Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: Implications for rapid susceptibility testing

Past genotypic studies of Mycobacterium tuberculosis may have incorrectly e stimated the importance of specific drug resistance mutations due to a numb er of sampling biases including an overrepresentation of multidrug-resistan t (MDR) isolates. An accurate assessment of resistance mutations is crucial for understanding basic resistance mechanisms and designing genotypic drug resistance assays. We developed a rapid closed-tube PCR assay using fluoro genic reporter molecules called molecular beacons to detect reportedly comm on M. tuberculosis mutations associated with resistance to isoniazid and ri fampin. The assay was used in a comparative genotypic investigation of two different study populations to determine whether these known mutations acco unt for most cases of clinical drug resistance. We analyzed samples from a reference laboratory in Madrid, Spain, which receives an overrepresentation of MDR isolates similar to prior studies and from a community medical cent er in New York where almost all of the resistant isolates and an equal numb er of susceptible controls were available. The ability of the molecular bea con assay to predict resistance to isoniazid and rifampin was also assessed . The overall sensitivity and specificity of the assay for isoniazid resist ance were 85 and 100%, respectively, and those for rifampin resistance were 98 and 100%, respectively. Rifampin resistance mutations were detected equ ally well in isolates from both study populations; however, isoniazid resis tance mutations were detected in 94% of the isolates from Madrid but in onl y 76% of the isolates from New York (P = 0.02). In New York, isoniazid resi stance mutations were significantly more common in the MDR isolates (94%) t han in single-drug-resistant isolates (44%; P < 0.001). No association betw een previously described mutations in the kasA gene and isoniazid resistanc e was found. The first mutations that cause isoniazid resistance may often occur in sequences that have not been commonly associated with isoniazid re sistance, possibly in other as yet uncharacterized genes. The molecular bea con assay was simple, rapid, and highly sensitive for the detection of rifa mpin-resistant M. tuberculosis isolates and for the detection of isoniazid resistance in MDR isolates.