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Introduction 1

Introduction 1. Laboratory Diagnosis Tuberculosis. Dr. Kiarash Ghazvini Department for bacteriology and virology, Mashhad University of medical Sciences. Mycobacterium tuberculosis. long, slender, straight or curved, acid fast bacilli.

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Introduction 1

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  1. Introduction 1 Laboratory DiagnosisTuberculosis Dr. Kiarash Ghazvini Department for bacteriology and virology, Mashhad University of medical Sciences

  2. Mycobacterium tuberculosis long, slender, straight or curved, acid fast bacilli slow growers, obligate aerobes, intracellular bacterium structure composed of high molecular weight acidic waxes, mycolic acid, cord factor

  3. Diagnosis

  4. Epidemiology 2 Direct Microscopy identification M.tuberculosis is a acid–fast bacterium, rod-shaped bacterium measuring 2-4 x 0.2-0.5 μm. They appear as bright red rods against a contrasting background. The Ziehl-Neelsen stain is used to demonstrate the presence of the bacilli in a smear. The technique is simple, inexpensive and detects those cases of tuberculosis who are infectious. M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain

  5. Reporting on AFB Microscopy Number of bacilli seen Result reported None per 100 oil immersion fields Negative 1-9 per 100 oil immersion fields Scanty, report exact number 10-99 per 100 oil immersion fields 1+ 1-10 per oil immersion field 2+ > 10 per oil immersion field 3+

  6. AFB MICROSCOPY Advantages -Rapid - High specificity (AFB in sputum = TB) • All mycobacterium are acid fast, no exception ; • > 98% for AFB in high burden countries - Accurate diagnoses - Using simple and available equipment Disadvantage Low sensitivity; Reported sensitivity ranging 25 to 65% when compared to culture Species differentiation impossible. False positive; Saprophytic mycobacteria.

  7. Three sputum smears are optimal

  8. AFB MICROSCOPY: SENSITIVITY • Quality of sputum, morning sputum 10-100% more positives • Number of samples • Quality of smear, staining, quality of microscopes, • Effort in examining number of fields

  9. 3 smears = sensitivity of 1 culture About 95% of infectious cases

  10. Smear-positive patients are 4-20 times more infectious • Untreated, a smear-positive patient may infect 10-15 persons/year • Smear-positive patients are much more likely to die if untreated Rouillon A. Tubercle 1976;57:275-99

  11. Direct Microscopy identification • Fluorescent dye (Auramine O and Rhodamine B) • Good for labs with high workload. • Auramine O- Bright yellow • Auramine O-Rhodamine B- Yellow orange.

  12. Appropriate collection techniques • Time • Sputum -3 consecutive samples. • Early morning complete sputum. • Urine -3 or more consecutive samples • 24 h pooled ? • Volume • Pleural, peritoneal fluids • Cerebrospinal fluid

  13. Epidemiology 3 Culture M. tuberculosis grows in Lowenstein Jensen medium, which contains inhibitors to keep contaminants from outgrowing the organism. Because of its slow growth, it takes 4-6 weeks before small buff-coloured colonies are visible on the medium. Typical small, buff coloured colonies of M. tuberculosis on Lowenstein Jensen medium

  14. Culture • more sensitive, but relative • low income countries : only 25% gain over smear ? • and in these settings less specific ? • main problem: DELAYS • on classical media (Lowenstein-Jensen....) • newer commercial media (BACTEC..) • faster (about 10 days) • but expensive++, technical demands • first step for susceptibility testing

  15. Other Methods for Laboratory Diagnosis of Tuberculosis

  16. BACTEC –TB System • based on the principal that the organisms multiply in the broth and metabolize C 14-containing palmatic–acid, producing radioactively labeled 14CO2. *.Containing • Middle Brook 7H12 containing palmatic acid • PANTA (Polymyxin B , Amphotericin B , Nalidic acid , trimethoprim ,Azlocillin ) • OADC enrichment

  17. Mycobacterium Growth Indicator Tubes (MGIT) • A fluorescent compound is embedded in silicone on the bottom of 16 x 100 mm round-bottom tubes.

  18. Tests based on Immune responce • PPD test • Gama interferone response • Invitro Blood Test (ELISPOT) • Serodiagnostic Test (ELISA)

  19. Blood Tests for TB • New blood tests examine immune response to TB antigens • Draw blood from patient, expose white cells to TB antigens, look for signs of immune response • Two most common methods are ELISA and ELISPOT

  20. Lancet 356: 1099 2000

  21. Blood Tests for TB • One-time blood draw • Less inter-reader variability than PPD • Looks at response to 2 TB-specific antigens: ESAT-6 and CFP-10 • Antigens are not found in BCG, most non-TB mycobacteria • ELISA and ELISPOT technologies commercially available

  22. (Pai M et al, Lancet Inf Dis 4: 761 2004)

  23. T-SPOT (ELISPOT) test Meier T et al, Eur J Clin Microbiol Infect Dis 24:529 (2005)

  24. TSTBlood tests Test itself cheap Clearly correlated with development of TB Can be done anywhere Reliability fair Cross-reacts with BCG, NTM More expensive Unclear correlation with development of TB Blood must be sent quickly to lab Reliable Does not cross-react with BCG, most NTM

  25. TST vs. Blood Tests

  26. Summary • Have great potential to reduce false-positive results • Likely more sensitive for active TB, but clinical utility in this setting less clear • Logistical and cost barriers to implementation • Ideally will replace TST in many settings

  27. Adenosine deaminase (ADA) • Adenosine deaminase (ADA) and interferon gamma studied for dx of extrapulmonary TB • Both are markers of immune response to TB • Not that specific

  28. Immune dx of TB • ADA and interferon gamma may be useful in endemic areas • In low-incidence areas, specificity and sensitivity are not good enough for routine use • More specific markers needed

  29. PCR polymerase chain amplification 1)Diagnose tuberculosis rapidly by identifying DNA from M. tuberculosis in clinical samples. 2) Determine rapidly whether acid-fast organisms identified by microscopic examination in clinical specimens are M.tuberculosis 3) Identify the presence of genetic modifications known to be associated with resistance to some anti mycobacterial agents. 4) Determine whether or not isolates of M.tuberculosis from different patients have a common origin in the context of epidemiological studies.

  30. Molecular method

  31. Sensitivity and Specificity of PCR • Incase of smear and culture positive the sensitivity is ranging 80% to 90% with specificity of 97%-99%. • Incase of smear negative and culture positive the sensitivity is ranging 60% to 80% with specificity of 97%-99%. Disadvantages: • Identification of the target sequence of DNA doesn't imply organism viability. • Contamination of samples by product from previous PCR experiments

  32. NAA Summary • NAA is useful to distinguish TB from NTM in smear + specimens • Less sensitive in smear – specimens • Clinical judgement must always take priority • Relatively expensive tests; need data to support cost-effectiveness

  33. Real time PCR • Rapid analysis(typically under 90 min) • No post-amplification processing(no gels or autoradiographs) • Automated(data collection and analysis) • Objective (controls & standards can be built-in) • Precise, sensitive and reproducible

  34. TB/HIV MDR/XDR-TB

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