title: Rapid detection of isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis clinical isolates using high-resolution melting analysis.
abstract: A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG, mabA-inhA promoter, rpoB, and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhA promoter, rpoB, and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katG and/or mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.
authors.affiliation: Centre for Infectious Diseases and Microbiology-Public Health, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia.
authors.forename: Xiaoyou
authors.initials: X
authors.lastname: Chen
authors.forename: Fanrong
authors.initials: F
authors.lastname: Kong
authors.forename: Qinning
authors.initials: Q
authors.lastname: Wang
authors.forename: Chuanyou
authors.initials: C
authors.lastname: Li
authors.forename: Jianyuan
authors.initials: J
authors.lastname: Zhang
authors.forename: Gwendolyn L
authors.initials: GL
authors.lastname: Gilbert
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D015242:Ofloxacin / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D015242:Ofloxacin; D007538:Isoniazid; D012293:Rifampin