Detection of discordant isolates of drug resistant mycobacterium tuberculosis

Abstract

Early diagnosis of mycobacterium tuberculosis is critical for proper treatment. With a regimen of rifampin and isoniazid, a patient with active tuberculosis can become non-infectious within 2 weeks (5). However, as resistance to these two drugs develops, second line antibiotic treatment must be used which lasts an extra 6 to 18 months and patients remain infectious for a longer period of time, which can further propagate the spread of mycobacterium tuberculosis (6). Testing for antibiotic resistance in M. tuberculosis is a lengthy process due to its long generation time. The WHO standard guideline for the drug susceptibility test of M. tuberculosis is inoculating the bacteria through dilution on Löwenstein Jensen agar with a set concentration of test antibiotic and incubating it at 37 degrees Celsius. Colonies are then counted on the 28th day and a proportion is calculated by comparing the colony count of the test medium to a control. If the proportion exceeds a critical proportion or if no colonies appear in the lowest drug concentration medium with the highest inoculum, the isolate is determined to be resistant or sensitive, respectively. However, if neither criteria are matched, the incubation must continue until the 40th day where the final results are read through the same process (7). The development of drug resistance strains in mycobacterium tuberculosis ultimately relies on exposure to the resistant drug because the presence of drug resistance mutations does not confer any selective advantage over strains lacking those mutations until exposure occurs (15). Within each geographical region, strains of M. tuberculosis may be under unique and various selective pressures to develop specific types of polymorphisms. Therefore, it can be hypothesized that there are genetic differences in the drug resistant strains of M. tuberculosis found in different regions and that the proportion of polymorphisms in genes associated with drug resistance will be different between geographic locations. Study Objectives 1. To sequence the hot spot regions of nine genes (rpoB, inhA promoter, katG, ahpC promoter, gyrA, gyrB, rrs, eis promoter, and tlyA). 2. To identify discordant isolates through the comparison of drug susceptibility data and sequence data. 3. To further define the resistance patterns found in M. tuberculosis within and between different geographical regions represented by the widely dispersed study sites