1. Module 11: Clinical guide to Xpert MTB/RIF Global Laboratory Initiative – Xpert MTB/RIF Training Package

2. Global Laboratory Initiative Xpert MTB/RIF Training Package -2--2- Contents of this module  What is Xpert MTB/RIF? Procedure and technology  Diagnostic algorithm with Xpert MTB/RIF and interpretation of results  Sputum collection  How to record Xpert MTB/RIF results  Follow up testing  Referral system for samples, results and patients

3. Global Laboratory Initiative Xpert MTB/RIF Training Package -3--3- At the end of this module, you will be able to:  Describe the role of Xpert MTB/RIF testing in diagnosis of TB and MDR-TB  Interpret Xpert MTB/RIF results  Explain and discuss Xpert MTB/RIF algorithms for diagnosis and management of TB and MDR-TB

4. Global Laboratory Initiative Xpert MTB/RIF Training Package -4--4- 1. What is Xpert MTB/RIF ?

5. Global Laboratory Initiative Xpert MTB/RIF Training Package -5--5- A review: shortcomings of conventional diagnostic methods for detection of TB and rifampicin resistance  Sputum smear microscopy is most commonly used in most resource-limited settings to detect TB. However: ◦ Microscopy detects acid-fast bacilli (AFB) but does not differentiate M. tuberculosis from other AFB, including non-tuberculous mycobacteria (NTMs) ◦ It misses many cases of TB, particularly in people with HIV ◦ It does not detect drug resistance  Sophisticated laboratories have been required to detect rifampicin resistance ◦ Central-level laboratories with strict biosafety measures and highly trained staff are needed for performing culture or molecular line probe assays ◦ Culture can take up to 12 weeks for results

6. Global Laboratory Initiative Xpert MTB/RIF Training Package -6--6- Xpert MTB/RIF assay  Xpert MTB/RIF: ◦ A molecular test that is able to detect both TB and rifampicin resistance in a single test in less than 2 hours ◦ Much more sensitive than smear microscopy for detection of TB, including HIV-associated TB. Sensitivity is similar to solid LJ culture ◦ Does not detect non-tuberculosis mycobacteria (NTM), unlike smear microscopy ◦ Used on the multi-disease GeneXpert platform, which can be placed at lower-level laboratories and health centres  Single source provider: Manufactured by Cepheid (Sunnyvale, USA)

7. Global Laboratory Initiative Xpert MTB/RIF Training Package -7--7- Example of RIF-Sensitive Profile – 5 probes & sample processing control (SPC) show fluorescence Molecular Beacon Target Hybrid Each probe is labeled with a different fluorescent dye, permitting simultaneous detection The PCR target is the 81 bp region of the rpoB gene: 5 probes bind to wild-type, but not mutant target SPC Include additional slides from Module 6, if needed

8. Global Laboratory Initiative Xpert MTB/RIF Training Package -8--8- Boehme CC et al. N Engl J Med 2010;363:1005-1015. Procedure for the Xpert MTB/RIF test

9. Global Laboratory Initiative Xpert MTB/RIF Training Package -9--9- Symptoms Smear LJ- 40 days Reference Lab Surveillance Clinic / Health Post Regional Lab Peripheral Lab LED +10% sensitivity Xpert +40% sensitivity LPA 1 day More sensitive than microscopy Faster than solid culture Simpler than microscopy MGIT 15 days Xpert MTB/RIF can be placed at all levels of the network if basic conditions for electricity, placement and biosafety measures similar to smear microscopy can be assured, and machine throughput utilized effectively < 2 hours In-house DST (MODS, NRA, CRI)

10. Global Laboratory Initiative Xpert MTB/RIF Training Package -10- Strong recommendations  Xpert MTB/RIF should be used rather than conventional microscopy, culture and DST as the initial diagnostic test in adults presumed to have MDR-TB or HIV-associated TB (high-quality evidence)  Xpert MTB/RIF should be used rather than conventional microscopy, culture and DST as the initial diagnostic test in children presumed to have MDR-TB or HIV-associated TB (very low-quality evidence) Consider adaption /removal if not in accordance with NTP guidelines

11. Global Laboratory Initiative Xpert MTB/RIF Training Package -11- Conditional recommendations (recognizing major resource implications)  Xpert MTB/RIF may be used rather than conventional microscopy and culture as the initial diagnostic test in all adults presumed to have TB (high-quality evidence)  Xpert MTB/RIF may be used rather than conventional microscopy and culture as the initial diagnostic test in all children presumed to have TB (very low-quality evidence)  Xpert MTB/RIF may be used as a follow-on test to microscopy in adults presumed to have TB but not at risk of MDR-TB or HIV associated TB, especially in further testing of smear-negative specimens (high-quality evidence) Consider adaption /removal if not in accordance with NTP guidelines

12. Global Laboratory Initiative Xpert MTB/RIF Training Package -12- Remarks (1 of 2): These recommendations apply to the use of Xpert MTB/RIF in processed and unprocessed sputum specimens; These recommendations also apply to gastric lavage aspirates from adults and children based on the generalization of data from children; These recommendations support the use of a single sputum specimen for diagnostic testing, acknowledging that multiple specimens increase the sensitivity of Xpert MTB/RIF but have resource implications; Children presumed to have pulmonary TB but with a single Xpert MTB/RIF -negative result should undergo further diagnostic testing, and a child with high clinical suspicion for TB should be treated even if an Xpert MTB/RIF result is negative; Consider adaption /removal if not in accordance with NTP guidelines

13. Global Laboratory Initiative Xpert MTB/RIF Training Package -13- Remarks (2 of 2): Conventional microscopy and culture remains essential for monitoring of therapy and for DST other than rifampicin (including second-line anti-TB drugs); Expanding the scope of Xpert MTB/RIF use and its placement in diagnostic algorithms will have significant operational implementation implications and should be phased in within the context of TB national strategic plans; Emerging data show that Xpert MTB/RIF detects some rifampicin-resistant strains that are susceptible on phenotypic DST. Sequencing of these discordant results usually resolves in favour of Xpert MTB/RIF, and patients with these strains missed by phenotypic DST and on first-line treatment have poor treatment outcomes. Consider adaption /removal if not in accordance with NTP guidelines

14. Global Laboratory Initiative Xpert MTB/RIF Training Package -14-  Cerebrospinal Fluid (CSF): Xpert MTB/RIF should be used in preference to conventional microscopy, culture and DST as the initial diagnostic test in testing CSF specimens from patients presumed to have TB meningitis (strong recommendation given the urgency of rapid diagnosis, very low quality of evidence)  Lymph node and other tissues: Xpert MTB/RIF may be used as a replacement test for usual practice (including conventional microscopy, culture, and/or histopathology) for testing of specific non-respiratory specimens (lymph nodes or other tissue) from patients presumed to have extrapulmonary TB (conditional recommendation, very low quality of evidence) Consider adaption /removal if not in accordance with NTP guidelines

15. Global Laboratory Initiative Xpert MTB/RIF Training Package -15- Remarks (1 of 2): Individuals presumed to have extrapulmonary TB but with a single Xpert MTB/RIF -negative result should undergo further diagnostic testing, and those with high clinical suspicion for TB (especially children) should be treated even if an Xpert MTB/RIF result is negative; For CSF specimens, Xpert MTB/RIF should be preferentially used over culture if the sample volume is low or additional specimens cannot be obtained, in order to reach quick diagnosis. If sufficient volume of material is available, concentration methods should be used to increase yield; Pleural fluid is a suboptimal sample for the bacterial confirmation of pleural TB, using any method. A pleural biopsy is the preferred sample. The sensitivity of Xpert MTB/RIF in pleural fluid is very low. Nevertheless, any positive Xpert MTB/RIF result on pleural fluid should be treated for pleural TB, while those with a negative Xpert MTB/RIF result should be followed by other tests; Consider adaption /removal if not in accordance with NTP guidelines

16. Global Laboratory Initiative Xpert MTB/RIF Training Package -16- Remarks (2 of 2): Conventional microscopy and culture remains essential for monitoring of therapy and for DST other than rifampicin (including second-line anti-TB drugs); Emerging data show that Xpert MTB/RIF detects some rifampicin-resistant strains that are susceptible on phenotypic DST. Sequencing of these discordant results usually resolves in favour of Xpert MTB/RIF, and patients with these strains missed by phenotypic DST and on first-line treatment have poor treatment outcomes; These recommendations do not apply to stool, urine or blood, given the lack of data on the utility of Xpert MTB/RIF on these specimens. Consider adaption /removal if not in accordance with NTP guidelines

17. Global Laboratory Initiative Xpert MTB/RIF Training Package -17- Policy Update Accompanying Implementation manual, Guidance on updated diagnostic algorithms using complementary methods Budgeting guidance Annex of SOPs for processing extrapulmonary specimens Available at: http://www.who.int/tb/laboratory/mtbrifrollout which provides:

18. Global Laboratory Initiative Xpert MTB/RIF Training Package -18- 2. TB diagnostic algorithms with Xpert MTB/RIF and interpretation of results

19. Global Laboratory Initiative Xpert MTB/RIF Training Package -19-  Add your country’s diagnostic algorithm (flow diagram) incorporating Xpert MTB/RIF in relation to other diagnostic and screening tests, including microscopy, chest X-ray, culture and DST, for different patient groups  Indicate in the flow diagram what are the steps when Xpert gives a RIF resistant result, but a second test (Xpert or another DST method) gives a RIF susceptible result  Discuss which categories of patients will receive Xpert MTB/RIF testing  Add your country’s clinical guidelines on case and patient management after Xpert testing  Add your country’s diagnostic algorithm (flow diagram) incorporating Xpert MTB/RIF in relation to other diagnostic and screening tests, including microscopy, chest X-ray, culture and DST, for different patient groups  Indicate in the flow diagram what are the steps when Xpert gives a RIF resistant result, but a second test (Xpert or another DST method) gives a RIF susceptible result  Discuss which categories of patients will receive Xpert MTB/RIF testing  Add your country’s clinical guidelines on case and patient management after Xpert testing

20. Global Laboratory Initiative Xpert MTB/RIF Training Package -20- For detection of rifampicin resistance:  Sensitivity of Xpert MTB/RIF compared to phenotypic DST: 95%  Specificity of Xpert MTB/RIF compared to phenotypic DST: 98%  However, phenotypic DST also misses some truly resistant strains.  In case of Xpert MTB/RIF-detected rifampicin resistance but phenotypic rifampicin susceptibility, only sequencing can resolve the discrepancy to understand the true resistance  The detection by sequencing of a change in the amino acid sequence of the rifampicin resistance-determining region (RRDR) should be considered confirmation of clinically significant rifampicin resistance  A second Xpert MTB/RIF test may be used to ensure there were no pre- and post analytical errors (mistakes in labeling specimens or reporting results), especially for patients who were not considered at risk for rifampicin resistance

21. Global Laboratory Initiative Xpert MTB/RIF Training Package -21- Adapt according to NTP guidelines in your country  Person who has been treated with anti-TB drugs and in whom pulmonary TB is suspected, that is, all retreatment categories (failure, relapse, return after loss to follow-up)  Person suspected of having pulmonary TB at risk of harbouring MDR-TB bacilli (risk groups as defined in current WHO guidelines for DR-TB management)  Any other person suspected of having TB and considered at higher risk of MDR-TB as per national policy/guidelines

22. Global Laboratory Initiative Xpert MTB/RIF Training Package -22-  If MTB detected RIF resistance NOT detected: start or continue patient on Cat I treatment  If MTB NOT detected: conduct follow-on diagnostic evaluation or treatment according to the national algorithm for bacteriologically negative TB. (Especially for symptomatic presumptive TB cases, TB should still be considered a realistic option)  If test result is error, invalid or no result: collect 1 more sputum for repeat Xpert testing Adapt according to NTP guidelines in your country

23. Global Laboratory Initiative Xpert MTB/RIF Training Package -23-  If MTB detected RIF resistance detected: ◦ Start patient on MDR-TB regimen ◦ Send additional specimen for further DST, to determine complete drug resistance profile for further refinement of regimen ◦ If patient was not considered at risk for MDR-TB, an additional Xpert test may be performed as a confirmation, in order to be sure no clerical or administrative errors were made in handling and registering the specimen and reporting the result. Adapt according to NTP guidelines in your country

24. Global Laboratory Initiative Xpert MTB/RIF Training Package -24- Adapt according to NTP guidelines in your country

25. Global Laboratory Initiative Xpert MTB/RIF Training Package -25- 3. Sputum collection Additional slides from Module 3 can be inserted as needed

26. Global Laboratory Initiative Xpert MTB/RIF Training Package -26- - Use appropriate container for specimen collection (sterile, translucent, combustible, leak-proof, screw-capped, wide-mouthed). - Make sure that specimen details on container match the Lab Request Form - At least 1 mL sputum must be collected for Xpert MTB/RIF - Collection of sputum should be supervised Optimal quality Purulent Mucoid Suboptimal quality Salivary Blood- stained

27. Global Laboratory Initiative Xpert MTB/RIF Training Package -27- Add your local request for sputum examination form. Demonstrate to participants how to correctly complete the form. Add your local request for sputum examination form. Demonstrate to participants how to correctly complete the form.

28. Global Laboratory Initiative Xpert MTB/RIF Training Package -28- 4. How to record Xpert MTB/RIF results

29. Global Laboratory Initiative Xpert MTB/RIF Training Package -29- Results of sputum smear microscopy and other examination Before treatment 2 or 3 months5 monthsEnd of treatmentCulture and DST Results Smear Date /Lab No. / Result Xpert Date / Lab No./ Result Smear Date / Lab No. / Result Culture Date / No. / Result DST Date / No. / Result Adapt according to NTP guidelines and registers in your country No. Date regis-tration Suspect ID No. Name (full) Sex M/F Date of birth (Age) Address + phone number Name of referring facility Suspect ClassificationXpert Exam TB Regis-tration No. Treatment category & Date treatment start MDR-TB suspect HIV+ TB suspect Other Date sputum collected Date results released Results (tick box) MTB Neg MTB pos RIF sus MTB pos RIF res Indet./ error TB suspect register TB treatment register

30. Global Laboratory Initiative Xpert MTB/RIF Training Package -30- 5. Testing for treatment follow-up

31. Global Laboratory Initiative Xpert MTB/RIF Training Package -31-  Xpert MTB/RIF can not be used for follow up of patients during treatment ◦ Continue to use smear and/or culture according to guidelines for follow up of patients on treatment Adapt according to NTP guidelines in your country

32. Global Laboratory Initiative Xpert MTB/RIF Training Package -32- 6. Referral system for samples, results and patients

33. Global Laboratory Initiative Xpert MTB/RIF Training Package -33- Peripheral health center OPD/DOTS center Xpert MTB/RIF laboratory Culture/DST laboratory DOTS/MDR clinic Sample Patient Result 4 4 3 3 2 2 1 1 5 5 6 6 Adapt according to NTP guidelines in your country

34. Global Laboratory Initiative Xpert MTB/RIF Training Package -34-  Xpert MTB/RIF can rapidly and accurately diagnose TB and rifampicin resistance, but can not be used for follow-up testing (monitoring) of patients on treatment  Collection of good quality sputum is essential for good diagnosis, using Xpert and all other lab tests  Collect one sputum specimen only for Xpert MTB/RIF testing (repeat in case of an error, invalid result, or no result) ◦ Xpert is more sensitive than smear ◦ Risk assessment is an important step before sending samples for testing Adapt according to NTP guidelines in your country

35. Global Laboratory Initiative Xpert MTB/RIF Training Package -35-  For patients with rifampicin resistance detected by Xpert MTB/RIF, start patients immediately on an MDR-TB regimen including isoniazid, and collect another specimen for further DST to determine the full resistance profile.  While Xpert MTB/RIF is able to provide results rapidly, systems must be in place for efficient sample & patient referral and reporting of results, in order to allow for rapid initiation of appropriate treatment for patients Adapt according to NTP guidelines in your country

36. Global Laboratory Initiative Xpert MTB/RIF Training Package -36-Assessment  Is Xpert MTB/RIF as sensitive as smear microscopy for TB detection, and can it be used for follow-up testing (monitoring)?  What is the sensitivity of Xpert MTB/RIF compared to DST for detection of rifampicin resistance?  Why is collection of good quality sputum essential and how would you ensure proper sputum specimen is collected?  What are the WHO recommendations for TB detection in adults and children using the Xpert MTB/RIF?  What is your National diagnostic algorithm?  Describe the referral system for sample, result and patient in your country

37. Acknowledgements The Xpert MTB/RIF Training Package has been developed by a consortium of GLI partners, including FIND, KNCV, US CDC, USAID, TB CARE I and WHO, with funding from USAID. The modules are based on materials originally developed by FIND, KNCV and Cepheid.