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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
EISSN: 1678-8060
Vol. 111, No. 2, 2016, pp. 93-100
Bioline Code: oc16012
Full paper language: English
Document type: Research Article
Document available free of charge

Memórias do Instituto Oswaldo Cruz, Vol. 111, No. 2, 2016, pp. 93-100

 en 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 check for this species in other resources
Martínez, Luz Maira Wintaco; Castro, Gloria Puerto & Guerrero, Martha Inírida

Abstract

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis check for this species in other resources 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 the rpoB, 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 for rpoB, 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.

Keywords
tuberculosis; multidrug-resistant tuberculosis; extensively drug-resistant tuberculosis; molecular diagnostic; mutations; SNP; reverse hybridisation

 
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