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Genus MYCOBACTERIUM

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• Long, slender bacilli with branching

filamentous forms.

• Obligate pathogens, Saprophytic and Opportunistic.

• Lipid-rich waxy cell wall.

• Responsible for Chronic granulomatous lesions.

• In 1882 Robert Koch :

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CLASSIFICATION

1. Tubercle bacilli:a. Human : M. tuberculosis, M.

africanumb. Bovine : M. bovisc. Murine : M. microti.d. Avian : M. avium.e. Cold blooded: M.marinum

2. Lepra bacilli: a. Human : M. leprae b. Rat : M. leprae murium

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3. Mycobacteria from skin ulcers. M. ulcerans, M. balnei, M. buruli.

4. Atypical Mycobacteriae:

5. Jhone’s bacillus:M. para tuberculosis.

( Chronic specific enteritis in cattle )

6. Saprophytic Mycobacteria.M. butyricum. M. smegmatis, M.stercoris.

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Cell wall structure• Lipid-rich waxy cell wall

• Acid fast ,alcohol fast.• Slow growth in culture

medium.• Resistant to antiseptics

and antibiotics.• Clumps in liquid medium.• Clot formation

• Acid fast staining Ziehl - Neelsen method.

Kinyoun’s method.• Gram staining : No use. LIKE SERPENTINE

CORDS.

Straight / slightly curved rods with occasional branching. 3 x0.3µm. Non motile, Non-capsulated, Non- sporing

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CULTURAL CHARACTERS

Doubling time 14 – 15 hrs.Optimum temperature 37º C .

(No growth < 25º C & > 40º C) 1.Egg media: Lowenstein – Jensen medium. Dorset egg medium. L J medium : 2 - 6 weeks

Sterilized by Inspissation.Glycerol, asparagines.Malachite green as selective agent.Egg albumen as solidifying agent.

2. Blood (Tarshis medium ). 3. Serum (Loeffler’s serum slope ).

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LIQUID MEDIA :• Dubo’s medium.

• Middle –Brook medium.

• Bactec 12B medium. Uses :

• Sensitivity tests.• Chemical tests.• Preparation of antigens & vaccines.

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GROWTH :

M. tuberculosis M. bovis.

Heaped up & luxuriant Sparse (dysgonic) growth. (eugonic)

Dry, rough, tuff Moist, smooth flat buff colour. with white colour.

0.5 % glycerol Sodium pyruate ( L J medium) ( Stone brink's medium )

Grows on surface. Grows as band few mm.

(Aerobe) below the surface (Anaerobe)

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Growth on Lowenstein- Jensen (LJ)

medium.

SENSITIVITY

• Pasteurization.

• Formaldehyde.

• Glutaraldehyde.

• Tincture of Iodine

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1. To differentiate M.tuberculosis from M. bovis and Atypical mycobacteriae

2. To identify virulent &

avirulent strains.

•Niacin production.

•Catalase positive.

•Amidase positive.

•Nitrate reductase

positive.

•Aryl sulphatase

negative•Resistant to thiophen – 2 – Carboxylic acid hydrazide (TCH) which is related to INH.

BIOCHEMICAL TESTS

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Antigenic structure:

•Antigenically homogenous. (Human,Bovine,Murine spp).

•Two types:

1.Cell wall insoluble polysaccharide antigens.

Group specificity.

2.Cytoplasmic soluble protein antigens.

Type specificity.

TUBERCULIN PROTEIN

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PATHOGENESIS:

Pulmonary alveoli.

Taken up by Macrophages & multiplied.

Primary site of infection in lower part of upper lobes / upper

part of lower lobes - Ghon’s focus. With hilar

lymphadenopathy (PRIMARY COMPLEX)

Initiates CMI

InhalationDroplets, aerosols from patients & Cough spray from Animal

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Pathogenesis Contd……: Activation of specific T - cells (cytokines, gamma

interferon production)

DTH Immune response

Formation of Tubercle. Activate macrophages ( Avascular granuloma) (Inhibits multiplication).

Consumes much of O2 & produces acidosis.

Most of the bacilli are killed.

Resolution Some remain dormant.

Protective Immunity. Post – primary disease.

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Avascular granuloma Central Zone of Caseous (Cheese like ) material

dead T &B cells and macrophages surrounded by

different types of cells.

IL 2: Proliferation of

Ag-primed T cells

Gama INF: Enhances activity of macrophages & NK cells.

TNF-ά (Cachectin): Induces cytokine secretion in the inflammatory area. Muscle wasting, fever.

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Expanding large Avascular granuloma (Tuberculomata).

Erodes through wall of bronchus.

Liquified contents discharged into the bronchus.

CAVITY formed.

Shelter for huge number of bacilli.

Gets access to sputum Open case of TB. ( Transmissible case of

TB )

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2. Ingestion : Unpasteurised milk

Primary complex in tonsils, cervical LN & Ileocaecal region ( Mesenteric

LN ).

Initiates CMI.

3. Inoculation : Rare ( Occupational in anatomists ,

pathologists ).

Skin with involvement of regional LN.

Prosector’s warts.

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Clinical features

•Persistent dry cough

•Tiredness, weakness. 

•Weight loss 

•Poor appetite 

•Sweating at night, in spite of cold 

•Chest  pain.

•Shortness of breath 

•Coughing up blood

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Lesion breaks down

Bacilli released and spread

Through blood & lymph

Spleen, liver, lungs, BM, Kidney,Adrenal glands, Eyes ,CNS .

Chronic pneumonitis,Tuberculous osteomyelitis, Tuberculous meningitisMilliary tuberculosis

Progression of disease

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Lymphadenitis:

• Children.

• Painless neck swelling.

Genitourinary TB:• Renal TB: Symptoms of UTI.

Sterile pyuria.• Scrotal mass.

• Tubo-ovarian mass ( Sterility ).

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POST-PRIMARY CUTANEOUS TB

1. Lupus vulgaris

Affects face & Neck.

Gross scarring & deformity.

2. Scrofuloderma

Sinus formation

between affected

lymph node & skin.

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IMMUNITY & DTH (Allergy):

CMI is useful.

6 – 8 weeks after infection : Tuberculin test reaction occurs.

Described by Koch’s Phenomenon.

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INJECTED with TU antigen SC 4-6 wks later.

GUINEA PIG INFECTED WITH TB BACILLI.

After 1 – 2 days Indurated lesion at the site of Injection.

Undergoes rapid necrosis.

Shallow ulcer

Heals rapidly without involvement of regional lymph nodes.

Koch’s phenomenon

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TUBERCULIN TEST

• Clemens von Pirquet (1907) : OT.

• Seibert (1939) : Purified Ag by

Ammonium sulphate fractionation (PPD).

• Strength of PPD expressed in TU.

• A measured amount ( 5 – 10 TU ) is injected.

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MONTOUX TEST 5 TU of PPD injected Intradermally in Flexor aspect of forearm. (Tuberculin syringe)

No scratch on itching.

Read after 48 - 72 hrs .

Induration.

Only erythema not considerable.

>10mm - Significant

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EPIDEMIOLOGY

• Transmission among households. • Dusty environment , Hill dwellers (Silica) . • Low Socio-economic status, Malnutrition. (A barometer of social welfare )• Asia & sub- Saharan nations are more prone.

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• Every minute someone dies of TB in India.

>5,00,000 die every year. If there is no

effective action, 5 million may die of TB in the

next few years. 1.8 million new cases every

year. India has the highest burden of the

disease in the world.

Dr L S Chauhan, Director General (TBCP) Nov’ 6 2006 India Together

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Initiative of the Government of India . It incorporates the principles of Directly observed treatment Short course (DOTS) - the global TB control strategy of the WHO. The program provides free of cost, quality Anti-Tubercular drugs through the PHCs and the private-sector DOTS-providers.

RNTCP or Revised National Tuberculosis Control Program is the State-run Tuberculosis Control

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The DOTS strategy is cost-effective and is

today the international standard for TB control programmes.

• To date, more than 180 countries are implementing the DOTS strategy. • India has adapted the DOTS strategy in various parts of the country since 1993, with excellent results, and by March 2006 nationwide DOTS coverage has been achieved.

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Rapid DOTS expansion in India

• In 2000 , 2001 and 2002 more than a million patients were treated in this way in India. As a result nearly 2,00,000 lives were saved.

• Extensively drug-resistant TB (XDR-TB) in > 30 countries since 2006, multidrug-resistant TB (MDR-TB) and XDR-TB have recently become a particular focus of international concern.

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1.Primary TB

If productive: Sputum If not productive: Bronchial washings/

brushings/ biopsy.2.Secondary/Post Primary TB

a. CSFb. Pleural fluidc. Synovial fluid.

LAB. DIAGNOSIS

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Decontamination & concentration methods.1. Petroff’s method:

• Equal volumes of sputum & 4% NaoH

Keep at 37º C with intermittent shaking

for 20 mts.

• Neutralized with (Potassium dihydrogen orthophosphate).

• Centrifuge at 3000 rpm for 30mts.

»Deposit: Microscopy

Culture.

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2.Non centrifugation & Non neutralization

method:

Equal volumes of sputum +

2% Cetrimonium bromide & 4%

NaoH

5 mts culture.

Materials used for Homogenization:

a. Diluted acids ( 6% H2 SO4, 3 % HCl

)

b. N-Acetyl Cystein with NaoH.

c. Pancreatin.

d. Cetrimide.

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MICROSCOPY Minimum of 10,000 bacilli / ml of sputum.

100 fields must be examined . 1.Kinyoun’s method

2.Ziehl - Neelsen technique.3.Fluorescent dye technique.

Auramine Phenol,

Auramine Rhodamine dye.

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INTERPRETATION

3-9 bacilli in entire smear:1+

10 or more /entire smear: 2+

10 or more / field: 3+Beaded forms: M.tuberculosis

Uniform: M.bovis

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2. CULTURE :

A. Conventional method:

• Concentrated sample.

• L -J medium 35 - 37º C.

• Inspect weekly up to 8 weeks.

B. Rapid diagnosis of growth:

Bactec system: Radiometric detection of

CO2.

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C. Fluorescent dye methods

Activation of fluorescent dye by the

released CO2

3.Nucleic acid technology:

1.Nucleic acid probes : Not

sensitive

2.PCR: Conventional PCR is best.

4. Tuberculin test:

5. Serology:

PHA .

Ig M, Ig G and Ig A estimation

( Specific but not sensitive tests).

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Primary complex in the hilar region

X-Ray findings of pulmonary TB

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1.General measures:Adequate nourishment. Good housing .Health education.Contact tracing.

2.Chemoprophylaxis:

INH

PREVENTIVE MEASURES

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•Preventing the release of the organism.

•Preventing the inhalation, through masks.

•Use of high-efficiency particulate air (HEPA) filters in the entry ways.

•Chemoprophylaxis for suspected cases.

The basic methods of preventing TB transmission

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3. IMMUNOPROPHYLAXIS: BCGLive attenuated vaccine.

Bovine strain (Danish 1331 by 239 serial subcultures on

Glycerin bile potato medium).

Freeze dried vaccine (Normal saline).

At birth / within 6 weeks of age.

Intradermally over deltoid region.

Dose : 0.1 mg in 0.1 ml. volume.

Efficacy : 0 – 80 %.

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EVENTS AFTER INJECTION :

• Papule within 2 – 3 weeks.

• Enlarges to 4 – 8 mm within 5 weeks.

• Subsides and broken into ulcer.

• Heals spontaneously with scar formation

within 6 - 12 weeks.

Complications:

Local : Abscess, indolent ulcer, Keloid.

Regional:

Local lymphadenopathy.

General :

Fever, mediastinal adenitis,

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CONTRAINDICATIONS

• Generalized eczema.

• Infective dermatosis.

• Hypo gamma globulinaemia.

• Immunodeficiency.

Protection not absolute after vaccination, May

suffer with milder form of disease.

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ANTI TUBERCULOSIS DRUGS First-line:

Rifampicin (R) Pyrazinamide (Z)

Isoniazid ( H ) Ethambutol

Second-line: Amikacin, Capreomycin, Kanamycin and Ofloxacin, Streptomycin (Since 2005)

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SHORT COURSE CHEMOTHERAPY Drugs Initial Drugs Continuation

phase phaseStandard regime.

RHZ 2M RH 4MIntermittent regime.

RHZ 2M R3 H3 4MRHZ 2M R2 H2 4MR3H3Z3 2M R3 H3 4M

Incase of high incidence of initial drug resistance.RHZE 2M RH 4MRHZS 2M RH 4M

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The challenge to Medical profession is to beprepared for all infectious diseases that may affect the practice.