal bat Central and
subject to a maximum of 3000 Units. South Africa
3. Mokola Shrew/ Nigeria/
ii. Human Rabies Immune Globulin cat/dog/ other African
human countries
Human rabies immune globulin (HRlG) has
now replaced equine antirabies serum in many 4. Duvenhage Human/ South Africa
countries. The dose recommended is a single bat
administration of 20 IU per kg of body weight. 5. European Bat/human Europe
bat
Vaccine for Animals lyssavirus:
Type I
Concentrated cell culture vaccines containing 6. European Bat/human Europe
inactivated virus are now available, which give bat
good protection after a single IM injection. lyssavirus:
Injections are given at 12 weeks of age and repeated Type II
at 1–3 year intervals. 7. Australian Bat/human Australia
bat
lyssavirus:
B. Preexposure Prophylaxis
This is indicated for persons at high risk of contact
with rabies virus or with rabid animals. It is rec
Rabies in India: Rabies is endemic in India.
ommended that antibody titers of vaccinated
Rabies is not a notifiable disease and the 30,000
individuals be monitored periodically and that
deaths reported by national authorities may not be
boosters be given when required.
complete picture. Every year approximately 1.1–1.5
million people receive post-exposure treatment with
Epidemiology either nerve tissue or cell culture rabies vaccine.
Rabies is the classic zoonotic infection, spread More than 95% of these cases are bitten by dogs.
from animals to humans. Rabies virus is present in
terrestrial animals in all parts of the world except Control
Australasia and Antartica, and some islands like Human rabies can be checked by control of rabies
Britain. in domestic animals, by registration, licensing and
All warm blooded animals including man are vaccination of pets and destruction of stray animals.
susceptible to rabies. Rabies in man is a dead-end
infection, and has no survival value for the virus. Rabies related viruses
Major source of virus is saliva in bite of rabid
animal. Direct person-to-person transmission of The genus Lyssavirus consists of the rabies virus and
rabies has not been recorded. An unusual mode other serologically related viruses. The relevance of
of transmission of rabies has occurred in some rabies related viruses in human diseases is not clear,
recipients of corneal grafts. Minor source is aerosols though some of them have caused illness and death
in bat caves containing rabid bats. in humans. They are considered to represent a
biological bridge between the rabies virus and other
Epidemiological forms of rabies: Rabies exists
rhabdoviruses. Lyssaviruses have been classified
in two epidemiological forms: a. Urban rabies: b.
into seven serotypes (Table 66.3).
Wild-life or sylvatic rabies:
In certain Latin American countries and parts Key Points
of U.S.A. the vampire bat is an important host
and vector of rabies. Vampire bats have not been Rhabdoviruses are bullet or rod shaped, enveloped
viruses with single stranded RNA genome
reported in India.
The rabies virus causes rabies in humans and a wide
Reservoir of rabies—Reservoir of rabies are variety of animals
wild animals.
www.ebook3000.com
464 | Section 4: Virology
The rabies virus isolated from natural human or 3. Describe the prophylaxis of rabies.
animal infection is termed the street virus 4. Write short notes on:
After several serial intracerebral passages in rabbits, a. Street virus vs. fixed virus or Differences between
the virus undergoes certain changes and becomes street virus and fixed virus.
what is called the fixed virus (or murant) virus b. Negri bodies.
Rabies virus is transmitted to humans by primarily c. Neural vaccines against rabies.
a bite of a rabid dog or by other infected animals.
The infection has also been acquired from aerosols d. Non-neural vaccines used against rabies.
in bats’ caves e. Cell culture vaccines.
Laboratory tests for human rabies—Viral antigens f. Rabies related viruses.
can be demonstrated in the corneal smear, skin
biopsy, and saliva (antemortem) or in the brain
tissue (postmortem) directly by direct fluorescent
Multiple choice questions
antibody (DFA) test 1. Which of the following species of animals is most
Isolation of rabies virus can be done by mouse susceptible to rabies infection?
inoculation and isolation in cell culture. Reverse a. Dog b. Man
transcription-polymerase chain reaction (RT-PCR) c. Cat d. Fowl
testing can be used for detection of rabies virus RNA
2. The inclusion bodies found in rabies are called:
Demonstration of Negri bodies in neural tissues by a. Bollinger bodies
microscopy is diagnostic of rabies
b. Councilman bodies
Postexposure prophylaxis is started in the persons c. Cowdry type a bodies
immediately after exposure to infection and consists d. Negri bodies
of (a) Local treatment of wound; (b) Antirabic
vaccines and (c) Hyperimmune serum 3. Negri bodies can be demonstrated in infection with:
a. Fixed rabies virus
Cell culture vaccines such as human diploid cell
b. Street rabies virus
vaccine (HDCS), purified chick embryo cell (PCEC)
c. Both of the above
vaccine, and purified vero cell (PVC) vaccines are now
d. None of the above
increasingly used. These are non-neural vaccines
Postexposure prophylaxis requires five or six doses, 4. Which of the following clinical specimens can be
on days 0, 3, 7, 14, 30 and optionally 90 used for the demonstration of rabies antigen by
Human rabies can be checked by control of rabies direct immunofluorescence antemortem?
in domestic animals, by registration, licensing and a. Salivary smears b. Corneal smears
vaccination of pets and destruction of stray animals. c. Conjunctival smears d. All of the above
5. All of the following antirabies vaccine are inactivated
Important questions vaccines except:
a. Human diploid cell strain vaccine
1. Describe the morphology and draw a labeled b. Purified vero cell vaccine
diagram of rabies virus. Discuss the laboratory c. Purified chick embryo cell culture vaccine
diagnosis of rabies. d. Chick embryo vaccine
2. Discuss the pathogenesis and clinical features of Answers (MCQs)
rabies. 1. c; 2. d; 3. b; 4. d; 5. d
6767
Chapter
Hepatitis Viruses
Learning Objectives
After reading and studying this chapter, you should modes of transmission of hepatitis B virus; hepatits
be able to: B carriers
∙∙ Classify various hepatitis viruses ∙∙ Describe laboratory diagnosis of hepatitis B virus
∙∙ Tabulate differences between various hepatitis ∙∙ Discuss prophylaxis of hepatitis B infections or
viruses hepatitis B vaccine
∙∙ Compare various features of hepatitis A virus (HAV) ∙∙ Describe the following:Hepatitis C virus or Type C
and hepatitis B virus (HBV) hepatitis; Hepatitis D virus or Delta agent; Hepatitis
∙∙ Describe the following: Morphology of hepatitis E virus; Hepatitis G virus.
B virus; antigenic structure of hepatitis B virus;
www.ebook3000.com
466 | Section 4: Virology
Table 67.1: Comparative features of hepatitis viruses
A B C D E
Virus structure HAV, 27 nm RNA, HBV, 47 nm DNA HCV, 30–60 nm HDV, 35–37 nm HEV, 32–34 nm RNA
Picornavirus (Hepadnavirus) RNA, Flavivirus Defective RNA Herpes virus
(Hepatovirus) (hepacivirus) Deltavirus
Modes of Fecal-oral Parenteral Parenteral Parenteral Fecal–oral
infection Vertical, sexual
Age affected Children Any age Adults Any age Young adults
Incubation 15–45 30–180 15–160 30–180 15–60
period (days)
Onset Acute Insidious Insidious Insidious Acute
Illness Mild Occasionally Moderate Occasionally severe Mild, except in preg-
severe nancy
Carrier state Nil Common Present Nil (only with HBV) Nil
Oncogenicity Nil Present specially Present Nil Nil
after neonatal
infection
Prevalence Worldwide Worldwide Probably Endemic areas Only developing
worldwide (Mediterranean, N, countriest lndia,
Europe, Central and Asia, Africa, Central,
N. America) America)
Fig. 67.1: The picornavirus structure of hepatitis A virus. The Laboratory Diagnosis
icosahedral capsid is made up of four viral polypeptides (VP1– Etiological diagnosis of type A hepatitis may be
VP4). Inside the capsid is a single-stranded, positive-sense RNA made by demonstration of the virus or its antibody.
single stranded RNA (ssRNA) that has a genomic viral protein
(VPg) on the 5’ end A. Demonstration of the virus: The virus can be
visualized by immune electron microscopy
(IEM) in fecal extracts during the late incubation
Natural infection with HAV is seen only in period and the preicteric phase.
humans. Though primates, such as chimpanzees B. Demonstration of antibody: The best way
have been shown to acquire the infection from to demonstrate an acute HAV infection is by
humans and transmit it to human contacts, there finding anti-HAV immunoglobulin M (IgM), as
is no evidence of any extrahuman source of the measured by an enzyme-linked immunosorbent
virus in nature. assay (ELISA) or radioimmunoassay. Antibody
IgM anti-HAV antibody appears during the
Clinical Features late incubation period, reaches peak levels in
The incubation period is 2–6 weeks. Disease in 2–3 weeks and disappears after 3–4 months.
children is generally milder than that in adults IgG antibody appears at about the same time,
and is usually asymptomatic. The clinical disease peaks in 3–4 months and persists much longer,
consists of two stages: the prodromal or preicteric perhaps for life (Fig. 67.2). Demonstration of
and the icteric stages. The onset may be acute or IgM antibody in serum indicates current or
insidious, with fever, malaise, anorexia, nausea, recent infection, while IgG antibody denotes
Chapter 67: Hepatitis Viruses | 467
Fig. 67.2: Typical course of hepatitis type A Fig. 67.3: Hepatitis B virus structure
recent or remote infection and immunity. ELISA virus (HBV) infects the liver and, to a lesser extent,
kits for detection of IgM and IgG antibodies are the kidneys and pancreas of only humans and
available. chimpanzees.
C. Virus isolation: It is not routinely performed.
Classification
Prophylaxis
HBV is assigned to a separate family Hepadnaviridae
A. General Measures (Hepatitis DNA viruses) which consists of two
The spread of HAV is reduced by interrupting the genera:
fecal–oral spread of the virus by avoiding potentially i. Orthohepadnavirus: It containing HBV as
contaminated water or food, water. well as the woodchuck and ground squirrel
hepatitis viruses. HBV is Hepadnavirus type 1.
B. Immunization ii. Avihepadnavirus: It containing the Pekin
1. Passive protection: Specific passive prophylaxis duck and gray heron hepatitis viruses.
by pooled normal human immunoglobulin
(16% solution in a dose of 0.2–0.12 mL/kg body Structure
weight) intramuscular (IM), before exposure
The HBV is a 42 nm DNA virus with an outer
or in early incubation period, can prevent or
envelope and an inner core, 27 nm in diameter,
attenuate clinical illness, while not necessarily
enclosing the viral genome and a DNA polymerase
preventing infection and virus excretion.
(Fig. 67.3) which is circular double stranded DNA.
2. Hepatitis A vaccine
i. Formalin inactivated, alum conjugaged Australia antigen: In 1965, Blumberg, studying
vaccine: A safe and effective formalin human serum lipoprotein allotypes, observed in
inactivated, alum conjugated vaccine the serum of an Australian aborigine, a new antigen
containing HAV grown in human diploid which gave a clearly defined line of precipitation
cell culture is available for use in children with sera from two hemophiliacs who had received
and adults at high risk for infection, multiple blood transfusions. This was named the
especially travelers to endemic regions. A Australia antigen. By 1968 the ‘Australia antigen’
full course consists of two intramuscular was found to be associated with serum hepatitis.
injections of the vaccine. Protection begins It was subsequently shown to be the surface
4 weeks after injection and lasts for 10–20 component of HBV. Therefore, the name Australia
antigen was changed to hepatitis B surface antigen
years.
(HBsAg).
ii. Live HAV vaccine: It has been developed
in China. Types of particles: Under the electron microscope,
sera from type B hepatitis patients show three types
Treatment of particles (Fig. 67.4).
Treatment is symptomatic. No specific antiviral i. Spherical particle: The predominant form is
drug is available. a small, spherical particle with a diameter of
22 nm.
ii. Tubular particle: The second type of particle
Hepatitis B Virus—serum hepatitis
is filamentous or tubular with a diameter of 22
Type B hepatitis is the most widespread and the nm and of varying length. The particles carry
most important type of viral hepatitis. Hepatitis B the hepatitis B surface antigen (HBsAg).
www.ebook3000.com
468 | Section 4: Virology
iii. Dane particle: The third type of particle, far 2. Hepatitis B core antigen (HBcAg): The antigen
fewer in number, is a double walled spherical expressed on the core is called the hepatitis B
structure, 42 nm in diameter. This particle core antigen (HBcAg).
is the complete hepatitis B virus. It was first 3. Hepatitis B e antigen (HBeAg): Hepatitis Be
described by Dane in 1970 and so is known antigen (HBeAg) is a soluble nonparticulate
as the Dane particle.The outer surface, or nucleocapsid protein. The HBeAg and HBcAg
envelope, contains HBsAg and surrounds a proteins share most of their protein sequence.
27-nm inner nucleocapsid core that contains 4. Viral genes and antigens: The genome has
HBcAg. The variable length of a single- a compact structure with four overlapping
stranded region of the circular DNA genome genes. These include structural proteins of the
results in genetically heterogeneous particles virion surface and core, a small transcriptional
with a wide range of buoyant densities. transactivator (X), and a large polymerase (P)
Genome: The nucleocapsid encloses the viral protein that includes DNA polymerase, reverse
genome consisting of two linear strands of DNA held transcriptase, and RNase H activities (Table
in a circular configuration. One of the strands (the 67.2, Fig. 67.5).
plus strand) is incomplete, so that the DNA appears
partially double stranded and partially single HBV Subtypes
stranded. Associated with the plus strand is a viral The particles containing HBsAg are antigenically
DNA polymerase, which has both DNA-dependent complex. It contains two different antigenic
DNA polymerase and RNA-dependent reverse components—the common group reactive antigen
transcriptase functions. Although a DNA virus, a, and two pairs of type specific antigens d-y and
it encodes a reverse transcriptase and replicates w-r, only one member of each pair being present at
through an RNA intermediate. This polymerase a time. HBsAg can thus be divided into four major
can repair the gap in the plus strand and render the antigenic subtypes: adw, adr, ayw and ayr.
genome fully double stranded (Fig. 67.3). They show a distinct geographical distribution.
(Table 67.3).
Antigenic Structure
1. Hepatitis B surface antigen (HBsAg): The Cultivation
envelope proteins expressed on the surface
HBV does not grow in any conventional culture
of the virion and the surplus 22 nm diameter
system. However, limited production of the virus
spherical and filamentous particles constitute
and its proteins can be obtained from several cell
the hepatitis B surface antigen (HBsAg).
lines transfected with HBV DNA. HBV proteins have
been cloned in bacteria and yeast. The chimpanzee
is susceptible to experimental infection and can be
used as a laboratory model.
Stability
The HBV is a relatively heat-stable virus. It remains
viable at room temperature for long periods. Heating
to 60°C for 10 hours inactivates virus by a factor of
100–1000-fold. Exposure to hypochlorite (10,000
ppm available chlorine) or 2% glutaraldehyde for 10
Fig. 67.4: Different types of particles of HBV: Spherical 22 minutes will inactivate virus 100,000-fold, though
nm particle, double shelled 42 nm particle (Dane particle), HBsAg may not be destroyed by such treatment.
tubular 22 nm particle
Epidemiology
The HBV is worldwide in distribution. Natural
infection occurs only in humans. There is no animal
reservoir. The virus is maintained in the large
pool of carriers whose blood contains circulating
virus for long periods, in some even lifelong.
The prevalence of HBV infection varies widely in
different parts of the world.
India falls in the intermediate group, with
Fig. 67.5: The HBV genes and gene products higher carrier rates in the southern part of the
country and lower rates in the northern part. The
Table 67.3: Antigenic types of HBsAg rich and the poor countries also differ in the age
and modes of infection.
Antigenic types Distribution
adw Worldwide Mode of Transmission
adr Asia
The HBV is a blood borne virus and there are three
ayw India, Africa, Russia important modes of transmission:
ayr India, Africa, Russia 1. Parenteral transmission
2. Perinatal transmission
Clinical Syndromes 3. Sexual transmission.
Acute Infection 1. Parenteral transmission: HBV is transmitted
The clinical presentation of HBV in children is less only in blood and other body fluids, including
severe than that in adults, and infection may even cervical secretions, semen, and breast milk. Many
be asymptomatic. Clinically apparent illness occurs other therapeutic, diagnostic, prophylactic and
in as many as 25% of those infected with HBV. even nonmedical procedures are now the main
1. Preicteric phase: HBV infection is characterized modes of infection. HBV is very highly infectious
by a long incubation period (about 1–6 months) far more that HIV.
and an insidious onset. Symptoms during the 2. Perinatal transmission: Vertical transmission
prodromal period may include fever, malaise, from mother to child is one of the most
and anorexia, followed by nausea, vomiting, important routes. HBV can be transmitted to
abdominal discomfort, and chills. babies through contact with the mother’s blood
2. Icteric phase: The classic icteric symptoms of at birth and in mother’s milk.
liver damage (e.g. jaundice, dark urine, pale 3. Sexual transmission: Since HBV is present in
stools) follow soon thereafter. semen and vaginal secretions, therefore, it can
3. Convalescent phase: About 90–95% of adults be transmitted by sexual contact. The risk of
with acute hepatitis B infection recover within transmission by heterosexual and homosexual
1–2 months of onset and eliminate the virus from contact increases with the number of partners
the body within about six months, remaining and the duration of such relationships.
immune thereafter. Mortality is about 0.5–2%.
Chronic infection: A proportion of cases (1–10%) Hepatitis B Carriers
remain chronically infected. They may be asympto Carriers are of two types:
matic carriers or may progress to recurrent or chronic 1. Super carriers: They have HBeAg, high titters
liver disease or cirrhosis. A few of them may develop of HBsAg and DNA polymerase in their blood.
hepatocellular carcinoma after many decades. HBV may also be demonstrable in their blood.
Very minute amount of serum or blood from
Pathogenesis such carriers can transmit the infection. About a
The pathogenesis of hepatitis appears to be immune quarter of the carriers in India are HBeAg positive.
mediated. HBV replicates in the hepatocytes, 2. Simple carriers: These are more common
reflected in the detection of viral DNA and HBcAg types of carriers who have low titer of HBsAg
in the nucleus and HBsAg in the cytoplasm and at in blood, with negative HBeAg, HBV and DNA
the hepatocyte membrane. polymerase. They transmit the infection only
www.ebook3000.com
470 | Section 4: Virology
when large volumes of blood are transferred indicative of viral replication. HBcAg is not
as in blood transfusion. Many super carriers in demonstrable in circulation. It is the earliest
time become simple carriers. antibody marker to be seen in blood, long
before anti-HBe or anti-HBs. As anti-HBc
HBV Markers remains lifelong, it serves as a useful indicator
The main antigens HBsAg, HBcAg, and HBeAg of prior infection with HBV; even after all the
each induce corresponding antibodies. With other viral markers become undetectable.
the exception of HBcAg, all these antigens and Initially, anti-H Bc is predominantly IgM,
antibodies, together with the viral DNA polymerase, but after about 6 months, it is mainly IgG.
can be detected in the blood at various times after Selective tests for IgM or IgG anti-HBc
infection and are referred to as ‘markers’ (Table therefore enable distinction between recent
67.4). HBcAg is readily detectable only in the or remote infection respectively.
hepatocyte nuclei. iii. HBeAg: HBeAg presence denotes high
infectivity and its absence, along with the
Laboratory Diagnosis presence of anti-HBe, indicates low infectivity.
HBeAg appears in blood concurrently with
Specific Diagnosis HBsAg, or soon afterwards. Circulating HBeAg is
Specific diagnosis of hepatitis B tests on serological an indicator of active intrahepatic viral replication,
demonstration of the viral markers and can be and the presence in blood of DNA polymerase,
carried out by detection of HBsAg, anti-HBs, HBV DNA and virions, reflecting high infectivity.
HBeAg, anti-HBe, IgM anti-HBc, IgG anti-HBc Before HBsAg disapp ears, HBeAg is replaced
and HBV DNA in the serum. The sequence of by anti-HBe, signaling the start of resolution of
appearance of viral markers in the blood is shown the disease. Anti-HBe levels often are no longer
in Figure 67.6. These can be detected by sensitive detectable after 6 months.
and specific tests like ELISA and RIA.
2. Viral DNA Polymerase
1. Detection of Viral Markers
DNA polymerase activity, HBV DNA, and HBeAg,
i. HBsAg: HBsAg is the first marker to appear which are representative of the viremic stage of
in blood after infection. HBsAg is usually hepatitis B, occur early in the incubation period,
detectable 2–6 weeks in advance of clinical concurrency or shortly after the first appearance
and biochemical evidence of hepatitis and of HBsAg.
persists throughout the clinical course of
the disease. In the typical case, it disappears
3. Polymerase Chain Reaction (PCR)
within about 2 months of the start of clinical
disease, but may sometimes last for 6 months Like HBeAg, HBV DNA is also an indicator of viral
and even beyond but typically disappears by replication and infectivity. Molecular methods,
the sixth month after exposure. such as DNA:DNA hybridization and PCR, at
ii. HBcAg: High levels of IgM-specific anti-HBc present used for HBV DNA testing are highly
are frequently detected at the onset of clinical sensitive and quantitative. HBV DNA level in serum
illness. Because this antib ody is directed reflects the degree of viral, replication in the liver
against the 27 nm internal core compo and so helps assess the progress of patients with
nent of HBV, its appearance in the serum is chronic hepatitis under antiviral chemotherapy.
www.ebook3000.com
472 | Section 4: Virology
70 subtypes. Because of this diversity there is little HEPATITIS D virus (HDV) (DELTA agent)
heterologous or even homologous postinfection
immunity in hepatitis C. This curious little agent was first detected in
people undergoing exacerbations of chronic HBV
The virus has not been grown in culture, but
infections. In 1977, Rizzetto et al. in Italy identified
has been cloned in Escherichia coli
a new viral antigen in the liver cell nuclei of patients
Mode of infection: Infection is mainly by blood infected with hepatitis B virus. This has been
transfusion and other modes of contact with infected shown to be due to the hepatotropic virus delta or
blood or blood products. Injectable drug abusers, hepatitis D. HDV is unique in that it uses HBV and
transplant recipients and immunocompromised target cell proteins to replicate and produce its one
persons are at high risk. Sexual transmission protein. Delta is a defective RNA virus dependent
is probably less important. The virus can be on the helper function of HBV for its replication
transmitted from mother to infant. and expression. It acquires an HBsAg coat for trans
Infections by HCV are extensive throughout the mission. Therefore, it has no independent existence
world. HCV infection is seen only in humans. The and can survive and replicate only as long as HBV
groups at risk are broadly similar to those listed infection persists in the host. It is often associated
for hepatitis B but their relative proportions are with the most severe forms of hepatitis in HBsAg-
different. positive patients. It is a viral parasite, proving that
“even fleas have fleas.”
Clinical Features The HDV is enclosed within the hepatitis B
The incubation period is long, 15–160 days, with a surface antigen, HBsAg, and has no recognizable
mean of 50 days. morphology of its own.
HCV causes three types of disease: The HDV is a defective satellite virus requiring
i. Acute hepatitis with resolution of the infect HBV as helper virus. HDV is a spherical, 36 nm
ion and recovery in 15% of cases. particle with an outer coat composed of the
ii. Chronic persistent infection with possible hepatitis B surface antigen surrounding the circular
progression to disease much later in life for 70%. single stranded RNA genome. The HDV RNA
iii. Severe rapid progression to cirrhosis in 15% of genome is very small, singlestranded RNA and
patients—Many patients develop cirrhosis and circular. Delta agent is thus an incomplete virus,
are at high risk for hepatocellular carcinoma reminiscent of the Dependoviruses (Fig. 67.7).
(5–25%) decades later. It has been classified in a new genus Deltavirus,
because of its special features.
Laboratory Diagnosis
Pathogenesis
A. Antobody detection: The diagnosis and
detection of HCV infection are based on ELISA Its mode of transmission is the same as for HBV.
recognition of antibody. The antigens used are Similar to HBV, the delta agent is spread in blood,
various structural and nonstructural proteins semen, and vaginal secretions. However, it can
cloned in E. coli. Confirmation by immunoblot replicate and cause disease only in people with
assay is therefore recommended. In HCV active HBV infections.
infection antibodies appear irregularly and late,
limiting their diagnostic utility.
B. HCV RNA identification: Reverse transcriptase-
polymerase chain reaction, branched-chain
DNA, and other genetic techniques can detect
HCV RNA in seronegative people and have
become key tools in the diagnosis of HCV
infection.
Prophylaxis
Only general prophylaxis, such as screening of
blood and blood products prior to transfusion, is
possible. No specific active or passive immunizing
agent is available.
Treatment
Recombinant interferon-alpha, alone or with
ribavarin is the only known treatment for HCV. Fig. 67.7: Delta hepatitis virion
Chapter 67: Hepatitis Viruses | 473
Types of infection: Two types of infection are young to middle aged adults (15–40 years old). The
recognized: symptoms and course of HEV disease are similar
1. Coinfection: In coinfection, delta and HBV are to those of HAV disease; it causes only acute dis
transmitted together at the same time. ease. However, the symptoms for HEV may occur
2. Superinfection: In superinfection, delta later than those of HAV disease, and response
infection occurs in a person already harboring to serum immunoglobulin G may be poor. The
HBV. More rapid, severe progression occurs in mortality rate associated with HEV disease is 1–2%.
HBV carriers superinfected with HDV than in HEV infection is especially serious in pregnant
people coinfected with HBV and the delta agent. women and during pregnancy may cause a high
No association has been noted between HDV rate of abortion, intrauterine death and increased
and hepatocellular carcinoma. In simultaneous perinatal mortality in babies born to women with
acute HBV and HDV infections, IgM anti-HBc fulminant hepatitis.
will be detectable, while in acute HDV infection
superimposed on chronic HBV infections, anti- Epidemiology
HBc will be of IgG class. HEV is predominantly spread by the feco-oral
route, especially in contaminated water. Hepatitis
Laboratory Diagnosis E has been shown to occur in epidemics, endemics
The only way to determine the presence of the and sporadic forms almost exclusively in the
agent is by detecting the delta antigen or antibodies. less developed parts of the world. A substantial
ELISA and radioimmunoassay procedures are proportion of cases of acute viral hepatitis occurring
available for doing this. Anti-delta antibodies in young to middle-aged adults in Asia and the
appear in serum and can be identified by ELISA. Indian subcontinent appear to be caused by HEV.
The IgM antibody appears 2–3 weeks after infection The largest such epidemic occurred in Delhi
and is soon replaced by the IgG antibody in acute during the winter of 1955–56, following a breakdown
delta infection. in the water supply and sewage systems of Delhi,
For the rapid identification of delta particles in affecting over 30,000 persons. The cause was
circulation, RNA sequences have been cloned and eventually identified as a new agent, HEV. A similar
DNA probes have been developed. The woodchuck epidemic of hepatitis E occurred between December
has been found to be a suitable experimental 1975 and January 1976 in Ahmedabad city, India.
model for the study of HDV infection.
Laboratory Diagnosis
Treatment Several antibody assays are being developed,
There is no known specific treatment for HDV mostly based on ELISA methods.
hepatitis. 1. Exclusion of hepatitis A and hepatitis B:
Exclusion of hepatitis A by IgM serology and
Prophylaxis hepatitis B by absence of HBsAg and IgM anti-
Because the delta agent depends on HBV for repli HBc.
cation and is spread by the same routes, prevention 2. Immunoelectron microscopy: Immunoelectron
of infection with HBV prevents HDV infection. microscopic examination of patient feces for
Immunization with HBV vaccine protects against aggregated calicivirus-like particles using
subsequent deltavirus infection. monoclonal antibodies.
3. ELISA tests: for IgM and IgG anti-HEV.
Hepatitis E Virus (HEV) 4. Western blot assay: A Western blot assay for
IgM and/or IgG antiHEV.
Hepatitis E virus (HEV) has been provisionally
5. Polymerase chain reaction (PCR): Polymerase
classified in the genus Hepevirus under the family
chain reaction (PCR) assay for the detection of
Caliciviridae. HEV is a spherical nonenveloped
HEV RNA (as cDNA) in patient feces or in acute-
virus, 32–34 nm in diameter, with a single
phase sera.
stranded RNA genome. The surface of the virion
shows indentation and spikes. HEV (E-NANBH)
(The E stands for “Enteric” or “epidemic”) is Prophylaxis
predominantly spread by the feco-oral route, General measures: These depend on the mainten
especially in contaminated water. ance of a clean water supply, and generally
resemble those used to control HAV.
Clinical Features Immunization: Vaccines based on recombinant
The incubation period ranges from 2–9 weeks with antigens are under development, and show some
an average of six weeks. Most cases occur in the promise.
www.ebook3000.com
474 | Section 4: Virology
Hepatitis G Virus encloses an inner icosahedral 27 nm nucleocapsid
(core), which contains hepatitis B core antigen
Two flavivirus-like isolates were’ obtained in 1995
(HBcAg). Inside the core is the genome, a circular
from Tamarin monkeys inoculated with blood from double stranded DNA and a DNA polymerase
a young surgeon (GB) with acute hepatitis. It was Hepatitis B surface antigen (H BsAg) is also named
termed GB, the patient’s initials. A similar virus was as Australia antigen
isolated from another human specimen the same Mode of transmission: HBV is a blood borne
year. These isolates were called GB viruses A, B virus and there are three important modes of
and C respectively. transmission: 1. Parenteral transmission; 2.
Perinatal transmission; 3. Sexual transmission
In 1996, an isolate closely resembling GBV-C Laboratory diagnosis: Specific diagnosis of hepatitis
was obtained from a patient with chronic hepatitis. B rests on serological demonstration of the viral
This has been called hepatitis G virus (HGV). markers and can be carried out by detection of
Hepatitis G virus resembles HCV in many ways. HBsAg, anti-HBs, HBeAg, anti-HBe, IgM anti-HBc, IgG
HGV is a flavivirus, is transmitted in blood, and has anti-HBc and HBV DNA in the serum. These can be
detected by sensitive and specific tests like ELISA
a predilection for chronic hepatitis disease. It has not
and RIA. Molecular methods such as DNA: DNA
been grown, but its RNA genome has been cloned. hybridization and PCR are used for HBV DNA testing
The HGV RNA has been found in patients with are highly sensitive and quantitative
acute, chronic and fulminant hepatitis, hemophiliacs, Prophylaxis: General prophylaxis consists in
patients with multiple transfusions and hemodialysis, avoiding risky practices
intravenous drug addicts and blood donors. HGV Immunization: I. Passive immunization: Hyper
appears to be a blood borne virus resembling HCV. immune hepatitis B immune globulin (HBIG)
administered 1 M in a dose of 300–500 iu soon after
Its role in hepatitis is yet to be clarified. exposure to infection
The virus is present worldwide. Majority of the II. Active immunization: such as plasma-derived
individuals with HGV infection have no detectable hepatitis B vaccine, recombinant yeast hepatitis
evidence of liver disease. There have been, however, B vaccine (Three doses given at 0, 1 and 6 months
cases of acute, fulminant and chronic hepatitis constitute the full course), recombinant Chinese
hamster ovary (CHO) cell hepatitis vaccine,
where HGV is presently the only explanation for
synthetic peptide vaccines and hybrid virus vaccine
their liver disease. There is no evidence of a causal
Hepatitis C Virus
relationship between HGV infection and hepato
HCV can cause acute HCV infection, chronic HCV
cellular carcinoma. HGV infection results frequently infection, and cirrhosis and other complications
in chronic viraemia. It often subsides after several induced by hepatitis
years and anti-HGenv antibody develops. Blood or blood products and also organs of infected
patients are the major sources of infection
For diagnosis, antibody to HCV antigen can be
Laboratory Diagnosis detected by enzyme immunoassay
1. HGV is identified by detection of the genome by The HCV RNA can be amplified by RT-PCR
reverse transcriptase polymerase chain reaction Hepatitis D Virus
(RT-PCR) or other RNA detection methods. The hepatitis D virus (HDV) is unique in being an
2. An immunoassay has been developed to detect incomplete virus, that requires hepadnavirus helper
anti-HG env. Serum HGV RNA indicates viremia, functions for propagation in hepatocytes
Transmission of HDV occurs parenterally
whereas anti-HGenv is associated with recovery.
HDV superinfections in patie nts with HBV results in
Key Points chronic HDV infection
Vaccination with HBV vaccine protects against
At least six viruses, A through G (A, B, C, D, E and G.) subsequent HDV infection
are hepatitis viruses Hepatitis E virus (HEV)
All the hepatitis viruses are RNA viruses, except Hepatitis E virus is the primary cause of enterically
the hepatitis B virus (HBV), which is a DNA virus transmitted non-A, non-B hepatitis
belonging to the family Hepadnaviridae It usually causes an acute, self-limiting disease similar
Hepatitis A virus (HAV) to HAV. Pregnant women—high mortality associated
Hepatitis A virus (HAV) can be demonstrated in the with HEV
stool by immunoelectron microscopy (IEM). ELISA is Hepatitis G virus (HGV) is a flavivirus, is transmitted
the method for detection of IgM and IgG antibodies in blood, and has a predilection for chronic hepatitis
in the serum disease.
Prevention of HAV infection depends on: vaccines
containing formalin-inactivated HAV
Hepatitis B Virus
HBV is associated with hepatocellular carcinoma Important questions
Hepatitis B virus (HBV) is a double-walled spherical
1. Name the hepatitis viruses. Describe the morphology
structure and measures 42 nm in diameter (Dane
and antigenic structure of hepatitis B virus.
particle). The outer surface or envelope of virus
2. Classify hepatitis viruses. Discuss the laboratory
contains hepatitis B surface antigen (HBsAg). It
diagnosis of infections caused by hepatitis B virus.
Chapter 67: Hepatitis Viruses | 475
3. Write short notes on: 8. Which of the following viral markers is first marker to
a. Hepatitis A virus (HAV) or type A hepatitis or appear in biood after infection with hepatitis B virus?
infectious hepatitis. a. HBsAg
b. Hepatitis B virus or Dane’s particle. b. HBcAg
c. Hepatitis B surface antigen (HBsAg) or Austral c. HBeAg
ia antigen. d. Antibody to HBsAg
d. Draw a neat labeled diagram of hepatitis B virus. 9. Which of the following viral markers is/are positive
e. Hepatitis B virus markers. in simple carriers of hepatitis B?
f. Hepatitis C virus or type C hepatitis. a. HBsAg
g. Hepatitis D virus or Delta agent. b. HBeAg
h. Non-A, Non-B hepatits c. Both of the above
i. Hepatitis E virus d. None of the above
j. Hepatitis G virus 10. In super carriers of hepatitis B which of the following
k. Pathogenesis of HBV or type B hepatitis or viral markers is/are positive?
serum hepatitis or serum jaundice or transfu a. HBsAg
sion hepatitis. b. HBeAg
l. Laboratory diagnosis of type B hepatitis. c. Both of the above
m. Prophylaxis of hepatitis B or hepatitis B vaccine. d. None of the above
11. High infectivity of hepatitis B virus is indicated by
which of the following markers when positive?
Multiple choice questions (mcqs) a. HBsAg
1. Which of the following hepatitis viruses is DNA b. HBeAg
c. HBcAg
virus?
d. None of the above
a. Hepatitis A virus
12. For preparation of recombinant hepatitis B vaccine
b. Hepatitis B virus
which of the following hepatitis B genes are used?
c. Hepatitis C virus
a. HBsAg gene b. HBeAg gene
d. Hepatitis E virus
c. HBcAg gene d. All of the above
2. Which of the following hepatitis viruses can be
13. The carrier state in hepatitis B virus infection is
transmitted by sexual route?
demonstrated by:
a. Hepatitis A virus a. Persistent presence of HBsAg in blood for at
b. Hepatitis C virus’ least 6 months
c. Hepatitis E virus b. Persistent presence of H and Ab in blood for at
d. All of the above least 6 months
3. Which of the following hepatitis viruses may cause c. Persistent presence of HBeAg in blood for at
hepatic carcinoma? least 6 months
a. Hepatitis A virus d. Persistent presence of H and Ag in blood for at
b. Hepatitis C virus least 6 months
c. Hepatitis E virus 14. All the following statements are true for hepatitis C
d. Hepatitis G virus virus except:
4. Which of the following hepatitis viruses is non a. It is closely related to hepatitis D virus
enveloped? b. It is a leading cause of cirrhosis
a. Hepatitis A virus c. Blood transfusion is the most important mode
b. Hepatitis B virus of transmission of HCV
c. Hepatitis C virus d. A live vaccine is available against HCV
d. Hepatitis D virus 15. Which statement is true for hepatitis D virus:
5. All the following statements are true for hepatitis A a. An incomplete virus
virus (HA V) except: b. Related to hepatitis B
a. It is most commonly transmitted by fecal-oral c. A single-stranded DNA
route d. Transmitted by fecal-oral route
b. It causes chronic disease or fatal disease 16. Hepatitis E virus is:
c. t has a short incubation period of 3–4 weeks a. Related to hepatitis C virus
d. There is only one serotype of HAV b. Yet to be cultured
6. Which of the following methods is preferred for c. Associated with chronic liver infection
diagnosis of hepatitis A virus (HAV) infection? d. A major cause of hepatitis transmitted by blood
a. Alanine aminotransferase levels 17. Which of the following statement is true for hepatitis
b. Detection of IgM antibody to HAV by ELISA G virus (HGV):
c. Detection of IgG antibody to HAV by ELISA a. HGV is a flavivirus
d. Cell cultures for growing HAV b. Hepatitis G virus resembles hepatitis C virus
(HCV)
7. Which of the following antigens is present in
c. Hepatitis G virus is transmitted in blood
the envelope of hepatitis B virus?
d. All of the above
a. HBsAg
b. HBcAg Answers (MCQs)
c. HBeAg 1. b; 2. b; 3. b; 4. a; 5. b; 6. b; 7. a; 8. a; 9. a; 10. c; 11. b; 12.
d. None of the above a; 13. a; 14. d; 15. a; 16. b; 17. d
www.ebook3000.com
68
Chapter
Retroviruses: Human
Immunodeficiency Virus (HIV)
Learning Objectives
After reading and studying this chapter, you should ∙∙ D iscuss opportunistic infections associated with
be able to: human immunodeficiency virus infection
∙∙ Classify retroviruses. ∙∙ D escribe the laboratory diagnosis of human
∙∙ Describe morphology of human immunodeficiency immunodeficiency virus infection
virus ∙∙ D escribe the laboratory tests for detection of
∙∙ Describe the antigenic structure of human immuno specific antibodies in human immunodeficiency
deficiency virus (HIV) and draw labeled diagram of virus infection
HIV-I ∙∙ Describe strategies of human immunodeficiency
∙∙ Describe modes of transmission of human immuno virus testing
deficiency virus ∙∙ Describe the following: Antiviral therapy for HIV;
∙∙ Describe types of exposure and their relative risks postexposure prophylaxis.
www.ebook3000.com
478 | Section 4: Virology
A. Genes Coding for Structural Proteins primate species. There are two distinct types of
(Table 68.2) human AIDS viruses: HIV-l and HIV-2.
1. The gag (group-specific antigen) gene: The HIV-1 groups: Based on env gene sequences, HIV-1
gag gene determines the core and shell of the comprises three distinct virus groups—M (for
virus. It is expressed as a precursor protein, ‘major’), O (for ‘outlier’) and N (for new).
P55. This precursor protein is cleaved into M group: The predominant M group, which cause
three proteins, P15, p18 and p24. The four the large majority of HIV-1 infections worldwide,
proteins coded for by the gag gene of HIV are all contains nine subtypes or “clades” (A-K, omitting
found in the virion , including p24. The major E and I).
core antigen is p24 which can be detected in
serum during the early stages of HIV infection O group: A few HIV-l strains isolated from West
before antibodies appear. Late in the course of Africa (Cameroon, Gabon) do not fall within the
infection, the decline of free anti-p24 antibody Group M and have been designated group O.
and reappearance of p24 antigen in circulation N group: Some recent isolates of HIV-1 from
point to exacerbation of the illness. Cameroon, distinct from M and O groups have
2. The env: The env determines the synthesis of been called group N.
envelope glycoprotein gp160, which is cleaved
into the two envelope components—gp120 Human immunodeficency virus-2 (HIV-2): HIV
which forms the surface spikes and gp41, the strains, first isolated from West Africa in 1986,
transmembrane anchoring protein. The spike which react with HIV type 1 antiserum very weakly
glycoprotein gp120 is the major envelope or not at all have been termed HIV type 2. The
antigen, and antibodies to gp120 are present envelope antigens of the two types are different,
in circulation till the terminal stage of the though their core polypeptides show some cross
infection. reactivity. HIV-1 and the chimpanzee virus carry
3. The pol gene: The pol gene codes for the a vpu gene, whereas HIV-2 and most SIVs have a
protease, endonuclease, integrase and reverse vpx gene. The sequences of the gag and pol genes
transcriptase. It is expressed as a precursor are highly conserved. It is much less virulent than
protein, which is cleaved into proteins p31, p51 HIV-1. It is largely confined to West Africa, though
and p66. isolations have been reported from some other
areas, including western and southern India.
B. Nonstructural and Regulatory Genes HIV-2 subtypes: Six subtypes of HIV-2 (A-F) have
There are at least six regulatory genes in HIV and been identified. HIV-2 has only 40% genetic identity
at least two in HTLV-I (Fig. 68.3). with HIV-I. It is more closely related to simian
1. tat (transactivating gene) enhancing the expres immunodeficiency virus than to HIV-1.
sion of all viral genes. HIV-1 subtypes show a geographical distribu
2. nef (negative factor gene) down regulating viral tion. All known HIV virus groups and subtypes are
replication. present in Cameroon, West Africa. Subtype A is the
3. rev (regulator of virus gene) enhancing most prevalent, being found worldwide, while B
expression of structural proteins. is the most common in the Americas and Europe.
4. vif (viral infectivity factor gene) influencing The common subtypes in Africa are A, C and D,
infectivity of viral particles. while in Asia the common subtypes are E, C and
5. vpu (only in HIV-1) and vpx (only in HIV-2) B. Subtype E is the most common in Thailand. In
enhancing maturation and release of progeny India and China, subtype C is the most prevalent.
virus from cells. (Detection of the type-specific
sequences vpu and vpx is useful in distinguishing Resistance
between infection by HIV-1 and 2). 1. Temperature: HIV is inactivated by heat, in the
6. vpr stimulating promoter region of the virus. autoclave or hot air oven. HIV is thermolabile,
The Vpr protein increases transport of the viral being inactivated in 10 minutes at 60°C and in
preintegration complex into the nucleus and seconds at 100°C.
also arrests cells in the G2 phase of the cell cycle. 2. Lyophilization: It withstands lyophilization.
7. LTR (long terminal repeat) sequences, one 3. Disinfectants: HIV is completely inactivated by
at either end, containing sequences giving treatment for 10 minutes at room temperature
promoter, enhancer and integration signals. with any of the following: 10% household bleach,
50% ethanol, 35% isopropanol, 1% Nonidet
Classification P40, 0.5% Lysol, 0.5% paraformaldehyde, or
Lentiviruses have been isolated from many species, 0.3% hydrogen peroxide. The virus is also
including at least 26 different African nonhuman inactivated by extremes of pH (pH 1.0, pH
Chapter 68: Retroviruses: Human Immunodeficiency Virus | 479
13.0). For treatment of contaminated medical The presence of other sexually transmitted
instruments, a 2% solution of glutaraldehyde is diseases such as syphilis, gonorrhea, or herpes
useful. simplex type 2 increases the risk of sexual HIV
The virus is not inactivated by 2.5% Tween 20. transmission. Transmission may be more likely
from male to female. Asymptomatic viruspositive
Routes of Transmission individuals can transmit the virus.
Virus is present in the blood, semen, and cervical
and vaginal secretions, and these sources are 2. Blood and Blood Products
important in transmission. HIV is spread only by Transfusion of infectious blood or blood products
three modes (Table 68.4): is an effective route for viral transmission.
1. Sexual contact with infected persons (hetero
sexual or homosexual). Contaminated needle: It can transmits the
infection. This is particularly relevant in drug
2. By blood and blood products.
addicts. The use of unsterile syringes and needles
3. From infected mother to babies (intrapartum, by qualified and unqualified health workers makes
perinatal, postnatal). iatrogenic infection likely.
The modes of transmission of HIV and their
relative risks are shown in Table 68.5. 3. Mother-to-child Transmission
1. Sexual Intercourse Transmission of infection from mother to baby can
HIV is primarily a sexually transmitted infection. take place before, during or after birth. Mother-to-
Both sexes are affected equally. Transmission in the infant transmission rates vary from 13 to 40% in
developing countries is almost always heterosexual untreated women. Infants can become infected in
and can take place in both directions. utero, during the birth process, or, more commonly,
through breastfeeding. Transmission during
breastfeeding usually occurs early (by 6 months).
Table 68.4: Transmission of HIV infection
Routes Specific transmission Pathogenesis
Known routes of transmission Infection is transmitted when the virus enters
1. Inoculation in blood Transfusion of blood and the blood or tissues of a person and comes into
blood products contact with a suitable host cell, principally the
Needle sharing among
CD4 lymphocyte. The major determinant in the
intravenous drug abusers
Needlestick, open wound, pathogenesis and disease caused by HIV is the
and mucous membrane virus tropism for CD4-expressing T cells and cells
exposure in healthcare of the macrophage lineage.
workers HIV genome is uncoated and internalized into
Tattoo needles
the cell after fusion of the virus with the host cell
2. Sexual transmission Anal and vaginal intercourse membrane Viral reverse transcriptase mediates
3. Mother to baby Intrauterine transmission transcription of its RNA into double stranded
Peripartum transmission DNA, which is integrated into the genome of
Breast milk
the infected cell through the action of the viral
Routes not involved in transmission enzyme integrase, causing a latent infection. HIV
Close personal contact Household members causes lytic and latent infection of CD4 T cells
Healthcare workers not and persistent infection of cells of the monocyte
exposed to blood
macrophage family and disrupts neurons. The
outcomes of these actions are immunodeficiency
and acquired immunod eficiency syndrome
(AIDS) dementia. From time to time, lytic
Table 68.5: Efficiency of different routes of trans
infection is initiated releasing progeny virions
mission of HIV
which infect other cells. The long and variable
Route Efficiency incubation period of HIV infection is because of
Blood transfusion >90% the latency. HIV can be isolated from the blood,
Perinatal 13−40%
lymphocytes, cell-free plasma, semen, cervical
secretions, saliva, tears, urine and breast milk in
Sexual intercourse
an infected individual.
• Anal intercourse 1% per episode The primary pathogenic mechanism in HIV
• Vaginal intercourse 0.1% per episode
infection is the damage caused to the CD4+ T
Intravenous drug use 0.5−1% lymphocyte. The T4 cells decrease in numbers
www.ebook3000.com
480 | Section 4: Virology
and the T4:T8 (helper: suppressor) cell ratio is Table 68.7: Summary of classification system for
reversed. An important feature in HIV infection is HIV infection (Centers for Disease Control, USA)
the polyclonal activation of B lymphocytes leading
Group I Acute HIV syndrome
to hypergammaglobulinemia.
In peripheral blood, lymphoid tissue and other Group II Asymptomatic infection
tissues such as brain where HIV replication occurs, Group III Persistent generalized
HIV targets CD4 positive (CD4+) cells and cells of lymphadenopathy
the monocyte-macrophage lineage. The principal Group IV Other diseases:
immunological abnormalities seen in HIV infection Subgroup A Constitutional disease—ARC
are listed in Table 68.6.
Subgroup B Neurologic diseases
Clinical manifestations in HIV infections are
Subgroup C Secondary infectious diseases
due not primarily to viral cytopathology but are:
secondary to the failure of immune responses. This Category C1 Specified infectious
renders the patient susceptible to opportunistic diseases listed in the CDC
surveillance definition
infections and malignancies. for AIDS, such as P. carinii
pneumonia, cryptosporidiosis,
Clinical Features of HIV Infection toxoplasmosis, generalized
strongyloidiasis,
The Centers for Disease Control (USA) have classified cryptococcosis CMV or herpes
the clinical course of HIV infection under various diseases.
groups (Table 68.7). The natural evolution of HIV Category C2 Other specified secondary
infection can be considered in the following stages. diseases, such as oral hairy
leukoplakia, salmonella
Group I—Acute HIV Infection bacteremia, nocardiosis,
tuberculosis, thrush
The acute seroconversion illness: It resembles
Subgroup D Secondary cancers, such as
glandular fever, with adenopathy and flu-like Kaposi’s sarcoma, lymphomas
symptoms.Within 3–6 weeks of infection with HIV,
Subgroup E Other conditions.
about 50% of persons experience low grade fever,
malaise, headache, lymphadenopathy, sometimes
with rash and arthropathy resembling glandular fever. Group III—Persistent Generalized
Tests for HIV antibodies are usually negative at lymphadenopathy
the onset of the illness but become positive during This has been defined as the presence of enlarged
its course. HIV antigenemia (p24 antigen) can be lymph nodes, at 1east 1 cm in diameter, in two
demonstrated at the beginning of this phase. or more noncontiguous extrainguinal sites, that
persist for at least three months, in the absence of
Group II—Asymptomatic or latent infection any current illness or medication that may cause
A clinically asymptomatic or “latent” period follows lymphadenopathy.
the acute infection. They show positive HIV anti
body tests during this phase and are infectious. Group IV—AIDS Related Complex
This group includes patients with considerable
Table 68.6: Immunological abnormalities in HIV immunodeficiency, suffering from various
infection constitutional symptoms or minor opportunistic
I. Features that characterize AIDS
infections. The typical constitutional symptoms
1. Lymphopenia. are fatigue, unexplained fever, persistent diarrhea
2. Selective T cell deficiency—Reduction in number and marked weight loss of more than 10% of body
of T4 (CD4) cells, inversion of T4:T8 ratio. weight. The common opportunistic infections
3. Decreased delayed hypersensitivity on skin are oral candidiasis, herpes zoster, hairy cell
testing.
4. Hypergammaglobulinemia—predominantly IgG
leukoplakia, salmonellosis or tuberculosis.
and IgA; and IgM also in children. Generalized lymphadenopathy and splenomegaly
5. Polyclonal activation of B cells and increased are usually present. ARC patients are usually
spontaneous secretion of Ig. severely ill and many of them progress to AIDS
II. Other consistently observed features: in a few months. With no treatment, the interval
1. Decreased in vitro lymphocyte proliferative
response to mitogens and antigens.
between primary infection with HIV and the first
2. Decreased cytotoxic responses by T cells and NK appearance of clinical disease is usually long in
cells. adults, averaging about 8–10 years. Death occurs
3. Decreased antibody response to new antigens. about 2 years later.
4. Altered monocyte/macrophage function. The acquired immunodeficiency syndrome
5. Elevated levels of immune complexes in serum.
(AIDS) presents in many ways, all due to the
Chapter 68: Retroviruses: Human Immunodeficiency Virus | 481
underlying severe loss of the ability to respond is probable that transmission can also take place
to infectious agents and to control tumors. The during delivery or from breast milk.
features classified as group IV include what was
known as the AIDS-related complex or ARC. Laboratory Diagnosis
Laboratory procedures for the diagnosis of HIV
AIDS: This is the end-stage disease representing
infection include specific tests for HIV and tests for
the irreversible breakdown of immune defence
immunodeficiency as well as. Evidence of infection
mechanisms, leaving the patient prey to progressive
by HIV can be detected in three ways:
opport unistic infections and malignancies.
1. Specific tests for HIV infection.
AIDS may be manifested in several different
2. Nonspecific tests.
ways, including lymphadenopathy and fever,
3. Tests for opportunistic infections and tumor.
opportunistic infections, malignancies, and AIDS-
related dementia (Table 68.8).
A. Specific Tests for HIV Infection (Table 68.9)
Pediatric AIDS: About one-third to half the 1. Antigen Detection
number of babies born to infected mothers are
The virus antigens may be detectable in blood
infected with HIV. In most cases, infection is
after about two weeks following a single massive
transmitted to the baby in the perinatal period. It
infection. The major core antigen p24 is the earliest
virus marker to appear in blood and is the one
Table 68.8: Opportunistic infections and malign tested for IgM antibodies appear in about 4–6
ancies commonly associated with HIV infection weeks, to be followed by IgG antibodies (Fig. 68.2).
Afterwards, free p24 antigen disappears from
I. BACTERIAL circulation and remains absent during the long
1. M. avium complex
asymptomatic phase, to reappear only when severe
2. Mycobacterium tuberculosis—disseminated or
extrapulmonary clinical disease sets in. The p24 antigen capture
3. Salmonella—recurrent septicemia assay (ELISA) which uses anti-p24 antibody as the
II. VIRAL solid phase can be used for this antigen.
1. Cytomegalovirus
2. Herpes simplex virus
3. Varicella-zoster virus 2. Virus Isolation
4. Epstein-Barr virus The virus is present in circulation and body fluids,
5. Human herpesvirus 6 within lymphocytes or cell-free. It can be isolated
6. Human herpesvirus 8
III. FUNGAL
from CD4 lymphocytes of peripheral blood, bone
1. Candidiasis marrow and serum. The technique of isolation
2. Cryptococcosis is by cocultivation of the patient’s lymphocytes
3. Aspergillosis with uninfected lymphocytes in the presence of
4. Pneumocystis carinii pneumonia interleukin-2. Virus presence is detected by assays
5. Histoplasmosis
6. Coccidioidomycosis
for reverse transcriptase and p24 antigen in the
IV. PARASITIC culture fluids.
1. Toxoplasomosis Virus titers parallel p24 titers, being high soon
2. Cryptosporidiosis after infection, low and antibody bound during
3. lsosporiasis the asymptomatic period, and again high towards
4. Microsporidiosis
5. Generalized strongyloidiasis
the end.
V. MALIGNANCIES
1. Kaposi’s sarcoma 3. Detection of Viral Nucleic Acid
2. B cell lymphoma or non-Hodgkin’s lymphoma
VI. SLIM DISEASE
As the most sensitive and specific test, PCR has
become the gold standard for diagnosis in all stages
www.ebook3000.com
482 | Section 4: Virology
HIV-1 ELISA tests were the earliest approved
serologic tests for HIV infection and remain the
most sensitive approved commercial assays for
infection. Direct solid phase antiglobulin ELISA
is the method most commonly used. The antigen
is obtained from HIV grown in continuous T
lymphocyte cell line or by recombinant techniques
and should represent all groups and subtypes
of HIV-1 and HIV-2. The antigen is coated on
Fig. 68.2: Sequence of appearance of p24 antigen and
antibodies after a massive HIV infection (as by blood
microtiter wells or other suitable solid surface.
transfusion) The test serum is added, and if the antibody is
↑ exposure: A = p24 antigen; B = IgM antibody; C = IgG present, it binds to the antigen. After washing away
antibody; 2, 4, 8, 12 = week after exposure. the unbound serum, antihuman immunoglobulin
↓ n ↓ = virus readily isolated from blood. linked to a suitable enzyme is added, followed by a
color-forming substrate. If the test serum contains
of HIV infection. Two forms of PCR have been used, anti-HIV antibody, a photometrically detectable
DNA PCR and RNA PCR. color is formed which can be read by special ELISA
i. DNA PCR: In the DNA PCR, peripheral readers. Conversion of substrate to product is
lymphocytes from the subject are lysed and quantitated by spectrophotometry.
the proviral DNA amplified using primer Modifications of ELISA in which the antibody
pairs from relatively constant regions of HIV in test serum either competes with enzyme
genome. The test is highly sensitive and conjugated anti-HIV antibody, or is captured by
specific when done with proper controls. antihuman immunoglobulin onto solid phase are
ii. RNA PCR: A related test, HIV RNA PCR can more specific. Thirdgeneration assays employ
be used for diagnosis as well as for monitoring a sandwich technique using enzyme-coup led
the level of viremia. HIV antigens and take advantage of the bi- or
multivalent nature of antibodies to improve
4. Antibody Detection specificity.
Demonstration of antibodies is the simplest and ELISA specific for IgM antibody is also available.
most widely employed technique for the diagnosis Immunometric assays are highly sensitive and
of HIV infection. The mean time to seroconversion specific.
after HIV infection is 3–4 weeks. Most individuals b. Rapid tests: These tests take less than 30
will have detectable antibodies within 6–12 weeks minutes and do not require expensive equipment.
after infection, whereas virtually all will be positive A number of ‘rapid tests’ have been introduced for
within 6 months. this purpose:
Serological tests for anti-HIV antibodies are of
∙∙ Dot blot assays
two type-screening and confirmatory tests (Table
∙∙ Particle agglutination (gelatin, RBC, latex,
68.10).
microbeads)
∙∙ HIV spot and comb tests
A. Screening Tests (Table 68.10)
∙∙ Fluorometric microparticle technologies
a. Enzyme-linked immunosorbent assays (ELISA) Tests using finger-prick blood, saliva and urine
tests have also been developed.
Table 68.10 Laboratory tests for detection of c. Simple tests
specific antibodies in HIV infection They take 1–2 hours. They also do not require
expensive equipment.
1. Screening (E/R/S) tests
a. ELISA B. Confirmatory Tests
b. Rapid tests
– Dot blot assays a. Western blot test: In this test, HIV proteins
– Particle agglutination (gelatin, RBC, latex, separated by polyacrylamide gel electrophoresis
microbeads) are blotted onto strips of nitrocellulose paper. The
– HIV spot and Comb tests
antigen impregnated nitrocellulose is then cut into
– Fluorometric microparticle technologies
c. Simple tests strips. Each strip is then incubated with a dilution
These are also based on ELISA principle but take 1–2 of patient serum. Antibodies which attach to the
hours separated viral antigens on the strip are detected
2. Supplemental tests by antihuman immunoglobulin antibody to which
a. Western blot assay
enzyme has been attached followed by application
b. Immunofluorescence test
of a substrate. The substrate changes color in
Chapter 68: Retroviruses: Human Immunodeficiency Virus | 483
the presence of enzyme and permanently stains detection of true reactivity (window period)
the strip. The location or position on the strip at or false reactivity with principally single-band
which a patient’s antibodies attach to viral antigens reactivity. In such cases the Western blot may
indicates whether antibody is specific for viral be repeated a later. If no definitive result can
antigens or directed against nonviral material from be given even then, it may be necessary to
the cells in which the virus was grown. have p24 assay done.
b. Immunofluorescence test: In this test, HIV
Interpretation of Western Blot Test Results
infected cells are acetone fixed on to glass slides and
Western blot results are scored as negative, positive, then reacted with test serum followed by fluorescein
or indeterminate (Fig. 68.3). conjugated antihuman gammaglobulin. A positive
i. Negative result: The WHO has suggested that reaction appears as apple-green fluorescence of
a weakly reactive p17 band may be considered cell membrane under fluorescence microscope.
negative. Antibodies to viral core protein p24
or envelope glycoproteins gp41, gp120, or Strategies of HIV Testing
gp160 are most commonly detected.
There are three strategies of HIV testing:
ii. Positive result: In a positive serum, bands will
be seen with multiple proteins, typically with Strategy I: The serum is tested with one E/R/S
p24 (gag gene, core protein), p31 (pol gene, test and if reactive, sample is considered positive
reverse transcriptase) and gp41, gp120 or and if non-reactive it is considered negative. This
gp160 (env gene, surface antigens). A positive strategy is used for transfusion safety. For this pur
reaction with proteins representing the three pose, a highly sensitive and very reliable test kit
genes gag, pol, env is conclusive evidence of must be used.
HIV infection. The test may be considered
positive even if it shows bands against at Strategy II: The serum reactive with one E/R/S test
least two of the following gene products: p24, is retested with a second E/R/S test with higher
gp41, gp120/160. However, interpretation specificity, based on a different antigen preparation
becomes difficult when bands that appear do and/or different test principle. If found reactive
not satisfy these criteria. This may happen in on second E/R/S test also, it is reported as posi
early infection but may also be nonspecific. tive, otherwise as negative. This strategy is used
for HIV surveillance. The majority of individuals
iii. Indeterminate result : Indeterminate
seroconvert within 2 months after viral exposure.
results are not uncommon. Indeterminate
HIV infection for longer than 6 months without a
results may arise from either insensitive
detectable antibody response is very uncommon
Strategy III: The serum reactive with two E/R/S
tests is retested with a third E/R/S test. The third
test should again be based on different antigen
preparation or test principle. A serum testing
reactive with all three E/R/S tests is reported
positive. A serum sample nonreactive in third
E/R/S is considered equivocal/borderline. Such
individuals should be retested after three weeks. If
this sample also provides an equivocal result, the
person is considered to be HIV antibody negative.
For asymptomatic HIV infection, it is necessary to
confirm the diagnosis with three tests. Symptomatic
infections with opportunistic infections, however,
may be subjected to two tests.
The first test selected for any of the strategies
should be of highest sensitivity and second and
third E/R/S test selected should be of highest
specificity.
B. Nonspecific Tests
Immunological Tests
Fig. 68.3: Diagrammatic representation of Western blot The following parameters help to establish the
test for HIV immunodeficiency in HIV infection.
www.ebook3000.com
484 | Section 4: Virology
a. Total leukocyte and lymphocyte count to tests are negative during the early stage of HIV
demonstrate leukopenia and a lymphocyte infection when the individual is infectious,
count usually below 2000/mm3. screening may not detect all dangerous donors
b. T cell subset assays. Absolute CD4+ T cell count but can still eliminate a large majority of them.
will be usually less than 200/mm3. T4:T8 cell Screening for p24 antigen can detect those in
ratio is reversed. the window period also.
c. Platelet count will show thrombocytopenia. A person found positive for HIV antigen or
d. Raised IgG and IgA levels. antibody should never donate blood or other
e. Diminished CMI as indicated by skin tests. biological materials. As the infection can be
f. Lymph node biopsy showing profound transmitted from mother to baby before, during
abnormalities. or after birth, antenatal screening is useful.
2. Seroepidemiology: Antibody surveys have been
C. Tests for Opportunistic Infections and most useful in identifying the geographical extent
Tumors of HIV infection and in other epidemiological
Apart from diagnosing HIV infection, the laboratory studies.
would be called upon to identify the opportunistic 3. Diagnosis: Serology is almost always positive
infections that are a feature of AIDS. Routine in persons with clinical features of AIDS. It
microbiological methods would suffice for this. may, however, be negative in acute illness
However, serological diagnosis of infections may and sometimes in the very late cases where
not always be reliable in AIDS as antibody formation the immune system is nonreactive. Routine
may be affected by the immune deficiency. serology may also be negative when the
infection is with a different AIDS virus. For
Applications of Serological Tests (Table example, HIV-2 infections are likely to be
68.11) missed if antibody testing is done with the HIV-l
antigen alone.
Serological tests for HIV infection are employed in
4. Prognosis: In a person infected with HIV,
the following situations:
loss of detectable anti-p24 antibody indicates
1. Screening: Screening is defined as the systematic
clinical deterioration. This is also associated
application of HIV testing, whether voluntary or
with HIV antigenemia and increased virus titer
mandatory, to entire populations or selected
in circulation.
target groups. It should be mandatory that all
donors of blood, blood products, semen, cells,
tissues and organs be screened. As antibody Laboratory Monitoring of HIV Infection
Some laboratory tests are important in monitoring
Table 68.11: Potential antiviral therapies the course of HIV infection.
for HIV infection 1. CD4+ T cell count: When the count falls below
500, it is an indication of disease progression
Nucleoside analog reverse transcriptase inhibitors
Azidothymidine (AZT) (Zidovudine) and the need for antiretroviral therapy. Counts
Dideoxycytidine (ddC) (Zalcitabine) below 200 denote risk of serious infections.
Dideoxyinosine (ddI) (Didanosine) 2. Direct measurement of HIV RNA: Direct
d4T (Stavudine) measurement of HIV RNA becomes necessary,
3TC (Lamivudine)
ABC (Abacavir) particularly in the course of treatment. This
is done usually by two methods, the reverse
Non-nucleoside reverse transcriptase inhibitors
Nevirapine (Viranune)
transcriptase PCR (RT-PCR) assay and the
Delavirdine (Rescriptor) branched DNA (bDNA) assay.
Efavirenz (Sustiva) 3. Beta-2-microglobulin and Neopterin: Beta-2-
Protease inhibitors microglobulin and Neopterin can be measured
Saquinavir (Invirase/Fortovase) in serum or urine. Their concentrations are
Ritonavir (Norvir) low in asymptomatic infection and rise with
Indinavir (Crixivan)
advancing disease.
Nelfinavir (Viracept)
Amprenavir (Agenerase)
Highly active antiretroviral therapy (HAART) Epidemiology
(combination) Routes of Transmission
Indinavir/AZT/3TC
Ritonavir/AZT/3TC HIV is spread only by three modes—sexual
Nelfinavir/AZT/3TC contact with infected persons; by blood and blood
Nevirapine/AZT/ddI products; and from infected mother to babies
Nevirapine/indinavir/3TC (intrapartum, perinatal, postnatal).
Chapter 68: Retroviruses: Human Immunodeficiency Virus | 485
Geographic distribution: HIV -1 infections are non-nucleoside reverse transcriptilse inhibitors, or
spreading worldwide, with the largest number protease inhibitors (Table 68.11). Other anti-HIV
of AIDS cases in sub-Saharan Africa but with drugs being developed
a growing number of cases in Asia, the United In the current guidelines, AZT is recommended
States, and the rest of the world. HIV-2 is more for the treatment of asymptomatic or mildly
prevalent in Africa (especially West Africa) than symptomatic people with CD4 counts of less than
in the United States. Heterosexual transmission is 500/µL and for the treatment of infected pregnant
the major means of spread of HIV-1 and HIV-2 in women to reduce the likelihood of transmission
Africa, HIV-2 produces a disease similar to but less of the virus to the fetus. Unfortunately, the high
severe than AIDS. mutation rate of HIV promotes the development
AIDS in the developing countries differs from of resistance to these drugs.
the disease in the western countries clinically too. A cocktail of several antiviral drugs (e.g. AZT,
In Africa, the major manifestation is pronounced 3TC, protease inhibitor) termed highly active
wasting so that it has been called the ‘slim disease’. antiretroviral treatment (HAART), each with
The high prevalence of tuberculosis and parasitic different mechanisms of action, has less potential to
infections complicate the clinical picture. breed resistance and has become a recommended
HIV infection in India: HIV infection was detected therapy. Multidrug therapy can reduce blood levels
rather late in India, the first cases having been of virus to nearly zero and reduce morbidity and
found in female sex workers in Madras (Chennai) mortality in many patients with advanced AIDS.
in 1986 and the first AIDS patient the same year Although HAART is a difficult drug regimen, many
from Bombay (Mumbai). Since then in every high patients return to nearly normal on this therapy.
risk group, the rate of infection has been mounting. Apart from specific antiretroviral therapy,
By the end of 2003, India is believed to have about other measures in the treatment of AIDS include
5 million HIV-infected people, the second largest (i) treatment and prophylaxis of opportunistic
such population after South Africa. infections and tumors (ii) general management
and (iii) immunorestorative measures.
Control
i. Sexual transmission: The best method of Postexposure Prophylaxis (Pep)
checking sexual transmission of infection is If an accidental exposure occurs, any wound
health education The use of condoms and should be washed with soap and water, or mucous
vaginal antiseptics could have an impact. membranes flushed with water. The accident must
ii. Exposure to blood: Drug injectors can avoid be reported so that, if necessary, prophylaxis can
risk by not injecting, or can reduce risk by be started as soon as possible. Knowledge of the
using only clean equipment. status of the source patient is essential.
iii. Mother to child transmission: This can be Exposure to blood, body fluid, other potentially
reduced by identifying infected mothers and infected material or an instrument contaminated
giving specific therapy in the later stages of with one of these materials may lead to risk of
pregnancy and to the baby after birth. All acquiring HIV infection. The risk of infection varies
women who have been potentially exposed with the type of exposure and other factors. Most
should seek HIV antibody testing before exposures do not result in infection. Health workers
becoming pregnant and, if the test is positive, are normally at very low risk of acquiring infection
should consider avoiding pregnancy. HIV during management of infected patients. Following
infected mothers should avoid breastfeeding exposure, postexposure prophylaxis (PEP) may be
to reduce transmission of the virus to their required depending upon the category of exposure
children. and HIV status of exposure source (Table 68.12).
Prophylaxis Basic PEP regimen consists of two drug
combination while expanded PEP regimen is a
The prevention of AIDS rests at present on general
combination of three drugs. Zidovudine 300 mg
measures, such as health education, identification
BD and Lamivudine 150 mg BD are used in basic
of sources and elimination of high risk activities. No
two drug regimen. In expanded three drug PEP
specific vaccine is available.
regimen, a protease inhibitor is added to this
Vaccine development: No vaccine against HIV is combination of drugs. Among protease inhibitors,
available despite several trials. lopinavir 400 mg BD or 800 mg OD or ritonavir 100
mg BD or 200 mg OD are preferred as third drug.
Treatment To be effective these drugs must be started within
The anti-HIV drugs approved can be classified as the first 72 hours and ideally within 2 hours. The
nucleoside analog reverse transcriptase inhibitors, PEP should be continued for a period of four weeks.
www.ebook3000.com
486 | Section 4: Virology
Table 68.12: PEP regimen according to exposure and status of source
Category of exposure HIV positive and HIV positive HIV staus not known
asymptomatic and clinically
symptomatic
i. Mild exposure (Mucous Consider two drug PEP regimen Start two drug PEP Usually no PEP or
membrane/non-intact skin with regimen consider two drug
small volumes). PEP regimen
ii. Moderate exposure (Mucous Start two drug PEP regimen Start three drug PEP Usually no PEP or
membrane/ nonintact skin with regimen consider two drug
large volume or percutaneous PEP regimen
superficial exposure with solid
needle)
iii. Severe exposure (Percutaneous Start two drug PEP regimen Start three drug PEP Usually no PEP or
with large volume) regimen consider two drug
PEP regimen
Both risk of infection and possible side-effects of Laboratory diagnosis of HIV infection includes specific
antiretroviral drugs should be carefully considered tests for HIV as well as tests for immunodeficiency.
when deciding to start PEP. Besides PEP, injured Specific tests include antigen (P24) detection, virus
isolation, detection of viral nucleic add and antibody
site on the wound should be thoroughly washed
detection
with soap and water. Antiseptics may also be used.
The p24 antigen is the earliest virus marker to appear
Therapy should be continued for 4 weeks and in the blood. HIV infected persons remain negative
the victim followed with testing for virus for the for antibodies during window period. Viral isolation,
next 6 months. Exposed persons should have post- detection of viral nucleic acid by polymerase chain
PEP HIV testing, at three months and at 6 months. If reaction (PCR) and p24 antigen detection are useful
for diagnosis in window period
the test at six months is negative, no further testing
HIV can be cultured by co-cultivation of lymphocytes
is required. with potentially infected and uninfected mono
Key Points nuclear cells
The diagnosis of HIV infection is made by detecting
Human immunodeficiency virus (HIV) causes AIDS, serum antibodies to viral proteins. There are two
belongs to retroviruses types of serological tests for anti-HIVantibodies are of
HIV is a spherical enveloped virus measuring up to two types—screening tests and supplementary tests
120 nm in diameter and consists of two identical Screening tests—ELISA, rapid test, and simple test
copies of single-stranded positive sense RNA (E/R/S) are usually highly sensitive tests
genome There are three strategies (strategy I to III) for HIV
The three important enzymes contained in the virion testing in India
are reverse transcriptase, protease and integrase.In Prevention and control include health education,
association with viral RNA is the reverse transcriptase screening of blood and blood products for HIV
enzyme antibodies or antigens, and infection control
Important structural components of the virus include Antiretroviral treatment (ART) is the mainstay in HIV
the surface antigen gp120, the transmembrane treatment
antigen gp41, the matrix protein p17 and the capsid
Postexposure prophylaxis (PEP) may be required
antigen p25
when there is exposure to blood, body fluid, other
The genome of HIV contains the three structural potentially infected material or an instrument
genes (gag, pol and env) characteristic of all contaminated with HIV.
retroviruses, as well as other nonstructural and
regulatory genes specific for the virus (tat, rev, nef,
vif, vpr and vpu). both Important questions
HIV shows two distinct antigenic types—HIV-1 and
HIV-2 1.
Describe the structure and laboratory
There are three modes of transmission of HIV diagnosis of human immunodeficiency virus.
infection. Sexual contact, parenteral and perinatal 2. Describe the antigenic structure of human
HIV infects principally the CD4 lymphocytes. immunodeficiency virus and draw labeled
The infection causes damage to T helper (T4)
lymphocytes. T4 cells are depleted in numbers and
diagram of HIV-1.
the T4:TB (helper: suppressor) ratio is reversed 3. Discuss the modes of transmission and
HIV causes acute infection, AIDS related complex pathogenesis of human immunodeficiency
(ARC), and AIDS virus.
When CD4+ cells fall below 200 per mm3, the titer 4. Write short notes on:
of virus increases markedly and there is irreversible a. Human immunodeficiency virus.
breakdown of immune defence mechanisms
b. Antigens of human immunodeficiency virus.
Chapter 68: Retroviruses: Human Immunodeficiency Virus | 487
c. Opportunistic infections associated with hu- 7. Which type of cells are most often infected by HIV?
man immunodeficiency virus infection. a. CD4+T lymphocytes
d. Strategies of human immunodeficiency virus b. CD8+T lymphocytes
testing. c. Null cells
e. Control HIV d. None of the above
f. Antiviral therapy for HIV 8. Which of the following opportunistic parasitic
infections is/are associated with HIV infection?
g. Postexposure prophylaxis
a. Toxoplasmosis
b. Cryptosporidiosis
Multiple choice questions (mcqs) c. Isosporiasis
d. All of the above
1. All of the following statements are true for the 9. Which of the following tests may be used for
viral genome in HIV except that they: diagnosis of HIV infection?
a. Are diploid a. P24 antigen detection
b. Consist of DNA-dependent DNA polymerase b. Virus nucleic acid detection
activity c. Antibody detection
Consist of three major genes gag, pol, and env;
c. d. All of the above
characteristic of all retroviruses 10. All of the following are the examples of screening
d. Are most complex of human retroviruses tests in HIV except:
2. What is the characteristic feature of viruses a. Western blot
b. ELISA
belonging to family Retroviridae?
c. HIV spot test
a. Presence of enzyme reverse transcriptase d. Latex agglutination test
b. Presence of envelope
11. Which of the following tests is/are confirmatory
c. Presence of nucleic acid
test for HIV infection?
d. Presence of lipid
a. Virus isolation.
3. Which structural genes are present in the genome b. Detection of p24 antigen.
of human immunodeficiency virus-I and not in c. Detection of viral nucleic acid.
immunodeficiency virus-2? d. All of the above.
a. gag gene 12. In HIV testing in India, the strategy I is used for:
b. pol gene a. Diagnosis of HIV infection in asymptomatic
c. env gene persons
d. All of the above b. Diagnosis of HIV infection in symptomatic per-
4. Which is the principal envelope spike antigen of sons
HIV-1? c. HIV surveillance
a. gp120 d. Ensuring safety of blood, organ, and tissue do-
b. gp140 nations
c. gp41 13. Which of the following antiviral agents has been
d. gp38 most widely used to inhibit HIV replication?
5. Which is the transmembrane pedicle antigen of a. Azidothymidine b. Zintevir
HIV-1? c. Nevirapine d. Indinavir
a. gp120 14. All of the following statements are true for highly
b. gp140 active antiretroviral therapy (HAART) except:
c. gp41 a. Inhibition of HIV replication
d. gp38 b. Improved efficacy of the therapy
c. Prevents emergence of drug resistance
6. HIV cannot be transmitted by: d. Minimize toxicity following thearpy)
a. Sexual contact
b. Kissing Answers (MCQs)
c. Intravenous drug abuse 1. b; 2. a; 3. d; 4 a; 5. c; 6. b; 7. a; 8. d; 9; d; 10. a; 11. d; 12.
d. Blood transfusion d; 13. a; 14. c
www.ebook3000.com
69
Chapter
Learning Objectives
After reading and studying this chapter, you should (CJD); Kuru; Bovine spongiform encephalopathy
be able to: (BSE) or mad cow disease; subacute sclerosing
∙∙ Describe slow virus and prion diseases panencephalitis (SSPE); progressive multifocal
∙∙ Describe the following: Creutzfeldt-Jakob Disease leukoencephalopathy (PML).
The virus can be grown in sheep choroid plexus ii. Transmissible mink encephalopathy: Trans
tissue cultures, from the CSF, blood and saliva of missible mink encephalopathy is a scrapie-
affected animals. like disease of farm-raised mink. It is believed
to have spread to mink by feeding them on
Maedi (Progressive Pneumonia) scrapie infected sheep meat.
Maedi (progressive pneumonia) is a slowly pro iii. Bovine spongiform encephalopathy (BSE)
gressive fatal hemorrhagic pneumonia of sheep, or mad cow disease:
with an incubation period of 2–3 years. A disease similar to scrapie, designated bovine
Visna and maedi (progressive pneumonia) spongiform encephalopathy (BSE), or “mad
viruses are closely related and are classified as cow disease,” emerged in cattle in Great
retroviruses (genus Lentivirus). Britain in 1986. This outbreak was traced to the
use of cattle feed that contained contaminated
Group B (Prion Diseases) bone meal from scrapie-infected sheep and
BSE-infected cattle carcasses.
Transmissible Spongiform Encephalopathies The epidemic of “mad cow disease” peaked
(Prion Diseases) in Great Britain in 1993.
Prion diseases of animals It is now accepted that the new variant forms
i. Scrapie: Scrapie is the prototype prion disease of CJD and BSE are caused by a common
and shows marked differences in susceptibility agent, indicating that the BSE agent had
of different breeds of animal. The incubation infected humans. Through 2002, of 129 people
period is about two years. The affected animals who had been diagnosed with new variant CJD
are irritable and develop intense pruritus, in England, 121 had died.
scraping themselves against trees and rocks, iv. Chronic wasting disease: A scrapie-like
hence the name scrapie. Emaciation and disease, designated chronic wasting disease, is
paralysis set in, leading to death. found in mule deer and elk in the United States.
The causative agent has been maintained in Human prion diseases
brain tissue explant cultures through several 1. Kuru: Kuru was a fatal neurologic disease
serial passages. with cerebellar signs exclusive to the Fore
www.ebook3000.com
490 | Section 4: Virology
tribes in Papua New Guinea. Kuru (means 3. Gerstmann–Strauss ler–Scheinker (GSS)
“shivering” or “trembling,”) was first described disease: It is rare neurologic diseases of humans
by Gajdusek and Zigas in 1957 as a mysterious due to prion-type agent.
disease seen only in the Fore tribe inhabiting 4. Fatal familial insomnia (FFI): It is also a rare
the eastern highlands of New Guinea. The dis neurologic diseases of humans are due to prion-
ease had an incubation period is 5–10 years type agent. In fatal familial insomnia there is loss
and led to progressive cerebellar ataxia and of ability to sleep and death within 1 to 2 years.
tremors, ending fatally in 3 to 6 months. A 5. Alzheimer’s disease: There are some neuro
total of 3700 cases occurred in a population of pathologic similarities between CJD and
35,000. Alzheimer’s disease. However, the disease has
Transmission from human to human was not been transmitted experimentally to primates
associated with ritual cannibalism involving or rodents, and the amyloid material in the
the consumption of the body of a dead member brains of Alzheimer’s patients does not contain
of the family after ritual nonsterilizing cooking. Prpsc protein.
When Gajdusek began his study, he noted
that women and children in particular were
the most susceptible to the disease, and he
Group C
deduced that the reasons were that the women i. Subacute Sclerosing Panencephalitis (SSPE)
and children prepared the food and they were
given the less desirable viscera and brains to Subacute sclerosing panencephalitis (SSPE) is an
eat. Their risk for infection was higher because extremely serious, very late neurologic sequela
they handled the contaminated tissue, making of measles. This disease occurs when a defective
it possible for the agent to be introduced measles virus persists in the brain and acts as a
through the conjunctiva or cuts in the skin, and slow virus. It has been reported that SSPE may
they ingested the neural tissue, which contains also develop as a very rare late complication of
the highest concentrations of the kuru agent. live measles virus vaccination. A similar picture
No cases have been seen in those born since has also been described as a rare complication of
1957, when cannibalism ceased. rubella infection.
Carlton Gajdusek was awarded the Nobel The SSPE is most prevalent in children who
Prize for Medicine in 1976 for his important were initially infected when younger than 2 years.
contributions on Kuru. The disease begins insidiously 5 to 15 years after a
case of measles. It is characterized by progressive
2. Creutzfeldt–Jakob disease(CJD): Creutzfeldt-
mental deterioration, involuntary movements,
Jakob disease is a rare chronic encephalopathy
muscular rigidity, and coma. Death occurs 1 to 3
of humans with associated dementia. CJD in
years after onset of symptoms.
humans develops gradually, with progressive
dementia, ataxia, and myoclonus, and leads to
death in 5–12 months. The disease was known ii. Progressive Multifocal
as both sporadic and inherited forms. leukoencephalopathy (PML)
The natural mode of transmission of Progressive multifocal leukoencephalopathy (PML)
Creutzfeldt–Jakob disease (GJD) is unknown, is a rare, degenerative CNS infection that usually
and it does not appear to be acquired from occurs in persons with severe immunodeficiency
sheep. Iatrogenic CJD has been transmitted disorders. The disease is characterized by
accidentally by contaminated growth hormone, memory loss, difficulty in speaking, and a loss of
by corneal transplant, by contaminated surgical coordination. Death occurs in 3–4 months.
instruments, and by cadaveric human dura
mater grafts. Causative agent: The causative agent is a human
A protein very similar to scrapie PrPsc is polyomavirus (JC virus), a member of the family
present in brain tissue infected with classic CJD. Polyomaviridae. PML has been seen in several
It has been speculated that the agent of CJD patients with AIDS-associated neurologic compli
was derived originally from scrapie-infected cations.
sheep and transmitted to humans by ingestion
of poorly cooked sheep brains. Key Points
An epidemic of mad cow disease in the United
Kingdom and the unusual incidence of CJD The term ‘slow virus disease’ is applied to a group
of infections in animals and human beings,
in younger people (younger than 45 years) characterized by a very long incubation period and
prompted concern that contaminated beef was a slow but relentless course, terminating fatally
the source of this new variant of CJD.
Chapter 69: Slow Virus and Prion Diseases | 491
d. Subacute sclerosing panencephalitis (SSPE)
Slow virus diseases are classified into: e. Progressive multifocal leukoencephalopathy
– Group A; such as visna and medi. (PML)
– Group B; prion diseases of the CNS (subacute
spongiform viral encephalopathies). Human
diseases include kuru, Creutzfeldt–Jakob disease Multiple choice questions (mcqs)
(CJD), Gerstmann–Straussler–Scheinker (GSS)
1. The prion-mediated disease in humans is
disease and fatal familial insomnia (FFI) Scrapie,
bovine spongiform encephalopathy (BSE) (mad cow a. Progressive multifocal leukoencephalopathy
disease), chronic wasting disease, and transmissible b. Subacute sclerosing panencephalitis
mink encephalopathy occur in animals. c. Gerstmann–Straussler–Scheinker syndrome
– Group C diseases include subacute sclerosing d. Bovine spongiform encephalopathy
panencephalitis and progressive multifocal 2. Variant Creutzfeldt–Jakob disease is transmitted by
leukoencephalopathy. a. Cuts in the skin
SSPE is a very rare delayed sequel to infection with
b. Transplantation of contaminated cornea
measles virus, many years after the initial infection.
Papova virus is responsible for PML. c. Ingestion of infected tissue
d. Blood transfusion
3. All the following are slow diseases transmitted by
Important Questions conventional viruses in humans except
a. Subacute sclerosing panencephalitis
1. Write briefly on slow virus diseases. b Progressive multifocal leukoencephalopathy
2. Write short notes on: c. Acquired immunodeficiency syndrome
a. Creutzfeldt–Jakob disease d. Creutzfeldt–Jakob disease
b. Kuru
c. Bovine spongiform encephalopathy (BSE) or Answers (MCQs):
mad cow disease 1. c; 2. d; 3. d
www.ebook3000.com
70
Chapter
Miscellaneous Viruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe structure of rotavirus
be able to: ∙∙ Discuss laboratory diagnosis of rotavirus infections
∙∙ Describe Rubella virus ∙∙ List the viruses causing diarrhea.
∙∙ Describe the following: Lassa fever; severe acute
respiratory syndrome (SARS)
www.ebook3000.com
Chap-70.indd 493 15-03-2016 11:23:52
494 | Section 4: Virology
Rodents act as reservoirs and transmission is in Marburg, Frankfurt (Germany) and Belgrade
believed to occur through rodent excreta. Serious (Yugoslavia) in 1967.In each case the infection
human pathogens are the closely related Junin, arose from tissues of African green monkeys to
Machupo, Guanariro, and Sabia viruses. which the laboratory workers had been exposed
a. Junin hemorrhagic fever (Argentine hemo imported to laboratories from Uganda via London.
rrhagic fever).
b. Machupo hemorrhagic fever (Bolivian hemo Ebola Virus
rrhagic fever). In 1976, two severe epidemics of hemorrhagic fever
c. Guanarito virus (the agent of Venezuelan occurred in Sudan and Zaire with high fatality. The
hemorrhagic fever). causative virus was morphologically identical to
the Marburg virus but antigenically distinct. The
Laboratory Diagnosis of Arenaviruses virus responsible was named Ebola virus after the
1. Virus Isolation: Blood, CSF, throat washings, Ebola river. In 1979 Ebola re-emerged in Sudan,
pleural fluid and urine are the specimens used with serial persont o-person spread. Another
for diagnosis. Arenaviruses can be isolated from epidemic occurred in Kikwit, Zaire, in 1995.
specimens by inoculation of suckling mice, Three distinct strains of Ebola virus have been
hamsters and guinea pigs. Virus can also be recognized.
isolated by inoculation of Vero E6 and BHK-21 1. Zaire strain (EBO- Z)
cells. 2. Sudan strain (EBO-S)
2. Antigen Detection: Viral antigen can be 3. Reston strain (EBO-R)
detected by Indirect immunofluorescence is
used for detection of viral antigen in the blood. Pathogenesis
3. Serology: For detection of IgM antibody and/or
It is probable that Marburg and Ebola viruses have
a rising titre of antibody in paired sera indirect
a reservoir host, perhaps a rodent or a bat, and
immunofluorescence test may be used.
become transmitted to humans only accidentally.
FILOVIRUSES Transmission among humans generally requires
direct contact with blood or body fluids, although
They have been classified as filoviridae.These are droplet infections can occur.
long threadlike viruses, hence the name (filum
means thread). They range in size from 80 to Laboratory Diagnosis
800–1000 nm. Marburg and Ebolaviruses causing
1. Electron microscopy: In the blood and in the
hemorrhagic fever belong to the genus Filovirus.
cytoplasm of the affected cells filamentous
Morphology: Marburg and Ebola viruses are viruses can be seen by electron microscopy.
enveloped negative sense single stranded RNA 2. Isolation of virus: Virus can be cultured in
viruses with long tubular or filamentous forms vero cells from the blood during the febrile
(Fig. 70.1). phase. Virus isolate is identified by electron
microscopy and direct immunofluorescence.
Marburg Virus Virus culture must only be attempted in
Marburg disease is a hemorrhagic fever that laboratories with required biosafety level.
occurred simultaneously in laboratory workers 3. Serology: Indirect immunofluorescence can be
used for detection of antibodies in the serum.
CORONAVIRUSES
A group of spherical or pleomorphic enveloped RNA
viruses, carrying petal or club shaped peplomers on
their surface has been classified as coronaviruses
(Corona, meaning crown) resembling the solar
corona (Fig. 70.2). It has been classified in the
family coronaviridae with two genera: Torovirus
and Coronavirus.
Torovirus: The toroviruses are widespread in ungul
ates and appear to be associated with diarrheas.
Coronavirus: There seem to be two serogroups of
human coronaviruses. Coronaviruses of domestic
animals and rodents are included in these two
Fig. 70.1: Ebola virus (long thin filamentous form) groups. There is a third distinct antigenic group
www.ebook3000.com
Chap-70.indd 495 15-03-2016 11:23:54
496 | Section 4: Virology
1. Reverse transcription PCR—Reverse trans without the RNA core are also seen. The genome
cription PCR (RT-PCR has been used for early of rotaviruses is located inside the inner core and
diagnosis). consists of 11 segments of double-stranded RNA.
2. Virus culture: Virus in clinical specimens can All segments, except one, code for only one virus-
be cultured on Vero cell lines. specific protein (VP).
3. Serology: Demonstration of rise in titer of
antibodies by “ELISA or indirect immuno Classification
fluorescent test in paired serum samples are Rotaviruses share a common group antigen situated
useful later. However, for early diagnosis PCR is in the inner capsid layer. So far, rotaviruses have
preferred. been classified into at least seven antigenic groups
(A to G) based on antigenic epitopes on the internal
Treatment and Prophylaxis structural protein VP6. For groups A-E complete
lack of serological cross-reactivity has been proven.
No specific therapy or prophylaxis has been
These can be detected by immunofluorescence,
identified. The virus is highly mutable and so
ELISA, and immune electron microscopy (IEM).
vaccine prophylaxis may not be easy. Control has
Multiple serotypes have been identified among
been achieved by strict isolation and quarantine.
human and animal rotaviruses.
However, ribavirin and steroids have been shown
to be useful in treatment of critical patients. Group A strains: Cause the majority of human
infections have been classified into subgroups
REOVIRIDAE (I and II) by ELISA, CF or immune adherence
agglutination, and into many serotvpes (1, 2, 3, etc.)
Reoviridae constitutes a diverse family of viruses that by neutralisation tests.
infect humans, many mammals, other vertebrates Group B strain: The Chinese ADRV is of group B.
(including birds), plants and insects. The family
Reoviridae derives its name from the prototype Group C rotaviruses: Cause occasional outbreaks
virus which was known as the Respiratory enteric in humans.
orphan virus, because it could be isolated fre
Animal Susceptibility
quently from the respiratory and enteric tracts,
and it was considered ‘orphan’ because it was not Rotaviruses have a wide host range. Human
associated with any disease. rotavirus infection has been transferred to piglets,
calves and monkeys. Swine rotavirus infects both
Classification newborn and weanling piglets.
The family Reoviridae is divided into nine genera. Propagation in Cell Culture
Four of the genera are able to infect humans and
Rotaviruses are fastidious agents to culture. As calf
animals: Orthoreovirus, Rotavirus, Coltivirus, and
and simian viruses grow readily in cell cultures, they
Orbivirus. Four other genera infect only plants and
have been used as antigens for serological studies.
insects, and one infects fish.
Morphology: All reoviruses have a double- Epidemiology
shelled capsid, no envelope and measure 75–80 Rotaviruses are the most common cause of
nm in diameter. The genome consists of double diarrhea in infants and children the world over.
stranded RNA in 10–12 pieces, a feature unique Rotavirus infections usually predominate during
among animal viruses. They are nonenveloped and the winter season. In tropical areas infections occur
resistant to lipid solvents. evenly throughout the year.
Rotavirus diarrhea is usually seen in children
Rotaviruses below the age of five years but symptomatic
Morphology: Morphologically, rotaviruses are infections are most common in children between
polyhedrons of 75 nm diameter displaying char ages 6 months and 2 years. By the age of five years,
acteristic sharp-edged double shelled capsids, most children have had clinical or subclinical
which in electron micrographs look like spokes infection. Transmission appears to be by the fecal–
grouped around the hub of a wheel. The name is oral route. Nosocomial infections are frequent.
derived from rota, in Latin, meaning wheel. Both
‘complete’ and ‘incomplete’ particles are seen. Clinical Features
The complete or ‘double shelled’ virus measures Infection is by the fecal-oral route. The incubation
about 70 nm in diameter and has a smooth surface. period is 2–3 days. Vomiting and diarrhea occur
The incomplete or ‘single shelled” virus is smaller, with little or no fever. Stools are usually greenish
about 60 nm, with a rough surface and is rota yellow or pale, with no blood or mucus. The disease
virus that has lost the outer shell. ‘Empty’ particles is self-limited and recovery occurs within 5–10 days.
www.ebook3000.com
Chap-70.indd 497 15-03-2016 11:23:54
498 | Section 4: Virology
2. Which of the following can cause congenital
Severe acute respiratory syndrome (SARS) refers
infections?
to a severe atypical pneumonia that assumed
pandemic proportions in 2003 a. Toxoplasma gondii
Reoviridae: There are three genera in this family: b. Rubella virus
Reovirus, Orbivirus and Rotavirus: c. Cytomegalovirus
– Orbivirus: The only known orbivirus infection of d. All of the above
human beings is Colorado tick fever, a minor febrile
3. All of the following viruses can cause haemorrhagic
illness.
fevers except:
– Rotaviruses are the most common cause of
diarrhea in infants and children. The human a. Junin virus
rotavirus is related to the viruses of epidemic b. Machupo virus
diarrhea of infant mice (EDIM), Nebraska c. Ebola virus
calf diarrhea and the simian virus SAIl. Swine
d. Norwalk virus
rotavirus infects both newborn and weanling
piglets. 4. Severe acute respiratory syndrome (SARS) can be
Laboratory diagnosis is by demonstrating of virus by diagnosed by which of the following tests?
IEM, latex agglutination tests, or ELISA. On clarified a. Electron microscopy
fecal samples. Polymerase chain reaction is the b. Growth in Vero cell culture
most sensitive detection method. Development of
c. Cloning
a rotavirus vaccine has been difficult.
Viruses causing diarrhea
d. All of the above
Viruses that are important causes of diarrhea include 5. Which of the following viral agents can cause
rotaviruses, the Norwalk virus, certain adenoviruses diarrhea?
and coronaviruses, and astroviruses. a. Norwalk agent
b. Astroviruses
c. Rotaviruses
IMPORTANT QUESTION
d. All of the above
Write short notes on:
6. All the followings are the causative agents of South
a. Rubella or German measles
American hemorrhagic fever except:
b. Viral hemorrhagic fevers
c. Lymphocytic choriomeningitis virus (LCM) a. Lassa virus
d. Ebola virus b. Junin virus
e. Coronaviruses c. C. Machupo virus
f. Severe acute respiratory syndrome (SARS) d. Ebola virus
g. Viruses causing diarrhea
h. Rotaviruses. 7. Which of the following techniques can be used for
detecting rotavirus infection?
MULTIPLE CHOICE QUESTIONS (MCQs) a. Latex agglutination test.
b. ELISA.
1. Which of the following viruses can cause cervical
c. Immune electron microscopy.
cancers?
a. Norwalk virus d. All of the above.
b. Herpes simplex virus
ANSWERS (MCQs)
c. Epstein-Barr virus
d. Human papilloma virus 1. d; 2. d; 3. a; 4. d; 5. d; 6. d; 7. d
Oncogenic Viruses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe oncogenes and mechanism of viral
be able to: oncogenesis
∙∙ Describe oncogenic viruses ∙∙ List of viruses associated with human cancers.
∙∙ Classify oncogenic viruses
www.ebook3000.com
Chap-71.indd 499 15-03-2016 11:24:18
500 | Section 4: Virology
Table 71.2: List of oncogenic viruses Marek’s disease can be prevented by vaccination
with an attenuated strain of Marek’s disease
A. DNA viruses
I. Papovavirus
virus.
1. Papillomaviruses of human beings, rabbits and 2. Lucke’s tumor of frogs: A herpesvirus is consi
other animals dered to be the etiological agent of a renal
2. Polyomavirus adenocarcinoma in frogs.
3. Simian virus 40 3. Herpesvirus saimiri: It causes fatal lymphoma
4. BK and JC viruses or reticulum cell sarcoma when injected into
II. Poxvirus owl monkeys or rabbits.
1. Yaba virus (Yaba monkey tumor virus)
4. Epstein–Barr virus: Epstein–Barr (EB) herpes
2. Shope fibroma virus
3. Molluscum contagiosum virus virus causes acute infectious mononucleosis
III. Adenovirus when it infects B lymphocytes of susceptible
Many human and nonhuman types humans. EB virus is etiologically linked to Burkitt’s
IV. Herpes virus lymphoma; to nasopharyngeal carcinoma
I. Marek’s disease virus (NPC); to post-transplant lymphomas; and to
2. Lucke’s frog tumour virus Hodgkin’s disease. Malaria may be a cofactor of
3. Herpes virus pan, papio, ateles and saimiri African Burkitt’s lymphoma.
4. Epstein–Barr virus 5. Herpes simplex and cancer cervix: An associ
5. Herpes simplex Virus types 1 and 2
ation has been proposed between herpes
6. Cytomegalovirus
simplex type 2 infection and cancer of the
V. Hepatitis Band C viruses
B. RNA viruses
uterine cervix, though not proved.
I. Retroviruses Kaposi’s sarcoma-associated herpesvirus is also
1. Avian leukosis viruses known as human herpesvirus 8 (KSHV/HHV8).
2. Murine leukosis viruses It is suspected of being the cause of Kaposi’s
3. Murine mammary tumor virus sarcoma, primary effusion lymphoma, and a
4. Leukosis-sarcoma viruses of various animals particular lymphoproliferative disorder.
5. Human T-cell leukemia viruses 6. Cytomegalovirus: Cytomegalovirus infection
has been associated with carcinoma of the
prostate and Kaposi’s sarcoma.
2. Polyoma virus: The polyoma virus causes
natural latent infection in laboratory and
domestic mice. However, it induces a wide V. Hepatitis B Virus
variety of histologically diverse tumors, when Hepatocellular carcinoma (HCC) following
injected into infant mice or other rodents. chronic infection with hepatitis B virus is one of
3. Simian virus 40 (SV 40): See Chapter 60. the most prevalent forms of cancer. HBV has been
4. BK and JC virus: The papovaviruses BK and claimed to be directly or indirectly involved in the
JC, which cause widespread asymptomatic etiology of hepatocellular carcinoma.It is also the
human infection, can induce tumors in only malignant disease of humans preventable by
immunodeficient subjects (see Chapter 60). immunization against the causal agent.
www.ebook3000.com
Chap-71.indd 501 15-03-2016 11:24:18
502 | Section 4: Virology
DNA, incorporate them into their genome and DNA of the infected host cell, and it is from this
express them. This method of gene transfer is called integrated DNA copy that all proteins of the virus
transfection. are translated.
72
Chapter
General Properties, Classification and
Laboratory Diagnosis of Fungi
Learning Objectives
After reading and studying this chapter, you should be ∙∙ Classify fungi
able to: ∙∙ Describe laboratory diagnosis of fungal infections
∙∙ Differntiate between fungi and bacteria ∙∙ Discuss diseases caused by fungi.
www.ebook3000.com
504 | Section 5: Medical Mycology
Flowchart 72.1: Classification of Fungi
B. Direct Microscopy
I. Potassium Hydroxide (KOH) Preparation
A portion of the treated specimen should be
examined microscopically to determine whether
hyphal elements are present. Most specimens
can be examined satisfactorily in wet mounts
after partial digestion of the tissue with 10–20%
potassium hydroxide. The alkali digests cells
and other tissue materials, enabling the fungus
elements to be seen clearly. Newer preparations
for the KOH test may provide easier and more
reliable results.
III. Gram-staining
It is used for the diagnosis of yeast infections of
mucous membranes.
C. Culture
1. Culture Media: The commonest culture media
used in mycology are Sabouraud’s dextrose agar
(SDA, pH 5.4), SDA with antibiotics, potato
dextrose or the slightly modified potato flakes
agar (PFA), and brain heart infusion (BHI) agar
with blood and antibiotics. The antimicrobials
usually included in SDA with antibiotics are
Fig. 72.4: Aerial spores
chloramphenicol and gentamicin to inhibit
bacterial growth and cycloheximide (actidione)
Laboratory Diagnosis to inhibit saprophytic fungi. The pH of Emmons’
modification of SDA is close to neutral and is
A. Collection and Processing of more efficient medium for primary isolation
Specimens than the original formulation.
The sampling procedures vary according to the 2. Incubation: Cultures are routinely incubated
area and type of tissue involved. Causative agents in parallel at room temperature 25°C (room
www.ebook3000.com
506 | Section 5: Medical Mycology
temperature for weeks) and at 37°C for days.
Cultures should be retained for at least 2-3
weeks before being discarded.
D. Identification of Fungi
Once an organism has grown, it is examined for
characteristic gross and microscopic structures,
so that identification can be made (Fig. 72.5).
1. Gross or macroscopic examination of
cultures: Growth characteristics useful for
identification are the rapidity of growth, colour
and morphology of the colony on the obverse
and pigmentation on the reverse.
2. Microscopic examination
i. Tease mount: For microscopic examination
slide mounts should be made in lactophenol Fig. 72.5: Asexual forms and asexual spores of fungi
cotton blue (LCB) from fungal colony (in
teased mounts or slide cultures) to study
the morphology of hyphae, spores and other Mycoses (Fungus Infections)
structures. Teased mounts are prepared Infection caused by fungus is known as mycosis
in lactophenol cotton blue (LCB) which (plural mycoses).
contains lactic acid, phenol and cotton blue.
Microscopic characteristics that should be Classification of mycoses
obtained as the following:
a. Septate versus sparsely septate hyphae Classification of fungal disease according to
b. Spores or conidia primary sites of infections is as follows (Table 72.1):
ii. Cellophane tape preparation: It should be A. Superficial mycoses: These infections are
read within 30 minutes and then discarded. limited to the outermost layers of the skin and
iii. Slide culture: Slide culture provides a hair. These include:
useful technique for demonstrating the a. Infection of skin caused by Malassezia
natural morphology of fungal structures. furfur (pityriasis versicolor) and Exophiala
3. Tests for the identification of molds: werneckii (tinea nigra), and
i. Hair perforation test: It is done to differtiate b. Infection of hair caused by Piedraia hortae
T. rubrum, from T. mentagrophytes,. (black piedra) and Trichosporon beigelii
ii. Urease test: It is done to help differentiate T. (white piedra).
mentagrophytes from T. rubrum B. Cutaneous mycoses: Infections that extend
iii. Thiamine requirement deeper into the epidermis as well as invasive
iv. Trichophyton agars hair and nail diseases.
v. Growth on rice grains.
4. Tests for the identification of yeasts Examples
Germ tube production, carbohydrate assimilation, a. Infection of skin, hair and nail caused by
chromogenic substrates, cornmeal agar, potassium dermatophytes.
nitrate assimilation, temperature studies and urease. b. Infection of skin, nail and mucous membrane
caused by C. albicans and other Candida
E. Serologic Tests specicies.
The most common tests for C. Subcutaneous mycoses: These infections
a. Fungal antibodies: involve the dermis, subcutaneous tissues,
1. Immunodiffusion; 2. countercurrent immuno- muscle, and fascia.
electrophoresis (CIE); 3. whole cell agglutination; Examples: Mycetoma, chromomycosis, sporotri
4. complement fixation; 5. Enzyme-linked chosis and rhinosporidiosis.
immunosorbent assay (ELISA). D. Systemic mycoses: Systemic mycoses-infections
b. Antigen detection: that originate primarily in the lung but that
1. Latex particle agglutination; 2. ELISA. may spread to many organ systems. Systemic
mycoses are caused by inhalation of airborne
F. Polymerase Chain Reaction (PCR) spores.
Polymerase chain reaction (PCR) for detection of Examples: Blastomycosis, histoplasmosis, cocci
fungal DNA in clinical material. dioidomycosis and paracoccidioidomycosis.
Chapter 72: General Properties, Classification and Laboratory Dignosis of Fungi | 507
Table 72.1 The major mycoses and causative fungi
Type of mycosis Mycosis Causative Fungal Agents
A. Superficial Pityriasis versicolor Malassezia species
Tinea nigra Hortaea werneckii
White piedra Trichosporon species
Black piedra Piedraia hortae
B. Cutaneous Dermatophytosis Microsporum species, Trichophyton species, and
Epidermophyton floccosum
Candidiasis of skin, mucosa, or nails Candida albicans and other candida species
C. Subcutaneous Sporotrichosis Sporothrix schencki
Chromoblastomycosis Phialophora verrucosa, Fonsecaea pedrosoi, others
Mycetoma Pseudallescheria boydii, Madurella mycetomatis, others
Phaeohyphomycosis Exophiala, bipolaris, exserohilum, and others
D. Systemic (prlmary, Coccidioidomycosis Coccidioides immitis, C. posadasii
endemic) Histoplasmosis Histoplasma capsulatum
Blastomycosis Blastomyces dermatitidis
Paracoccidioidomycosis Paracoccidioides brasiliensis
E. Opportunistic Systemic candidiasis Candida albicans and other candida species
Cryptococcosis Cryptococcus neoformans
Aspergillosis Aspergillus fumigatus and other aspergillus species
Mucormycosis (zygomycosis) Species of Rhizopus, Absidia, Mucor, and other
zygomycetes
Penicilliosis Penicillium marneffei
www.ebook3000.com
73
Chapter
Superficial, Cutaneous and
Subcutaneous Mycoses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe causative agets of ectothrix and endothrix
be able to: ∙∙ Describe the following: mycetoma; chromoblasto-
∙∙ List superficial, cutaneous and subcutaneous mycosis; sporotrichosis; rhinosporidiosis.
mycoses
A. SUPERFICIAL MYCOSES
These infections are limited to the outermost layers
of the skin and hair. These include:
a. Infection of skin: Caused by Malassezia furfur
(pityriasis versicolor) and Exophiala werneckii
(tinea nigra)
b. Infection of hair: Caused by Piedraia hortae
(black piedra) and Trichosporon beigelii (white
piedra).
a. Infection of Skin
Pityriasis Versicolor (Tinea Versicolor) Fig. 73.1: Morphology of pityriasis versicolor
Pityriasis versicolor (Tinea versicolor) is a chronic, Creamy colony develop within 5–7 days at 37°C.
usually asymptomatic, involvement of the stratum Lactophenol cotton blue mount of the colonies
corneum, characterized by discrete or confluent show budding yeast cells along with a number
macular areas of discoloration or depigmentation of bottleshaped cells. Hyphae are occasionally
of the skin. The areas involved are mainly the chest, seen in culture.
abdomen, upper limbs and back. The disease is
worldwide in distribution. Tinea Nigra
Causative agent: A lipophilic, yeast-like fungus Tinea nigra (or tinea nigra palmaris) is a localized
Pityrosporum orbiculare (Malassezia furfur). infection of the stratum corneum, particularly of
the palms, producing black or brownish macular
Laboratory Diagnosis lesions. It is caused by the dematiaceous fungus
A. Direct microscopy: The diagnosis is confirmed Hortaea (Exophiala) werneckii.
by direct microscopic examination of scrapings
of infected skin, treated with 10–20% KOH or Laboratory Diagnosis
stained with calcofluor white. Demonstration of A. Direct microscopy: Microscopic examination
clusters of the characteristic round yeast cells with of skin scrapings from the periphery of the
short, stout hyphae, which may be curved and lesion will reveal brownish, branched, septate
occasionally branched, is diagnostic (Fig. 73.1). hyphae and budding cells (Fig. 73.2).
B. Culture: The fungus can be grown on Sabour B. Culture: On Sabouraud agar the fungus
aud’s agar, covered with a layer of olive oil. develops as grey, yeast-like colonies, which
www.ebook3000.com
Chap-73.indd 509 15-03-2016 11:25:05
510 | Section 5: Medical Mycology
M. canis
M. canis forms numerous thick-walled, 8–15 celled
macroconidia with curved or hooked spiny tips. A
Fig. 73.3: Characteristic macroconidia of dermatophytes. (1) lemon-yellow or yellow-orange pigment develops
Cylindrical in Trichophyton; (2) Fusiform in Microsporum; and on the reverse of the colony. It is zoophilic.
(3) Club-shaped in Epidermophyton
M. gypseum
characters, such as spiral hyphae, racquet mycelium M. gypseum colonies grow rapidly producing a flat,
and favic chandeliers (Fig. 73.4). spreading, powdery surface that is cinnamonbuff to
brown which consists of abundant macroconidia.
Important Species They are boat-shaped, rough walled with 4 to 6
T. rubrum, T. mentagrophyte, T. tonsurans, T. septa. It is geophilic.
schoenleinii, T. violaceum.
Pathogenicity
T. rubrum: The typical colony of T. rubrum has a Dermatophytes grow only on the keratinised
white, cottony surface and a deep red, nondiffusible layers of the skin and its appendages and do not
pigment when viewed from the reverse side of the ordinarily penetrate the living tissues. Lesions vary
colony. The microconidia are small and piriform considerably according to the site of the infection
(pear-shaped) and clubshaped thinwalled macro and the species of fungus involved.
conidia (scanty).
Dermatophytids (or ‘id’ reaction): Hypersensitivity
T. mentagrophyte: T. mentagrophyte has grape- to fungus antigens may play a role in pathogenesis
like clusters of microconidia (Fig. 73.4) and cigar and is probably responsible for the sterile
shaped macroconidia.It has no red pigment and vesicular lesions, sometimes seen in sites distant
urease positive. Hair perforation test is positive. from the ringworm. These lesions are called
T. tonsurans: T. tonsurans produces a flat, dermatophytids (or ‘id’ reaction). Hypersensitivity
powdery to velvety colony on the obverse surface can be demonstrated by skin testing with the fungus
that becomes reddish-brown on reverse. The antigen, trichophytin.
microconidia are mostly elongate (Fig. 73.4). Types of hair infections: Three types of hair
infection can be seen in 10% KOH wet mounts:
Microsporum 1. Ectothrix: In this, the hyphae are sparsely
Colonies are cotton-like, velvety or powdery, with distributed within hair shaft with a sheath
white to brown pigmentation. Microconidia are of arthrospores on the surface of hair shaft.
relatively scanty and are not distinctive. Macroconidia It is caused by M. audouinii, M. canis and T.
are the predominant spore form. They are large, mentagrophytes (Fig. 73.5A).
multicellular, spindle shaped structures, borne singly 2. Endothrix: In this, the arthrospore formation
on the ends of hyphae. Both types of conidia are occurs entirely within the hair completely filling
borne singly in these genera. Microsporum species the hair shaft. This is caused by T. tonsurans and
infect only hair and skin but usually not the nail. T. violaceum (Fig. 73.5B).
Important species are M. audouinii, M. canis, 3. Favus: In this, there is sparse hyphal growth and
M. gypseum formation of air spaces within the hair shaft.
This is caused by T. schoenleinii (Fig. 73.5C).
M. audouinii
M. audouinii rarely forms conidia in the culture but Clinical Findings
many thick-walled chlamydospores (chlamydo Dermatophyte infections were mistakenly termed
conidia) may be present. It is anthropophilic. ringworm or tinea. Table 73.1 lists the clinical
www.ebook3000.com
Chap-73.indd 511 15-03-2016 11:25:07
512 | Section 5: Medical Mycology
1. Mycetoma Table 73.2 Important causative agents and color
Mycetoma is a chronic, granulomatous infection of the grains of various types of mycetoma
of the skin, subcutaneous tissues, fascia and bone, Causative agent Color of the grains
which most often affects the foot or the hand. A. Eumycetoma
The disease was originally reported by Gill (1842) • Madurella mycetomatis Black
from Madurai, South India, and Carter (1860)
• M. grisea Black
established its fungal etiology. It is, therefore,
• Exophiala jeanselmei Black
commonly known as Maduramycosis or Madura
foot. • Pseudallescheria boydii White-yellow
Mycetoma occurs worldwide. The disease is • Acremonium falciforme White-yellow
most prevalent in tropical and subtropical regions • Aspergillus nidulans White
of Africa, Asia and Central America. In India, it is B. Actinomycetoma
quite common in Tamil Nadu but rare in Kerala. • Actinomadura madurae White-yellow
• A. pellitieri Red-yellow
Etiology
• Nocardia asteroids White-yellow
It can be divided into three types, eumycetomas,
• N. brasiliensis White
actinomycetomas and botryomycosis. A similar
condition called ‘botryomycosis’ is caused by • Streptomyces somaliensis Yellow
Staphylococcus aureus and some other bacteria. C. Botryomycosis
Important causative agents of these are given in • Staphylococcus species White
Table 73.2. • Streptococcus species White
• Escherichia coli White
Pathogenesis • Proteus species White
Triad of symptoms: Infection follows traumatic • Pseudomonas aeruginosa . White
inoculation of the organism into the subcutaneous Actinobacillus lignieresi Yellow
tissue from soil or vegetable sources, usually on
thorns or splinters and results in tumefactions,
Treatment
deformities and draining sinuses discharging
fungal colonies called grains or granules (triad of The management of eumycetoma is difficult,
symptoms). Subcutaneous tissues of the feet, lower involving surgical debridement or excision and
extremities, hands, and exposed areas are most chemotherapy. Actinomycetoma responds well to
often involved. rifampicin in combination with sulfonamides or
cotrimoxazole.
Grains: Within host tissues the organisms develop
to form compacted colonies (grains) 0.5–2 mm 2. Chromoblastomycosis
in diameter, the color of which depends on the This disease, also known as chromomycosis, is a
organism responsible. The color and consistency chronic, localized disease of the skin and subcutan
of the grains vary with the different agents causing eous tissues. The disease is mainly encountered in
the disease (Table 73.2). the tropics.
Laboratory Diagnosis Etiological agents: The etiological agents are soil
inhabiting fungi of the family Dematiaceae.
A. Direct Examination All are dematiaceous fungi which are darkly
The presence of grains in pus collected from pigmented fungi. These include: Phialophora
draining sinuses or in biopsy material is diagnostic. verrucosa, Fonsecaea pedrosoi, Rhinocladiella
The grains are visible to the naked eye and their aquaspersa, Fonsecaea compacta, and Cladophial
color may help to identify the causal agent. Grains ophora carrionii.
should be examined microscopically to differentiate They enter the skin by traumatic implantation.
between actinomycetoma and eumycetoma. The lesion develops slowly around the site
Material from actinomycetoma grains may be Gram of implantation. The infection is chronic and
stained to demonstrate the gram-positive filaments. characterized by the slow development of progres
sive granulomatous lesions that in time induce
B. Culture hyperplasia of the epidermal tissue.
Samples should also be cultured, at both 25–30°C
and 37°C, on brain-heart infusion agar or blood Laboratory Diagnosis
agar for actinomycetes and on Sabouraud agar A. Microscopic examination: Histologically, the
(without cycloheximide) for fungi. The fungi that lesions show the presence of the fungus as
cause eumycetoma are all septate molds that round or irregular, dark brown, yeastlike bodies
appear in culture within 1–4 weeks. with septae, called sclerotic cells (Fig. 73.6).
Diagnosis can be established by demonstration on delicate sterigmata (Fig. 73.7). Conidia are
of these sclerotic bodies in KOH mounts or in also produced along the sides of the hyphae.
sections, and by culture on Sabouraud’s agar. C. Serology: A latex agglutination test is of value
B. Culture: Culture on Sabouraud agar at 25–30°C for the diagnosis of the extracutaneous forms of
yields slow-growing, greenish grey to black, sporotrichosis.
compact, folded colonies. Cultures should be D. Skin test: A skin test with sporotrichin antigen
incubated for 4–6 weeks. is positive in almost all patients with cutaneous
sporotrichosis.
Phaeohyphomycosis E. Animal inoculation: Rats are highly susceptible
Phaeohyphomycosis is a term applied to infections and can be infected by intraperitoneal or
characterized by the presence of darkly pigmented intratesticular inoculation.
septate hyphae in tissue. The sites of lesions may be
cutaneous, subcutaneous, deeper tissues, or organs 4. Rhinosporidiosis
like the brain or lung. Sclerotic cells or granules are Rhinosporidiosis is a chronic granulomatous
not found. The fungi appear in lesions as distorted disease characterized by the development of large
hyphal strands. polyps or wart-like lesions in the nose, conjunctiva
and occasionally in ears, larynx, bronchus, penile
3. Sporotrichosis urethra, vagina, rectum and skin. More than 90% of
Sporotrichosis is a chronic, pyogenic granulomatous the cases have been reported from India, Sri Lanka
infection of the skin and subcutaneous tissues and South America.
which may remain localized or show lymphatic
Etiology: The causative fungus Rhinosporidium
spread. The disease is worldwide and is rare in
seeberi.
Europe.
Causative agent: Sporothrix schenckii, a saprophyte Mode of infection: The mode of infection is not
in nature. known, though infection is believed to originate
from stagnant water or aquatic life. It is believed
Pathogenesis that fish may be the natural hosts of R. seeberi.
The fungus is a saprophyte found widely on plants,
thorns and timber. Infection is acquired through Laboratory Diagnosis
thorn pricks or other minor injuries. It has not been cultured and animal inoculation is
also not successful.
Laboratory Diagnosis
Diagnosis is made by culture as frequently the Demonstration of Sporangia
fungus may not be demonstrable in pus or tissues. Diagnosis depends on the demonstration of
A. Microscopic examination: Direct microscopy sporangia. Direct examination of the surface of
is of little value. the polypoid growth and histologic examination
B. Culture: SDA or blood agar are the media used. are the only ways to make a diagnosis. R. seeberi
S. schenckii is a dimorphic fungus occurring can be identified in hematoxylin and eosin-stained
in the yeast phase in tissues and in cultures at sections, but sometimes one may need special
37°C, and in the mycelial phase in nature and stains. Histologically, the lesion is composed of
in cultures at room temperature. The septate large numbers of fungal spherules embedded in
hyphae are very thin (1-2 mm diameter) and a stroma of connective tissue and capillaries. The
carry flower-like clusters of small conidia borne spherules are 10–200 mm in diameter and contain
www.ebook3000.com
Chap-73.indd 513 15-03-2016 11:25:08
514 | Section 5: Medical Mycology
IMPORTANT QUESTIONS
1. Discuss the morphology, cultural characters and
Fig. 73.8: Rhinosporidiosis: Sporangium with numerous pathogenicity of dermatophytes.
endospores 2. Describe the etiology, pathogenesis and laboratory
diagnosis of mycetoma.
thousands of endospores (6–7 μm in diameter) 3. Discuss different kinds of subcutaneous mycoses.
(Fig. 73.8). These spores develop into new 4. Write short riotes on:
sporangia when released. a. Superficial mycoses
b. Dermatophytes
5. Subcutaneous Phycomycosis c. Mycetoma or Madura foot or maduramycosis
In this condition, a painless subcutaneous nodule d. Chromoblastomycosis or verrucous dermatitis
develops which enlarges to involve a whole limb e. Sporotrichosis
or large areas of the body. f. Rhinosporidiosis.
Causative agent: is Basiodiobolus haptosporus,
a saprophytic phycomycete. MULTIPLE CHOICE QUESTIONS (MCQs)
Key Points 1. Black piedra is a superficial infection of the hair
caused by:
Superficial Mycoses a. Malassezia furfur
Pityriasis versicolor or tinea versicolor is caused by
b. Cladosporium werneckiib
Malassezia furfur (Pityrosporum orbiculare)
Tinea nigra is an infection of keratinized layer of skin
c. Trichosporon beigelli
caused by Hortaea (Exophiala) werneckii d. Piedraia hortae
Black piedra is a superficial infection of the hair 2. All the following are anthropophilic dermatophytes
caused by Piedroiohortae, a dematiaceous fungus except:
White piedra is an infection of the hair caused by a. Trichophyton violaceum
yeast-like organism, Trichosporon beigelii b. Trichophyton rubrum
Cutaneous Mycoses c. Epidermophyton floccosum
The dermatophytes infect only superficial keratinized d. Microsporum audouinii
structure such as skin, hair and nail but not deeper 3. Urease test is positive by:
tissues
a. Trichophyton violaceum
Dermatophytes belong to three genera: Trichophyton,
Microsporum and Epidermophyton
b. Trichophyton rubrum
Clinical findings: Dermatophyte infections were c. Trichophyton mentagrophytes
mistakenly termed ringworm or tinea because of d. Epidermophyton floccosum
the raised circular lesions 4. The major causative agent of mycetoma in India is:
Laboratory diagnosis is based on microscopy and a. Actinomadura madurae
culture. The differentiation of the three genera is b. Streptomyces somaliensis
based mainly on nature of macroconidia c. Nocardia brasiliensis
Subcutaneous Mycosis d. Actinomyces pelletieri
Mycetoma is a chronic, granulomatous infection
5. The sclerotic bodies are useful for diagnosis of:
of the skin, subcutaneous tissues, fascia and bone,
a. Sporotrichosis b. Mycetoma
which most often affects the foot or the hand
It can be divided into three types, eumycetomas,
c. Chromoblastomycosis d. Rhinosporidiosis
actinomycetomas and botryomycosis 6. Which of the following fungi has not been cultured:
Chromoblastomycosis or chromomycosis is caused a. Sporothrix b. Rhinosporidium
by fungi collectively called dematiaceous fungi c. Blastomyces d. Acremonium
Sporotrichosis is caused by Sporothrix schenckii, a
saprophyte in nature
ANSWERS (MCQs)
1. d; 2 a; 3. c; 4. a; 5. c; 6. b
Systemic Mycoses
Learning Objectives
After reading and studying this chapter, you should ∙∙ Descrbe histoplasmosis
be able to:
∙∙ List of fungi causing systemic infections
Introduction
These are the include blastomycosis, histoplasmosis,
coccidioidomycosis and paracoccidioidomycosis.
1. Blastomycosis
Blastomycosis is a chronic infection of the lungs
which may spread to other tissues, particularly
skin, bone and genitourinary tract.
It is caused by Blastomyces dermatitidis, a
dimorphic fungus. The disease has been called Fig 74.1: Blastomyces dermatitidis: yeast and mycelial
North American blastomycosis, because it is forms
endemic and most cases occur in the United States filamentous with septate hyphae and many round or
and Canada. oval conidia, and in older cultures, chlamydospores
also.
Pathogenesis
Laboratory Diagnosis
Soil is considered to be the source of infection,
which is acquired by inhalation. Human infection A. Specimens: Sputum or pus, exudates, urine
is initiated in the lungs. and biopsy lesions.
B. Microcopic examination: Potassium hydroxide
1. Asymptomatic
(10%) mount of specimens may show char
2. Chronic pneumonia acteristic thick-walled yeast cells with a single
3. Disseminated diseases: They form multiple broadbased bud.
abscesses in various parts of the body. C. Culture: Colonies usually develop on Sabouraud’s
4. Cutaneous blastomycosis: The cutaneous dextrose agar or enriched blood agar at 30°C
disease is usually on the skin of the face or other within 2 weeks.
exposed parts of the body. D. Serology: Antibodies can be measured by
complement fixation (CF) test, immunodiffusion
Morphology (ID) tests and enzyme immunoassay (EIA).
Blastomyces dermatitidis is a dimorphic fungus. In Treatment: Severe cases of blastomycosis are
tissue and in cultures at 37°C, the fungus appears treated with amphotericin B.
as budding yeast cells, which are large (7–20 μm)
and spherical, with thick, double-contoured walls. 2. Paracoccidioidomycosis
Each cell carries only a single broad-based bud This is a chronic granulomatous disease of the skin,
(Fig. 74.1). At room temperature, the culture is mucosa, lymph nodes and internal organs. As the
www.ebook3000.com
516 | Section 5: Medical Mycology
A B
Figs 74.3A and B: Coccidioides immitis: (A) Arthrospores
formation; (B) Spherule formation with endospores
Fig. 74.4: H. capsulatum: yeast cells in macrophage Fig. 74.5: H. capsulatum: mycelial form
www.ebook3000.com
518 | Section 5: Medical Mycology
identical to H. capsulatum in its mycelial phase but Important Questions
differs in the yeast phase both in vivo and in vitro 1. Enumerate dimorphic fungi. Describe the morphology,
and H. duboisii is morphologically identical to H. pathogenicity and lab diagnosis of dimorphic fungi.
capsulatum in its mycelial phase but the yeast phase 2. Write short notes on:
has larger cells (12–15 μm diameter). a. Blastomycosis
African histoplasmosis is mainly restricted to b. Coccidioidomycosis or Coccidioides immitis
c. Histoplasmosis or Darling’s disease
the continent of Africa. It involves mainly the skin,
d. Paracoccidioidomycosis or Paracoccidioides
subcutaneous tissues and bones. The lungs are not brasiliensis
commonly affected and the disseminated disease
is infrequent. Multiple choice questions (MCQs)
1. All the following statements are true for spherule
of Coccidioides immitis except:
Key Points
a. It is infective stage of the fungus
Blastomycosis: Blastomycosis is caused by Blasto b. It reproduces byendosporulation
myces dermatitidis, a dimorphic fungus. It is also c. It contains endospores
known as North American blastomycosis. d. It is not found in culture
Coccidioidomycosis is primarily an infection of the
2. Which of the following fungi infects reticuloendo
lungs caused by Coccidioides immitis, a dimorphic thelial system?
fungus. Infection is acquired by inhalation of dust a. Aspergillus fumigatus
containing arthrospores of the fungus. b. Histoplasma capsula tum
c. Trichophyton rubrum
Histoplasmosis: It is primarily a disease of reticulo
d. All the above
endothelial system caused by an intracellular fungus
3. The diagnostic form of Histoplasma capsulatum is:
Histoplasma capsulatum, a dimorphic fungus.
a. Arthrospore b. Spherule
H. capsulatum causes acute pulmonary histoplasmosis, c. Macroconidia d. Microconidia
chronic pulmonary histoplasmosis, and progressive
disseminated histoplasmosis. Answers (MCQs)
1. a; 2. b; 3. c
75
Chapter
Opportunistic Mycoses
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Discuss cryptococcosis
be able to: ∙∙ Discuss laboratory diagnosis of Cryptococcus
∙∙ Describe diseases caused by Candida albicans neoformans
∙∙ Describe the following: thrush or oral thrush; Germ ∙∙ Describe species of Asperillus
tube test or Reynaulds–Braude phenomenon ∙∙ Describe the following: Aspergillosis; mucormycosis;
∙∙ Discuss laboratory diagnosis of candidiasis pneumocystosis; otomycosis; mycotic keratitis.
∙∙ List opportunistic fungi
Morphology
In culture or tissue, Candida species grow as oval, Fig. 75.1: Sputum specimen illustrating budding yeast cells
budding yeast cells (3–6 μm in size). They also form and pseudohyphae of Candida species
www.ebook3000.com
Chap-75.indd 519 15-03-2016 11:25:32
520 | Section 5: Medical Mycology
Fig. 75.2: Candida albicans: yeast form and pseudohyphae Fig. 75.3: Gram stained smear of Candida albicans
Fig. 75.4: Candida albicans showing germ tubes Fig. 75.5: Formation of chlamydospores by Candida
albicans when cultured on cornmeal agar at 25°C
Treatment
Management of candidosis is mainly by removing the
predisposing causes. All Candida strains are sensitive
to Nystatin. Amphotericin B, 5-fluorocytosine
and clotrimazole may be used for disseminated
candidosis.
Cryptococcosis
Cryptococcosis (torulosis, European blastmycosis,
Busse–Buschke disease) is subacute or chronic
infection caused by the capsulate yeast Cryptococcus
neoformans. It is most frequently recognized as a
disease of the central nervous system (CNS), although
the primary site of infection is the lungs. The disease
Fig. 75.6: Cryptococcus neoformans: India ink preparation of
occurs sporadically throughout the world but it is now spinal fluid showing yeast cells surrounded by a large capsule
seen most often in patients with AIDS.
do not appear to become infected, probably
Morphology because of their high body temperature. In addition
In culture, C. neojormans produces a whitish mucoid to patients with AIDS or hematologic malignancies,
colony in 2–3 days. Microscopically, in culture patients being maintained on corticosteroids are
or clinical material, C. neoformans is a spherical highly susceptible to cryptococcosis.
budding yeast (5–10 μm in diameter), surrounded
by a thick polysaccharide capsule (Fig. 75.6). Pathogenesis
Infection is usually acquired by inhalation but may
Serotypes sometimes be through skin or mucosa. The primary
Adsorbed antisera have defined five serotypes pulmonary infection may be asymptomatic or may
(A–D and AD) and three varieties—C. neoformans mimic an influenza-like respiratory infection, often
vargrubii (serotype A), C. neoformans var neofor resolving spontaneously. Pulmonary cryptoccoc
mans (serotype D), and C. neoformans var gattii osisis may lead to a mild pneumonitis. In patients
(serotype B or C). Most infections are caused by C. who are compromised, the yeasts may multiply
neoformans var. neoformans, which is commonly and disseminate to other parts of the body but
found in the excreta of wild and domesticated birds preferent ially to the central nervous system,
throughout the world. causing cryptococcal meningoencephalitis. Other
common sites of dissemination include the skin,
Epidemiology eye, and prostate gland.
Cryptococcosis is worldwide in distribution.
Bird droppings (particularly pigeon droppings) Cryptococcal meningitis
enrich for the growth of C. neoformans and serve Cryptococcal meningitis is the most serious type
as a reservoir of infection. The organism grows of infection and can resemble tuberculous or other
luxuriantly in pigeon excreta. The birds themselves chronic types of meningitis.
www.ebook3000.com
Chap-75.indd 521 15-03-2016 11:25:34
522 | Section 5: Medical Mycology
Other Sites of Dissemination 3. Produce phenol oxidase—produce black colo
Although predominantly a disease of the CNS, nies on niger seed agar, bird seed agar and
lesions of the skin, mucosa, viscera and bones may caffeic acid agar.
also occur. Visceral forms simulate tuberculosis 4. Produce disease in mice on intracerebral and
and cancer clinically. Bones and joints may be intraperitoneal inoculation (animal inoculation
involved. Cutaneous cryptococcosis varies from test positive). Capsulated budding yeast cells can
small ulcers to large granulomas. be demonstrated in the brain of infected mice.
www.ebook3000.com
Chap-75.indd 523 15-03-2016 11:25:35
524 | Section 5: Medical Mycology
Fig. 75.8: Zygomycetes: (A) Mucor; (B) Rhizopus; (C) Absidia Fig. 75.9: Penicillium
Morphology
Treatment
P. carinii has three stages:
Treatment consists of aggressive surgical debride Trophozoites thinwalled (1–4 μm).
ment, rapid administration of amphotericin B, and Precyst: It is 5–8 μm.
control of the underlying disease.
Cysts: Cysts are thick-walled and spherical (4–6 μm)
and contains 4 to 8 nuclei.
Penicilliosis
Pathogenesis
There are more than 150 known species of the
genus Penicillium. Infections are caused by P. jiroveci is normally a commensal in the lung, spread
Penicillium marnefei. by respiratory droplets. In immunocompetent
individuals, infection is asymptomatic. However,
Pathogenesis in inimunocompromised patients, serious life-
threatening pneumonia can develop (Fig. 75.10).
It causes penicillosis, keratitis, otomycosis and Until the AIDS epidemic, human disease was
rarely deep infections. Penicillium marneffei causes confined to interstitial plasma cell pneumonia
serious disseminated disease with characteristic in malnourished or premature infants and
papular skin lesions in AIDS patients in South- immunosuppressed patients. Since the early 1980s,
East Asia. Cutaneous lesions and subcutaneous it has remained one of the primary opportunistic
abscesses have been reported. infections found in patients with AIDS.
The multiplication of the parasite in the lungs
Identification induces a hyaline or foamy alveolar exudate. In
Fungi belonging to this genus are characterized by stained sections, the exudate filling the alveoli
producing conidiophores at the tips of branching shows a characteristic honeycomb pattern. Chest
septate hyphae, which in turn may produce radiographs may be normal or show a diffuse
secondary structures termed metulae, from which interstitial infiltrate.
flask-shaped structures called phialides bearing
smooth or rough shaped conidia are produced in Laboratory Diagnosis
chains, giving the entire structure a brush-like or 1. Specimens: Traditionally, diagnosis was made
broom-like appearance (Fig. 75.9). by finding the cyst or trophozoite in tissue
www.ebook3000.com
Chap-75.indd 525 15-03-2016 11:25:37
526 | Section 5: Medical Mycology
www.ebook3000.com
Chap-75.indd 527 15-03-2016 11:25:37
76
Chapter
Mycotoxicosis
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: mycotoxicosis; aflatoxin
be able to:
∙∙ Describe mycotic poisoning
www.ebook3000.com
Section 6: Miscellaneous
77
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ L ist organisms present as normal microbial flora
be able to: in normal flora of upper respiratory tract and
∙∙ Describe the role of normal flora in a human body gastrointeinal tract
www.ebook3000.com
532 | Section 6: Miscellaneous
low in the small intestine. In the adult duodenum, activity of Doderlien’s bacilli, E. coli and yeasts.
there are 103–106 bacteria per gram of contents; in This appears to be an important mechanism in
the jejunum and ileum, 105–108 bacteria per gram; preventing the establishment of other, possibly
and in the cecum and transverse colon, 108–1010 harmful, microorganisms in the vagina. During
bacteria per gram. In the upper intestine, lactobacilli pregnancy, there is an increase in Staphylococcus
and enterococci predominate, but in the lower ileum epidermidis, Doderlien’s bacilli and yeasts.
and cecum, the flora is fecal. In the sigmoid colon Occasionally, other members of the intestinal
and rectum, there are about 1011 bacteria per gram flora may be present. After menopause, lactobacilli
of contents, constituting 10–30% of the fecal mass. again diminish in number and a mixed flora returns
Anerobes outnumber facultative organisms by 1000- and the flora resembles that found before puberty.
fold. In diarrhea, the bacterial content may diminish
greatly, whereas, in intestinal stasis, the count rises. Key Points
In the normal adult colon, 96–99% of the resident The term ‘normal microbial flora’ denotes the
bacterial flora consists of anerobes: Bacteroides population of microorganisms that inhabit the
species, especially B. fragilis; Fusobacterium skin and mucous membrane of normal healthy
species; anerobic lactobacilli, e.g. bifidobacteria; individuals
clostridia (C perfringens, 103–105/g); and anerobic The resident flora consists of relatively fixed types
of micro-organisms regularly found in a given area
gram-positive cocci (Peptostreptococcus species).
at a given age. and cannot be removed permanently
Only 1–4% are facultative aerobes (gram-negative The transient flora inhabit the skin or mucous
coliform bacteria, enterococci, and small numbers membranes and does not produce disease, and
of Proteus, pseudomonads, lactobacilli, candidae, does not establish itself permanently on the surface.
and other organisms). More than 100 distinct types The normal flora is composed principally of bacteria
of organisms occur regularly in normal fecal flora. A resident, actively proliferating, microbial flora
occurs in the skin, nose and oropharynx, the mouth
and large intestine, the anterior parts of the urethra
Normal Flora of the Genitourinary Tract and the vagina.
Mycobacterium smegmatis, a harmless commensal,
is found in the smegma of the genitalia of both
men and women. This may, by its presence in the Important questions
voided specimens of urine, cause confusion with 1. What is normal microbial flora of the human body?
tubercle bacilli. From apparently normal men, Write briefly on beneficial and harmful effects of
aerobic and anerobic bacteria can be cultured normal flora.
including, lactobacilli, Gard. vaginalis, alpha 2. Write short notes on:
hemolytic streptococci and Bacteroids species. Chlam. a. Normal bacterial flora.
trachomatis and Ureaplasma urealyticum may also
b. Difference between resident and transient flora.
be present. The female urethra is either sterile or
c. Normal flora of the skin.
contains a few gram-positive cocci.
d. Normal flora of the intestine.
At birth: At birth, the vagina is sterile. In the e. Normal flora in the mouth and upper respiratory
first 24 hours, it is invaded by micrococci, tract.
enterococci and diphtheroids. In 2–3 days, the f. Normal flora of the genitourinary tract.
maternal estrin induces glycogen deposition in
the vaginal epithelium. This facilitates the growth Multiple choice questions (mcqs)
of a Lactobacillus (Doderlien’s bacillus), which
produces acid from glycogen, and the flora for a 1. The majority of normal bacteria flora are present in:
few weeks is similar to that of the adult. After the a. Conjunctiva b. Skin
passively transfected estrin has been eliminated c. Nasopharynx d. Large intestine
2. Which bacteria is responsible for producing acidic
in the urine, the glycogen disappears, along with
pH in adult vagina:
Doderlien’s bacillus and the pH of the vagina
a. Lactobacillus
becomes alkaline. This brings about a change in b. Bacteroides species
the flora to micrococci, alpha and nonhemolytic c. Diphtheroids
streptococci, coliforms and diphtheroids. d. Gardnerella vaginalis
At puberty: At puberty, the glycogen reappears Answers (MCQs)
and the pH changes to acid due to the metabolic 1. d; 2. a
78
Chapter
Infective Syndromes*
Learning Objectives
After reading and studying this chapter, you should ∙∙ List microbial pathogens that cause pneumonia.
be able to: ∙∙ Define diarrhea and dysentery
∙∙ Define bacteremia, septicemia, pyemia and ∙∙ List causative organisms of diarrhea and dysentery
endotoxemia ∙∙ Discuss laboratory diagnosis of dirrhea
∙∙ List causative organisms of septicemia, infective ∙∙ Discuss laboratory diagnosis of dysentery
endocarditis and subacute endocarditis ∙∙ List causative organisms of food poisoning
∙∙ Discuss laboratory diagnosis of subacute endocarditis ∙∙ Discuss the laboratory diagnosis of food poisoning
∙∙ List causative organisms of meningitis ∙∙ List microbial pathogens that cause pneumonia
∙∙ Discuss the laboratory diagnosis of acute pyogenic ∙∙ List causative organisms of (i) sexually transmitted
meningitis diseases (STDs); (ii) painless genital ulcer; painful
∙∙ Describe characterstics features of cerebrospinal genital ulcer; urethral discharge
fluid (CSF) in different forms of meningitis ∙∙ Discuss the laboratory diagnosis of gonorrhea
∙∙ List causative organisms of urinary tract infection ∙∙ Describe the following; Non-gonococcal urethritis
∙∙ Discuss the laboratory diagnosis of urinary tract (NGU)
infection ∙∙ Discuss the laboratory diagnosis of syphilis
∙∙ Describe characterstics features of cerebrospinal ∙∙ List causative organisms of wound infection
fluid (CSF) in different forms of meningitis ∙∙ Discuss the laboratory diagnosis of wound infection
∙∙ Describe the following: aseptic meningitis; tuber ∙∙ List causative organisms of pyrexia of unknown origin
culous meningitis (PUO)
∙∙ List causative organisms of sore throat ∙∙ Discuss the laboratory diagnosis of pyrexia of
∙∙ Discuss the laboratory diagnosis of sore throat unknown origin (PUO)
*This chapter was contributed by Dr Savita Kumari, Professor, Department of Internal Medicine, Postgraduate
Institute and Medical Research (PGIMER), Chandigarh-160 012, India.
www.ebook3000.com
534 | Section 6: Miscellaneous
Table 78.1 Causative organisms of septicemia Table 78.3 Causative agents of subacute endocar
ditis
A. Gram-negative bacilli (60–70% cases)
Salmonella Typhi (A) Bacteria
S. Paratyphi A Viridans group of streptococci- responsible for 60–80%
S. Paratyphi B of cases?
S. Paratyphi C Streptococcus sanguis
Brucella spp. Str. mutans
Haemophilus influenzae Str. mitis
Escherichia coli Enterococcus faecalis
Klebsiella pneumoniae Staph. epidermidis
Proteus spp. Coxiella burnetii
Enterobacter spp. Chlamydia psittaci
Bacteroides spp. (B) Fungi
Pseudomonas spp. Candida albicans
B. Gram-positive bacilli Aspergillus spp.
Listeria monocytogenes
C. Gram-negative cocci
Neisseria meningitidis vegetations comprising of dense fibrin, platelet
D. Gram-positive cocci (20–40% cases) aggregates with bacterial colonies are formed.
Staphylococcus aureus It runs a chronic course. It is more common
Staph. epidermidis and comprises of almost 70% cases of bacterial
Streptococcus pyogenes
Str. pneumoniae
endocarditis. Causative agents of subacute
bacterial endocarditis are shown in Table 78.3.
2. Meningitis
Meningitis is an inflammation of the membranes 1. Purulent meningitis (acute
surrounding the brain and spinal cord. Most cases pyogenic meningitis)
of meningitis fall into one of the two categories:
purulent meningitis and aseptic meningitis. The In purulent meningitis, the CSF is typically turbid
causative agents of these types are given in Table due to the presence of large numbers of leukocytes,
78.4. from 100 to several thousands/mm3, most of which
www.ebook3000.com
536 | Section 6: Miscellaneous
Table 78.4 Causative agents of purulent and B. Laboratory Examination of CSF for Cells
aseptic meningitis and Microorganisms
Purulent meningitis Aseptic meningitis 1. Naked eye examination: Naked eye examin
• Neisseria meningitidis A. Viruses ation is done for the presence of turbidity and
• Streptococcus • Enteroviruses any sign of contamination with blood from the
pneumoniae (echoviruses, polioviruses, puncture wound.
• Haemophilus coxsackieviruses)
2. Cell count: The leucocytes in the CSF are
influenzae • Mumps
• Escherichia coli • Herpes simplex counted by microscopical observation. The
• Group B streptococci • Varicella-zoster relative numbers of polymorphs and lympho
• Pseudomonas • Measles cytes should be noted, and the number of
• Salmonellae • Adenoviruses erythrocytes in specimens contaminated with
• Staphylococcus aureus • Arboviruses
blood (Table 78.5).
• S. epidermidis B. Spiral bacteria
• Listeria • Syphilis (Treponema A wet film of centrifuged CSF deposit mixed
monocytogenes pallidum) with India ink will, when examined under oil-
• Klebsiella • Leptospira immersion, demonstrate the characteristic
• Anaerobic cocci Interrogans serovars capsulate yeast cells of Cryptococcus neofor
• Bacteroides Icterohaemorrhagiae and
mans and a wet film examined on a warm
In neonates and canicola
infants C. Other bacteria stage will show the slowly motile trophozoites
E. coli Tuberculosis (Mycobacterium of Acanthamoeba (Hartmanella) or Naegleria.
Group B streptococci tuberculosis)
Pseudomonads, Partially treated with Dilution
salmonellae antibiotics
Staph. aureus Brain abscess
CSF that is clear or only slightly turbid should be
H. influenzae D. Fungi examined undiluted but when the specimen is
Listeria monocytogenes Cryptococcus neoformans highly turbid and its cell count very high, it may
Streptococcus Candida albicans be necessary to dilute it 1 in 10 or 1 in 100 before
pneumoniae E. Protozoa examination.
Klebsiella spp. • Acanthameba
• Naegleria
• Toxoplasma gondli Gram Film of CSF
F. Noninfective After taking some CSF for the cell count, the
Lymphoma remaind er should be centrifuged to deposit
Leukemias
Metastatic and primary
any cells and bacteria and a film of the deposit
neoplasms should be stained by Gram’s method. The finding
Collagen-vascular disease of bacterial forms resemb ling meningococci,
pneumococci, haemophili, coliform bacilli,
streptococci or listeriae be reported.
The supernatant from the centrifuged CSF should
are polymorphs. The majority of cases are caused be tested for its content of glucose and protein.
by one or other of three bacteria: Meningococcus,
Pneumococcus and Haemophilus influenzae. In Culture
neonates and infants, coliform bacilli, group B i. CSF Culture
streptococci and, less commonly, pseudomonads, Immediately after centrifugation of the CSF and the
salmonellae and Listeria monocytogenes may be the removal of some of the deposit for the Gram film, the
cause (Table 78.4). remainder of the deposit should be seeded heavily
on to culture media, blood agar and chocolate agar
Laboratory Diagnosis for incubation in humid air plus 5–10%, CO2 and
A. Collection of Specimens a-tube of cooked-meat broth. A further blood agar
The principal specimen to be examined is of CSF plate should be seeded for incubation for 2–5 days
collected by lumbar puncture under strict aseptic in an anerobic atmosphere with 5-10% CO2.
conditions. Only 3–5 mL of fluid should be collected The cultures should be inspected after overnight
in a fresh sterile screw-capped containers. The incubation. The isolated organisms are identified
specimen must be dispatched to the laboratory as by colony morphology, Gram staining from
quickly as possible, for delay may result in the death colonies, biochemical reactions and/or serological
of delicate pathogens, such as meningococci, and the tests. Tests with appropriate antibiotics should be
disintegration of leukocytes. It should not be kept in a done on the isolate.
refrigerator, which tends to kill H. influenzae. If delay If no growth is apparent after overnight
for a few hours is unavoidable, the specimen is best incubation, the plates should at once be reincubated
kept in an incubator at 37°C. for another day and then again inspected for growth.
Chapter 78: Infective Syndromes | 537
Table 78.5 Typical CSF findings in different types of meningitis
CSF in
Characteristic Normal CSF Acute pyogenic Tuberculous meningitis Viral meningitis
meningitis
I. Pressure Normal Highly increased Moderately increased Slightly increased
II. Direct examination
A. Cell count
1. Total cell 1–3 1,000–20,000 50–500 10–500
(per cu. mm
2. Predominant Lymphocytes Neutrophils (90–95%) Lymphocytes (90%) Lymphocytes
B. Biochemical analysis
1. Total 30–45 100–600 (Highly 80–120 (moderately 60–80 (slightly
proteins increased) increased) increased)
(mg%)
2. Sugars 40–80 Diminished or absent Diminished (30–50) Normal
(mg%) (10–20)
III. Bacteriological examination
A. Microsocopy
1. Gram Nil Gram-negative cocci, — —
staining Gram-positive cocci,
Gram-negative bacilli
or gram-positive bacilli
may be found depending
upon the causative agent
responsible.
2. Ziehl– Nil Nil Acid-fast bacilli (AFB) —
Neelsen may be found
staining
B. Culture Nil According to the M. tuberculosis may grow Viruses may be
causative agent, specific on L–J media grown on cell
organism may grow on cultures
appropriate media.
If the plate cultures remain free from growth, tests are used for rapid diagnosis of meningitis and
and turbidity develops in the cooked-meat broth, are particularly useful in partially treated patients
the broth should be filmed and subcultured in whom smear and culture may be negative. These
on to blood agar and heated-blood agar plates, are available for N. meningitidis, Str. pneumoniae,
incubated aerobically and anerobically. H. influenzae type b and Group B streptococcus.
www.ebook3000.com
538 | Section 6: Miscellaneous
which are lymphocytes, except in the earliest stage. 2. Microscopy
The great majority of cases are due to viruses (viral The centrifuged deposit of the CSF should be
meningitis), particularly enteroviruses of the echo, examined in an auramine or Ziehl–Neelsen-stained
coxsackie and polio groups. (Table 78.4). film for acidfast bacilli.
A few cases with CSF findings resembling
those of viral meningitis are caused by leptospires 3. Culture
(serovars canicola and icterohemorrhagiae), fungi
(Cryptococcus neoformans or Candida albicans) Centrifuged deposit of CSF is inoculated on Lowen
and amoebae (Naegleria or Hartmanella) and an stein–Jensen (L–J) media and incubated at 37°C
underlying viral encephalitis may give a moderate for 6–8 weeks. Identification of M. tuberculosis
lymphocytic exudate in the CSF. depends on colony morphology, Z–N staining from
colonies and biochemical reactions.
It should also be noted that when antibiotic
If facilities are available, some of the CSF should
therapy is started at an early stage in a bacterial
be inoculated into a guinea-pig. As in purulent
meningitis, the CSF findings may be like those of
meningitis, the glucose cont ent of the CSF is
aseptic meningitis (Tables 78.4 and 78.5).
reduced and the protein content increased.
Laboratory Diagnosis Key Points
CSF is used for: Meningitis is an inflammation of the membranes
Cell count and biochemical tests, microscopy, surrounding the brain and spinal cord
culture and other tests according to suspected Most cases of meningitis fall into one of the two cat
causative agents (viruses, fungi or protozoa). egories: purulent meningitis and aseptic meningitis
Acute pyogenic or purulent meningitis. The majority
of cases are caused by one or the other of three
3. Tuberculous meningitis bacteria: Meningoc occus, Pneumococcus and
Haemophilus influenzae
The CSF findings often resemble those of aseptic Laboratory examination: CSF is the specimen of
meningitis, but the cell count is usually slightly choice. Direct microscopy by Gram staining, antigen
higher, e.g. 100–500 leucocytes/mm 3 , mostly detection and culture are key methods in laboratory
lymphocytes, and a veil clot (fibrin web) often diagnosis of acute pyogenic meningitis
develops when the CSF is allowed to stand Aseptic meningitis of the great majority of cases
are due to viruses (viral meningitis), a few cases are
undisturbed (Table 78.5).
caused by leptospires, fungi and amebae
When a tuberculous infection is suspected, the Tuberculous meningitis-The CSF findings often
centrifuged deposit of the CSF should be examined resemble those of aseptic meningitis
in an auramine or Ziehl–Neelsen-stained film for When a tuberculous infection is suspected, the
acidfast bacilli and cultured on one or two slopes CSF should be examined in an auramine or Ziehl–
Neelsen-stained film for acidfast bacilli and cultured
of Lowenstein–Jensen medium.
Lowenstein–Jensen medium.
Laboratory Diagnosis
Important Questions
1. Specimen
1. Name the various organisms causing meningitis.
CSF is collected by lumbar puncture in a sterile
Discuss the laboratory diagnosis of acute pyogenic
container under aseptic conditions. When CSF meningitis.
is allowed to stand, a fibrin web (cobweb) often 2. Write short notes on:
develops. Cell count and biochemical analysis can a. Aseptic meningitis
be done as described earlier. b. Tuberculous meningitis.
www.ebook3000.com
540 | Section 6: Miscellaneous
B. Semiquantitative methods iii. Triphenyltetrazolium chloride (TTC) test:
These are quicker mehods: It is based on the production of a pink-red
precipitate in the reagent caused by the
I. Standard loop Method respiratory activity of the growing bacteria.
Measured quantity of uncentricuged urine with iv. Glucose test paper: It is based on the
the help of standardized loop is inoculated on utilisation of the minute amounts of glucose
blood agar and MacConkey media and incubated present in normal urine, by bacteria causing
overnight at 37°C. The number of colonies is the infection. Hence, it indicates bacteriuria.
counted to get the bacterial count per mL of urine. v. Polymorphonuclear neutrophils (PMNs):
PMNs are counted in uncentrifuged urine
Interpretation of Results specimen with the help of hemacytometer. 8
Kass (1957) gave a criterion of active bacterial PMN/mm3 is indicative of infection.
infection of urinary tract as follows: vi. Leukocyte esterase: Presence of this enzyme
i. Count more than 10 5 bacteria of single in urine is indication of bacteriuria.
species per mL: significant bacteriuria which vii. Gram staining: Presence of at least one
indicates active UTI. bacteria per oil immersion field (examining 20
ii. Less than 104 organisms/mL and usually less fields) correlates with significant bacteriuria
than 103/mL accounts contamination. (>105 bacteria/mL).
viii. Dip-slide culture methods: Agar-coated
Identification and Sensitivity Tests slides are immersed in urine or even exposed
If similar colonies are found in numbers suggesting to the stream of urine during voiding,
significant bacteriuria, a separate colony or a incubated and the growth estimated by colony
portion of apparently pure growth should be counting or by color change of indicators.
subcultured for identification and testing of its None of the screening methods is as sensitive
sensitivity to antibiotics. or reliable as a culture.
II. Filter Paper Method Differentiation of Upper UTI and Lower UTI
This method of semiquantitative culture is rapid The antibody-coated bacteria test has been
and very economical in the use of culture medium. employed for the localization of the site of urinary
infection. This is based on the assumption that
bacteria coated with specific antibodies are present
III. Dip-slide Method
in the urine only when the kidneys are infected
The dip-slide is small plastic tray carrying a layer (upper UTI) and not when the infection is confined
of an appropriate agar culture medium. Opposite to the bladder (lower UTI). Antib ody-coated
sides of the tray may carry different media, e.g. bacteria are detected by immunofluorescence
cysteine lactose electrolyte-deficient (CLED) agar using fluorescent-tagged antihuman globulin or
medium on one side and MacConkey, brainheart by staphylococcal coagglutination.
infusion or pseudomonas selective agar on
the other. The dip-slide is withdrawn from the Tuberculosis of Kidney and Urinary Tract
container and briefly immersed in the urine. It is Tuberculosis of kidney is a blood-borne infection.
then incubated at 37°C overnight and examined The patient presents with frequency and painless
for a growth of colonies. This method is relatively hematuria and routine urine culture does not show
expensive. any pathogen. Tuberculosis must be considered in
cases where pyuria is present without bacteriuria.
Screening Techniques
Laboratory Diagnosis
Several screening techniques have been introduced
for the presumptive diagnosis of significant 1. Specimen
bacteriuria. These include the following: Excretion of M. tuberculosis from kidney is
i. Griess nitrite test: It is based on nitrite- intermittent. Hence, midstream urine specimen
reducing enzymes produced by bacteria is not useful. Early morning urine specimens
present in urine. The presence of nitrite, should be collected in sterile container on three
detectable by a simple test, indicates the consecutive days.
presence of nitrite-reducing bacteria in urine.
ii. Catalase test: The presence of catalase as 2. Direct Ziehl–Neelsen Staining
evidenced by frothing on addition of hydrogen Smear made from centrifuged deposit of urine
peroxide indicates bacteriuria, though a is stained with Ziehl–Neelsen staining and may
positive result is obtained also in hematuria. reveal acid-fast bacilli. Saprophytic mycobacteria
Chapter 78: Infective Syndromes | 541
(e.g. M. smegmatis) may be present in normal urine
Laboratory diagnosis: For specimen collection,
which may be excluded by using acid-alcohol various methods used are: 1. Midstream specimen
as decolorizing agent in staining procedure. M. of urine (MSU); 2. Catheter specimen of urine (CSU);
tuberculosis is acid-alcohol-fast while M. smegmatis 3. Suprapubic stab; 4. Noninvasive method; 5. Early
is only acid-fast. morning urine (EMU); 6. Initial flow of urine
Part of the specimen is used for bacteriological
culture and the rest is examined immediately under
3. Culture the microscope
Culture is performed on Lowenstein–Jensen Culture: Semi-quantitative methods (Standard loop
medium and incubated for 6–8 weeks. Growth technique) are used for culture
is identified by Ziehl–Neelsen staining and Count more than 105 bacteria of single species per ml
is called significant bacteriuria. It indicates active UTI
biochemical tests. None of the screening methods is as sensitive or
reliable as a culture.
Key Points
Urinary tract infection (UTI) may be defined as the
presence of bacteria undergoing multiplication in Important Questions
urine within the urinary drainage system
1. Name the various organisms causing urinary tract
Lower UTI includes) Urethritis, cystitis and prostatitis infection. Discuss the laboratory diagnosis of this
and due to ascending infection condition.
Upper UTI includes acute pyelitis and acute pyelone
2. Write short notes on:
phritis.
Midstream specimen of urine (MSU).
4. Sore throat
Sore throat is essentially an acute tonsillitis Viral pharyngitis or other causes of pharyngitis/
or pharyngitis. It is characterized by redness tonsillitis must be differentiated from that caused
and edema of mucosa, exudation of tonsils, by Streptococcus pyogenes since it is treatable with
pseudomembrane formation, edema of uvula, penicillin whereas viral infections are not.
grey coating of tongue and enlargement of cervical
lymph nodes. Causative agents of sore throat are A. Specimen
given in Table 78.7. For bacteriological sampling, a plain, albumen-
Pseudomembrane formation: Corynebacterium coated or charcoal-coated cotton-wool swab should
diphtheriae, Candida albicans, β-hemolytic group A be used to collect as much exudate as possible from
Streptococcus, Treponema vincentii and Leptotrichia the tonsils, posterior pharyngeal wall and any other
buccalis may lead to pseudomembrane formation. area that is inflamed or bears exudate. Two throat
swabs are collected. If it cannot be delivered to the
Laboratory Diagnosis laboratory within about 1 hour, it should be placed
in a refrigerator at 4°C until delivery.
The signs and symptoms of sore throat (pharyngitis)
caused by streptococci and viruses are similar.
B. Direct Microscopy
From one swab, make two smears for Gram
Table 78.7 Causative agents of sore throat staining and Albert-staining.
A. Bacteria 1. Gram Staining
• Streptococcus β-hemolytic group A and occasionally
groups C and G
It is not helpful unless Vincent’s organisms or
• Corynebacterium diphtheriae
Candida albicans are suspected. Vincent’s infection
• Haemophilus influenzae
shows gram-negative spirochaetes (Borrelia
• Bordetella pertussis
vincentii) and gram-negative fusiform bacilli
• Neisseria gonorrheae
(Fusobacterium spp.). When Candida albicans is
• Treponema vincentii
suspected, it appears as gram-positive oval budding
• Leptotrichia buccalis
yeast cells.
B. Fungi 2. Albert Staining
• Candida albicans It shows green-colored, V and L-shaped (Chinese
C. Viruses letter pattern) bacilli with bluish-black metachromatic
• Epstein–Barr virus granules in infection due to C. diphtheriae. Albert-
• Adenoviruses staining is helpful for presumptive diagnosis of C.
• Coxsackievirus A diphtheriae.
www.ebook3000.com
542 | Section 6: Miscellaneous
C. Culture Elek’s gel precipitation test .
Culture media are selected according to the Animal inculation test
organism suspected to be the causative agent iii. For Candida albicans
of sore throat. Following media may be used for a. Germ tube test
culture. b. Carbohydrate fermentation and assimil
ation tests.
Blood agar: All the organisms will grow on this iv. For other causative agents
medium. Special culture media and different biochemical
reactions or serological tests may be required
Crystal violet blood agar: It is selective for Str.
as described in respective chapters. For
pyogenes especially when incubated anaerobically.
details of tests mentioned above, refer to the
Loeffler’s serum slope: For isolation of C. corresponding chapters.
diphtheriae, grow very rapidly (in 6–8 hours).
Potassium tellurite blood agar: Selective media E. Antibiotic Sensitivity
for isolation of C. diphtheriae. All β-hemolytic group A streptococci are sensitive to
Sabouraud’s dextrose agar (SDA): When penicillin G, and most are sensitive to erythromycin.
suspecting Candida albicans, SDA should be C. diphtheriae is sensitive to penicillin.
included.
These culture media should be incubated at 37°C Pneumonia
for overnight. In case of potassium tellurite blood
agar, it should be incubated for 48 hours. After 6–8 Pneumonia may be defined as inflammation and
hours, a subculture should be made from Loeffier’s consolidation of the lung substance. Bacterial
serum slope onto potassium tellurite blood agar, causes for pneumonia are listed in Table 78.8.
which is then incubated at 37°C for 48 hours.
Table 78.8 Microbial pathogens that cause pneu
D. Identification monia
1. Morphology A. Community-acquired pneumonia
i. Str. pyogenes: Colnies are small (pin-point), Common organisms
circular, semitransparent, low convex discs Streptococcus pneumoniae
having β-hemolysis. Chlamydophila pneumoniae
ii. C. diphtheriae: On potassium tellurite blood Mycoplasma pneumoniae
agar, gray or black coloured round colonies Legionella pneumophila
Uncommon organisms
are seen.
Haemophilus influenzae
iii. Candida albicans: White or cream colored
Staphylococcus aureus
colonies may be seen.
Chlamydophila psittaci
Coxiella burnetii
2. Gram Staining or Albert Staining Kleb. pneumoniae
i. Gram Staining Actinomyces israillae
Primary viral pneumonia
∙∙ Small Gram: Positive spherical cocci occurring Influenza, parainfluenza virus and measles
in chains is characteristic of Str. pyogenes. Respiratory syncytial virus in infancy
∙∙ Candida albicans reveals gram-positive budding Varicella (chickenpox)
yeast cells. B. Hospital-acquired pneumonia
C. Diphtheriae is seen as gram-positive bacilli. Gram-negative bacilli (60%)
Escherichia, Pseudomonas, Klebsiella species
ii. Albert Staining Gram-positive organisms (16%)
Staph. aureus
Diphtheriae shows green-colored V-or L-shaped
Anaerobes
bacilli with bluish-black metachromatic granules.
Legionella spp.
C. Pneumonia in immunocompromised patients
3. Other Tests for Confirmation Pnemocystis carinii
i. For β-hemolytic streptococci Gram-negative bacteria—Ps. aeruginosa
a. Bacitracin sensitivity test—for Str. pyogenes Mycobacteruim tuberculosis
b. Lancefield grouping—for all β-hemolytic Streptococcus pneumoniae
streptococci Haemophilus influenzae
ii. For C. Diphtheriae Candida albicans
Aspergillus fumigatus
a. Biochemical tests
Viruses–Cytomegalovirus
b. Toxigenicity tests
Chapter 78: Infective Syndromes | 543
1. Classification 3. Giemsa staining: Giemsa stain of sputum.
is useful to detect cysts and trophozoites of
A. Community-acquired Pneumonia Pneumocystis carinii.
Community-acquired pneumonia has been thought
to present as either of the two syndromes—typical C. Culture
or atypical. Sputum, blood and pleural fluid can be cultured
on blood agar and chocolate agar. Purulent portion
Typical Pneumonia Syndrome of sputum is best for culture. If the sputum is too
Typical pneumonia syndrome is usually caused viscid, it may be homogenized.
by the most bacterial pathogen in community- 1. Blood agar: It is incubated aerobically at 37°C
acquired pneumonia, Streptococcus pneumoniae, under 5–10% CO2.
but can also be due to other bacterial pathogens. 2. Chocolate agar: It is also incubated at 37°C
with 5–10 percent CO2.
Atypical Pneumonia Syndrome 3. Lowenstein–Jensen (L–J) medium: Three
Atypical pneumonia is classically produced by specimens of sputum are collected on three
Mycoplasma pneumoniae, Legionella pneumophila. successive days for culture on L–J media which
Chlamydophila pneumoniae. There is patchy are then incubated at 37°C for 6–8 weeks.
consolidation of lungs. 4. Selective media are required for culture of L.
pneumophila (see Chapter 37).
Lobar Pneumonia 5. Isolation of chlamydia can be done on cell-lines.
It is an acute inflammation characterized by homo
geneous consolidation of one or more lobes. It is D. Detection of Bacterial Antigens
caused by Streptococcus pneumoniae.
Direct Immunofluorescence Test
Bronchopneumonia Direct immunofluorescence examination of sputum
It is almost always a secondary infection and is done to detect antigens in L. pneumophila.
generally follows viral infections of the respiratory
tract. It is an acute inflammation of bronchi and E. Serology
the consolidation is scattered. It is caused by 1. Mycoplasma pneumoniae
Streptococcus pneumoniae Haemophilus influenzae –– Complement fixation test (CFT)
(rarely Kleb. pneumoniae, Staph. aureus). –– Cold agglutinin test
Respiratory syncytial virus, Mycoplasma 2. Chlamydia pneumoniae
pneumoniae, Chlamydophila pneumoniae and
–– Microimmunofluorescence
Bordetella pertussis cause bronchitis and bronchiolitis.
–– TWAR antigen
B. Hospital-acquired Pneumonia –– CFT
–– Immunofluorescent antibody test
Hospital-acquired or nosocomial pneumonia
4. Coxiella burnetii
refers to a new episode of pneumonia occurring
–– CFT
at least two days after admission in the hospital.
Key Points
C. Pneumonia in lmmunocompromized
Sore throat is essentially an acute tonsillitis or
Patients pharyngitis
The common causative agents include Pneumocystis Laboratory diagnosis of sore throat caused by
carinii, Staph. aureus, Ps. aeruginosa, viral bacteria depends on direct microscopy and culture.
infections (CMV and herpes) and M. tuberculosis. For bacteriological sampling, two throat swabs are
collected
Pneumonia may be defined as inflammation and
Laboratory Diagnosis consolidation of the lung substance, which may
A. Specimen be classified as community-acquired pneumonia,
hospital-acquired pneumonia and pneumonia in
Sputum; blood; pleural fluid; blood for serological immunocompromized patients
tests. Laboratory diagnosis of pneumonia depends on
direct microscopy and culture.
B. Direct Microscopy
1. Gram Staining: Adequate number of pus Important Questions
cells alongwith the presence of predominant 1. Name the various organisms causing sore throat.
organisms gives a clue to the probable pathogen. How will you diagnose it in the laboratory?
2. Ziehl–Neelsen staining: Presence of acid-fast 2. Name the various bacterial causes of pneumonia.
bacilli (AFB) gives a presumptive diagnosis of Discuss the laboratory diagnosis of pneumococcal
tuberculosis. pneumonia.
www.ebook3000.com
544 | Section 6: Miscellaneous
www.ebook3000.com
546 | Section 6: Miscellaneous
red-polymyxin agar (MYPA) medium may also D. Fungus
be used. There have been reports of diarrhea associated
iii. Identification: Colonies have curled hair with Candida albicans.
appearance. Gram-staining shows gram-
positive spore bearing bacilli. Dysentery
Dysentery is a disease marked by frequent
11. Other Bacteria watery stools, often with blood and mucus, and
Aeromonas hydrophila and Plesiomonas shigelloides characterized clinically by cramping abdominal
have been reported to cause diarrheal diseases. pain, tenesmus (painful straining when passing the
stools), fever and dehydration. Dysentery results
from ‘enteroinvasive’ microorganisms (Table 78.10).
B. Viruses
that penetrate through the mucosa and cause
1. Rotavirus inflammation of the intestinal wall. The differences
2. Norwalk virus between protozoal (amebic) and bacterial (bacillary)
3. Adenoviruses dysentery are given in Table 78.10. For laboratory
4. Other viruses: diagnosis, see under “Diarrhea”.
Astroviruses and caliciviruses. Table 78.10 Microorganisms causing dysentery
A. Bacteria
Laboratory Diagnosis • Shigella dysenteriae
• S. fIexneri
i. Specimen: Feces • S. boydii
ii. Electron microscopy • S. sonnei
iii. Fluorescent antibody test and ELISA. • Escherichia coli (EIEC, EPEC, EHEC)
B. Protozoa
• Entamoeba histolytica
C. Protozoa • Balantidium coli
1. Entamoeba histolytica
Key Points
2. Giardia lamblia
3. Cryptosporidium parvum Diarrhea is defined as the passage of loose, liquid or
watery stools. These liquid stools are usually passed
4. Balantidium coli: This is a rare cause of chronic more than three times a day
recurrent diarrhea, with dysenteric episodes in Gastroenteritis may be defined as inflammation of
some. the mucous membrane of stomach and intestine,
resulting in frequent loose motions with or without
Laboratory Diagnosis mucus and with or without blood, pain abdomen
and with or without fever. It is often used as a
i. Specimen: Feces synonym for acute diarrhea, especially when
associated with vomiting
ii. Microscopy
Bacterial diarrhea Vibrio cholerae, E. coli, Salmonellae
are some important bacterial causes of diarrheal
a. Saline Preparation and Iodine Mount diseases. Rotavirus is the most important viral
etiology of diarrhea
In saline preparation, motility of trophozoites Dysentery is a disease marked by frequent
can be observed while cysts take up iodine and watery stools, often with blood and mucus, and
appear distinct in iodine mount. Cysts and motile characterized clinically by cramping abdominal pain,
trophozoites of E. histolytica can be observed in feces tenesmus, fever and dehydration
of amebic dysentery. Giardia lamblia cysts in formed Laboratory diagnosis of diarrhea, and dysentery
stools, or trophozoites in fresh diarrheal stools can be depends on isolation of organism from the relevant
specimen.
seen in giardiasis. Trophozoites of Balantidium coli
are found in liquid stool of this parasitic infection.
Rarely cysts may be seen in formed stools. Important questions
1. Enumerate the different causes of diarrhea. How will
b. Acid-fast Staining you diagnose a case of diarrhea in the laboratory.
2. Enumerate the etiological agents of dysentery.
Feces smear shows acid-fast oocyst of Crypto
Discuss in detail the laboratory diagnosis of
sporidium parvum.
dysentery.
iii. Serological tests: Indirect hemagglutination 3. Write short notes on:
assay (lHA) and ELISA are used to detect a. Traveller’s diarrhea
antibody titer in sera of patients with amebiasis. b. Viral diarrhea
Chapter 78: Infective Syndromes | 547
6. Food poisoning
The term bacterial food poisoning is restricted to 8–24 hours. The typical example of this type of food
acute gastroenteritis due to the presence of bacteria, poisoning is by salmonellae.
usually in large numbers, or their products in food.
It is of three types (Table 78.11). B. Toxic Type
In this type, the disease follows ingestion of food
A. Infective Type with preformed toxin. Incubation period is short
In this type, multiplication of bacteria occurs in (2 to 6 hours).Example is staphylococcal food
vivo when infective doses of microorganisms are poisoning.
ingested with food. Incubation period is generally
C. Infective-toxic Type
Table 78.11 Causative agents of food poisoning In this type, bacteria release the toxin in the bowel.
The incubation period is 6–12 hours. The typical
1. Infectlve type example is Cl. perfringens food poisoning.
• Salmonella Typhimurium For the laboratory diagnosis, refer to the corres
• S. Enteritidis ponding chapters. It has also been described under
• S. Heidelberg “Laboratory diagnosis of diarehea”.
• S. Indiana
Key Points
• S. Newport
The term ‘bacterial food poisoning’ acute in is of
• S. Dublin
three types A. Infective type, B. Toxic type, and
• Vibrio parahaemolyticus Infective-toxic type.
• Campylobacter jejuni Laboratory diagnosis of food poisoning depends on
isolation of organism from the relevant specimen.
2. Toxic type
• Staphylococcus aureus
Bacillus cereus Important questions
• Clostridium botulinum 1. Name the various organisms causing food
poisoning. Describe briefly the laboratory diagnosis
3. Infectlve-toxic type
of this condition.
Clostridium perfringens
2. Write short notes on food-borne botulism.
www.ebook3000.com
548 | Section 6: Miscellaneous
Table 78.12 Organisms causing sexually trans 2. Isolation: Isolation of the Chlamydia by
mitted diseases and their clinical presentations intracerebral inoculation into mice and into yolk
sac of eggs has been replaced by cell cultures.
Sexually transmitted Organisms
diseases (STDs) 3. Serological tests: LGV patients develop high
titers of circulating antibodies, with titers of 1:64
A. Painless genital ulcers
or more in CF test and 1:512 or more in micro-IF.
• Syphilis Treponema pallidum
• Lymphogranuloma Chlamydia trachomatis Serological diagnosis is, therefore, feasible.
venereum (LGV) 4. Frei test: It is a skin test using LGV antigen and
• Donovanosis Calymmatobacterium shows delayed type of hypersensitivity.
granulomatis
B. Painful genital ulcers C. Donovanosis
• Chancroid Haemophilus ducreyi
• Herpes genitalis Herpes simplex viruses Donovanosis is caused by Calymmatobacterium
(HSV) type 2 and 1 granulomatis.
1. Specimen: Tissue smear from the ulcer
C. Urethral discharge
• Gonorrhea Neisseria gonorrhoeae 2. Staining: Diagnosis can be made by demon
• Nongonococcal Chlamydia trachomatis stration of Donovan bodies in Wright-Giemsa-
urethritis (NGU) (types D-K) stained impression smears from the lesions.
Ureaplasma urealyticum They show bipolar condensation of chromatin,
Mycoplasma genitalium
giving a closed safety pin appearance in stained
M. hominis
smears. Capsules are usually seen as dense
D. Vaginal discharge acidophilic areas around the bacilli.
• Gonorrhea N. gonorrhoeae
• NGU C. trachomatis
M. hominis D. Chancroid
• Trichomoniasis Trichomonas vaginalis Chancroid or soft chancre is caused by H. ducreyi.
• Vaginitis Gardnerella vaginalis
Mobiluncus sp.
1. Specimen: Exudate
• Vulva-vaginal Candida albicans 2. Gram staining: Gram-negative coccobacilli are
candidiasis seen in Gram staining.
E. Genital warts Human papilloma viruses 3. Culture: Exudate is cultured onto chocolate agar
enriched with isovitalex and fetal calf serum,
F. No genital lesions HIV-1 and HIV-2
but only systemic Hepatitis B virus (HBV)
and containing vancomycin as a selective agent.
manifestations Hepatitis C virus (HCV) Culture plates are incubated at 35°C under 10
percent CO2 and in high humidity in 2–8 days.
G. Miscellaneous Group B streptococci
Molluscum contagiosum 4. Identification: For identification of the organism,
virus colony morphology and Gram staining are
Cytomegalovirus useful.
Phthirus pubis i. Colony morphology: H. ducreyi forms small,
Sarcoptes scabei
Shigella sp. gray, translucent colonies.
Campylobacter sp. ii. Gram-staining: It shows gram-negative
Giardia lamblia coccobacilli.
Entamoeba histolytica
E. Herpes genitalis
Herpes simplex virus (HSV), types 1 and 2, is the
b. FTA-ABS (fluorescent treponemal antibody etiological agent but type 2 strains are more com
absorption test). monly associated.
c. TPI (Treponema pallidum immobilization 1. Specimens
test) i. Scrapings from base of the lesions
VDRL and TPHA are two most commonly
ii. Blood for serology
used tests.
2. Microscopy: Intranuclear type A inclusion
bodies may be seen in Giemsa-stained smears.
B. Lymphogranuloma Venereum (LGV) The virus particle may also be demonstrated
It is caused by C. trachomatis serotypes L1, L2 and L3. under the electron microscope.
1. Direct microscopy: Smears of material aspirated 3. Virus isolation: Diagnosis is confirmed by
from the bubos may show the elementary tissue culture in human diploid fibroblast cells.
bodies (Miyagawa’s granulocorpuscles). The Typical cytopathic changes may appear as early
sensitivity of microscopy is very low. as in 24–48 hours.
Chapter 78: Infective Syndromes | 549
4. Serology: ELISA, neutralization or complement immunofluorescence test with a mono
fixation tests are used for antibody detection clonal antibody or by ELISA.
and are useful in the diagnosis of primary 3. Culture: The exudate is inoculated on McCoy or
infection. HeLa cell cultures treated with cycloheximide.
Intracytoplasmic glycogen-rich inclusions
F. Gonorrhea are detected by Giemsa stain or by immuno
It is caused by Neisseria gonorrhoeae. fluorescence. These are suggestive of C.
1. Specimens: Specimens used are: (i) Urethral trachomatis.
discharge; (ii) An endocervical swab; (iii) In 4. Serology
chronic cases; (iv) Rectal swab. i. Complement fixation test (CFT)
2. Direct Gram-staining: Gram-stain of the penile ii. Microimmunofluorescence or ELISA is useful
exudate shows characteristic and diagnostic for detection of serovar-specific antibody.
gram-negative intracellular diplococci (GNID)
in granulocytes.
H. Trichomoniasis
3. Culture: Commonly used selective media are
modified Thayer–Martin and Martin–Lewis It is caused by Trichomonas vaginalis.
plates. Inoculated plates should immediately 1. Specimen: Swab of vaginal discharge is exami
be placed in an incubator at 37°C for 24–48 ned freshly. Specimen should be collected in
hours in the presence of CO2. Cultures with no stuart’s transport medium if delay in transport
visible growth must be held for 72 hours before is inevitable.
discarding. 2. Direct microscopy: Direct wet film shows
4. Identification: Identification of an organism motile trichomonads. Direct microscopy is at
is based on colony morphology, Gram staining least 80 percent as positive as culture.
from colonies and biochemical reactions. 3. Culture: Fineberg’s medium is used for culture
i. Colony morpholo g y: Small, gray, of specimen and it is incubated for 5 days and
translucent, raised colonies. examined for motile protozoa.
ii. Gram-staining: Gram-negative diplococci
iii. Biochemical reactions: They are oxidase I. Bacterial Vaginosis-associated Organisms
positive and produce acid from glucose but The diagnosis of bacterial vaginosis does not depend
not lactose, maltose or sucrose. on the isolation of a particular microorganism, e.g.
iv. Direct detection methods, such as Gardnerella vaginalis or Mycoplasma hominis, but on
fluorescent antibody and agglutination the replacement of the predominantly lactobacillary
tests, are also available. flora with a mixture of aerobes; and anaerobic
v. Nucleic acid probes for the direct detection organisms, a shift of pH to neutral or alkaline, and
of N. gonorrhoeae from clinical specimens. the presence of ‘clue’ cells. These changes are most
easily assessed from the Gram stained film.
G. Nongonococcal Genital Infection For diagnosis of Shigella sp, Campylobacter
Symptoms of discharge and dysuria clinically sp, Group B streptococci refer to corresponding
indistinguishable from gonorrhea caused by chapters.
organisms other than N. gonorrhoeae is called
nongonococcal urethritis (NGU). Causative agents J. Vulvovaginal Candidiasis
are shown in Table 78.13. A significant proportion It is caused by various species of Candida but C.
of nongonococcal genital infection in women is albicans accounts for 80% of cases.
generally due to chlamydia trachomatis. 1. Specimen: Swab from vaginal secretions
2. Direct microscopy
Laboratory Diagnosis
i. KOH mount: It shows yeast cells.
1. Specimens
(i) Swabs from exudate of urethra; (ii) Cervical ii. Gram-staining: Gram staining shows
discharge. characteristic gram-positive budding yeast
2. Direct examination cells and pseudohyphae.
i. Giemsa stain: It shows intracytoplasmic 3. Culture: Sabouraud’s dextrose agar (SDA) is
inclusion bodies suggestive of C. tracho inoculated with the specimen and incubated
matis. at 37°C for 48 hours.
ii. Antigen detection: For detection of ele 4. Identification
mentary bodies of C. trachomatis, smears i. Colony morphology: Colonies are creamy
are made from exudate and examined by white and smooth.
www.ebook3000.com
550 | Section 6: Miscellaneous
ii. Gram staining: Gram-stained smear shows Key Points
budding gram-positive yeast cells.
The sexually transmitted diseases (STDs) are a group
Germ tube formation: C. albicans forms
iii.
of communicable diseases which are transmitted
germ tube within two hours when incubated predominantly or entirely by sexual contact. The
in human serum at 37°C. causative organisms include a wide range of
iv. Chlamydospores formation: On cornmeal bacterial, viral. protozoal and fungal agents
For laboratory diagnosis and treatment according
agar C. albicans forms chlamydospores.
to the suspected organism responsible for the
manifestations of that particular STD.
K. Genital Warts
Genital warts, also known as condyloma acuminata, Important Questions
are common in sexually active adults. These are 1. Name the various organisms causing sexually
usually due to human papillomavirus (HPV) types transmitted diseases. Discuss the laboratory
6 and 11. diagnosis of syphilis.
For detection of inclusion bodies of HPV, 2. Write short notes on:
cytological or histological examination of cells in a. Laboratory diagnosis of gonorrhea
b. Nongonococcal urethritis (NGU)
urine is used.
c. Chancroid
For diagnosis of Shigella sp, Campylobacter spp, d. Lymphogranulum venereum (LGV)
Group B streptococci, refer to the corresponding e. Donovanosis
chapters. f. Vulvovaginal candidiasis.
8. Wound Infection
Wound infections may be endogenous or exogenous. Gas chromatography may be performed directly
It may be caused by a variety of aerobic and on liquid specimens to indicate the presence of
anerobic species of bacteria (Table 78.13). anerobes.
www.ebook3000.com
552 | Section 6: Miscellaneous
Table 78.14 Causes of pyrexia of unknown origin (PUO)
A. Infective causes B. Noninfective causes
a. Bacterial a. Neoplasms
• Urinary tract infections • Hodgkin’s lymphoma
• Lung, subdiaphragmatic, appendix and other deep • Non-Hodgkin’s lymphoma
abscesses • Leukemia
• Septicemia associated with cryptic abscesses, • Hypernephroma
pneumonia, pyelonephritis, biliary tract infection, • Hepatoma
infective endocarditis and immunodeficiencies • Disseminated malignancy
• Enteric fever b. Connective tissue disorders
• Tuberculosis • Systemic lupus erythematosus (SLE)
• Brucellosis • Polyarteritis nodosa
• Syphilis • Temporal arteritis
• Relapsing fever c. Granulomatous diseases
• Rheumatic fever • Sarcoidosis
• Leptospirosis without jaundice or meningitis • Crohn’s disease
• Typhus fever • Granulomatous hepatitis
• Nonmeningitic meningococcal infection d. Drug reactions
• Q fever Drug-induced fevers
b. Parasitic
• Malaria
• Hepatic amebiasis
• Leishmaniasis
• Trypanosomiasis
• Toxoplasmosis
• Filariasis
c. Viral
• EBV infection
• CMV infection
• HIV infection
• Rubella and other infectious fevers without typical
rash
C. Viral Infections
4. Identification
Peripheral blood smear may be helpful in infectious
Organisms may be identified on the basis of
mononucleosis. Viral infections may be detected
colony morphology, Gram-staining, biochemical
either by tissue culture or by serology. Paul–Bunnel
reactions and agglutination, etc. Ziehl–Neelsen
test is useful in infectious mononucleosis.
(Z–N) staining is performed to detect acid-fast
bacilli (AFB) for M. tuberculosis. This is further
confirmed by culture and biochemical reactions. D. Fungal Infections
For details of individual organisms, refer to Specimens may be cultured on Sabouraud’s
corresponding chapters. dextrose agar or brain–heart infusion agar.
Chapter 78: Infective Syndromes | 553
E. Other Tests for Diagnosis 4. The most common viral agent causing aseptic
meningitis is:
1. Skin Tests a. Enteroviruses
Mantoux test: A tuberculin test and a chest X-ray b. Adenoviruses
should be done to detect tuberculosis. c. Cytomegalovirus
Skin tests for histoplasmosis, coccidioido d. Parainfluenza virus
mycosis, sarcoidosis should also be done. 5. Which of the following bacteria is the most
comrnon cause of UTI:
2. Hematological Investigations a. E. coli
Hematological investigations should be done b. Ps. aeruginosa
to detect leukocytosis, suggestive of a cryptic c. Staph. aureus
abscess; eosinophilia, suggestive of helminthiasis; d. Staph. saprophyticus
and atypical lymphocytes, suggestive of infectious 6. All the following are causative agents of sore throat
mononucleosis. except:
a. Streptococcus pyogenes
3. Immunologic Tests b. Haemophilus influenzae
LE cell phenomenon and antinuclear antibody c. Borrelia vincenti
test in SLE. d. Staphylococcus aureus
7. Which of the following organisms may lead to
4. Biopsy pseudomembrane formation in throat:
a. C. diphtheriae b. Strep. pyogenes
Biopsy of liver and bone marrow and other tissues
c. Candida albicans d. All the above
such as skin, lymph nodes and kidney.
8. Which of the following agents can cause diarrhea?
Key Points a. Rotavirus b. Vibrio cholerae
c. S. Typhimurium d. All the above
Pyrexia of unknown origin (PUO) may be defined
as (a) any febrile illness (body temperature greater 9. Which of the following protozoa can cause diarrhea:
than 38°C) on several occasions, (b) duration of a. Entamoeba histolytica
fever of more than 3 weeks, and (c) failure to reach a b. Cryptosporidium parvum
diagnosis despite 1 week of inpatient investigation.
c. Giardia lamblia
It is also known as ‘fever of unknown origin’ (FUO)
d. All the above
The causes of PUO include infections bacterial, parasitic
and viral) neoplasms, connective tissue disorders, 10. Which of the following agents can cause dysentery:
granulomatous diseases and drug reactions a. Escherichia coli (EIEC)
Tests should first be done for the more likely infec b. Shigella dysenteriae
tions and then, if these are negative, tests for the less
c. Entamoeba histolytica
likely should be done.
d. All the above
11. Which of the following bacteria can cause infective
Important Question type of food poisoning:
Define and enumerate the causes of pyrexia of unknown a. S Enteritidis
origin (PUO). Discuss the laboratory diagnosis of PUO. b. Clostridium botulinum
c. Staph. aureus
Multiple choice questions (MCQs) d. All the above
12. Infective-toxic type of food poisoning is caused by:
1. Presence of bacteria in blood without multiplication
a. Staph. aureus
is known as:
b. Salomonella Enteritidis
a. Bacteremia b. Septicemia
c. Clostridium perfringens
c. Pyemia d. Endotoxemi
d. Salmonella Dublin
2. Which of the following bacteria can cause purulent
meningitidis: 13. Ulcer/s is/are painless I n which of the following
a. H. influenzae sexually transmitted diseases:
b. Strep. pneumoniae a. Syphilis b. Chancroid
c. N. meningitis c. Herpes genitalis d. All of the above
d. All the above 14. Genital ulcer is painful in:
3. Which of the following agents cause/s aseptic a. Syphilis
meningitis? b. Chancroid
a. Herpes simplex b. Enteroviruses c. lymphogranuloma venereum
c. Arboviruses d. All of the above d. Donovanosis
www.ebook3000.com
554 | Section 6: Miscellaneous
15. Which of the following bacteria is/are implicated 17. Which of the following anerobes can cause wound
as causative agents of nongonococcal urethritis: infection:
a. C. trachomatis a. Bacteroides spp
b. Peptostreptococci
b. Ureaplasma urealyticum
c. Clostridium perjringens
c. Mycoplasma hominis. d. All the above
d. All of the above 18. Which of the following conditions can result in PUO:
16. Urethral discharge is present in: a. Urinary tract infection b. Enteric fever
a. Chancroid c. Malaria d. All the above
b. Gonorrhea Answers (MCQs)
c. Herpes genitalis 1. a; 2. d; 3. d; 4. a; 5. a; 6. d; 7. d; 8. d; 9. d; 10. d; 11. d;
d. Lymphogranuloma venereum 12. c; 13. a; 14. b; 15. d; 16. b; 17. d; 18. d.
79
Chapter
Hospital-acquired Infection
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe common hospital-associated infections
be able to: and causative organisms responsible for these
∙∙ Define hospital-associated infection conditions
∙∙ List of routes of transmission of hospital-associated ∙∙ Describe diagnosis and control of hospital-
infections associated infections.
www.ebook3000.com
556 | Section 6: Miscellaneous
the hospital environment for long periods and skin scales, spread to the susceptible site,
develop resistance to antibiotics and disinfectants e.g. Ps aeruginosa, Staph. aureus.
are particularly important in this respect. Though iii. Aerosols: Aerosols produced by nebulizers,
protozoal infections are rare. Strep. pyogenes was, humidifiers and air conditioning apparatus
perhaps, the most important cause of hospital transmit certain pathogens to the
infection formerly but is hardly ever encountered respiratory tract. Occurrence of legionellae
now as it is highly susceptible to antibiotics. in hospital water supply and a number of
1. Staph. aureus: Staph. aureus strains, resistant persons with an impaired immune system
to multiple antibiotics and belonging to has led to outbreaks of infection mainly
phage type 80/81, spread globally in the 1950s
with Legionella pneumophila.
and 1960s, colonizing hospitals and causing
nosocomial infection. Subsequently, epidemic 3. Oral route: Hospital food contains gram-
or pandemic strains characterized by resistance negative bacilli which are most often antibiotic
to methicillin-resistant Staphylococcus aureus resistant (P. aeruginosa, E. coli, Klebsiella spp.
(MRSA) have been found in many hospitals and others), which may colonize the gut and
worldw ide.Staph. epidemidis and Group D later cause infection in susceptible patients.
streptococci also are sometimes responsible 4. Parenteral route (inoculation): Certain
for hospital infections. infections may be transmitted by blood
2. Pseudomonas species: Ps. aeruginosa and transfusion or tissue donation, contaminated
other Pseudomonas species have always been blood-products (factor VIII), contaminated
important causes of hospital infection. infusion fluids and from accidental injury
with contaminated sharp instruments (HIV,
3. Tetanus spores: Hospital tetanus is usually due
hepatitis B and C).
to faulty sterilization techniques or other lapses
in asepsis. 5. Self-infection and cross-infection: Self-
4. Viral infections: Viral infections probably infection may occur due to transfer into the
account for more hospital-acquired infections wound of staphylococci (or occasiona lly
than previously realized. streptococci) carried in the patient’s nose and
distributed over the skin, or of coliform bacilli
i. HIV and hepatitis B and C viruses are
and anaerobes released from the bowel during
transmitted by contaminated blood or
surgery. Alternatively, cross-infection may result
blood products. Screening of blood donors from staphylococci or coliform bacilli derived
has reduced the risk to a large extent. from other patients or healthy staff carriers.
However, HIV escapes detection during
the window period. Common hospital-acquired
ii. Viral diarrhea and chickenpox are other infection
viral infections that spread in hospitals.
iii. Cytomegalovirus, herpesvirus, influenza, 1. Urinary Tract Infection
enteroviruses and arenaviruses may also Most hospital-acquired infections of the urinary tract
cause hospital infection. are associated with urethral catheterization. Urinary
5. Fungus and parasites: The range of hospital tract infection is caused by Esch. coli, Klebsiella,
pathogens also includes yeasts (Candida Proteus, Serratia, Pseudomonas, Providencia, coagu
albicans), moulds, (Aspergillus, Mucor) and lase negative staphylococci, enterococci and Candida
protozoa (Entamoeba histolytica, plasmodia, albicans.
Pneumocystis carinii, Toxoplasma gondii).
2. Respiratory Infections
ROUTES OF TRANSMISSION Aspiration in unconscious patients and pulmonary
1. Contact ventilation or instrumentation may lead to
Direct contact: Spread from person to person nosocomial pneumonia, particularly in those
(staphylococcal and streptococcal sepsis). with preexisting cardiopulmonary disease. The
Indirect contact: Spread via contaminated major pathogens include Staph. aureus, Klebsiella
hands or equipment (enterobacterial diarrhoea, spp., Enterobacter, Serratia, Proteus, Esch. coli,
Pseudomonas aeruginosa sepsis). Pseudomonas aeruginosa, Acinetobacter, Legionella
2. Airborne spread pneumophila and respiratory viruses.
i. Droplets
ii. Dust: Dust from bedding, floors; exudate 3. Wound and Skin Sepsis
dispersed from a wound during dressing The incidence of postoperative infection is higher
and from the skin by natural shedding of in elderly patients. Most wound infections manifest
Chapter 79: Hospital-aquired Infection | 557
within a week of surgery. Staph. aureus is the medical microbiologist who will usually serve
predominant pathogen, followed by Pseudomonas as chairman, a physician, a surgeon, nurse
aeruginosa and then Esch. coli, Proteus, enterococci teachers, nurse representatives for surgery,
and coagulase negative staphylococci. obstetrics, gynecology and medicine, and
sterile service manager. The ICC should meet
4. Gastrointestinal Infections regularly to formulate and update policy for the
Food poisoning and neonatal septicemia in hospital whole hospital matters having implications for
have been reported. These infections are mainly infection control and to manage outbreaks of
associated with salmonella and Shigella sonnei. no nosocomial infection.
Roles of the infection control committee
5. Burns i. The surveillance of hospital infection.
Staph. aureus, Pseudomonas aeruginosa , ii. The establishment and monitoring of policies
Acinetobacter and Strept. pyogenes are responsible and procedures designed to prevent infection,
for hospital-acquired infections in cases of burns. e.g. catheter care policy, antibiotic policy,
disinfectant policy.
iii. The investigation of outbreaks.
6. Bacteremia and Septicemia
2. Infection control team: An infection control
These may be conseq uences of infections at team of workers, headed by the infection
any site but are commonly caused by infected control doctor (usually the microbiologists).
intravenous cannulae. Gram-negative bacilli are The functions of this team include surveillance
the common pathogens. and control of infection and monitoring of
Staph. epidermidis bacteremia is seen hygiene practices. The infection control nurse
commonly in patients with artificial heart valves. is a key member of this team.
Bacteremia in those with valvular defects may lead
to endocarditis. Prevention
Diagnosis and control of The hospital-acquired infections can be prevented
by following means:
hospital infection
1. Sterilization: The provision of sterile
The most important steps in’ preventing instruments, dressings, surgical gloves, face-
nosocomial infections are to first recognize their masks, theater clothing and fluids.
occurrence and then establish policies to prevent 2. Cleaning and disinfection: The general
their development. Hospital infection may occur hospital environment can be kept in good
sporadically or as outbreaks. order by attention to basic cleaning, waste
Etiological diagnosis is by the routine bacterio disposal and laundry. The use of chemical
logical methods of smear, culture, identification disinfectants for walls, floors and furniture is
and sensitivity testing. When an outbreak occurs, necessary only in special instances.
the source should be identified and eliminated. 3. Skin disinfection and antiseptics: Procedures
This requires the sampling of possible sources of for preoperative disinfection of the patient’s
infection. Typing of isolate—phage, bacteriocin, skin and for surgical scrubs are mandatory
antibiogram or biotyping—from cases and sites within the operating theater.
may indicate a causal connection. 4. Rational antibiotic prophylaxis
The provision of sterile instruments, dressings 5. Protective clothing
and fluids is of fundamental importance in hospital 6. Isolation source isolation and protective
practice. The cause of infection may be a defective isolation.
autoclave or improper techniques, such as boiling 7. Hospital building and design: The routine
infusion sets in ward sterilizers. A careful analysis of maintenance.
the pattern of infection may often reveal the source 8. Equipment
but sometimes it eludes the most diligent search. 9. Personnel: Hepatitis B vaccine should be
given to all health care workers.
Infection control policy 10. Monitoring: Monitoring of the physical
The establishment of an effective infection performance of air-conditioning plants
control organization is the responsibility of good and machinery used for disinfection and
management of any hospital. There will normally sterilization is essential.
be two parts: 11. Surveillance and the role of the laboratory:
1. Infection control committee (ICC): Every The detection and characterization of hospital
hospital should have an infection control com infection incidents or outbreaks rely on
mittee (ICC). The committee will consist of a laboratory data.
www.ebook3000.com
558 | Section 6: Miscellaneous
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Explain the meaning of sensitive, intermediate and
be able to: resistant applied to antimicrobial susceptibility
∙∙ List different methods of antibiotic sensitivity test results
testing ∙∙ Describe the following: Epsilometer or E-Test;
∙∙ Describe disk diffusion methods Minimum inhibitory concentration of antimicrobial
∙∙ Explain Stokes disk diffusion method and its reporting agents;Minimum bactericidal concentration of
∙∙ Differentiate between Stokes disk diffusion and antimicrobial agents.
modified Stokes disk diffusion method
INTRODUCTION Medium
It is essential to determine the susceptibility of Mueller–Hinton broth and agar may be used for
isolates of pathogenic bacteria to antibiotics that testing aerobic and facultative anaerobic isolates.
are likely to be used in treatment. As strains of most The medium is and poured to a depth of 4 mm
pathogenic organisms differ from one another (25 mL medium) in flat-bottomed 9 cm Petri dishes
within their species in their antibiotic sensitivities, on a level surface. When set, the plates may be
sensitivity tests are required as a routine. stored for up to a week at 4°C and their surfaces
should be dried with their lids ajar before use. The
ANTIBIOTIC SENSITIVITY TESTS pH of the medium must be close to 7.3. A more acid
Antibiotic sensitivity tests are of two types: reaction decreases the activity of aminoglycoside
A. Diffusion methods and macrolide antibiotics. A more alkaline pH
1. Kirby–Bauer disk diffusion method favors the action of tetracyclines, novobiocin
2. Stokes disk diffusion method and fusidic acid, but interferes seriously with the
B. Dilution methods activity of nitrofurantoin.
1. Broth dilution method The addition of 5% lysed horse blood is needed
2. Agar dilution method to support the growth of fastidious species such
Diffusion methods: Here the drug is allowed as Haemophilus influenzae. Lysed horse blood
to diffuse through a solid medium so that a should also be added for tests with sulfonamides
gradient is established, the concentration and trimethoprim; its content of thymidine
being highest near the site of application of phosphorylase is needed to neutralize the inhibitory
the drug and decreasing with distance. The effect of thymidine in the medium on the action of
test bacterium is seeded on the medium and these drugs. Low levels of free Ca2+ and Mg2+ ions in
its sensitivity to the drug determined from the the medium increase the action of aminoglycoside
inhibition of its growth. Several methods have antibiotics against Pseudomonas aeruginosa.
been used for the application of the drug. The The addition of 5% NaCl to the medium
method most commonly employed is to use is needed in one of the methods for detecting
filter paper disks, impregnated with antibiotics. resistance to methicillin in strains of staphylococci .
Disk Diffusion Methods Inoculum: Prepare the inoculum from material
These methods are suitable for organisms that picked up with a loop from five to ten colonies
grow rapidly overnight at 35°C. of the species to be tested. Inoculate them in a
www.ebook3000.com
Chap-80.indd 559 15-03-2016 11:26:48
560 | Section 6: Miscellaneous
Table 80.1 Control strains for Kirby–Bauer and
Stokes disk diffusion methods
Test bacteria Control strain
Kirby–Bauer Stokes
Coliform E. coli ATCC 25922 E. coli NCTC
organisms 10418
Pseudomonas P. aeruginosa P. aeruginosa
ATCC 27853 NCTC 10662
Haemophilus H. influenzae H. influenzae
spp ATCC 49247 NCTC 11931
Gonococci N. gonorrhoeae N. gonorrhoeae
Fig. 80.1: Principle of antibiotic diffusion in agar. The ATCC 49226 (sensitive strain)
concentration of antibiotic decreases as the distance from Enterococci E. faecalis ATCC E. faecalis
the disk increases 29212 NCTC 12697
Other organisms S. aureus ATCC S.aureus
that can grow 25923 NCTC 6571
suitable broth medium. Incubate at 35–37°C for
aerobically
4–6 hours when the growth is considered to be in
logarithmic phase. The density of the organisms is
adjusted to approximately 108 colony-forming units Table 80.2 Diluents used for various antibiotics
(cfu)/mL by comparing its turbidity with that of 0.5
Antibiotic Diluent
McFarland opacity standard (Fig. 80.1).
Chloramphenicol Ethanol
www.ebook3000.com
Chap-80.indd 561 15-03-2016 11:26:49
562 | Section 6: Miscellaneous
Table 80.4 Interpretation chart used in Kirby–Bauer disk diffusion method
Antibiotic* Diameter of zone inhibition (in mm)
Resistant Intermediate sensitive Sensitive
Benzylpenicillin ≤28 — ≥29
Ampicillin
Methicillin ≤9 10–13 ≥14
Carbenicillin
Gentamicin ≤12 — ≥13
Amikacin ≤14 15–16 ≥17
Erythromycin ≤13 14–17 ≥18
Tetracycline ≤14 15–18 ≥19
Chloramphenicol ≤12 13–17 ≥18
Nalidixic acid ≤13 14–18 ≥19
Trimethoprim ≤10 11–15 ≥16
Ciprofloxacin ≤15 16–20 ≥21
* Only limited antibiotics have been shown in the table.
www.ebook3000.com
Chap-80.indd 563 15-03-2016 11:26:50
564 | Section 6: Miscellaneous
3. Reference points in the evaluation and com in controls against many variables and therefore
parison of new and existing antimicrobial provides dependable results.
agents. Dilution tests: There are two types of dilution tests:
Broth dilution method and agar dilution method.
Agar Dilution Method
Here, serial dilutions of the drug are prepared in IMPORTANT QUESTIONS
agar (Mueller–Hinton agar) and poured into plates.
1. Name different methods of antibiotic sensitivity
The ‘agar dilution’ method is more convenient when testing. Discuss in detail Kirby–Bauer disk diffusion
several strains are to be tested at the same time. The method for antimicrobial sensitivity testing.
advantage is that many strains can be inoculated on 2. Write short notes on:
each plate containing an antibiotic dilution. a. Kirby–Bauer disk diffusion method
Automated versions of sensitivity tests are b. Stokes disk diffusion method
available and are in use in large laboratories. c. Minimum inhibitory concentration of antimi
crobial agents.
ANTIBIOTIC ASSAYS IN BODY FLUIDS d. Minimum bactericidal concentration of antimi-
crobial agents.
These are required to verify whether adequate drug
concentrations are achieved in blood and other MULTIPLE CHOICE QUESTIONS (MCQs)
body fluids. Likewise, new drugs must be tested to
determine achievable levels in the blood, urine, or 1. Which of the following media is most suitable for
antibiotic sensitivity testing?
other body fluids.
The assays are generally done by making serial a. Mueller–Hinton medium b. Nutrient agar
dilutions of the specimen and inoculating standard c. Blood agar d. MacConkey agar
suspensions of bacteria of known MIC. Assays 2. The pH of the medium for antibiotic sensitivity
by the agar diffusion method can also be done. testing should be:
A technique called the diffusion assay is used to a. 6.0–6.2 b. 6.8–7.0
measure the concentration of an antimicrobial c. 7.2–7.4 d. 7.6–7.8
in a fluid specimen. The test relies on the same 3. In Stokes disk diffusion method, if the zone size
principle as the Kirby–Bauer test, except in this case is larger than, equal to or not more than 3 mm
it is the concentration of drug, not the sensitivity of smaller than the control a strain is considered:
organism, being assayed. This depends on the direct a. Sensitive b. Intermediate
relationship between antibiotic concentration c. Resistant d. None of the above
and the diameter of the zone of inhibition with a 4. Addition of 5% salt in the medium is done when
standard sensitive strain of bacterium. testing antibiotic susceptibility of :
a. Methicillin resistant staphylococci
Key Points b. Pneumococci
Antibiotic susceptibility tests are of two types: c. Coliforms
Diffusion tests and dilution tests d. Non fermenting gram-negative bacilli
Diffusion tests consists of Kirby–Bauer and Stokes
disk method. Stokes disk method incorporates built- ANSWERS (MCQs)
1. a; 2. c; 3. a; 4. a
Antimicrobial Chemotherapy
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe mechanism of drug resistance
be able to: ∙∙ List cephalosporins of first, second, third and fourth
∙∙ Describe mechanism of action of antibacterial generation.
drugs
www.ebook3000.com
Chap-81.indd 565 15-03-2016 11:28:09
566 | Section 6: Miscellaneous
MECHANISMS OF ACTION OF 2. Those with high activity against both gram-
ANTIBACTERIAL DRUGS positive and gram-negative organisms
but destroyed by b-lactamases: Ampicillin;
Mechanisms of action of antibacterial agents can amoxicillin; carbenicillin; ticarcillin; piperacillin.
be placed under the headings: 3. Those stable in gastric acid and suitable for
1. Inhibition of bacterial cell wall synthesis. oral administration: Penicillin-V; cloxacillin;
2. Inhibition of bacterial cytoplasmic membrane ampicillin.
function. 4. b-Iactamase resistant penicillins: Methicillin;
3. Inhibition of bacterial nucleic acid synthesis. nafcillin; oxacillin; cloxacillin; dicloxacillin;
flucloxacillin.
4. Inhibition of bacterial protein synthesis.
5. Penicillins active against Pseudomonas:
Apalcillin, carbenicillin; ticarcillin; azlocillin;
1. Inhibititon of Bacterial Cell Wall mezlocillin; piperacillin.
Synthesis
Inhibitors of bacterial cell wall synthesis act on Cephalosporins
the formation of the peptidoglycan layer. Bacteria Cephalosporins are a family of antibiotics and
that lack peptidoglycan, such as mycoplasmas, are their b-lactam structure is very similar to that of
resistant to these agents. the penicillins (Figs 81.1A and B). In place of
The antib iotics which inhibit cell wall 6-aminopenicillanic acid, they have a nucleus of
synthesis are b-Iactam antibiotics (penicillins and 7-aminocephalosporanic acid. They are broad-
cephalosporins), glycopeptides, bacitracin, cyclo spectrum drugs frequently given to patients with
serine, fosfomycin and isoniazid. penicillin allergies.
They are grouped as the first, second, third, and
A. b-Iactam Agents fourth generation cephalosporins. These include
This group includes penicillins, cephalosporins cephalexin and cephradine (first generation),
and other compounds that feature a b-lactam ring cefaclor and cefprozil (second gene ration),
in their structure. All these compounds bind to cefixime and ceftbuten (third generation), and
proteins situated at the cell wall–cell membrane cefepime (fourth generation) (Table 81.2).
interface. These penicillin-binding proteins (PBPs) First-generation cephalosporins are more
are involved in cell wall construction, including effective against Gram-positive than gram-negative
the cross-linking of the peptidoglycan strands that
gives the wall its strength. Opening of the b-lactam
ring by hydrolytic enzymes, collectively called
b-lactamases, abolishes antibacterial activity. Many
such enzymes are found in bacteria.
Penicillins
Penicillins are a group of antimicrobial substances,
all of which possess a common chemical nucleus
(6-aminopenicillanic acid) which contains a
b-lactam ring essential to their biologic activity. A
side chain is attached to the b-lactam ring which
determines many of the antibacterial and pharma
cological characteristics of a particular type of
penicillin.
All b-lactam antibiotics inhibit formation of
bacterial cell wall. They particularly block the
final transpeptidation reaction in the synthesis of
cell wall peptidoglycan and also activate autolytic
enzymes in the cell wall.
Penicillins can be divided into several groups:
1. Those with highest activity against gram-posi
tive organisms but susceptible to hydrolysis by Figs 81.1A and B: The β-lactam ring of penicillins and
b-lactamases: cephalosporins the core chemical structure of (A) a penicillin
(B) a cephalosporin. The β-lactam rings are marked by an
• Penicillin-G orange circle. The R groups vary among different penicillins
• Penicillin-V and cephalosporins
pathogens. Second generation drugs act against subunit of DNA gyrase, an enzyme that engineers
many gram-negative as well as Gram-positive the breaking and rejoining of super coiled DNA.
pathogens. Third-generation drugs are particularly Nalidixic acid and its early congeners are narrow-
effective against gram-negative pathogens, and spectrum agents active only against gram-negative
often also reach the central nervous system. bacteria.
Newer quinolones like ciprofloxacin, norflox
Other b-Lactam Antibiotics acin, ofloxacin, pefloxacin and lomefloxacin are
Two other groups of β-lactam drugs, carbapenems broad spectrum quinolones. These have been
and monobactams, are very resistant to β-lactamases. successfully used in a wide variety of infections, but
resistance is becoming more prevalent.
Carbapenems
The carbapenems are effective against a wide range Rifamycins
of gram-negative and gram-positive bacteria. Two Rifamycins inhibit bacterial growth by binding
types are available, imipenem and meropenem. strongly to the DNA-dependent RNA polymerase of
Glycopeptides bacteria thus inhibiting transcription of RNA from
DNA. This group of antibiotics is characterized
Two glycopeptides, vancomycin and teicoplanin,
by excellent activity against mycobacteria.
are in clinical use. Their chief importance resides
Staphylococci in particular are exquisitely sensitive.
in their action against gram-positive cocci with
Rifampicin and rifabutin are most widely used.
multiple resistance to other drugs. They are mainly
used in serious infections with staphylococci and
Nitroimidazoles
enterococci that are resistant to other drugs.
Azole derivatives have wide-ranging antimicrobial
Other Inhibitors of Bacterial Cell Wall activity against fungi, protozoa, helminths, as well
Synthesis as bacteria. Those that exhibit antibacterial activity
These include bacitracin, cycloserine, fosfomycin are 5-nitroimidazoles which are active only against
and isoniazid. anaerobic bacteria and anaerobic protozoa. The
representative of the group most commonly used
2. Inhibition of Bacterial Cytoplasmic clinically is metronidazole, but similar derivatives
Membrane Function include tinidazole, ornidazole and nimorazole.
They are primarily antiprotozoal agents, but they
Only polymyxins have been regularly used
exhibit potent activity against anaerobic bacteria.
systemically among membrane active agents used
in human medicine. Two members of the family Nitrofurans
are in therapeutic use: polymyxin B and colistin
(polymyxin E). Various nitrofuran derivatives are in use around
They exhibit potent antipseudomonal activity, the world as antibacterial agents. These include
but toxicity has limited their usefulness, except in nitrofurantoin and furazolidone.
topical preparations and bowel decontamination Nitrofurantoin: It is bactericidal to most urinary
regimens. pathogens at concentrations achievable in urine.
Furazolidone: Furazolidone is used in enteric
3. Inhibitors of Nucleic Acid Synthesis
infections. The mode of action of nitrofurans
Quinolones has not been elucidated, but it is probable that
The quinolones are synthetic drugs that contain a reduced metabolite acts on DNA in a manner
the 4-quinolone ring. Quinolones act on the α analogous to that of the nitroimidazoles
www.ebook3000.com
Chap-81.indd 567 15-03-2016 11:28:10
568 | Section 6: Miscellaneous
Novobiocin Macrolides
Novobiocin acts on the β subunit of bacterial DNA The macrolides reversibly bind to the 50S ribosomal
gyrase. It is quite active against staphylococci and subunit and prevent the continuation of protein
streptococci, but is no longer favored because of synthesis. Macrolides include erythromycin,
problems of resistance and toxicity. Staphylococcus azithromycin, clarithromycin, dirithromycin and
saprophyticus is novobiocin resistant. spiramycin.
Macrolides as a group are effective against a
4. Inhibition of Bacterial Protein Synthesis variety of bacteria, including many gram-positive
organisms as well as the most common causes of
The major classes of antibiotics that inhibit atypical pneumonia (walking pneumonia). They
protein synthesis are the aminoglycosides, the often serve as the drug of choice for patients who
tetracyclines, and the macrolides. Others include are allergic to penicillin.
the lincosamides and chloramphenicol. Of these,
only the aminoglycosides are bactericidal; the Lincosamides
others are all bacteriostatic. Two classes of drugs
Lincosamides bind to the 50S ribosomal subunit
that have recently been approved for use are
and resemble macrolides in binding site,
the oxazolidinones and the streptogramins. A
antibacterial activity, and mode of action.
synergistic combination of two streptogramins is
bactericidal against some organisms.
Fusidic Acid
It blocks factor G, which is involved in peptide
Aminoglycosides
elongation. It has excellent activity against
The aminoglycosides irreversibly bind to the staphylococci, good activity against Corynebacterium
30S ribosomal subunit, causing it to distort and diphtheriae and modest activity against streptococci,
malfunction. This blocks the initiation of translation Gram-negative anaerobes, Nocardia asteroides, and
and causes misreading of mRNA by ribosomes that Mycobacterium tuberculosis.
have already passed the initiation step. Examples of
aminoglycosides include streptomycin, kanamycin, Streptogramins
neomycin, gentamicin, tobramycin, amikacin, etc.
Derivatives suitable for parenteral administration,
Use: They are bactericidal compounds, and some, quinupristin and dalfopristin, have been developed
notably gentamicin and tobramycin, exhibit good as a combination product. The combination is
activity against Pseudomonas aeruginosa. The effective against a variety of gram-positive bacteria,
group also has in common a tendency to damage including some of those that are resistant to
the eighth cranial nerve (ototoxicity) and the b-lactam drugs and vancomycin.
kidney (nephrotoxicity).
Mupirocin
Chloramphenicol This is an antibiotic, produced by Pseudomonas
fluorescens. Its useful activity is restricted to
Chloramphenicol binds to the 50S ribosomal
staphylococci and streptococci and used only in
subunit. It is mainly bacteriostatic.
topical preparations.
Use: Use of chloramphenicol has been limited to
typhoid fever, meningitis and a few other clinical 5. Metabolic Antagonism
indications because of the occurrence of a rare but Antibacterial medications that inhibit metabolic
fatal side-effect, aplastic anemia. pathways.
ANTIBIOTIC RESISTANCE
Understanding the mechanisms and the spread
of antimicrobial resistance is an important step in
curtailing the problem.
www.ebook3000.com
Chap-81.indd 569 15-03-2016 11:28:11
570 | Section 6: Miscellaneous
to each drug is extremely low. If any organism
Antimicrobial resistance can be due to either
spontaneously develops resistance to one drug, spontaneous mutation, or acquisition of new genes
the other drug will still kill it. The most common mechanism of transfer of resis
tance is through the conjugative transfer of R
2. Gene Transfer plasmids (resistance plasmids).
i. Conjugation: R plasmids frequently carry
several different resistance genes, each one
mediating resistance to a specific antimicrobial
IMPORTANT QUESTIONS
drug. Thus, when an organism acquires an R 1. Define the terms antimicrobial agent, chemothera
plasmid, it acquires resistance to several different peutic agent and antibiotic. Describe the various
medications simultaneously. mechanisms of action of antibiotics with examples.
ii. Transduction: Acquisition of resistance by 2. Describe the structure and functions of bacterial
cell wall. Name various antibiotics which affect cell
transduction is common in staphylococci.
wall synthesis.
The penicillin plasmids (carrying gene for 3. Write short notes on:
β-lactamase production) enclosed in a a. Antibiotics inhibiting bacterial cytoplasmic
bacteriophage is transferred from a penicillin- membrane function.
resistant staphylococcus to a susceptible b. Antibiotics inhibiting bacterial nucleic acid
staphylococcus. synthesis.
iii. Transformation: Resistance transfer can c. Antibiotics that act as metabolic antagonists.
be demonstr ated experimentally but its 4. Discuss genetic basis of drug resistance in bacteria.
significance is not known.
iv. Transposons: Many of the resistance genes MULTIPLE CHOICE QUESTIONS (MCQs)
on R plasmids are carried on transposons that 1. Which of the following antibiotics act/s by
can move from a plasmid to the chromosome, inhibiting cell wall synthesis?
from one plasmid to another, or from the a. Penicillins b. Vancomycin
chromosome to a plasmid. Thus, if one organism c. Bacitracin d. All of the above
has two different plasmids, an antibiotic- 2. Which of the following antibiotics acts by inhibiting
resistance gene can move from one to the other. cytoplasmic membrane:
a. Polymyxin b Thyrocidine
Key Points c. Gramicidin d. All of the above
Antimicrobial agent is a chemical substance 3. Which antibiotic/s act/s by inhibiting bacterial
inhibiting the growth or causing the death of a mi nucleic acid synthesis?
croorganism a. Quinolones b. Novobiocin
Antibiotic as originally defined was a chemical c. Nitrofurans d. All of the above
substance produced by various species of 4. Which of the following antibiotics act/s by
microorganisms that was capable of inhibiting the inhibiting bacterial protein synthesis?
growth or causing death of other microorganisms a. Aminogycosides b. Tetracycline
in low concentration c. Macrolides d. All of the above
Chemotherapeutic agents are the chemical 5. Which antibiotic/s act/s as metabolic antagonists
substances used to kill or inhibit the growth of
for their action?
microorganisms already established in the tissues
a. Sulphones b. Trimethoprim
of the body
c. Isoniazid d. All of the above
Mechanisms of action’ of antibacterial agents can
be: 1. Inhibition of bacterial cell wall synthesis; 6. What is the genetic basis of drug resistance in
2. Inhibition of bacterial cytoplasmic membrane Mycobacterium tuberculosis?
function; 3. Inhibition of bacterial nucleic acid a. Transformation b. Transduction
synthesis; 4. Inhibition of bacterial protein synthesis c. Mutation d. Conjugation
and 5. Metabolic Antagonism 7. Staphylococcus aureus acquire drug resistance by:
Mechanisms of drug resistance are: 1. Drug- a. Transformation b. Transduction
inactivating enzymes; 2. Alteration in the target c. Mutation d. Conjugation
molecule; 3. Decreased uptake of the drug; 4.
Increased elimination of the drug ANSWERS (MCQs)
1. d; 2. d; 3. d; 4. d; 5. d; 6. c; 7. b
Immunoprophylaxis
Learning Objectives
After reading and studying this chapter, you should ∙∙ Explain immunization schedule
be able to: ∙∙ Discuss national immunization schedule
∙∙ List of immunizing agents ∙∙ Describe passive immunization.
∙∙ Describe the following: live attenuated vaccines;
killed vaccines; toxoids
www.ebook3000.com
572 | Section 6: Miscellaneous
vaccine from the polysaccharide antigen of the based on active immunization, e.g. polio,
cell wall, the pneumococcal vaccine from the tetanus, diphtheria and measles. Vaccination
polysaccharide contained in the capsule of the against these diseases is given as a routine
organism and hepatitis B polypeptide vaccines. during infancy and early childhood, with
periodic boosters to maintain adequate levels
5. Mixed or Combined Vaccine of immunity.
If more than one kind of immunizing agent is 2. Immunization of individuals or selected
included in the vaccine, it is called a mixed or groups: There are immunizations against certain
combined vaccine. The aim of combined vaccines diseases which are offered to high-risk groups or
is to simplify administration, reduce costs and restricted to definite geographic areas where the
minimize the number of contacts of the patient disease is endemic or a public health problem
with the health system. The following are some of (e.g. yellow fever).
the well-known combinations:
∙∙ DPT (Diphtheria-pertussis-tetanus) Immunization Schedules
∙∙ DT (Diphtheria-tetanus) National Immunization Schedule
The National Immunization Schedule is given in
∙∙ DP (Diphtheria-pertussis) .
Table 82.1. The first visit may be made when the
∙∙ DPT and typhoid vaccine infant is 6 weeks old; the second and third visits,
∙∙ MMR (Measles, mumps and rubella) at intervals of 1–2 months. Oral polio vaccine may
∙∙ DPTP (DPT plus inactivated polio) be given concurrently with OPT. BCG can be given
Plain vaccines are less efficacious than adjuvant with any of the three doses but the site for the
vaccines. Freeze-dried vaccines (e.g. BCG, yellow injection should be different. The schedule also
fever measles) are more stable preparations than covers immunization of women during pregnancy
liquid vaccines. against tetanus.
www.ebook3000.com
574 | Section 6: Miscellaneous
2. An example of killed inactivated vaccine is:
influenza vaccines are the examples of killed inacti
vated vaccines a. MMR vaccine
Toxoids: Tetanus toxoid and diphtheria toxoids are b. Influenza vaccines
two most widely used toxoids for immunization c. Oral polio vaccine
Subunit vaccines: Hepatitis B subunit vaccine d. BCG vaccine
Passive immunization is carried out by administration 3. An example of live attenuated vaccine is:
of human and animal sera a. Hepatitis B vaccine
Combined active and passive immunization is often b. Rocky Mountain spotted fever vaccine
carried out to confer slowly developing immunity
c. Yellow fever vaccine
and immediate passive immunity, respectively,
against certain diseases, such as diphtheria, tetanus,
d. Rabies vaccine
and rabies. 4. Which of the following vaccines is/are subunit
vaccine/s?
a. Hepatitis B vaccine (plasma derived)
Important question b. Vi typhoid fever vaccine
rite short notes on:
W c. Meningococcal vaccine
a. Vaccines d. All of the above
b. Live attenuated vaccines 5. Toxoid is used for active immunization against:
c. Killed vaccines a. Pertussis b. Diphtheria
d. Toxoids c. Typhoid fever d. Tuberculosis
e. National Immunization Schedule 6. Specific immunoglobulins are available for passive
f. WHO EPI Immunization Schedule. immunization against:
a. Tetanus b. Rabies
Multiple choice questions (MCQs) c. Hepatitis B d. All of the above
7. Combined passive and active immunization is
1. Which of the following vaccines is/are killed available against all the diseases except:
vaccines? a. Poliomyelitis b. Tetanus
a. Cholera vaccine c. Rabies d. Diphtheria
b. Pertussis vaccine
c. Japanese encephalitis vaccine Answers (MCQs)
d. All of the above 1. d; 2. b; 3. c; 4. d; 5. b; 6. d; 7. a
8383
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Water-borne pathogens;
be able to: presumptive coliform count; Eijkman test
∙∙ Describe bacteriological examination of water ∙∙ Describe settle plate method and slit sampler
∙∙ Discuss bacteriological examination of milk method for bacteriology of air.
www.ebook3000.com
576 | Section 6: Miscellaneous
Table 83.2 Water-borne diseases BACTERIOLOGICAL EXAMINATION OF
1. Those caused by the presence of an infective agent: WATER
a. Viral: Viral hepatitis A, hepatitis E, poliomyelitis,
The following tests are generally done for routine
rotavirus diarrhea in infants
b. Bacterial: Typhoid and paratyphoid fever, bacteriological analysis of water.
bacillary dysentery, Escherichia coli diarrhea,
cholera a. Multiple Tube Test
c. Protozoal: Amebiasis, giardiasis 1. Total coliform count or presumptive coliform
d. Helminthic: Roundworm, threadworm, hydatid
count.
disease.
e. Leptospiral: Weil’s disease 2. Fecal coliform and confirmed Escherichia coli
2. Those due to the presence of an aquatic host count: Eijkman test.
a. Snail: Schistosomiasis 3. Count of fecal streptococci.
b. Cyclops: Guinea worm, fish tape worm 4. Count of Clostridium perfringens.
www.ebook3000.com
578 | Section 6: Miscellaneous
its feces, or from the environment or from milk i. Methylene Blue Reduction Test
handlers such as dairy workers. It depends on the reduction of methylene blue
by bacteria in milk when incubated at 37°C in
Milk-borne Diseases complete darkness.
Milk is a goodmedium for bacteria and good Procedure: The test is performed by adding 1 ml of
vehicle for many pathogens. Milk-borne diseases standard methylene blue solution to 10 ml of milk
are shown in Table 83.4. in a test tube. The tube is closed with a sterile rubber
stopper, inverted once or twice and incubated in the
Bacteriological Examination of Milk dark at 37°C. The milk is considered satisfactory, if
1. Viable Count it fails to reduce the dye in 30 minutes.
This is estimated by doing plate counts with serial Resazurin test: The Resazurin test is similar but
dilutions of the milk sample. Raw milk always the dye resazurin, on reduction, passes through a
contains bacteria, varying in number from about series of color changes.
500 to several million per ml.
ii. Phosphatase Test
The enzyme phosphatase normally present in milk
2. Test for Coliform Bacilli
is inactivated if pasteurization has been carried
This is tested by inoculating varying dilutions out properly. The test depends upon the ahility of
of milk into three tubes of MacCon key’s fluid the enzyme to liberate p-nitrophenol from diso
medium with Durham tube and noting the dium p-nitrophenyl phosphate and thereby pro
production of acid and gas after incubation at 37°C duce a yellow color that can be quantitated by a
for 48 hours. All coliforms are killed by adequate colorimeter. Residual phosphatase activity indicates
pasteurization and their presence in pasteur that pasteurization has not been adequate.
ized milk indicates faults in pasteurizer or post
pasteurization contamination. iii. Turbidity Test
This is the definitive test for sterilized milk. If milk
3. Chemical Tests has been boiled or heated to the temperature
prescribed for ‘sterilization’, all heat coagulable
Table 83.4 Milk-borne diseases proteins are precipitated. If ammonium sulfate
1. Infections of animals that can be transmitted to is then added to the milk, no turbidity results.
man Absence of turbidity indicates that the milk has
Primary importance been heated to at least 100°C for at least 5 min.
• Tuberculosis
• Brucellosis 4. Examination for Specific Pathogens
• Streptococcal infections
a. Tubercle bacillus
• Staphylococcal enterotoxin poisoning
Centrifuge 100 ml of milk at 3,000 rpm for 30
• Salmonellosis
minutes and inoculate two guinea pigs. Keep the
• Q fever
Lesser importance animals under observation for signs of tuberculous
• Cowpox lesions. Tubercle bacilli may also be isolated in
• Foot and mouth disease culture. Microscopic examination for tubercle
• Anthrax bacilli is unsatisfactory.
• Leptospirosis b. Brucella
• Tick-borne encephalitis—transmitted through goat Brucella may be isolated by inoculating cream
milk
from the milk sample on serum dextrose agar or
2. Infections primary to man that can be transmitted
by injecting centrifuged deposit of the milk sample
through milk
intramuscularly into guinea pigs. The animals are
• Typhoid and paratyphoid fevers
sacrificed after six weeks and the serum tested for
• Shigellosis
agglutinins and the spleen inoculated in culture
• Cholera
media for brucellae. Brucellosis in animals can be
• Enteropathogenic Escherichi coli (EEC)
detected also by demonstrating the antibodies in
• Nondiarrheal diseases
milk, by the milk-ring or the whey agglutination tests.
a. Streptococcal infections
b. Staphylococcal food poisoning
c. Diphtheria EXAMINATION OF AIR
d. Tuberculosis
A person inhales over 15 cubic meters of air in the
e. Enteroviruses
course of a day. Hence the bacterial content of the
f. Viral hepatitis
air one breathes is important, particularly when it
Chapter 83: Bacteriology of Water, Milk and Air | 579
contains pathogens. The bacterial content of air Important question
depends on the location, i.e. whether it is outdoor
rite short notes on:
W
air or indoor air.
a. Bacterial flora in water
Essential conditions for bacteriological examin b. Water-borne pathogens
ation of air c. Bacteriological analysis of water
1. Surgical operation theater. d. Bacteriology of milk
e. Presumptive coliform count
2. In hospital wards in which there is an outbreak
f. Eijkman test
of cross-infection.
g. Bacteriology of air.
3. Premises where food articles are prepared and
packed.
Multiple choice questions (MCQs)
4. Premises where pharmaceutical materials are
prepared. 1. Which of the following organisms can serve as
indicators of fecal pollution of drinking water?
a. E. coli
Measurement of Air Contamination b. Bacillus subtilis
The methods for bacteriological examination of air c. Corynebacterium diphtheriae
d. All of the, above
are of two types.
2. All the following bacteria are indicators of bacterial
contamination of water except:
1. Settle Plate Method a. Escherichia coli
Petri dishes containing an agar medium of known b. Enterococcus species
c. Staphylococcus aureus
surface area are left open for a measured period d. Clostridium perfringens
of time. Large bacteria-carrying dust particles
3. Drinking water is considered satisfactory if the
settle on to the medium. The plates are incubated presumptive coliform count of water is:
at 37°C for 24 hours and a count of the colonies a. 0/100 mL b. 1–3/100 mL
formed shows the number of settled pastulle. Blood c. 4–10/100 mL d. > 10/100 mL
agar medium may be used for the count of the 4. The test done for the routine bacteriological
pathogenic, commensal and saprophytic bacteria analysis of water is:
in the air. The method is specially used for testing A. Slit sampler method b. Turbidity test
the air in surgical theatres and hospital wards. C. Phosphatase test d. Eijkman test
5. All the following bacteria can be normally present
2. Slit Sampler Method in the milk except:
a. Streptococcus lactis
By this method, the number of bacteria in a meas b. Enterococcus species
ured volume of air is determined. c. Lactobacillus bulgaricus
d. Streptococcus cremoris
Procedure: In this, a known volume of air is 6. All the following tests are used for detection of
directed onto a plate through a slit 0.33 mm wide bacterialcontamination of milk except:
and 27.5 mm long with vertical parallel sides about a. Eijkman test
3 mm deep. At the correct negative pressure, air will b. Methylene blue reduction test
enter through a slit of these dimensions. The culture c. Phosphatase test
medium is incubated and colonies counted which d. Turbidity test
gives the number of bacteria present in the air. 7. The method used for estimation of number of
bacteria in a measured volume of air is:
a. Settle plate method
Key Points b. Phosphatase test
c. Turbidity method
Methods of water analysis include presumptive
d. Methylene blue reduction test method
coliform count, differential coliform count,
membrane filtration method, and detection of fecal 8. In operation theaters bacterial count of air should
streptococci and C. perfringens not exceed:
Pathogenic microorganisms in milk can transmit
a. 50 per cubic foot
milk-borne dise ases such as tuberculosis, and b. 10 per cubic foot
typhoid fever c. 5 per cubic foot
d. 1 per cubic foot
Bacteriological examination of milk can be carried
out by colony counts, coliform counts; chemical tests 9. What is the acceptable limit of bacterial count in
such as methylene blue reduction test, phosphatase air in operation theatre for neurosurgery?
test, and turbidity test; and detection of specific a. 50 per cubic feet b. 10 per cubic feet
pathogens c. 4 per cubic feet d. 1 per cubic feet
Settle plate method and slit sampler methods are Answers (MCQs)
used for bacteriological examination of air. 1. a; 2. c; 3. b; 4. d; 5. b; 6. a; 7. a; 8. b; 9. d
www.ebook3000.com
84
Chapter
LEARNING OBJECTIVES
After reading and studying this chapter, you should waste; Waste egregation; Waste treatment and
be able to: disposal
∙∙ Describe universal precautions ∙∙ Describe treatment and technologies for health
∙∙ Describe the following: Categories of biomedical care waste.
Let the wastes of “the sick” not contaminate the lives of the healthy”
Table 84.1 Color coding and type of container for disposal of biomedical wastes
Color coding Type of container Waste category Treatment option as per
schedule 1
Yellow Plastic bag 1, 2, and 5, Cat. 6 Incineration/deep burial
Red Disinfected container/plastic 3, 4, 7 Autoclaving/microwaving/
bag chemical treatment
Blue/white translucent Plastic bag/puncture proof Autoclaving/microwaving/
container chemical treatment and
destruction/shredding
Black Plastic bag Cat. 5 and Cat. 9 and Cat. Disposal in secured landfill
10 (solid)
www.ebook3000.com
Chap-84.indd 581 15-03-2016 11:28:48
582 | Section 6: Miscellaneous
Types of incinerators chlorine required is 0.1% and for dirty conditions
Three basic kinds of incineration technology are of available chlorine should be 1.0%.
interest for treating health-care waste:
i. Double-chamber pyrolytic incinerators which 4. Wet and Dry Thermal Treatment
may be especially designed to burn infectious Wet thermal treatment
healthcare waste. Wet thermal treatment or steam disinfection is
ii. Single-chamber furnaces with static grate, based on exposure of shredded infectious waste
which should be used only if pyrolytic to high temperature, high pressure steam, and is
incinerators are not affordable, and similar to the autoclave sterilization process.
iii. Rotary kilns operating at high temperatures, Screw-feed technology
capable of causing decomposition of genotoxic Screw-feed technology is the basis of a non-burn,
substances and heat-resistant chemicals. dry thermal disinfection process in which waste is
Double chambered incineration shredded and heated in a rotating auger.
An incinerator should consist of 2 chamb ers,
5. Microwave Irradiation
primary and secondary. Waste is burnt in one
chamber (primary chamber) at 800°C. Combustion Most microorganisms are destroyed by the
of gases emitted from the first chamber, occurs in action of microwave of a frequency of about 2450
the second or secondary chamber which has a MHz and a wave length of 12.24 cm. The water
high temperature of 1000°C. The negative pressure contained within the waste is rapidly heated by
is maintained inside the incinerator by the system, the microwaves and the infectious components
thereby forcing the end-gases out of the chimney. are destroyed by heat conduction. This cannot be
used for animal or human body parts, metal items
2. Autoclaving or toxic or radioactive material.
Autoclaving, at 121°C for 60 minutes, is an effective 6. Inertization
method for treating infectious waste before disposal. The process of “inertization” involves mixing waste
A separate autoclave dedicated for waste treatment with cement and other substances before disposal,
should be used. Waste arising from microbiology in order to minimize the risk of toxic substances
and biotechnology laboratories including cultures contained in the wastes migrating into the surface
and stocks must be autoclaved before disposal by water or ground water.
incineration. Plastic disposables including blood
bags, urine bags, etc. should be autoclaved followed DISPOSAL
by shredding. It is not recommended for pathological
Land filling, deep burial and sewage are used for
waste. Autoclaved material is typically land-filled,
disposal. Infectious waste after treatment can be
therefore, it has a large strain on land fill capacity.
disposed of by land-filling or deep burial. Liquid
Types of autoclaves: There are two kinds of waste can be disposed in sewage drains. Besides
autoclaves: treatment, incineration is also a method of disposal.
i. Prevacuum type: In the prevacuum type, Treatment methods used for different types of
steam is created outside the chamber loaded infectious wastes are shown in Table 81.1.
with waste. Air in the chamber is then gradually
removed and steam is injected in. This type of Waste Management Program
autoclave eliminate ‘cold spots’ and ‘air pockets’ All laboratories should develop waste management
(where the steam is unable to penetrate) by program according to the specific needs of the
creating this vacuum. This ensures quicker individual laboratory. The policies and procedures
heating. A temperature of 121°C and pressure should be incorporated in the laboratory’s
of 15 pounds per square inch is used. operating manuals. Emphasis should be on waste
ii. Gravity autoclave: For gravity autoclave, minimization (by reducing waste, reuse and
the waste material should be subjected to recycling), proper segregation, and health and
autoclave residence time of not less than 60 safety of the workers. All personnel generating,
minutes, while in a vacuum autoclave time collecting, transporting and storing infectious
period should not be less than 45 minutes. waste must be trained under the programme.
Biological (Bacillus stearothermophilus
spores) or chemical indicators (strips/tapes) Key Points
should be used for validation test of autoclave. Biomedical waste (BMW) means any waste, which
is generated during the diagnosis, treatment or
3. Chemical Disinfection immunization of human beings or animals or in
The most economical and effective disinfectant research activities pertaining there to or in the
production or testing of biologicals
is hypochlorite. For clean conditions available
www.ebook3000.com
Chap-84.indd 583 15-03-2016 11:28:48
85
Chapter
Learning Objectives
Introduction ∙∙ Cholera
∙∙ Poliomyelitis
To maintain an active infectious disease in a human ∙∙ Hepatitis A virus infection
population, the pathogen must be transmitted ∙∙ Food poisoning
from one host or source to another. Transmission ∙∙ Intestinal parasitic infestation.
is the third link in the infectious disease cycle and
occurs by four main routes: airborne, contact, B. Diseases Transmitted by Blood
vehicle, and vector-borne.
1. Viruses
Vehicles and vectors ∙∙ Hepatitis B
∙∙ Human immunodeficiency viruses (HIV)
The agents of transmission that bring the ∙∙ Human T cell lymphotropic viruses
microorganism from the reservoir to the host may ∙∙ Infectious mononucleosis
be a living entity, in which case they are called ∙∙ Cytomegalovirus
vectors, or they may be a nonliving entity referred
to as a vehicle or fomite. 2. Bacteria
Modes of transmission ∙∙ Syphilis
A. Direct: Transmitted by direct contact between ∙∙ Brucellosis
reservoir and host.
B. Indirect: Transmitted to host (human host) 3. Parasites
via intervening agent(s) such as vectors ∙∙ Malaria
(animals, insects, other humans and vehicles ∙∙ Trypansomiasis (Chaga’s disease)
(water, food, air, medical devices, various other ∙∙ Trypanosoma cruzi.
inanimate objects).
2. Vector-borne
1. Vehicle-borne In infectious disease epidemiology, vector is
Vehicle-borne transmission implies transmission defined as an arthropod or any living carrier (e.g.
of the infectious agent through the agency of water, snail) that transports an infectious agent to a
food, ice, blood, serum plasma or other biological susceptible individual. Transmission by a vector
products such as tissues and organs. Of these may be mechanical or biological. In the latter case,
water and food are the most frequent vehicles of the disease agent passes through a developmental
transmission, because they are used by everyone. cycle multiplication in the vector.
www.ebook3000.com
86
Chapter
Emerging and Re-emerging
Infectious Diseases
Learning Objectives
After reading and studying this chapter, you should be able to:
∙∙ Describe the following: Emerging infectious diseases; re-emerging infectious diseases.
www.ebook3000.com
588 | Section 6: Miscellaneous
Table 86.2 Re-emerging infectious diseases and vaccines; and above all by strengthening
epidemiological surveillance and drug-resistance
Disease Causative agent
surveillance mechanisms and procedures with
A. Bacterial appropriate laboratory support for early detection,
tuberculosis Multidrug resistant
confirmation and communication. The laboratory
Mycobacterium tuberculosis
technologist must relearn information once
Typhoid fever Multidrug resistant
thought to be out of date. Media selection,
Salmonella Typhi
identification techniques, and safety precautions
Leptospirosis Leptospira interrogans must all be re-examined and implemented.
Melioidosis Burkholderia Interaction with the infection control program
(Pseudomonas) strengthens the establishment of prevention and
pseudomallei
control strategies.
Anthrax Bacillus anthracis The category of diseases—“new diseases—
Plague Yersinia pestis new problems”—such as Ebola and other viral
B. Parasitic hemorrhagic fevers, is probably the most frightening.
Malaria Drug resistant Much of this already applies to HIV/AIDS, one of the
Plasmodium falciparum most serious diseases to emerge in recent decades.
Leishmaniasis Leishmania donovani
Lymphatic filariasis Wuchereria bancrofti, Key Points
Brugia malayi and B. timori
Emerging infectious diseases are those whose
incidence in humans has increased during the last
international challenge. An outbreak anywhere two decades or which threaten to increase in the
must now be seen as a threat to virtually all near future
Re-emerging, or resurging, diseases are those that
countries, especially those that serve as m have been around for decades or centuries, but have
come back in a different form or a different location.
Antimicrobial Resistance Resistance by disease-causing organisms to
Resistance by disease-causing organisms to antimicrobial drugs and other agents is a major
antimicrobial drugs and other agents is a major public health problem worldwide.
public health problem worldw ide. It is having
a deadly impact on the control of diseases Important Questions
such as tuberculosis, malaria, enterococci,
staphylococci, streptococci, pneumococci and 1. Write an essay on emerging and re-emerging
infections.
Haemophilus influenzae, Neisseria gonorrhoeae,
2. Discuss various factors responsible for the emer
Shigella dysenteriae, Salmonella Typhi. gence and re-emergence of infectious diseases.
Factors responsible for emergence and
re-emergence of infectious diseases Multiple Choice Questions (MCQs)
1. Economic development and land use, 1. Factors responsible for the emergence and
unplanned and under-planned urbanization. re-emergence of communicable diseases is/are:
2. Overcrowding and rapid population growth. a. Unhygienic living conditions
b. Encroachment by humans to areas so far unin-
3. Poor sanitation.
habited leading to ecological changes.
4. Inadequate public health infrastructure. c. Development of insecticide resistance in vec-
5. Resistance to antibiotics. tors.
6. Increased exposure of humans to disease d. All of the above
vectors and reservoirs of infection in nature, 2. The problem of drug resistance has assumed
and alarming proportions in:
a. Tubercle bacilli
7. Rapid and intense international travel.
b. Pseudomonas aeruginosa
c. Klebsiella pneumoniae
Responding to Epidemics d. All of the above
The strategy for controlling re-emerging diseases is 3. Following are the examples of re-emerging
through available cost-effective interventions such infections except:
as early diagnosis and prompt treatment, vector a. Plague
control measures and the prevention of epidemics, b. Malaria
for malaria ; and DOTS—directly observed c. AIDS
treatment, short-course—for tuberculosis; by d. Ebala hemorrhagic fever
launching research initiatives for treatment Answers (MCQs)
regimens and improved diagnostics, drugs 1. d; 2. d; 3. c
Section 7: Diagnostic Medical Microbiology
87
Chapter
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Nucleic acid probes,
be able to: polymerase chain reaction (PCR); Transcription
∙∙ Describe molecular methods for microbial mediated amplification ( TMA); Nucleic acid
identification sequence based amplification (NASBA); Ligase
chain reaction (LCR).
www.ebook3000.com
590 | Section 7: Diagnostic Medical Microbiology
i. Reverse-transcriptase PCR (RT-PCR): B o t h T M A a n d NA S BA a re e x a mp l e s
Reverse transcriptase PCR (RT-PCR) of transcription-mediated amplification.
amplifies an RNA target. The unique step These isothermal assays use three enzymes:
to this procedure is the use of the enzyme transcriptase (RT), RNAase H, and T7 DNA
reverse transcriptase that directs synthesis dependent RNA polymerase. Like TMA, it is also
of DNA from the viral RNA template. Once an isothermal RNA amplification method. The
the DNA has been produced, relatively rou method is similar to TMA. RNA target is reverse
tine PCR technology is applied to obtain transcribed into cDNA and then RNA copies are
amplification. synthesized with the help of RNA polymerase.
ii. Nested PCR: Nested PCR involves the It also does not require thermal cycler. NASBA
based kits for detection and quantitation of
sequential use of two primer sets. The first
HIV-1 RNA and CMV RNA are available.
set is used to amplify a target sequence.
4. Ligase chain reaction (LCR): Ligase chain
The amplicon obtained is then used as the
reaction (LCR) is an amplification of probe
target sequence for a second amplification
nucleic acid rather than target nucleic acid. By
using primers internal to those of the first this approach an amplified probe is the final
amplicon. Essentially this is an amplification reaction product to be detected, while the target
of a sequence internal to an amplicon. sequence is neither amplified nor incorporated
The advantage of this approach is extreme into this product.
sensitivity and confirmed specificity The LCR uses two pairs of probes that span
without the need for using probes. the target sequence of interest, Once annealed
iii. Multiplex PCR: Multiplex PCR is a method to the target sequence, a space remains between
by which more than one primer pair is the probes that is enzymatically closed using a
included in the PCR mixture. This will help ligase (i.e. a ligation reaction). On heating, the
in amplification of more than one target joined probes are released as a single strand that
sequence in a clinical specimen. The control is complementary to the target nucleic acid. These
amplicon should always be detectable after newly synthesized strands are then used as the
PCR. Mutliplex PCRs are usually less sensi- template for subsequent cycles of probe annealing
tive than PCRs with single set of primers. and ligations. Through the process, probe DNA is
iv. Arbitrary primed PCR: Arbitrary primed amplified to a level readily detectable using assays
PCR uses short primers that are not similar to those described for the biotin-avidin
specifically complementary to a particular system. Like PCR, LCR also requires thermal cycler.
sequence of a target DNA. By comparing frag The LCR based amplification has been used
to detect Chlamydia trachomatis and Neisseria
ment migration patterns following agarose
gonorrhoeae.
gel electrophoresis, strains or isolates can be
judged to be the same, similar, or unrelated. C. Sequencing and Enzymatic Digestion
v. Real-time PCR: Real-time PCR combines
of Nucleic Acids
rapid thermocycling with the ability to detect
target by fluorescently labeled probes as the 1. Nucleic acid sequences.
hybrids are formed, i.e. in real time. This 2. High density DNA probes.
3. Enzyme digestion and electrophoresis of
technology allows for high throughput of
nucleic acids.
samples, multiplexing reactions, quantitation
of target, and on-line monitoring. Applications of molecular
2. Transcription mediated amplification
methods in clinical laboratory
(TMA): Transcription mediated amplification
(TMA) is an isothermal RNA amplification Molecular methods have a significant role in
method and use three enzymes; reverse the following situations in clinical microbiology
transcriptase (RT), RNAase H, and T7 DNA laboratory.
dependent RNA polymerase. RNA target is 1. Detection of uncultivable growing micro
reverse transcribed into cDNA and then RNA organisms.
copies are synthesized with the help of RNA 2. Role in clinical virology.
polymerase. A 109 fold amplification of the 3. Disease prognosis.
target RNA can be achieved in about 2 hours. 4. Response to treatment.
TMA based assays are available for detection of
M. tuberculosis, C. trachomatis, N. gonorrhoeae, 1. Detection of Uncultivable and Slow
HCV and HIV-l. Growing Microorganisms
3. Nucleic acid sequence-based amplification Molecular methods have been used to detect
(NASBA) or self-sustained sequence replicat previously unknown agents directly in clinical
ions (3SR). specimens by using broad-range primers for a
Chapter 87: Molecular Detection of Microorganisms | 591
number of microorganisms. HCV, Sin nombre virus genes, which mediate vanc omycin resistance
and Human herpes virus 8 (HHV-8), Bartonella among enterococci, and the mec gene, which
henselae are some examples of human pathogens encodes resistance among staphylococci and
first identified from clinical specimens using rifampicin resistance in Mycobacterium tuberculosis.
molecular methods. Molecular techniques have a significant role
These methods are also useful for fastidious in predicting and monitoring patient responses to
microorganisms which may die in transit or may antiviral therapy. HIV-1 viral load assays have been
be overgrown by contaminants when cultured. N. developed to monitor the response of antiretroviral
gonorrhoeae is one such example whose nucleic therapy. Viral load assays have also been used in
acid can be detected under circumstances in which monitoring the response to therapy in patients who
it cannot be cultured. The use of improper collection, are chronically infected with HBV and HCV.
inappropriate transport conditions or delay in Molecular methods can be used to detect drug
transport can reduce the viability of the organism but resistance mutations in RT and protease genes
do not affect the nucleic acid detection. of HIV-1. These mutations lead to lower levels of
These can detect and identify organisms sensitivity to antiretroviral drugs and are important
that cannot be grown in culture or are extremely causes of treatment failure. This helps to determine
difficult to grow (e.g. hepatitis B virus and the an appropriate treatment in patients who do not
agent of Whipple’s disease) and also more rapid respond to therapy.
detection and identification of organisms that grow
slowly (e.g. mycobacteria, certain fungi). Key Points
Molecular methods are classified into three
2. Role in Clinical Virology categories: A. Hybridization; B. Amplification; C.
Sequencing and enzymatic digestion of nucleic acids
Molecular methods are limited to replace culture Amplified methods: 1. Polymerase chain reaction
for detection of bacteria in routine practice (PCR); 2. Transcription mediated amplification (TMA);
because of need to isolate the organisms for 3. Nucleic acid sequence amplification (NASBA); 4.
antibiotic sensitivity testing. The culture can Ligase chain reaction (LCR).
Molecular methods have a significant role in 1.
actually be replaced by these methods only in
Detection of uncultivable growing microorganisms;
those microorganisms which have predictable 2. Role in clinical virology; 3. Disease prognosis; 4.
antibiotic susceptibility, and consequently, routine Response to treatment
susceptibility testing is not performed.
Molecular approaches are often faster, more
sensitive, and more cost-effective than the Important Question
conventional approaches in clinical virology. rite short notes on:
W
Enteroviral meningitis, HSV encephalitis and CMV a. Detection of microorganisms by molecular methods
infections in immunocompromised patients are b. Nucleic acid probes
examples for which nucleic acid-based tests are c. Polymerase chain reaction (PCR) in diagnosis of
infectious agents.
relevant and cost effective for diagnosis.
d. RT-PCR
e. Applications of molecular methods in clinical
3. Disease Prognosis microbiology
Molecular methods are able to quantitate infectious
agent burden directly in patient specimens, an Multiple choice questions (MCQs)
application that has particular importance for 1. DNA probes can be applied for:
managing human immunodeficiency virus (HIV a. Detection of microbes in specimens
infections). Thus, it provides important information b. Identification of resistant markers
which may predict disease progression. c. Identification of culture isolates
Molecular methods can be used for subtyping d. All of the above
of certain viruses which may provide information 2. All of the following molecular methods is/are
about the severity of infection. HPV causes amplified methods for detection of microorganisms
except:
dysplasia, neoplasia and carcinoma of cervix in
a. Polymerase chain reaction (PCR)
women. HPV types 16 and 18 are associated with a b. Ligase chain reaction (LCR)
high risk of progression to neoplasia, whereas HPV c. RT-PCR
types 6 and 11 have a low risk. d. ELISA
3. How many pairs of amplification primers are used
4. Response toTreatment and Drug Resistance in nested PCR?
Molecular methods have been developed to detect a. One b. Two
the genes responsible for drug resistance that c. Three d. Five
may not always readily be detected by phenotypic Answers (MCQs)
methods. Examples include detection of the van 1. d; 2. d; 3. b
www.ebook3000.com
88
Chapter
Staining Methods
Learning Objectives
After reading and studying this chapter, you should ∙∙ Describe the following: Simple stains; Differential
be able to: stains; Gram stain; Acid fast stain (Ziehl–Neelsen
∙∙ Describe common staining techniques staining of acidfast bacilli); Albert’s stain.
www.ebook3000.com
594 | Section 7: Diagnostic Medical Microbiology
Procedure-See chapter 89 page 598. the stain carbol fuchsin to build up the diameters
of the flagella until they become visible under the
Fluorochrome Staining for
light microscope.
Acid Fast Bacteria
A stain containing the fluorescent dye, auramine Staining of Volutin-containing Organisms
O, is substituted for the hot carbol fuchsin in
Certain bacilli possess volutin granules in the
the Ziehl–Neelsen method. Tubercle bacilli
protoplasm which aid in the identification of
are rendered fluorescent and becomes easy to
diphtheria bacilli. Well developed granules of
detect by fluorescence microscopy. Heating is
volutin (polyphosphate) may be seen in unstained
unnecessary.
wet preparations as round refractile bodies within
Tubercle bacilli are seen as yellow luminous the bacterial cytoplasm. They tend to stain more
rods in a dark field. When detected under low strongly than the rest of the bacterium with basic
power, the morphology of the bacilli is confirmed dyes, and with toluidine blue or methylene blue
with an oil-immersion objective. they stain metachromatically, a reddish-purple
color. They are demonstrated most clearly by
C. Special Stains special methods, such as Albert’s and Neisser’s,
Special stains are used to color and isolate specific which stain them dark purple but the remainder
parts of microorganisms, such as endospores and of the bacterium with a contrasting counterstain.
flagella, and to reveal the presence of capsules.
Special Stains for Corynebacterium
1. Negative Staining for Capsules diphtheriae (Stains to Demonstrate
Here, bacteria are mixed with dyes such as Indian Metachromatic Granules)
ink or nigrosin that provide a uniformly colored Certain bacilli possess volutin granules in the
background against which the unstained bacteria protoplasm which aid in the identification of
stand out in contrast. This is particularly useful in diphtheria bacilli. Several special and differential
the demonstration of bacterial capsules which do stains are used to stain diphtheria bacilli and to
not take simple stains. demonstrate their metachromatic granules.
Capsules stain poorly, a characteristic exploited 1. Albert’s stain
with a capsule stain, an example of a negative stain. 2. Neisser’s stain
It colors the background, allowing the capsule to 3. Ponder’s stain
stand out as a halo around an organism. 4. Pugh’s stain.
To observe capsules, a liquid specimen is
placed on a slide next to a drop of India ink. A thin 1. Albert’s Stain
glass coverslip is then placed over the two drops, It is used for routine use (See chapter 89, page 599).
causing them to flow together. At the optimum
concentration of India ink, the fine dark particles
of the stain color the background enough to allow
2. Neisser’s Stain
the capsule to be visible. By this method the bacilli exhibit deep blue
granules and the bacterial protoplasm takes pink
2. Endospore (Spore) Staining color.
Endospores cannot be stained by ordinary methods,
such as simple staining and Gram staining, because 3. Ponder’s Stain
the dyes do not penetrate the wall of the endospore. The volutin granules stain dark blue, whereas the
A spore stain is used to make endospores more Bacillus pale blue.
readily noticeable. Generally, malachite green is
used as a primary stain. Its uptake by the endospore
4. Pugh’s Stain
is facilitated by gentle heat. When water is then
used to rinse the smear, only endospores retain the The volutin granules stain reddish purple and the
malachite green. The smear is then counterstained, remainder of the organism light blue.
most often with the red dye safranin. The spores
appear green amid a background of pink cells. Key Points
www.ebook3000.com
89
Chapter
LEARNING OBJECTIVES
After reading and studying this chapter, you should ∙∙ Describe the following: Gram staining; Ziehl–
be able to: Neelsen staing; Alber staining
∙∙ Describe various spots for practical examination ∙∙ Discuss identification of bacterial culture
with identification with relevant questions to answer ∙∙ Describe stool/feces examination for isolation and
∙∙ Prepare exercises for practical in microbiology for identification of pathogenic findings.
undergraduate students
Table 89.1 Specimen Practical question paper of Microbiology for MBBS 2nd Professional examination
Total Marks:25
Ques. No. 1 Identify the spots displayed. (2 × 2 + 3 × 1) = 7
Ques. No. 2 (a) Stain smears “A” by Ziehl–Neelsen technique. Draw and comment on this smear and show your findings
to the examiner. (3)
(b) Stain smear “B” by Albert’s technique. Draw and comment on this smear and show your findings tto the examiner. (2)
Ques. No. 3 You have been provided with a pure bacterial culture on plate and in broth. Do Gram’s staining from plate
and hanging drop preparation for motility from broth. Comment on the growth and mention other tests needed to
identify this bacterium. Show your findings to the examiner. 9(3+3+3)
Ques. No. 4 Identify and draw two abnormal findings (ova and cysts) in the given specimen of feces. Show your findings
to the examiner. 4(2+2)
Contd...
www.ebook3000.com
Chap-89.indd 597 15-03-2016 11:29:17
598 | Section 7: Diagnostic Medical Microbiology
Contd...
VI. Immunology
1. Latex agglutination tile 5. Counter immunoelectrophoresis
2. Microtitre plate 6. HIV-Comb test
3. VDRL slide 7. HIV-Tri-dot test
4. Radial immunodiffusion
VII. Disposable items
1. Presterilized disposable container 3. Presterilized disposable swab
2. Presterilized disposable syringe
VIII. Equipments/instruments
1. Incubator 5. Water bath
2. Hot air oven 6. VDRL rotator
3. Autoclave 7. Lovibond comaparator
4. Centrifuge
IX. Miscellaneous spots
1. Agar-Agar shreds 8. West’s postnasal swab
2. Gas-Pak 9. Stoke’s method of antibiotic sensitivity testing
3. Craigie's tube 10. Kirby-Bauer’s method of antibiotic sensitivity
4. McIntosh-Filde's jar testing
5. Nichrome wire loop 11. Tissue culture bottle
6. Seitz filter 12. Candle jar
7. NIH swab 13. Epsilometer or E-test
XI. Medical entomology
1. Mosquito 6. Ticks
i. Anopheles i. Hard tick
ii. Aedes ii. Soft tick
iii. Culex 7. Mite
2. Housefly i. Trombiculid mite
3. Sandfly ii. Itch mite
4. Louse 8. Cyclops
5. Fleas
B. Albert’s stain
A fixed smear is provided for Albert’s staining. If the Fig. 89.2: Albert’s stain
smear is unfixed, it is fixed by passing the dried slide,
film downwards, three times slowly through the Gram Staining (See Also Chapter 88)
flame, or by heating through the glass slide (the slide Gram staining is done for bacterial culture (solid
is held, film upwards) in the top of the Bunsen flame material, such as cultures on agar) which is provided
for a few seconds so that the slide becomes hot. to the student along with liquid culture of the same
material in a test tube which is used for hanging
Staining Solution drop preparation to observe the motility of bacteria.
A. Albert 1 or Albert stain Preparing film or Smear for Staining
1. Toluidine blue 1.5 g 1. Film preparations are made on clean and free
2. Malachite green 2g from grease glass slides.
3. Glacial acetic acid 10 mL 2. Place a loopful of clean water on the slide and
4. Alcohol (95% ethanol) 20 mL the loop is then sterilized.
5. Distilled water 1 liter 3. A minute quantity of material, obtained by
just touching the growth (with solid material,
B. Albert II or Albert’s iodine
such as cultures on agar), is transferred to the
Iodine 6g
drop, thoroughly emulsified to make a smooth
Potassium iodide 9g suspension, and the mixture is spread evenly
Distilled water. 900 mL on the slide. A thin film of material containing
the microorganisms is spread over the surface
Procedure of the slide with the help of wire loop. This film,
1. Make film, dry in air, and fix by heat. called a smear, is allowed to air dry.
2. Cover slide with Albert’s stain (Albert’s solution 4. The slide is then held in the palm of the hand
A) and allow to act for 3–5 min. high over a Bunsen flame and dried. The
www.ebook3000.com
Chap-89.indd 599 15-03-2016 11:29:18
600 | Section 7: Diagnostic Medical Microbiology
film is fixed by passing the dried slide, film Interpretation of Gram Staining
downwards, three times slowly through the Two broad groups
flame, or by heating through the glass slide. ∙∙ Gram-positive
In the latter method the slide is held, film ∙∙ Gram-negative
upwards, in the top of the Bunsen flame for a
few seconds so that the slide becomes hot. Care Gram-positive: Gram-positive bacteria are those
must be taken not to char the film, and when that resist decolorization and retain the primary
the slide is just too hot to be borne on the back stain, appearing violet.
of the hand, fixation is complete. Air drying and Gram-negative: Gram-negative bacteria are
flaming fix the microorganisms to the slide. decolorized by organic solvents (acetone/alcohol)
and, therefore, take the counterstain, appearing red.
Reagents Gram reactivity is of considerable importance
as the gram-positive and -negative bacteria
A. Violet Dye differ not merely in staining characteristics and
Crystal violet or methyl violet is used at concentra in structure but also in several other properties
tions of 0.5–2%. Solution is facilitated if the dye is first such as growth requirements, susceptibility to
dissolved in alcohol and then added to the water. antibiotics and pathogenicity.
1. Crystal violet or methyl violet 6B 10 g
2. Absolute alcohol (100% ethanol) 100 mL Gram Staining Mechanism
3. Distilled water 1L The exact mechanism is not understood. It may,
however, be attributed to following:
B. Gram’s Iodine 1. Protoplasm: There is a more acidic protoplasm
1. Iodine 10 g (pH 2–3) in the gram-positive cells, which is
2. Potassium iodide 20 g responsible for retaining the basic dye more
3. Distilled water 1L strongly than the Gram-negative bacteria.
2. Cell wall structure: Different kinds of bacteria
C. Decolorizer react differently to the Gram stain, because
structural differences in their cell walls affect
i. Acetone.
the retention or escape of a combination of
ii. Absolute alcohol (100% ethanol).
crystal violet and iodine, called the crystal
iii. Acetone-alcohol. This is a mixture of 1 volume
violet-iodine (CV-I) complex.
of acetone with 1 volume of 95% ethanol. It
Iodine makes the protoplasm more acidic and
requires application for about 10 seconds.
serves as mordant, i.e. iodine combines with dye
to form a dye-iodine complex and fixes the dye in
D. Safranine Counterstain bacterial cell. When applied to both gram-positive
Safranine 0.5% in distilled water. and gram-negative cells, crystal violet and then
iodine readily enter the cells. Inside the cells, the
Procedure crystal violet and iodine combine to form crystal
1. Heat-fixed smear is covered with a basic purple violet-iodine (CV-I) complex.
dye, usually crystal violet (primary stain) for
one minute. (Other dyes such as gentian violet or Gram-positive Cells
methyl violet may also be used as primary stain). Gram-positive bacteria have a thicker peptido
2. Wash the smear thoroughly with water. glycan cell wall than Gram-negative bacteria. The
3. Cover the smear with Gram’s iodine for one complex crystal violet-iodine (CV-I) complex is
minute. larger than the crystal violet molecule that entered
4. Wash again with water. the cells, and because of its size, it cannot be washed
5. Decolorize the smear with acetone for 10 out of the intact peptidoglycan layer of gram-positive
seconds or less taking care not to overdecolorise. cells by acetone/alcohol. Consequently, gram-
(Alcohol can be substituted for acetone). positive cells retain the color of the crystal violet dye.
6. Immediately wash with water to remove the
decoloriser. Gram-negative Cells
7. Cover the slide with dye safranin (counterstain) Gram-negative bacterial cell walls are thinner, have
for 1 minute. (Dilute carbol fuchsin or neutral a smaller amount of peptidoglycan, less exten
red may also be used as counterstain). sively cross-linked and contain a high percentage
8. Wash off the smear with water, blot and dry. of lipids. There is a layer of lipopolysaccharide
9. Examine the stained smear under the 100 × as part of their cell wall. They dissolve during
(oil) immersion objective of the microscope treatment with acetone alcohol, forming larger
(Fig. 89.3). pores in the cell wall, and the CV-I complex is
www.ebook3000.com
Chap-89.indd 601 15-03-2016 11:29:19
602 | Section 7: Diagnostic Medical Microbiology
Table 89.3 Description of appearance of growth Table 89. 4 Description of appearance of growth
on solid media in liquid media
1. Shape—circular, irregular, or rhizoid. 1. Degree: None, scanty, moderate abundant or profuse
2. Size—in millimeters. Pin-head size is characteristic 2. Turbidity: Present or absent; if present, slight,
of staphylococci and pin-point size is a feature of moderate or dense; uniform, granular or flocculent.
streptococcal colonies. Most of the gram-negative bacteria grow in uniform
3. Elevation—effuse, elevated, convex; concave, turbidity form.
umbonate or umbilicate. 3. Deposit: Present or absent: if present, slight,
4. Margins—bevelled or otherwise. moderate or abundant; powdery, granular, flocculent,
5. Surface—smooth, wavy, rough, granular, papillate or membranous or viscid; disintegrating completely or
glistening. incompletely on shaking, e.g. Streptococci growth
6. Edges—entire, undulate, crenated, fimbriate or occurs as a granular turbidity with a powdry deposit.
curled. 4. Surface growth: Present or absent; All aerobes
7. Color—pigment production have tendency to grow on surface of media due to
8. Structure—opaque, translucent or transparent more content of oxygen present on the surface, e.g.
9. Consistency—membranous, friable, butyrous or Pseudomonas sp.
viscid.
10. Emulsifiability—easy or difficult.
11. Differentiation—Differentiated into a central and a
peripheral portion
Identification Scheme
The following scheme of identification should be
adopted.
A. Culture Plate
Culture medium is identified and cultural
characteristics of bacterial growth are noted.
1. Culture Medium
Culture media provided may be nutrient agar,
blood agar or MacConkey’s agar. A
i. Cultural characteristics: Then the cultural
characteristics (Table 89.3) of bacterial
growth is described. This may help in some
provisional identification of the bacteria.
ii. Gram staining: Gram staining is done from
culture plate and notes the following:
i. Gram reaction—Gram-positive or gram- B
negative Figs 89.4: (A) Hanging drop preparation;
ii. Morphology—Cocci, bacilli or coccobacilli, (B) Hanging drop examination
approximate size. If bacilli, then describe
Various characterstics are noted such as degree,
whether these are thin, stout, long or short
turbidity, deposit and surface growth (Table 89.4).
bacilli.
Hanging drop preparation—is done to find out.
i. Motility—Motile or non-motile.
B. Liquid Medium ii. Morphology—Cocci, bacilli or coccobacilli.
Liquid medium is prepared from the same culture
plate which is given for examination and has the same Hanging Drop Preparation
bacteria. Therefore, first identify the liquid culture
This experiment is done to demonstrate motility
medium and describe the characteristics of growth
and to study morphology of bacteria (Fig. 89.4). It
(Table 89.4). Hanging drop preparation is made
is prepared from the bacterial growth provided in
from this liquid media for motility and morphology
liquid culture medium in a test tube.
of the bacteria.
Requirements: 1. A clean cavity (depression) slide;
i. Liquid Culture Medium 2. A clean coverslip; 3. Young broth culture; 4. Wire
Liquid culture medium may be in peptone water loop; 5. Bunsen burner/spirit lamp; 6. Microscope.
or glucose broth. Glucose broth is darker and more
yellowish in color as compared to peptone water. Procedure
Generally, streptococci are provided in glucose broth 1. A ‘hollow ground’ slide, (a glass slide with a
whiles all other bacteria are given in peptone water. shallow, circular concavity in its center) is used
www.ebook3000.com
Chap-89.indd 603 15-03-2016 11:29:20
604 | Section 7: Diagnostic Medical Microbiology
Confirmatory Biochemical Reactions for
Pseudomonas aeruginosa
1. Oxidative–fermentation
medium of Hugh
and Leifson
(OF medium) Oxidative
2. Glucose Oxidatively with the
production of acid
only
3. Lactose Negative
4. Maltose Negative
5. Indole Negative
6. MR Negative
Fig. 89.5: Green pigmented growth of Pseudomonas 7. VP Negative
aeruginosa on the nutrient agar
H2S Negative
8. Oxidase reaction (within 30 seconds)
Observations (Fig. 89.5) 9. It reduces nitrates to nitrites and further to
A. Solid Media gaseous nitrogen.
i. Medium: Nutrient agar. 10. It is catalase, arginine dihydrolase and gelatinase
ii. Colony morphology: The colonies are large, positive
2–3 mm in diameter, smooth, translucent, 11. Lysine decarboxylase Negative
irregularly round and emit a characteristic 12. Aesculian hydrolysis Negative
fruity or grape-like smell. Note your observations on your answer sheet
iii. Pigment : It is a bluish-green diffusible with well labeled diagrams of your observations
pigment. This pigment diffuses into the with color pencils and show to the examiner.
surrounding medium. Demonstration of
the presence of the pigment is absolute 2. Blood Agar
confirmation of a strain as Pseudomonas
aeruginosa and thus the major diagnostic test. Blood agar supports the growth of a variety of
B. Liquid media (Peptone water) fastidious and nonfastidious organisms, gram-
Growth in broth: Dense turbidity with a surface positive bacteria and gram-negative bacteria. Gram-
pellicle green pigment. Surface pellicle gives’ a positive cocci (Staphylococcus aureus or Streptococcus
clue that bacteria are aerobic nature because pyogenes) or gram-negative bacilli such as Proteus
bacteria have tendency to accumulate at the species may also be provided on blood agar plate
top due to more oxygen content available at
surface. A. Gram-positive cocci Observations
C. Gram staining and Hanging Drop Preparation A. Solid Media
Proceed for gram staining (from solid media) i. Medium: Blood agar
and hanging drop preparation (from liquid ii. Colony morphology: In case of a bacterial
media). Note the following finding in case of growth with hemolysis on blood agar,
Pseudomonas aeruginosa proceed further as under Staphylococcus and
Gram staining—gram-negative bacilli Streptococcus below.
Hanging drop preparation (motiliy): Actively
Staphylococcus and Streptococcus are further
motile.
processed as follows.
D. Presumptive diagnosis-Pseudomonas sp.
E. Confirmatory test: P. aeruginosa is identified by B. Liquid media-Growth in broth.
colonial characteristics and simple biochemical C. Gram staining and Hanging Drop Preparation
tests (e.g. positive oxidase reaction). All strains Proceed for gram staining (from solid media) and
Pseudomonas euruginosa give a rapid positive hanging drop preparation (from liquid media). Note
oxidase reaction (within 30 seconds) and this the following finding.
is a useful preliminary test for non-pigmented Gram staining: Gram-positive cocci in clusters
strains. Pseudomonas are nonfermenter and (Staphylococcus)-gram-positive cocci in chains
break down glucose oxidatively with acid (Streptococcus).
production. They reduce nitrates to nitrites and Hanging drop: Non-motile cocci
further to gaseous nitrogen and utilize citrate D. Presumptive diagnosis: Staphylococcus and
as sole source of carbon. Streptococcus are further processed as follows.
Fig. 89.6: Growth of Staphylococcus aureus showing beta Fig. 89.7: Growth of Streptococcus pyogenes showing beta
hemolysis and golden pigment on blood agar hemolysis on blood agar
www.ebook3000.com
Chap-89.indd 605 15-03-2016 11:29:21
606 | Section 7: Diagnostic Medical Microbiology
ii. Insoluble in 10% bile unlike Streptococcus. suspected because swarming does not occur
pneumoniae. To differentiate Streptococcus on MacConkey’s medium, on which smooth
at species level, following tests are to be colorless (NLF) formed.
performed: the
1. Lancefield technique: Hemolytic strepto B. Liquid Media
cocci are grouped by the Lancefield Growth in broth (in peptone water): Uniform
technique using serologic methods, or turbidity’ with slight powdery deposit and an
biochemical tests can be performed. ammonical odor in liquid medium (peptone water).
2. Fluorescent antibody technique: For the
rapid identification of group A streptococci.
C. Gram Staining and Hanging Drop
3. Str. pyogenes (Group A) Sensitive to
Preparation
bacitracin (0.04 unit disk).
A key test that should be done is bacitracin i. Gram staining: Gram-negative coccobacilli,
susceptibility or PYR hydrolysis. 1–3 mm long and 0.6 mm wide. Pleomorphism is
4. Pyrrolidonyl naphthylamine (PYR) frequent – short coccobacilli to long filaments.
hydrolysis –Positive. ii. Hanging drop preparation: Actively motile.
iii. Group ‘B’ streptococci-CAMP reaction and
hippurate hydrolysis is positive. D. Presumptive Diagnosis
iv. Group ‘D’ streptococci-Heat resistant test is Proteus sp.
positive. The enterococci grow in the presence
of 6.5% NaCl, 40% bile, at ph 9.6, at 45°C and in E. Confirmatory Tests
0.1% methelyne blue and survives heating at Keeping in view all the observations your
60°C for 30 min. On MacConkey medium they presumptive diagnosis is Proteus sp. It is confirmed
produce deep pink colonies. Enterococci are with the following tests.
PYRase test positive. They do not hydrolyze i. Phenyl pyruvic acid (PPA) test: Positive
hippurate.
ii. Urease test: Positive
E. Draw well labeled diagrams of your observations
iii. Glucose: A/G (acid/gas)
with color pencils to show Gram reaction.
iv. Lactose: Negative
v. Malonate utilization: Negative
BACILLUS PROTEUS SP.
vi. Indole test : Positive in P. vulgaris but is
If the student is provided with a culture media with negative in P. mirabilis.
swarming growth on noninhibitory solid media vii. MR: Positive
such as nutrient agar and blood agar, the same viii. VP: Negative.
procedure is adopted for identification of bacteria. ix. H2S: Positive in P. vulgaris and P. mirabilis.
x. Ornithine decarboxylase: Positive in P.
A. Solid Media mirabilis but negative in P. vulgaris
1. Culture medium: Blood agar (Fig. 89.8)
2. Colony morphology: Swarming appearance on F. Final Diagnosis
noninhibitory solid media, such as nutrient agar The given bacterium may be identified as P. vulgaris
and blood agar with characteristic putrefactive or P. mirabilis depending on biochemical reactions.
odor (‘fishy’ or ‘seminal’) then genus Proteus is Typing methods (phage typing, bacteriocin typing
and serotyping schemes) have been developed for
Proteus species. Swarming Proteus strains exhibit the
Dienes phenomenon and this forms the basis for a
precise method of differentiation among such strains.
3. MacConkey’s Agar
1. Gram-negative rods are better described on
MacConkey agar because these organisms
produce similar-looking colonies on blood agar
plate and chocolate agar media. MacConkey
is best used, however, to differentiate lactose
fermenters (LF) from nonlactose ferment
ers (NLF). Therefore, on MacConkey’s agar,
Fig. 89.8: Growth of Proteus sp. showing swarning on colonies may be either pink (lactose fermen
blood agar ter) or pale (non-lactose fermenter).
Fig. 89.9: MacConkey agar with smooth, lactose fermenter Fig. 89.10: MacConkey‘s agar with lactose fermenter
(non-mucoid) colonies of Escherichia coli (mucoid) colonies of Klebsiella sp.
2. The LF colonies may be E. coli or Klebsiella Escherichia coli: Red or pink, nonmucoid colony,
sp., while NLF colonies generally given are gram-negative straight, rod and motile.
Salmonella sp. or Shigella sp. Klebsiella sp: 2 Red or pink, mucoid colonies, short
3. Examine the colony characters on solid and plump bacilli on Gram staining and nonmotile.
medium and growth in liquid medium.
4. Gram staining from culture plate and hanging F. Confirmatory Tests by Various
drop preparation from liquid broth are to be Biochemical Reactions (Table 89.5)
made.
Escherchia coli: Biochemical reactions
5. The following features may be observed on
i. E. coli ferments glucose, lactose, mannitol,
Gram’s staining and hanging drop preparation.
maltose and many other sugars with the
production of acid and gas. Typical strains do
Lactose Fermenter (LF)
not ferment sucrose.
A. Solid Media ii. Indole and MR positive, and VP and citrate
1. Culture medium: MacConkey’s agar (Figs negative (IMViC + + – –)
89.9 and 89.10). iii. It is negative for phenylalanine deaminase
2. Colony morphology: red or pink, nonmucoid test, urease test, H 2S production, gelatin
colony—Escherichia coli. liquefaction, growth in the presence of KCN,
Large, mucoid pink colonies—Klebsiellaj sp. and malonate utilization.
B. Klebsiella sp: Biochemical reactions (Table 89.3)
B. Liquid Broth They ferment sugars (glucose, lactose, sucrose,
Growth in broth: General turbidity and a
i. mannitol) with production of acid and gas, urease
heavy deposit or uniform turbidity. positive, indole negative, MR negative, VP positive
and citrate positive (IMViC – – ++). These reactions
C. Gram Staining and Hanging Drop are typical of K. pneumoniae subsp. aerogenes.
Preparation Final diagnosis of Esch. coli or Klebsiella sp. can be
made based on the above-mentioned biochemical
i. Gram staining: Gram-negative straight rod- reactions. Further tests, such as serotyping should
Escherichia coli; gram-negative short and be done for confirmation. Typing mehods, such as
plump bacilli Klebsiellaj sp. bacteriocin typing, phage typing, resistotyping and
ii. Hanging drop preparation: Motile or non molecular typing methods (Plasmid analysis, DNA
motile. profiling by random amplified polymorphic DNA
(RAPD) and pulsed-field gel electrophoresis) can
D. Identification be used for Klebsiella strains.
The bacteria may be identified by correlating the
colony characters, Gram staining and hanging B. Nonlactose Fermenter (NLF) Colonies
drop preparation and then by various biochemical 1. For undergraduate practical examination the
reactions. students are provided either of two important
NLF bacteria—Salmonella sp. or Shigella sp
E. Presumptive Diagnosis (nonlactose-fermenting bacteria are pale).
Presumptive diagnosis of Esch. coli or Klebsiella sp. Liquid broth of the same bacteria is also given
can be made on the basis of above features. alongwith it.
www.ebook3000.com
Chap-89.indd 607 15-03-2016 11:29:23
608 | Section 7: Diagnostic Medical Microbiology
Table 89.5 Differentiating features of Escherichia coli and Klebsiella sp
Characteristics Escherichia coli Klebsiella sp
1. Colony morphology on MacConkey’s Colonies are red or pink due to lactose Colonies are red or pink in color due
agar fermentation (LF or lactose fermenter to lactose fermentation (LF or lactose
colonies), nonmucoid, circular moist, fermenter colonies), large and mucoid.
smooth with entire margin (Large, mucoid and red in color)
2. Growth in broth (peptone water) Growth occurs as general turbidity and Uniform turbidity.
a heavy deposit.
3. Gram staining Gram-negative, straight, rod Gram-negative, short, plump,
measuring 1–3 x 0.4–0.7 µm nonsporing, capsulated, 1–2 µm long
arranged singly or in pairs. and 0.5–0.8 µm wide.
4. Motility (Hanging drop preparation) Motile bacilli Nonmotile bacilli
5. Biochemical reactions
1. Acid from glucose Acid and gas (AG) Acid and gas (AG)
2. Lactose + +
Mannitol + +
Sucrose Variable +
Indole + –
Methyl red (MR) + –
Voges–Proskauer (VP) – +
Citrate – +
Urease – +
Growth in KCN – +
Fig. 89.11: Nonlactose fermenter colonies of Salmonella sp Fig. 89.12: Nonlactose fermenter colonies of Shigella sp
on MacConkey‘s agar on MacConkey‘s agar
Table 89.7 Distinguishing features of Shigella vi. They reduce nitrates to nitrites, do not form
species H2S and are inhibited by KCN.
Subgroup A B C D vii. Catalase is produced, except by S. dysenteriae
type 1 which are catalase negative.
Species Sh. Sh. Sh. Sh.
dysenteriae flexneri boydii sonnei viii. Members of groups A, B and C fail to decar
Mannitol – A A A boxylate lysine and ornithine. S. sonnei decar
boxylates ornithine but not lysine.
Lactose – – – A (Late)
Salmonella sp. Biochemical Reactions (Table
Sucrose – – – A (Late)
89.8)
Dulcitol – – d
Salmonellae will be indole and urease negative,
Indole d d d
catalase positive, oxidase negative and ferment
Ornithine – – – + glucose, mannitol and maltose but not lactose or
decarboxylase
sucrose. S. Typhi will be anaerogenic and ferments
Serotypes 10 6+ 15 Only glucose and mannitol with production of acid only,
variants one
while paratyphoid bacilli (S. Paratyphi A, B and C)
A = Acid; d = Variable will form acid and gas from sugars. Identification
of the isolate is by slide agglutination.
biochemical reactions are very important to
F. Show your findings to the examiner after
differentiate these Salmonella sp. and Shigella sp.
drawing well labeled diagrams of your observations
up to species level (Tables 89.6 to 89.8).
of Salmonella sp. or Shigella sp.
Shigella sp: Biochemical reactions (Table 89.7)
i. The shigellae are divided into four groups, or IV. Bacterial Growth on any Other Medium
species, by their biochemical reactions and
antigenic structure (Table 89.7). The groups Usually bacterial growth is given on the medium
A, B, C and D correspond to the species S. such as nutrient agar, blood agar and MacConkey’s
dysenteriae, S. flexneri, S. boydii and S. sonnei. agar. If the media other than nutrient agar, blood
agar and MacConkey’s agar is provided for
ii. Glucose is fermented with the production of
examination, then proceed using the same basic
acid, without gas, except for two serotypes:
method of processing, i.e. colony morphology,
Sh. flexneri serotype 6 (Newcastle and
liquid broth, Gram staining, hanging drop
Manchester varieties) and S. boydii serotype
preparation, presumptive diagnosis and further
14. Fermentation of mannitol in Sh. flexneri,
confirmatory tests. In vivo examiner may ask any
Sh. boydii and Sh. sonnei
question related to the specific bacteria along with
Mannitol nonfermenting—Sh. dysenteriae questions related to culture media, Gram staining,
iii. Lactose and sucrose are not fermented, except hanging drop preparation.
by Sh. sonnei which ferments them late.
iv. Dulcitol is not fermented by the majority of
shigellae. Adonitol, inositol and salicin are 4. STOOL/FECES EXAMINATION
not fermented. In this exercise, stool specimen is provided to
v. IMV, C––– + +–– (S. dysenteriae serotype 1, S. isolate two pathogenic parasitic findings. Stool
flexneri serotype 6 and S. sonnei are always examination is done to find out ova and cysts of
indole negative). different parasites.
www.ebook3000.com
Chap-89.indd 609 15-03-2016 11:29:24
610 | Section 7: Diagnostic Medical Microbiology
Table 89.8 Biochemical reactions of Salmonella species
Subgroup S. Typhi S. Paratyphi A S. Paratyphi B S. Paratyphi C
Catalase + + + +
Oxidase – – – –
Urease – – – –
Glucose A AG AG AG
Mannitol A AG AG AG
Maltose A AG AG AG
Lactose – – – –
Sucrose – – – –
Indole – – – –
Methyl red + + + +
Voges- Proskauer(VP) – – – –
Citrate – – + +
H2 S production + – + +
+, positive; negative; A, acid; AG, acid and gas
B
Figs 89.15A and B: (A) Fertilized egg of Ascaris lumbricoides; Fig. 89.16: Egg of Trichuris trichiura
(B) Unfertilized egg of Ascaris lumbricoides (Roundworm)
www.ebook3000.com
Chap-89.indd 611 15-03-2016 11:29:26
612 | Section 7: Diagnostic Medical Microbiology
www.ebook3000.com
Chap-89.indd 613 15-03-2016 11:29:30
614 | Section 7: Diagnostic Medical Microbiology
www.ebook3000.com
616 | Essentials of Microbiology
B Bacteriophages 4, 64, 65f, 79, 402 Bordetella species, differentiatial
lysogenic cycle 403, 404f characterstics of 324
Bacillary dysentery 272 lytic cycle 403, 403f Borrelia 342, 347
Bacilli/bacillus 12f, 12, 205 morphology 402, 402f laboratory diagnosis 344
arrangement 13 role of 402 morphology 342
general characterstics of 205 significance of 404 Borrelia burgdorferi 342, 343
Bacillus anthracis 205, 210 Bacteroides spp 223, 534t Borrelia recurrentis 342; 342
antigenic structure 207 Bartonella 368 Borrelia vincenti 342
differentiating features between Bartonella bacilliformis 368 Botulinum toxin 218
B. anthracis and anthracoid Bartonella henselae 369 Botulism 218
bacilli 209 Bartonella quintana 368 food-borne 218
morphology 205 Batch culture 22 infant 218
resistance 207 B-cell 119, 121
wound 218
Bacillus cereus 205, 209 activation 127
Bovine spongiform
infections 210 maturation 121
encephalopathy 489
Bacteremia 80, 533, 535 BCG vaccination 236, 245
Brazilian purpuric fever/BPF 320
Bacteria 11-20 Bence–Jones proteins 95
Brill–Zinsser disease 363, 369
aerobic 23 Benzyl penicillin 168
Beta (β) hemolysin 165 Broth dilution method 563, 564f
anaerobic 23, 221
Betapropiolactone/BPL 33 Broth, nutrient 38t
capsulated 16
fermentative 50 Bifidobacterium 222 Brucella 327, 534
genotypic characteristics of 48, 48t Biomedical waste /BMW 580 antigenic structure 327
gram-positive and gram- black bags 581 classification of 328
negative 14t blue/white translucent bags 581 differential characteristics of 328
methods used to identify 48t disposal 582 laboratory diagnosis 329
phenotypic characteristics 48t, 48 red bags 581 morphology 327
shape of 12, 12f segregation 581 pathogenesis 328
transformation in 64 treatment of 581 resistance 327
mechanism of genetic 64f yellow bags 581 Brucella abortus 23, 326
Bacteria capsule 15 BK polyomaviruses 422 Brucella bacteriophage 328
demonstration of 16 Blastomyces dermatitidis 515 Brucella melitensis 328
Bacteria cell 11 Blastomycosis 515, 518 Brucella suis 328
anatomy of 13, 13f Bleaching powder 32 Brucella tularensis 315
cellular appendages 13, 15 Blood group systems 160 Brucellin skin test 331
chemical structure of 13 ABO 159 Brucellosis 329
components 13 Lewis 160 blood culture in 329
cytoplasm 17 MN system 160 course of disease 329
gram-positive and gram- Rh (rhesus) 160 prophylaxis 331
Blood transfusion 160 treatment 331
negative 13
complications of 160 types of infection 329
mesosomes 17
nonimmunological
ribosomes 17 Bruton’s hypogammaglobulinemia
complications of 161
RNA 17 137
Blotting 71
Bacteria cell wall Burkholderia cepacia 304, 306
northern 71
chemical structure 13, 14f pathogenicity 304
southern 71
comparison of treatment 304
western 71
gram-positive bacteria 13, 14t Burkholderia mallei 304
Bone marrow 117, 123
gram-negative bacteria 13, 14t Burkholderia pseudomallei 305
Bordetella ansorpic 323
functions 13, 14 Burkitt’s lymphoma 414, 500
Bordetella avium 323, 326
gram-negative 14, 15f Bordetella bronchicanis 326 Buruli ulcer 250
gram-positive 14, 14f Bordetella bronchiseptica 323, 326 Busse–Buschke disease 521
Bacterial count 21 Bordetella holmesii 323 C
Bacterial cultures Bordetella petrii 323
identification of 601, 603 Bordetella trematum 323 California encephalitis virus 454
methods of isolating 46 Bordetella hinzii 323 Calymmatobacterium
Bacterial division 21 Bordetella parapertussis 323, 326 granulomatis 360
Bacterial flora, in water 575 Bordetella pertussis 323, 326, 541 Camp test 355
Bacterial growth curve 21, 22f adhesins 324 Campylobacter 297
Bacterial metabolism 23 antigenic constituents 323 Campylobacter jejuni 545
Bacterial nucleus 18 morphology 323 Candida 519
Bacterial nutrition 22 prophylaxis 325 C. albicans 519, 520, 525
Bacterial spore 18, 19f resistance 323 C. glabrata 519
Bacteriology of milk 577 treatment 325 C. guilliermondii 519
Bacteriolysis 98, 100 virulence factors 323 C. krusei 519
Index | 617
C. parapsilosis 519 Cholera toxin 291 fixation test 102, 108, 109f
C. stellatoidea 519 Christensen’s urease medium 52 indirect 109
C. tropicalis 519 Chromobacterium uses 109
C. viswanathii 519 violaceum 357, 360 inhibitor deficiencies 140
Candidiasis 519, 525 Chromoblastomycosis 512 system 96
superficial 519 Chronic granulomatous disease 140 Conjugation 65, 68
systemic 519, 520 Chronic mucocutaneous chromosomal transfer 66
treatment 521 candidiasis 520 in E.coil 66f
Candle Jar 44 Chronic wasting disease 489 plasmid and chromosomal
Capnocytophaga 360 Ciprofloxacin, for Legionella transfer 66
Capsule-swelling reaction 16 pneumophila 308 Contagious pustular dermatitis 408
Carbapenems 567 Citrate utilization test 51, 51f Continuous culture 22
Cardiobacterium hominis 360 Koser’s citrate medium 51 Cooked meat broth/CMB 42, 46
Carrier 76 Simmons’ citrate medium 51 Coombs’ test
Cellulitis, due to H. inflenzae 319 C1 esterase 99 direct 107
Central dogma of molecular Clostridial endometritis 214 indirect 107
biology 60 Clostridial myonecrosis 214 types of 107
Cephalosporins Clostridium 211 Coronaviruses 494, 495f
antibacterial spectrum 567t classification 212 severe acute respiratory
for lyme disease 344 diseases produced 212 syndrome 495
for Pneumococcus 186 general features of 211 Counter immunoelectrophoresis 105
Chlamydophila psittaci 542t Clostridium botulinum 218, 219, Cowpox 408
220, 404 Coxiella 367
Chediak-Higashi syndrome 140
treatment 219 antigenic variation 367
Chemotactic factors 145
Clostridium difficile 219, 220
eosinophil 145 Coxiella burnetii 367, 370, 542t
antibiotic associated
neutrophil 145 prophylaxis 368
diarrhea 219
Chemotherapy 5 Coxsackievirus 429
pseudomembranous colitis 219
Chicken cholera vaccine 2 antigenic characters 429
toxins 219
Chickenpox 412 clinical syndromes associated
treatment and prophylaxis 219
Chick-Martin test 34 with 431t
Clostridium perfringens 212, 214,
Chigger-borne typhus 365 features of 429, 430t
220, 545, 577
Chikungunya virus 449, 455 group a viruses 430
colitis 214
Chlamydia 371 group B viruses 430
food poisoning 214
antigenic structure 373 morphology 212 C-reactive protein/CRP 83
classification 371 prophylaxis 215 Cresols 31
C. pecorum 371 resistance 212 Creutzfeldt–Jakob disease/CJD 490
C. pneumoniae 371 soft tissue infections 213 Cryoglobulinemia 95
C. psittaci 371 toxins 213 Cryotococcus neoformans 526
differences between treatment 215 Cryptococcal meningitis 521
chlamydiae and viruses 371 Clostridium tetani 215, 220 Cryptococcosis 521, 526
growth cycle 372, 372f Cluster of differentiation 120 Culex tritaeniorhynchus 451, 455
Chlamydia and chlamydophila; Coagglutination 108f Culture media
diseases caused by 372t Coagulase 79 anaerobic media 41
Chlamydia pneumonia 375, 543 Cocci 12, 12f classification of 37, 37t
Chlamydia trachomatis 371, 373 anaerobic 221 complex media 38
biovars of 373 arrangement 12 differential media 41
genital infections 374 Coccidioides immitis 516, 518 enrichment media 37t, 39, 40
inclusion conjunctivitis 373 Coccidioidomycosis 515, 516, 518 indicator media 37t, 40
infections 377 Code 59 ingredients of 37
trachoma 373 Codon 59 liquid media 37, 37t, 38t
Chlamydophila pneumoniae 372, Cold agglutinin test 89 selective media 40
375, 377 Colicinogenic/Col factor 66 semisolid media 38
Chlamydophila psittaci 371, 375, 377 Collagenase 79 simple media 38
Chloramphenicol Collagen-vascular inflammatory solid media 38
for Neisseria meningitidis 190 diseases, complement sugar media 41
for plague 313 deficiencies in 99 transport media 41
for Pneumococcus 186 Colony-stimulating factors 132t, 132 Cystic fibrosis 72
Chlorhexidine 31 Commensals 75 Cytokines 131, 132t, 135
Chlorine 32 Complement 96 Cytolysins 79
Chloroxylenol 31 activation 96, 100 Cytolysis 100
Cholera 291 principle pathways 96 Cytolytic/cytocidal tests 109
prophylaxis 294 biological effects of 98 Cytomegalovirus 393, 413, 416, 500
treatment 293 complement deficiencies 99t, 140 Cytopathic effects 385
www.ebook3000.com
618 | Essentials of Microbiology
D Ectothrix 510 Epidermolytic toxins/exfoliative
Edward jenner 7 toxins 166
Dapsone; for leprosy 245 Ehrlichia 366 Epiglottitis, due to H. influenzae 319
Delta (δ) hemolysin 166 Ehrlichia chaffeensis 366 Epitome 87
Dengue 451 Ehrlichia ewingii 366 Epsilometer or E-test 563
clinical findings 452 Eijkman test 577 Epstein–barr virus 413, 416, 500
Dengue hemorrhagic fever 99, 452, Eikenella corrodens 359 360 associated malignancies 414
453, 455, 585 treatment 360 infectious mononucleosis 414
Dental caries 181 Electroimmunodiffusion 105 specific antibodies 415
Deoxyribonucleic acid (DNA) 58 countercurrent eletrophoresis/ Erwinia 269
structure 58 CIEP 105 Erysipelas 177
Deoxyribonulease (DNAase) counterimmuno- Erysipelothrix 357
test 164 electrophoresis/CIE 105 Erysipelothrix rhusiopathiae 356,
Dependo virus 425 one-dimensional double 105 360
Dermatophytes 509 one-dimensional single 105 human infection 357
classification 509 Electrophoresis Erythro virus 424
Diarrhea 544 Laurell’s two-dimensional 106, Erythromycin, for Pneumococcus 186
causative agents of 544t Escherichia coli 534t
106f
traveller’s 544 differentiating features of
Elisa 112f
Dienes phenomenon 268 Escherichia coli and
cassette-based
Digeorge’s syndrome 138 Klebsiella sp 608t
membrane-bound 114
Dilute carbol fuchsin 593 Ethylene oxide 33
competitive 112
Dimorphic fungi 503 Eubacterium 222
indirect 112, 113
Diphthericin 66 Eukaryotic cells 11
sandwich 112, 113
Diplococci 12 differentition from
uses of 113
Diplococcus pneumoniae 183 prokaryotes 11t
Endemic 80
Disinfectants Exaltation 78
Endemic typhus 363, 369
categories of 30 Exons 59
Endocarditis
characteristics of 30 Exotoxins 78t, 78
acute 534
low-level 30
subacute 534
skin 32
used in hospitals 34t
Endospore (spore) staining 594 F
Endospores 19
Disinfection 25, 35 Fab fragments 90
Endothrix 510
high-level 30 Farmer’s lung 147
Endotoxemia 533
methods of 25, 26t Fatal familial insomnia/FFI 490
Endotoxins 78t, 79
Disseminated histoplasmosis 517 Favus 510, 511
Entamoeba histolytica, cysts 612
DNA probes 70 FC fragment 90, 91
Donovania granulomatis 357 Enteric fever 279
functions of 92
Donovanosis 358, 548 Enterobacter spp 534
Fertility factor 66
treatment 358 Enterobius vermicularis 597,
Fever 83
Double helix 58 611,612
Filamentous fungi 503, 519
DPT vaccine 325 Enterococcus/enterococci 174,
Filamentous hemagglutinin
Drug resistance 180,181
adhesin/FHA 324
in bacteria 68 characteristics of 180
Filoviruses 494
mutational CE 68, 68t clinical infections 180
Filters 29
transferable 68, 68t differences between group d
asbestos 29
Duchenne’s muscular streptococci and earthware 29
dystrophy 72 Enterococcus spp 180 membrane 29
Dyes identification 180 sintered glass 29
acridine 33 treatment 180 Filtration 29
aniline 32 Enterococcus avium 180 Fimbria 17
Dysentery 546 Enterococcus casseliflavus 180 Fixed virus 458, 460, 464
microorganisms causing 546t Enterococcus durans 180 Flagella 16
Enterococcus faecalis 174, 180 demonstration of 17
Enterococcus gallinarum 180 Flagellin 16
E Enterococcus raffinosus 180 Flagellum 16f
Eastern equine encephalitis/EEE Enterotoxins, of Staphylococcus Flavobacterium meningosepticum
448t, 455 aureus 166 357, 360
Ebola virus 494 Enteroviruses 394, 398, 426 Flocculation 102
Sudan strain 494 Enzyme immunoassay/EIA 338, 339 Flocculation tests, types of 103
Zaire strain 494 Enzymes 79 Fluctuation test 63, 63f
Echoviruses 430 Epidemic 80 Fluorescent treponemal antibody/
clinical features 430 Epidemic typhus 363 FTA test 339
Index | 619
Fluorochrome staining 594 Grafts 154 Hepatitis A virus 465, 474
Food poisoning 547 Graft-versus-host reaction 156 antigenic structure 468
Foot-and-mouth disease of cattle 4 Gram stain 48, 593 immunization 467
Formaldehyde 33 Gram staining 599, 601f prophylaxis 467
Formalin 33 interpretation of 600 treatment 467
Fort bragg fever 345t mechanism 600 Hepatitis B carriers 469
F-prime 66 Gram-negative bacteria 601 Hepatitis B virus 467, 474
Fragment-crystallizable See FC active immunization 471
Gram-positive bacteria 601
Frambesia 341 acute infection 469
Granuloma inguinale 358
Francisella tularensis 315 avihepadnavirus 467
Granulomatosis infantiseptica 356
morphology 315 chronic infection 469
Griffith typing 173
prophylaxis 315 hepatocellular carcinoma 500
Gut-associated lymphoid tissue 119 interpretation of serological
Frei’s test 375, 377
French neurotropic vaccine 451 markers 470t
Fungal keratitis 525, 526 H markers 470
Fungi 503 orthohepadnavirus 467
H antigen 159, 302
classification of 503, 503fc passive immunization 471
Hacek group bacteria 321
general properties of 503 prophylaxis 471
Haemophilus aphrophilus 317 structure 467
identification of 506 Haemophilus ducreyi 317, 320, 322 subtypes 468
Fusidic acid 568 Haemophilus haemolyticus 321, 322 treatment 471
Fusobacterium 223 Haemophilus influenzae 317, 318t, Hepatitis c virus 471, 474
322, 534 clinical features 472
G antigenic structure 318 prophylaxis 472
characters of six biotypes of 318t treatment 472
Gamma (γ) hemolysin 165
cultural characteristics 317 Hepatitis D virus/HDV 472, 474
Gardnerella vaginalis 360
growth characteristics of 317 Hepatitis E virus/HEV 473, 474
Gas gangrene 214
invasive infections 319 clinical features 473
Gaspak 45
Gastroenteritis 544 laboratory diagnosis 319 prophylaxis 473
due to Salmonella infection 280 morphology 317 Hepatitis G virus 474
Gastrointestinal tract 82 noninvasive disease 319 Hepatitis viruses, comparative
prophylaxis 320 features of 466t
Gene expression 59
resistance 318 Herpangina 430, 431t
Gene therapy 72
treatment 320 Herpes genitalis 548
applications 72
Haemophilus parainfluenzae 321 Herpes gladiatorum 410
Genetic engineering 68
Halberstaedter-Prowazek/HP Herpes simplex virus 393, 409
basic tools of 69
bodies 373 association with cervical
procedure 69, 69f
Halophilic vibrios 294 cancer 500
Genetics 58
Hand-foot-and-mouth clinical features 410
Genital chlamydiasis 374
disease 430, 431t encephalitis 410
Genitourinary tract, role in
Hansen’s disease 228, 242 See also sporadic encephalitis 410
immunity 82
leprosy treatment 411
Genome 58, 59 Herpes virus 409, 411, 500
Genomics 58 Hantavirus 454
classification of 410
Gentamicin, for plague 313 Haptens 87, 89
structure 409
German measles 492 complex 87
typical structure of 409f
Gerstmann–Straussler–Scheinker/ simple 87
Herpes zoster 412
GSS disease 490 HbcAg 470
Herpis febrilis 410
Giardia lamblia 613 HbeAg 470
Heterotrophs 22
Glomerulonephritis 177 HbsAg 470, 474 Hexachlorophane 31
distinguishing features Heavy chain disease 95 Hib PRP vaccine 320
of rheumatic fever and Hemolysins 175 Histamine 144
glomerulonephritis 177t Hemolytic disease of newborn 146, Histocompatibility antigens 155
Glomerulonephritis, due to Str. 160, 162 Histocompatibility complex 122
pyogene 176 Hemophilia 72 Histoplasma capsulatum 516, 518
Glucose nonfermenters 305 Hemorrhagic fever with renal Histoplasmin skin test 517
characteristics of 306t syndrome 454, 455 Histoplasmosis 515, 516, 518
Glutaraldehyde 33 Hemorrhagic viral fevers 497 HIV infection
Glycopeptides 567 Hendra viruses 444 clinical features of 480
Gonorrhea 192 Henipavirus 440 confirmatory tests 482
prophylaxis 193 Hepadnaviruses 382 infections and malignancies
treatment 193 Heparin 144 associated 481t
www.ebook3000.com
620 | Essentials of Microbiology
postexposure prophylaxis 485 Immune response 125 Immunodiffusion 103
prophylaxis 485 cell-mediated 130 Immunodiffusion tests, types of 104
treatment 485 deficiencies of humoral and Immunoelectron microscopic
screening tests 482 cellular 141 tests 102
serological markers 481 primary 131 Immunoelectrophoresis 104
HIV virus 393, 476 secondary 131 Immunoenzyme test 115
major antigens of 477t type of 117, 125 Immunofluorescence 110
resistance 478 Immunity 4, 5, 81, 125 direct 111, 111f
routes of transmission 479, 479t acquired 81, 83, 86 indirect 111, 111f
HIV-1 476, 486 active 83, 84, 84t Immunoglobulin A/IgA 92
HIV-2 476, 486 artificial 84, 86 Immunoglobulin classes 92, 92t
HLA complex 122 natural 84, 86 Immunoglobulin D/IgD 94
HLA molecules 123 adoptive 85 Immunoglobulin deficiencies,
HLA typing 123 antibody-mediated 125 selective 138
Hookworm 544t, 597t, 610, 611, 613 cell-mediated 117, 125, 135 Immunoglobulin domain 92
Hortaea werneckii 509f humoral 125 Immunoglobulin E/IgE 94
Hospital-acquired infection 555 primary (central) organs 117 Immunoglobulin G/IgG 92
factors influencing 555 secondary (peripheral) Immunoglobulin M/IgM 94, 94f
Hot air oven 26, 26f, 27 organs 117 Immunoglobulin preparations 85
sterilization controls 27 cell-mediated 137 Immunoglobulin/Ig 90, 91
Human herpesvirus 6 415 cellular concept of 5 abnormal 94
Human herpesvirus 7 415 HERD 86 H-chains 91
Human herpesvirus 8 416 humoral or antibody- human 85
mediated 117 human serum 93t
Human T cell lymphotropic
individual 81
virus-III/HTLV-III 476 L-chains 91
innate 86
Human T-cell leukemia nonhuman 85
cellular factors in 83
(lymphotropic) Immunological response 395
mechanisms of 82
viruses/HTLV 501 Immunological tolerance 134; 135
local 86
Human virus infection, acquired tolerance 134
mechanical barriers 82
transmission of 393 natural tolerance 134
natural 81, 86
Humoral response Immunosuppressive agents 130
nonspecific 81
primary 126 Immunotherapy
passive 84t, 85
secondary 126 active 157
passive artificial 85
Hyaluronidase 79, 176 of cancer 157
passive natural 85
Hybridization 70, 589 racial 81 passive 158
Hydrogen peroxide 32 species 81 Impetigo 177
Hymenolepis nana 613 specific 81 Inactivated polio vaccine/IPV 428
Hyperimmune hepatitis B immune Immunization 4, 572 Inactivators 99
globulin/HBIG 471 active 572 Incineration 26
Hypersensitivity 142 combined 85 Incomplete immunogen 87
cell-mediated 143 influenza 86 Indole test 50f
classification of 142 National Immunization Kovac’s reagent 50
delayed 142, 148 Schedule 572, 573t Indonesian Weil’s disease 345t
granulomatous 149 passive 572 Infant death syndrome 444
immediate 142 indications of 85 Infections 75
type I 143, 146 Immunochromatographic tests atypical 76
type II 146 102, 114 cross 76
type III 147 Immunodeficiencies 137; 138 iatrogenic 76
type IV 148 cellular 138 inapparent 76
type V 149 combined 139 latent 76
Hypochlorites 32 common variable local 76
immunodeficiency 138 modes of transmission of 77
I humoral 137 nosocomial 76
primary 137, 141 primary 75
IgA, functions of 93 secondary 140, 141 route of 80
IgA, secretory 93 severe combined secondary 76
IgA, serum 93 immunodeficiency/SCID 139 subclinical 76
IgG, functions of 92 with thymoma 139 Infectious disease 75
Ilheus virus 450 Immunodeficiency diseases 137 types of 80
Immune complex disease classification of 137 Infective endocarditis 533, 535
local 147 Immunodeficiency syndromes, treatment 535
systemic 147 primary 138 Inflammation 83
Index | 621
Influenza Lachrymal fluid, role in Listeriosis 355
chemoprophylaxis 438 immunity 83 clinical features 356
complications 436 Lactobacillus 222 Live-virus vaccines 399
diagnosis of; immunity Lactoperoxidase 83 advantages of 399
against 437 Lactose fermenter 607 disadvantages 399
pandemic 586 Lamivudine 485 Loeffler’s methylene blue 593
vaccines 438 Lassa fever 493 Lopinavir 485
uncomplicated 436 Latex agglutination test 107 Louis Pasteur 2, 7
Inhibitors 99 Lawn culture 43 contributions in microbiology 2
Inspissation 27 Lazy leukocyte syndrome 140 Louse-borne typhus 363
Interferons 132t, 133, 395 LDL-receptor deficiency 72 Lowenstein-Jensen medium 40, 40f
biological effects of 396 Lecithinase-C 79 Lyme disease 343
synthesis of 395 Lectin pathway 96, 100 stages of 343
types of 395 Legionella pneumophila 307 treatment 344
Interleukins 131, 132t clinical diseases 308 Lymphadenitis 250
Intestinal candidosis 520 morphology 307 Lymphatic recirculation 119
Introns 59 treatment 308 Lymphocytes 119
Iodophors 32 Legionnaire’s disease 308 Lymphocytic choriomeningitis
Isoantigens 88 Lens protein 88, 152 virus 493
Isograft 154, 158 Lepromin test 243, 246 Lymphogranuloma venereum 374,
Isolation, virus 398 Leprosy 241 548
Isospecificities 88 borderline 242 clinical manifestations 374
classification system of Ridley Lymphoid cells 119
and Jopling 241
J forms of 241
Lymphokine-activated killer/LAK
cells 121
Japanese ‘B’ encephalitis 450 immunoprophylaxis 245
Lysogenic conversion 65
Japanese encephalitis 446t, 448t, indeterminate type 242
Lysozyme 15
449, 450 lepromatous 242
Lyssavirus sera 463t
Jarisch–Herxheimer reaction 341 prophylaxis 245
JC virus 422,490, 500t tuberculoid (TT) 242
Job’s syndrome 140 Leptospira 344, 347 M
Joseph Lister 3 antigenic properties 345
MacConkey’s agar 606
classification 344
Macrolides 568
resistance 344
K Leptospiral infections 345
Macrophages 83, 121
functions of 122
Kaposi’s sarcoma 476 Leptospirosis 345
polymorphonuclear 122
Kaposi’s sarcoma-associated treatment 346
Maedi 489
herpesvirus 500 Leptotrichia 223
Magic bullet 5
Kawasaki syndrome 89 Leptotrichia buccalis 541
Major histocompatibility
Kelsey-Sykes test 34 Leukemia 4
complex 88
Keratomycosis 525, 526 Leukemia inhibitory factor 133
Leukocidin 166 Malignancy
Kerion 511
Leukocyte G-6-PD deficiency 140 immune response in 157
Killed viral vaccines 399
Leukocyte-associated antigen 88 immunology of 156
Kingella 195
Leukosis-sarcoma viruses 501 Mallein test 305
Kirby–Bauer disk diffusion
Leukotrienes 145 MALT See mucosa-associated
method 559, 560, 561f
LFNS See interferons 132t lymphoid tissue
interpretation chart 562t
Kissing disease 414, 416 LGV biovar 373 Marburg virus 494
Klebsiella granulomatis 357 LIF See leukemia inhibitory Marek’s disease 500
Klebsiella pneumoniae 534, 542t factor McCrady probability table 577t
Koch and Arnold steamer 27 Ligase chain reaction/LCR 589 Measles 443
Koch’s phenomenon 3, 235 Lincosamides 568 clinical features 443
Koch’s postulates 3 Liquid culture 44 complications 443
Koch-Weeks bacillus 320 Listeria ivanovii 355 morphology; prophylaxis 444
treatment 320 Listeria monocytogenes 355, Membrane attack complex 98
Kuru 489 360, 534 Meningitis 535
Kyasanur forest disease 453 laboratory diagnosis 357 aseptic 537
morphology 355 causative agent 536t, 537t
treatment 356 CSF findings in 537
L Listeria seeligeri 355 due to H. influenzae 319
LA crosse virus 454 Listeria, distinguishing examination of CSF 536
Lac operon 61 characters of 356t purulent 535, 538
of Escherichia coli 60f Listeriolysin-O 356 tuberculous 538
www.ebook3000.com
622 | Essentials of Microbiology
Meningococcal septicemia 189 Morbillivirus 440, 441t Nasopharyngeal carcinoma 414
Mercuric chloride 32 Morganella 269 Negative staining for capsules 594
Messenger RNA 59 Morganella morganii 269 Neisser’s stain 594
Metapneumovirus 445 Mouth or oral cavity, role in Neisseria 188
Methyl red/MR test 50, 50f immunity 82 commensal 194, 195
Metronidazole; for Clostridium Mucormycoses 523 differential characteristics of 194r
difficle 219 clinical manifestations 523 Neisseria gonorrhoeae 191, 195
Microbial growth, requirements rhinocerebral 523 cultural characterstics 191
for 22 thoracic 523 morphology 191
Microbiology 2 treatment 524 opa protein 191
contributions of Louis Mucosa-associated lymphoid pili 191
Pasteur in 2 tissue 119 por proteins 191
scientific development of 2 Multiple myeloma 95 proctitis 192
Micrococci 170 Multiresistant salmonellae 285 resistance 192
Microorganisms 2 Mumps 441t, 442 rmp 191
role in diseases 2 Murray valley encephalitis types of 191
Microphages 122 virus 450 Neisseria meningitidis 188, 195, 534
Microscope 8 Mutagens 62 cultural characteristics 188
electron 10 Mutation 62 laboratory diagnosis 189
scanning 10 conditional 62 prophylaxis 190
transmission 10 frameshift 61f, 62 resistance 189
Microscopy 8 induced 62 stages of infections 189
lethal 62 treatment 190
atomic force 10
missense 62 Neorickettsia 366
compound light 8
nonsense 62 Neorickettsia sennestu 367
confocal 9
recognition of 62 Neosalvarsan 4, 5
darkground 9
spontaneous 62 Neutralization tests 110
differential interference
types of 62 toxin neutralization 110
contrast 9
Mycetism 512, 528 viral neutralization 110
electron 9, 10, 397
causative agents 512 Nichol’s strain 338
epifluorescence 9
Mycobacteria 227 Nicotinamide adenine
fluorescence 9, 397
classification of 227t dinucleotidase 176
immunoelectron 397
Mycobacteriophages 230 Nipah virus 440, 444
light 8, 397 Mycobacterium gordonae 248 Nitrate reduction test 51, 52f
phase-contrast 9 Mycobacterium leprae 240, 246 Nitrofurans 567
scanned-probe 10 morphology 240 Nitroimidazoles 567
scanning tunneling 10 Mycobacterium lepraemurium 245 NK-cells 121, 123
scanning-probe 10 Mycobacterium marinum 248 Nonsporing anaerobes
Microsporum 510 Mycobacterium szulgai 248 classification of 221t
M. audouinii 510 Mycobacterium tuberculosis 70, Nongonococcal urethritis 193, 195
M. canis 510 228, 238 Nonlactose fermenter/NLF
M. gypseum 510 cell wall of 230f colonies 607
Mild virus-like syndrome 345 cultural characteristics 228 Nonphotochromogens 248
Milk ring test 331 distinguishing features of Nonsporing anaerobes 221
Milk mycobacterium tuberculosis Nontuberculous mycobacteria 247
bacteriological and M Bovis 229t differentiating characterstics
examination of 578 multidrug resistant 237 of M. tuberculosis and
pasteurization of 27 Mycoplasma 12, 12f nontuberculous
Milk-borne diseases 578, 578t pneumoniae 542, 542t, 543 mycobacteria 247
Milker’s node 408 Mycoses 506 Runyon classification
Minimum infecting dose 80 classification of 506 scheme 248
Minimum inhibitory cutaneous 509 treatment 251
concentration/MIC 34 subcutaneous 511 Normal flora 530
Minimum lethal dose 80 superficial 508 harmful effects of 530
Mitogens 130 Mycotoxicosis 528 of conjunctiva 531
MMR vaccine 84, 444, 445, 493, 571 Mycotoxins 528t of gastrointestinal tract 531
Mobiluncus 222 Myeloperoxidase/MPO of genitourinary tract 532
Molluscipoxvirus 406t deficiency 140 of nose, nasopharynx and
Molluscum contagiosum 408 accessory sinuses 531
Monkeypox 408 of upper respiratory tract 531
N
Monocytosis 355 role of 530
Mononuclear cells 121 Nagler’s reaction 220, 213f Novobiocin 568
Moraxella lacunata 195 Nairovirus 389, 453, 454 Nucleic acid probes 70
Index | 623
Nucleic acid sequence based Paul Ehrlich 3, 6t, 7 differential characters of
amplification/NASBA 589 Paul-Bunnel test 89, 107, 416 pneumococci and viridans
Nucleic acid structure 58 PCR, applications of 71, 73t streptococci 186t
Nucleotides 58 Penicillin laboratory diagnosis 185
Null cells 121 for Clostridium perfringens 215 meningitis 185
Nutrient agar 38 for lyme disease 344 pathogenesis 185
Nutrient broth 38 for Pneumococcus 186 pneumonia 185
for syphilis 341 prophylaxis 186
mechanisms of action 566 resistance 184
O neisseria meningitidis 190 treatment 186
Oncogenic viruses 499, 500t, 502 Penicilliosis 524 virulence factors 185
DNA viruses 499 Peplomers 380 Pneumocystis carinii
RNA viruses 500 Pepsin digestion 90 pneumonia 476
Onychomycosis 520 Peptococcus 221 Pneumocystis jiroveci 519, 524, 526
Onyong-nyong virus 449 Peptone water 38 life cycle of 525f
Ophthalmic neonatorum 192 Peptostreptococcus 222 Pneumonia 542
Opportunist mycobacterial Pertussis toxin/PT 324, 326 community-acquired 543
disease 249t Pertussis vaccine, acellular 325 due to H influenzae 319
Opportunistic fungi 519 Pfeiffer phenomenon 96 hospital-acquired 542
Opsonization 99, 102, 100, 110 Phaeohyphomycosis 513 in immunocompromized
Oral hairy leukoplakia 414 Phagocyte, disorders of 140 patients 543
Oral polio vaccine/OPV 428 Phagocytic cells 121 Pneumonia syndrome
differences between killed/IPV Phagocytosis 83 atypical 543
and live polio Phenylalanine deaminase test 53 typical 543
vaccines/OPV 429 Phlebovirus 389, 446t, 453, 454 Pneumovirus 440, 444
Orbivirus 498 Phosphorylation 23 Poliomyelitis
Orthomyxovirus 434 oxidative 23 abortive 427
differences between substrate-level 23 global eradication 429
orthomyxovirus and Photochromogens 248 nonparalytic 427
paramyxovirus 434t Picornaviridae 389, 426, 432 paralytic 427
properties of434 classification 426 progressive
Orthopoxvirus 406t properties of 426t postpoliomyelitis 427
Orthoreovirus 454, 496 Piedra 509 Poliovirus 427, 432
Oseltamivir, for influenza 438 Piedraia hortae 508, 509 antigenic properties 427
Otomycosis 525, 526 Pigeon fancier’s disease 147 clinical features 427
Oxidase test 52 Pili 17 resistance 427
Pinta 334, 341 Polychrome methylene blue 593
Pityriasis versicolor 508 Polymerase chain reaction 71, 72f,
P morphology of 508f 589
Pandemic 80 Plague 311, 315 Polyomaviruses 388, 421, 422
Papillomatosis bubonic 311 Ponder’s stain 594
oral 421 chemoprophylaxis 313 Pontiac fever 308
recurrent respiratory 421 laboratory diagnosis 312 Porphyromonas 223
Papillomaviruses 421 pathogenesis 311 Potassium cyanide test 53
Papovaviruses 499 pneumonic 311 Pour-plate culture 44
Paracoccidioides brasiliensis 516 prophylaxis 312 Poxvirus 382, 406, 500
Paracoccidioidomycosis 515 septicemic 311 antigenic structure 407
Parainfluenza viruses 441 treatment 313 morphology 406
Paramyxoviruses 440 vaccine 313 Prausnitz–Kustner/PK reaction 146
morphology 440 Plasma cells 121 Precipitation 102
Parapoxvirus 406t Plasmid transfer 65 mechanism of 103
Parasites 75 Plasmids 18, 60, 79 reaction 103
Paratyphoid bacilli 275 conjugative 60, 67 types of 103
Paratyphoid fever 279 nonconjugative 60 Primary sensitivity tests 563
Parvovirus 424, 587t Platelet-activating factor 145 Primary tuberculosis 231
Pasteurella multocida 314, 315 Pleomorphism 19 Prion diseases 489
morphology 314 Plesiomonas 295 Prions 35, 390
Pasteurization 2 Pneumococcus 183 Progressive multifocal
Pathogens bacteremia 185 leukoencephalopathy/
opportunist 75 biochemical reactions 184 PML 490
primary 75 bronchopneumonia 185 Prokaryotes 11
types of 75 cultural characteristics 183 differentiation from eukaryotic 11t
www.ebook3000.com
624 | Essentials of Microbiology
Properdin pathway 96 Reoviridae 496 Rose-Waaler test 107
Propionibacterium 222 classification 496 Ross river virus 449
Prostaglandin 145 Reovirus 498 Rotavirus 496, 498
Proteases 145 Replica plating 63 Rotavirus diarrhea 496
Proteinase 176 Resistance factors/R factors 67 treatment 497
Proteus bacilli 267 characteristics of 67 Rubella 492
Proteus sp 267, 534t, 606 features of 67 clinical features 492
Providencia 269 Resistance transfer factor /RTF 67 prophylaxis 493
Prozone phenomenon 330 Resolution 8 Rubella vaccines 493
Pseudomonas aeruginosa 301, 306 Respiration Rubella virus 449, 492, 497
antigenic characteristics 302 aerobic 23 Rubivirus 492
pathogenesis 303 anaerobic 23
pigment production 302 Respiratory syncytial virus 440, S
resistance 303 441, 444
treatment 303 clinical features 444 S. delphini 169
Psoriasis 89 treatment 445 S. dysenteriae 271
Psychotropic agents 529 Respirovirus 440, 441t S. epidermidis 163
Pugh’s stain 594 Retroviruses 476 clinical infection 169
Purines 58 genes 477, 478t S. haemolyticus 169
Pyemia 80, 533, 535 structure 476 S. hyicus 169
Pyocin 66 Retroviruses 477t, 501 S. intermedius 168
Pyocyanin 302 Reye’s syndrome 436 S. lutrae 169
Pyomelanin 302 Rh (rhesus) blood group S. saprophyticus 163
Pyorubrin 302 system 160 S. sonnei 271
Pyoverdin 302 Rh antigens 162 Salmonella 274, 285
Pyrimidines 58 Rh compatibility 160 antigenic structure of 275, 275f
Pyrogallic acid 45 Rh factor 160 morphology 274
Rhabdoviridae 455 syndromes due to 279
Rhabdoviruses 457 Salmonella gastroenteritis 285
Q
Rheumatic fever 89 Salmonella septicemia 285
Q fever 367 due to STR Pyogene 176 Salmonella Typhi 534t
Quaternary ammonium Rhinosporidiosis 513 Salvarsan 4, 5
compounds/QUATS 31 Rhinoviruses 432 Saprophytes 75
Quellung-Ger swelling 16 clinical syndromes 432 Saprophytic mycobacteria 249
Rh-isoimmunization 161 Sarcoma, in fowls 4
R prevention of 161 SARS See Severe acute
Ribavirin 496 respiratory syndrome
R plasmids 67, 79, 570 Ribonucleic acid 58 Satellitism 318, 318f, 322
Rabies 4, 459 structure 59 Scarlet fever 176
clinical features 459 Ribosomes 59 Schultz-Dale phenomenon 144
postexposure prophylaxis 460, Rickettsia 362 Schwartzman reaction 149
462, 463 antigenic structure 363 Scotochromogens 248
preexposure prophylaxis 460, morphology 362 Scrapie 489
462, 463 resistance 363 Semliki forest virus 449
vaccines 3, 461 Rickettsia akari 365, 369 Sensitivity 102
Rabies virus 457, 459f Rickettsia conorii 365, 369 Septic shock syndrome 149
antigenic properties 458 Rickettsia mooseri 369 Septicemia 80, 533
resistance 458 Rickettsia prowazekii 369 Serotherapy 5
Radiations 29 Rickettsia rickettsii 369 Serotonin 144
ionizing 30 Rickettsia typhi 363 Serum opacity factor 176
non-ionizing 29 differences between R. typhi Serum sickness 147
Radioimmunoassay 111 (R. mooseri) and Severe acute respiratory
Rapid plasma reagin/RPR R. prowazekii 364 syndrome 495, 497, 498
tests 337, 338 Rickettsial diseases 365 Severe combined
Rat-bite fever 358 treatment 366 immunodeficiency disease/
Reagents 600 Rickettsial pox 365 SCID 72
Redox potential 24 Rideal-Walker test 34 Sexduction 66
Reiter protein complement Rifamycins 567 Sexually transmitted diseases/
fixation/RPCF test 338 Rimantadine 400 STDs 547
Reiter’s syndrome 374 Ring test 103 organisms causing 548t
Relapsing fever 342 Ritonavir 485 Shanghai fever 303
endemic Robert Koch 3, 5t, 7 Shigella 270
or louse-borne 342 Rocky mountain spotted cultural characteristics 270
or tick-borne 342 fever 364, 365 laboratory diagnosis 272
Index | 625
Shigella boydii 271 Staphylococcus aureus 534t Stroke culture 44
Shigella flexneri 271 Staphylococcus epidermidis 169 Subacute endocarditis 181
Shock organs 143 Staphylococcus saprophyticus 169 causative agents of 534t
Shwachman’s disease 140 Stenotrophomonas maltophilia 304 Subacute sclerosing
Silver nitrate 32 Sterilization 25, 35 panencephalitis/SSPE 393, 490
Sindbis virus 449 dry heat 26, 26f Subcutaneous phycomycosis 514
Skin, role in immunity 82 intermittent 28 Sugar fermentation 49, 49f
Slide test 103 low temperature steam Sulfonamides, mechanism of
Slides 592 formaldehyde/LTSF 27 action of 568
Slot-blot and dot-blot assays 114 methods of 25, 26t Superantigens 89, 130
Slow virus disease 488, 490 moist heat 27 Superinfection immunity 404
classification 488 procedures of 34t Suppurative streptococcal disease 176
Smallpox 406 Sterilizing cycle 26 Surface-active agents 31
control of 407 Stokes disk diffusion Swimming pool granuloma 250
Sore mouth 408 method 559, 561 Swineherd’s disease 345t
Sore throat 541 Streak culture 43, 43f Syphilis 334, 335, 547
causative agents of 541t Street virus 458 acquired nonvenereally 336
South American hemorrhagic Streptobacillus moniliformis 358 associated with human
fevers 493 morphology 358 immunodeficiency virus/
Specificity 102 treatment 359 HIV infections 340
antigenic 88 Streptococcal gangrene 177 congenital 336
autospecificity 88 Streptococcal toxic shock diagnosis 109
heterogenetic 88 syndrome 177 diagnostic tests for 337t
organ 88 Streptococci 172, 174 endemic 334
species 88 immunity in 340
Alpha (α) hemolytic 173
Spirilla 12, 12f latent 335
beta (β) hemolytic 173
Spirillum minus 359
classification 172 primary 335
morphology 359
gamma (γ) or prophylaxis in 340
treatment 359
non-hemolytic 173 secondary 335
Spirochetes 12, 12f, 333
group B 179 tertiary 336
classification 333
group C 179 treatment 341
diseases caused by 334
group D 180 venereal 334
Spleen 119
group G 179
functions of 119
Spontaneous generation 1
Streptococcus agalactiae 174, 179, 181 T
Sporotrichosis 513 Streptococcus bovis 174
Streptococcus equisimilis 174 Taenia sp. 612
Spotted fever group 364
Streptococcus mitis 174 Taxonomy 56
Stab culture 44
Streptococcus mutans 174 T-cell activation 127
Stain
Streptococcus pneumoniae 183, T-cells 119, 120
differential 593
534t, 535 basis of functions 120
special 594
Streptococcus pyogenes 173, 174t, basis of surface markers 120
types of 592, 593
181, 534t, 605 blast transformation 119
Staphylococcal diseases 167
acute suppurative infections 178 cytotoxic 120
cutaneous infections 167
deep infections 167 antigenic structure 174, 175f delayed type-
toxin-mediated diseases 167 cultural characteristics 173 hypersensitivity 120
Staphylococcus 163, 605 enzymes 176 differentiation from B-cells 119
bacteriophage typing 167 laboratory diagnosis 178 regulatory 120
differentiation between morphology 173 suppressor 120
staphylococci and non-suppurative types of 120
micrococci 170 complications 178 TEGO compounds 31
epidemiology 167 nonsuppurative sequelae 177 Tetanolysin 216
Staphylococcus aureus 163, 170, 535 pathogenesis 176 Tetanospasmin 216
biochemical reactions 164 prophylaxis 179 Tetanus 216
cell surface proteins 165 pyrogenic exotoxins 175 laboratory diagnosis 216
cell-associated polymers 165 resistance 174 prophylaxis 217
cultural characteristics 164 toxins 175 treatment 217
enzymes 165 treatment 179 Tetanus toxin 216, 220
methicillin-resistant 170 Streptococcus salivarious 174 Tetracycline, for plague 313
morphology 163, 163f Streptokinase 79, 176 TGF See tumor necrosis factors
resistance 165 Streptolysin O 175 Th1 cells 120
sensitivity to antibiotics 169 Streptolysin S /SLS 175 Th2 120
toxins 165 Streptomycin, for plague 313 Thioglycollate broth 41, 46
www.ebook3000.com
626 | Essentials of Microbiology
Thymus 117 Treponema pallidum immune Vancomycin
Tick typhus 364 adherence/TPIA test 339 for Clostridium difficle 219
Tick-borne encephalitis viruses/ Treponema pallidum particle for Pneumococcus 186
TBEV 452 agglutination/TP-PA 340 Varicella 412
Tick-borne hemorrhagic fevers 453 Treponema vincentii 541 Varicella-zoster
Tinea barbae 511 Treponemal tests 338, 347 immunoglobulin 413
Trichophyton 509 Varicella-zoster virus 393, 411, 416
Tinea corporis 511
Trichosporon beigelii 508 properties of 412
Tinea cruris 511
Trichuris trichiura 597t, 610, 611 prophylaxis and treatment 413
Tinea nigra 508 Triple-sugar iron/TSI agar 53, 54t Vectors 76, 77, 584
Tinea pedis 511 Tsutsugamushi disease 365 Vectors-borne diseases 584
Tinea unguium 511 Tube flocculation test 103 Vehicles 584
Tissue culture 384, 390 Tuberculin test 235, 238 Veillonella 222
Tobacco mosaic disease 4 Tuberculosis 231 Veneral disease research
Togaviruses 382, 447, 453, 492 extrapulmonary 234 laboratory/VDRL test 337
Toluidine red unheated serum test/ postprimary (secondary) 231 Venezuelan equine encephalitis/
TRUST 338 pulmonary 231 VEE 446t, 449, 455
Torch agents 77 Tuberculosis complex 227 Vertical transmission 77
Toxic shock syndrome toxin-I/ Tumor antigens 156 Vesicoureteral reflux 539
TSTT-1 166 oncofetal 156 Vibrio cholerae 287, 290, 544
Tumor associated carbohydrate morphology 287
Toxoids 79
antigens 156 pathogenesis 291
Trachoma 373
Tumor necrosis factors 132t, 133 resistance 288
Trachoma biovar 373, 377
Tumor-associated transplantation subtypes of 290
Transcobalamin II deficiency 138 antigens/TATAs 156 Vibrio parahaemolyticus 294, 544
Transcription 59 Tyndallization 28. 35 Vibrio vulnificus 295
Transcription mediated Typhoid fever 279 Vibrios 12, 12f
amplification/TMA 589, 590 laboratory diagnosis of 280 Vincent’s angina 343
Transduction 64, 68 prophylaxis 283 Viral assay 386
generalized 65 vaccines against 283 Viral capsid 379
role of 65 Typhus fever 369 Viral diseases
specialized 65 immunoprophylaxis of 399
types of 65 U pathogenesis of 393
Transfer factor 133, 135 Viral envelope 379
Ulcerative gingivostomatitis 343 Viral genetics 387
Transfer RNA/TRNA 60
Urease test 52, 52f Viral hemagglutination 381
Transformation 68
Urinary tract infection 538 Viral hemorrhagic fevers 493
Transforming growth factor causes of 539t Viral hepatitis 465
beta 132t, 133 clinical features 539 Viral infections
Transient differentiation of upper UTI and indications for 397
hypogammaglobulinemia of lower UTI 540 laboratory diagnosis of 397
infancy 137 lower 538 Viral nucleic acids 380
Transmissible spongiform tuberculosis of kidney and Viral oncogenes 502
encephalopathies 489 urinary tract 540 Viral oncogenesis 4
Transplantation 5, 158 upper 538 Viral proteins, detection of 398
Transplants 154 Viral replication 381
types of 154 V Viral vaccines, in common use 399t
Transposons 68 Viridans streptococci 180
structure of 68, 69f V factor 318 classification 181
Treponema 334 334 Vaccines 2, 84, 571 clinical infections 181
attenuated anthrax 2 Viroids 390
nonpathogenic 341
bacterial 84 Virulence 78
Treponema carateum 334
cellular fractions 571 determinants of 78
Treponema endemicum 334
chicken cholera 2 Virus diseases
Treponema pallidum 9, 334 killed 84, 85, 571 chemoprophylaxis and
agglutination/TPA test 338, 339 live 4, 84, 571 chemotherapy of 400
antigenic structure 335 mixed or combined 572 Virus infections 395
hemagglutination/TPHA multiple 129 antibody-mediated
107, 338, 339 recombinant-vector 572 immunity 395
immobilisation/TPI test 338 subunit 84 cell-mediated immunity 395
morphology 334 toxoids 571 immunological response 395
pathogenesis 335 viral 84 nonimmunological
resistance 335 Vaccinia virus 407 responses 395
Index | 627
Virus neutralization 98 Water, peptone 38t Yaws 334
Virus symmetry 379 Water-borne diseases 576t Yeast 519, 503
complex symmetry 379 Waterhouse–Friderichsen Yeast-like fungi 503, 519
helical symmetry 379 syndrome 149 Yeasts 503
icosahedral symmetry 379 Watson-Crick structure, of Yellow fever 4
Viruses 4, 390, 378 DNA 59f Yellow fever virus 449, 451, 455
baltimore classification of 382 Weil-Felix reaction 89, 107, 366, 369 Yersinia 309
cultivation of 383 West nile virus 450 Yersinia enterocolitica 314, 315, 545
direct detection of 397 Western blot test 482 Yersinia pestis 309
discovery of 4 Western equine encephalitis/WEE biotypes of 310f
DNA, double stranded 382 448t, 449, 455 morphology 309
double stranded 382 White piedra 509 resistance 310
incubation period of 394 Whooping cough 324 toxins 310
morphology of 378 complications 324 virulence factors 310
properties of 378 stages of 324 Yersinia pseudotuberculosis 313
RNA 389, 389f Widal test 107, 282 treatment 314
single stranded 382 Wilson and Blair medium 40 Yersiniosis 313
Visna 488 Wiskott–Aldrich syndrome 139
Vitronectin 99
Voges-Proskauer/VP test 50, 51f Z
X
Zaire strain 494
W Xenograft 154, 158
Zanamivir, for influenza 439
X-linked agammaglobulinemia 137
Waldenstrom’s X-linked hyper-IgM syndrome 138 Zidovudine 400, 485
macroglobulinemia 95 Ziehl-Neelsen staining 48, 593, 598
Warts 421 Zone phenomenon 102
Y Zoonosis 76
anogenital 421
cutaneous 421 Yatapoxvirus 406t Zygomycosis 523, 526
www.ebook3000.com