title: Performance Assessment of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of Extensively Drug-resistant Tuberculosis in Clinical Isolates of Mycobacterium tuberculosis.
abstract: BACKGROUND
  The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis.
  
  
  METHODS
  From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced.
  
  
  RESULTS
  Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001).
  
  
  CONCLUSIONS
  Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance consisting of MDR and XDR forms to anti-tuberculosis drugs using this method.
authors.affiliation: Infectious Diseases Research Center and Department of Microbiology, Arak University of Medical Sciences, Arak, Iran. mmatinam81@yahoo.com; arjomandzadegan@arakmu.ac.irn.
authors.forename: M
authors.initials: M
authors.lastname: Arjomandzadegan
authors.affiliation: Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran.
authors.forename: R
authors.initials: R
authors.lastname: Nazari
authors.affiliation: Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran.
authors.forename: M R
authors.initials: MR
authors.lastname: Zolfaghari
authors.affiliation: Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Taherahmadi
authors.affiliation: Department of Biology, Payame Noor University, Tehran, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Sadrnia
authors.affiliation: Research Institute for Epidemiology and Microbiology, Minsk, Belarus.
authors.forename: L P
authors.initials: LP
authors.lastname: Titov
authors.affiliation: Infectious Diseases Research Center (IDRC), Arak University of Medical Sciences, Arak, Iran.
authors.forename: A
authors.initials: A
authors.lastname: Ahmadi
authors.affiliation: Research Center of Molecular Medicine, Arak University of Medical Sciences, Arak, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Shojapoor