MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era

Abstract

Background: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini.

Methods: We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009-2010; (2) MDR strains from the Nhlangano region, 2014-2017).

Results: MDR strains in collection 1 had a 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, which likely emerged around 1993 (95% highest posterior density 1987-1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2.

Conclusion: The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Keywords: Diagnostice escape; MDR outbreak strains; Multidrug resistance; Resistance evolution; Treatment escape; Treatment failure; Tuberculosis.

Fig. 1

Phylogenetic diversity of 273 M. tuberculosis complex isolates from Eswatini. Data are presented in a maximum likelihood tree (MLT) based on 12,062 SNP positions. Phylogenetic lineages are displayed. Cluster genome: isolates in one cluster share the same colour. Occurrence of resistance mediating and putative compensatory mutations in RpoB and RpoC are highlighted; identical mutations share the same colour

Fig. 2

Maximum clade credibility tree of the S-type lineage. gCL10, the largest MDR cluster in Eswatini, has been circulating for 17.2 years prior to the DRS conducted in 2009–2010. Three different Rv0678 mutations occurred within this cluster. The M146T mutation was the most common (n = 21) and likely arose 6.7 years prior to the DRS. The N98D (n = 2) and G121R (n = 1) must have evolved within 5.5 and 7.8 years prior to the DRS, respectively. Isolates, for which BDQ and CFZ MICs were measured, are highlighted (Fig. 3 and Additional file 1: Table S3)

Fig. 3

Effect of Rv0678 mutations on BDQ and CFZ MICs. The MICs for the DS Beijing mutant with the Rv0678 A110V mutation were compared with those of 24 gCL10-outbreak MDR isolates with either the Rv0678 N98D, G121R, or M146T mutations (Fig. 2). The NCOFFs for the Rv0678 M146T distributions for CFZ (part a) and BDQ (part b) were set using the eyeball method, and a corresponding idealized representation of the MIC distributions of the gWT and Rv0678 M146T populations for both drugs (and their area of overlap in grey) was prepared for illustrative purposes (part c) [25]. The MIC increases for M146T relative to gWT isolates were statistically significant for both CFZ and BDQ. The Rv0678 G121R and A110V mutations could not be evaluated statistically as the mutations only appeared once