GeneXpert-evading MDR M. tuberculosis outbreak clone in Eswatini has elevated bedaquiline and clofazimine resistance levels dated to the pre-treatment era. Eswatini GeneXpert-evading outbreak clone

Multidrug resistant (MDR) Mycobacterium tuberculosis complex (Mtbc) strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to a particular resistance mutation (RpoB I491F) are threatening the control of MDR tuberculosis (MDR TB) in Eswatini. Here, we investigate the evolution and spread of MDR-Mtbc strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole genome sequencing in two collections (1: drug resistance survey 2009-2010, 2: MDR strains 2014-2017). We found that MDR-strains in collection 1 had an extraordinarily high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n=28; gCL10, n=40). All gCL10 isolates carried the RpoB I491F mutation missed by commercial mDST. In addition, 24 (60%) gCL10 isolates had mutation in the BDQ and CFZ resistance gene Rv0678 and had increased BDQ/CFZ mMinimal inhibitory concentrations (MIC) compared to wild type isolates. These strains could be detected also in the later cohort . The high clustering rate suggests that transmission is driving the MDR-TB epidemic in Eswatini over decades. The high and further increasing prevalence of gCL10 I491F isolates threatens the use of commercial mDST assays (e.g. Xpert MTB/RIF), and of new drugs such as BDQ and CFZ. Measures containing the spread of these outbreak isolates need to be urgently implemented.