This study demonstrates that the molecular beacon assay effectively detects rifampin resistance in clinical M. tuberculosis
isolates from countries with a high incidence of tuberculosis. Other advantages of this assay include the single-well format, the ability to combine PCR and post-PCR analysis into a single step, and the virtual elimination of cross-contamination conferred by the ability to perform assays in closed tubes. The assay detected mutations in the rpoB
core region targeted by the molecular beacons every time a mutation was present. Conversely, the assay identified the core region as containing the wild-type sequence every time that mutations were absent from this region. The main limitation of the assay is that it is unable to detect rifampin resistance caused by mutations outside of the rpoB
core region. Indeed, the assay had a sensitivity of 100% for the isolates from Delhi, but it had a sensitivity of 86% for isolates from Mexico, where it mistakenly identified 9 out of 64 rifampin-resistant isolates as being rifampin susceptible because these isolates did not have mutations in the rpoB
core region. Screening assays should be highly specific, and the molecular beacon assay had an overall specificity of 99%. The assay identified 37 out of 37 rifampin-susceptible Indian isolates as being rifampin susceptible, and it identified 125 of 126 rifampin-susceptible Mexican isolates as being rifampin susceptible. Furthermore, the sole rifampin-susceptible isolate that was misidentified as being rifampin resistant contained an S531W mutation that has previously been shown to be associated with rifampin resistance (11
). Thus, it is likely that this susceptible isolate was, in fact, rifampin resistant. Unfortunately, this sample was not viable for repeat susceptibility testing. A potential problem of the assay is that it would be expected to detect silent rpoB
mutations when they were present and would falsely identify such mutants as rifampin resistant. However, silent mutations are exceedingly rare in M. tuberculosis
), thus, this problem does not have an important effect on specificity. In fact, it is worth noting that in the present study, the assay correctly identified five rifampin-susceptible isolates from India that had been initially classified as rifampin resistant by conventional susceptibility testing (but later confirmed to be susceptible by repeat testing). This observation suggests that the specificity of the molecular beacon assay may sometimes be higher than that of conventional susceptibility testing.
We found that four isolates from Delhi gave negative signals with probe A. Three of these isolates had mutations in codon 511, and the fourth had a novel deletion spanning codons 511 to 514. Mutations at codon 511 have been found by other workers in India (8
). No rifampin-resistant isolate from Mexico contained a mutation in this region. This difference could reflect regional strain variations or differences in host factors. None of the 243 isolates showed a negative fluorescence signal with probe C, which targets rpoB
codons 518 to 522. Earlier studies from India have reported mutations in codon 518, but only when they were accompanied by mutations in codon 531 (8
). Other studies have reported mutations at codons 518, 521, and 522 at frequencies of only 0.8, 1.5, and 3%, respectively (18
). Thus, it is possible that probe C could be omitted from future assays without a major effect on assay sensitivity. We also compared the results from the molecular beacon assays to results obtained with SSCP PCR in 14 isolates from Mexico City. Our results show that the molecular beacon assays were much more sensitive in detecting rifampin resistance in this group of isolates.
In summary, the molecular beacon assay was as effective at detecting mutations associated with rifampin resistance in M. tuberculosis
isolates from northern India and Mexico as has previously been reported for isolates from the United States and Spain (15
). The assay also identified rifampin-susceptible isolates that had previously been misidentified as resistant, further supporting the utility of a genetic approach to susceptibility testing. The assay was also more effective than SSCP PCR at detecting rifampin-resistant isolates. The assay is not dependent on probes hybridizing to specific mutant codons; hence, new and unknown mutations arising in a population, such as those identified in this study, can be easily detected with the same set of probes. With real-time instruments becoming more affordable, we anticipate that the assay can become economically feasible for developing countries in the near future. Ultimately, this assay will enable more rapid diagnosis, earlier treatment, and prompt implementation of infection control procedures to reduce the morbidity, mortality, and the spread of drug-resistant tuberculosis.