An emergence of Mycobacterium abscessus in blood cultures of Thai patients

@#Aims: Tuberculosis and other mycobacterial infections occur worldwide especially in patients with immunodeficiency. Typically, an empirical treatment for disseminated disease is required for initial therapy due to slow growing nature of most mycobacterial species. Therefore, species distribution and average time to positivity of blood culture is crucial. However, such information is limited for blood culture and, therefore, were determined. Methodology and results: The blood culture data using the BACTEC FX system and drugs susceptibility testing (DST) pattern was recovered during 2012-2017 from a large teaching hospital in Bangkok, Thailand. Overall, 7.8% of 4,838 blood and 6.4% of 1,056 bone marrow (BM) samples were positive for mycobacterial growth. Mycobacterium tuberculosis complex (MTBC), M. avium, and M. abscessus, were the most three common species to be isolated from blood (3.8%, 2.1%, and 0.9%, respectively) and BM (2.4%, 2.4%, and 0.9%, respectively). The average time to positivity for MTBC, M. avium, and M. abscessus was 25.7, 16.1, and 3.8 days, respectively. From 209 antimycobacterial susceptibility testing (AST)-available MTBC strains, 6 (2.87%) strains were multi-drugs resistant (MDR-TB). From 35 AST-available M. avium complex (MAC) isolates, 6 (17.14%), 33 (94.29%), and 28 (80%) isolates were resistant to clarithromycin, moxifloxacin, and linezolid, respectively. BM MAC isolates were significantly more resistant to clarithromycin than the blood isolates (44.5% vs 7.69%; p= 0.027). Conclusion, significance and impact of study: In summary, an emergence of M. abscessus and unusually high moxifloxacin and linezolid resistance of MAC isolates were reported in this study. Additional information of this study benefits physicians for anti-mycobacterial drug selection for initial treatment of mycobacteremia while blood and BM culture is pending.

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.

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.