Decoding antibiotic resistance in tuberculosis: Role of RRDR and non-RRDR mutations in diagnostic perspectives

Rifampicin resistance continues to pose a challenge to successful tuberculosis (TB) control, mainly driven by mutations in the rpoB gene of Mycobacterium tuberculosis. Although most diagnostic tools, such as Xpert MTB/RIF, Xpert Ultra, and line probe assays, focus on the standard 81-bp rifampicin resistance-determining region (RRDR), recent findings emphasize the critical role of non-RRDR mutations in the development of rifampicin resistance. Although less frequent, these mutations either directly modify drug-binding sites or drive compensatory evolution that restores bacterial fitness impaired by RRDR mutations. Clinically significant variants such as I491F and V170F have been detected in various high-burden areas, often with elevated minimum inhibitory concentrations; however, they are frequently missed by routine molecular assays, leading to false-susceptible results, delayed diagnosis, and inappropriate treatment. This gap is particularly alarming in regions experiencing outbreaks of non-RRDR-harbouring isolates, like Eswatini, Myanmar, and specific areas of South Africa. Emerging tools such as whole-genome sequencing (WGS), targeted next-generation sequencing (tNGS), multiplex allele-specific PCR, high-resolution melting analysis, and nanopore sequencing enable comprehensive detection of both RRDR and non-RRDR mutations with improved accuracy. Integrating these high-resolution tools with existing diagnostic approaches can bridge these diagnostic blind spots and enhance detection and genomic surveillance. This review focuses on the molecular basis, epidemiology, and diagnostic challenges associated with non-RRDR mutations, highlighting the need to upgrade diagnostic platforms and strengthen genomic surveillance to ensure early, accurate detection of rifampicin-resistant TB.