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Burton's Microbiology for the Health Sciences

Chapter 13. Diagnosing Infectious Diseases

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Chapter 13 Outline
• Introduction • Clinical Specimens • Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases • The Pathology Department (“The Lab”) – – Anatomical Pathology Clinical Pathology

Role of Healthcare Professionals in the Submission of Clinical Specimens
Importance of High-Quality Clinical Specimens Proper Selection, Collection, and Transport of Clinical Specimens Contamination of Clinical Specimens with Indigenous Microflora

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• The Clinical Microbiology Laboratory – – Organization Responsibilities

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Introduction
The proper diagnosis of an infectious disease requires: 1. Taking a complete patient history 2. Conducting a thorough physical examination of the patient 3. Carefully evaluating the patient’s signs and symptoms 4. Implementing the proper selection, collection, transport, and processing of appropriate clinical specimens
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Clinical Specimens
• Specimens collected from patients such as blood, urine, feces, and cerebrospinal fluid (CSF), are known as clinical specimens.

• Specimens commonly submitted to the hospital’s Clinical Microbiology Laboratory (CML) include: blood, bone marrow, bronchial washings, sputum, CSF, cervical and vaginal swabs, feces, hair and nail clippings, pus, skin scrapings, sputum, synovial fluid, throat swabs, tissue specimens, urethral discharge material, urine, and urogenital secretions.
• All specimens should be of the highest possible quality!
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Clinical Specimens, cont.
• The Role of Healthcare Professionals in the Submission of Clinical Specimens: – There should be a close working relationship among the members of the healthcare team to ensure a proper diagnosis of infectious diseases. – Healthcare professionals who collect and transport specimens should exercise extreme caution during the collection and transport of specimens. – In the laboratory, all specimens are handled carefully, exercising Standard Precautions.
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Role of Healthcare Professionals in the Submission of Clinical Specimens, cont.
• Although laboratory professionals do not themselves make diagnoses, they make laboratory observations and generate test results that assist clinicians to correctly diagnose infectious diseases and initiate appropriate therapy.

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Importance of High-Quality Clinical Specimens
• High-quality clinical specimens are required to achieve accurate, clinically relevant laboratory results. • The 3 components of specimen quality are:

• Proper specimen selection
• Proper specimen collection • Proper transport of the specimen to the laboratory

• The laboratory must provide written guidelines (“Laboratory Policies and Procedures Manual”).
• The person who collects the specimen is ultimately responsible for its quality.
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Proper Selection, Collection, and Transport of Clinical Specimens
• Specimens must be properly selected. • Specimesn must be collected properly. • Material (i.e., specimens) should be collected from a site where the suspected pathogen is most likely to be found. • Specimens should be obtained before antimicrobial therapy, if possible. • The acute stage of the disease is the most appropriate time to collect a specimen. • Specimen collection should be performed with care and tact to avoid harming the patient.

• A sufficient quantity of the specimen must be obtained to provide enough material for all required diagnostic tests.

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Proper Selection, Collection, and Transport of Clinical Specimens, cont.
• All specimens should be placed or collected into a sterile container to prevent contamination. • Specimens should be protected from heat and cold and promptly delivered to the laboratory. • Whenever possible, a sterile, disposable specimen container should be used. • The specimen container must be properly labeled and accompanied by an appropriate request slip with adequate instructions.

• Hazardous specimens must be handled with even greater care to avoid contamination of couriers, patients, and healthcare professionals.

• Specimens should be collected and delivered to the lab as early in the day as possible to allow sufficient processing time.

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Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases
• Blood – Usually sterile

– The presence of bacteria in the bloodstream is known as bacteremia.
– Septicemia is a serious disease involving chills, fever, prostration, and the presence of bacteria or their toxins in the bloodstream. – To prevent contamination of a blood specimen with indigenous skin flora, extreme care must be taken to use aseptic technique.
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Proper Method of Preparing the Venipuncture Site When Obtaining Blood for Culture

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Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.
• Urine – Normally sterile in the bladder; becomes contaminated by indigenous microflora of the distal urethra during voiding. – Contamination is reduced by collecting a clean-catch, midstream urine. – Urine culture involves 3 parts: • A colony count (using a calibrated loop) • Isolation and identification of the pathogen • Antimicrobial susceptibility testing
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Urine Colony Count
• The colony count is a way of estimating the number of viable bacteria that are present in a urine specimen. • A calibrated loop, either 0.01 mL or 0.001 mL, is used to inoculate the entire surface of a blood agar plate. • After incubation at 37oC overnight, the colonies are counted and the number is multiplied by the dilution factor (either 100 for the 0.01 mL loop, or 1000 for the 0.001 mL loop) to determine the number of colonyforming units (CFUs). – # Colonies x dilution factor = # CFUs/mL
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Obtaining a Urine Colony Count

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Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.
• Cerebrospinal Fluid (CSF) – Meningitis is inflammation or infection of the membranes (meninges) that surround the brain and spinal column. – Encephalitis is inflammation or infection of the brain. – Meningoencephalitis is inflammation or infection of both the brain and meninges. – CSF is collected by lumbar puncture into a sterile tube; this is a surgically aseptic procedure performed by a physician. – CSF is considered a STAT (emergency) specimen in the lab! Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Technique of Lumbar Puncture

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Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.
• Sputum – Sputum is pus that accumulates deep within the lungs of a patient with pneumonia, tuberculosis, or other lower respiratory infection. – Often, specimens labeled “sputum” are actually just saliva; saliva specimens don’t provide clinically relevant information. – If TB is suspected, extreme care should be taken! – Better specimens can be obtain by bronchial aspiration or transtracheal aspiration.
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Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.
• Throat Swabs – Routine throat swabs are used to determine whether a patient has strep throat – Specific cultures may be necessary when Neisseria gonorrhoeae or Corynebacterium diphtheriae are suspected • Wound Specimens – Whenever possible, a wound specimen should be an aspirate (i.e., pus collected by needle and syringe), rather than a swab

– Specimens collected by swab are frequently contaminated with indigenous microflora

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Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.
• GC Cultures (for Neisseria gonorrhoeae) – N. gonorrhoeae is a fastidious, microaerophilic, and capnophilic bacterium. – Only Dacron, calcium alginate, or nontoxic cotton swabs should be used to collect GC specimens. – Specimens (e.g., vaginal, cervical, urethral, throat, and rectal swabs) are cultured on special medium (e.g., Thayer-Martin medium) and incubated in a CO2 incubator. – Special transport media are available and GC swabs should never be refrigerated.
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Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.
• Fecal Specimens

– Ideally, fecal (stool) specimens should be collected at the laboratory and processed immediately to prevent a decrease in temperature, which would allow the pH to drop and cause the death of many Shigella and Salmonella species. – Bacteria in fecal flora are obligate-, aerotolerant-, and facultative anaerobes.
– A combination of direct microscopic examination, culture, biochemical tests, and immunologic tests may be performed to identify Gram-negative and Gram-positive bacteria, fungi, intestinal protozoa, and intestinal helminths isolated from fecal specimens.
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The Pathology Department (“The Lab”)
• Clinical specimens are submitted to the Clinical Microbiology Laboratory (CML), which is a part of the Pathology Department.

• The Pathology Department (often referred to as “the Lab”) is under the direction of a pathologist (a physician who has specialized training in pathology).
• The pathology department is divided into 2 major divisions: – Anatomical Pathology – Clinical Pathology
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The Pathology Department, cont.
• Anatomical Pathology – Diseased organs, stained tissue sections, and cytology specimens are examined here. – Cytogenetic technologists, cytotechnologists, histologic technicians, histotechnologists, and pathologist’s assistants are employed in this division.

– In addition, autopsies are performed in the morgue and some Pathology Departments have an Electron Microscopy Laboratory.

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The Pathology Department, cont.
• Clinical Pathology – Consists of several laboratories in addition to the Clinical Microbiology Laboratory: Clinical Chemistry, Urinalysis, Hematology/Coagulation, Blood Bank, and Immunology. – Personnel include pathologists, chemists and microbiologists, clinical laboratory scientists (also known as medical technologists - MTs), and clinical laboratory technicians (also known as medical laboratory technicians - MLTs).

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The Clinical Microbiology Laboratory

The CML may be under the direction of a pathologist, a microbiologist, or a senior clinical laboratory scientist.
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The Clinical Microbiology Laboratory, cont.
• Responsibilities – Primary mission of the CML is to assist clinicians in the diagnosis and treatment of infectious diseases.

– The 4 major day-to-day responsibilities are to:
• Process various clinical specimens that are submitted to the CML

• Isolate pathogens from those specimens
• Identify (speciate) the pathogens • Perform antimicrobial susceptibility testing, when appropriate to do so
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Isolation and Identification (Speciation) of Pathogens
• Bacteriology Section – Bacterial pathogens are isolated from specimens, tests are performed to identify the bacterial pathogens, and antimicrobial susceptibility testing is performed whenever appropriate to do so. – CML professionals are very much like detectives and crime scene investigators, in that they gather clues about a pathogen until they are able to identify it. – Numerous phenotypic characteristics are used to identify the bacteria (e.g., Gram reaction, cell shape, motility, presence and location of spores, presence or absence of various enzymes, etc.)
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CML professionals are very much like detectives and crime scene investigators -- gathering clues about a pathogen until they have enough information to identify it.

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Minisystems Used to Identify Bacteria

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API-20E for identification of Enterobacteriaceae

Enterotube II for identification of Enterobacteriaceae

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Diagram Ilustrating the 3 types of Hemolysis That Can be Observed on a Blood Agar Plate

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Isolation and Identification (Speciation) of Pathogens, cont.
• Mycology Section – Responsibility is to assist clinicians in the diagnosis of fungal infections (mycoses)

– The specimens processed here are the same as those that are processed in the Bacteriology Section, with the addition of hair and nail clippings and skin scrapings. – A variety of procedures are used to identify fungal pathogens including special media, KOH preps, tease mounts, biochemical tests (for yeasts), and a combination of microscopic and macroscopic observations (for moulds).
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A Colony (Mycelium) of the Mould Aspergillus fumigatus, a Common Cause of Pulmonary Infections in Immunosuppressed Patients

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Colonies (Mycelia) of a Penicillium Species

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Isolation and Identification (Speciation) of Pathogens, cont.
• Parasitology Section – Assists clinicians in the diagnosis of parasitic diseases – Parasites are identified by observing and recognizing various parasite life cycle stages (e.g., trophozoites, cysts, microfilariae, eggs, larvae, adult worms) in specimens – identified primarily by their physical appearance (e.g., size, shape, internal details) • Virology Section – Assists clinicians in the diagnosis of viral diseases – Techniques used in the identification of viruses include immunodiagnostic tests, cytologic or histologic examination, electron microscopy, molecular techniques, virus isolation by cell cultures, and cytopathic effect (CPE)

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Isolation and Identification (Speciation) of Pathogens, cont.
• Mycobacteriology Section (also called the TB Lab) – Assists clinicians in the diagnosis of tuberculosis (TB)

– Various types of specimens are submitted, but sputum is the most common type
– Mycobacterium spp. are identified by the acid-fast staining procedure and by using a combination of growth characteristics (e.g., growth rate, colony pigmentation, photoreactivity, and morphology) and a variety of biochemical tests

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