MYCOBACTERIA

1. MYCOBACTERIA Dr. Sudheer Kher

2. Acid Fast Ziehl-Neelsen Stain Mycolic acids Tuberculosis (Koch’s disease) M. tuberculosis M. bovis M. leprae Tubercle Lowenstein Jensen medium PPD Tuberculin BCG Polymerase chain reaction Runyon groups KEYWORDS

3. Tubercle bacilli Human – MTB Bovine – M. bovis Murine – M. microti Avian – M. avium Cold blooded – M. marinum Lepra bacilli Human – M. leprae Rat – M. leprae murium Mycobacteria causing skin ulcers M. ulcerans M. belnei Atypical Mycobacteria (Runyon Groups) Photochromogens Scotochromogens Nonphotochromogens Rapid growers Johne’s bacillus M. paratuberculosis Saprophytic mycobacteria M. butyricum M. phlei M. stercoralis M. smegmatis Others Classification of Mycobacteria

5. Tuberculosis (TB, Consumption, Koch’s Disease) • M. tuberculosis • major human disease • healthy people • problems • association with AIDS • multiple drug-resistance • Chronic disease • Prolonged treatment

6. Slender rods Resist staining but once stained, resist decolorization by dilute mineral acids; hence called ACID FAST BACILLI (AFB) Aerobic, Non-motile, Non-sporing, Non-capsulated. Growth generally slow Genus includes Obligate parasites Opportunist pathogens Saprophytes General characters of the genus

7. Mycobacterium tuberculosis • One of the most serious infectious diseases in the developing world • One third of world’s population infected with M. tuberculosis • Thirty million people have active disease • Nine million new cases occur • Three million people die of the disease, each year.

8. Mycobacterium tuberculosis (MTB) • Morphology – • Ziehl – Neelsen stain – Once stained by Carbol fuchsin, resist decolorization by 20% Sulphuric acide and absolute alcohol. Acid & Alcohol Fast (AFB) • Fluorescent dyes like Auramine O or Rhodamine also stain and the decolorization is resisted. • Reason for Acid & Alcohol fastness – • Presence of unsaponifiable wax Mycolic acid • Semi permeable membrane around the cell • Property of cell wall and related to integrity of the cell wall • Staining may be uniform or granular

9. Grow slowly. Generation time 14-15 hrs Colonies appear after 2 weeks or at 6-8 weeks MTB - Obligate aerobe MTB grows more luxuriantly (eugonic) than M. bovis (dysgonic). Addition of 0.5% Glycerol supports growth of human strains. No effect or inhibitory effect on bovine strains. MTB : Cultural characters

10. Solid media – Egg containing Lowenstein-Jensen Medium Petragnini medium Dorset Blood containing – Tarshi’s Serum containing – Loeffler’s serum slope Potato containing – Pawlowsky’s Liquid media – Dubos’ Middlebrooks Proskauer & Beck’s Sula’s Sauton’s MTB : Culture media

11. Resistance • Not specifically resistant to heat. 60 C x 20 min destroys. • In sputum can survive 20-30 hrs • Relatively resistant to disinfectants. Survives exposure to • 5 % Phenol • 15 % Sulphuric acid • 3 % Nitric acid • 5 % Oxalic acid • 4 % NaOH

12. Biochemical reactions • Niacin test – Human MTB produces niacin when grown in egg medium. • Aryl Sulphatase test – Enzyme Aryl sulphatase formed by only atypical mycobacteria.

13. Biochemical reactions • Neutral red test – Virulent strains of tubercle bacilli bind neutral red in alkaline solution while avirulent strains can not. • Catalase–paroxidase test – Most atypical mycobacteria are strongly catalase positive while MTB is only weakly positive. MTB is strongly peroxidase positive while atypical mycobacteria are negative. • Nitrate reduction test – Positive in MTB and negative in M. bovis

14. Morphology – Straight or slightly curved rods Mycobacterium tuberculosis (MTB)

15. Modes of infection 1- Droplet infection Person to person by inhalation aerosols Mycobacterium tuberculosis (Pulmonary tuberculosis) 2- Ingestion of milk Infected cattle Mycobacterium bovis (Intestinal tuberculosis) 3- Contamination of abrasion Laboratory workers (Skin infection)

16. Pathogenesis of tuberculosis • infects lung • distributed within macrophages • facultative intracellular pathogen • inhibits phagosome-lysosome fusion • resists lysosomal enzymes

17. Other minor pathogenesis factors • tuberculosis • mycobactin • siderophore • cord factor • damages mitochondria

18. Cell-mediated immunity -tuberculosis • infiltration • macrophages • lymphocytes • granulomas • tubercles

19. Classical tubercular lesion – Granuloma with typical Langhan’s giant cells, epithelioid cells, lymphocytes and fibrosis.

20. There are multiple light areas (opacities) of varying size that run together (coalesce). Arrows indicate the location of cavities within these light areas. The appearance is typical for chronic pulmonary tuberculosis.

21. Tuberculosis Clinical picture : * Low grade fever * Weight loss * Night sweats * Fatigue * Cough & haemoptysis

22. Laboratory diagnosis M. tuberculosis • Acid fast bacteria in sputum • Culture on L J media • Biochemical identification • Antibiotic sensitivity test • Tuberculin test • PCR

23. Lowenstein Jensen Medium – Selective. Always in screw capped bottle. Bluish Green. Contains – Egg protein – Solidifying agent Mineral salts – Mg sulphate, Mg citrate Asparagine Malachite Green – Selective agent Sterilized by - Inspissation

24. Laboratory diagnosis - tuberculosis • skin testing • delayed hypersensitivity • tuberculin • protein purified derivative, PPD • X-ray

25. Tuberculin Test (Mantoux test) • Delayed hypersensitivity skin test to assay: cell mediated immunity to tubercle bacillius Material: A purified protein derivative (PPD)Dose : 0.1 ml of (PPD) is injected intradermalReading : Positive test is defined as - Induration equal or greater than 10 mm - Develop 48-72 hours after injection

26. Positive skin test -tuberculosis • indicates exposure to organism • does not indicate active disease

27. Tuberculin Test Interpretation: * A positive test indicates previous exposure and carriage of T.B. * A negative tuberculin test excludes infection in suspected persons * Tuberculin positive persons may develop reactivation type of T.B. * Tuberculin negative persons are at risk of gaining new infection * False positive reactions are mainly due to: - Infection with nontuberculous mycobacteria * False negative reactions may be due to: - Sever tuberculosis infection (Miliary T.B.) - Hodgkin’s disease - Corticosteroid therapy - Malnutrition - AIDS * Children below 5 years of age with no exposure history: - Positive test must be regarded suspicious

28. Laboratory Diagnosis • Demonstration of bacilli • Culture & isolation or Animal inoculation • Demonstration of hypersensitivity to tubercular protein • Serological tests limited value

29. Specimen * According to site of infection : - Sputum - Urine - Body fluids - Gastric lavage - Blood - Tissue biobsy * Specimens need appropriate processing Liquefaction with N-acetyl-L- cysteine Sputum Decontamination with NaOH Centrifugation

30. Laboratory Diagnosis • Pulmonary TB – • Specimen – • Sputum – Early morning, if scanty 24 hrs, three consecutive day samples. Laryngeal swabs or gastric lavage in children. • Microscopy –See at least 100 field / 10 minutes. • Grading – • 1+ -> 3-9 bacilli in entire smear • 2+ -> 10 or more in entire smear • 3+ -> 10 or more bacilli seen in most oil immersion fields • Culture & AST • Animal inoculation – Animal of choice Guinea Pig.

31. Laboratory Diagnosis • Extra -Pulmonary TB – • Specimen – • CSF – in suspected meningitis • Pleural fluid & other exudates • 2-3 days urine in renal TB • Biopsy material.

32. Concentration methods • Purpose – Homogenization & Concentration in sputum & other specimens. • Methods –

33. Concentration methods • Useful for microscopy, culture & animal inoculation • Petroff’s method • Most widely used • Equal volumes of Sputum + 4% NaOH incubated at 37C X 20 min.Centrifuge at 3000 rpm X 30 min. Sediment neutralized by N/10 HCl. • Can be used for smear, culture, animal inoculation • Simpler method • To avoid centrifugation & Neutralization • Equal volumes of sputum + solution of Cetrimoniom bromide 20 g + NaOH 40 g in one litre. Allow to stand for five minutes. Inoculate Acid Buffered LJ Medium with swab.

34. Laboratory diagnosis M. tuberculosis (culture) • grows very slowly • several weeks • non-pigmented colonies • niacin production • differentiates from other mycobacteria

35. Tuberculosis • polymerase chain amplification • rapid diagnosis

36. Pathogenesis * Inhalation of tubercle bacilli * They multiply in the alveolar macrophages * An early tubercle (granuloma) is formed

37. Pathogenesis * Lesions, healing or progression of infection depend upon 1- Dose of infecting mycobacteria 2- Resistance and hypersensitivity of host * Virulence : Glycolipids on the outer surface of bacteria - Enhance granuloma formation - Inhibit migration of polymorphnuclear leucocytes - Help survival of tubercle bacilli inside macrophages

38. Pathogenesis A- Primary infection: * An exudative lesion : - spread to regional lymph nodes - A scar of healing may later calcify (Ghon’ focus) - Lymph nodes caseate and then calcify - Bacilli in the lesion slowly die - Tuberculin test becomes positive - The person immune & hypersensitive

39. Pathogenesis B- Reactivation type : * Activation of tubercle bacilli due to immunity * Formation of tubercles that caseate - fibrosis - open into a bronchus (open tuberculosis) * Tubercle bacilli erode a blood vessels - Infect any organ (Miliary T.B.)

40. Treatment • Chemoprophylaxis – INH for one year • Domicilliary treatment preferred • Drugs – • Rifampicin • Isoniazide Bactericidal • Pyrazinamide • Streptomycin • Ethambutol • Ethionamide • Thiacetazone Bacteriostatic • Paraminosalicylic acid • Cycloserine

41. Treatment • Short term chemotherapy of six months is sufficient • Problem area – Development of resistance by mutant selection • Solution – Treatment by two to three drug combination, adequate treatment.

42. Treatment Use multiple drug therapy to prevent emergence of resistant mutants * Long duration treatment (6-18 months) * Four drugs are usually started in initial therapy due to: - Intracellular location of bacilli - Slow growth rate of bacilli - Caseous material blocks penetration of drugs - Some bacilli persist in a metabolically inactive state * Sputum becomes non-infective 2-3 weeks after starting therapy

43. Treatment Drugs used : 1- First line drugs : - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective): - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin * Noncompliance (failure to complete the course): Directly observed therapy (DOT) Health care workers observe the medication

44. Immuno-prophylaxis • Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG). • The strain causes self limited lesion and induces hypersensitivity & immunity. • Coverts tuberculin negative person to positive reactor. • Immunity lasts for 10-15 years. Immunity 60-80%

45. BCG • Given at birth without tuberculin testing • Protects against TB, the disease runs milder course in protected, prevents skeletal, meningeal & miliary forms. • Also found useful in leprosy, leukaemias and other malignancies by non-specific stimulation of RE system.