1. Role of Laboratory Services in TB Control Dr Mohammad Rahbar Professor of Microbiology Department Microbiology Reference health Laboratory,Tehran iran

2. 2 National Reference Laboratory Province Laboratories District Laboratories Health Centers Ministry of Health National Laboratory Infrastructure

3. 3 Desirable Features of Diagnosis Methods in TB Desirable Features of Diagnosis Methods in TB  Sensitivity  Specificity  Predictive value  Speed  Reliability  Reproducibility  Cost  Safety  Easy to use  Robustness  Widest usage What is new in the diagnosis of TB TRC/ICMR 3

4. 4 AFB smear AFB (shown in red) are tubercle bacilli

5. 5 Aims of sputum microscopy  Diagnosis of patients with infectious tuberculosis  Monitoring progress of patients on treatment

6. 6 Advantages of sputum microscopy  More reliable than x-ray for the diagnosis of infectious TB  Simple to perform  Easy to read  Minimal infrastructure required  Inexpensive  Quick  Only tool to monitor and declare patients as “cured’’

7. 7 Diagnosis of pulmonary tuberculosis Three specimens optimal  Spot specimen on first visit;sputum container given to patient  Early morning collection next day  Spot specimen during second visit

8. 8 Three sputum smears are optimal

9. 9 A good smear  Made from mucopurulent sputum  Spread evenly  3 cm x 2 cm in size  Not too thick  Thin enough to read newsprint through  Air dried before fixing

10. 10 False negative results  Inadequate sputum collection avoid – saliva, nasal discharge collect – bronchial sputum from depth of chest  Inadequate storage of sputum / stained smears exposure to direct sunlight radiation ( UV light ) excessive heat / humidity  Not taking mucopurulent portion of sputum  Inadequate smear preparation  Inadequate smear examination  Administrative & recording errors

11. 11 Consequences of false negative smear results  Patients with TB will not be treated, resulting in suffering, spread of TB and death.  Intensive phase of treatment will not be extended for the required duration, resulting in inadequate treatment  Patient may lose confidence in the programme

12. 12 False positive results  Food particles  Precipitated stains  Saprophytic AFB  Spores of B.subtlis  Fibres and pollen  Scratches on slide  Contamination through carry over of AFB from one smear to another

13. 13 Consequences of false positive results  Patients are started on treatment unnecessarily  Treatment is continued longer than necessary, in follow-up examinations  Medications will be wasted  Patients lose confidence in the programme

14. QUALITY ASSURANCE PROGRAM (QAP )IN SPUTUM SMEAR MICROSCOPY

15. 15 Quality Assessment Also called External Quality Assessment (EQA) A process that allows participant laboratories to assess their capabilities by comparing their results with those in other laboratories in the network.

16. 16 EQA Activities –On-site evaluation of the laboratory, –Panel testing and –Blinded cross checking or Random Blinded Cross Checking (RBCC)

17. 17 Feedback A process of communicating results of EQA to the original laboratory, including suggestions for possible causes of errors and remedies.

18. 18 High False Results High False Negative (HFN). –A 1+ to 3+ positive smear that is misread as negative. This is a major error. High False Positive (HFP). –A negative smear that is misread as 1+ to 3+ positive. This is a major error.

19. 19 Low False Results This type of minor error occurs occasionally even in laboratories that are performing well.

20. 20 Low False Results Low False Negative (LFN). –A scanty (1- 9 AFB / 100 fields) positive smear that is misread as negative. Low False Positive (LFP). –A negative smear that is misread as a scanty (1- 9 AFB / 100 fields) positive.

21. 21 Quantification Error (QE) Errors seen in grading of positive slides. Difference of more than one grade in reading a positive slide between examinee and controller. This is considered as a minor error that generally has no impact on case management.

22. 22 EQA Three methods to evaluate laboratory performance: On-site evaluation Panel testing Blinded rechecking

23. 23 EQA Three methods to evaluate laboratory performance: On-site evaluation Panel testing Blinded rechecking

24. 24 Contents of a On-site evaluation checklist. General Information. Action required to be taken in the previous visit. Current visit particulars. Panel testing of slides, if required. Assessment of EQA responsibilities of lower level supervisor. On-site Evaluation

25. 25 On-site Evaluation.. Availability of patient slides. Proper storage for EQA. Adequate training of staff with refresher courses.

26. 26 Panel Testing  For assessment of proficiency in staining and or reading.  Least expensive and resource intensive of the 3 methods of EQA.

27. 27 Panel Testing Preparation from known patients’ specimen. Manufacture of slides with required grade of smears.

28. 28 Manufacture of Panel Testing Slides To be done at National ReF Lab. Collection of negative and high grade positive patients specimen. Addition of Formalin to stabilize the pus cells and to render the specimen safe for handling. Liquefaction of specimen using NALC.

29. 29 Manufacture.. Mixing of positive and negative specimens to obtain the a known number of AFBs and pus cells in the specimen. Preparation of slides. Validation of a sample of specimen for consistency of the results. –Results to match to an extent of at least 95%.

30. 30 Panel Testing.. Limitation: Checks only technicians ability to stain and/ or read smears not a useful means to assess routine laboratory performance Not required as a routine if Blinded Rechecking is done.

31. 31 Panel Testing.. Panel testing is useful to: –Supplement rechecking program. –Provide some preliminary data on peripheral laboratory capabilities prior to implementing a rechecking program. –Asses current status of performance or to quickly detect problems associated with very poor performance.

32. 32 Panel Testing.. –Evaluate proficiency of laboratory technicians following training. –Monitor performance of individuals when adequate resources are not available to implement a rechecking program.

33. 33 Issues Regarding to Panel Testing Proper preparation of test smears. Number of slides to be included in the test panel set. Types of smears to include (stained and unstained, low positive, smears that are too thick or thin, poorly stained smears). Mechanism for sending slides to the peripheral laboratories (post, courier, OSE).

34. 34 Issues Regarding to Panel Testing Form for test laboratories to record results. Time allowed for technicians in the test laboratories to complete panel and report result. Evaluation criteria for acceptable performance. Plan for reporting results to the test laboratory and implementing corrective action if needed. Mechanism to resolve discrepant results.

35. 35 Random Blinded Rechecking The most important method in EQA. Controller and reading from high level lab for peripheral microscopy centers (MCs). A country wide program for blinded rechecking of slides at regular intervals. A long term goal for optimal EQA.

36. 36 Lot Quality Assurance Sampling A blinded rechecking sampling method. Sample size depends on; –Total number of negatives slides processed each year. –Slide positivity rate. –Expected performance (sensitivity and specificity) compared to the controllers. –Maximum number of errors allowed before action is taken.

37. 37 Summary  Establishment of a Laboratory network is an important function of a National level Program Manager  Internal Quality Control and External Quality Assurance are essential for ensuring proper diagnosis by sputum smear microscopy