Detection of resistance to isoniazid, rifampin, and streptomycin in clinical isolates of Mycobacterium tuberculosis by molecular methods

Abstract

Molecular techniques can detect drug resistance in Mycobacterium tuberculosis, but whether these methods are practical for clinical laboratory use and the management of tuberculosis is unclear. We evaluated several available molecular methods (restriction fragment length polymorphism [RFLP], heteroduplex, and direct DNA sequence analyses) for detecting resistance to isoniazid, rifampin, and streptomycin and compared these methods with conventional methods for susceptibility testing. RFLP analysis detected the mutation at position S315T in katG in 12 (44.4%) of 27 isoniazid-resistant strains. Heteroduplex analysis of rpoB, detected 16 (76.2%) of 21 rifampin-resistant strains, whereas direct DNA sequencing detected all rifampin-resistant strains. RFLP analysis of the rpsL gene detected only nine (28.1%) of 32 streptomycin-resistant strains, while direct DNA sequencing detected nearly 68% of streptomycin-resistant strains. The specificity of all of the above-mentioned methods was excellent. RFLP analysis for katG and direct DNA sequencing of rpoB and rpsL may be practical methods for routine use in clinical microbiology laboratories or molecular pathology laboratories with good molecular capabilities and autosequencers. Despite the less than optimal sensitivity for some assays, resistance can be detected rapidly. However, molecular methods are not yet capable of replacing more traditional methods of susceptibility testing for M. tuberculosis.