Application of Mycobacterium tuberculosis whole-genome sequencing to address epidemiologically and clinically relevant issues and improve tuberculosis control strategies. Summary of the Doctoral Thesis
No Thumbnail Available
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Rīga Stradiņš University
Abstract
Despite significant advancements in tuberculosis (TB) diagnostics, treatment, and prevention, the disease remains a major global public health challenge, with only limited reductions in incidence rates observed. In recent years, whole-genome sequencing (WGS) of Mycobacterium tuberculosis isolates has become a key approach for studying various aspects of TB, providing high-resolution insights into strain phylogenetic diversity, drug resistance mechanisms, disease transmission dynamics, and recurrence causes. Although WGS offers a promising alternative to widely implemented molecular and culture-based M. tuberculosis analysis methods, further improvements in sequencing data analysis strategies are needed to fully unlock its potential for addressing critical scientific, epidemiological, and clinical challenges in TB control. This doctoral thesis aimed to apply WGS to M. tuberculosis isolates from Latvian patients to address unresolved epidemiologically and clinically relevant issues impacting TB control and propose improvements for current WGS data analysis strategies. This research focused on two key subsets of patients with TB: those who experienced recurrent TB episodes and those suspected of being part of the same TB clusters based on epidemiological data and conventional genotyping results. The studied population included adults and children with pulmonary or extrapulmonary TB. M. tuberculosis isolates were subjected to WGS for genetic-variant-based strain genotyping, phylogenetic analyses, determination of the causes of recurrent TB episodes, identification of epidemiologically relevant TB clusters, delineation of transmission chains, detection of mixed-strain infections, and WGS-based drug susceptibility testing (DST). Relevant clinical and epidemiological data and results from previous M. tuberculosis isolate analyses were retrieved from patients’ medical records. Conventional genotyping (spoligotyping and insertion sequence 6110 restriction fragment length polymorphism analysis) and phenotypic DST results were compared with WGS findings. Patients’ diagnoses, sputum smear microscopy results, specimen collection timelines, contact tracing and geospatial data were integrated to interpret the WGS results according to the analysis objective. WGS-assigned lineages and sub-lineages generally agreed with the spoligotyping results of the M. tuberculosis isolates studied, with both methods revealing significant variability in M. tuberculosis genotypes in Latvia, even within specific TB patient subsets. The isolates within the two most prevalent M. tuberculosis genotype families in Latvia, Beijing and LAM, exhibited greater genetic relatedness than isolates from other genotype families, with varying drug resistance distribution frequencies observed among different M. tuberculosis spoligotypes. Distinguishing between the two possible causes of TB recurrence (endogenous reactivation and exogenous reinfection) was more precise when performed as a part of the TB cluster transmission chain delineation. This approach integrated WGS data with relevant clinical and epidemiological information, as identifying pairwise single nucleotide variant (SNV) distances and analysing differing SNVs between isolates proved insufficient. Using genetic distance thresholds of 12 SNVs for identifying epidemiologically relevant TB clusters and 5 SNVs for inferring recent transmission events proved effective in a low-to-moderate TB incidence setting. However, the close genetic relatedness of isolates belonging to Beijing and LAM genotype families, which are highly prevalent in Latvia, should be considered during TB cluster identification to avoid the risk of false clustering. Limitations in TB transmission chain delineation included unidentified active TB cases, variability in M. tuberculosis mutation rate, low genetic variability of the pathogen, and tight specimen collection timeline of genetically identical isolates. The proposed integrated approach significantly complemented epidemiological investigation and conventional genotyping results, enabling more accurate identification of source cases and evaluation of index cases. However, its application may not be necessary for all epidemiologically relevant cases. Lastly, discordances between phenotypic and WGS-based DST among the studied isolates were attributed to the technical complexities of phenotypic tests, the variable effects of genetic variants on the minimal inhibitory concentration of drugs, and insufficient data on drug-resistance-conferring variants. While WGS-based DST cannot fully replace pDST, combining these methods provides the most accurate assessment of drug resistance patterns. To conclude, this research provided valuable insights into the TB transmission dynamics in Latvia, the genetic diversity of M. tuberculosis genotypes circulating in the country, and the distribution of drug-resistance-conferring variants among these genotypes. It also highlighted challenges in phenotypic and WGS-based DST, evaluated different WGS-based strategies for determining the causes of recurrent TB, and proposed an integrative approach for delineating TB transmission chains. These findings support the potential of integrating WGS into routine practice as part of a comprehensive local TB surveillance program.
Description
The Doctoral Thesis was developed Latvian Biomedical Research and Study Centre, Laboratory of Molecular Microbiology. Defence: at the public session of the Promotion Council of Basic Medicine on 28 May 2025 at 14.00 in the Hippocrates Lecture Theatre, 16 Dzirciema Street, Rīgas Stradiņš University and remotely via online platform Zoom.
Keywords
Summary of the Doctoral Thesis, tuberculosis, Mycobacterium tuberculosis, whole-genome sequencing, genotyping, drug resistance, drug susceptibility testing, transmission, endogenous reactivation, exogenous reinfection
Citation
Sadovska, D. 2025. Application of Mycobacterium tuberculosis whole-genome sequencing to address epidemiologically and clinically relevant issues and improve tuberculosis control strategies: Summary of the Doctoral Thesis: Sub-Sector – Clinical Pharmacy. Rīga: Rīga Stradiņš University. https://doi.org/10.25143/prom-rsu_2025-11_dts