title: Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoniazid and rifampicin using fluorogenic probes.
abstract: BACKGROUND
  This study evaluates a method based on real-time PCR for direct detection in clinical samples of the common mutations responsible for isoniazid and rifampicin resistance of Mycobacterium tuberculosis.
  
  
  METHODS
  Six pairs of fluorogenic 5' exonuclease probes (Taqman), mutated and wild-type, were designed for six targets: codon 315 of katG, substitution C209T in the regulatory region of inhA, and codons 513, 516, 526 and 531 of rpoB.
  
  
  RESULTS
  A total of 98 clinical samples harbouring resistant bacilli from 55 patients and 126 samples harbouring susceptible bacilli from 126 patients were processed. The isolates from samples were tested for drug susceptibility with the radiometric method and sequenced for the same genetic targets. Among the samples, 93 harboured isoniazid-resistant bacilli. According to the sequencing results, 30 had mutations in katG, 30 in inhA and 33 (35.4%) had no mutations in these targets. All 27 clinical specimens harbouring rifampicin-resistant bacilli showed mutations in rpoB. The detection threshold of this method in detecting target genes in serial dilutions of artificial samples was 1.5 x 10(3) cfu/mL. In clinical samples, the sensitivity ranged from 30.4 to 35.3% for smear-negative samples and from 95.1 to 99.2% for smear-positive samples, with a specificity of 100%. In this study, the overall sensitivity in detecting patients having the target mutations was 74.3%.
  
  
  CONCLUSIONS
  The main advantage of the described method is the possibility of detecting rifampicin and isoniazid resistance within 48-72 h after sample collection, with a sensitivity of nearly 100% in smear-positive samples if the chosen target is responsible for the resistance.
authors.affiliation: Departament de Microbiologia, Centre de Diagnòstic Biomèdic, Hospital Clinic-IDIBAPS.
authors.forename: Mateu
authors.initials: M
authors.lastname: Espasa
authors.forename: Julián
authors.initials: J
authors.lastname: González-Martín
authors.forename: Fernando
authors.initials: F
authors.lastname: Alcaide
authors.forename: Lina Marcela
authors.initials: LM
authors.lastname: Aragón
authors.forename: Joan
authors.initials: J
authors.lastname: Lonca
authors.forename: Joxe Mari
authors.initials: JM
authors.lastname: Manterola
authors.forename: Margarita
authors.initials: M
authors.lastname: Salvadó
authors.forename: Griselda
authors.initials: G
authors.lastname: Tudó
authors.forename: Pilar
authors.initials: P
authors.lastname: Orús
authors.forename: Pere
authors.initials: P
authors.lastname: Coll
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D005456:Fluorescent Dyes; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity
chemical_list: D000995:Antitubercular Agents; D005456:Fluorescent Dyes; D007538:Isoniazid; D012293:Rifampin