Molecular Epidemiology and Genetic Diversity of Multidrug-Resistant Mycobacterium tuberculosis Isolates in Bangladesh.

Although the number of multidrug-resistant (MDR) tuberculosis (TB) cases is high overall, a major gap exists in our understanding of the molecular characteristics and transmission dynamics of the MDR Mycobacterium tuberculosis isolates circulating in Bangladesh. The present study aims to characterize the MDR-TB isolates of Bangladesh and to investigate the mode of transmission. A total of 544 MDR-TB isolates were obtained from a nationwide drug-resistant TB surveillance study conducted between October 2011 and March 2017 covering all geographic divisions of Bangladesh. The isolates were characterized using TbD1 deletion analysis, spoligotyping, and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Deletion analysis showed that 440 (80.9%) isolates were the modern type, while the remainder were the ancestral type. The largest circulating lineage was the Beijing type, comprising 208 isolates (38.2%), followed by T, EAI, and LAM with 93 (17.1%), 58 (10.7%), and 52 (9.5%) isolates, respectively. Combined MIRU-VNTR and spoligotyping analysis demonstrated that the majority of the clustered isolates were of the Beijing and T1 lineages. The overall rate of recent transmission was estimated at 33.8%. In conclusion, the MDR M. tuberculosis isolates circulating in Bangladesh are mostly of the modern virulent type. The Beijing and T lineages are the predominant types and most of the transmission of MDR-TB can be attributed to them. The findings also suggest that, along with the remarkable transmission, the emergence of MDR-TB in Bangladesh is largely due to acquired resistance. Rapid and accurate diagnosis and successful treatment will be crucial for controlling MDR-TB in Bangladesh. IMPORTANCE Multidrug-resistant TB is considered to be the major threat to tuberculosis control activities worldwide, including in Bangladesh. Despite the fact that the number of MDR-TB cases is high, a major gap exists in our understanding of the molecular epidemiology of the MDR-TB isolates in Bangladesh. In our study, we characterized and classified the MDR-TB isolates circulating in Bangladesh and investigated their mode of transmission. Our results demonstrated that the MDR M. tuberculosis isolates circulating in Bangladesh are mostly of the modern virulent type. The Beijing and T lineages are the predominant types and are implicated in the majority of MDR-TB transmission. Our findings reveal that, along with the remarkable transmission, the emergence of MDR-TB in Bangladesh is largely due to acquired resistance, which may be due to nonadherence to treatment or inadequate treatment of TB patients. Rapid diagnosis and adherence to an appropriate treatment regimen are therefore crucial to controlling MDR-TB in Bangladesh.

Pyrazinamide Susceptibility and pncA Mutation Profiles of Mycobacterium tuberculosis among Multidrug-Resistant Tuberculosis Patients in Bangladesh.

Pyrazinamide (PZA) is a frontline antituberculosis (anti-TB) drug used in both first- and second-line treatment regimens. However, due to complex laboratory requirements, the PZA susceptibility test is rarely performed, leading to a scarcity of data on susceptibility to PZA. Bangladesh is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB), but to our knowledge, published data on rates of PZA susceptibility (PZAs), especially among MDR-TB patients, are limited. We aimed to analyze the PZA susceptibility patterns of Mycobacterium tuberculosis isolates from MDR-TB patients and to correlate the pncA mutation with PZA resistance in Bangladesh. A total of 169 confirmed MDR M. tuberculosis isolates from a pool of specimens collected in a nationwide surveillance study were included in this analysis. All the isolates were tested for phenotypic PZA susceptibility in Bactec mycobacterial growth indicator tube (MGIT) culture medium, and the pncA gene was sequenced. We also correlated different types of clinical information and treatment outcomes with PZA susceptibility. We found that 45% of isolates were phenotypically PZA resistant. Sequencing of the pncA gene revealed a high concordance (82.2%) between the pncA gene sequence and the phenotypic assay results. A total of 64 different mutations were found, and 9 isolates harbored multiple mutations. We detected 27 new pncA mutations. We did not find any significant correlation between the different clinical categories, the genetic lineage, or treatment outcome group and PZA susceptibility. Considering the turnaround time, sequencing would be the more feasible option to determine PZA susceptibility, and further studies to investigate the MIC of PZA should be conducted to determine an effective dose of the drug.

Performance of TaqMan array card to detect TB drug resistance on direct specimens.

Culture based phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis (TB) is time consuming therefore rapid genotypic methods are increasingly being utilized. We previously developed and evaluated on TB isolates a rapid genotypic TaqMan array card (TAC) that detects mutations in several resistance-associated genes using dozens of primer pairs, probes, and high resolution melt analysis, with >96% accuracy versus Sanger sequencing. In this study we examined the performance of TAC on sputum, comparing results between 71 paired sputum and TB isolates of which 62 were MDR-TB. We also adapted the TAC to include wild-type probes and broadened coverage for rpoB and gyrA mutations. TAC was 89% successful at detecting wild-type or mutations within inhA, katG, rpoB, eis, gyrA, rplC, and pncA on smear positive sputa and 33% successful on smear negative sputa. The overall accuracy of these detections as compared to the TAC results of the paired isolate was 95% ± 7 (average sensitivity 98% ± 3; specificity 92% ± 14). Accuracy of sputum TAC results versus phenotypic DST for isoniazid, rifampin, ofloxacin/moxifloxacin, and pyrazinamide was 85% ± 12. This was similar to that of the isolate TAC results (accuracy 88% ± 13), thus inaccuracies primarily reflected intrinsic genotypic-phenotypic discordance. The TAC is a rapid, modular, comprehensive, and accurate TB DST for the major first and second line TB drugs and could be used for supplemental testing of GeneXpert resistant smear positive sputum.

Quantitative drug-susceptibility in patients treated for multidrug-resistant tuberculosis in Bangladesh: implications for regimen choice.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) treatment in Bangladesh is empiric or based on qualitative drug-susceptibility testing (DST) by comparative growth in culture media with and without a single drug concentration. METHODS: Adult patients were enrolled throughout Bangladesh during the period of 2011-2013 at MDR-TB treatment initiation. Quantitative DST by minimum inhibitory concentration (MIC) testing for 12 first and second-line anti-TB drugs was compared to pretreatment clinical characteristics and treatment outcomes. MIC values at or one dilution lower than the resistance breakpoint used for qualitative DST were categorized as borderline susceptible, and MIC values one or two dilutions greater as borderline resistant. RESULTS: Seventy-four patients were enrolled with a mean age of 35 ± 15 years, and 51 (69%) were men. Of the rifampin isolates with MIC >1.0 µg/ml, 12 (19%) were fully susceptible or borderline susceptible to rifabutin (MIC ≤ 0.5 µg/ml). Amikacin was fully susceptible in 73 isolates (99%), but kanamycin in only 54 (75%) (p<0.001). Ofloxacin was borderline susceptible in 64%, and fully susceptible in only 14 (19%) compared to 60 (81%) of isolates fully susceptible for moxifloxacin (p<0.001). Kanamycin non-susceptibility and receipt of the WHO Category IV regimen trended with interim treatment failure: adjusted odd ratios respectively of 5.4 [95% CI 0.82-36.2] (p = 0.08) and 7.2 [0.64-80.7] (p = 0.11). CONCLUSIONS: Quantitative MIC testing could impact MDR-TB regimen choice in Bangladesh. Comparative trials of higher dose or later generation fluoroquinolone, within class change from kanamycin to amikacin, and inclusion of rifabutin appear warranted.

Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes.

UNLABELLED: Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. IMPORTANCE: Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.

Discordance across several methods for drug susceptibility testing of drug-resistant Mycobacterium tuberculosis isolates in a single laboratory.

Given the increases in drug-resistant tuberculosis, laboratory capacities for drug susceptibility testing are being scaled up worldwide. A laboratory must decide among several endorsed methodologies. We evaluated 87 Mycobacterium tuberculosis isolates for concordance of susceptibility results across six methods: the L-J proportion method, MGIT 960 SIRE AST, Gene/Xpert MTB/RIF, GenoType MTBDRplus line probe assay, MycoTB MIC plate, and a laboratory-developed mycobacteriophage quantitative PCR (qPCR)-based method. Most (80%) isolates were multidrug resistant. Of the culture-based methods, the mycobacteriophage qPCR method was fastest, the L-J proportion method was the slowest, and the MGIT method required the most repeat testing (P < 0.05). For isoniazid (INH), 82% of isolates were susceptible by all methods or resistant by all methods, whereas for rifampin (RIF), ethambutol (EMB), and streptomycin (STR), such complete concordance was observed in 77%, 50%, and 51% of isolates, respectively (P < 0.05 for INH or RIF versus EMB or STR). The discrepancies of EMB and STR stemmed largely from diminished concordance of the MGIT EMB results (kappa coefficient range, 0.26 to 0.30) and the L-J STR result (kappa range, 0.35 to 0.45) versus other methods. Phage qPCR and the MycoTB MIC plate were the only methods that yielded second-line susceptibilities and revealed significant quantitative correlations for all drugs except cycloserine, as well as moderate to excellent kappa coefficients for all drugs except for para-aminosalicylic acid. In summary, the performance of M. tuberculosis susceptibility testing differs by platform and by drug. Laboratories should carefully consider these factors before choosing one methodology, particularly in settings where EMB and STR results are clinically important.