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Open Access Highly Accessed Research article

Pyrosequencing for rapid detection of Tuberculosis resistance in clinical isolates and Sputum samples from re-treatment Pulmonary Tuberculosis patients

Ruijuan Zheng1, Changtai Zhu3*, Qi Guo1, Lianhua Qin1, Jie Wang1, Junmei Lu1, Haiyan Cui1, Zhenling Cui1, Baoxue Ge1, Jinming Liu2* and Zhongyi Hu1*

Author Affiliations

1 Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, No. 507 Zhengmin Rd, Shanghai 200433, People's Republic of China

2 Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, No. 507 Zhengmin Rd, Shanghai 200433, People’s Republic of China

3 Department of Laboratory Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, No. 600 Yishan Rd, Shanghai 200233, China

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BMC Infectious Diseases 2014, 14:200  doi:10.1186/1471-2334-14-200

Published: 13 April 2014

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.