No phenotypic resistance observed for most group-3 and -4 variants in Mycobacterium tuberculosis genes related to bedaquiline, clofazimine, delamanid, and pretomanid in a Central and West African context

The interpretation of genetic variants' association (or not) with phenotypic resistance to newly introduced and repurposed antituberculosis drugs remains challenging, as many mutations detected by whole-genome sequencing (WGS) are classified as of uncertain significance (group 3) or not associated with resistance-interim (group 4) by the World Health Organization (WHO) mutation catalog v2. We evaluated the phenotypic impact of such variants on minimum inhibitory concentrations (MICs) for bedaquiline (BDQ), clofazimine (CFZ), delamanid (DLM), and pretomanid (PA) in Mycobacterium tuberculosis complex isolates from the multi-country DIAMA cohort in sub-Saharan Africa (SSA), which recruited RR/RS-TB patients naïve to these drugs. Among 1,475 isolates with available WGS data, 163 variants met eligibility criteria; due to viable strain unavailability, 89 isolates carrying 29 unique BDQ/CFZ-related and 60 unique DLM/PA-related variants were tested for MIC determination using broth microdilution. Additional structural modeling was performed to explore potential effects of amino-acid substitutions on protein stability. Among BDQ/CFZ-related variants, MICs above the critical concentrations (CCs) were consistently associated with mmpR5 variants, whereas variants in atpE, pepQ, and Rv1979c were not. DLM/PA variants (ddn, fbiA-D, and fgd1) were frequently detected as non-fixed populations, yet rarely yielding MIC values above the CC. Predicted structural destabilization showed no consistent association with MIC values or variant fixation status. Under the conditions tested, phenotypic resistance was not detected for most group 3 and 4 variants detected by WGS. Our data provide evidence from SSA to support improved interpretation of resistance-associated mutations for new and repurposed antituberculosis drugs.IMPORTANCEWhole-genome sequencing increasingly detects Mycobacterium tuberculosis complex mutations classified by the World Health Organization (WHO) mutation catalog v2 as group 3 variants of uncertain significance or group 4 variants not associated with resistance-interim, limiting reliable prediction of resistance to new and repurposed antituberculosis drugs. By generating minimum inhibitory concentration (MIC) data for such variants identified in a multi-country sub-Saharan African cohort, this study provides phenotypic evidence to support future refinement and expansion of the WHO mutation catalog v2. Notably, mmpR5 variants associated with elevated bedaquiline/clofazimine MICs were identified in eight isolates, suggesting that some patients in this cohort may have harbored pre-existing resistance-associated variants yet remained potentially eligible for bedaquiline-containing regimens. These findings contribute to improving the interpretation of genomic resistance data and strengthening surveillance of resistance to bedaquiline, clofazimine, delamanid, and pretomanid.