Detection of resistance to rifampicin and isoniazid inMycobacterium Tuberculosis isolates by High Resolution Melting(HRM) Real-Time PCR to set-up this method in Tuberculosis ReferenceLaboratory

Background and Aim : Drug resistance to isoniazid (INH) and rifampin (RIF) as two main drugsof the first line of tuberculosis (TB) treatment in Mycobacterium tuberculosis (Mtb) isolates isincreasing due to the excessive use of these drugs. Isoniazid resistance is mainly caused bymutations in the catalase-peroxidase (katG) gene and the mabA-inhA gene regulatory regions, andmutations in the rpoB gene are responsible for RIF resistance. Detection of Mtb drug-resistantisolates by conventional methods is time-consuming. Thus, developing rapid molecular techniquesseems vital for detecting drug resistance and preventing the spread of drug-resistant bacteria.Therefore, this study was carried out to evaluate antibiotic susceptibility to RIF and INH in Mtbisolates using High-Resolution Melting Analysis (HRMA).Methods : Twenty rifampin and isoniazid-resistant Mtb isolates were evaluated by standardproportional method from ۴۳۱ tuberculosis patients who were referred to the NortheastTuberculosis Reference Laboratory in Mashhad from ۲۰۱۶ to ۲۰۱۸. All drug-resistant Mtb clinicalisolates were screened for genetic mutations in rpoB, katG, and promoter region of the inhA geneusing PCR and real-time PCR amplification. Then, the presence of mutations in these genes wasinvestigated by the HRMA method.Results : Proportional resistance analysis showed that ۱۱ out of the ۲۰ studied isolates (۲.۵۵%)were resistant to both isoniazid and rifampin, ۷ (۱.۶۲%) of which were only isoniazid-resistant and۲ (۰.۴۶%) were only rifampin resistant. HRMA assay identified katG gene mutations and themabA-inhA promoter region in ۱۵ of ۱۸ isoniazid-resistant samples, and rpoB gene mutationswere successfully evaluated in ۱۱ out of ۱۳ RIF-resistant samples. The sensitivity and specificityof the HRMA method were ۸۳.۳% and ۹۱% for isoniazid and ۸۴.۶% and ۸۵.۱% for rifampin,respectively. In this study, ۱۰۰% of rifampin-resistant samples had mutations in the rifampinresistance determining region (RRDR). Also, ۸۸.۸% of isoniazid-resistant samples had mutationsin the katG gene and the mabA-inhA promoter region.Conclusion : The results of this study showed that HRM assay is a rapid, accurate, and costeffectivemethod possessing high sensitivity and specificity for determining antibiotic resistanceamong Mtb clinical isolates, screening of their associated mutations, and preventing the emergenceof possible MDR strains.

کلیدواژه ها:

Mycobacterium tuberculosis ، Drug resistance ، HRMA ، Isoniazid ، Rifampin