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Role of Laboratory Services in TB Control Part - II Role of Culture, PCR & Serology

Role of Laboratory Services in TB Control Part - II Role of Culture, PCR & Serology C.N. PARAMASIVAN Tuberculosis Research Centre Indian Council of Medical Research Chennai Indications for Culture in DOTS Failures of re-treatment cases Seriously ill cases; extra-pulmonary cases

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Role of Laboratory Services in TB Control Part - II Role of Culture, PCR & Serology

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  1. Role of Laboratory Servicesin TB ControlPart - IIRole of Culture, PCR & Serology C.N. PARAMASIVAN Tuberculosis Research Centre Indian Council of Medical Research Chennai

  2. Indications for Culture in DOTS • Failures of re-treatment cases • Seriously ill cases; • extra-pulmonary cases • smear negative cases • childhood TB & HIV-TB • For DRS • Not for New Smear Positive Cases

  3. MYCOBACTERIAL CULTURE Advantages: • Increases number of cases found • Detects cases among smear negative patients • Establishes viability of organisms • Distinguishing between Mycobacterial species • Helps in performing DST • Helps in diagnosing cases of failure Limitations: • Expensive • Require enriched media • Require considerable expertise • Time consuming

  4. Decontamination Procedures • 1915 – Petroff’s NaOH • 1946 – Trisodium Phosphate • 1955 – Pancreatin Desogen • 1958 – Pancreatin + 1% cetrimide • 1962 – Zephiran Trisodium PO4 • 1963 – N-acetyl L-cysteine + 2%NaOH • 1969 – Swab culture technique + 1% cetrimide • 1975 – CPC + NaCl2

  5. Culture Media : Solid • LJ • LJ with Na pyruvate • LJ with out asparagine • Middlebrook’s 7H10 & 7H11 • Selective 7H10 & 11 • Ogawa • Tarshi’s Blood Agar

  6. PETROFF’S METHOD Advantages: • Simple, inexpensive & control the growth of contaminants • Twenty samples can be processed in 2 Hrs, with centrifuge capacity being the limiting factor • Sterilized NaOH can be kept for several weeks Limitations: • The specimen exposure times must be strictly followed to prevent over kill of tubercle bacilli. The initial kill is independent of additional contributory factors such as heat build-up in the centrifuge and centrifugal efficiency

  7. Processing of sputum with CPC Method • If delay of more than 48 hours between collection and processing is anticipated, the sputum should be collected with 1%CPC and 2%NaCl2 • CPC acts as homogenizing and decontaminating agent • It helps in retaining viability of Tubercle bacilli up to 7 days • These specimens should not be treated with NaOH ( Petroff’s)

  8. Colony Morphology of M.tuberculosis • Dry wrinkled warty growth. • Eugonic

  9. Reading and Reporting Characteristics of Tubercle bacilli • Growth of Primary culture takes 2 – 4 weeks to obtain visible colonies • Colonies are buff colored and rough, having the appearance of bread crumbs or cauliflower • Not easily emulsified but give a granular suspension • Microscopically frequently arranged in serpentine cords of varying length or show linear clumping

  10. Other Culture Methods • Septi-check AFB • MGIT 960 • Backtec/MB/Bact • ESP Culture ii • Microscopic Observation of Broth Culture • MODS: Micro Colony Detection System

  11. Nucleic acid amplification for mycobact. diagnosis Genus specific protocols Targeting genes code for 16S rRNA 65KDa hsp M.TB Complex specific is 6110 Other targets: Genes encoding 38 KDa MPB 64 mtp 40 PMT 64 Methods: Target amplification - PCR (TMA, LCR, SDA or signal amplification EG: QB amplification) Current status: adjunct to standard procedure What is new in the diagnosis of TB PFYFFER G.E. J.INF. 1999, 39, 21-26. TRC/ICMR 30

  12. Diagnostic performance of NAA for Direct detection of MTB complex What is new in the diagnosis of TB 31 Pfyffer . G.E. J.Inf 1999 39 21-26 TRC/ICMR

  13. Evaluation of in-house PCR for the detection of M.TB. • PCR Results from 6 labs • Samples reconstituted with defined amount of M.TB cells • Each lab used specific conditions of • Sample processing • NA Amplification • Amplicon detection • Large differences observed in sensitivity & specificity Conclusion: in house PCR can not be used as a single diagnostic tool What is new in the diagnosis of TB TRC/ICMR 32 Suffs. P. et al Int. J. Tuberc. Lung Dis 2000, 4(2) 179-183.

  14. Serological diagnosis of TB • Advantages • Low turn around time • High NPV • Useful as a screening test • Limitation • Low sensitivity in Smear Negative • In HIV positive - Low NPV - Low sensitivity • Disease Endemic Countries  Latency - Low PPV • High Cost • Extensive Personnel Training • Difficulty in distinguishing MTB / NTM What is new in the diagnosis of TB Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000,89,131-140 TRC/ICMR 34

  15. Antigens used in serological diagnosis of TB • Mycobacterial sonicates • Extracted glycolipids • PPD • Ag5 (38KDa Ag) • A60 • 45 / 47 – KDa Ag • Ag Kp 90 • 30 KDa Ag • P32 Ag • Cord Factor (trehalase dimycolate) • LAM TRC/ICMR What is new in the diagnosis of TB 33 Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000, 89, 131-140

  16. Antigens used in serological diagnosis of TB • Mycobacterial sonicates • Extracted glycolipids • PPD • Ag5 (38KDa Ag) • A60 • 45 / 47 – KDa Ag • Ag Kp 90 • 30 KDa Ag • P32 Ag • Cord Factor (trehalase dimycolate) • LAM TRC/ICMR What is new in the diagnosis of TB 33 Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000, 89, 131-140

  17. Sensitivity(%) of smear-negative vs smear-positive TB What is new in the diagnosis of TB Chan,E.D.,Heifets,L.,Iseman,M.D., Tubercle & Lung Dis.,2000,89,131-140 TRC/ICMR 35

  18. SummaryRole of culture in DOTS • Culture has no role in the diagnosis of TB in DEDC. • Indicated in; • Failures of re-treatment cases • Seriously ill cases; • extra-pulmonary cases • smear negative cases • childhood TB & HIV-TB • For DRS

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