No abstract

The GenoType MTBDRsl test and DNA sequencing were used to rapidly detect second-line drug-and ethambutol (EMB)-resistant Mycobacterium tuberculosis. The ability of these two assays to detect the presence of mutations associated with resistance to fluoroquinolones (FLQ), aminoglycosides/cyclic peptide (AG/CP), and EMB in the gyrA, rrs, and embB genes (for the GenoType MTBDRsl test) and gyrA, gyrB, rrs, eis, embC, embA, embB, and embR genes (for DNA sequencing) was compared to that of conventional agar proportion drug susceptibility testing (DST). We evaluated 234 multidrug-resistant (MDR) M. tuberculosis isolates. The two molecular methods had high levels of specificity (95.8 to 100%). The sensitivities for FLQ resistance detection for both methods were 85.1%. For AG (kanamycin [KM] and amikacin [AM]) and CP (capreomycin CAP]), the sensitivities of resistance detection using the GenoType MTBDRsl test were 43.2%, 84.2%, and 71.4%, respectively, while with the inclusion of an extra gene, eis, in sequencing, the sensitivity reached 70.3% for detection of KM resistance. The sensitivities of EMB resistance detection were 56.2% and 90.7% with the GenoType MTBDRsl test and sequencing, respectively. We found that the GenoType MTBDRsl test can rapidly detect resistance to FLQ, CAP, and AM. The accuracy of the GenoType MTBDRsl test for the detection of FLQ and AM resistance was comparable to that of conventional DST; however, the test was less accurate for the detection of KM and EMB resistance and demonstrated a poor predictive value for CAP resistance. We recommend including new alleles consisting of the eis promoter and embB genes in molecular analysis. However, conventional DST is necessary to rule out false-negative results from molecular assays.

The dN/dS ratio provides evidence of adaptation or functional constraint in protein-coding genes by quantifying the relative excess or deficit of amino acid-replacing versus silent nucleotide variation. Inexpensive sequencing promises a better understanding of parameters, such as dN/dS, but analyzing very large data sets poses a major statistical challenge. Here, I introduce genomegaMap for estimating within-species genome-wide variation in dN/dS, and I apply it to 3,979 genes across 10,209 tuberculosis genomes to characterize the selection pressures shaping this global pathogen. GenomegaMap is a phylogeny-free method that addresses two major problems with existing approaches: 1) It is fast no matter how large the sample size and 2) it is robust to recombination, which causes phylogenetic methods to report artefactual signals of adaptation. GenomegaMap uses population genetics theory to approximate the distribution of allele frequencies under general, parent-dependent mutation models. Coalescent simulations show that substitution parameters are well estimated even when genomegaMap’s simplifying assumption of independence among sites is violated. I demonstrate the ability of genomegaMap to detect genuine signatures of selection at antimicrobial resistance-conferring substitutions in Mycobacterium tuberculosis and describe a novel signature of selection in the cold-shock DEAD-box protein A gene deaD/csdA. The genomegaMap approach helps accelerate the exploitation of big data for gaining new insights into evolution within species.

Most drugs demonstrated efficacy against M. tuberculosis . When these MICs are compared with the published pharmacokinetic/pharmacodynamic profiles of the respective drugs in humans, trimethoprim/sulfamethoxazole, meropenem/clavulanate, linezolid, clofazimine and nitazoxanide appear promising and warrant further clinical investigation.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-resistant-tuberculosis (MDR-TB) (DREAM) was a 5-year (2015–2019) phenotypic drug-resistance surveillance study across 11 countries. DREAM assessed the susceptibility of 5036 MDR-TB isolates of bedaquiline-treatment-naïve patients to bedaquiline and other anti-tuberculosis drugs by the 7H9 broth microdilution (BMD) and 7H10/7H11 agar dilution (AD) minimal inhibitory concentration (MIC) methods. Bedaquiline AD MIC quality control (QC) range for the H37Rv reference strain was unchanged, but the BMD MIC QC range (0.015–0.12 μg/ml) was adjusted compared with ranges from a multilaboratory, multicountry reproducibility study conforming to Clinical and Laboratory Standards Institute Tier-2 criteria. Epidemiological cut-off values of 0.12 μg/ml by BMD and 0.25 μg/ml by AD were consistent with previous bedaquiline breakpoints. An area of technical uncertainty or Intermediate category was set at 0.25 μg/ml and 0.5 μg/ml for BMD and AD, respectively. When applied to the 5036 MDR-TB isolates, bedaquiline-susceptible, intermediate and bedaquiline-resistant rates were 97.9%, 1.5% and 0.6%, respectively, for BMD, and 98.8%, 0.8% and 0.4% for AD. Resistance rates were: ofloxacin 35.1%, levofloxacin 34.2%, moxifloxacin 33.3%, 1.5% linezolid and 2% clofazimine. Phenotypic cross resistance between bedaquiline and clofazimine was 0.4% in MDR-TB and 1% in pre-extensively drug-resistant (pre-XDR-TB)/XDR-TB populations. Co-resistance to bedaquiline and linezolid, and clofazimine and linezolid, were 0.1% and 0.3%, respectively, in MDR-TB, and 0.2% and 0.4% in pre-XDR-TB/XDR-TB populations. Resistance rates to bedaquiline appear to be low in the bedaquiline-treatment-naïve population. No treatment-limiting patterns for cross-resistance and co-resistance have been identified with key TB drugs to date.