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Mycobacteria
Mycobacteria are slender bacilli which may show branching filamentous forms resembling
fungal mycelium (‘myces’ means fungus). They are difficult to stain but once stained, resist
decolourisation with dilute mineral acids and are called acid- fast bacilli or AFB.

Species –

M. tuberculosis (human tubercle bacilli),[Link], M. microti, [Link]

These together form Mycobacterium tuberculosis complex.

M. tuberculosis and [Link] are typical tubercle bacilli affecting humans.

Mycobacterium tuberculosis
Morphology

-[Link] is a slender, straight or curved bacillus occurring singly, in pairs or in small

clumps.

-They are acid- fast, nonsporing, non capsulated.

In Ziehl- Neelsen stain,

Mycobacterium tuberculosis

Pus cell

Tubercle bacilli are seen bright red (acid fast),

while the tissue cells and other organisms are stained blue.

beaded or barred forms are seen. (m. Bovis stains more uniformly)

Another method of staining is Kinyoun’s method also called as cold staining.

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Culture

M. tuberculosis is an obligate aerobe.

The bacilli grow slowly and colonies appear only in about two weeks.

Optimum temperature is 370C.

Commonly used media: Lowenstein- Jensen (LJ) medium. Colonies of [Link] are dry,
rough, buff coloured raised with a wrinkled surface.

Other solid media employed: Dorset egg medium, Middlebrook 7H10.

Liquid media: Dubos’ medium, Sula’s and Sauton’s

Newer methods: BacTAlert, BACTEC, MGIT.

Positive Biochemical reactions of mycobacterium tuberculosis

1. Niacin test:
2. Nitrate reduction test:
3. Pyrazinamidase test:
4. Neutral red test:

Pathogenesis

Source of infection:

An open case of pulmonary tuberculosis.

Mode of transmission: Acquired by inhalation of infected droplets coughed or sneezed into the
air by a patient with pulmonary tuberculosis.

In bovine tuberculosis, infected cows develop lesions in the udder and bacilli are excreted in
milk which can infect people who drink it raw.

The inhaled bacilli are arrested by the natural defense of the upper respiratory tract.

Tubercle bacilli that escape reach the lungs and are engulfed by macrophages but they survive
and multiply in macrophages. The cell mediated immunity (CMI) plays a major role to interact
with these macrophages whereas humoral immunity appears to be irrelevant.

The production of a characteristic lesion called the tubercle in infected tissues is an essential
feature of tuberculosis.

Human tuberculosis is divided into primary and secondary forms.

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Primary tuberculosis is the initial infection by tubercle bacilli in a host.

In young children the bacilli engulfed by alveolar macrophages multiply and located in the lower
lobe or the lower part of the upper lobe (Ghon focus). The hilar lymph lodes are involved. The
Ghon focus together with the enlarged hilar lymph node constitutes the primary complex. In
majority of cases, the lesion heals in 2-6 months, leaving behind a calcified nodule. A few bacilli
may survive in the healed lesion and remain latent. In a few children with impaired immunity,
the primary lesion may enlarge and cause other forms of disseminated tuberculosis.

Post primary (Secondary or adult) type of tuberculosis is due to reactivation of latent infection
or exogenous reactivation. It affects mainly the upper lobes of the lungs, the lesions undergoing
necrosis and tissue destruction leading to cavitation. The necrotic material break out into the
airways leading to expectoration of bacteria-laden sputum. In the immunodeficient, there is
widespread dissemination of lesions in the lungs and other organs.

Clinically tuberculosis presents as low grade fever, chronic cough, hemoptysis, weight loss &
chronic wasting disease.

The two types of clinical manifestation of tuberculosis (TB) are pulmonary TB (PTB) and
extrapulmonary TB (EPTB). The former is most common. EPTB refers to TB involving organs
other than the lungs (e.g., pleura, lymph nodes, abdomen, genitourinary tract, skin, joints and
bones, or meninges).

Examples: Extra pulmonary(EPTB)

Tuberculous lymphadenitis, Pleural tuberculosis, Renal tuberculosis, genital tuberculosis,
Skeletal tuberculosis (Pott’s disease), Tuberculosis of CNS, Miliary tuberculosis/disseminated
tuberculosis

Koch’s phenomenon

The response of a tuberculous animal to reinfection was originally described by Koch.

Subcutaneous injection of virulent tubercle bacillus in a normal guinea pig produces no
immediate response, but after 10-14 days a nodule develops at the site which forms an ulcer that
persists till the animal dies of progressive tuberculosis.

On the other hand, when virulent tubercle bacillus is injected in a guinea pig which had received
a prior injection of the tubercle bacillus 4-6 weeks earlier, a lesion appears at the site in a day or
two. This undergoes necrosis in a day to form a shallow ulcer that heals rapidly. This is known as
the Koch phenomenon and is a combination of hypersensitivity and immunity.

Lab diagnosis

Specimens:Specimen collection depends on the site of involvement.

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Pulmonary tuberculosis: sputum.

A morning specimen on three consecutive days is [Link] sputum is not available,

laryngeal aspirates or bronchial washings are collected. In small children, gastric lavage is used.

Meningitis: CSF ; shows a spider web clot on standing.

Renal tuberculosis: three consecutive days’ morning urine samples

Bone and joint tuberculosis: aspirated fluid.

Tissue: Biopsy of tissue

Blood: from those with HIV co- infection.

Direct Microscopy

[Link]- Neelsen staining (Acid fast stain)

Smears should be prepared from the thick purulent part of the sputum. Direct or concentrated
smears of sputum are examined for AFB.

Steps

 Strong carbol fuchsin is poured on a slide containing heat fixed smear. This is gently
heated to steaming for 5-7 minutes without letting the stain boil and become dry.
 The slide is then washed with water and decolorized with 20% sulphuric acid till no more
stain comes off. Then washed with water. (In case of lepra bacilli, 5% sulphuric acid is
used as [Link] is less acid- fast)
 The smear is counterstained with 2% methylene blue, The smear is then washed with
water and air dried.
Under the oil immersion objective, acid fast bacilli appear red (colour of carbolfuchsin)
in blue Acid-fastness is due to the high content of lipids and fatty acids found in the cell
wall of Mycobacterium. Mycolic acid (a wax) is found in all acid- fast bacteria. A
negative report should not be given till at least 300 fields have been examined, taking
about 10 minutes. A positive report can be given only if two or more typical bacilli have
been seen. Smears are graded based on Revised National Tuberculosis Control
Program(RNTCP).

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ZN smear evaluation and AFB report as per RNTCP guidelines

Result Grading No. of fields

˃10/field +ve 3+ 20
1-10/ field +ve 2+ 50
10-99/100 field +ve 1+ 100
1-9/100 field +ve Scanty* 100
No. of AFB in 100 -ve 1000
fields
*Record actual [Link] bacilli seen in 100 fields

1. Kinyoun’s modification of acid-fast staining

This is a modified cold method where heating of the stain is not employed. It requires increasing
the concentration of phenol acid and duration of staining.

2. Auramine rhodamine: Smears are stained with auramine phenol or auramine rhodamine
fluorescent dyes and examined with fluorescent microscopy. Bacilli appear as bright rods
against a dark background. Advantage: rapid method.

I. Culture:
On Lowenstein- Jensen medium (two bottles).
The culture media are inoculated and incubated at 370C in the dark and in the light.
The tubercle bacilli usually grow in 2 to 8 weeks. If positive, characteristic colonies
appear on culture medium and it is stained with Ziehl- Neelsen technique. The slow
growing acid- fast bacillus (AFB), non pigmented and niacin positive is regarded as
M. tuberculosis.
II. Automated systems
BACTEC, Mycobacterial growth indicator tube (MGIT), BacT/Alert 3D system. All
these systems are slowly replacing solid culture methods because of rapid indication
of growth.

III. Animal inoculation-In guinea pigs.

IV. Molecular methods
Nested PCR
Cartridge based nucleic acid amplification test(CBNAAT)-Automated real time PCR,
GeneXpert
Line Probe assay(LPA)-Probe based detection of amplified DNA for positive culture

V. Immunodiagnosis(Tuberculin skin test/)

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Demonstration of hypersensitivity to tuberculoprotein by tuberculin testing is a standard

procedure to detect exposure to the bacilli.

Mantoux test

Method: In theMantoux test, 0.1ml of PPD is injected intradermally on the flexor aspect of the
forearm. The site is examined 48- 72 hours later. In a positive reaction, there is induration (local
oedema) of 10mm diameter at the site of inoculation.

A positive tuberculin test indicates hypersensitivity to tuberculoprotein denoting infection with

tubercle bacillus or BCG immunization, recent or past, with or without clinical disease. It does
not indicate the presence of active stage of the disease.

Uses:

 As an aid in diagnosing active infections in infants and young children.

 To determine prevalence of infection in an area.
 As an indication of successful vaccination.

Treatment

Now the treatment is provided by RNTCP which follows the DOTS. A serious consequence of
unchecked drug resistance has been the emergence and spread of multidrug- resistant
tuberculosis (MDR-TB). The Directly Observed Treatment, Short Course (DOTS) is used to
prevent resistance problem.

Prophylaxis -Protection from tuberculosis is done by public health measures, BCG vaccination
and by chemoprophylaxis.

 General measures: Good nutrition and health education have to be adopted.

 Immunoprophylaxis:

BCG vaccine: Calmette and Guerin prepared an attenuated strain of [Link] by growing it on
potato medium. The strain was attenuated by repeated subcultures. When the strain became
incapable of producing tuberculosis in the susceptible guinea pig, it was named BacilleCalmette
Guerin (BCG)Vaccine is given intradermally in a dose of 0.1ml. BCG vaccine should be given
soon after birth.

 Chemoprophylaxis-Only in some risk groups.

MDRTB-Resistance to Isoniazid ,rifampicin with or without first line antibiotics

EXDRTB-MDRTB ,resistant to Fluoroquinolones and any aminoglycosides(Amikacin)

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RNTCP

Revised National Tuberculosis Control Program (RNTCP) is the state-run tuberculosis control
initiative of the Government of India. RNTCP India was implemented in 1997 based on WHO
recommended strategy of Directly Observed Treatment, Short Course (DOTS). The aim was to
provide standardized treatment and proper diagnostic facilities. Treatment is provided under
direct observation by a DOT provider at the DOTS center near patient’s home. Under RNTCP,
any patient with cough for 2 weeks or more is included for diagnosis of pulmonary tuberculosis.
Here the diagnosis is mainly based on smear examination of sputum.

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