Defense of the dissertation of Akhmetova Ainur Zharmukhambetovna for the degree of Doctor of Philosophy (PhD) in the educational program «8D05107 - Biology»
L.N. Gumilyov Eurasian National University, a dissertation defense for the degree of Doctor of Philosophy (PhD) by Akhmetova Ainur Zharmukhambetovna on the topic «Molecular genetic characteristics of Mycobacterium tuberculosis Beijing genotype in Kazakhstan: Distribution and association with drug resistance» to the educational program «8D05107 – Biology».
The dissertation was carried out at the «General Biology and Genomics education department» of L.N. Gumilyov Eurasian National University.
The language of defense is russian
Official reviewers:
Gaipov Abduzhappar Erkinovich – Candidate of Medical Sciences, Associate Professor of the School of Medicine of Nazarbayev University (Astana, Republic of Kazakhstan).
Abeldenov Sailau Kasenovich - PhD, Head of the Laboratory of Molecular Biotechnology of the National Center for Biotechnology (Astana, Republic of Kazakhstan).
Temporary members of the Dissertation Committee:
Kistaubayeva Aida Serikovna – Candidate of Biological Sciences, Professor, Head of the Department of Biotechnology of Al-Farabi Kazakh National University (Almaty, Republic of Kazakhstan).
Chesov Dumitru Ivanovich - MD, PhD, Assosiate Professor of the Department of Pulmonology and Allergology of Nicolae Testemitanu State University of Medicine and Pharmacy (Kishinev, Republic of Moldova).
Shaikhin Serik Murzakhmetovich – Doctor of Biological Sciences, Leading Researcher of the Laboratory of Genetics and Biochemistry of Microorganisms of the Republic Collection of Microorganisms (Astana, Republic of Kazakhstan).
Scientific advisors:
Akilzhanova Ainur Rakhmetulovna - Doctor of Medical Sciences, PhD, Professor, Head of the Laboratory of Genomic and Personalized Medicine of the PI «National Laboratory Astana» (Astana, Republic of Kazakhstan).
Filipenko Maxim Leonidovich – Doctor of Biological Sciences, Head of the Laboratory of Pharmacogenomics of the Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of Russian Academy of Sciences (Novosibirsk, Russian Federation).
The defense will take place on June 23, 2025, at 11:00 in the Dissertation Council for the training direction «8D051 – Biological and related sciences» in the educational program «8D05107 – Biology» of L.N. Gumilyov Eurasian National University. Conducting a meeting of the dissertation council in a mixed (offline and online) format.
Link: https://us06web.zoom.us/j/84013448647?pwd=MyvzA4yp0DVgPiB1OmiOKaRCrlgLFl.1
Conference ID: 840 1344 8647
Access code: 633666
Address: Astana, Kazhymuqana str., 13, building №3, room №333.
Abstract (English): General characteristics of the dissertation. The dissertation is devoted to a comprehensive study of the molecular-genetic and clinical-epidemiological characteristics of the Beijing genotype of M. tuberculosis. Relevance of the research topic. Tuberculosis (TB) remains a major public health problem worldwide and is the second leading cause of death from a single infectious agent after coronavirus disease (COVID-19). According to the World Health Organization (WHO), 10.8 million people fell ill with TB in 2023, including 8.2 million among new cases. In the same year, 1.25 million people died from TB, including 161 000 cases among HIV-infected people. The main obstacle in the fight against tuberculosis is the widespread of drug-resistant M. tuberculosis strains. The most dangerous resistance strains are multidrug-resistant (MDR) strains and strains with more complex forms of MDR – pre-extensive drug resistance (pre-XDR) and XDR. Currently, rifampicin resistance (RR) is used as an indicator of MDR-TB, among patients with rifampicin resistance, MDR-TB is detected in ~80% of cases. According to WHO, in 2023, 400 000 cases of MDR/RR-TB were detected worldwide. In Kazakhstan, despite the fact that over the past 21 years (2002-2023) the incidence and mortality rates from tuberculosis have significantly decreased from 165.1 to 34.7 cases per 100 000 population and from 24.2 to 1.12 cases per 100 000 population, respectively, the number of cases of MDR-TB is growing every year. Over 13 years (2010-2023), the MDR-TB rates in the country have doubled from 14 to 28 cases per 100 000 population. According to WHO, Kazakhstan is among the top 30 countries with the highest indicator of MDR/RR-TB in the world. According to various studies, MDR-TB and its widespread distribution are associated with the Beijing genotype of M. tuberculosis. However, the increased ability to acquire drug resistance is not characteristic of all Beijing isolates and varies among different subgroups and clusters of the Beijing genotype. Since different isolates of the Beijing genotype circulate in different geographic regions, it is relevant to study the structure of the Beijing genotype and its clinically and epidemiologically significant clusters in Kazakhstan to monitor drug-resistant, including multidrug-resistant tuberculosis among new and recurrent cases. Previous studies show that the Beijing genotype is the most spread genotype among M. tuberculosis strains in Kazakhstan and was identified in 70.4%-79% of cases. However, in these studies, M. tuberculosis genotypes (families) were mainly characterized on a small sample size. In most studies, the results were described in the general sample collection without distinguishing between new and recurrent cases. In studies where the analysis was conducted among samples isolated from new and recurrent cases separately, the samples included only drug-resistant isolates or mainly consisted of samples from recurrent cases of tuberculosis. Collection of M. tuberculosis isolates mostly only from recurrent patients makes the sample limited for assessing drug resistance in the general population. Therefore, to identify the Beijing genotype and its features among the Kazakhstani population of M. tuberculosis, it is necessary to study this genotype among both new and recurrent TB cases, including all currently available samples (resistant and susceptible). In general, previous studies have not conducted a detailed study of the Beijing genotype in Kazakhstan. The association of this genotype and its main clusters with clinical forms of the disease, as well as with the age and gender of TB patients have not been studied previously, information on genomic studies of Kazakhstani drug-resistant samples of the Beijing genotype is limited. Therefore, it is necessary to conduct a comprehensive molecular-genetic and clinical-epidemiological characterization of Beijing genotype isolates circulating in Kazakhstan. The object of the study is DNA of clinical isolates of M. tuberculosis, DNA of the reference strain M. tuberculosis H37Rv. The subject of the study is genetic markers associated with drug resistance to anti-tuberculosis drugs and genetic loci used to identify M. tuberculosis genotypes. Objectives and tasks of the study The aim of the study is to conduct a comprehensive molecular genetic characterization of the M. tuberculosis Beijing genotype in Kazakhstan among new and recurrent cases of tuberculosis. To achieve the goal, the following tasks were set: 1. To determine the frequency of the M. tuberculosis Beijing genotype among new and recurrent cases in Kazakhstan. 2. To investigate the association of the Beijing genotype and its main clusters with various drug-resistant forms of TB (monoresistant TB, polyresistant TB and MDR-TB), as well as with the most common mutations Ser531Leu of rpoB gene and Ser315Thr of katG gene associated with resistance to the most effective first-line anti-TB drugs - rifampicin and isoniazid, respectively. 3. To identify the features of the clinical and epidemiological parameters of tuberculosis in patients with the M. tuberculosis Beijing genotype. 4. To study the whole genomes of Kazakhstani isolates of the Beijing genotype with MDR, pre-XDR and XDR and test various genomic databases for determining drug susceptibility based on next-generation sequencing. Research methods. Comprehensive molecular-genetic and clinical-epidemiological assessment of the Beijing genotype was carried out using methods of microbiology, molecular biology, bioinformatics and statistics. Scientific novelty of the study. For the first time in Kazakhstan, a comprehensive molecular-genetic characterization of the Beijing genotype was carried out among new and recurrent cases of TB and a comparative analysis of data from samples obtained from new cases, recurrent cases and in the general sample collection was performed. The frequency of distribution of the Beijing genotype and its dominant clusters was determined. Clinical and epidemiological data of patients infected with the Beijing genotype were studied in detail. For the first time in Kazakhstan, the relationship between the Beijing genotype and its main clusters with clinical forms of TB, various drug resistance (monoresistance and polyresistance, MDR) and the most common mutations Ser531Leu of rpoB gene and Ser315Thr of katG gene, associated with resistance to the most effective first-line anti-TB drugs - rifampicin and isoniazid, respectively were studied. For the first time, we characterized and published the whole genomes of drug-resistant Beijing isolates (MDR, pre-XDR and XDR) from Kazakhstan. Theoretical significance of the study. The results obtained in this study will expand knowledge about the molecular-genetic and clinical-epidemiological features of the most spread M. tuberculosis Beijing genotype and its main clusters among new and recurrent cases of TB in Kazakhstan and can be used in medicine and phthisiology to improve strategic measures for monitoring and preventing drug-resistant TB, including MDR-TB. The methodological recommendations ‘Determination of Mycobacterium tuberculosis families in clinical isolates according to the developed genotyping scheme using MIRU-VNTR analysis’ developed within the framework of the dissertation in Kazakh and Russian languages can be used in the educational process in medical universities, in scientific laboratories and laboratories of the anti-tuberculosis service for M. tuberculosis genotyping. The M. tuberculosis genomes uploaded as a result of our study to the international NCBI database (under numbers PRJNA503963 and PRJNA481625) make the protein and gene sequences of Kazakhstani Beijing isolates with MDR, pre-XDR and XDR available to the biomedical community and provide an opportunity to gain new knowledge for the creation of vaccines and new anti-tuberculosis drugs, as well as for studying the evolution of mycobacteria. Practical significance of the study. In the framework of our study, we developed and tested a MIRU-VNTR genotyping scheme consisting of 7 polymorphic loci specific to Kazakhstani M. tuberculosis strains and implemented it in the work of the National Scientific Center of Phthisiopulmonology of the Ministry of Health of the Republic of Kazakhstan (Almaty) for preliminary screening studies of M. tuberculosis populations and identification of clusters circulating in Kazakhstan. Standard MIRU-VNTR genotyping schemes, including the scheme we propose, can also be used in the TB control program for the rapid identification of closely related strains and possible transmission chains, identification and differentiation of cases of tuberculosis reactivation from reinfection, monitoring secondary cases of TB and identification of epidemiologically and clinically important genotypes during outbreaks of the disease in certain foci of infection. As a result of the dissertation work, DNA libraries were prepared for whole-genome sequencing on next-generation sequencing platforms, six genomic databases were tested and the most optimal databases were recommended for determination of the drug susceptibility profile and identification of mutations in genes associated with resistance to first- and second-line anti-TB drugs. This knowledge can be applied in clinical practice for rapid determination and analysis of drug susceptibility of M. tuberculosis strains in combination with microbiological methods, which in turn will allow to select the most effective, personalized antibiotic therapy. Main conclusions of the study 1. The frequency of the M. tuberculosis Beijing genotype among new and recurrent TB cases in Kazakhstan was determined - 60% and 78%, respectively. The main clusters of this genotype (94-32, 96-32, 94-33, 100-32, 96-145 and 99-32) were identified, accounting for 65.2% of all Beijing isolates. Of these, 52.5% were classified as the Central Asian/Russian cluster 94-32. 2. A statistically significant relationship was found between the Beijing genotype and drug-resistant tuberculosis, including MDR-TB in all study groups, with a two-fold prevalence in the group of recurrent TB cases compared to new cases. The most spread cluster 94-32 of the Beijing genotype (52.5% among all Beijing) was associated with drug-resistant M. tuberculosis strains in the general sample collection and among recurrent cases, and with MDR-TB among new cases. And the Beijing 94-15 cluster was associated with drug-susceptible TB infection in the general sample collection and among recurrent cases. Among non-Beijing isolates, in the general sample collection, the LAM genotype showed a statistically significant association with drug-resistant TB, including MDR-TB. The prevalence of the LAM genotype among resistant isolates was observed among new cases. Among rifampicin-resistant, as well as MDR samples with confirmed mutations in rpoB gene, a statistically significant association was observed between the most common mutation Ser531Leu in codon 531 of rpoB gene and the Beijing genotype. For the first time in Kazakhstan, we showed that the His526Leu mutation in codon 526 of rpoB gene was associated with the LAM genotype. 3. The features of clinical and epidemiological parameters of tuberculosis among patients infected with the M. tuberculosis Beijing genotype were identified. Among age groups, the Beijing genotype was statistically associated with young age from 18 to 44 years in the general sample collection and among recurrent cases (p0.05). In the general sample collection, women fell ill earlier than men (mean age 36.3 years vs. 38.1 years, p=0.02642). Among patients with recurrent cases of tuberculosis, the mean age of patients infected with the Beijing genotype was significantly lower than the mean age of patients with the non-Beijing genotypes (39.7 years vs. 46.6 years, p=0.00544). And among patients infected with the Beijing genotype, women were statistically younger, than men in the general sample collection (35.1 years vs. 37.6 years, p=0.0232) and among recurrent cases (35.7 years vs. 41 years, p=0.00854). Among the Beijing genotype clusters, this feature was observed among women infected with the most spread Central Asian/Russian cluster 94-32 in the general sample collection (p=0.01928). In the general sample collection, patients with fibro-cavernous tuberculosis (FCT) were statistically more often infected with drug-resistant strains of M. tuberculosis (p=0.0019). Among patients infected with drug-resistant M. tuberculosis, the relationship between FCT and MDR was found (p=0.0054). And infection with drug-susceptible strains of M. tuberculosis was determined to a greater extent in patients with focal TB among new cases (p<0.05). Tuberculous pleurisy was associated with non-Beijing genotypes among new and recurrent TB cases (p=0.0430). 4. As a result of whole-genome sequencing of Beijing isolates with MDR, pre-XDR and XDR, various types of mutations (missense mutations, frameshift variants, nonsense variants, etc.) associated with resistance to anti-TB drugs were identified, including new mutations that require further investigation and validation. The obtained genomes of Kazakhstani Beijing isolates were uploaded to the international NCBI database under numbers PRJNA503963 and PRJNA481625 and are available for use by the biomedical community. Six genomic databases (ResFinder, CARD, CASTB, TGS-TB, TB-Profiler and Mykrobe) were tested for rapid diagnosis of drug susceptibility and analysis of mutations associated with first and second-line anti-TB drugs. We recommend three databases, namely ResFinder, TB-Profiler and Mykrobe for primary screening of drug susceptibility in M. tuberculosis genomes and two databases, specifically ResFinder and TB-Profiler for extensive and detailed analysis of the spectrum of mutations in genes associated with resistance to first and second-line anti-TB drugs. Publications. In the framework of the dissertation 35 works were published, including 5 articles in Journals included in the Web of Science and Scopus databases; 5 articles in Journals recommended by the Committee on Quality Assurance of Higher Education of the Ministry of Science and Higher Education of the Republic of Kazakhstan; 10 abstracts in editions included in the Web of Science and Scopus databases; 13 abstracts in the materials of International Conferences; 2 methodological recommendations. Structure of the dissertation. The dissertation is presented on 149 pages. The work consists of the introduction, literature review, materials and methods, results and discussions, conclusion, a list of references, including 240 sources and 4 appendices. The dissertation consists of 26 tables and 10 figures.
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