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  1. 1. MYCOBACTERIUM Fungus Like Bacteria
  2. 2. <ul><li>Slendr rods—smtms show branching-resembling fungal mycelium. </li></ul><ul><li>In liquid cultures—mould like pellicle—donot stain readily—if staind—retains evn against dil.mineral acids—ACID FAST BACILLI/AFB </li></ul><ul><li>AEROBIC,NONMOTILE,NONSPORING,NONCAPSULATED </li></ul><ul><li>Slow growth— </li></ul><ul><li>Genus includes-obligate parasites,oppurtunistic pathogens—and saprophytes </li></ul>
  3. 3. <ul><li>Hansen-identified first membr—lepra bacillus </li></ul><ul><li>Koch-isolated mammalian Tubercle bacillus—proved its causitive role in TB </li></ul><ul><li>TB in humans –M.tuberculosis & M.bovis </li></ul><ul><li>M.tuberculosis—M.africanum &M.microti </li></ul><ul><li>Lepra bacillus-Leprosy </li></ul><ul><li>Also atypical mycobacteria—saprophytic ones –M.butrycum,phlei,stercosis,smegmatis </li></ul>
  4. 4. <ul><li>Morphology-straight/slightly curved </li></ul><ul><li>Gram +(not strictly correct) </li></ul><ul><li>Acid and alcohol fast </li></ul><ul><li>Acid fastness-mycolic acid </li></ul><ul><li>Culture-grows slowly </li></ul><ul><li>Generation time-14-15 hrs </li></ul><ul><li>Colonies appear by 2-8wks </li></ul>M.tuberculosis
  5. 5. Niacin test and nitrate reduction test -ve Niacin test &Nitrate reduction test &PO+ve no effect 0.5%glycerol-improves growth Dysgonic-sparsely Eugonic-grows luxuriantly microaerophilic primary isolation,aerobic on subculture obligate aerobe M.bovis M.tuberculosis
  6. 6. In liquid media-growth begins at bottom,creeps up the sides,forms prominent surface pellicle Flat smooth,white,moist,breakup easily when touched Solid medium-dry,rough,raised,irregular,wrinkled colonies-creamy white to yelow on incubation inc growth Sodium pyruvate-inc growth
  7. 7. <ul><li>Human tubercle bacilli do not grow in presence of P-nitrobezoic acid </li></ul><ul><li>Highly susceptible to fatty acids---can b neutralised with serum albumin /charcoal </li></ul><ul><li>Solid media- </li></ul><ul><li>With egg-LJ medium </li></ul><ul><li>Blood –Tarshis </li></ul><ul><li>Serum-Loeffler </li></ul><ul><li>Potato-Pawlowsky </li></ul>
  8. 8. <ul><li>LJ medium,without starch is used – </li></ul><ul><li>Coagulated hen’s eggs </li></ul><ul><li>Mineral salt solution </li></ul><ul><li>Asparagine </li></ul><ul><li>Malachite green (inhibits other bacteria) </li></ul><ul><li>Liquid media-(sensitivity tst,chemical analysis,antigen & vacc. Prep) </li></ul><ul><li>Dubos’ </li></ul><ul><li>Middlebrook’s </li></ul><ul><li>Proskauer </li></ul>
  9. 9. <ul><li>Resistance-destroyed by tincture of iodine by 5 min, ethanol is needed disinfectant for skin, gloves, clinical thermometers </li></ul><ul><li>Biochemical reactions- </li></ul><ul><li>Niacin test </li></ul><ul><li>Nitrate reduction test </li></ul><ul><li>Peroxidase test </li></ul><ul><li>Aryl sulphatase test </li></ul><ul><li>Catalase test </li></ul><ul><li>Neutral red test-+-virulent </li></ul>
  10. 10. <ul><li>Antigenic properties-group specificity is due to polysaccharides ,type specificity is due to protein antigens </li></ul><ul><li>Infection---delayed hypersensitivity against tuberculin (bacillary protein) </li></ul><ul><li>Bacteriophage-phages not truly lysogenic-genome lies freely as plasmid-pseudolysogeny </li></ul><ul><li>Host range-Mtb causes natural infection in humans,other primates, dogs. </li></ul><ul><li>M.bovis –pathogenic for animals- </li></ul><ul><li>BCG-attenuated vaccine of bovis </li></ul>
  11. 11. <ul><li>Pathogenesis-source of infection-case of pulm.tb </li></ul><ul><li>One case—25 inf. </li></ul><ul><li>Bacilli—macrophages— </li></ul><ul><li>Only 10% of infected -dvlp d/s </li></ul><ul><li>Only immunity effective is cell mediated </li></ul><ul><li>Release of –IFN ,IL1,2,TNF </li></ul><ul><li>Tubercle formation-avascular granuloma,central zone of giant cells,+/- caseation and peripheral zone of lymphocytes &fibroblasts </li></ul>
  12. 13. <ul><li>Tuberculous lesions- </li></ul><ul><li>Exudative-A/c inf. Reaction-more in nature of DTH </li></ul><ul><li>Productive-cellular—more in nature of protective immunity </li></ul><ul><li>Depending on time and type of infection,TB may b classified as </li></ul><ul><li>Primary </li></ul><ul><li>Post primary </li></ul>
  13. 15. <ul><li>Lab dig-bacillus micro-culture-inoculation-demonstration of hypersenstivity to tuberculin protein </li></ul><ul><li>Pulmonary tuberculosis </li></ul><ul><li>Specimen-sputum,best collected at morning b4 any meal-if scanty-24hr sample taken </li></ul><ul><li>If not—laryngeal swabs/bronchial washings/gastric lavage(in swallowers) </li></ul>
  14. 16. <ul><li>Microscopy </li></ul><ul><li>Direct/concentrated smears examined </li></ul><ul><li>Sputum microscopy-single most reliable method in cotrolling TB infection </li></ul><ul><li>Smear-stained by Ziehl-Neelson method-under oil immersion-AFB seen as bright red rods- </li></ul><ul><li>Atleast 10’000 AFB /ml should be present to demonstrate in direct smears </li></ul><ul><li>A negative report should be given only after looking atleast 300 fields,taking about 10 fields </li></ul>
  15. 17. ZN smear evln. & AFB report 4 + 1 F >= 10 3 + 1 F 1-9 2 + 10 F 1-9 1 + 100 F 1-9 Repeat smear 300 F 1-2 AFB not seen 300 F 0 report OIL imm.field No. of AFB
  16. 18. <ul><li>Concentration methods-which donot kill org. and used for culture and animal inoculation inccludes </li></ul><ul><li>Petroff’s method- </li></ul>
  17. 19. <ul><li>Culture-very sensitive d/g tool for tubercle bacilli-detecting as few as 10-100 bacilli /ml </li></ul><ul><li>Conc. Material—LJ medium—37C –4days—twice weekly afterwards </li></ul><ul><li>A negative report—no growth after 8-12 weeks </li></ul><ul><li>Sensitivity tests- </li></ul><ul><li>Absolute concentration method </li></ul><ul><li>Resistance ratio method </li></ul><ul><li>Proportion method </li></ul>
  18. 20. <ul><li>Extra pulmonary TB-micro-culture-inoculation---but few no. of bacilli </li></ul><ul><li>CSF –from tb.meningitis—spider web clot on standing </li></ul><ul><li>Pleural effusion, pus, blood, etc used as specimen </li></ul><ul><li>Intermittent urinary excretion – test 3-6 samples of urine ,morning samples-each sample is centrifuged at 3000 rpm for 30 min---sediment for culture after concentration </li></ul>
  19. 21. <ul><li>Allergy and immunity </li></ul><ul><li>Old tuberculin-protein extract of tubercle bacillus(OT) </li></ul><ul><li>Purified protein derivative-(PPD)- </li></ul><ul><li>1 Batch of PPD-tuberculin=50’000 TU/mg </li></ul><ul><li>1 TU=0.01ml OT=0.00002mg PPD-S </li></ul><ul><li>Mantoux test-0.1 ml of PPD containing 5 TU –injected intra dermally-flexor aspect of forearm with a tuberculin syringe raising a wheal </li></ul><ul><li>Should b intra dermal not sub cu. </li></ul><ul><li>Site is examined after 48-72 hrs later,induration is measured at its widest point transversely to the long axis of the forearm. </li></ul><ul><li>Induration of diametre-10mm/more is +ve </li></ul><ul><li>Less than 5mm—ve </li></ul><ul><li>Between-equivocal </li></ul>
  20. 22. <ul><li>A PPD idose of 1 TU is used when extreme hypersensitivity is suspected- </li></ul><ul><li>Dose of 10/100-used when test is –ve </li></ul><ul><li>A positive tuberculin test-indicates hypersensitivity to tubercular proteins-denoting infection with tubercle bacillus/BCG immunisation,recent ,or past,with or without clinical illness </li></ul><ul><li>Test become =ve 4-6wks after infection/immunisation---disappears after 4-5 yrs </li></ul>
  21. 23. <ul><li>Persons who hav never had contact with the tubercle bacilli are tuberculin neg. </li></ul><ul><li>False negative test-Miliary Tb, convalescence of some viral infections like measles,lymphoreticular malignancy,sarcoidosis,severe malnutrition,immunosuppressive therapy,impaired cell mediated immunity </li></ul><ul><li>Repeated testing-no change in normal </li></ul><ul><li>Increased response in reactive </li></ul><ul><li>So in equivocal--repeat </li></ul>
  22. 24. Prophylaxis <ul><li>BCG I/D-is a strain of M.bovis, attenuated by 239 serial sub cultures in glycerine-bile potato medium over a period of 13 yrs </li></ul><ul><li>Immunity for 10-15 yrs- similar to the immunity after natural infection except for the fac6t that there is no chance of any reinfection due to reactivation </li></ul><ul><li>Infants of mother’s with active TB-Chemopx.-INH-5mg/kg-daily for 6-12 months </li></ul>
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