Xpert MTB/RIF Ultra resistant and MTBDRplus susceptible rifampicin results in people with tuberculosis: utility of FluoroType MTBDR and deep sequencing

BackgroundXpert MTB/RIF Ultra (Ultra)-detected rifampicin-resistant tuberculosis (TB) is often programmatically confirmed using MTBDRplus. There are limited data on discordant results, including re-tested using newer methods like FluoroType MTBDR (FT-MTBDR) and targeted deep sequencing. MethodsMTBDRplus rifampicin-susceptible isolates from people with Ultra rifampicin-resistant sputum were identified from a South African programmatic laboratory. FT-MTBDR and single molecule-overlapping reads deep (SMOR; rpoB, inhA, katG) on isolate DNA were done (SMOR reference standard). FindingsBetween 01/04/2021-30/09/2022, 8% (109/1347) of Ultra rifampicin-resistant specimens were MTBDRplus-susceptible. Of 89% (97/109) isolates with a sequenceable rpoB, SMOR resolved most in favour of Ultra [79% (77/97)]. Sputum with lower mycobacterial load was associated with Ultra false-positive resistance [46% (11/24) of "very low" Ultras had false-resistance vs. 12% (9/73; p=0.0004) in those [≥]"low"], as were Ultra heteroresistance calls (all wild type probes, [≥]1 mutant probe) [62% (23/37 vs. 25% (15/60) for Ultra without heteroresistance calls; p=0.0003]. Of the 91% (88/97) of isolates successfully tested by FT-MTBDR, 55% (48/88) were FT-MTBDR rifampicin-resistant and 45% (40/88) susceptible, translating to 69% (47/68) sensitivity and 95% (19/20) specificity. In the 91% (99/109) of isolates with inhA and katG sequenced, 62% (61/99) were SMOR isoniazid-susceptible. InterpretationWhen Ultra and MTBDRplus rifampicin results are discordant, Ultra is more likely to be correct and FT-MTBDR agrees more with Ultra than MTBDRplus, however, lower load and the Ultra heteroresistance probe pattern were risk factors for Ultra false rifampicin-resistant results. Most people with Ultra-MTBDRplus discordant resistance results were isoniazid-susceptible. These data have implications for drug-resistant TB diagnosis. FundingThis work was supported by European & Developing Countries Trial Partnerships (EDCTP2; RIA2020I-3305, CAGE-TB), National Institutes of Health (D43TW010350; U01AI152087; U54EB027049; R01AI136894).