Enhancing Tuberculosis Management: Addressing Gaps in Molecular Detection and Resistance

rpoB variants outside canonical hotspots and low-frequency substitutions often disrupt probe binding and melting-curve signals, lowering detection of rifampicin resistance by assays focused on RRDR hotspots. The consequence is reduced assay sensitivity and a measurable risk of missed rifampicin-resistant TB on initial molecular testing—a diagnostic blind spot supported by current evidence.

Low-frequency substitutions and mutations at the margins of the rifampicin-resistance–determining region frequently lie beyond the coverage or melting ranges of many assays. Xpert MTB/RIF and several line-probe assays target a limited set of RRDR codons; noncanonical mismatches reduce hybridization efficiency or shift melt profiles and can produce false-negative molecular calls, particularly at low variant allele frequency. These detection gaps can meaningfully alter initial diagnostic classification and downstream management.

That diagnostic gap prompts escalation to sequencing when molecular and phenotypic results disagree or clinical response is unexpected. Indications for reflex sequencing include persistent molecular–phenotypic discordance, atypical resistance patterns, treatment failure despite apparent susceptibility, or epidemiologic links suggesting transmitted resistance. Targeted rpoB sequencing offers faster, locus-focused depth and higher sensitivity for specific variants, while whole-genome sequencing provides comprehensive resistance profiling and phylogenetic context at the cost of longer turnaround. Sequencing therefore functions as a problem-solving tool to clarify resistance mechanisms and guide further testing.

Phenotypic drug-susceptibility testing and MIC determination can reclassify disputed variants and reveal low-level resistance missed by molecular assays. Lowering MIC breakpoints increases sensitivity for some disputed substitutions but can reduce inter-laboratory reproducibility and lengthen turnaround time. Integrating molecular calls with phenotypic MIC results improves clinical interpretability and can change treatment choices—such as continuing rifampicin in borderline cases versus switching to a second-line regimen—so phenotypic methods are valuable when molecular results are ambiguous.

Together, these findings support focused thresholds for heightened diagnostic suspicion and clearer lab–clinician workflows for discordant results. Pragmatic approaches include reflex sequencing for discordance or treatment failure, routine phenotypic follow-up for disputed variants, and multidisciplinary case review. These steps help close diagnostic gaps and inform practice and policy refinement.