Whole-Genome Sequencing for Resistance Prediction and Transmission Analysis of Mycobacterium tuberculosis Complex Strains from Namibia

Abstract

Namibia is among 30 countries with a high burden of tuberculosis (TB), with an estimated incidence of 460 per 100,000 population and around 800 new multidrug-resistant (MDR) TB cases per year. Still, data on the transmission and evolution of drug-resistant Mycobacterium tuberculosis complex (Mtbc) strains are not available. Whole-genome sequencing data of 136 rifampicin-resistant (RIFr) Mtbc strains obtained from 2016 to 2018 were used for phylogenetic classification, resistance prediction, and cluster analysis and linked with phenotypic drug susceptibility testing (pDST) data. Roughly 50% of the strains investigated were resistant to all first-line drugs. Furthermore, 13% of the MDR Mtbc strains were already pre-extensively drug resistant (pre-XDR). The cluster rates were high, at 74.6% among MDR and 85% among pre-XDR strains. A significant proportion of strains had borderline resistance-conferring mutations, e.g., inhA promoter mutations or rpoB L430P. Accordingly, 25% of the RIFr strains tested susceptible by pDST. Finally, we determined a potentially new bedaquiline resistance mutation (Rv0678 D88G) occurring in two independent clusters. High rates of resistance to first-line drugs in line with emerging pre-XDR and likely bedaquiline resistance linked with the ongoing recent transmission of MDR Mtbc clones underline the urgent need for the implementation of interventions that allow rapid diagnostics to break MDR TB transmission chains in the country. A borderline RIFr mutation in the dominant outbreak strain causing discrepancies between phenotypic and genotypic resistance testing results may require breakpoint adjustments but also may allow individualized regimens with high-dose treatment. IMPORTANCE The transmission of drug-resistant tuberculosis (TB) is a major problem for global TB control. Using genome sequencing, we showed that 13% of the multidrug-resistant (MDR) M. tuberculosis complex strains from Namibia are already pre-extensively drug resistant (pre-XDR), which is substantial in an African setting. Our data also indicate that the ongoing transmission of MDR and pre-XDR strains contributes significantly to the problem. In contrast to other settings with higher rates of drug resistance, we found a high proportion of strains having so-called borderline low-level resistance mutations, e.g., inhA promoter mutations or rpoB L430P. This led to the misclassification of 25% of the rifampicin-resistant strains as susceptible by phenotypic drug susceptibility testing. This observation potentially allows individualized regimens with high-dose treatment as a potential option for patients with few treatment options. We also found a potentially new bedaquiline resistance mutation in rv0678.

Keywords: Namibia; drug resistance; low-level resistance; whole-genome sequencing.

FIG 1

Maximum likelihood phylogeny based on the concatenated SNP sequence from 136 Mtbc strains. The concatenated SNP sequence consists of 3,402 parsimony-informative sites, 2,029 singleton sites, and 0 constant sites; mutations related to the respective drugs and resistance status are color-coded and expressed as annotation rings on the tree. RR, rifampicin resistant; MDR, multidrug resistant; pre-XDR, pre-extensively drug resistant; INH, isoniazid; RIF, rifampicin; d12, distance of ≤12 SNPs.

FIG 2

Maximum likelihood phylogeny of SNP group 22 based on the concatenated SNP sequence from 24 Mtbc strains of group 22. The concatenated SNP sequence consists of 21 parsimony-informative sites, 69 singleton sites, and 0 constant sites; mutations related to the respective drugs and resistance status are color-coded and expressed as annotation columns on the tree. pDST, phenotypic drug susceptibility testing; d5, distance of ≤5 SNPs; RIFs, rifampicin sensitive.

FIG 3

Map of Namibian districts with the appearance of the mutation rpoB L430P. The mutations appear in all districts; however, the frequency is higher in the northern parts of the region.