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. 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. Conventional Methods for Laboratory Diagnosis of Tuberculosis

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. 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. Microscopy

5. Sputum collection

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

12. Negative. Borderline. Positive. Mantoux test. Purified protein derivative (P.P.D.). 5 T.U. 48 - 72 hours. Induration / Erythema. Positive: >15 mm. Converter/asso. Risk :10mm. Contacts/H.I.V./ fibrotic lesions/drug users : 5mm. Tuberculin Test.

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) + Mitchison’s 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.

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.