Anthwal Divya, Gupta Rakesh Kumar, Bhalla Manpreet, Bhatnagar Shinjini, Tyagi Jaya Sivaswami, Haldar Sagarika
Center for Bio-Design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, Mehrauli, New Delhi, India.
J Clin Microbiol. 2017 Jun;55(6):1755-1766. doi: 10.1128/JCM.02104-16. Epub 2017 Mar 22.
Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of , , and an promoter and used as positive controls in HRM. The assay was first validated in 25 MDR clinical isolates. The assay was evaluated on DNA isolated from 99 culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-, and INH- assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.
耐多药结核病是全球结核病控制工作面临的重大威胁。在全球范围内，2015年估计在34万例通报的结核病患者中，仅40%的患者被检测出患有耐多药结核病（MDR-TB）。本研究旨在评估高分辨率熔解曲线分析（HRM）在痰液样本中快速直接检测MDR-TB的效用。首先构建了一个参考质粒文库，其中包含在rpoB、katG和inhA启动子耐药决定区域中最常观察到的突变，并用作HRM中的阳性对照。该检测方法首先在25株MDR临床分离株中进行了验证。使用DNA测序作为金标准，对从99份培养阳性痰液样本（包括84份涂片阴性痰液样本）中提取的DNA进行了评估。通过使用HRM软件将熔解曲线模式与对照质粒的模式进行比较，区分突变体和野生型。HRM分别在11份和21份样本中检测到利福平（RIF）单耐药和异烟肼（INH）单耐药。通过测序，6份样本被分类为MDR-TB，其中1份被HRM漏检。HRM-RIF、INH-和INH-检测方法在涂片阴性样本中的敏感性分别为89%（95%置信区间[CI]，52,100%）、85%（95%CI，62,97%）和100%（95%CI，74,100%），而所有检测方法在涂片阳性样本中的敏感性均为100%。所有检测方法的特异性均为100%。测序与HRM之间的一致性为97%至100%（κ值，0.9至1）。通过测序在99份样本中的5份中观察到异质性耐药。总之，HRM检测是一种经济高效（400印度卢比/6美元）、快速且封闭管法，可直接检测痰液中的MDR-TB，尤其是直接涂片阴性病例。
