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Tuberculosis in the World

ADVANCES in BACTERIOLOGICAL DIAGNOSIS of TUBERCULOSIS Assoc. Prof. Alpaslan Alp Hacettepe University Faculty of Medicine Department of Medical Microbiology. Tuberculosis in the World. WHO 2009: 14 million cases 1.68 million death. Undetected probable cases in 2009 (WHO). WHO 2009:.

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Tuberculosis in the World

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  1. ADVANCES in BACTERIOLOGICALDIAGNOSIS of TUBERCULOSIS Assoc. Prof. Alpaslan AlpHacettepe University Faculty of MedicineDepartment of Medical Microbiology

  2. Tuberculosis in the World • WHO 2009: • 14 million cases • 1.68 million death

  3. Undetected probable cases in 2009 (WHO)

  4. WHO 2009: • WHO; case detection rate in 2009: 63% • Half of TB cases are detectable in Africa. • Patients do not admit • Inadequate diagnostic tools

  5. Laboratory Diagnosis • Smear microscopy: 20-80% sensitivity in culture-confirmed cases. • At least two sputum specimens are necessary. • Problem in HIV-infected and pediatric patients.

  6. Laboratory Diagnosis • Culture: Results can be obtained in several weeks. • Well-equipped laboratories are necessary.

  7. Rate limiting step in TB control: Case Detection • 625.000 life/year could be saved by using tests with 100% accuracy. • 392.000 life/year could be saved by using tests with 85% sensitivity and 97% specificity… 22.4% of deaths due to TB.

  8. For the laboratory diagnosis of TB;Is there an IDEAL TEST? • with high sensitivity, • with high specificity, • easy to perform, • rapid, • cheap…

  9. For the laboratory diagnosis of TB;There is NO IDEAL TEST ! • Molecular methods • Diagnostic tests • Tests detecting resistance • Point of care tests Possible candidates could arise from which test group?

  10. Drug Susceptibility Tests • Phenotypic Methods: • Solid Media • Löwenstein-Jensen medium • Middlebrook agar • Rapid culture systems (liquid media) • Bactec 460 • MGIT • Mycolor-TK • Genotypic Methods: • Mutation detection methods • Reverse hybridization (Strip tests) • Real-time PCR • DNA sequencing

  11. Molecular Methods • 1-2 days • Well-standardized commercial methods • Inno-Lipa Rif TB • Hain MDRTBplus • Cepheid Xpert MTB/RIF

  12. Direct susceptibility testing • Should be performed in smear-positive cases. • Rapid; reflects bacteria population better; cheaper. • Calibration of inoculum is more difficult; normal respiratory flora can grow despite decontamination process • Overall rate of failure can be 10-15% or more.

  13. Direct susceptibility testing • MTBDR strip test • MTBDRplus strip test • Nitrate reduction assay • MODS: Microscopic observation drug susceptibility

  14. Direct Susceptibility TestsMeta-Analysis Study • The studies in which INH and RIF resistance detected by direct method were selected. • Eighteen studies out of 64, were included in meta-analysis.

  15. Direct Susceptibility TestsMeta-Analysis Study Detection of rifampicin resistance:

  16. Direct Susceptibility TestsMeta-Analysis Study Detection of isoniazide resistance:

  17. Direct Susceptibility TestsMeta-Analysis Study

  18. WHO Recommendations for Strip Tests-2008 • Adoption of line probe assays for rapid detection of MDR-TB should be decided by Ministries of Health. • Direct testing of sputum smear-positive specimens and indirect test on isolates grown from smear-negative and smear-positive specimens are adequately validated. • Direct use on smear-negative specimens is not recommended. • Adoption of line probe assays does not eliminate the need for conventional culture and DST.

  19. Point of Care Tests • Tests performed instantly, near the patient • Should be cheap, accurate, practical • Should be performed in any treatment site without any need for a specialized laboratory. • Should be performed on different specimens like blood, urine and breath • Duration:<3saat (even 15min…5min)

  20. Lipoarabinomannan • Lipoarabinomannan (LAM): within Mtb cell wall, • 17.5 kDa, heat-stable, glycolipid • 15% of bacterial weight • Immunogenic, virulence factor

  21. Detection of LAM in Urine

  22. LAM ..Clerview-TB-ELISA Dheda 2010: 440 TB, 31% infected with HIV

  23. Determine TB-LAM (Alere) Will be sold within 2012 LAM Ag in urine 30 minutes 3-3.5 USD

  24. Detection of LAM in Urine Peter 2011: Active TB, HIV (+) patients Determine TB-LAM Overall sensitivity: 74% Sensitivity in CD4<100cells/ml: 85% Clearview TB-ELISA • Overall sensitivity: 58% • Sensitivity in CD4<100cells/ml: 72%

  25. “TB-Breathalyzer” (Rapid Biosensor Systems Inc.) • Mtb Ag85B in cough • Collection tube containing Ab; fluorescent response • Sensitivity 74%, specificity 79% (31 TB patients) • Sensitivity together with smear microscopy: 93.5%

  26. Xpert MTB/RIF Assay • Product of a collaborative study: • FIND (Foundation for Innovative New Diagnostics) • Cepheid (USA) • University of Medicine and Dentistry of New Jersey • Detection of M. tuberculosis and rifampicin resistance in two hours. • A “hemi-nested real-time PCR” that uses three primers and five probes.

  27. Xpert MTB/RIF Assay Satisfactory results were obtained in the studies that were performed in 2010: • Sensitivity in smear-positive culture-positive specimens: 99% • Sensitivity in smear-negative culture-positive specimens: 90%

  28. Xpert MTB/RIF Assay Meta-Analysis Study • Eighteen study had been chosen among 90 studies. • A total of 10.224 specimen • 2983 bacteriological TB diagnosis • 6183 smear and culture-negative • Average sensitivity for diagnosis of TB: 90.4% • Average specificity for diagnosis of TB : 98.4%

  29. Xpert MTB/RIF Assay Meta-Analysis Study • Average sensitivity for detection of rifampicin resistance: 94.1% • Average specificity for detection of rifampicin resistance: 97.0% • Average sensitivity in extra-pulmonary specimens: 80.4% • Average specificity in extra-pulmonary specimens : 86.1%

  30. Xpert MTB/RIF AssayMeta-Analysis Study COMMENT: • Xpert MTB/RIF assay fulfills the requirements of rapidly and effectively diagnosing TB and RIF resistance. • Since the mutation points in 5% RIF-resistant Mtb isolates occur outside core gene region, they would not be identified by Xpert MTB/RIF assay.

  31. TB Scent • Giant African pouched rats trained to detect TB evaluated sputum samples from 10.523 patients whose sputum had previously been evaluated by smear microscopy. • Microscopists found 13.3% of the patients to be positive. • Screening of the samples by rats revealed 620 new TB-positive patients, increasing the case detection rate by 44%.

  32. TB Scent • 10 well-trained giant African pouched rats (Cricetomys gambianus) • 23.101 sputum samples • Smear microscopy: 2.487 positive sputum • Rats identified 2.274 of these samples; an additional 927 samples were also found to be positive by the rats, increasing the case detection rate by 44%.

  33. TB Scent • Daily sensitivity of rats to detect Mtb: 72-100% • Daily false positivity: 0.7-8.1%

  34. TB Scent • Metil fenilasetat • Metil p-anisat • Metil nikotinat • o-fenilanizol

  35. Electronical Nose !

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