Detection of Rifampin-resistance in Mycobacterium tuberculosis

Control of tuberculosis is threatened by widespread emergence of drug resistance in Mycobacterium tuberculosis. Understanding the molecular basis of resistance might lead to development of novel rapid methods for diagnosing drug resistance. Rifampin is a key component among therapeutic regimens for the tuberculosis; therefore, patients who have drug resistance do not convalesce satisfactorily. The molecular mechanism of resistance to rifampin in M. tuberulosis has been elucidated. Substitutions of a limited number of highly conserved amino acids encoded by the rpoB gene are responsible for the ""single-step"" high-level resistance of M. tuberculosis to rifampin. Currently, two genotype-based protocols allow drug test from minimally grown cultured materials: (i)mutation identification by direct sequencing of PCR-amplified material. and (ii)mutation screening by PCR-SSCP. The purpose of this study is to evaluate the usefulness of the both methods. A sample of 75 isolates of M. tuberculosis was studied, and it inculded 36 rifampin-resistant strains and 39 rifampin-sensitive strains by conventional methods. Mutaions were identified in 36 rifampin-resistant isolates but in none of 39 sensitive isolates. All mutations were clustered within a region of 23 amino acids. Both methods allow detection of rifampin resistance in 2 to 3 days and will thus help in the early management of infection by M. tuberculosis.

The Detection of Rifampin-Resistant Mycobacterium tuberculosis by Polymerase Chain Reaction and Single - Strand Conformation Polymorphism Analysis

Control of tuberculosis is threatened by widesread emergence of drug resistant Mycobacterium tuberculosis. Rifampin is a key component among therapeutic regimens for the tuberculosis; therefore patients in whom resistance to this drug develop have a poor outlook, particularly if rifampin resistance is associated with resistance to other tuberculosis drugs. The purpose of this study was to detect the mutation in rpoB gene of rifampin resistant M. tuberculosis in Korea and to evaluate the usefulness of the method in clinical aspects. A sample of 80 M. tuberculosis was studied, and it included 40 rifampin resistance isolates and 40 rifampin sensitive isolates by conventional methods. The detection method involved the amplification by polymerase chain reaction (PCR) of the Rif' region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products (157 bp). Mutation were identified in 39 of 40 rifampin resistant isolates, and in 1 of 40 rifampin sensitive isolates.