Detection of discordant isolates of drug resistant mycobacterium tuberculosis

M.S. University of Hawaii at Manoa 2013.Includes bibliographical references.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 site

Predicting extensively drug-resistant Mycobacterium tuberculosis phenotypes with genetic mutations.

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity