CONVENTIONAL METHODS FOR LAB. DIAGNOSIS OF TUBERCULOSIS

1,663 views
1,417 views

Published on

1 Comment
1 Like
Statistics
Notes
No Downloads
Views
Total views
1,663
On SlideShare
0
From Embeds
0
Number of Embeds
109
Actions
Shares
0
Downloads
83
Comments
1
Likes
1
Embeds 0
No embeds

No notes for slide

CONVENTIONAL METHODS FOR LAB. DIAGNOSIS OF TUBERCULOSIS

  1. 1. CONVENTIONAL METHODSCONVENTIONAL METHODS FOR LAB. DIAGNOSIS OFFOR LAB. DIAGNOSIS OF TUBERCULOSIS.TUBERCULOSIS. DR. MADHUWANTI ABHYANKAR.DR. MADHUWANTI ABHYANKAR. ( M.D.) ( Micro.)( M.D.) ( Micro.) CONSULTANT MICROBIOLOGISTCONSULTANT MICROBIOLOGIST..  NISHNAT MICROBIOLOGY SERVICES.NISHNAT MICROBIOLOGY SERVICES.  GOLWILKAR LABORATORIES.GOLWILKAR LABORATORIES.  ANAMOL LABORATORIES PVT. LTD.ANAMOL LABORATORIES PVT. LTD.
  2. 2. Conventional Methods for LaboratoryConventional Methods for Laboratory Diagnosis of TuberculosisDiagnosis of Tuberculosis Rapid Methods:Rapid Methods:  Demonstration of acid fastDemonstration of acid fast bacilli in smearbacilli in smear  Demonstration of antigensDemonstration of antigens in sample eg.CSFin sample eg.CSF  Chemical testsChemical tests  HaematologicalHaematological parametersparameters  Serum- acute phaseSerum- acute phase reactantsreactants  Mycobacteriophages.Mycobacteriophages. Slower Methods:Slower Methods:  In vivo tests-In vivo tests- - human(TT).- human(TT). - Animal inoculation.- Animal inoculation.  Culture.Culture. - conventional L.J.- conventional L.J. - Rapid slide culture.- Rapid slide culture.
  3. 3. Sample Collection.Sample Collection.  Early morning complete sample.Early morning complete sample.  Sputum-3 consecutive samples.Sputum-3 consecutive samples.  Urine-3 consecutive samples pooled.Urine-3 consecutive samples pooled.  Concentration/Decontamination for smear.Concentration/Decontamination for smear.  Transport medium-1% cetyl pyridiumTransport medium-1% cetyl pyridium chloride in 2% saline.chloride in 2% saline.
  4. 4. MicroscopyMicroscopy  Ziehl-Neelsen stain.Ziehl-Neelsen stain.  Acid fastness.Acid fastness.  Hot strong carbolHot strong carbol fuschinfuschin  ResistsResists decolourisation bydecolourisation by strong mineral acids.strong mineral acids.  Myth of acid andMyth of acid and alcohol fast.alcohol fast.  Fluorescent stainFluorescent stain  Auramine O andAuramine O and Rhodamine B.Rhodamine B.  Calcofluor white.Calcofluor white.  Less eye strain.Less eye strain.  More expensive.More expensive.  Good for labs withGood for labs with high workload.high workload.
  5. 5. Sputum collectionSputum collection
  6. 6. Microscopy-problems.Microscopy-problems.  least count 5000-10000 bacilli per ml.(Some books-least count 5000-10000 bacilli per ml.(Some books- 1,00,000/mL.).1,00,000/mL.).  Species differentiation impossible.Species differentiation impossible.  Specimen contamination.Specimen contamination.  False positive.False positive.  Saprophytic mycobacteria.Saprophytic mycobacteria.
  7. 7. Microscopy-precautions.Microscopy-precautions.  To use sterile containers/collection pots.To use sterile containers/collection pots.  Sterile reagents,slides and equipment.Sterile reagents,slides and equipment.  Do not use tap water forDo not use tap water for staining(saprophytic mycobacteria).staining(saprophytic mycobacteria).  New slides only-fungal spores.New slides only-fungal spores. -scratches.-scratches.  In children gastric aspirates-centrifuged v/sIn children gastric aspirates-centrifuged v/s uncentrifuged.uncentrifuged.
  8. 8. Concentration methods forConcentration methods for smears.smears.  Autoclaving.Autoclaving.  1% Sodium hypochlorite.1% Sodium hypochlorite.  Sputum treated with 2%-4% NaOH.Sputum treated with 2%-4% NaOH.  Urine,C.S.F.,fluids centrifuged and depositUrine,C.S.F.,fluids centrifuged and deposit  Treated and/ or stained.Treated and/ or stained.
  9. 9. Reporting Smears.Reporting Smears.  ?+ : 1-3 bacilli in whole smear.?+ : 1-3 bacilli in whole smear.  + : 3-9 bacilli per 100 fields.+ : 3-9 bacilli per 100 fields.  ++ : 1-9 bacilli per 10 fields.++ : 1-9 bacilli per 10 fields.  +++ : 1-9 bacilli per field.+++ : 1-9 bacilli per field.  ++++ : 10 or more bacilli per field.++++ : 10 or more bacilli per field.  CDC or I.U.A.T. classification.CDC or I.U.A.T. classification.
  10. 10. Mycobacteriophages.Mycobacteriophages. 24-48 hour detection.24-48 hour detection.  Selectively targetSelectively target mycobacteria.mycobacteria.  Genus /speciesGenus /species specific.specific.  Can identify drugCan identify drug resistant strains.resistant strains.  Back in vogue as aBack in vogue as a rapid diagnosticrapid diagnostic procedure.procedure.
  11. 11. Tuberculin Test.Tuberculin Test.  Negative.Negative.  Borderline.Borderline.  Positive.Positive.  Mantoux test.Mantoux test.  Purified protein derivativePurified protein derivative (P.P.D.).(P.P.D.).  5 T.U.5 T.U.  48 - 72 hours.48 - 72 hours.  Induration / Erythema.Induration / Erythema.  Positive: >15 mm.Positive: >15 mm.  Converter/asso. Risk :Converter/asso. Risk : 10mm.10mm.  Contacts/H.I.V./ fibroticContacts/H.I.V./ fibrotic lesions/drug users : 5mm.lesions/drug users : 5mm.
  12. 12. Concentration methods forConcentration methods for culture.culture.  4% NaOH in equal amount –incubate at4% NaOH in equal amount –incubate at 37C with shaking-neutralise with HCl/wash37C with shaking-neutralise with HCl/wash with dist. Water/inoculate on L.J.withwith dist. Water/inoculate on L.J.with pH5.5.pH5.5.  2% NaOH with-N acetyl L-cysteine.2% NaOH with-N acetyl L-cysteine.  -Sodium citrate.-Sodium citrate.  Urine samples-oxalic acid +/- SodiumUrine samples-oxalic acid +/- Sodium citrate as urinary saprophytes are resistantcitrate as urinary saprophytes are resistant to alkali.to alkali.
  13. 13. CultureCulture  Strict aerobe.Strict aerobe.  Growth enhanced by 5-10% CO2.Growth enhanced by 5-10% CO2.  Slow grower : doubling time 18 hours.Slow grower : doubling time 18 hours.  Conventional Lowenstein Jensen medium.Conventional Lowenstein Jensen medium.  Egg yolk,mineral salts,malachite green.Egg yolk,mineral salts,malachite green.  Colonies visible in 2-6 weeks in case ofColonies visible in 2-6 weeks in case of M.tuberculosis.M.tuberculosis.  May be 8 weeks or more if pt. Has receivedMay be 8 weeks or more if pt. Has received A.T.T.A.T.T.
  14. 14. Culture -2Culture -2  Additives to Lowenstein-Jensen mediumAdditives to Lowenstein-Jensen medium  Glycerol-prevents drying of media.Glycerol-prevents drying of media.  Pyruvate-helpful for growth of M.bovis.Pyruvate-helpful for growth of M.bovis.  P-nitro benzoic acid-differentiate betweenP-nitro benzoic acid-differentiate between typical and atypical mycobacteria.typical and atypical mycobacteria.  Antimicrobials-for susceptibility testing.Antimicrobials-for susceptibility testing.
  15. 15. Culture-contd.Culture-contd.  Slow growers-M.xenopi,M.malmoense.Slow growers-M.xenopi,M.malmoense.  By increasing incubation time from 6 to 12By increasing incubation time from 6 to 12 weeks isolation rate increases 4.1% forweeks isolation rate increases 4.1% for M.tuberculosis,10.5% for otherM.tuberculosis,10.5% for other mycobacteria,and 70% for M.malmoense.mycobacteria,and 70% for M.malmoense.  Temperature-35C-ordinary.Temperature-35C-ordinary.  -33C-skin lesions.-33C-skin lesions.  Sensitivity of culture 10-100 organisms/mL.Sensitivity of culture 10-100 organisms/mL.
  16. 16. Rapid slide cultureRapid slide culture  Banked blood + distilled water ( forBanked blood + distilled water ( for haemolysis) + Mitchison’s cocktailhaemolysis) + Mitchison’s cocktail (Carbenicillin, Amphotericin B,(Carbenicillin, Amphotericin B, Trimethoprim, Polymyxin B).Trimethoprim, Polymyxin B).  Sterile slides -SmearSterile slides -Smear  -Susceptibility testing.-Susceptibility testing.
  17. 17. Identification of M.tuberculosis.Identification of M.tuberculosis.  Slow growth rate.Slow growth rate.  Failure to grow at 25C.Failure to grow at 25C.  Susceptibility to p-nitro-benzoic acid(PNB).Susceptibility to p-nitro-benzoic acid(PNB).  No pigment production.No pigment production.  Other tests-Nitrate reduction.Other tests-Nitrate reduction.  -10ug/mL thiacetazone.-10ug/mL thiacetazone.  -Tween 80 hydrolysis.-Tween 80 hydrolysis.  -Arylsulphatase test.-Arylsulphatase test.
  18. 18. Increasing success rate of cultureIncreasing success rate of culture  Early morning complete sample.Early morning complete sample.  3 consecutive samples/pooled urine sample.3 consecutive samples/pooled urine sample.  Sputum unavailable-laryngeal swab.Sputum unavailable-laryngeal swab.  -gastric aspirate.-gastric aspirate.  -induced sputum.-induced sputum.  BronchoscopyBronchoscopy - bronchial lavage- bronchial lavage - transbronchial lung biopsy.- transbronchial lung biopsy.
  19. 19. Increasing culture success-2Increasing culture success-2  Biopsy-homogenised by grinding withBiopsy-homogenised by grinding with sterile phosphate buffered saline and sand insterile phosphate buffered saline and sand in Griffith tube or mortar and pestle.Griffith tube or mortar and pestle.  Fluid-citrated sample.(2 drops 2% SodiumFluid-citrated sample.(2 drops 2% Sodium citrate per 10 mL sample.citrate per 10 mL sample.  Urine-membrane filtration / centrifugation.Urine-membrane filtration / centrifugation.
  20. 20. Animal InoculationAnimal Inoculation  Guinea pig or rabbit.Guinea pig or rabbit.  Animal house.Animal house.  Animal is sacrificed.Animal is sacrificed.  Time consuming.Time consuming.  Species identificationSpecies identification not possible.not possible.  Culture is safer,Culture is safer, simpler, cheaper,simpler, cheaper, humane.humane.
  21. 21. Antimicrobial Susceptibility TestingAntimicrobial Susceptibility Testing  Resistance ratio method.Resistance ratio method.  Proportion method.Proportion method.  Absolute concentration method.Absolute concentration method.  Diffusion method.Diffusion method.  Radiometric method.Radiometric method.  Resistance ratio:Resistance ratio: – Standardised suspension of culture.Standardised suspension of culture. – LJ media incorporating doubling dilutions ofLJ media incorporating doubling dilutions of antibiotics.antibiotics.
  22. 22. AST-2AST-2 Drug concentration inhibiting test stDrug concentration inhibiting test st Drug concn. Inhibiting reference st.Drug concn. Inhibiting reference st.  Proportion method:- measure proportion ofProportion method:- measure proportion of bacteria growing on drug containing mediabacteria growing on drug containing media compared to drug free media.compared to drug free media.  Colony count required (at least 18-20).Colony count required (at least 18-20).  Expensive,time consuming,quality control.Expensive,time consuming,quality control.  On treatment – first culture negative , then smear.On treatment – first culture negative , then smear. R.Ratio =
  23. 23. BACILLUS CALMETTE GUERINBACILLUS CALMETTE GUERIN  BCG : avirulent strain with capacity toBCG : avirulent strain with capacity to induce immune response and thus resistanceinduce immune response and thus resistance to subsequent infection with virulentto subsequent infection with virulent tubercle bacilli. This reduces morbidity andtubercle bacilli. This reduces morbidity and mortality among those at risk.mortality among those at risk.  1921-25 : Oral vaccine.1921-25 : Oral vaccine.  1927 : intradermal vaccine.1927 : intradermal vaccine.  1948 : globally accepted.1948 : globally accepted.
  24. 24. BCG-2BCG-2  Liquid / freeze dried.Liquid / freeze dried.  WHO: Danish 1331WHO: Danish 1331 strain.strain.  In India at GuindyIn India at Guindy  Reconstituted vaccineReconstituted vaccine stable 3hrs.protectedstable 3hrs.protected from light.from light.  New born: 0.05ml.New born: 0.05ml.  Adult: 0.1ml.Adult: 0.1ml.  Protection 15-20 yrs.Protection 15-20 yrs.

×