Browsing by Author "Ranka, Renāte"

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    Advantages of analysing both pairwise SNV-distance and differing SNVs between Mycobacterium tuberculosis isolates for recurrent tuberculosis cause determination.
    (2023-03-01) Sadovska, Darja; Nodieva, Anda; Pole, Ilva; Ķimsis, Jānis; Vīksna, Anda; Ozere, Iveta; Norvaiša, Inga; Bandere, Dace; Ranka, Renāte; Department of Infectology; Department of Pharmaceutical Chemistry
    Endogenous reactivation and exogenous reinfection are two possible causes of recurrent tuberculosis (TB). However, in some cases, precise cause determination can be challenging. In this study, we used whole genome sequencing to determine pairwise SNV distances and detect differing SNVs in initial and subsequent isolates for recurrent TB cases when the first and second episodes were caused by Mycobacterium tuberculosis ( Mtb) strains with an identical spoligotype pattern. In total, 104 Mtb isolates from 36 recurrent TB and 16 single TB episode patients were included in the study. Most isolate pairs belonged to the SIT1 (n=21), SIT42 (n=9), SIT53 (n=9), and SIT254 (n=7) spoligotypes, and in 27 cases, resistance to at least one anti-TB drug was found in either isolate. Drug susceptibility was more common in the recurrent TB patient cohort, and longitudinal single TB episode isolates were more prone to be drug-resistant (p=0.03), while the association between patient cohort and spoligotype was not statistically significant (p=0.07). The pairwise SNV-distance between the longitudinal single TB episode isolates was small (0-7 SNVs). Among the recurrent TB isolates, based on the high SNV-distance (38-273 SNVs), six reinfection cases (16.7%) were identified. This distance was small (<10 SNVs) in the remaining 30 isolate pairs. Further analysis of differing SNVs revealed that 22 (61.1%) cases could be classified as possible reactivation. Notably, despite the small distance of 2-7 SNVs, initial isolates of eight patients (22.2%) had several SNVs that were not found in the second isolates; therefore, these cases were classified as reinfection with a closely related Mtb strain. No statistically significant difference in the time interval between specimen collection in the reactivation and reinfection Mtb sample groups (p=0.13) or an association between recurrence cause and drug resistance status (p=0.62) or spoligotype (p=0.79) could be detected. The mycobacterial median mutation rate of longitudinal single TB episodes and possible reactivation isolate pairs (n=37) was 0.12 SNVs/genome/year (IQR 0-0.39), and in 18 cases (48.6%), it was equal to zero. No statistically significant differences in mutation rate were found between recurrent TB and longitudinal single TB episode isolates (p=0.087), drug-susceptible and resistant isolates (p=0.37) or isolates of Beijing and other genotype families (p=0.33). Furthermore, four cases of fluoroquinolone resistance development through the acquired SNVs in the gyrA gene were identified. To conclude, this study highlighted the complexity of recurrent episode cause determination and showed the usefulness of differing SNV identification in both Mtb isolates in such cases. Expected drug susceptibility was the only discriminative factor for recurrent TB episode-causing mycobacterial strains, while no differences between reactivation and reinfection sample groups could be identified.
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    Application of Mycobacterium tuberculosis whole-genome sequencing to address epidemiologically and clinically relevant issues and improve tuberculosis control strategies. Doctoral Thesis
    (Rīga Stradiņš University, 2025) Sadovska, Darja; Ranka, Renāte; Bandere, Dace
    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.
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    Application of Mycobacterium tuberculosis whole-genome sequencing to address epidemiologically and clinically relevant issues and improve tuberculosis control strategies. Summary of the Doctoral Thesis
    (Rīga Stradiņš University, 2025) Sadovska, Darja; Ranka, Renāte; Bandere, Dace
    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.
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    Assessment of Amikacin- and Capreomycin-Related Adverse Drug Reactions in Patients with Multidrug-Resistant Tuberculosis and Exploring the Role of Genetic Factors
    (2023-04) Freimane, Lauma; Barkāne, Linda; Kivrane, Agnija; Sadovska, Darja; Ulanova, Viktorija; Ranka, Renāte; Faculty of Pharmacy
    Following the introduction of all-oral treatment regimens for patients with drug-resistant tuberculosis (TB), second-line injectable drug applications have been reduced in the last few years. However, they are still important for anti-TB therapy. This study aims to analyze the occurrence of amikacin- and capreomycin-related adverse drug reactions (ADR) in patients with multidrug-resistant tuberculosis (MDR-TB) and evaluate the role of multiple patient-, disease-, and therapy-related factors on the frequency of the observed adverse events. In addition, the possible role of genetic risk factors was studied by full-length mitochondrial DNA sequencing. Toward this aim, we retrospectively evaluated 47 patients with MDR-TB who received amikacin and/or capreomycin. In total, 16 (34.0%) patients developed ototoxicity and 13 (27.7%) developed nephrotoxicity, including 3 (6.4%) patients who experienced both adverse events. Ototoxicity development was more common in patients who received amikacin. No other factors showed a significant impact. Nephrotoxicity was likely associated with previous renal health impairment. Full mitochondrial genome sequencing did not reveal any specific ADR-associated variants, and results showed no differences in adverse event occurrence for any specific variants, mutation count, or mitochondrial haplogroup. The absence of the previously reported ototoxicity-related mtDNA variants in our patients with ototoxicity and nephrotoxicity highlighted the complex nature of the ADR occurrence.
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    Changes in TP53 Gene, Telomere Length, and Mitochondrial DNA in Benign Prostatic Hyperplasia Patients
    (2024-10-15) Zole, Egija; Baumanis, Edgars; Freimane, Lauma; Dāle, Rolands; Leiše, Andrejs; Lietuvietis, Vilnis; Ranka, Renāte; Research Professor (Tenured Professor) Group at the Faculty of Pharmacy
    Background: Benign prostatic hyperplasia (BPH) is a growing issue due to an ageing population. Our study investigated the possible associations between BPH and ageing hallmarks, including the telomere length (TL) and mitochondrial genome copy number (mtDNA CN), along with genetic variations in the TP53 gene and mtDNA. Methods: Prostate tissue samples were obtained from 32 patients with BPH, together with 30 blood samples. As a healthy control group, age-matching blood DNA samples were used. For the comparison of mtDNA sequence data, 50 DNA samples of the general Latvian population were used. The full mtDNA genome was analyzed by using Next-Generation Sequencing (NGS), the TP53 gene by Sanger sequencing, and the mtDNA copy number (mtDNA CN) and telomere length (TL) byqPCR assay. Results: The results showed that in BPH patients, telomeres in the prostate tissue were significantly longer than in blood cells, while the TL in blood cells of the healthy controls was the shortest. Also, the mtDNA amount in the prostate tissue of BPH patients was significantly greater in comparison with blood cells, and controls had the smallest mtDNA CN. We did not find any mutations in the TP53 gene that could be linked to BPH; however, in mtDNA, we found several unique mutations and heteroplasmic changes, as well as genetic changes that have been previously associated with prostate cancer. Conclusions: In conclusion, prolonged telomeres and changes in the mtDNA amount might be involved in the molecular mechanisms of BPH. Some of the heteroplasmic or homoplasmic mtDNA variants might also contribute to the development of BPH. Additional studies are needed to substantiate these findings.
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    Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia
    (2023-12) Vīksna, Anda; Sadovska, Darja; Berge, Iveta; Bogdanova, Ineta; Vaivode, Annija; Freimane, Lauma; Norvaiša, Inga; Ozere, Iveta; Ranka, Renāte; Rīga Stradiņš University
    Background: Multidrug-resistant tuberculosis (MDR–TB) remains a major public health problem in many high tuberculosis (TB) burden countries. Phenotypic drug susceptibility testing (DST) take several weeks or months to result, but line probe assays and Xpert/Rif Ultra assay detect a limited number of resistance conferring gene mutations. Whole genome sequencing (WGS) is an advanced molecular testing method which theoretically can predict the resistance of M. tuberculosis (Mtb) isolates to all anti-TB agents through a single analysis. Methods: Here, we aimed to identify the level of concordance between the phenotypic and WGS-based genotypic drug susceptibility (DS) patterns of MDR–TB isolates. Overall, data for 12 anti-TB medications were analyzed. Results: In total, 63 MDR–TB Mtb isolates were included in the analysis, representing 27.4% of the total number of MDR–TB cases in Latvia in 2012–2014. Among them, five different sublineages were detected, and 2.2.1 (Beijing group) and 4.3.3 (Latin American-Mediterranean group) were the most abundant. There were 100% agreement between phenotypic and genotypic DS pattern for isoniazid, rifampicin, and linezolid. High concordance rate (> 90%) between phenotypic and genotypic DST results was detected for ofloxacin (93.7%), pyrazinamide (93.7%) and streptomycin (95.4%). Phenotypic and genotypic DS patterns were poorly correlated for ethionamide (agreement 56.4%), ethambutol (85.7%), amikacin (82.5%), capreomycin (81.0%), kanamycin (85.4%), and moxifloxacin (77.8%). For capreomycin, resistance conferring mutations were not identified in several phenotypically resistant isolates, and, in contrary, for ethionamide, ethambutol, amikacin, kanamycin, and moxifloxacin the resistance-related mutations were identified in several phenotypically sensitive isolates. Conclusions: WGS is a valuable tool for rapid genotypic DST for all anti-TB agents. For isoniazid and rifampicin phenotypic DST potentially can be replaced by genotypic DST based on 100% agreement between the tests. However, discrepant results for other anti-TB agents limit their prescription based solely on WGS data. For clinical decision, at the current level of knowledge, there is a need for combination of genotypic DST with modern, validated phenotypic DST methodologies for those medications which did not showed 100% agreement between the methods.
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    Identification of Factors Determining Patterns of Serum C-Reactive Protein Level Reduction in Response to Treatment Initiation in Patients with Drug-Susceptible Pulmonary Tuberculosis
    (2024-12) Kivrāne, Agnija; Ulanova, Viktorija; Grinberga, Solveiga; Sevostjanovs, Eduards; Vīksna, Anda; Ozere, Iveta; Bogdanova, Ineta; Simanovica, Ilze; Norvaisa, Inga; Pahirko, Leonora; Bandere, Dace; Ranka, Renāte; Research Professor (Tenured Professor) Group at the Faculty of Pharmacy; Department of Infectology; Department of Pharmaceutical Chemistry
    Background: Serum C-reactive protein (CRP) levels vary depending on radiological and bacteriological findings at the time of tuberculosis (TB) diagnosis. However, the utility of this biomarker in monitoring response to anti-TB treatment and identifying patients at risk of treatment failure is not well established. Objectives: This study evaluated the impact of patients’ baseline characteristics and anti-TB drug plasma exposure on the early reduction in serum CRP levels and its relationship with treatment response. Methods: We enrolled 42 patients with drug-susceptible pulmonary TB, who received a standard six-month regimen. The plasma concentrations of four anti-TB drugs were analysed using LC-MS/MS. Clinically relevant data, including serum CRP levels before and 10–12 days after treatment initiation (CRP10–12d), were obtained from electronic medical records and patient questionnaires. Results: In 10–12 days, the median serum CRP level decreased from 21.9 to 6.4 mg/L. Lower body mass index, positive sputum-smear microscopy results, and lung cavitations at diagnosis were related to higher biomarker levels at both time points; smoking had a more pronounced effect on serum CRP10–12d levels. Variability in anti-TB drug plasma exposure did not significantly affect the reduction in serum CRP levels. The serum CRP10–12d levels, or fold change from the baseline, did not predict the time to sputum culture conversion. Conclusions: Disease severity and patient characteristics may influence the pattern of early CRP reduction, while anti-TB drug plasma exposure had no significant effect at this stage. These early changes in serum CRP levels were not a predictor of response to anti-TB therapy.
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    Mycobacterium tuberculosis pilna genoma sekvencēšanas lietojums epidemioloģiski un klīniski nozīmīgu jautājumu risinājumiem un tuberkulozes kontroles stratēģiju uzlabošanai. Promocijas darba kopsavilkums
    (Rīgas Stradiņa universitāte, 2025) Sadovska, Darja; Ranka, Renāte; Bandere, Dace
    Neskatoties uz būtiskiem sasniegumiem tuberkulozes (TB) diagnostikā, ārstēšanā un profilaksē, slimība joprojām ir globāli nozīmīgs sabiedrības veselības izaicinājums un novērota tikai ierobežota incidences samazināšanās. Pēdējos gados Mycobacterium tuberculosis izolātu pilna genoma sekvencēšana (whole-genome sequencing, WGS) ir kļuvusi par vadošo pieeju dažādu aspektu izpētē TB gadījumos, nodrošinot detalizētus ieskatus celmu filoģenētiskajā daudzveidībā, kā arī atziņas par zāļu rezistences mehānismiem, slimības transmisijas dinamiku un recidīvu cēloņiem. Lai gan M. tuberculosis WGS sniedz daudzsološu alternatīvu plaši lietotām molekulāro un fenotipisko analīžu metodēm, papildu uzlabojumi ir nepieciešami sekvencēšanas datu analīzes stratēģijās, lai maksimāli izmantotu WGS potenciālās iespējas, risinot nozīmīgas zinātniskas, epidemioloģiskas un klīniskas TB kontroles problēmas. Šī promocijas darba mērķis bija veikt M. tuberculosis izolātu WGS no pacientiem Latvijā, lai izpētītu TB kontroli ietekmējošas epidemioloģiski un klīniski nozīmīgas neatrisinātās problēmas un piedāvātu uzlabojumus esošajās datu analīzes stratēģijās. Šajā pētījumā galvenokārt tika analizētas divas TB pacientu izlases: pacienti ar TB recidīviem un potenciāli iesaistītie TB transmisijas klasteros, pamatojoties uz epidemioloģiskajiem datiem un konvencionālās genotipēšanas rezultātiem. Pētāmajā populācijā tika iekļauti pieaugušie un bērni ar plaušu vai ārpusplaušu TB. M. tuberculosis izolātiem tika veikta WGS uz ģenētiskajiem variantiem balstītai celmu genotipēšanai, filoģenētiskajām analīzēm, TB recidīvu cēloņu noteikšanai, epidemioloģiski nozīmīgu TB klasteru identifikācijai, transmisijas ķēžu analīzei, vairāku celmu koinfekciju noteikšanai un uz WGS datiem balstītai zāļu jutības pārbaudei. Pētījumam nepieciešamie klīniskie un epidemioloģiskie dati, kā arī iepriekš veikto M. tuberculosis izolātu analīžu rezultāti tika atlasīti no pacientu medicīniskās dokumentācijas. Konvencionālās genotipēšanas (spoligotipēšanas un insercijas sekvences 6110 restrikcijas fragmentu garuma polimorfisma analīzes) un fenotipiskās zāļu jutības pārbaudes dati tika salīdzināti ar WGS rezultātiem. Pacientu diagnozes, krēpu mikroskopiskās izmeklēšanas rezultāti, paraugu iegūšanas datumi, kā arī kontaktpersonu izsekošanas un ģeotelpiskie dati tika integrēti WGS rezultātu interpretācijā atbilstoši analīzes mērķim. WGS datu analīzes rezultātā noteiktās celmu līnijas (lineage) un apakšlīnijas (sub-lineage) bija atbilstošas pētāmo M. tuberculosis izolātu spoligotipēšanas rezultātiem. Iegūtie dati liecināja par ievērojamu M. tuberculosis genotipu daudzveidību Latvijā, ņemot vērā specifiskās pētāmās TB pacientu izlases. Divu Latvijā izplatītāko M. tuberculosis genotipu – Beijing un LAM – izolātiem bija tuvāka ģenētiskā radniecība nekā citu identificēto genotipu izolātiem, turklāt tika novērots atšķirīgs zāļu rezistences sastopamības biežums starp dažādiem M. tuberculosis spoligotipiem. TB recidīvu cēloņu noteikšana, kas paredz endogēnas reaktivācijas diferencēšanu no eksogēnas reinfekcijas, bija precīzāka, kad tika veikta kā daļa no TB klastera transmisijas ķēdes analīzes. Šī pieeja apvieno WGS datu analīzi ar būtisku klīnisko un epidemioloģisko informāciju, jo ar viena nukleotīda variantu (single nucleotide variant, SNV) distances noteikšanu un atšķirīgu SNV analīzi starp izolātiem nebija pietiekami informatīvi. Ģenētiskās distances sliekšņu piemērošana epidemioloģiski nozīmīgu TB klasteru (12 SNV) un nesenas transmisijas gadījumu (5 SNV) noteikšanai izrādījās efektīva zemas līdz vidējas TB incidences apstākļos. Tomēr, identificējot TB klasterus, ir jāņem vērā Latvijā izplatītāko Beijing un LAM genotipu izolātu tuvā ģenētiskā radniecība, lai novērstu kļūdainu klasterizāciju. TB transmisijas ķēžu analīzi ierobežoja neidentificēti aktīvas TB gadījumi, M. tuberculosis mutācijas ātruma mainīgums, zems patogēna ģenētiskais mainīgums un īss laika intervāls starp paraugu iegūšanas datumiem ģenētiski identisku izolātu gadījumos. Piedāvātā integrētā pieeja būtiski papildināja epidemioloģiskās izmeklēšanas un konvencionālās genotipēšanas rezultātus, nodrošinot precīzāku infekcijas avotu noteikšanu un indeksa gadījumu (index case) izvērtēšanu. Taču šī pieeja var nebūt nepieciešama visu epidemioloģiski nozīmīgu gadījumu analīzei. Visbeidzot, nesakritības starp pētāmo izolātu fenotipisko un uz WGS datiem balstīto zāļu jutības pārbaudi tika pamatotas ar fenotipisko testu tehnisko sarežģītību, ģenētisko variantu mainīgo ietekmi uz medikamentu minimālo inhibējošo koncentrāciju, kā arī nepietiekamo informāciju par zāļu rezistenci izraisošiem variantiem. Lai gan uz WGS datiem balstītā zāļu jutības pārbaude nespēj aizstāt fenotipisko metodi pilnībā, abu pieeju kombinācija nodrošina visprecīzāko zāļu rezistences profila novērtējumu. Noslēgumā – pētījums ir sniedzis vērtīgas atziņas par TB transmisijas dinamiku Latvijā, valstī cirkulējošo M. tuberculosis genotipu ģenētisko daudzveidību un zāļu rezistenci izraisošu ģenētisko variantu izplatību šo genotipu starpā. Tika akcentēti izaicinājumi zāļu jutības pārbaudes metodēs, izvērtētas trīs uz WGS datu analīzi balstītās stratēģijas recidīvu cēloņu noteikšanai un piedāvāta integrētā pieeja transmisijas ķēžu analīzei. Pētījuma rezultāti apstiprina WGS ieviešanas potenciālu rutīnas praksē kā daļu no vietējās TB uzraudzības programmas.
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    Pharmacogenetic Aspects of Anti-Tuberculosis Therapy in Latvian Population. Doctoral Thesis
    (Rīga Stradiņš University, 2025) Ulanova (Igumnova), Viktorija; Ranka, Renāte; Bandere, Dace
    Pharmacogenetics (PGx) plays a crucial role in personalised medicine by revealing how genetic variations influence drug response, guiding initial doses, and working in conjunction with therapeutic drug monitoring (TDM) to adjust dosing according to drug metabolism for each individual. Together, PGx and TDM work synergistically to optimise treatment, as using the patient’s PGx profile and up-to-date patient data on drug levels in the systemic circulation enables precision, effectiveness and safety of treatment, moving away from a one-size-fits-all approach to a more individualised strategy. Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which is transmitted between humans through the respiratory tract, most commonly affecting the lungs. TB is a major global public health challenge and social threat worldwide. With the increasing population mobility and travel, TB strains, including drug-resistant variants, are easily disseminated worldwide. The implementation of personalised medicine strategies may help to ensure the effectiveness of existing treatments, promptly identify those patients most likely to develop adverse drug reactions (ADRs), and reduce the likelihood of acquired drug resistance in Mtb, thus playing an important role in the global fight against TB. This Thesis describes the PGx aspects of TB treatment focusing on the Latvian population. Isoniazid (INH) is a cornerstone of the standard regimen for the treatment of drug-susceptible TB (DS-TB). However, the variability in pharmacokinetic (PK) parameters and drug plasma levels may affect drug response and lead to ADRs including a drug-induced hepatotoxicity (DIH). This Thesis primarily investigates the impact of changes in INH PK parameters due to genetic variability in three genes encoding INH-metabolising enzymes, together with a number of patient-related factors, on the response to anti-TB treatment and the risk of DIH in patients with DS-TB. In particular, the N-acetyltransferase 2 (NAT2) haplotype, referred to as acetylator status, and the genotype of glutathione S-transferase M1 class (GSTM1) were assessed in TB patients. Additionally, a new next-generation sequencing-based method for full-length cytochrome P450 family 2 subfamily E member 1 (CYP2E1) gene analysis was designed and evaluated to determine whether CYP2E1 gene variants are of clinical significance. Aminoglycosides (AGs) are potent, broad-spectrum antibiotics, which remain important drugs in the treatment of multidrug-resistant TB. AG-induced hearing loss (AIHL) is a well-recognised ADR with rapid, profound, and irreversible hearing loss that can occur in predisposed individuals. The MT-RNR1 gene, encoding the mitochondrial ribosomal 12S subunit, is particularly susceptible to gene variants associated with AIHL. In this Thesis, the prevalence of AIHL-related MT-RNR1 gene variants was investigated in the Baltic-speaking ethnic Latvian population in comparison to the general population worldwide in order to improve the accuracy and safety of treatment with AGs. This Thesis is presented as a comprehensive compilation of peer-reviewed publications, summarising advancements in personalised TB medicine. Specifically, it focuses on identifying and characterising the factors that contribute to variations in drug response and safety through comprehensive analysis of the PGx and PK data in TB patients. This approach could provide us with an opportunity to advance therapeutic decision-making. The inclusion of profiling of INH and its two main metabolites in clinical studies, together with PGx screening, could be beneficial in determining optimal dosing strategies, tailoring treatment regimens, and fostering a patient-centred healthcare approach for TB patients, challenging the current paradigm of modern public health.
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    Pharmacogenetic Aspects of Anti-Tuberculosis Therapy in Latvian Population. Summary of the Doctoral Thesis
    (Rīga Stradiņš University, 2025) Ulanova (Igumnova), Viktorija; Ranka, Renāte; Bandere, Dace
    Pharmacogenetics (PGx) plays a crucial role in personalised medicine by revealing how genetic variations influence drug response, guiding initial doses, and working in conjunction with therapeutic drug monitoring (TDM) to adjust dosing according to drug metabolism for each individual. Together, PGx and TDM work synergistically to optimise treatment, as using the patient’s PGx profile and up-to-date patient data on drug levels in the systemic circulation enables precision, effectiveness and safety of treatment, moving away from a one-size-fits-all approach to a more individualised strategy. Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which is transmitted between humans through the respiratory tract, most commonly affecting the lungs. TB is a major global public health challenge and social threat worldwide. With the increasing population mobility and travel, TB strains, including drug-resistant variants, are easily disseminated worldwide. The implementation of personalised medicine strategies may help to ensure the effectiveness of existing treatments, promptly identify those patients most likely to develop adverse drug reactions (ADRs), and reduce the likelihood of acquired drug resistance in Mtb, thus playing an important role in the global fight against TB. This Thesis describes the PGx aspects of TB treatment focusing on the Latvian population. Isoniazid (INH) is a cornerstone of the standard regimen for the treatment of drug-susceptible TB (DS-TB). However, the variability in pharmacokinetic (PK) parameters and drug plasma levels may affect drug response and lead to ADRs including a drug-induced hepatotoxicity (DIH). This Thesis primarily investigates the impact of changes in INH PK parameters due to genetic variability in three genes encoding INH-metabolising enzymes, together with a number of patient-related factors, on the response to anti-TB treatment and the risk of DIH in patients with DS-TB. In particular, the N-acetyltransferase 2 (NAT2) haplotype, referred to as acetylator status, and the genotype of glutathione S-transferase M1 class (GSTM1) were assessed in TB patients. Additionally, a new next-generation sequencing-based method for full-length cytochrome P450 family 2 subfamily E member 1 (CYP2E1) gene analysis was designed and evaluated to determine whether CYP2E1 gene variants are of clinical significance. Aminoglycosides (AGs) are potent, broad-spectrum antibiotics, which remain important drugs in the treatment of multidrug-resistant TB. AG-induced hearing loss (AIHL) is a well-recognised ADR with rapid, profound, and irreversible hearing loss that can occur in predisposed individuals. The MT-RNR1 gene, encoding the mitochondrial ribosomal 12S subunit, is particularly susceptible to gene variants associated with AIHL. In this Thesis, the prevalence of AIHL-related MT-RNR1 gene variants was investigated in the Baltic-speaking ethnic Latvian population in comparison to the general population worldwide in order to improve the accuracy and safety of treatment with AGs. This Thesis is presented as a comprehensive compilation of peer-reviewed publications, summarising advancements in personalised TB medicine. Specifically, it focuses on identifying and characterising the factors that contribute to variations in drug response and safety through comprehensive analysis of the PGx and PK data in TB patients. This approach could provide us with an opportunity to advance therapeutic decision-making. The inclusion of profiling of INH and its two main metabolites in clinical studies, together with PGx screening, could be beneficial in determining optimal dosing strategies, tailoring treatment regimens, and fostering a patient-centred healthcare approach for TB patients, challenging the current paradigm of modern public health.
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    Prettuberkulozes terapijas farmakoģenētiskie aspekti Latvijas populācijā. Promocijas darba kopsavilkums
    (Rīgas Stradiņa universitāte, 2025) Ulanova (Igumnova), Viktorija; Ranka, Renāte; Bandere, Dace
    Farmakoģenētikai (angl. PGx) ir izšķiroša nozīme personalizētajā medicīnā, jo tā palīdz atklāt, kā ģenētiskās variācijas ietekmē pacienta reakciju uz zālēm. Tas ļauj precīzāk noteikt sākotnējās zāļu devas, un apvienojot ar zāļu terapeitisko uzraudzību (angl. TDM) pielāgot zāļu devu katram pacientam, balstoties uz viņu individuālo zāļu metabolismu. PGx un TDM darbojas sinerģiski, lai optimizētu ārstēšanu, nodrošinot ārstēšanas precizitāti, efektivitāti un drošumu. Pacienta PGx profila kombinēšana ar aktuāliem datiem par zāļu līmeni sistēmiskajā cirkulācijā ļauj pāriet no pieejas "viens izmērs der visiem" uz individualizētām terapijas stratēģijām. Tuberkuloze (TB) ir infekcijas slimība, ko izraisa mikobaktērija Mycobacterium tuberculosis (Mtb). TB izplatās gaisa pilienu ceļā no cilvēka uz cilvēku un visbiežāk skar plaušas. Šī infekcija ir nozīmīga globāla sabiedrības veselības problēma un sociāls drauds. Pieaugot iedzīvotāju mobilitātei un ceļotāju migrācijai, Mtb celmi, tostarp pret zālēm rezistenti varianti, viegli izplatās visā pasaulē. Personalizētās medicīnas stratēģiju ieviešana var būtiski uzlabot esošās ārstēšanas efektivitāti, savlaicīgi identificējot pacientus ar augstu zāļu nevēlamu blakusparādību (angl. ADRs) risku, kā arī samazināt zāļu rezistences rašanās iespējas pret Mtb. Šāda pieeja spēlē nozīmīgu lomu globālajā cīņā pret TB. Šajā disertācijā ir aprakstīti TB ārstēšanas PGx aspekti ar īpašu uzsvaru uz Latvijas iedzīvotājiem. Izoniazīds (angl. INH) ir standarta ārstēšanas shēmas stūrakmens pret zālēm jūtīgās TB (angl. DS-TB) gadījumos. Tomēr farmakokinētisko (angl. PK) parametru un zāļu līmeņa mainīgums asins plazmā var ietekmēt TB ārstēšanas efektivitāti vai izraisīt ADRs, piemēram, zāļu izraisītu aknu bojājumu (angl. DIH). Šajā promocijas darbā galvenā uzmanība tika pievērsta INH PK parametru izmaiņām, ko izraisa INH metabolizējošo enzīmu kodējošo gēnu ģenētiskā variabilitāte un dažādi ar pacientu saistīti faktori, analizējot to ietekmi uz terapijas atbildes reakciju un DIH attīstības risku pacientiem ar DS-TB. Īpašs uzsvars tika likts uz N-acetiltransferāzes 2 (NAT2) acetilatora statusu, glutationa S-transferāzes M1 klases (GSTM1) genotipu. Turklāt tika izstrādāta un izvērtēta jauna metode, kas balstīta uz nākamās paaudzes sekvencēšanu, lai veiktu pilna garuma citohroma P450 2. saimes E apakšsaimes 1. locekļa (CYP2E1) gēna analīzi. Šī pieeja tika izmantota, lai novērtētu, vai vairāku reģionu CYP2E1 gēna variantiem ir klīniskā nozīme. Aminoglikozīdi (AG) ir spēcīgas plaša spektra antibiotikas, kurām joprojām ir būtiska loma multirezistentās TB ārstēšanā. Tomēr AG izraisīts dzirdes zudums (angl. AIHL) ir labi pazīstama un atzīta ADR. Tas izpaužas kā ātrs, dziļš un neatgriezenisks dzirdes zudums, kas īpaši bieži novērojamas pacientiem ar ģenētisku predispozīciju. MT-RNR1 gēns, kas kodē mitohondriālo ribosomālo 12S apakšvienību, ir īpaši uzņēmīgs pret gēna variantiem, kas saistīti ar AIHL. Šajā promocijas darbā tika pētīta ar AIHL saistītu MT-RNR1 gēna variantu prevalence Latvijas populācijā un salīdzināta ar globālajām populācijām. Pētījuma mērķis bija sekmēt AG lietošanas drošumu un veicināt ārstēšanas precizitāti. Šī disertācija ir veidota kā visaptveroša recenzētu publikāciju kopa, kurā apkopoti pētījumi personalizētās medicīnas jomā. Tajā īpaša uzmanība pievērsta faktoriem, kas ietekmē zāļu atbildes reakcijas un drošuma atšķirības, kā arī šo faktoru identificēšanai un raksturošanai, izmantojot visaptverošu PGx un PK datu analīzi pacientiem ar TB. Šāda pieeja var sniegt nozīmīgu ieguldījumu terapeitisko lēmumu pieņemšanā. INH un tā divu galveno metabolītu koncentrācijas uzraudzība un PGx skrīninga iekļaušana klīniskajos pētījumos varētu būt lietderīga optimālu dozēšanas stratēģiju noteikšanai, ārstēšanas režīmu pielāgošanai un pacientu orientētas veselības aprūpes pieejas veicināšanai pacientiem ar TB. Šis darbs izaicina pašreizējo modernās sabiedrības veselības aprūpes paradigmu, piedāvājot personalizētākas un efektīvākas ārstēšanas iespējas.
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    Unraveling tuberculosis patient cluster transmission chains : integrating WGS-based network with clinical and epidemiological insights
    (2024) Sadovska, Darja; Ozere, Iveta; Pole, Ilva; Ķimsis, Jānis; Vaivode, Annija; Vīksna, Anda; Norvaiša, Inga; Bogdanova, Ineta; Ulanova, Viktorija; Čapligina, Valentīna; Bandere, Dace; Ranka, Renāte; Department of Infectology; Department of Pharmaceutical Chemistry
    BACKGROUND: Tuberculosis remains a global health threat, and the World Health Organization reports a limited reduction in disease incidence rates, including both new and relapse cases. Therefore, studies targeting tuberculosis transmission chains and recurrent episodes are crucial for developing the most effective control measures. Herein, multiple tuberculosis clusters were retrospectively investigated by integrating patients' epidemiological and clinical information with median-joining networks recreated based on whole genome sequencing (WGS) data of Mycobacterium tuberculosis isolates. METHODS: Epidemiologically linked tuberculosis patient clusters were identified during the source case investigation for pediatric tuberculosis patients. Only M. tuberculosis isolate DNA samples with previously determined spoligotypes identical within clusters were subjected to WGS and further median-joining network recreation. Relevant clinical and epidemiological data were obtained from patient medical records. RESULTS: We investigated 18 clusters comprising 100 active tuberculosis patients 29 of whom were children at the time of diagnosis; nine patients experienced recurrent episodes. M. tuberculosis isolates of studied clusters belonged to Lineages 2 (sub-lineage 2.2.1) and 4 (sub-lineages 4.3.3, 4.1.2.1, 4.8, and 4.2.1), while sub-lineage 4.3.3 (LAM) was the most abundant. Isolates of six clusters were drug-resistant. Within clusters, the maximum genetic distance between closely related isolates was only 5-11 single nucleotide variants (SNVs). Recreated median-joining networks, integrated with patients' diagnoses, specimen collection dates, sputum smear microscopy, and epidemiological investigation results indicated transmission directions within clusters and long periods of latent infection. It also facilitated the identification of potential infection sources for pediatric patients and recurrent active tuberculosis episodes refuting the reactivation possibility despite the small genetic distance of ≤5 SNVs between isolates. However, unidentified active tuberculosis cases within the cluster, the variable mycobacterial mutation rate in dormant and active states, and low M. tuberculosis genetic variability inferred precise transmission chain delineation. In some cases, heterozygous SNVs with an allelic frequency of 10-73% proved valuable in identifying direct transmission events. CONCLUSION: The complex approach of integrating tuberculosis cluster WGS-data-based median-joining networks with relevant epidemiological and clinical data proved valuable in delineating epidemiologically linked patient transmission chains and deciphering causes of recurrent tuberculosis episodes within clusters.