The GenoType MTBDRplus on the other hand, detects resistance to isoniazid and
rifampicin in culture samples and smear-positive specimens, based on the
detection of the most common mutations in the kalG, m/iA and [HA1] rpoB genes
respectively. Use of such assays in low prevalence countries is now routine.
However, implementation in high-burden settings has been questioned due to
issues of feasibility, technical capacity, specialized equipment and facilities
and cost. The MTBDRplus assay is currently being evaluated in a large-scale demonstration project in South Africa under programmatic conditions, by FIND and local partners.
Bacteriophage Based Assays M'20
Phage-based assays have been evaluated for diagnosis of
TB, as well as drug susceptibility testing. They use mycobacteriophages to infect live M.
tuberculosis and detect the bacilli using either phage-amplification method or detection of light. In the first method, the key principle is amplification of phages after their infection of M.
tuberculosis, followed by detection of progeny phages as plaques on a lawn of M. smegmatis (Figure 3). In the second method, the principle is detection of light (using luminometry or photographic films) produced by luciferase reporter phages (LRP), phages with fire-fly luciferase genes inserted within their genome, after their infection of live A/I.
tuberculosis.
Phage-based assays are available as commercial kits. For diagnosis, the FASTPlaque-
TB assay can be directly used on sputum specimens. A variant, the FASTPlaque-TB-MDRi kit, is designed to detect rifampicin resistance in culture isolates. An advanced version of this kit, FASTPIaque-TB-Response, has been developed for the detection of
drug resistance directly from sputum specimens. In general, phage assays have a turn around time of 48 hours, and require a laboratory infrastructure similar to that required for performing mycobacterial cultures.
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Molecular Beacon Assays
Molecular beacons are single-stranded oligonucleotides hybridization probes that form a stem-and-loop structure. The loop contains a probe sequence that is complementary to a target sequence of interest and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence. A fluorophore moiety is linked to the end of one arm, and a non-fluorescent quencher moiety is linked to the end of the other arm. When the target sequence is absent, the probe cannot fluoresce. When the targetsequence is present, the probe and the target hybridize, and the beacon undergoes a spontaneous conformational change that forces the fluorophore and the quencher to dissociate and move away from each other, causing fluorescence that can be detected in a real-time PCR assay.'
Although few studies have evaluated molecular beacons, available data suggest that these tests have high sensitivity (89-98%) and specificity (99-100%) for the detection of rifampicin resistance. Sensitivity for the detection of isoniazid resistance is lower because of the multiplicity of mutations that may lead to resistance to isoniazid. Molecular beacons are not available as commercial kits and are not FDA approved. They require sophisticated technology that limits their widespread use. Their use is mostly restricted to research and reference laboratories.