Targeted next-generation sequencing implementation in Eswatini identifies rifampicin and bedaquiline resistance undetected by routine diagnostic testing.

Summary

Targeted next-generation sequencing revealed widespread rifampicin diagnostic escape due to rpoB I491F and high rates of Rv0678-mediated bedaquiline resistance in Eswatini. Routine tests under-classified resistance; tNGS informed treatment changes in 53% of patients with an 88% treatment success rate among those with outcome data, supporting regimen revision and expanded sequencing-based surveillance.

Key Findings

• tNGS detected rifampicin resistance in 159 strains; 101/159 (64%) carried rpoB I491F.
• Rv0678 mutations indicating bedaquiline resistance were found in 87 strains; 55% of all RR strains and 85% of rpoB I491F strains were genotypically BDQ-resistant.
• Routine diagnostics (Xpert Ultra, LPA, MGIT pDST) substantially under-classified resistance.
• tNGS-informed regimen changes occurred in 53% (31/59) of patients with detailed data; treatment success was 88% (52/59).

Clinical Implications

Programs in high-burden settings should incorporate tNGS to detect rpoB I491F and Rv0678 mutations, adapt DST algorithms, and reconsider BPaLM regimens where bedaquiline resistance is prevalent. Results argue for updating global drug resistance classifications and routine surveillance.

Limitations

• Observational design with potential selection bias toward complex cases
• Detailed clinical outcomes available for a subset (n=59); limited phenotypic confirmation for all detected mutations

Future Directions

Scale tNGS in high-burden regions, integrate with rapid triage algorithms, and conduct prospective studies to evaluate regimen adaptations (e.g., alternatives to BPaLM) in settings with high BDQ resistance.