Beyond Hotspot Mutations: Diagnostic Relevance of High Frequency, Low Frequency, and Disputed rpoB Variants in Rifampicin-Resistant Mycobacterium tuberculosis

Rifampicin-resistant tuberculosis (RR-TB) remains a major threat to global TB control, primarily driven by mutations in the <i>rpoB</i> gene of <i>Mycobacterium tuberculosis</i> (Mtb). Most resistance-conferring mutations occur within the 81-base pair RIF resistance determining region (RRDR), particularly at codons S450L, H445Y/D, and D435V, which are strongly associated with high level resistance. However, increasing evidence of low-frequency and disputed variants both within and beyond the RRDR reveals a broader genetic spectrum that contributes to diagnostic uncertainty and variable phenotypic outcomes. This review summarizes current knowledge of high frequency, low frequency, and disputed <i>rpoB</i> mutations and their implications for molecular detection of RIF resistance. Structural analyses show that specific amino acid substitutions alter key hydrogen bonds or create steric hindrance in the RIF-binding pocket, leading to diverse resistance levels. Despite the success of molecular platforms such as Xpert MTB/RIF and line probe assays, their hotspot-based detection limits sensitivity to noncanonical variants. Lowering the minimum inhibitory concentration (MIC) breakpoint and integrating sequencing-based approaches, such as targeted and whole-genome sequencing, can enhance detection accuracy. A combined genomic and phenotypic framework will be essential to close existing diagnostic gaps and advance precision guided management of RIF-resistant and multidrug-resistant tuberculosis.