Genotypic characterization of ‘inferred’ rifampin mutations in GenoType MTBDRplus assay and its association with phenotypic susceptibility testing of Mycobacterium tuberculosis

Abstract In GenoType MTBDRplus assay [line probe assay (LPA)], when Mycobacterium tuberculosis (M. tuberculosis) sample DNA fails to hybridize to atleast one rpoB wild-type probe and any mutation probe, it is inferred as rifampin (RIF)-resistant. In this study, we sought to identify such ‘inferred’ mutations in M. tuberculosis isolates (n = 203) by rpoB gene sequencing and determined their association with phenotypic resistance. D516Y, H526N, L511P mutations were associated with both phenotypically-sensitive (59%, n = 38/64) and resistant (23.7%, n = 33/139) antimicrobial susceptibility testing (AST) results; whereas, S531 W mutation was associated with only RIF-resistant isolates (33%, n = 46/139). These results demonstrated that at standard drug concentrations, some ‘inferred’ mutations may be missed by RIF-AST (phenotypically-sensitive). The use of LPA permits identification of these RIF-resistant isolates and incorporation of additional mutation probes (e.g. S531 W) could further increase LPA specificity. Further studies are needed to establish the significance of the type of ‘inferred’ mutation with clinical/treatment outcomes.