Development and Assessment of a Novel Whole-Gene-Based Targeted Next-Generation Sequencing Assay for Detecting the Susceptibility of Mycobacterium tuberculosis to 14 Drugs.

Targeted next-generation sequencing (tNGS) has emerged as an alternative method for detecting drug-resistant tuberculosis (DR-TB). To provide comprehensive drug susceptibility information and to address mutations missed by available commercial molecular diagnostics, we developed and evaluated a tNGS panel with 22 whole-gene targets using the Ion Torrent platform to predict drug resistance to 14 drugs, namely, rifampicin (RIF), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), moxifloxacin (MFX), levofloxacin (LFX), amikacin (AMK), capreomycin (CM), kanamycin (KM), streptomycin (SM), bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), and delamanid (DLM). We selected 50 and 35 Mycobacterium tuberculosis isolates with various DR profiles as the training set and the challenge set, respectively. Comparative variant analyses of the DR genes were performed using Sanger sequencing and whole-genome sequencing (WGS). Phenotypic drug susceptibility testing (pDST) results were used as gold standards. Regarding the limit of detection, the tNGS assay detected 2.9 to 3.8% minority variants in 4% mutant mixtures. The sensitivity and specificity of tNGS were 97.0% (95% confidence interval [CI] = 93.1 to 98.7%) and 99.1% (95% CI = 97.7 to 99.7%), respectively. The concordance of tNGS with pDST was 98.5% (95% CI = 97.2 to 99.2%), which was comparable to that of WGS (98.7%, 95% CI = 97.4 to 99.3%) and better than that of Sanger sequencing (96.9%, 95% CI = 95.3 to 98.0%). The agreement between tNGS and pDST was almost perfect for RIF, INH, EMB, MFX, LFX, AMK, CM, KM, SM, BDQ, and LZD (kappa value = 0.807 to 1.000) and substantial for PZA (kappa value = 0.791). Our customized novel whole-gene-based tNGS panel is highly consistent with pDST and WGS for comprehensive and accurate prediction of drug resistance in a strengthened and streamlined DR-TB laboratory program. IMPORTANCE We developed and validated a tNGS assay that was the first to target 22 whole genes instead of regions of drug resistance genes and comprehensively detected susceptibility to 14 anti-TB drugs, with great flexibility to include new or repurposed drugs. Notably, we demonstrated that our custom-designed Ion AmpliSeq TB research panel platform had high concordance with pDST and could significantly reduce turnaround time (by approximately 70%) to meet a clinically actionable time frame. Our tNGS assay is a promising DST solution for providing needed clinical information for precision medicine-guided therapies for DR-TB and allows the rollout of active pharmacovigilance.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-Resistant Tuberculosis (MDR-TB): a 5-Year Prospective In Vitro Surveillance Study of Bedaquiline and Other Second-Line Drug Susceptibility Testing in MDR-TB Isolates.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-resistant tuberculosis (MDR-TB) (DREAM) was a 5-year (2015 to 2019) phenotypic drug resistance surveillance study across 11 countries. DREAM assessed the susceptibility of 5,036 MDR-TB isolates of bedaquiline treatment-naive patients to bedaquiline and other antituberculosis drugs by the 7H9 broth microdilution (BMD) and 7H10/7H11 agar dilution (AD) MIC methods. Bedaquiline AD MIC quality control (QC) range for the H37Rv reference strain was unchanged, but the BMD MIC QC range (0.015 to 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 cutoff 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 5,036 MDR-TB isolates, bedaquiline-susceptible, -intermediate, and -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 the following: 35.1% ofloxacin, 34.2% levofloxacin, 33.3% moxifloxacin, 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. Coresistance to bedaquiline and linezolid and clofazimine and linezolid were 0.1% and 0.3%, respectively, in MDR-TB and 0.2% and 0.4%, respectively, in pre-XDR-TB/XDR-TB populations. Resistance rates to bedaquiline appear to be low in the bedaquiline-treatment-naive population. No treatment-limiting patterns for cross-resistance and coresistance have been identified with key TB drugs to date.

Performance assessment of the GenoType MTBDRsl test and DNA sequencing for detection of second-line and ethambutol drug resistance among patients infected with multidrug-resistant Mycobacterium tuberculosis.

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.

Performance assessment of the GenoType MTBDRplus test and DNA sequencing in detection of multidrug-resistant Mycobacterium tuberculosis.

To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDRplus test and DNA sequencing, were assessed for the rapid detection of drug-resistant Mycobacterium tuberculosis for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the rpoB, katG, inhA regulatory region, inhA, and oxyR-ahpC genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible M. tuberculosis isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDRplus test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDRplus test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDRplus test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDRplus test has the advantage of a short turnaround time for drug-resistant M. tuberculosis detection. Overall, the two assays performed equally well in detecting RIF resistance (P = 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (P < 0.001) and MDR tuberculosis (P < 0.001). We suggest that new alleles of INH resistance genes should be evaluated to improve the sensitivity of the GenoType MTBDRplus test, especially for different geographic areas with genetically diverse M. tuberculosis strains.