Rapid diagnosis of Isoniazid resistant Mycobacterium Tuberculosis, isolated from East Azerbaijanian patients by PCR-RFLP method

Abstract

Objectives: The efforts on control of tuberculosis have been encountered with serious problems by emergence of drug resistant Mycobacterium Tuberculosis (MT), and rapid diagnosis of resistance can play an essential role in control and prevention of the disease. Isoniazid is a first line drug in treatment of TB and development of resistance against this drug are increasingly reported. In this study, rapid diagnosis of Isoniazid resistant MT was investigated by PCR-RFLP method. Methods: A total of 25 Isoniazid - resistant and 25 Isoniazid - susceptible MT isolated at Tuberculosis and lung disease research center were screened for Isoniazid resistance. In the first step MIC of isolates to INH was determined by proportion method. After that, genomic DNA was extracted from all isolates and a fragment of Kat G gene was amplified by PCR using specific primers. Screening for mutation on Ser 315 and Arg 463 codons in the Kat G gene was carried out by digestion of PCR product with restriction enzyme Msp I. Results: Among 25 INH- resistant isolates, 14 isolates (56%) had mutation in Ser315 locus and 5 isolates (20%) showed mutation in Arg 463 locus, whereas 6 strains (24%) didn't show any mutation in these codons. Mutation in both 315 and 463 codons were not found in any isolates. In the susceptible isolates, no any mutation was detected in the studied codons. Conclusion: The results of this study indicated that PCR-RFLP method is able to detect resistance to INH in 76% cases and so, it can be used for rapid diagnosis of Isoniazid resistant MT isolates.