1 / 18

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

johana
Télécharger la présentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related