Revision of new diagnostics for TB Churchyard GJ

Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM Diagnostics pipeline Conclusion

Introduction The global burden of TB is declining slowly The lack of a rapid & accurate diagnostics is compromising progress towards TB elimination Sputum microscopy remains the mainstay of TB diagnosis in many resource poor countries Globally <10% of MDR TB patients are diagnosed & treated HIV associated TB Is more difficult to diagnose If undiagnosed, is associated with a high mortality

Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM Diagnostics pipeline Conclusion

Xpert MTB/RIF Detects M. tuberculosis and common mutations that confer resistance to rifampin Is a hemi-nested real-time PCR of MTB- specific region of rpoB gene, which is then probed with molecular beacons for mutations Fully automated Uses GeneXpert platform (Cepheid, CA)

Assay Procedure for the MTB/RIF Test 2 manual steps: addition of bactericidal buffer to sputum then transfer of defined volume to cartridge Integrated sample processing and PCR; disposable plastic cartridge contains all reagents

Evaluation Study of Xpert MTB/RIF Cross-sectional study of diagnostic test accuracy Population and Procedures Peru, Azerbaijan, South Africa x 2, India Adults with pulmonary TB symptoms 3 sputa obtained (2 spot, 1 morning) Results 1730 eligible participants 976 with HIV status known; 40.2% HIV-positive (C. Boehme et al. NEJM 2010;363:1005)

Evaluation Study of Xpert MTB/RIF # Xpert tests per participant Sensitivity Specificity All CX POS SM POS, CX POS SM NEG, CX POS 1 sputum 675/732 92.2% 551/561 98.2% 124/171 72.5% 604/609 99.2% 3 sputa 723/741 97.6% 566/567 99.8% 157/174 90.2% 604/616 98.1% Among culture positive patients, a single direct Xpert test correctly detected TB in 551/561 amear positive patients (sensitivity of 98.2%), and in 124/171 smear neg patients. A single Xpert test was specific in 604/609 participants (specificity 99.2%). Two additional Xpert tests slightly increased the overall yield for smear positive patients, but more substantially increased the yield for smear-negative patients (from 72.5% to 90.2%). (C. Boehme et al. NEJM 2010;363:1005)

Evaluation Study of Xpert MTB/RIF Xpert SENSITIVITY for Rifampin Resistance # correct/# total % Xpert SPECIFICITY for Rifampin Resistance Phenotypic DST 200/205 97.6% 505/515 98.1% and Discrepant Resolution by Sequencing 209/211 99.1% 506/506 100.0% (C. Boehme et al. NEJM 2010;363:1005)

Xpert MTB/RIF: false positives WHO estimates Positive Predictive value at >90% if greater than 15% of isolates are Rif resistant <70% if less than 5% of isolates are Rif resistant Further culture based DST to first and second line drugs recommended Patients should receive MDR TB treatment pending further results The software & cartridge have been redesigned to address these limitations

Xpert MTB/RIF: Operational performance Detection of MTB Sensitivity Specificity Decentralised implementation 90.3% 99% HIV-associated TB 58%-73% 99.2%-93.9% Extrapulmonary TB 53%-95% 98.2%-100% Active case finding 62.6% 99.6% (Lawn, Nicol. Future Micobiol. 2012; Dorman et al. PLoS ONE. 2012)

Xpert MTB/RIF Attributes & Advantages Simple to perform, minimal training required Not prone to cross-contamination Requires minimal biosafety facilities “Near-care” Shortcomings & Disadvantages Complex instrument (calibration, power supply) Cost for instrument Cost of cartridges reduced to ~$10 Single supplier

Xpert MTB/RIF WHO expert group recommendations: “Xpert should be used as the initial diagnostic test in individuals suspected of having MDR-TB or HIV- associated TB” (strong recommendation) “Xpert may be used as a follow-on test to microscopy where MDR and/or HIV is of lesser concern, especially in smear-negative specimens” (conditional recommendation, recognizing resource implications)

Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM Diagnostics pipeline Conclusion

GenoType MTBDRplus (Hain) Is a molecular line probe assay with probes for MTB Almost all of the common rifampicin resistance-conferring mutations A subset of the mutations conferring resistance to isoniazid

GenoType MTBDRplus (Hain) Ling D. 2008 Sensitivity Specificity Rifampicin 98.4% 98.9% Isoniazid 88.7% 99.2% Validation study* showed that performance on Smear positive specimens was good Smear negative specimens was reasonable (* Barnard M. Am J Respir Crit Care Med 2008;177:787-792)

Genotype MTBDRplus vs MGIT for detection of MTB, by smear status Missed 15% of RIF resistant and 37% of INH resistant TB (Dorman S. PLoS One. 2012. In press)

Genotype MTBDRplus 2.0 vs MGIT & clinical TB Crudu. JCM, 2012 Sensitivity Specificity MTB Overall 87.6% 99.2% Smear negative 79.8% Rifampicin resistance 94.3% 96.0% 90.7% Isoniazid resistance 95.8% 88.9% 93.5% 82.3%

GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011 Fluoroquinolone 86.2% 100% Kiet,V.S. JCM. 2010 75.6% kanamycin Ethambutol 64.2% Hillemann,D. JCM. 2009 88.9% Amikacin 75.0% 38.5%

GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011 Fluoroquinolone 86.2% 100% Kiet,V.S. JCM. 2010 75.6% kanamycin Ethambutol 64.2% Hillemann,D. JCM. 2009 88.9% Amikacin 75.0% 38.5%

GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011 Fluoroquinolone 86.2% 100% Kiet,V.S. JCM. 2010 75.6% kanamycin Ethambutol 64.2% Hillemann,D. JCM. 2009 88.9% Amikacin 75.0% 38.5%

GenoType MTBDRsl Drug Sensitivity Specificity Kontsevaya,I. JCM. 2011 Fluoroquinolone 86.2% 100% Kiet,V.S. JCM. 2010 75.6% kanamycin Ethambutol 64.2% Hillemann,D. JCM. 2009 88.9% Amikacin 75.0% 38.5%

Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM Diagnostics pipeline Conclusion

Urine Assays for Mycobacterial Lipoarabinomannan (LAM) A lipopolysaccharide component of MTB cell wall Released from metabolically active or degraded MTB Urine-based test Urine easy to obtain Lacks infection control issues of blood, sputum Inverness: ELISA format Alere: lateral flow

Urine Assays for Mycobacterial Lipoarabinomannan (LAM) A lipopolysaccharide component of MTB cell wall Released from metabolically active or degraded MTB Urine-based test Urine easy to obtain Lacks infection control issues of blood, sputum Inverness: ELISA format Alere: lateral flow

Determine LAM lateral flow assay (Alere) uses Determine testing platform No sample processing; results in 25 minutes Analytical sensitivity reported to be 0.25 ng/ml Reporting scale: no band (neg), 1+ to 5+ (pos) Analytical sensitivity reported by Alere to be 0.25 ng/ml Please note reporting scale of 1+ to 5+ instead of +/- to 4+ sample application pad patient result window control window

Determine TB-LAM Author/ Year N Setting Sensitivity Specificity Overall CD4<100 Peter, 2012 335 Inpatients 45% 96% Lawn, 2012 516 ART clinic 28% 52% 99% Dorman S, 2012 * 561 Outpatients 80% 90% (* Dorman S, Interim unpublished data)

Overview Introduction Xpert MTB/RIF Line probe assays Urine LAM Diagnostics pipeline Conclusion

Global TB diagnostics pipeline

Conclusion In many high burden countries sputum microscopy remains the mainstay of TB diagnosis New diagnostics can substantially reduce the time to diagnosis of TB and drug resistant TB A point of care test is urgently required

Acknowledgements Susan Dorman