Report of Expert Consultations on Rapid Molecular Testing to Detect Drug-Resistant Tuberculosis in the United States

Background on molecular drug-resistance (DR) tests

Recent advances in the understanding of the molecular basis or genetics of drug resistance have enabled development of rapid, DNA-based, molecular tests to detect mutations associated with drug resistance. If a mutation thought to be associated with resistance is detected in such a rapid test, the bacteria are considered to be drug resistant. If no mutation is detected, the bacteria are assumed to be drug susceptible. The key advantage of the molecular tests is that they can provide results within 24 to 48 hours, because they take advantage of the speed of nucleic acid amplification. These tests have been referred to in various publications as genetic or molecular drug-susceptibility tests, genetic or molecular detection of drug resistance tests, molecular tests to detect drug (or antimicrobial or antibiotic)-resistance mutations, or tests to detect molecular or genetic markers of drug resistance. In this report, the tests will be referred to simply as molecular drug-resistance (DR) tests.

Mutations associated with resistance to many of the anti-TB drugs have been described (8,9). For example, ~95% of rifampin-resistant M. tuberculosis strains carry mutations within the rifampin resistance-determining region (RRDR), an 81-bp region of the rpoB gene. Because of the strong association between the presence of mutations in the RRDR and rifampin resistance, several molecular genetic tests to detect RRDR mutations have been developed and evaluated for their ability to detect resistance in clinical isolates. Genetic or molecular tests for detecting mutations are, in general, variations of nucleic acid amplification (NAA) tests. Typically, the polymerase chain reaction (PCR) is used to amplify a target sequence followed by a second assay to determine if the sequence contains a mutation associated with resistance, such as DNA sequencing or hybridization assays.

1. For hybridization assays such as the INNO-LiPA® Rif.TB (Innogenetics) and GenoType® MTBDR(plus) (Hain LifeScience GmbH) line-probe assays, the region of a gene associated with resistance is PCR amplified, and the labeled PCR products hybridized to oligonucleotide probes immobilized on a nitrocellulose strip. Mutations are detected by lack of binding to wild-type probes or by binding to probes specific for commonly occurring mutations. Compared to culture-based DS tests, the MTBDR(plus) line probe assay displays a pooled sensitivity of 0.98 and a pooled specificity of 0.99 for detecting rifampin resistance in isolates or directly from clinical specimens (10–12).
2. Molecular beacons are hybridization probes which emit fluorescence only when hybridized to their target and which can discriminate between targets differing only by a single nucleotide. In the California Microbial Diseases Laboratory, molecular beacon assays were designed to detect mutations in the rpoB gene directly from clinical specimens and from cultures. The results of rpoB molecular beacons tests showed 96% to 97% agreement with culture-based results in a series of ~1,000 clinical specimens and cultures (E. Desmond, personal communication).
3. Validation studies conducted at the Wadsworth Center of an approach that combines PCR-amplification of the entire 81 bp RRDR with pyrosequencing revealed that the test displayed a sensitivity of <1 colony forming unit, 100% specificity, and 99% agreement in the 188 cultures and specimens tested (13–15; K. Musser, personal communication).

Molecular DR tests for other anti-TB drugs are much less developed than the tests for rifampin resistance. A meta-analysis of the performance of the Hain MTBDR(plus) assay for detecting isoniazid resistance revealed a pooled sensitivity of 0.85 (95%CI 0.77– 0.90) and a pooled specificity of 0.99 (95%CI 0.98–1.00) (11,12). Validation studies conducted in the California Microbial Diseases Laboratory using archived cultures revealed that the molecular beacon test displayed 82.7% sensitivity, 100% specificity, 100% positive predictive value, and 98.1% negative predictive value for detecting isoniazid resistance (16, E. Desmond, personal communication).

The critical contribution of molecular DR tests for TB treatment and control is earlier detection of resistance: they can reliably detect mutations associated with drug resistance in 1 to 2 days. Not only does this reduce the time to detect rifampin resistance, but for MDR TB patients this also reduces the time from TB diagnosis to the start of MDR TB treatment and from the first positive culture to culture conversion by six weeks and improves patient outcomes (2,3). The reduction of the estimated infectious period after diagnosis by six weeks should have a large impact on public health measures to stop the spread of MDR TB.