Rezaei Faranak, Haeili Mehri, Fooladi Abbasali Imani, Feizabadi Mohammad Mehdi
Department of Microbiology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran.
Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
Maedica (Bucur). 2017 Dec;12(4):246-257.
Development of molecular techniques for rapid detection of drug resistant tuberculosis allows for the prompt initiation of appropriate anti-TB treatment. We aimed to assess high-resolution melting (HRM) analysis for the detection of rpsL, rrs and embB mutations to identify streptomycin and ethambutol resistance in Mycobacterium tuberculosis.
A total of 76 clinical isolates of M. tuberculosis including 25 SM-R, 21 EB-R and 30 drug susceptible - determined by the proportion method of drug susceptibility testing (DST) - were analyzed by HRM analysis, and the results were confirmed using DNA sequencing.
The sensitivity and specificity of the HRMA compared to phenotypic DST were 88% and 100.0%, respectively for the detection of streptomycin resistance (SM-R), and 90.4% and 96.6%, respectively for ethambutol resistance (EB-R). Three SM-R and two EB-R isolates had no mutations in the studied regions of rpsL, rrs and embB genes determined by DNA sequencing and therefore were not identified as resistant by HRM assay. Interestingly, one phenotypic EM-S isolate was found by sequencing to have a mutation at codon 423 (Met->Ilu) of embB gene and was clustered as resistant by HRM as well.
The sensitivity and specificity of HRM curve assay was consistent with DNA sequencing, which is the gold standard method for genotypic DST. This assay can be utilized as a screening method for detection of drug-resistant tuberculosis, offering the advantages of a high throughput, single step, cost effectiveness, and rapid work flow method.
开发用于快速检测耐药结核病的分子技术，以便能迅速开始适当的抗结核治疗。我们旨在评估高分辨率熔解（HRM）分析用于检测rpsL、rrs和embB基因突变，以鉴定结核分枝杆菌中的链霉素和乙胺丁醇耐药性。
通过HRM分析对总共76株结核分枝杆菌临床分离株进行分析，其中包括25株链霉素耐药（SM-R）、21株乙胺丁醇耐药（EB-R）以及30株药物敏感株（通过药物敏感性试验（DST）比例法确定），并使用DNA测序对结果进行确认。
与表型DST相比，HRMA检测链霉素耐药（SM-R）的敏感性和特异性分别为88%和100.0%，检测乙胺丁醇耐药（EB-R）的敏感性和特异性分别为90.4%和96.6%。通过DNA测序确定，有3株SM-R和2株EB-R分离株在rpsL、rrs和embB基因的研究区域没有突变，因此HRM检测未将其鉴定为耐药。有趣的是，通过测序发现1株表型乙胺丁醇敏感（EM-S）分离株在embB基因的423密码子处发生突变（Met->Ilu），并且HRM也将其聚类为耐药。
HRM曲线检测的敏感性和特异性与DNA测序一致，而DNA测序是基因型DST的金标准方法。该检测可作为检测耐药结核病的筛查方法，具有高通量、单步、成本效益高和工作流程快速的优点。
