Molecular detection of rifampin, isoniazid, and ofloxacin resistance in Iranian isolates of Mycobacterium tuberculosis by high-resolution melting analysis

Mehrandokht Sirous,1,2 Azar Dokht Khosravi,1,2 Mohammad Reza Tabandeh,3 Shokrollah Salmanzadeh,1 Nazanin Ahmadkhosravi,4 Sirus Amini5 1Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 2Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 3Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran; 4Khuzestan Tuberculosis Regional Reference Laboratory, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; 5Tehran Tuberculosis Regional Reference Laboratory, Tehran University of Medical Sciences, Tehran, Iran Background: The emergence of drug resistance among Mycobacterium tuberculosis (MTB) strains is a serious health concern worldwide. The development of rapid molecular diagnostic methods in recent years has a significant impact on the early detection of resistance to major anti-TB drugs in MTB isolates, which helps in employing appropriate treatment regimen and prevents the spread of drug-resistant strains. This study was designed to evaluate the efficacy of real-time PCR and high-resolution melting (HRM) curve analysis for the determination of resistance to rifampin (RIF), isoniazid (INH), and ofloxacin (OFX) in MTB isolates and to investigate their resistance-related mutations. Methods: HRM analysis was performed to screen 52 (32 drug-resistant and 20 fully susceptible) MTB clinical isolates for mutations in rpoB, katG, mab-inhA, and gyrA genes. The HRM results were then confirmed by DNA sequencing. Results: In total, 32 phenotypically resistant isolates, comprising 18 RIF-, 16 INH-, and five OFX- resistant strains, were investigated. HRM analysis successfully identified 15 out of 18 mutations in rpoB, 14 out of 16 mutations in katG and mab-inhA , and four out of five mutations in gyrA conferring resistance to RIF, INH, and OFX, respectively. The obtained sensitivity and specificity, respectively, for HRM in comparison with phenotypic susceptibility testing were found to be 83.3% and 100% for RIF, 87.5% and 100% for INH, and 80% and 100% for OFX. In five resistant strains (12.8%), no mutation was detected by using HRM and DNA sequencing. Conclusion: HRM assay is a rapid, accurate, and cost-effective method possessing high sensitivity and specificity for the determination of antibiotic resistance among MTB clinical isolates and screening of their associated mutations. This method can generate results in a shorter period of time than taken by the phenotypic susceptibility testing and also allows for timely treatment and prevention of the emergence of possible MDR strains. Keywords: Mycobacterium tuberculosis, drug resistance, high-resolution melting curve analysis, isoniazid, rifampin, ofloxaci