Pyrosequencing for rapid detection of tuberculosis resistance in clinical isolates and sputum samples from re-treatment pulmonary tuberculosis patients.

Ruijuan Zheng,Haiyan Cui,Junmei Lu,Qi Guo,Zhenling Cui,Lianhua Qin,Jinming Liu,Baoxue Ge,Jie Wang,Zhongyi Hu,Changtai Zhu

doi:10.1186/1471-2334-14-200

Abstract

BackgroundMultidrug-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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.

Highlights

Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem
There were four kinds of codons including 12 types of mutations in the regions of the quinolone resistance-determining regions (QRDR) of the gyrA gene, and 62% (88/142) of the OFL-resistant clinical samples had a mutation at codon 94
The 77.3% (34/ 44) of the AMK-resistant clinical samples showed a mutation in position 1401 in the rrs gene, and no mutation in this position was found in AMK-susceptible clinical samples

Summary

Introduction

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. Due to the severity of MDRTB, the WHO convened a international meeting focused on MDR-TB in April 2009 in Beijing, China, urging member states to take detailed and effective action in diagnosing and treating M/XDR-TB [4]. Another survey of WHO in 2010 showed that the estimated MDR-TB among the notified re-treatment cases (n = 7,795) was 2,200 (28%) [5]. In order to control the MDR-TB,it was important for retreatment PTB patients to develop a rapid, reliable and high-throughput drug susceptibility testing to obtain firstand second-line anti-tuberculosis drugs susceptibility result simultaneously, as it was essential to decrease patient morbidity and mortality as well as treatment-associated costs

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