MYCOBACTERIUMMYCOBACTERIUM

Fungus Like BacteriaFungus Like Bacteria

Slendr rods—smtms show branching-Slendr rods—smtms show branching-resembling fungal mycelium.resembling fungal mycelium.

In liquid cultures—mould like pellicleIn liquid cultures—mould like pellicle—donot stain readily—if staind——donot stain readily—if staind—retains evn against dil.mineral acidsretains evn against dil.mineral acids—ACID FAST BACILLI/AFB—ACID FAST BACILLI/AFB

AEROBIC,NONMOTILE,NONSPORING,AEROBIC,NONMOTILE,NONSPORING,NONCAPSULATEDNONCAPSULATED

Slow growth—Slow growth— Genus includes-obligate Genus includes-obligate

parasites,oppurtunistic pathogens—parasites,oppurtunistic pathogens—and saprophytesand saprophytes

Hansen-identified first membr—lepra Hansen-identified first membr—lepra bacillusbacillus

Koch-isolated mammalian Tubercle Koch-isolated mammalian Tubercle bacillus—proved its causitive role in TBbacillus—proved its causitive role in TB

TB in humans –M.tuberculosis & M.bovisTB in humans –M.tuberculosis & M.bovis M.tuberculosis—M.africanum &M.microtiM.tuberculosis—M.africanum &M.microti Lepra bacillus-LeprosyLepra bacillus-Leprosy Also atypical mycobacteria—saprophytic Also atypical mycobacteria—saprophytic

ones –ones –M.butrycum,phlei,stercosis,smegmatisM.butrycum,phlei,stercosis,smegmatis

Morphology-straight/slightly curvedMorphology-straight/slightly curved Gram +(not strictly correct)Gram +(not strictly correct) Acid and alcohol fastAcid and alcohol fast Acid fastness-mycolic acidAcid fastness-mycolic acid Culture-grows slowlyCulture-grows slowly Generation time-14-15 hrsGeneration time-14-15 hrs Colonies appear by 2-8wksColonies appear by 2-8wks

M.tuberculosisM.tuberculosis

M.tuberculosisM.tuberculosis M.bovisM.bovis

obligate aerobeobligate aerobe microaerophilic primary microaerophilic primary isolation,aerobic on isolation,aerobic on subculturesubculture

Eugonic-grows Eugonic-grows luxuriantlyluxuriantly

Dysgonic-sparselyDysgonic-sparsely

Niacin test &Nitrate Niacin test &Nitrate reduction test &PO+vereduction test &PO+ve

Niacin test and nitrate Niacin test and nitrate reduction test -vereduction test -ve

0.5%glycerol-improves 0.5%glycerol-improves growthgrowth

no effectno effect

Sodium pyruvate-inc Sodium pyruvate-inc growthgrowth

inc growthinc growth

Solid medium-Solid medium-dry,rough,raised,irregdry,rough,raised,irregular,wrinkled ular,wrinkled colonies-creamy colonies-creamy white to yelow on white to yelow on incubationincubation

Flat Flat smooth,white,moist,bsmooth,white,moist,breakup easily when reakup easily when touchedtouched

In liquid media-In liquid media-growth begins at growth begins at bottom,creeps up the bottom,creeps up the sides,forms sides,forms prominent surface prominent surface pelliclepellicle

Human tubercle bacilli do not grow Human tubercle bacilli do not grow in presence of P-nitrobezoic acidin presence of P-nitrobezoic acid

Highly susceptible to fatty acids---Highly susceptible to fatty acids---can b neutralised with serum can b neutralised with serum albumin /charcoalalbumin /charcoal

Solid media-Solid media-

1.1. With egg-LJ mediumWith egg-LJ medium

2.2. Blood –TarshisBlood –Tarshis

3.3. Serum-LoefflerSerum-Loeffler

4.4. Potato-PawlowskyPotato-Pawlowsky

LJ medium,without starch is used –LJ medium,without starch is used –

1.1. Coagulated hen’s eggsCoagulated hen’s eggs

2.2. Mineral salt solutionMineral salt solution

3.3. AsparagineAsparagine

4.4. Malachite green (inhibits other Malachite green (inhibits other bacteria)bacteria)

Liquid media-(sensitivity tst,chemical Liquid media-(sensitivity tst,chemical analysis,antigen & vacc. Prep)analysis,antigen & vacc. Prep)

1.1. Dubos’Dubos’

2.2. Middlebrook’sMiddlebrook’s

3.3. ProskauerProskauer

Resistance-destroyed by tincture of Resistance-destroyed by tincture of iodine by 5 min, ethanol is needed iodine by 5 min, ethanol is needed disinfectant for skin, gloves, clinical disinfectant for skin, gloves, clinical thermometersthermometers

Biochemical reactions-Biochemical reactions-1.1. Niacin testNiacin test2.2. Nitrate reduction testNitrate reduction test3.3. Peroxidase testPeroxidase test4.4. Aryl sulphatase testAryl sulphatase test5.5. Catalase testCatalase test6.6. Neutral red test-+-virulentNeutral red test-+-virulent

Antigenic properties-group specificity Antigenic properties-group specificity is due to polysaccharides ,type is due to polysaccharides ,type specificity is due to protein antigensspecificity is due to protein antigens

Infection---delayed hypersensitivity Infection---delayed hypersensitivity against tuberculin (bacillary protein)against tuberculin (bacillary protein)

Bacteriophage-phages not truly Bacteriophage-phages not truly lysogenic-genome lies freely as lysogenic-genome lies freely as plasmid-pseudolysogenyplasmid-pseudolysogeny

Host range-Mtb causes natural Host range-Mtb causes natural infection in humans,other primates, infection in humans,other primates, dogs.dogs.

M.bovis –pathogenic for animals-M.bovis –pathogenic for animals- BCG-attenuated vaccine of bovisBCG-attenuated vaccine of bovis

Pathogenesis-source of infection-case of Pathogenesis-source of infection-case of pulm.tbpulm.tb

One case—25 inf.One case—25 inf. Bacilli—macrophages—Bacilli—macrophages— Only 10% of infected -dvlp d/sOnly 10% of infected -dvlp d/s Only immunity effective is cell mediatedOnly immunity effective is cell mediated Release of –IFN ,IL1,2,TNFRelease of –IFN ,IL1,2,TNF Tubercle formation-avascular Tubercle formation-avascular

granuloma,central zone of giant cells,+/- granuloma,central zone of giant cells,+/- caseation and peripheral zone of caseation and peripheral zone of lymphocytes &fibroblastslymphocytes &fibroblasts

Tuberculous lesions-Tuberculous lesions-

1.1. Exudative-A/c inf. Reaction-more in Exudative-A/c inf. Reaction-more in nature of DTHnature of DTH

2.2. Productive-cellular—more in nature Productive-cellular—more in nature of protective immunityof protective immunity

Depending on time and type of Depending on time and type of infection,TB may b classified asinfection,TB may b classified as

1.1. PrimaryPrimary

2.2. Post primaryPost primary

Lab dig-bacillus micro-culture-Lab dig-bacillus micro-culture-inoculation-demonstration of inoculation-demonstration of hypersenstivity to tuberculin proteinhypersenstivity to tuberculin protein

Pulmonary tuberculosisPulmonary tuberculosis Specimen-sputum,best collected at Specimen-sputum,best collected at

morning b4 any meal-if scanty-24hr morning b4 any meal-if scanty-24hr sample takensample taken

If not—laryngeal swabs/bronchial If not—laryngeal swabs/bronchial washings/gastric lavage(in swallowers)washings/gastric lavage(in swallowers)

MicroscopyMicroscopy Direct/concentrated smears examinedDirect/concentrated smears examined Sputum microscopy-single most Sputum microscopy-single most

reliable method in cotrolling TB reliable method in cotrolling TB infectioninfection

Smear-stained by Ziehl-Neelson Smear-stained by Ziehl-Neelson method-under oil immersion-AFB seen method-under oil immersion-AFB seen as bright red rods-as bright red rods-

Atleast 10’000 AFB /ml should be Atleast 10’000 AFB /ml should be present to demonstrate in direct present to demonstrate in direct smearssmears

A negative report should be given only A negative report should be given only after looking atleast 300 fields,taking after looking atleast 300 fields,taking about 10 fieldsabout 10 fields

ZN smear evln. & AFB reportZN smear evln. & AFB report

No. of AFBNo. of AFB OIL imm.fieldOIL imm.field reportreport

00 300 F300 F AFB not seenAFB not seen

1-21-2 300 F300 F Repeat smearRepeat smear

1-91-9 100 F100 F 1 +1 +

1-91-9 10 F10 F 2 +2 +

1-91-9 1 F1 F 3 +3 +

>= 10>= 10 1 F1 F 4 +4 +

Concentration methods-which donot kill Concentration methods-which donot kill org. and used for culture and animal org. and used for culture and animal inoculation inccludesinoculation inccludes

Petroff’s method-Petroff’s method-

Culture-very sensitive d/g tool for Culture-very sensitive d/g tool for tubercle bacilli-detecting as few as tubercle bacilli-detecting as few as 10-100 bacilli /ml10-100 bacilli /ml

Conc. Material—LJ medium—37C –Conc. Material—LJ medium—37C –4days—twice weekly afterwards4days—twice weekly afterwards

A negative report—no growth after 8-A negative report—no growth after 8-12 weeks12 weeks

Sensitivity tests-Sensitivity tests-

1.1. Absolute concentration methodAbsolute concentration method

2.2. Resistance ratio methodResistance ratio method

3.3. Proportion methodProportion method

Extra pulmonary TB-micro-culture-Extra pulmonary TB-micro-culture-inoculation---but few no. of bacilliinoculation---but few no. of bacilli

CSF –from tb.meningitis—spider web CSF –from tb.meningitis—spider web clot on standingclot on standing

Pleural effusion, pus, blood, etc used as Pleural effusion, pus, blood, etc used as specimenspecimen

Intermittent urinary excretion – test 3-6 Intermittent urinary excretion – test 3-6 samples of urine ,morning samples-samples of urine ,morning samples-each sample is centrifuged at 3000 rpm each sample is centrifuged at 3000 rpm for 30 min---sediment for culture after for 30 min---sediment for culture after concentrationconcentration

Allergy and immunityAllergy and immunity Old tuberculin-protein extract of tubercle Old tuberculin-protein extract of tubercle

bacillus(OT)bacillus(OT) Purified protein derivative-(PPD)-Purified protein derivative-(PPD)- 1 Batch of PPD-tuberculin=50’000 TU/mg1 Batch of PPD-tuberculin=50’000 TU/mg 1 TU=0.01ml OT=0.00002mg PPD-S1 TU=0.01ml OT=0.00002mg PPD-S Mantoux test-0.1 ml of PPD containing 5 TU –Mantoux test-0.1 ml of PPD containing 5 TU –

injected intra dermally-flexor aspect of forearm injected intra dermally-flexor aspect of forearm with a tuberculin syringe raising a whealwith a tuberculin syringe raising a wheal

Should b intra dermal not sub cu.Should b intra dermal not sub cu. Site is examined after 48-72 hrs later,induration Site is examined after 48-72 hrs later,induration

is measured at its widest point transversely to is measured at its widest point transversely to the long axis of the forearm.the long axis of the forearm.

Induration of diametre-10mm/more is +veInduration of diametre-10mm/more is +ve Less than 5mm—veLess than 5mm—ve Between-equivocalBetween-equivocal

A PPD idose of 1 TU is used when A PPD idose of 1 TU is used when extreme hypersensitivity is suspected-extreme hypersensitivity is suspected-

Dose of 10/100-used when test is –veDose of 10/100-used when test is –ve A positive tuberculin test-indicates A positive tuberculin test-indicates

hypersensitivity to tubercular hypersensitivity to tubercular proteins-denoting infection with proteins-denoting infection with tubercle bacillus/BCG tubercle bacillus/BCG immunisation,recent ,or past,with or immunisation,recent ,or past,with or without clinical illnesswithout clinical illness

Test become =ve 4-6wks after Test become =ve 4-6wks after infection/immunisation---disappears infection/immunisation---disappears after 4-5 yrsafter 4-5 yrs

Persons who hav never had contact with Persons who hav never had contact with the tubercle bacilli are tuberculin neg.the tubercle bacilli are tuberculin neg.

False negative test-Miliary Tb, False negative test-Miliary Tb, convalescence of some viral infections convalescence of some viral infections like measles,lymphoreticular like measles,lymphoreticular malignancy,sarcoidosis,severe malignancy,sarcoidosis,severe malnutrition,immunosuppressive malnutrition,immunosuppressive therapy,impaired cell mediated therapy,impaired cell mediated immunityimmunity

Repeated testing-no change in normalRepeated testing-no change in normal Increased response in reactiveIncreased response in reactive So in equivocal--repeatSo in equivocal--repeat

Prophylaxis Prophylaxis

BCG I/D-is a strain of M.bovis, attenuated BCG I/D-is a strain of M.bovis, attenuated by 239 serial sub cultures in glycerine-bile by 239 serial sub cultures in glycerine-bile potato medium over a period of 13 yrspotato medium over a period of 13 yrs

Immunity for 10-15 yrs- similar to the Immunity for 10-15 yrs- similar to the immunity after natural infection except for immunity after natural infection except for the fac6t that there is no chance of any the fac6t that there is no chance of any reinfection due to reactivationreinfection due to reactivation

Infants of mother’s with active TB-Infants of mother’s with active TB-Chemopx.-INH-5mg/kg-daily for 6-12 Chemopx.-INH-5mg/kg-daily for 6-12 monthsmonths