Zheng Ruijuan, Zhu Changtai, Guo Qi, Qin Lianhua, Wang Jie, Lu Junmei, Cui Haiyan, Cui Zhenling, Ge Baoxue, Liu Jinming, Hu Zhongyi
Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, No, 507 Zhengmin Rd, Shanghai 200433, People's Republic of China.
BMC Infect Dis. 2014 Apr 13;14:200. doi: 10.1186/1471-2334-14-200.
Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. Early diagnosis of MDR-TB patients is essential for minimizing the risk of Mycobacterium tuberculosis (MTB) transmission. The conventional drug susceptibility testing (DST) methods for detection of drug-resistant M. tuberculosis are laborious and cannot provide the rapid detection for clinical practice.
The aim of this study was to develop a pyrosequencing approach for the simultaneous detection of resistance to rifampin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and amikacin (AMK) in M. tuberculosis clinical isolates and sputum samples from re-treatment pulmonary tuberculosis (PTB) patients. We identified the optimum conditions for detection mutation of rpoB, katG, rpsl, embB, gyrA and rrs gene by pyrosequencing. Then this approach was applied to detect 205 clinical isolates and 24 sputum samples of M. tuberculosis from re-treatment PTB patients.
The mutations of rpoB and gyrA gene were detected by pyrosequencig with the SQA mode, and the mutations of katG, rpsl, embB, gyrA and rrs gene were detected by pyrosequencing with SNP mode. Compared with the Bactec MGIT 960 mycobacterial detection system, the accuracy of pyrosequencing for the detection of RIF, INH, EMB, SM, AMK and OFL resistance in clinical isolates was 95.0%, 79.2%, 70.3%, 84.5%, 96.5% and 91.1%, respectively. In sputum samples the accuracy was 83.3%, 83.3%, 60.9%, 83.3%, 87.5% and 91.7%, respectively.
The newly established pyrosequencing assay is a rapid and high-throughput method for the detection of resistance to RIF, INH, SM, EMB, OFL and AMK in M. tuberculosis. Pyrosequencing can be used as a practical molecular diagnostic tool for screening and predicting the resistance of re-treatment pulmonary tuberculosis patients.
耐多药结核病（MDR-TB）是一个主要的公共卫生问题。早期诊断MDR-TB患者对于将结核分枝杆菌（MTB）传播风险降至最低至关重要。用于检测耐药性结核分枝杆菌的传统药物敏感性试验（DST）方法费力，且无法为临床实践提供快速检测。
本研究的目的是开发一种焦磷酸测序方法，用于同时检测结核分枝杆菌临床分离株以及复治肺结核（PTB）患者痰液样本中对利福平（RIF）、异烟肼（INH）、乙胺丁醇（EMB）、链霉素（SM）、氧氟沙星（OFL）和阿米卡星（AMK）的耐药性。我们确定了通过焦磷酸测序检测rpoB、katG、rpsl、embB、gyrA和rrs基因变异的最佳条件。然后将该方法应用于检测205株临床分离株以及复治PTB患者的24份结核分枝杆菌痰液样本。
通过焦磷酸测序以SQA模式检测到rpoB和gyrA基因变异，以SNP模式检测到katG、rpsl、embB、gyrA和rrs基因变异。与Bactec MGIT 960分枝杆菌检测系统相比，焦磷酸测序检测临床分离株中RIF、INH、EMB、SM、AMK和OFL耐药性的准确率分别为95.0%、79.2%、70.3%、84.5%、96.5%和91.1%。在痰液样本中，准确率分别为83.3%、83.3%、60.9%、83.3%、87.5%和91.7%。
新建立的焦磷酸测序检测法是一种快速、高通量的方法，用于检测结核分枝杆菌对RIF、INH、SM、EMB、OFL和AMK的耐药性。焦磷酸测序可作为一种实用的分子诊断工具，用于筛查和预测复治肺结核患者的耐药性。
