1. Evaluation of Xpert MTB/RIF Assay for the Rapid Identification of TB and Rifampin Resistance in HIV Infected & HIV uninfected Pulmonary TB suspects: ACTG 5295/TBTC 34 AF Luetkemeyer, C Firnhaber, MA Kendall, X Wu, D Benator, GH Mazurek, B Metchock, P Johnson, S Swindells, I Sanne, DV Havlir, B Grinsztejn, D Alland, on behalf of the ACTG A5295/TBTC 34 Study teams

2. Disclosures Instruments, cartridges and research grant support from Cepheid Funding from NIH AIDS Clinical Trials Group (ACTG) and the CDC Tuberculosis Trials Consortium (TBTC)

3. Background GeneXpert MTB/RIF: automated real time PCR that diagnoses M. tuberculosis & identifies rifampin resistance in < 2 hours Performance in high TB prevalence settings well characterized –Sensitivity of 1 Xpert: AFB+ 98-99%, AFB- ~60-70% –Specificity: 98-99% Limited data for use in lower TB prevalence regions and in HIV coinfection

4. Study Aims Determine sensitivity and specificity of Xpert MTB/RIF assay overall and by AFB smear status Evaluate Xpert performance by: –low TB prevalence (US) vs. higher TB prevalence (Brazil and South Africa) –HIV-infected vs. HIV-uninfected Evaluate by Xpert performance for RIF resistance detection Performance in setting of non-tuberculosis mycobacterial growth

5. Study population Pulmonary TB suspects –AFB+ sputum within 7 days of entry, or –Clinical suspicion of TB as evidenced by decision to send sputum for AFB smear/culture in addition to cough, fever, night sweats, or weight loss TB treatment < 48 hours Regions by TB prevalence –Low prevalence (US: target enrollment ≥ 70%) –Higher prevalence (Brazil & South Africa) HIV-infected and uninfected –All participants underwent HIV testing

6. Methods 2 sputum samples for Xpert MTB/RIF testing (G4 cartridges) –Method of sputum collection (induced or expectorated) and processing (unprocessed or sedimented) prior to Xpert testing determined by local standard of care and not assigned by study –Xpert conducted within 7 days of sputum collection Xpert testing at 2 central labs in US (Emory, UCSF) and at 1 lab in Rio de Janeiro, Brazil and 1 in Johannesburg, South Africa –All labs participated in external Xpert Quality Assurance program Xpert results compared to 2 sputum specimens, each evaluated with AFB smear, liquid and solid mycobacterial culture Rifampin resistance by proportions method on Middlebrook agar rpoB sequencing conducted on a specimen from each participant with ≥1 culture with TB growth

7. Results 720 of 994 participants had results for 2 AFB smears, 2 liquid and solid media mycobacterial cultures, and 2 Xpert available at time of analysis Median Age (Q1,Q3) 46.5 (454/720) Male63% Site of enrollment US: 71% South Africa: 19% Brazil: 10% HIV+ 48% (343/720) US 41% (211/512) South Africa 79% (107/139) Brazil 34% (25/72) Median CD4 + cells/mm 3 for 330 HIV+ participants (Q1,Q3) 157 (44,369)

8. Results ≥ 1 culture TB+15 % (109/720 ) US: 53% (58/109) South Africa: 40% (44/109) Brazil: 6% (7/109) AFB+ 63% (69/109) HIV+ 39% (42/109) Rifampin susceptibility (agar proportions method) Rifampin resistant 2.8% (3/109) Rifampin sensitive 91.7% (100/109) Contaminated or no growth on DST media 5.5% (6/109) Non Tuberculosis Mycobacterial(NTM) Growth 9.3% (67/720) M. avium complex (MAC): 67% (45) US (45), S. Africa (0), Brazil (0) M. kansasii: 6% (4) US (3), S. Africa (0), Brazil (1) Other NTM: 27% (18) US (11), S. Africa (3), Brazil (4)

9. Sensitivity of 1 Xpert No significant impact of region on sensitivity in AFB-/TB culture+ Sensitivity (95% CI)Xpert +/ TB culture + Overall 85.8% (78.0%, 91.2%)91/106 AFB+/TB culture +100% (94.6%, 100%)67/67 AFB-/TB culture +61.5% (45.9%, 75.1%)24/39

10. Specificity of 1 Xpert Specificity (95% CI)Xpert - / TB culture - All Participants 98.8% (97.6%, 99.4%)591/598 AFB+100% (51.0%, 100 %)4/4 AFB-98.8% (97.6%, 99.4%)587/594 US only99.3% (98.0%, 99.8%)441/444 AFB+100% (51%, 100%)4/4 AFB-99.3% (98.0%, 99.8%)437/440 No significant impact of region on specificity

11. Xpert Performance in HIV infection HIV status did not significantly impact sensitivity or specificity HIV+Sensitivity (95% CI)Specificity (95% CI) AFB+100% (84.5,% 100%)100% (20.7%, 100%) AFB-57.9% (36.3%,76.9%) 98.3% (96.1%, 99.3%) HIV- AFB+100% (92.3%, 100%)100% (43.9%, 100%) AFB-65.0% (43.3%, 81.9%) 99.3% (97.6,% 99.8%)

12. Xpert in setting of NonTuberculosis Mycobacteria(NTM) Growth nXpert TB positive AFB+/Non tuberculosis Mycobacterial Culture + 3 (all MAC) 0/3 AFB-/Non tuberculosis Mycobacterial Culture + 64 1/64 (this specimen with MAC growth)

13. Discordance between Xpert & Culture All discordance occurred in AFB smear negative specimens 10 with Xpert TB+/ TB Culture negative: n1 st Xpert2 nd XpertTB culture 4positive negative 3positivenegative 2 positivenegative 1failurepositivenegative

14. Xpert TB Negative /TB culture + Of the 15 with 1 st Xpert TB negative, 2 nd Xpert was TB+ in 3 out of 15 –20% incremental yield of 2 nd Xpert test In AFB-/TB culture+: –Sensitivity of 2 Xperts: 69.2% (27/39) –Sensitivity of 1 Xpert: 61.5% (24/39)

15. Rifampin susceptibility 3 Rifampin resistant specimens by culture-based DST: Xpert detected 3 out of 3 Specificity: 98.8% (81/82) One participant: Xpert RIF resistant / Culture RIF sensitive –2 Xpert tests: 1 RIF resistant, 1 RIF sensitive –rpoB sequencing: wild type NPV: 100% PPV: 75% (n=4)

16. Conclusions Excellent performance for detection of TB including in low prevalence settings –Sensitivity AFB+100%, AFB- 61.5% –Specificity 98.9% overall, 99.3% in low prevalence Performance not significantly impacted by region nor HIV status Xpert detected 3 of 3 specimens rifampin resistant specimens by culture Data support use of Xpert MTB/RIF in low prevalence settings and in HIV infection

17. Acknowledgements Study participants Support from NIH, CDC, & Cepheid ACTG 5295 Team Tuberculosis Trials Consortium (TBTC) S34 Team CDC Tuberculosis Lab Participating ACTG & TBTC sites & site investigators ACTGTBTC 603 UCLA11101 WITS31475 VCU22 Denver62 Houston 701 UCSD12101 FIOCRUZ31477 UMDNJ40 South Texas63 San Antonio 801 UCSF30329 Columbia31472 Henry Ford53 Washington VA70 Vanderbilt 1201 USC31468 Bronx Lebanon31473 Houston54 DukeUCSF TBTC 5802 Emory 7804 Cornell