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

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

4. Diagnosis

5. Epidemiology 2Epidemiology 2Epidemiology 2Epidemiology 2 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. Direct Microscopy identification M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain

9. Reporting on AFB Microscopy Number of bacilli seenResult reported None per 100 oil immersion fieldsNegative 1-9 per 100 oil immersion fieldsScanty, report exact number 10-99 per 100 oil immersion fields1+ 1-10 per oil immersion field2+ > 10 per oil immersion field3+

10. 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.

11. Three sputum smears are optimal

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

13. 3 smears = sensitivity of 1 culture About 95% of infectious cases

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

15. 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.

17. 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

19. Culture Epidemiology 3Epidemiology 3Epidemiology 3Epidemiology 3 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

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

21. Other Methods for Laboratory Diagnosis of Other Methods for Laboratory Diagnosis of Tuberculosis

22. 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

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

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

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

26. Lancet 356: 1099 2000

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

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

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

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

33. TST vs. Blood Tests TSTBlood Tests Sensitivity (in pts with active TB) 60-80%67-96% Specificity (in low-risk pts) As low as 35%95-100%

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

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

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

37. 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.

40. Molecular method

41. Sensitivity and Specificity of PCR l Incase of smear and culture positive the sensitivity is ranging 80% to 90% with specificity of 97%-99%. l 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

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

43. 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

44. TB/HIV MDR/XDR-TB