title: Rapid Detection of Rifampicin- and Isoniazid-ResistantMycobacterium tuberculosisUsing Real-Time PCR.
abstract: BACKGROUND
  Accurate and rapid detection of drug-resistantMycobacterium tuberculosisis fundamental for the successful treatment of tuberculosis (TB).
  
  
  OBJECTIVE
  The aim of this study was to determine the frequency of common mutations leading to isoniazid (INH) and rifampicin (RMP) resistance.
  
  
  METHODS
  In a cross-sectional study carried out in 2014, 90 patients withM. tuberculosisfrom five border provinces of Iran were selected. After a full clinical history and physical evaluation, real-time polymerase chain reaction (PCR) technique was performed for the detection of mutations in the patients'katGandrpoBgenes. The results were compared with results of a standard proportion method as well as a multiplex allele-specific PCR (MAS-PCR).
  
  
  RESULTS
  A total of 23 mutations were found in isolates among which, codonkatG315, rpoBP1 (511 - 519 sequence) andrpoBP2 (524-533 sequence) were responsible for seven, nine and seven cases, respectively. The mean (standard deviation (SD)) of melting temperature (Tm) inkatG315 codon,rpoBP1 and P2 sequences in susceptible and mutant isolates was as follows:katG85.4°C (0.18) and 87.54°C (0.62);rpoΒP1 84.6°C (0.61) and 82.9°C (0.38);rpoΒP2 83.4°C (0.18) and 85.3°C (0.19), respectively. In comparison to the standard proportion test, the sensitivity of real-time PCR in detecting INH- and RMP-resistant mutations was 75% and 83.3%, respectively. In comparison to the MAS-PCR test, 100% ofkatG315 mutations and 80% ofrpoBmutations were determined. Overall, 10% of the patients were diagnosed with a recurrence of TB. Age and previous history of TB treatment increased mutation odds inrpoBsequences (P = 0.046, P = 0.036, respectively).
  
  
  CONCLUSIONS
  Detection of drug resistance associated with mutations through real-time PCR by melting analysis technique showed a high differentiating power. This technique had high concordance with the standard proportion test and MAS-PCR results.
authors.affiliation: PhD of Molecular Epidemiology, Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors.forename: Leyla
authors.initials: L
authors.lastname: Sahebi
authors.affiliation: PhD of Molecular Epidemiology, Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors.forename: Khalil
authors.initials: K
authors.lastname: Ansarin
authors.affiliation: Department of Hematology, Faculty of Medicine, Tabriz Branch, Islamic Azad University, Tabriz, IR Iran.
authors.forename: Amir
authors.initials: A
authors.lastname: Monfaredan
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors.forename: Safar
authors.initials: S
authors.lastname: Farajnia
authors.affiliation: PhD Student of Epidemiology, Health Deputy, Kurdistan University of Medical Sciences, Sanandaj, IR Iran.
authors.forename: Seiran
authors.initials: S
authors.lastname: Nili
authors.affiliation: Medical Philosophy and History Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Khalili
keywords: Drug Resistance; Melt Curve Technique; Mycobacterium tuberculosis; Real-Time PCR