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

Developing a fast, inexpensive, and specific test that reflects the mutations presentin Mycobacterium tuberculosis isolates according to geographicregion is the main challenge for drug-resistant tuberculosis (TB) control. Theobjective of this study was to develop a molecular platform to make a rapid diagnosisof multidrug-resistant (MDR) and extensively drug-resistant TB based on singlenucleotide polymorphism (SNP) mutations present in therpoB,katG, inhA,ahpC, andgyrA genes from Colombian M. tuberculosisisolates. The amplification and sequencing of each target gene was performed. Captureoligonucleotides, which were tested before being used with isolates to assess theperformance, were designed for wild type and mutated codons, and the platform wasstandardised based on the reverse hybridisation principle. This method was tested onDNA samples extracted from clinical isolates from 160 Colombian patients who werepreviously phenotypically and genotypically characterised as having susceptible orMDR M. tuberculosis. For our method, the kappa index of thesequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625forrpoB, katG,inhA,ahpC, and gyrA,respectively. Sensitivity and specificity were ranked between 90-100% compared withthose of phenotypic drug susceptibility testing. Our assay helps to pave the way forimplementation locally and for specifically adapted methods that can simultaneouslydetect drug resistance mutations to first and second-line drugs within a fewhours.