Diagnostics For Rapid Detection Of Drug Resistance
Line Probe Assays
Line probe assays are a family of novel DMA strip tests that use PCR and reverse hybridization methods for the rapid detection of drug resistance. There are currently two commercially available techniques: the INNO-LiPA Rif. -
TB Assay
(Innogenetics, Ghent, Belgium) and the GenoType MTBDRplus assay (Main
Lifescience, Nehren, Germany).
The INNO-LiPA Rif. -
TB (LiPA) is a commercial line probe assay designed to rapidly detect rifampicin resistance, a marker of MDR-TB. LiPA identifies the M. tuberculosis complex and simultaneously detects genetic mutations in the rpoB gene region related to rifampicin-resistance. The LiPA kit contains 10 oligonucleotide probes (one specific for the M. tuberculosis
complex, five overlapping wild-type S probes, and four R probes for detecting specific mutations of resistant genotypes) immobilized on nitrocellulose paper strips. LiPA is performed by extracting DNA from cultures or directly from clinical samples and amplifying the rifampicin -resistance-determining region of the rpoB gene using PCR. Biotinylated PCR products are then hybridized with the immobilized probes, and results are determined by colorimetric development.
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The LiPA assay is highly sensitive and specific for detecting rifampicin-resistant M. tuberculosis
in culture and, to a slightly lesser degree, in clinical specimens. The majority
of studies had sensitivity estimates of 95% or greater, and nearly all were 100%
specific.
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.
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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 target
sequence 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.
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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.