CONVENTIONAL METHODS FOR LAB' DIAGNOSIS OF TUBERCULOSIS'

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CONVENTIONAL METHODS FOR LAB. DIAGNOSIS OF
TUBERCULOSIS.

• DR. MADHUWANTI ABHYANKAR. ( M.D.) (
  Micro.)
• CONSULTANT MICROBIOLOGIST.
• NISHNAT MICROBIOLOGY SERVICES.
• GOLWILKAR LABORATORIES.
• ANAMOL LABORATORIES PVT. LTD.

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Conventional Methods for Laboratory Diagnosis of
Tuberculosis

• Rapid Methods
• Demonstration of acid fast bacilli in smear
• Demonstration of antigens in sample eg.CSF
• Chemical tests
• Haematological parameters
• Serum- acute phase reactants
• Mycobacteriophages.

• Slower Methods
• In vivo tests- - human(TT).-
  Animal inoculation.
• Culture.- conventional L.J.- Rapid slide
  culture.

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Sample Collection.

• Early morning complete sample.
• Sputum-3 consecutive samples.
• Urine-3 consecutive samples pooled.
• Concentration/Decontamination for smear.
• Transport medium-1 cetyl pyridium chloride in 2
  saline.

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Microscopy

• Ziehl-Neelsen stain.
• Acid fastness.
• Hot strong carbol fuschin
• Resists decolourisation by strong mineral acids.
• Myth of acid and alcohol fast.

• Fluorescent stain
• Auramine O and Rhodamine B.
• Calcofluor white.
• Less eye strain.
• More expensive.
• Good for labs with high workload.

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Sputum collection
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Microscopy-problems.

• least count 5000-10000 bacilli per ml.(Some
  books-1,00,000/mL.).
• Species differentiation impossible.
• Specimen contamination.
• False positive.
• Saprophytic mycobacteria.

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Microscopy-precautions.

• To use sterile containers/collection pots.
• Sterile reagents,slides and equipment.
• Do not use tap water for staining(saprophytic
  mycobacteria).
• New slides only-fungal spores.
• -scratches.
• In children gastric aspirates-centrifuged v/s
  uncentrifuged.

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Concentration methods for smears.

• Autoclaving.
• 1 Sodium hypochlorite.
• Sputum treated with 2-4 NaOH.
• Urine,C.S.F.,fluids centrifuged and deposit
• Treated and/ or stained.

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Reporting Smears.

• ? 1-3 bacilli in whole smear.
• 3-9 bacilli per 100 fields.
• 1-9 bacilli per 10 fields.
• 1-9 bacilli per field.
• 10 or more bacilli per field.
• CDC or I.U.A.T. classification.

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Mycobacteriophages.

• 24-48 hour detection.

• Selectively target mycobacteria.
• Genus /species specific.
• Can identify drug resistant strains.
• Back in vogue as a rapid diagnostic procedure.

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Tuberculin Test.

• Negative.
• Borderline.
• Positive.

• Mantoux test.
• Purified protein derivative (P.P.D.).
• 5 T.U.
• 48 - 72 hours.
• Induration / Erythema.
• Positive gt15 mm.
• Converter/asso. Risk 10mm.
• Contacts/H.I.V./ fibrotic lesions/drug users
  5mm.

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Concentration methods for culture.

• 4 NaOH in equal amount incubate at 37C with
  shaking-neutralise with HCl/wash with dist.
  Water/inoculate on L.J.with pH5.5.
• 2 NaOH with-N acetyl L-cysteine.
• -Sodium citrate.
• Urine samples-oxalic acid /- Sodium citrate as
  urinary saprophytes are resistant to alkali.

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Culture

• Strict aerobe.
• Growth enhanced by 5-10 CO2.
• Slow grower doubling time 18 hours.
• Conventional Lowenstein Jensen medium.
• Egg yolk,mineral salts,malachite green.
• Colonies visible in 2-6 weeks in case of
  M.tuberculosis.
• May be 8 weeks or more if pt. Has received A.T.T.

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Culture -2

• Additives to Lowenstein-Jensen medium
• Glycerol-prevents drying of media.
• Pyruvate-helpful for growth of M.bovis.
• P-nitro benzoic acid-differentiate between
  typical and atypical mycobacteria.
• Antimicrobials-for susceptibility testing.

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Culture-contd.

• Slow growers-M.xenopi,M.malmoense.
• By increasing incubation time from 6 to 12 weeks
  isolation rate increases 4.1 for
  M.tuberculosis,10.5 for other mycobacteria,and
  70 for M.malmoense.
• Temperature-35C-ordinary.
• -33C-skin lesions.
• Sensitivity of culture 10-100 organisms/mL.

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Rapid slide culture

• Banked blood distilled water ( for haemolysis)
  Mitchisons cocktail (Carbenicillin,
  Amphotericin B, Trimethoprim, Polymyxin B).
• Sterile slides -Smear
• -Susceptibility testing.

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Identification of M.tuberculosis.

• Slow growth rate.
• Failure to grow at 25C.
• Susceptibility to p-nitro-benzoic acid(PNB).
• No pigment production.
• Other tests-Nitrate reduction.
• -10ug/mL thiacetazone.
• -Tween 80 hydrolysis.
• -Arylsulphatase test.

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Increasing success rate of culture

• Early morning complete sample.
• 3 consecutive samples/pooled urine sample.
• Sputum unavailable-laryngeal swab.
• -gastric
  aspirate.
• -induced sputum.
• Bronchoscopy - bronchial lavage -
  transbronchial lung biopsy.

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Increasing culture success-2

• Biopsy-homogenised by grinding with sterile
  phosphate buffered saline and sand in Griffith
  tube or mortar and pestle.
• Fluid-citrated sample.(2 drops 2 Sodium citrate
  per 10 mL sample.
• Urine-membrane filtration / centrifugation.

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Animal Inoculation

• Guinea pig or rabbit.
• Animal house.
• Animal is sacrificed.
• Time consuming.
• Species identification not possible.
• Culture is safer, simpler, cheaper, humane.

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Antimicrobial Susceptibility Testing

• Resistance ratio method.
• Proportion method.
• Absolute concentration method.
• Diffusion method.
• Radiometric method.
• Resistance ratio
• Standardised suspension of culture.
• LJ media incorporating doubling dilutions of
  antibiotics.

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AST-2

• Drug concentration inhibiting test st
• Drug concn. Inhibiting reference st.
• Proportion method- measure proportion of
  bacteria growing on drug containing media
  compared to drug free media.
• Colony count required (at least 18-20).
• Expensive,time consuming,quality control.
• On treatment first culture negative , then
  smear.

R.Ratio
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BACILLUS CALMETTE GUERIN

• BCG avirulent strain with capacity to induce
  immune response and thus resistance to subsequent
  infection with virulent tubercle bacilli. This
  reduces morbidity and mortality among those at
  risk.
• 1921-25 Oral vaccine.
• 1927 intradermal vaccine.
• 1948 globally accepted.

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BCG-2

• Liquid / freeze dried.
• WHO Danish 1331 strain.
• In India at Guindy
• Reconstituted vaccine stable 3hrs.protected from
  light.
• New born 0.05ml.
• Adult 0.1ml.
• Protection 15-20 yrs.