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

In Eswatini, multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains harbouring the rifampicin-resistance (RR) rpoB I491F mutation are missed by routine diagnostics, including GeneXpert MTB/RIF Ultra (Xpert Ultra), line probe assays (LPA), and mycobacterium growth indicator tube (MGIT) phenotypic drug susceptibility testing (pDST). To address this diagnostic gap, Eswatini introduced targeted next-generation sequencing (tNGS) in 2019. We analysed 234 patient samples enrolled from June 2021 to December 2024 with isoniazid and/or rifampicin resistance detected by routine diagnostics, or suspected treatment failure, and obtained detailed clinical and outcome data from 59 patients. tNGS detected RR in 159 strains, of which 101 (64%) carried the rpoB I491F mutation. Bedaquiline (BDQ) resistance, conferred by Rv0678 mutations, was identified in 87 strains, rendering 55% (87/159) of RR and 85% (86/101) of rpoB I491F strains genotypically BDQ-resistant. Routine tests substantially under-classified resistance, particularly in strains reported as isoniazid-resistant and rifampicin-susceptible. tNGS-informed treatment changes occurred in 53% (31/59) of patients, with 88% (52/59) treatment success. tNGS is therefore an essential tool to detect rpoB I491F "diagnostic escape" strains with additional BDQ resistance, and underscores the urgent need to reconsider current BPaLM regimens and global drug-resistance classifications.