Banu Sayera, Pholwat Suporn, Foongladda Suporn, Chinli Rattapha, Boonlert Duangjai, Ferdous Sara Sabrina, Rahman S M Mazidur, Rahman Arfatur, Ahmed Shahriar, Houpt Eric R
Mycobacteriology Laboratory, Infectious Diseases Division, International Center for Diarrhoeal Diseases Research, Dhaka, Bangladesh.
Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, United States of America.
PLoS One. 2017 May 4;12(5):e0177167. doi: 10.1371/journal.pone.0177167. eCollection 2017.
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.
基于培养的结核分枝杆菌（TB）表型药物敏感性试验（DST）耗时较长，因此快速基因型方法的应用越来越广泛。我们之前开发并在结核分枝杆菌分离株上评估了一种快速基因型TaqMan阵列卡（TAC），该阵列卡使用数十对引物、探针和高分辨率熔解分析来检测多个耐药相关基因中的突变，与桑格测序相比，准确率>96%。在本研究中，我们检测了TAC在痰液上的性能，比较了71对痰液和结核分枝杆菌分离株的结果，其中62株为耐多药结核（MDR-TB）。我们还对TAC进行了改进，加入了野生型探针，并扩大了对rpoB和gyrA突变的覆盖范围。TAC在涂片阳性痰液中检测inhA、katG、rpoB、eis、gyrA、rplC和pncA内野生型或突变的成功率为89%，在涂片阴性痰液中的成功率为33%。与配对分离株的TAC结果相比，这些检测的总体准确率为95%±7（平均敏感性98%±3；特异性92%±14）。痰液TAC结果与异烟肼、利福平、氧氟沙星/莫西沙星和吡嗪酰胺的表型DST的准确率为85%±12。这与分离株TAC结果相似（准确率88%±13），因此不准确主要反映了基因型与表型的内在不一致。TAC是一种针对主要一线和二线抗结核药物的快速、模块化、全面且准确的结核DST，可用于对GeneXpert耐药涂片阳性痰液进行补充检测。
