Xpert Ultra Can Unambiguously Identify Specific Rifampin Resistance-Conferring Mutations

In this work, we validated raw results of the new version of Xpert MTB/RIF, Xpert Ultra, and found that unique combinations of Ultra probe and melting temperature shift (ΔTm) can discriminate between rifampicin-resistance (RR) conferring rpoB mutations and identify specific RR mutations including disputed mutations commonly missed in rapid phenotypic drug susceptibility tests. Negative ΔTm was associated with all mutations except those in codon 450. The combination of ΔTm values with the capturing probes enabled to differentiate mutations in codons 428, 430, 431, 432, 434, 435, 441, 445, 446, and 452, including disputed mutations. Mutation Asp435Tyr was unambiguously distinguished from Asp435Val through probe rpoB2 |ΔTm|, while mutations Ser441Gln and Ser441Leu were discriminated from the rest by |ΔTm| values of probes rpoB2 and rpoB3. Mutations His445Asp and His445Tyr were distinguished from disputed mutations His445Leu and His445Asn through probe rpoB3 |ΔTm|. Ser450Leu was distinguished from Ser450Trp by probe rpoB4A |ΔTm| except for one strain with an outlier rpoB4A Tm of 70.9°C in contrast to the other 13 strains with rpoB4A Tm of 73.3-73.8°C. The indeterminate result associated with His445Arg may be caused by its |ΔTm|=1.8°C compared with |ΔTm| typically exceeding 2°C for other mutations. With these findings, we propose potential full utilization of Ultra results for RR tuberculosis (RR-TB) surveillance in association with the growing number of digitally linked Xpert MTB/RIF machines in more than half of high TB burden countries worldwide. Our findings may guide end-users to rapidly identify underlying RR mutations that will flag the national TB control program to investigate suspected cases of RR-TB transmission based on epidemiologic linking. This, in turn, may rule-out transmission between RR-TB patients in a specific setting. Discriminatory results of Ultra could also distinguish relapse from reinfection, and resolve discordance between an RR Ultra result and a low-level RS phenotypic result due to a disputed mutation.