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CONVENTIONAL METHODS FOR LAB' DIAGNOSIS OF TUBERCULOSIS'

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Conventional Methods for Laboratory Diagnosis of Tuberculosis. Rapid Methods: ... scratches. In children gastric aspirates-centrifuged v/s uncentrifuged. ... – PowerPoint PPT presentation

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Title: CONVENTIONAL METHODS FOR LAB' DIAGNOSIS OF TUBERCULOSIS'


1
CONVENTIONAL METHODS FOR LAB. DIAGNOSIS OF
TUBERCULOSIS.
  • DR. MADHUWANTI ABHYANKAR. ( M.D.) (
    Micro.)
  • CONSULTANT MICROBIOLOGIST.
  • NISHNAT MICROBIOLOGY SERVICES.
  • GOLWILKAR LABORATORIES.
  • ANAMOL LABORATORIES PVT. LTD.

2
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.

3
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.

4
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.

5
Sputum collection
6
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7
Microscopy-problems.
  • least count 5000-10000 bacilli per ml.(Some
    books-1,00,000/mL.).
  • Species differentiation impossible.
  • Specimen contamination.
  • False positive.
  • Saprophytic mycobacteria.

8
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.

9
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.

10
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.

11
Mycobacteriophages.
  • 24-48 hour detection.
  • Selectively target mycobacteria.
  • Genus /species specific.
  • Can identify drug resistant strains.
  • Back in vogue as a rapid diagnostic procedure.

12
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.

13
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.

14
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.

15
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.

16
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.

17
Rapid slide culture
  • Banked blood distilled water ( for haemolysis)
    Mitchisons cocktail (Carbenicillin,
    Amphotericin B, Trimethoprim, Polymyxin B).
  • Sterile slides -Smear
  • -Susceptibility testing.

18
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.

19
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20
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.

21
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.

22
Animal Inoculation
  • Guinea pig or rabbit.
  • Animal house.
  • Animal is sacrificed.
  • Time consuming.
  • Species identification not possible.
  • Culture is safer, simpler, cheaper, humane.

23
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.

24
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
25
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.

26
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.
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