A rapid susceptibility test of Mycobacterium tuberculosis using hybridization protection assay

The conventional drug susceptibility tests of Mycobacterium tuberculosis are time-consuming and usually require 2 to 4 weeks to obtain final results. In this study, we attempted to develop a novel method for rapid detection of drug-resistant M. tuberculosis using hybridization protection assay (HPA). Clinically isolated strains of M. tuberculosis including seven isoniazid (INH) susceptible strains and six resistant strains were used. The organisms grown on Ogawa egg medium were inoculated into Middlebrook 7H9 broth and cultured at 37°C for one week. Then, the inoculum of each strain was prepared as a tenfold dilution of bacterial suspension at McFarland No. 0.5. The inocula were mixed with INH solutions to yield final concentrations of 0.1 and 1.0 μg/ml, and the resulting bacterial suspensions with or without test drug were cultured on the swaying plate at 37°C for up to 5 days. At intervals, 50 μl of each sample was withdrawn and subjected to the protocol of the HPA using acridinium-ester (AE) labeled DNA probe, and then the relative light unit (RLU) was read in a luminometer. In the case of the susceptible strains, a significant difference in the mode of increase in RLU ratio (% of RLU on day x/RLU on day 0) was observed between the INH-treated and drug-free control sets within three days of cultivation, while no such differences were seen in the case of the resistant strains. Since this method was not only rapid, safe and simple, but gives the results well correlated to those by the conventional methods, it is expected that the method will be used widely in many laboratories.