A molecular platform for the diagnosis of multidrug-resistant and
pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism
mutations present in Colombian isolates of Mycobacterium
tuberculosis
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Authors: | Luz Maira Wintaco Martínez Gloria Puerto Castro Martha Inírida Guerrero |
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Institution: | Instituto Nacional de Salud, Dirección de Investigación en Salud Pública, Grupo de Micobacterias, Bogotá, Colombia |
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Abstract: | Developing a fast, inexpensive, and specific test that reflects the mutations present
in Mycobacterium tuberculosis isolates according to geographic
region is the main challenge for drug-resistant tuberculosis (TB) control. The
objective of this study was to develop a molecular platform to make a rapid diagnosis
of multidrug-resistant (MDR) and extensively drug-resistant TB based on single
nucleotide polymorphism (SNP) mutations present in therpoB,
katG, inhA,ahpC, and
gyrA genes from Colombian M. tuberculosis
isolates. The amplification and sequencing of each target gene was performed. Capture
oligonucleotides, which were tested before being used with isolates to assess the
performance, were designed for wild type and mutated codons, and the platform was
standardised based on the reverse hybridisation principle. This method was tested on
DNA samples extracted from clinical isolates from 160 Colombian patients who were
previously phenotypically and genotypically characterised as having susceptible or
MDR M. tuberculosis. For our method, the kappa index of the
sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625
forrpoB, katG,
inhA,ahpC, and gyrA,
respectively. Sensitivity and specificity were ranked between 90-100% compared with
those of phenotypic drug susceptibility testing. Our assay helps to pave the way for
implementation locally and for specifically adapted methods that can simultaneously
detect drug resistance mutations to first and second-line drugs within a few
hours. |
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Keywords: | tuberculosis multidrug-resistant tuberculosis extensively drug-resistant tuberculosis molecular diagnostic mutations SNP reverse hybridisation |
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