UNIVERSITY OF SANTO TOMAS GRADUATE SCHOOL PAGE Genetic Characterization of Rifampin-Resistant Mycobacterium tuberculosis Isolates from the Research Institute for Tropical Medicine

Rifampin plays a key component of the first line antitubercular drugs. However, resistance to rifampin in tuberculosis is gaining a foothold in the Philippines. Studies show that resistance to rifampin involves mutation in the rpoB gene which encodes the β subunit of the RNA polymerase. In this pilot study, DNA sequences of a 305-bp fragment of the rpoB gene were determined from 40 clinical isolates of Mycobacterium tuberculosis where 22 rifampin-resistant and 18 rifampinsusceptible isolates were obtained. All of the rifampinsusceptible isolates showed no mutations in the rpoB gene fragment, hi contrast, 20 out of 22 (90%) rifampinresistant isolates showed missense mutations. Two isolates showed 2step point mutations each and 1 isolate had a point mutation and a deletion. The rest showed one-step point mutations. The missense mutations reported here were similar to those reported by Telenti et al (Lancet 1993; 341:647-50), and Williams et al (Antimicrob. Agents Chemother. 1994; 38:2380-86). However, the discovery of an isolate with a point mutation and a deletion has not been reported elsewhere and is, therefore, considered novel. Because the study was limited to the analysis of mutations in the Cluster I region, it is probable that the 2 rifampin resistant isolates without mutations could have substitutions in other clusters of the rpoB gene. With the number of rifampin-resistant cases on the rise, it is important to monitor the development and spread of rifampinresistant isolates to enable clinicians to prescribe the appropriate antitubercular drug. The discovery of the mutant rpoB gene has led to the development of a molecular approach for the detection of rifampin resistant isolates from clinical specimens. This study utilizes the Polymerase Chain ReactionHeteroduplex Formation (PCR-HDF), a molecular approach, to demonstrate for the first time, the utility of this tool for the rapid and accurate detection of rifampin-resistant M. tuberculosis in the Philippines.