title: Pyrosequencing for rapid detection of tuberculosis resistance in clinical isolates and sputum samples from re-treatment pulmonary tuberculosis patients.
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.
authors.forename: Ruijuan
authors.initials: R
authors.lastname: Zheng
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, No, 507 Zhengmin Rd, Shanghai 200433, People's Republic of China. zct101@163.com.
authors.forename: Changtai
authors.initials: C
authors.lastname: Zhu
authors.forename: Qi
authors.initials: Q
authors.lastname: Guo
authors.forename: Lianhua
authors.initials: L
authors.lastname: Qin
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Junmei
authors.initials: J
authors.lastname: Lu
authors.forename: Haiyan
authors.initials: H
authors.lastname: Cui
authors.forename: Zhenling
authors.initials: Z
authors.lastname: Cui
authors.forename: Baoxue
authors.initials: B
authors.lastname: Ge
authors.forename: Jinming
authors.initials: J
authors.lastname: Liu
authors.forename: Zhongyi
authors.initials: Z
authors.lastname: Hu
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D058889:Molecular Typing / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods; D013183:Sputum / Q000382:microbiology*; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial
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