Lpa and Genexpert/CBNAAT/Xpert MTB/Rif

Seminar on methods, advantages, disadvantages, new developments in LPA and Genexpert/CBNAAT/Xpert MTB/Rif

1. LPA & GeneXpert Dr.Kalaiyarasan 15-Dec-2015

2. Topics Introduction – MDR/XDR global & Indian scenario Importance of detecting DR-TB Why Xpert MTB/RIF & LPA? LPA-Method, advantages, disadvantages, evidence base, newer developments Xpert MTB/RIF-Method, advantages, disadvantages, evidence base , newer developments Conclusion

3. Introduction Drug-resistant TB poses a major threat to control of TB worldwide. Among notified pulmonary TB patients in 2014, an estimated 300 000 (range: 220 000–370 000) had MDR-TB . Globally in 2014, 123 000 patients with MDR -TB or rifampicin resistant tuberculosis (RR-TB) were notified.

4. MDR TB Globally, 3.3% of new cases (95% CI: 2.2–4.4%) and 20% of previously treated cases (95%CI: 14–27%) have MDR-TB. In India (2014), an estimated 2.2% of new cases (95% CI: 1.9–2.6%) and 15% of previously treated cases (95%CI: 11–19%) have MDR-TB. WHO Global tuberculosis report 2015

5. WHO Global tuberculosis report 2015

6. WHO Global tuberculosis report 2015

7. XDR TB Extensively drug-resistant TB (XDR-TB) has been reported by 105 countries. 1 On average, an estimated 9.7% of people with MDR-TB have XDR-TB (95% CI: 7.4–12%). 1 Sub-national drug resistance surveys (2005-09) showed 4-7% XDR & 21-24% Ofx resistance in MDR isolates. 1. WHO Global tuberculosis report 2015

8. WHO Global tuberculosis report 2015

9. Importance of detecting drug resistant TB Earlier treatment initiation Improved patient outcomes Reducing transmission Reducing the emergence MDR-TB and XDR-TB

10. Why LPA or Xpert MTB/RIF?

11. Drug-susceptibility testing Phenotypic methods involve culturing M.tuberculosis in the presence of anti-TB agents to detect growth or inhibition of growth. Genotypic methods target specific molecular mutations associated with resistance against individual anti-TB agents. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

12. Advantages of Culture methods Bacterial growth can be identified visually or by automated detection of its metabolism . Provides a definitive diagnosis of TB. Significantly increases the number of cases identified (30–50%) when compared with microscopy. Provides the necessary isolates for conventional DST. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

13. Disadvantages of culture methods More complex and expensive. Takes longer time. Needs appropriate lab infrastructure & bio-safety precautions. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

14. Advantages of molecular methods May be applied directly on clinical specimens . Can also be used on specimens not suitable for growth-based assays. Simplification and reduction of bio-safety requirements for both laboratory testing and specimen transport. Large number of specimens can be processed at the same time.

15. Advantages of molecular methods contd. Automation provides standardization & quality assurance of testing. Able to detect mutations associated with lower levels of phenotypic resistance . Detection of specific mutations allows to predict drug resistance to an entire family of drugs ( eg : Aminoglycosides ) 1 . 1. Evaluation of Genetic Mutations Associated with Mycobacterium tuberculosis Resistance to Amikacin , Kanamycin and Capreomycin : A Systematic Review - Sophia B. Georghiou http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033275

16. Why Genexpert /LPA? Phenotypic DST Solid LJ media (around 84 days) Liquid Culture (MGIT) (around 42 days) Molecular DST LPA (around 72 hours) CB-NAAT (around 2 hours) PMDT guidelines May 2012

17. Polymerase chain reaction (PCR) Amplifies a single copy or a few copies of a piece of DNA, generating thousands to millions of copies of a particular DNA sequence. Denaturation (DNA melting): Two strands of the DNA double helix are physically separated at a high temperature. Annealing step : Temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. Extension/elongation step Final elongation Final hold

18. Three steps of PCR: Denaturation , annealing and extension

19. Primers are target sequence specific oligonucleotides that serve as template for the DNA polymerase Forward and reverse primers flanking the target sequence allow both DNA strands to be copied simultaneously in both directions. Primers

20. Line Probe Assay (LPA)

21. HAINs test: MTB DR plus: Line probe assay Based on PCR line probe assay Identifies M.tuberculosis and detects Rif & INH resistance in a day 21

22. Principle of Reverse Hybridization 23

23. Interpretation 24

24. Advantages Rapid detection of resistance to rifampicin (in less than 48 hours, alone or in combination with resistance to isoniazid ). Up to 48 specimens to be processed simultaneously. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

25. Disadvantages Does not eliminate the need for conventional culture and DST. Available LPAs are recommended for use only on smear-positive sputum specimens and isolates of M. tuberculosis . Current LPAs cannot replace phenotypic DST for second-line anti-TB agents. LPAs cannot identify resistance to specific second-line injectable agents; thus, they cannot be used to guide the choice of second-line agents included in individualized MDR-TB regimens. 1 The sensitivity of LPAs to detect resistance to INH is lower (approximately 85%) than that of culture methods. 1. The use of molecular line probe assay for the detection of resistance to second-line anti-tuberculosis drugs: expert group meeting report. Geneva, World Health Organization, 2013 (WHO/HTM/ TB/2013.01) (available at http://apps.who.int/iris/bitstream/10665/78099/1/WHO_HTM_TB_2013.01.eng.pdf).

26. Evidence base Line Probe Assay

27. A systematic review and meta-analysis. After the literature searches, 14 comparisons for rifampicin and 15 comparisons for isoniazid were identified in 10 articles that used GenoType MTBDR assays.

28. Results The pooled sensitivity ( 98.1% , 95% confidence interval (CI) 95.9–99.1) and specificity ( 98.7% , 95% CI 97.3–99.4) estimates for rifampicin resistance were very high and consistent across all subgroups, assay versions and specimen types. The accuracy for isoniazid was variable, with lower sensitivity ( 84.3% , 95% CI 76.6–89.8) and more inconsistent than specificity ( 99.5% , 95% CI 97.5–99.9). While specificity is excellent for isoniazid , sensitivity estimates were modest and variable.

29. Conclusion The LPA test provides an early diagnosis of resistance to isoniazid and rifampicin and is highly sensitive and specific for an early diagnosis of MDR-TB. Sensitivity for isoniazid is suboptimal Based on these findings, it is concluded that the LPA test is to be implemented as first line test for diagnosis of drug resistant TB among sputum smear positive patients in high TB burden countries

30. Results: Overall concordance of RIF and INH result between Genotype MTBDR plus and MGIT 960 was found to be 96.6 and 84.7 per cent, respectively. The sensitivity, specificity, positive and negative predictive values for detection of RIF resistance by Genotype MTBDR plus was found to be 97.6, 94.4, 97.6 and 94.4 per cent respectively.

31. Newer LPA In 2009, Hain Lifescience introduced a new LPA, the Genotype MTBDR sl ® test For the rapid determination of genetic mutations associated with resistance to fluoroquinolones , aminoglycosides ( kanamycin , amikacin ), cyclic peptides ( capreomycin ), ethambutol and streptomycin. Allows for testing and reporting results within 48 hours. Recommended by WHO to be used as a rule-in test for XDR-TB. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

32. Objectives: To assess the diagnostic performance of the GenoType MTBDR sl LPA for the rapid detection of mutations conferring resistance to ofloxacin (OFX), amikacin (AMK), and ethambutol and to determine the impact of implementation on the turnaround time in a high-throughput diagnostic laboratory. 657 patients smear-positive specimens resistant to isoniazid , rifampin , or both according to the GenoType MTBDR plus assay were consecutively tested, using the GenoType MTBDR sl LPA.

33. A total of 516 of 657 patient specimens had valid results for both tests. Implementation of this test significantly reduced the turnaround time by 93.3% ( P < 0.001), calculated from the date that the specimen was received at the laboratory to reporting second-line results. sensitivity (95%CI) specificity (95%CI) OFX 90.7% (80.1–96.0%) 98.1% (95% CI, 96.3–99.0%) AMK 100% (91.8–100%) 99.4% (95% CI, 98.2–99.8%) XDR 92.3% (75.9–97.9%) 99.6% (95% CI, 98.5–99.9%)

34. Newer LPA in research mode In 2011, Nipro Corporation completed development of their LPA-based NTM+MDRTB Detection Kit 2 Four strips - NTM/MDR-TB, INH, PZA, and FQ strips. The NTM/MDR-TB strip was designed to identify four M ycobacterium species — M. tuberculosis , M. avium , M. intracellulare , and M. kansasii —and to detect mutations associated with RIF and INH resistance in M. tuberculosis . The INH, PZA, and FQ strips were designed to detect mutations associated with INH, PZA, and FQ resistance of M. tuberculosis , respectively.

35. RESULTS In the sputum specimens, sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates were both 100% & for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. Sensitivity Specificity Rifampicin 98.9 97.3 INH 90.6 100 PZA 89.7 96 Levofloxacin 93 100 A Japanese multi-center study conducted at 6 hospitals assessed this new line probe assay in 554 isolates (316 from MTB) and 163 sputum specimens.

36. Using microscopy and line-probe assays in conjunction with drug-susceptibility testing (with solid or liquid media) to diagnose TB WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

37. Xpert MTB/RIF

38. Xpert MTB/RIF Automated, cartridge-based nucleic acid amplification test (NAAT) that uses the multidisease GeneXpert platform. Performed directly on sputum, processed sputum sediment and selected extrapulmonary specimens from adults and children. Capacity to have 1, 2, 4, 16, 48 or 80 independently functioning modules. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

39. Rifampicin Resistant Sample At least 2 of 5 rpoB probes positive within 2 cycles and amplification control positive= M. tuberculosis complex Failure of proper hybridization of one or more rpoB probes= rifampin resistance 41

40. Advantages - Xpert MTB/RIF Simultaneously detects M. tuberculosis and rifampicin resistance in less than 2 hours . The sensitivity for detecting TB is similar to that of to liquid culture (sensitivity 88%); the specificity is also high (99%). The superior performance of Xpert MTB/RIF in detecting TB over that of microscopy makes it a particularly useful tool for case-finding among people living with HIV. 1. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF assay for the diagnosis of pulmonary and extrapulmonary TB in adults and children. Policy update. Geneva, World Health Organization, 2013 (WHO/HTM/TB/2013.16) http://www.who.int/tb/laboratory/xpert_policyupdate/en/

41. Advantages - Xpert MTB/RIF As a tool for detecting rifampicin resistance, Xpert MTB/RIF has a sensitivity of 95% and specificity of 98% when compared with phenotypic reference standards. Biosafety precautions and the training required are minimal. For smear-negative culture-positive TB, the pooled sensitivity of Xpert MTB/RIF has been found to be 68%. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

42. Disadvantages - Xpert MTB/RIF A stable uninterruptable electrical supply is needed. The ambient operating temperature of the instrument cannot exceed 30 °C, cartridges must be stored at less than 28 °C. The shelf-life of the cartridges must be monitored to prevent them from expiring before they are used. Security measures must be put in place to prevent the theft of the accompanying laptop or desktop computer. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

43. Disadvantages - Xpert MTB/RIF The modules require annual calibration. Does not eliminate the need for conventional microscopy, culture and DST. In patients who are not at risk for drug resistance but tested positive for Rif resistance, a second Xpert MTB/RIF test should be performed to control for preanalytical and postanalytical errors, and to improve the clinician’s confidence in the diagnosis. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

44. Infrequent occurrence of false-positive results may be linked to the detection by Xpert MTB/RIF of strains that are truly resistant to rifampicin but for which resistance is not detected by phenotypic culture-based DST. In cases of discordant results obtained from Xpert MTB/RIF Vs phenotypic DST or LPA, the culture isolate should be referred to a reference laboratory for DNA sequencing. while awaiting the results, a clinical decision should be made whether to continue the MDR-TB regimen. WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

45. Research & Development GeneXpert Omni At just 9 inches tall (about 23 cm) and weighing just 2.2 pounds (about 1kg), the GeneXpert Omni will run the same PCR-based cartridge tests. Additionally, the GeneXpert Omni is battery-operated, wireless and web-enabled, able to transmit instrument & test information in real-time. WHO expects to evaluate the platform in 2016. GLOBAL TUBERCULOSIS REPORT 2015

46. Research & Development Xpert MTB/RIF Ultra The current Xpert MTB/RIF test has a limit of detection (LOD) of 130 cfu /ml. New innovations, including a larger DNA reaction chamber in the cartridge, will bring the LOD down ten-fold, to approximately10 cfu /ml (across all strains) — a level similar to or potentially better than liquid culture. Both GeneXpert Omni & Xpert MTB/RIF Ultra is expected to be available in the first half of 2016.

47. Summary of WHO’s 2013 policy recommendations for using Xpert MTB/RIF Should be used rather than conventional microscopy, culture and DST as the initial diagnostic test in adults and children suspected of having MDR-TB or HIV-associated TB. May be used rather than conventional microscopy and culture as the initial diagnostic test in all adults and children suspected of having TB. Should be used in preference to conventional microscopy and culture as the initial diagnostic test for CSF specimens from patients suspected of having TB meningitis . Xpert MTB/RIF implementation manual: technical and operational “how-to”; practical considerations. Geneva: World Health Organization; 2014 (WHO/HTM/TB/2014.1). http://apps.who.int/iris/bitstream/10665/112469/1/9789241506700_eng.pdf.

48. May be used as a follow-on test to microscopy in adults suspected of having TB who are not at risk of MDR-TB or HIV-associated TB, especially when further testing of smear-negative specimens is necessary. May be used as a replacement test for usual practice (including conventional microscopy, culture or histopathology) for testing specific non respiratory specimens from patients suspected of having extrapulmonary TB . Xpert MTB/RIF implementation manual: technical and operational “how-to”; practical considerations. Geneva: World Health Organization; 2014 (WHO/HTM/TB/2014.1). http://apps.who.int/iris/bitstream/10665/112469/1/9789241506700_eng.pdf.

49. Using the Xpert MTB/RIF assay as an initial diagnostic test for TB followed by drug susceptibility testing for second-line anti-TB agents when necessary WHO IMPLEMENTING TUBERCULOSIS DIAGNOSTICS Policy framework, April 2015

50. Evidence base XPERT MTB/RIF

51. A review commissioned by WHO included all of the evidence on the use of Xpert MTB/RIF published until the end of February 2013. The reviews focused on four specific areas of interest: using Xpert MTB/RIF to diagnose pulmonary TB and rifampicin resistance in adults using Xpert MTB/RIF to diagnose extrapulmonary TB in adults and children using Xpert MTB/RIF to diagnose pulmonary TB and rifampicin resistance in children the affordability and cost effectiveness of Xpert MTB/RIF for diagnosing TB. WHO Xpert MTB/RIF implementation manual 2014

52. Pooled Sensitivity Pooled Specificity Studies / Population Initial diagnostic test replacing smear microscopy 88% (95% credible interval [ CrI ], 84-92%) 99% (95% CrI , 98-99%) 22 studies, 9008 participants add-on test following a negative smear microscopy result 68% (95% CrI , 61-74%) 99% (95% CrI, 98-99%) 23 studies, 7151 participants cases of smear-positive, culture-positive TB 98% (95% CrI , 97-99%) - 23 studies, 1952 participants people living with HIV 79% (95% CrI, 70-86%) - 7 studies, 1789 participants for people without HIV infection 86% (95% CrI , 76-92%) - 7 studies, 1470 participants to detect rifampicin resistance in adults 95% (95% CrI , 90-97%) 98% (95% CrI , 97-99%) 24 studies, 2414 specimens WHO Xpert MTB/RIF implementation manual 2014 Using Xpert MTB/RIF to diagnose pulmonary TB & Rif resistance in adults

53. Using Xpert MTB/RIF to diagnose extrapulmonary TB in adults & children A total of 15 published studies and 7 unpublished studies (5922 samples) were included in the review. WHO Xpert MTB/RIF implementation manual 2014 Pooled sensitivity Studies/Samples Lymph node/aspirate 84.9% (95% confidence interval [ CI], 72.1-92.4%) 14 studies, 849 samples Pleural fluid 43.7% (95% CI, 24.8-64.7%) 17 studies, 1385 specimen CSF 79.5% (95% CI, 62.0-90.2%) 16 studies, 709 specimens Gastric fluid 83.8% (95% CI, 65.9-93.2%) 12 studies, 1258 samples Other tissue specimens 81.2% (95% CI, 67.7-89.9%) 12 studies, 699 specimens

54. Using Xpert MTB/RIF to diagnose pulmonary TB and rifampicin resistance in children A total of 16 studies (12 published, 4 unpublished) were included in the review. The diagnosis of pulmonary TB was evaluated in 13 studies that included 2603 participants. WHO Xpert MTB/RIF implementation manual 2014 Sensitivity Specificity To detect pulmonary TB in children (overall) Sputum - 66% ( 95% CrI , 52-77% Gastric lavage - 66% (95% CrI , 51-81%) 98% with narrow confidence interval against culture 13 studies, 2603 participants To detect rifampicin resistance in children 86% (95%CrI, 53-98%) - -

55. PMDT guidelines May 2012

56. Conclusion LPAs are currently limited to detecting resistance to rifampicin and isoniazid and are suitable only on smear positive Specimens . Conventional DST is needed to detect resistance to anti-TB agents other than rifampicin and isoniazid & to detect XDR-TB.

57. Conclusion Overall the LPA test is a reliable, rapid and easy to perform for the simultaneous detection of RMP and INH resistance in M.tuberculosis It is recommend that the LPA should serve as an early guidance of therapy , which should be followed by a phenotypic DST confirmation for all suspected MDR-TB patients.

58. Conclusion Although Xpert MTB/RIF is suitable for use at all levels of the health system & does not require additional laboratory equipment, it requires care in handling, a stable and uninterrupted electrical supply, security against theft, adequate storage space & bio-safety measures. Xpert MTB/RIF does not eliminate the need for capacity for conventional TB microscopy, culture and DST. The Xpert MTB/RIF assay is suitable for diagnosing TB and detecting RR-TB . A negative result accurately excludes the possibility of rifampicin resistance and no further testing is needed to confirm the negative results. (NPV>99%)

59. Microscopy or culture, or both, remain essential for monitoring treatment since molecular tests based on DNA detection have not been shown to be suitable. Liquid culture, molecular LPAs and the Xpert MTB/RIF assay should be phased in to programmes in a way that ensures that the existing capacity for solid culture and DST is maintained.

60. Thank You..!