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

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.

Clinical Evaluation of a Line-Probe Assay for Tuberculosis Detection and Drug-Resistance Prediction in Namibia.

Treatment of tuberculosis requires rapid information about Mycobacterium tuberculosis (Mtb) drug susceptibility to ensure effective therapy and optimal outcomes. At the tuberculosis referral hospital in Windhoek, Namibia, a country of high tuberculosis incidence, we evaluated the diagnostic accuracy of a line-probe-assay (LPA), GenID, for the molecular diagnosis of Mtb infection and drug resistance in patients with suspected tuberculosis (cohort 1) and confirmed rifampin (RIF)-resistant tuberculosis (cohort 2). GenID test results were compared to Xpert MTB/RIF and/or Mtb culture and antimicrobial suceptibilty testing. GenID LPA was applied to 79 and 55 samples from patients in cohort 1 and cohort 2, respectively. The overall sensitivity of GenID LPA for the detection of Mtb DNA in sputum from patients with detectable and undetectable acid-fast bacilli by sputum smear microscopy was 93.3% (56/60; 95% confidence interval = 83.8-98.2) and 22.7% (5/22; 7.8-45.4). The sensitivity/specificity for the detection of drug resistance was 84.2% (32/38; 68.7-94.0)/100% (19/19; 82.4-100.0) for RIF, 89.7% (26/29; 72.6-97.8)/91.7% (22/24; 73.0-99.0) for isoniazid, and 85.7% (6/7; 42.1-99.6)/94.7% (18/19; 74.0-99.9) for fluoroquinolones; 23.6% of tests for second-line injectable resistance were invalid despite repeat testing. The diagnosis of tuberculosis by detection of Mtb DNA in sputum by GenID LPA depends strongly on the detection of acid-fast bacilli in sputum specimen. Prediction of drug resistance by GenID did not reach the World Health Organization (WHO) target product profile. IMPORTANCE Mycobacterium tuberculosis (Mtb) drug-resistance detection is crucial for successful control of tuberculosis. Line-probe assays (LPA) are frequently used to detect resistance to rifampin, isoniazid, fluoroquinolones (FQs), and second-line injectables (SLIs). GenID RIF/isoniazid (INH), FQ, and SLI LPA have not been widely tested and used so far. This study tested the diagnostic performance of the GenID LPA in a high-incidence TB/HIV, real-world setting in Namibia. The LPA demonstrates only an acceptable diagnostic performance for Mtb and drug-resistance detection. The diagnostic sensitivity and specificity fall short of the WHO suggested target product profiles for LPA.