Projects_laboratory_gpm

Prevalence, drug resistance, and genotypic diversity of Beijing M. tuberculosis family in Kazakhstan.

2022-09-16 15:53
The goal of the project is to conduct a comprehensive molecular genetic genotype characterization of Beijing M. tuberculosis in Kazakhstan. This project is aimed at investigating the role of the Beijing M. tuberculosis family in the spread of drug resistant forms of tuberculosis in both newly diagnosed and repeated cases of diseases and to study the characterization of the complete genomes of antibiotic-resistant isolates of the Beijing resistant isolates of the Beijing M. tuberculosis family.
To achieve the goal of the project the following tasks are set:
  1. Molecular genetic characterization and genotype evaluation of Beijing Using genotyping methods and determination of mutations in genes responsible for drug resistance to basic first-line ARVs - rifampicin and isoniazid.
  2. To identify the frequency of Beijing M. tuberculosis genotype among newly diagnosed cases and relapses in Kazakhstan and to study the association of this genotype with antibiotic-resistant forms of tuberculosis.
  3. Characterization of complete genomes of antibiotic-resistant isolates of Beijing M. tuberculosis genotype in Kazakhstan by next-generation high-throughput sequencing.

Abstract:
Despite the global introduction of DOTS (strictly controlled short-course chemotherapy) therapy and reduction of morbidity and mortality from tuberculosis, the prevalence of drug-resistant tuberculosis, especially multidrug-resistant tuberculosis (MDR-TB), associated with resistance to the most effective antituberculosis drugs (ATT) of the first line - isoniazid and rifampicin, remains a pressing problem in the world. According to WHO (2018), Kazakhstan is one of the thirty countries with high MDR-TB rates, accounting for 87% of all global MDR-TB cases. Many studies have shown the association of M. tuberculosis strains of the Beijing family with antibiotic resistance and the massive spread of MDR-TB. Strains of the Beijing genotype are the most virulent and predominate in Asian and Eastern European countries. Strains of this family have a higher ability to resist TB treatment and are considered to be a significant risk factor for TB treatment failure or relapse. Different sublineages and clonal clusters of M. tuberculosis isolates of the Beijing family may be distributed in different geographic regions; therefore, it is important to study the molecular genetic features of the strains of this family distributed in Kazakhstan. In this project, sublineages and clonal clusters of Beijing family isolates involved in drug resistance among new cases and relapses, and characterization of complete genomes of antibiotic-resistant Beijing family isolates will be studied for the first time in Kazakhstan.
During the reporting period, preliminary work was done to collect clinical samples of M. tuberculosis from newly diagnosed cases and relapsed cases from different regions of Kazakhstan. MIRU-VNTR genotyping of collected clinical M. tuberculosis isolates from newly diagnosed cases and relapses was performed using 15 MIRU-VNTR loci. Genotyping results showed that the most common M. tuberculosis family among all clinical isolates was the Beijing Chinese genotype. Strains of the Beijing family were identified among new cases in 62% of cases and among relapsed cases in 88% of cases. This may be due to the fact that the Beijing genotype is associated with drug resistance and is the most virulent and more associated with chronic forms of tuberculosis.
Sequencing of katG and rpoB genes of collected clinical isolates of M. tuberculosis among first detected cases on ABI 3730 sequencer (Applied Biosystems) was performed. In the katG gene associated with drug resistance to first-line ARV isoniazid a mutation was found in the 315 codon with a substitution of the amino acid serine for threonine Ser315Thr (AGCACC) in 40% of cases. In the rpoB gene associated with drug resistance to Rifampicin, 2 mutation variants were identified in the so-called "hot spot" of the gene (81 bp RRDR region, 507-533 codons). Mainly, in the rpoB gene, mutations were found in the 531 codon. The replacement of the amino acid serine with leucine Ser531Leu (TCGTTG) was found in this codon; this mutation was detected in 21.3% of cases. In one sample of M. tuberculosis, a mutation was identified in the 516 codon Asp516Val (AGC→ACC). The absence of mutations in katG (60%) and rpoB (77.3%) genes is probably due to the fact that 62.6% of the isolates in this sample of new cases were drug-sensitive. In the future, fabG-inhA and oxyR-ahpC genes of M. tuberculosis clinical isolates that are associated with drug resistance to isoniazid will be sequenced; mutations in these promoter regions occur in up to 20% of isoniazid-resistant isolates.

Project publications:
  1. A. Akhmetova, U. Kozhamkulov, D. Yerezhepov, A. Akilzhanova. Preliminary study of association of tuberculosis forms with polymorphisms in IFN-γ, IL-1β, NOS2, MARCO and TLR8 genes. European Journal of Human Genetics. 2022. 30:539; https://doi.org/10.1038/s41431-021-01026-1.
  2. Ахметова А.Ж, Акильжанова А.Р, Кожамкулов У.А. Қазақстанда таралған изониазидке-төзімді M. tuberculosis клиникалық изоляттарының katG генінде мутацияларды анықтау. Сборник X Международной научно-практической конференции «Наука и образование в современном мире: вызовы XXI века». Нур-Султан, 5-10 февраля 2022 г. С.72-73.
  3. Asset Daniyarov, Askhat Molkenov, Saule Rakhimova, Ainur Akhmetova et.al. Genomic analysis of multidrug-resistant Mycobacterium tuberculosis strains from patients in Kazakhstan. Frontiers in Genetics. 2021. doi: 10.3389/fgene.2021.683515.
  4. Akhmetova A, Akilzhanova A, Bismilda V, Chingissova L, Kozhamkulov U. Use of 15 MIRU-VNTR genotyping for discriminating M. tuberculosis clinical isolates from Kazakhstan. Georgian Medical News. 2021. №7-8 (316-317): 129-135.
  5. Ахметова А.Ж, Акильжанова А.Р., Рахимова С.Е, Бисмилда В.Л., Чингисова Л.Т, Кожамкулов У.А. Секвенирование KATG, INHA-FABG, OXYR-AHPC и RPOB генов мультирезистентных M.tuberculosis изолятов в Казахстане. Российский ежемесячный научно-практический журнал «Клиническая лабораторная диагностика». XXVI Всероссийская научно-практическая конференция с международным участием «Клиническая лаборатория: от аналитики к диагнозу». Москва, 12-14 мая 2021 г. С.13.
  6. Ахметова А.Ж, Акильжанова А.Р, Бисмилда В.Л, Чингисова Л.Т, Токсанбаева Б, Кожамкулов У.А. MIRU-VNTR генотипирование клинических изолятов Mycobacterium tuberculosis из Казахстана. Сборник трудов Х Юбилейной международной научно-практической конференции «Молекулярная диагностика». Москва, 9-11 ноября 2021 г. С. 309-310.
  7. Ахметова А.Ж, Акильжанова А.Р, Чамойева А.Е, Нышан У, Бисмила В.Л, Чингисова Л.Т, Токсанбаева Б, Кожамкулов У.А. Генотипирование M. tuberculosis изолятов среди повторных случаев туберкулеза в Казахстане. Сборник трудов Х Юбилейной международной научно-практической конференции «Молекулярная диагностика». Москва, 9-11 ноября 2021 г. С. 322-323.
  8. A. Akhmetova, A. Akilzhanova, U. Kozhamkulov. Prevalence of multidrug resistant Beijing family isolates among recurrent tuberculosis cases in Kazakhstan. CHEST. 2021: 582A, 160#4S CHEST OCTOBER 2021. DOI: https://doi.org/10.1016/j.chest.2021.07.560
  9. Ахметова А.Ж, Акильжанова А.Р, Кожамкулов У.А. Генотипирование клинических изолятов M. tuberculosis среди новых случаев в Казахстане. Сборник тезисов VIII Международной научно-практической конференции молодых ученых: биофизиков, биотехнологов, молекулярных биологов и вирусологов. Новосибирк, Россия. 2021. Раздел 4. С.258.