Abstract
Evaluation of resistance to antituberculosis drugs is routinely performed with genotypic or phenotypic methods; however, discordance can be seen between these different methodologies. Our objective was to identify mutations that could explain discordant results in the evaluation of susceptibility to rifampicin and isoniazid between molecular and phenotypic methods, using whole genome sequencing (WGS). Peruvian strains showing sensitive results in the GenoType MTBDRplus v2.0 test and resistant results in the proportions in the agar-plaque test for isoniazid or rifampin were selected. Discordance was confirmed by repeating both tests, and WGS was performed, using the Next Generation Sequencing methodology. Obtained sequences were aligned “through reference” (genomic mapping) using the program BWA with the algorithm “mem”, using as a reference the genome of the M. tuberculosis H37Rv strain. Discordance was confirmed in 14 strains for rifampicin and 21 for isoniazid, with 1 strain in common for both antibiotics, for a total of 34 unique strains. The most frequent mutation in the rpoB gene in the discordant strains for rifampicin was V170F. The most frequent mutations in the discordant strains for isoniazid were katG R463L, kasA G269S, and Rv1592c I322V. Several other mutations are reported. This is the first study in Latin America addressing mutations present in strains with discordant results between genotypic and phenotypic methods to rifampicin and isoniazid. These mutations could be considered as future potential targets for genotypic tests for evaluation of susceptibility to these drugs.
Highlights
Peru is one of the 30 countries in the world with the highest burden of multidrug-resistant tuberculosis (MDR-TB). e public health system provides free diagnosis and drugsusceptibility testing with phenotypic [1] and molecular methods (Genotype, Hain LifesciencesTM (Genotype )) [2] for first- and second-line drugs
E Genotype method is based on the identification of mutations of the mutations of the rpoB gene in the Rifampicin-resistance determining region (RRDR), an 81 basepair segment of the gene
Whole Genome Sequencing (WGS) is a technology that can identify new sequences that could explain certain traits related to International Journal of Microbiology resistance, and could currently be used to investigate the genome of Mycobacterium tuberculosis strains showing this kind of discordant results [7]
Summary
Peru is one of the 30 countries in the world with the highest burden of multidrug-resistant tuberculosis (MDR-TB). e public health system provides free diagnosis and drugsusceptibility testing with phenotypic (proportions in 7H10) [1] and molecular methods Molecular diagnosis of drug resistance has been shown to significantly reduce the time to diagnosis, most of all to isoniazid and rifampicin, the pillars of antituberculosis regimens, and contribute to improve the clinical outcomes [3] the reference standard continue to be phenotypic methods, such as MGIT 960 and proportions in agar plaque, both currently used at the National Reference Laboratory of Mycobacteria of Peru. Ere are reports postulating novel mutations to explain discrepancies between molecular and phenotypic methods [5, 6], as these could explain the discordant results. E World Health Organization has issued recommendations on their interpretation [8] Knowledge on this topic continues to evolve, and it is very important to generate additional information about the presence of these novel mutations. Our aim was to identify mutations that could explain discordant results in the evaluation of susceptibility to rifampicin and isoniazid between molecular and phenotypic methods, using whole genome sequencing. Our aim was to identify mutations that could explain discordant results in the evaluation of susceptibility to rifampicin and isoniazid between molecular and phenotypic methods, using whole genome sequencing. is information could contribute in the future to develop more comprehensive diagnostic devices for evaluation of drug resistance in our country
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