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Mycobacterium
MYCOBACTERIUM TUBERCULOSIS Secondary TB
 Occurs in adults (latent infection – reactivation)
 Acid fast organism  It mainly affects the upper lobe
 Gram-positive bacilli  It leads to necrosis, tissue destruction and cavity formation
 Slender rods  They expectorate plenty of bacilli leading to spread of infec-
 Has characteristic presence of mycolic acid in cell wall tion
 Slow growing, nonmotile, and noncapsulated  Widespread dissemination occurs in immunodeficient indi-
viduals
 Mycobacterium tuberculosis differs from other organisms by
not being visualized in Gram staining
 It needs special staining called as acid-fast staining
 Discovered by Robert Koch
 Acid fastness is because of presence of unsaponifiable lipid-
rich waxy mycolic acid that is seen in the cell wall
 M. tuberculosis is both acid (20–25% sulphuric acid) and
alcohol (3% hydrochloric acid) fast

Acid fast organisms:

Mycobacterium
Nocardia
Rhodococcus
Legionella micdadei
Coccidiean parasites
Figure 1: HPE of a TB granuloma (Courtesy: Dr S.Jamuna Rani,
MD (Pathology), Associate professor, Tagore Medical College and
Pathogenesis Hospital, Chennai)
 Mode of infection is by droplet nuclei (mainly the smallest
droplets <5–10 μm in diameter)
 After entering the respiratory tract – the risk of developing Clinical Manifestations
disease mainly depends upon individual’s innate immunity  TB is classified based on the clinical features as pulmonary,
and cell-mediated immunity extrapulmonary or both.
 Clinical illness that develops first, mainly in children is called  It classically presents as low grade fever, cough with
primary TB expectorations, weight loss, loss of appetite, hemoptysis,
 Bacilli that remains in the body for years and being persist – cervical lymph nodes enlargement.
later in life can go for reactivation leading to secondary TB  Systemic extrapulmonary infections can affect any site and
 It is estimated that up to 10% of infected people will develop may present as:
TB in their lifetime.  Pleural TB
 Lymphadenopathy
Primary TB  Genitourinary TB
 Skeletal TB
 It is seen in children; the alveolar macrophages that engulf
 TB meningitis
the TB bacilli make it multiply intracellularly; this leads to
 Tuberculoma
localized subpleural infection affecting lower lobe or lower
 Gastrointestinal TB
part of upper lobe named as Ghon focus
 Pericardial TB
 Ghon focus along with the hilar lymphadenopathy is called
Primary complex. (Occurs after 1–2 months after infection).
 Around 2 to 6 months, the lesion heals leaving a calcified
nodule.
Laboratory Diagnosis
Sputum Microscopy
 Sputum should be collected and concentrated using 4% N
acetyl cysteine or 2% NaCl (Petroff’s method)
 To identify the bacilli in microscopy 104 bacilli/mL should be
 Liquid media used are Middlebrooks 7H9 media (used in
automated cultures like MGIT)
 Liquid media are also helpful for antimicrobial suscep-
tibility testing in Mycobacterial growth indicator tube
(MGIT)

present in the sputum; hence concentration techniques will
improve the chance of detection
 Ziehl-Neelsen staining method using strong carbol fuchsin
with intermittent heating, followed by 25% H2SO4 (according
to RNTCP) and counterstaining with methylene blue is done.
Mycobacterium
Culture media commonly used are:
Lowenstein Jensen media (solid media)
Middle brook 7H10 and 7H11 media (solid media)
Middle brook 7H9 broth (liquid media)

Figure 2: Image of Ziehl Neelsen staining (Courtesy: CDC/ Ronald

W. Smithwic)
 Fluorescent staining using Auramine rhodamine or
acridine orange and counting done by LED microscope is
most sensitive as it is done in high power field and it covers
many areas
Figure 3: Growth of M.tb colonies in LJ media
(Courtesy: Dr Vanathi S, Asst Prof, Dept of Microbiology KAP
Viswanathan medical college, Trichy)

Table 1: RNTCP grading of tuberculosis smear in AFB

Automated Systems of TB Culture
Number of AFB Grading Number of fields  BACTEC MGIT
examined  BACTEC 9000MB
≥10 AFB/ OIF 3+ 20  BacT/ALERT
1–10 AFB/OIF 2+ 50
High Yield
10–99 AFB/100 OIF 1+ 100
Newer method of cultivation of M.tb  BACTEC MGIT –
1–9 AFB/100 OIF Scanty 100
Automated Mycobacteria growth indicator tube – medium
No AFB Negative 100 used in this system is 7H9 Middlebrook medium with
fluorometric detection based on oxygen consumption by the
Sputum Culture microorganism for its growth
MGIT also detects resistance to Pyrazinamide – because this is
High Yield added in the medium

Collection of sputum: (RNTCP)  Another system used is ESP system – recently this helps to
Day 1: Patient should provide on the spot sputum sample detect drug susceptibility of mycobacteria also.
Day 2: Patient should bring an early morning sample
Antimicrobial Susceptibility Testing
 Gold standard for diagnosis of TB is culture which can detect  Phenotypic methods in LJ media:
even 10–100 bacilli/mL  Absolute concentration method
 Limitation of culture is generation time for TB bacilli is 14 – 15  Resistance ratio method
hours hence culture takes 1 month to show visible colonies  Proportion method
 Media used:  Automated methods
 Solid media like Lowenstein Jensen media, Dorset egg
 Genotypic methods – detect genes that code for resistance
media, Petragnani medium
(E.g. GeneXpert detects rifampicin resistance)
 Mostcommonlyusedin LJmedia(sterilizedbyinspissation
as it contains egg)
Molecular Methods  Time period to diagnose is less than 2 hours
 It helps to identify rifampicin resistance
 Polymerase chain reaction (PCR)
 WHO and RNTCP recommended diagnostic tool
 Ligase chain reaction (LCR)
 Transcription-mediated amplification (TMA) Table 3: Ideal method for diagnosis of Tuberculosis
 RFLP
 IS fingerprinting Tuberculosis Methods used to diagnose TB
 Line probe assay Pulmonary TB Sputum culture
Immunodiagnosis TB lymphadenopathy HPE
 Skin sensitivity testing: TB meningitis CSF culture
 Mantoux test: For screening and to estimate the preva- TB Genitourinary Early morning urine repeated
lence of M.tb collection and culture
 WHO advocates PPD-RT-23 with Tween 80
Disseminated / Miliary Biopsy and culture of bone marrow
 Mantoux test is the m/c used tuberculin test
TB and liver tissue and other sites
 Induration should be measured
TB with HIV Sputum microscopy is usually
≤ 5 mm Negative negative; Xpert MTB/RIF assay is
sensitive
6 – 9 mm Equivocal
≥ 10 mm Positive Treatment
 Uses of tuberculin test: For diagnosis of active infection in Table 4: Treatment as per RNTCP Guidelines
infants and young children; it measures prevalence and
incidence of infection but not the disease First-line drugs Second-line drugs
 Isoniazid (H)  Ethionamide
IGRA: Interferon Gamma Release Assays  Rifampicin (R)  Thiacetazone (T)
 It is a test done with whole blood to diagnose Mycobacterium  Pyrazinamide (Z)  Para aminosalicylic acid
tuberculosis infection  Ethambutol (E)  Bedaquiline
 They do not differentiate latent TB infection from active  Streptomycin (S)  Amikacin
tuberculous disease  Capreomycin
 It measures T cell release of IFN gamma  Cycloserine
 It is very useful in vaccinated populations as it is not affected
 Ciproloxacin
by vaccines like it happens in tuberculin test
 Rifabutin
 Two tests approved are:
 QuantiFERON – TB Gold test
 Kanamycin
 T-SPOT: TB test

DOTS–Short Term Regimens

Table 2: Difference between QuantiFERON TB Gold test
and T-SPOT
QFT-GT
Process whole blood within 16
Hours
Antigens used: ESAT-6, CFP-10
& TB7.7 (single mixture of all)
Concentration of IFN gamma is
measured
 Rapid response
 Lower failure rate
 Lesser chances for resistance
T-SPOT
 Better patient compliance
Process peripheral mononu-
clear cells within 8 hours Category I – new HRZE thrice a week for 2 months followed
Antigens used: ESAT-6 & CFP-10 patients by HR daily or thrice a week for 4 months
(Separate) Category II – HRZES thrice a week for 2 months
Number of IFN gamma Previously treated followed by HRZE for 1 month followed
producing cells are measured patients by HRE thrice a week for 5 months

GeneXpert MTB – RIF Assay Drug-Resistant TB

 It is a molecular rapid test method for the diagnosis of  DOTS plus refers to the programs that have components for
pulmonary and extrapulmonary TB MDR TB diagnosis and treatment
 Principle used in Real time PCR  Proper antimicrobial susceptibility testing should be done
 It is a cartridge-based assay to coin the term MDR TB or XDR TB
Table 5: Treatment of drug-resistant TB

Definition
MDR TB :
Multi drug resistant TB –
Resistant to rifampicin and
isoniazid
XDR TB:
Extensively drug resistant
TB – resistant to rifampicin,
isoniazid and any
fluoroquinolones and at least
one injectable second-line
drugs
Treatment
Kanamycin + ofloxacin +
Ethionamide + pyrazinamide +
ethambutol + cycloserine (6–9
months) followed by
Ofloxacin + ethionamide +
ethambutol + cycloserine for 18
months
Figure 4: Cigarbundle appearance of M.leprae (Courtesy: CDC)

High Yield Pathogenesis

Newer Drugs for MDR TB:  Virulence factor of lepra bacilli is – PGL – Phenolic glyco-
Bedaquiline lipid - 1
Delamanid  Spreads by droplet infection – low pathogenicity but highly
infectious
Immunoprophylaxis  Incubation period is very long may be up to 2–5 years
 Disease primarily infects skin, peripheral nerves and nasal
 BCG vaccine – Bacille Calmette Guerin vaccine
mucosa but any organ can be affected
 It is developed from live attenuated strain of M. bovis
 Based on the presentation, leprosy is classified into:
 Intradermal injection is given at birth; vaccine should not be
 Lepromatous
given to immunosuppressed babies and not be given above
 Tuberculoid
age of 2 years
 Dimorphic
 It undergoes series of reactions from papule to vesicle finally  Indeterminate
forming a scar
 Pure neuritic type (seen only in Indian type)
 The immunity ranges from 0–60% and may last for 10–15 years
 It mainly protects from miliary and disseminated serious TB
Table 6: Differences between tuberculoid leprosy and
 Complication of vaccine:
lepromatous leprosy
 Locally abscess or ulcerations may occur
 Enlargement and suppuration of draining lymph nodes Tuberculoid leprosy Lepromatous leprosy
 Generally fever with mediastinal adenitis may occur
Paucibacillary disease Multibacillary disease
Minimally infectious; less severe Highly infectious
Chemoprophylaxis
 Izoniazid is given for prophylais in those who has Skin lesions are asymmetric in Symmetrical skin lesions
 Children who have contact with active TB (usually in the
distribution
same house) Nerves involved are ulnar, All organs systems are
 Infants of mother with active TB posterior auricular, peroneal and involved except lungs and
 Latent TB posterior tibial nerve CNS
 HIV patients having contact with active TB.
CMI present CMI absent

MYCOBACTERIUM LEPRAE Granulomatous lesions Non granulomatous

Lepromin test positive Lepromin test negative
 Acid-fast organism but it differs from M.tb – needs 5% H2SO4
in acid-fast staining method (because it is less acid fast than
M.tb)
 Not alcohol fast
Lab Diagnosis
 Bacilli are seen singly or in groups sometimes the bacilli  Microscopy is the best method to diagnose in resource-poor
appears grouped together by a lipid-like substance called glia areas:
– this group is called globi;  Slit skin smear technique should be followed or skin

 Each globus appears as parallel rows and gives cigar-bundle punch biopsy or nerve biopsy specimens can be used
appearance;  Smears are graded based on the number of bacilli
Table 7: Grading of smears Treatment
Table 9: Doses of drugs in multibacillary and
Bacilli per field Grading
paucibacillary leprosy
1 – 10 bacilli in 100 fields 1+
Drug Multibacillary Paucibacillary
1 – 10 bacilli in 10 fields 2+ Rifampicin 600 mg once a month 600 mg once a month
1 – 10 bacilli per field 3+ Dapsone 100 mg daily self- 100 mg daily self-
administered administered
10 -100 bacilli per field 4+
Clofazimine 300 mg once a month
100 – 1000 bacilli per field 5+ or 50 mg daily
>1000 bacilli or clumps and globi in every field 6+ Treatment 12 months 6 months
duration
 Bacteriological index is calculated by adding all the grading in
all the smear divided by the number of smears ROM regimen: Rifampicin, Ofloxacin and Minocycline
 Morphological index is calculated as the percentage of solid
fragmented granular bacilli (SFGB) out of the total number
of bacilli
 Criteria for calling solid rods are:
 Uniform staining of entire organism
 Parallel sides
 Rounded ends
 Length 5 times than that of width
 Methods of cultivation:
 It is not possible to cultivate lepra bacilli in bacteriological
media or in tissue culture
 Generation time: Average: 12–13 days ATYPICAL MYCOBACTERIA
 Foot pad of mice: Intradermal inoculation granuloma  It is also called as nontuberculous mycobacteria or mycobac-
develops standard procedure for experimental work teria other than tuberculous (MOTT) or anonymous myco-
 Nine banded armadillo: Highly susceptible to infections bacteria
 Artificial culture media: ICRC, Bombay – human fetal
spinal ganglion cell culture, adapted for growth on LJ Table 10: Classification of atypical mycobacteria
media
 Test to detect CMI: Lepromin test Runyon classification Species
 0.1 mL of lepromin is injected in the forearm of patient and Photochromogens M. kansasii, M. marinum
observed on 48 hours and 21 days
 Two reactions are noted as follows:
Scotochromogens M. scrofulaceum, M. gordonae
Non-photochromogens M. avium, M. intracellulare,
Table 8: Early and late reactions in lepromin test M. ulcerans, M. xenopi
Rapid growers M. fortuitum, M. chelonae,
Early reaction Late reaction M. smegmatis, M. phlei
Fernandez reaction Mitsuda reaction
Seen within 24–48 hours Develops after 7–10 days Table 11: Important points to be remembered
Disappears in 3–4 days after injection and reaches
M. kansasii  Causes chronic pulmonary disease (DD: TB)
maximum in 3 to 4 weeks
M. marinum  Fish tank granuloma or swimming pool
Seen as redness and induration Test is read on 21st day
granuloma
in the inoculated site If a nodule of more than 5 mm
If the redness is more than is seen- then test is positive M. scrofulaceum  Cervical adenitis in children
10 mm – then the test is M. gordonae  Tap water scotochromogen
positive M. avium  Called as Battey bacillus
It indicates whether or not a It indicates cell-mediated intracellulare  Most common NTM isolated from lung disease
person has been previously immunity complex  Causes pulmonary disease and disseminated
sensitized to lepra bacilli lesions in AIDS patients
It is superior than late reaction M. fortuitum  Chronic abscess
This reaction is due to DTH to This reaction is due to bacillary M. chelonae  Injection site abscess
soluble constituents of lepra component of antigen M. ulcerans  Buruli ulcer
bacilli M. vaccae  Immunomodulator