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

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.

Characterization of katG, inhA, rpoB and pncA in Mycobacterium tuberculosis isolates from MDR-TB risk patients in Thailand

“…These results were similar to those of previous studies [26,32,33]. The frequency of this mutation in this study was 79% (41/52) whereas other studies reported frequencies of 27% [24], 64% [29] and 60% [30]. The second most common mutation was His526Tyr (7.8%).…”

“…From a previous report from the USA, Thailand and Korea, the frequency of -15 (C>T) point mutations is 39%, 14% and 25%, respectively [22,26,27]. Therefore, eight strains from this study without the mutation in katG_Ser315Thr inhA_and -15 (C>T) may carry mutations located in other katG or inhA loci [28,29] or other associated genes such as ahpC and ndh gene [30]. However, the mutations katG_Ser315Thr and inhA_-15 (C>T), both of which showed high sensitivity (89%) and high specificity (100%), could be used as indicators for detecting INH resistance.…”

Determinación de la mutación S315T del gen katG en aislados resistentes a Isoniacida de Mycobacterium tuberculosis mediante PCR-RFLP

“…En conclusión, las pruebas moleculares no reemplazan los métodos convencionales, sin embargo, son un eficiente mé-todo de tamizaje cuando se combinan con datos clínicos y epidemiológicos, impactando significativamente en una diagnosis temprana de la resistencia, lo que contribuye a estrategias de control de la resistencia de TB en los países 44 . De igual manera, investigaciones dirigidas al estudio de la resistencia por secuenciamiento del genoma completo ayudarían en el futuro a dilucidar los mecanismos moleculares de resistencia a isoniacida u otros medicamentos en aislados de Mycobacterium tuberculosis circulantes en el Ecuador [45][46][47] .…”