Mycobacterium tuberculosis

Dr. Pendru Raghunath Reddy

Mycobacteia are slender rods that sometimes show branching,filamentous forms resembling fungal mycelium

Classification

The genus Mycobacterium contains three groups

1.Obligate parasites

2.Opportinistic pathogens

3.Saprophytes

Obligate parasites

Mycobacterium tuberculosis complex

Contains M. tuberculosis, M. bovis, M. africanum, M. microti,M. canetti, M. caprae and M. pinnipedii

Mycobacterium leprae

Opportunistic pathogens

Non-tuberculous mycobacteria (NTM)

This group contains mixed group of isolates from diverse sources: birds, cold-blooded and warm-blooded animals, from skin ulcers, and from soil, water and other environmental sources

They are opportunistic pathogens and can cause many types of disease

Mycobacterium tuberculosis

• long, slender, straight or curved, about (3 x 0.3 µm in size)• Aerobe• Acid fast bacilli• Intracellular• Mycolic acid, waxes & lipids in cell wall• Slow growing (Doubling time: 15 – 20 hours)

In 1882 while working in Berlinhe discovered the tuberculosis bacteriaand the means of culturing it

The Nobel Prize in Physiology or Medicine 1905

Pathogenesis

Source of infection

Open case of pulmonary tuberculosis

Mode of infection

Direct inhalation of aerosolised bacilli contained in the droplet nuclei of expectorated sputum

Infection also occurs infrequently by ingestion for example,through infected milk, and rarely by inoculation

Transmission of M. tuberculosis

• One cough can release 3,000 droplet nuclei

• One sneeze can release tens of thousands of droplet nuclei

Millions of tubercle bacilli in lungs (mainly in cavities)

Coughing projects droplet nuclei into the air that contain tubercle bacilli

M. tuberculosis does not spread by:

• Sharing dishes and utensils

• Using towels and linens

• Handling food

• Sharing cell phones

• Touching computer keyboard

The initial infection with M. tuberculosis is referred to as a primary infection

Subsequent disease in a previously sensitized person, either from an exogenous source or by reactivation of a primary infection is known as postprimary tuberculosis

Both forms exhibit quite different pathological features

Primary tuberculosis

It is the initial infection by tubercle bacilli in a host

The site of the initial infection is usually the lung

These bacilli engulfed by alveolar macrophages, multiply and give rise to a subpleural focus of tuberculous pneumonia

Which is commonly located in the lower lobe or lower part of the upper lobe to form the initial lesion or Ghon focus

Some bacilli are carried to the hilar lymphnodes through macrophages, where additional foci of infection develops

The Ghon focus, together with the enlarged hilar lymphnodes, form the primary complex

M. tuberculosis multiply within the alveolar macrophages

Th-1 cells produce cytokines to activate these macrophages

Activated macrophages effectively destroy most of the tubercle bacilli

However, some bacilli escape the macrophage- mediated destruction and induce the hypersensitivity reaction

A hard tubercle or granuloma is formed due to the hypersensitivity reaction

When fully developed, tubercle/granuloma consists of 3 zones

1. A central area of large, multinucleated giant cells containing tubercle bacilli2. A mid zone of pale epitheloid cells, often arranged radially3. A peripheral zone of fibroblasts, lymphocytes and monocytes

Later, peripheral fibrous tissue develops, and the central area undergoes caseation necrosis

A caseous tubercle may break into a bronchus, empty its contents there, and form a cavity

It may subsequently heal by fibrosis or calcification

Tubercle or granuloma formation in tuberculosis

Postprimary (secondary) tuberculosis

It is due to reactivation of latent infection or exogenous reinfection and differs from the primary type in many respects

It is characterised by chronic tissue lesions, the formation of tubercles, caseation and fibrosis

Regional lymphnodes are only slightly involved, and they do not caseate

Postprimary tuberculosis always begins at the apex of the lung, where the oxygen tension is highest

The necrotic materials break out into the airways, leading to expectoration of bacteria-laden sputum, which is the main source of infection to contacts

Characteristics Primary PostprimarySite Any part of lung Apical region

Local lesion Small LargeCavity formation Rare Frequent

Lymphatic involvement

Yes Minimal

Infectivity* Uncommon UsualLocal spread Uncommon Frequent

*Pulmonary cases

Differences beween primary and postprimary tuberculosis

Immunology

Tubercle bacilli do not contain or secrete a toxin

The exact basis of their virulence is not understood, but seems to be related to their ability to survive and multiply in macrophages

Humoral immunity appears to be irrelevant

The only specific immune mechanism effective is the CMI

The key cell is the activated CD4+ helper T cell which can develop along two different paths: The Th1 and Th2 cells

Th1 dependent cytokines activate macrophages, resulting in protective immunity and containment of the infection

Th2 cytokines induce delayed type hypersensitivity (DTH), tissue destruction and progressive disease

Koch’s phenomenon

Koch’s phenomenon is a combination of hypersensitivity and immunity It is the response of a tuberculous animal to reinfection

When a healthy guinea pig is inoculated subcutaneously with virulent tubercle bacilli, the puncture site heals quickly

After 10-14 days, a nodule appears at the site of injection which ulcerates and the ulcer persists till the animal dies of progressive tuberculosis

If on the other hand, virulent tubercle bacilli are injected in a guinea pig, which had received a prior injection of tubercle bacilli 4-6 weeks earlier, an indurated lesion appears at the site of injection in a day or two which undergoes necrosis to form a shallow ulcer

This ulcer heals rapidly without involvement of the regional lymphnodes or tissues. This is called Koch’s phenomenon

Koch’s phenomenon has got three components

1. A local reaction of induration and necrosis

2. A focal response in which there occurs acute congestion and even hemorrhage around the tuberculous foci in tissues

3. A systemic response of fever that may sometimes be fatal

Laboratory diagnosisSpecimen collection

Early morning sputum samples should be collected for 3 consecutive days in a sterile container In case of renal tuberculosis, 3-6 morning urine samples should be collected

Type of lesion SpecimenPulmonary tuberculosis Sputum

Laryngeal swabs or bronchial washings

Gastric lavage

Renal tuberculosis Urine

Tuberculous meningitis CSF

Concentration of specimens

Concentration of a specimen is done to achieve;

1. Homogenisation of the specimen

2. Decontamination i.e. to kill commensal bacteria

3. Concentrate the bacilli in the specimen without inactivation

The concentrate is used for smear preparation, cultutre and animal inoculation

Petroff’s method is used to concentrate sputum specimens

Diagnostic Methods

Direct Methods

Direct Microscopy

Ziehl-Neelsen staining (hot staining method)

Kinyoun’s method (cold staining method)

Acid fast bacilli resist decolourisation with acid and alcohol once they have been stained with carbolfuchsin

AFB appear as pink, long, slender bacilli with beaded appearance

Fluorescent staining by Auramine O or auramine rhodamine

Mycobacterium spp. will fluoresce yellow against dark background under fluorescent microscope

Diagnosis of pulmonary tuberculosis under RNTCP

DOTS: Directly observed treatment short-course

Culture

Concentrated specimen is inoculated on Lowenstein – Jensen’s medium and incubated at 370C for 2 – 8 weeks

Colonies appear as buff coloured, dry, irregular colonies with wrinkled surface and not easily emulsifiable (Buff, rough and tough colonies)

Colonies are creamy white to yellow colour with smooth surface and easily emulsifiable

M. bovis

M. tuberculosis

Differentiating features of M. tuberculosis and M. bovis

Biochemical reactions Niacin test

M. tuberculosis lacks the enzyme that converts Niacin to Niacin ribonucleotide due to this large amount of Niacin accumulates in the culture medium

Niacin is detected by addition of 10% cyanogen bromide and 4% aniline in 96% ethanol

Positive reaction – canary yellow

M. tuberculosis – Positive

M. bovis - Negative

Nitrate reduction test

M. tuberculosis produce an enzyme nitro reductase which reduces nitrate to nitrite

This detected by colorimetric reaction by addition of sulphanilamide and n-naphthyl- ethylene diamine dihydrochloride

Positive reaction – pink or red colour

M. tuberculosis – Positive

M. bovis - Negative

M. tuberculosis is resistant to TCH (Thiophene - 2 - carboxylic acid hydrazide); hence, growth occurs

M. bovis is susceptible; therefore, does not grow

M. bovis

M. tuberculosis

Growth in presence of TCH

Rapid culture methods1. BACTEC

2. Mycobacterial growth indicator tube (MGIT)

3. Bac T/ Alert 3D system

BACTEC system

Average time to detect Mycobacterium growth is 8 days

Radio metric method

Detects the presence of Mycobacteria based on their metabolism rather than visible growth

0.5 ml of processed sample is added to 4 ml of Middlebrook 7H12 broth containing C14 radio labelled palmitic acid

Mycobacteria metabolises C14 radio labelled palmitic acid and release radio actively labeled 14CO2

BACTEC 460 instrument measures 14CO2 and reports in terms of growth index (GI)

A growth index of 10 or more is considered positive

More sensitive than traditional method

Problem of disposal of radio active waste

Animal inoculation

0.5 ml of concentrated specimen is inoculated intramuscularly into the thigh of two healthy guineapigs

The animals are weighed prior to inoculation and thereafter at weekly intervals

Tuberculin test is done after 3 – 4 weeks

Progressive loss of weight and positive tuberculin skin reaction indicates infection

One animal is killed after 4 weeks and autopsied, if it shows no evidence of tuberculosis the other animal is autopsied after 8 weeks

Autopsy shows

1. Caseous lesion at the site of inoculation

2. Enlarged caseous inguinal lymph nodes

3. Tubercles may be seen in spleen, lungs, liver, or peritoneum

4. Kidneys are not affected

Allergic tests

Tuberculosis infection leads to the development of delayed hypersensitivity to M. tuberculosis antigen, which can be detected by Mantoux test

Mantoux test (tuberculin test)

0.5 ml of PPD containing 5 TU is injected intradermally on flexor aspect of fore arm

Site is examined after 48 – 72 hrs

Induration of 10 mm or more is considered positive

Positive tuberculin test indicates hypersensitivity to tuberculoprotein denoting infection with tuercule bacilli or BCG immunisation, recent or past with or without clinical disease

Uses

1. To diagnose active infection in infants and young children

2. To measure the prevalence of infection in community

3. Indication of successful BCG vaccination

Detection of antibodies

Various methods such as enzyme linked immunosorbent assay (ELISA), radio immunoassay (RIA), latex agglutination assay have been employed for detection of antibodies in patient serum

However, diagnostic utility of these methods is doubtful

WHO has recommended that these tests should not be used for diagnosis of active tuberculosis

Quantiferon-Gold

Is an in vitro assay that measures the cell mediated immune -response in the infected individuals through the levels of interferon gamma (IFN-γ) released by the sensitised T- lymphocytes after stimulation by M. tuberculosis antigens

Molecular methods

1. Polymerase chain reaction (PCR)

2. LAMP

3. Ligase chain reaction

PCR

Rapid method to detect M. tuberculosis directly in clinical samples based on DNA amplification

IS6110 sequence is generally targeted for detection M. tuberculosis complex

Prophylaxis

General measures

Adequate nutrition, good housing and health education are as important as specific antibacterial measures

Immunoprophylaxis

The BCG (Bacille Calmette-Guerin) vaccine (0.1 ml), administered soon after birth by intradermal Injection failing which it may be given at any time during the first year of life

This is a strain of M. bovis attenuated by 239 serial subcultures in a glycerine-bile-potato medium over a period of 13 years

Bacille Calmette-Guérin = BCG!

Albert Calmette Camille Guérin

Chemoprophylaxis

This is the administration of antituberculous drugs (usually only isoniazid)

1. To persons with latent tuberculosis (asymptomatic tuberculin positive)

2. To persons with a high risk of developing active tuberculosis

3. To the infant whose mother with active tuberculosis

4. To the children living with a case of active tuberculosis in the house

Isoniazid 5 mg/kg daily for 6 – 12 months is the usual course

References:

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