Unraveling tuberculosis patient cluster transmission chains: integrating WGS-based network with clinical and epidemiological insights.

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.

Understanding the high-risk human papillomavirus prevalence and associated factors in the European country with a high incidence of cervical cancer.

BACKGROUND: High-risk human papillomavirus (HR-HPV) is a known cause of cervical cancer (CC). Latvia has a high incidence of CC compared with the average incidence in the European Union. This study aims to fill the data gap on the HR-HPV burden in Latvia, providing information on its prevalence and associated factors. METHODS: The cross-sectional study was conducted from February 2021 to April 2022. Participants 25-70 years old visiting a general practitioner (general population) or those referred to a colposcopy clinic with changes in their cervical cytology (colposcopy population) collected vaginal self-sample and completed a paper-based questionnaire. Samples were analyzed with Cobas 6800 System (Roche) for HPV16, HPV18 and other HR-HPV (HPV31/33/35/39/45/51/52/56/58/59/66/68). Descriptive statistics for categorical variables were performed. The Chi-square test was used to determine for the statistical significance of differences in the proportions of the dependent variable between subgroups of the independent variable. Univariate and multivariate binary logistic regression were used to identify factors associated with positive HR-HPV status. Results were considered statistically significant at P < 0.05. RESULTS: A total of 1274 participants provided a valid sample. The prevalence of any HR-HPV infection was 66.8% in the colposcopy group and 11.0% in the general population. Factors associated with positive HR-HPV status were marital status single/divorced/widowed (vs. married/cohabiting) [adjusted OR (aOR) 2.6; P = 0.003], higher number of lifetime sex partners [aOR 5.1 (P < 0.001) and 4.0 (P = 0.001)] for six or more and three to five partners in the general population; in the colposcopy group, the statistical significance remained only for Latvian ethnicity (vs. other) (aOR 1.8; P = 0.008) and current smoking (vs. never) (aOR 1.9; P = 0.01). CONCLUSION: We documented a comparison to European Union HR-HPV infection burden in Latvia. Any HR-HPV positivity was significantly associated with sexual and other health behavior.

Advantages of analysing both pairwise SNV-distance and differing SNVs between Mycobacterium tuberculosis isolates for recurrent tuberculosis cause determination.

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.

LC-MS/MS method for simultaneous quantification of the first-line anti-tuberculosis drugs and six primary metabolites in patient plasma: Implications for therapeutic drug monitoring.

The pharmacokinetic profiling of drug substances and corresponding metabolites in the biological matrix is one of the most informative tools for the treatment efficacy assessment. Therefore, to satisfy the need for comprehensive monitoring of anti-tuberculosis drugs in human plasma, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of first-line anti-tuberculosis drugs (ethambutol, isoniazid, pyrazinamide, and rifampicin) along with their six primary metabolites. Simple single-step protein precipitation with methanol was chosen as the most convenient sample pre-treatment method. Chromatographic separation of the ten analyte mixture was achieved within 10 minutes on a reverse-phase C8 column using mobile phase gradient mode. The multiple reaction monitoring mode (MRM) was used for analyte detection and quantification in patient samples. The chosen quantification ranges fully covered expected plasma concentrations. The method exhibited acceptable selectivity; the within- and between-run accuracy ranged from 87.2 to 113.6%, but within- and between-run precision was between 1.6 and 14.9% (at the LLOQ level CV < 20%). Although the response of the isonicotinic acid varied depending on the matrix source (CV 21.8%), validation results proved that such inconsistency does not affect the accuracy and precision of results. If stored at room temperature plasma samples should be processed within 4 h after collection, temporary storage at -20 °C up to 24 h is acceptable due to stability issues of analytes. The developed method was applied for the patient sample analysis (n = 34) receiving anti-tuberculosis treatment with the first-line drugs.

Implementation of a next-generation sequencing-based targeted approach for full-length CYP3A4 gene sequencing.

Aim: To evaluate the application of next-generation sequencing-based targeted protocol for full-length CYP3A4 gene sequencing analysis. Materials & methods: The developed sequencing protocol was applied to analyze human DNA samples (n = 7) obtained from tuberculosis patients admitted to the Riga East University Hospital, Center of Tuberculosis and Lung diseases. Results: The sequencing data quality was sufficient for the detection of already known genetic variants, as well as for identifying rare and novel variants dispersed throughout the CYP3A4 gene with a high degree of confidence. Conclusion: Developed protocol can be applied in subpopulation level association studies to determine whether specific genetic variants or variant combinations from multiple regions of the CYP3A4 gene are of clinical significance.

Application of whole-genome sequencing in a case study of renal tuberculosis in a child.

BACKGROUND: Urogenital tuberculosis (TB) is rare in children and usually develops due to reactivation of the foci in the genitourinary tract after the latency period following initial infection. Urogenital TB in children has no pathognomonic clinical features that can result in overlooking or misdiagnosing this clinical entity. Here, we report important findings regarding the pathogenesis and transmission of TB by using genotyping and whole-genome sequencing (WGS) in a study of renal TB case in a child. CASE PRESENTATION: A 13-year-old boy was admitted to the hospital because of high fever, severe dry cough, flank pain and painful urination. Abdominal ultrasonography and CT revealed an 8 mm calculus in the kidney, and clinical findings were initially interpreted as nephrolithiasis. Nevertheless, due to the atypical clinical presentation of kidney stone disease, additional investigations for possible TB were performed. The QuantiFERON®-TB Gold Plus test was positive, and the Mantoux test resulted in 15 mm of induration, confirming infection with Mycobacterium tuberculosis (Mtb). Chest X-ray was normal. Chest CT revealed calcified intrathoracic lymph nodes. The urine sample tested positive for acid-fast bacilli, and Mtb cultures were obtained from urine and bronchial aspirate samples, resulting in a final diagnosis of intrathoracic lymph node and renal TB. Contact investigation revealed that the child's father was diagnosed with TB when the child was 1 year old. Genotyping and WGS analysis of Mtb isolates of the child and his father confirmed the epidemiological link and pointed to the latency of infection in the child. CONCLUSIONS: This case report confirmed the development of active TB from calcified lesions in adolescent after 12 years of exposure, demonstrated the absence of microevolutionary changes in the Mtb genome during the period of latency, and proved the importance of appropriate evaluation and management to prevent the progression of TB infection to active TB disease. The use of WGS provided the ultimate resolution for the detection of TB transmission and reactivation events.

Analysis of Mycobacterium tuberculosis genetic lineages circulating in Riga and Riga region, Latvia, isolated between 2008 and 2012.

Although the number of new tuberculosis (TB) cases registered per year has decreased by 3-fold between 2001 and 2017 in Latvia, the TB incidence and rates of multidrug resistant TB in this Baltic country remain substantially higher than in most other European countries. Molecular typing methods of Mycobacterium tuberculosis (MTB) play an important role both in clinical studies of the disease and the epidemiological investigations, allowing to describe and characterize the pathogen's population structure and spread of particular genotypes. Aim of this study was to examine the prevalence of MTB lineages in Riga and Riga region of Latvia within a five-year period (2008-2012), and to evaluate the discriminatory power (DP) of spoligotyping, standard 24-locus MIRU-VNTR and IS6110-RFLP methods in this setting. The results showed that the main MTB spoligotype families were Beijing (25.3%) and LAM (24.3%), followed by T (22.1%), Ural (11.2%), Haarlem (6.6%) and X superfamily (3.4%). This distribution remained stable over the five consecutive years. 67.6% of MTB isolates were pan-susceptible, and 32.4% were resistant to any drug; multi-drug resistance was found in 5.8% of MTB strains, and 7.6% of MTB isolates were extensively drug-resistant. Drug resistance was associated with SIT1, SIT283 and SIT42 genotypes, while SIT1 and SIT42 were overrepresented among multi drug-resistant MTB strains. Overall, DP of spoligotyping method alone was 0.8953, while DP of both 24-locus MIRU-VNTR analysis and IS6110 RFLP was higher (DP = 0.9846 and 0.9927, respectively), mainly due to the improvement of the resolution for the Beijing strains. In conclusion, this work represents the first comprehensive molecular epidemiological description of TB in Latvia, highlighting the high genetic diversity of MTB strains circulating in Riga and Riga region. In combination with detailed epidemiological data this approach was helpful for the in-depth understanding of epidemiological processes in settings where the Next-Gen sequencing is not available as a routine method.

Mycobacterium tuberculosis RD-Rio Strain in Kazakhstan.

Mycobacterium tuberculosis RD-Rio strains are still rare in the former Soviet Union countries and Asia. We describe a strain in Kazakhstan that belongs to the RD-Rio secondary branch, which is endemic to northwest Russia and eastern Europe. Although RD-Rio strains are frequently multidrug resistant, this heterogeneous branch included only drug-susceptible isolates.

Mycobacterium tuberculosis Acquires Limited Genetic Diversity in Prolonged Infections, Reactivations and Transmissions Involving Multiple Hosts.

Background:Mycobacterium tuberculosis (MTB) has limited ability to acquire variability. Analysis of its microevolution might help us to evaluate the pathways followed to acquire greater infective success. Whole-genome sequencing (WGS) in the analysis of the transmission of MTB has elucidated the magnitude of variability in MTB. Analysis of transmission currently depends on the identification of clusters, according to the threshold of variability (<5 SNPs) between isolates. Objective: We evaluated whether the acquisition of variability in MTB, was more frequent in situations which could favor it, namely intrapatient, prolonged infections or reactivations and interpatient transmissions involving multiple sequential hosts. Methods: We used WGS to analyze the accumulation of variability in sequential isolates from prolonged infections or translations from latency to reactivation. We then measured microevolution in transmission clusters with prolonged transmission time, high number of involved cases, simultaneous involvement of latency and active transmission. Results: Intrapatient and interpatient acquisition of variability was limited, within the ranges expected according to the thresholds of variability proposed, even though bursts of variability were observed. Conclusions: The thresholds of variability proposed for MTB seem to be valid in most circumstances, including those theoretically favoring acquisition of variability. Our data point to multifactorial modulation of microevolution, although further studies are necessary to elucidate the factors underlying this modulation.

Genotype and allele frequencies of isoniazid-metabolizing enzymes NAT2 and GSTM1 in Latvian tuberculosis patients.

Pharmacogenomic testing of tuberculosis drug-metabolizing enzyme genes was proposed as a strategy to identify patients at risk for suboptimal responses to medications. However, variations of the genotype frequencies among ethnic groups exist and new alleles are been identified. The aim of this study was to identify polymorphisms of genes encoding metabolic enzymes NAT2 and GSTM1 in tuberculosis patients in Latvia and to estimate the frequency of NAT2 slow acetylator and GSTM1 null genotypes. In total, 85 DNA samples were genotyped, all individuals were Caucasian. An ethnic heterogeneity reflecting the multiethnic population of the country was observed. 49 patients were Latvians, 30 were Russians and 6 of other ethnicity. In total, 7 NAT2 alleles were identified: *4, *5, *6, *7, *11, *12, * and *13. The most frequent was the slow acetylation allele NAT2*6 (frequency 0.388) followed by the slow acetylation allele NAT2*5 and the rapid acetylation allele NAT2*4 (frequencies 0.306 and 0.194, respectively). The predominance of slow (51.8%) and intermediate (43.5%) acetylators compared with rapid acetylators (4.7%) was observed. The GSTM1 null genotype was detected in 48.2% of tuberculosis patients. When subgroup analysis was performed according to ethnicity, the results showed that neither NAT2 allele frequencies nor GSTM1 null genotype frequency did not differ significantly in TB patients of Latvian or Russian ethnicity. Overall, genotyping results were similar with previous reports of a NAT2 gene variation and GSTM1 null genotype frequency in Caucasians. Our findings have a contribution for the pharmacogenetics-based tuberculosis therapy in Latvia in future.

Latin-American-Mediterranean lineage of Mycobacterium tuberculosis: Human traces across pathogen's phylogeography.

Currently, Mycobacterium tuberculosis isolates of Latin-American Mediterranean (LAM) family may be detected far beyond the geographic areas that coined its name 15years ago. Here, we established the framework phylogeny of this geographically intriguing and pathobiologically important mycobacterial lineage and hypothesized how human demographics and migration influenced its phylogeography. Phylogenetic analysis of LAM isolates from all continents based on 24 variable number of tandem repeats (VNTR) loci and other markers identified three global sublineages with certain geographic affinities and defined by large deletions RD115, RD174, and by spoligotype SIT33. One minor sublineage (spoligotype SIT388) appears endemic in Japan. One-locus VNTR signatures were established for sublineages and served for their search in published literature and geographic mapping. We suggest that the LAM family originated in the Western Mediterranean region. The most widespread RD115 sublineage seems the most ancient and encompasses genetically and geographically distant branches, including extremely drug resistant KZN in South Africa and LAM-RUS recently widespread across Northern Eurasia. The RD174 sublineage likely started its active spread in Brazil; its earlier branch is relatively dominated by isolates from South America and the derived one is dominated by Portuguese and South/Southeastern African isolates. The relatively most recent SIT33-sublineage is marked with enigmatic gaps and peaks across the Americas and includes South African clade F11/RD761, which likely emerged within the SIT33 subpopulation after its arrival to Africa. In addition to SIT388-sublineage, other deeply rooted, endemic LAM sublineages may exist that remain to be discovered. As a general conclusion, human mass migration appears to be the major factor that shaped the M. tuberculosis phylogeography over large time-spans.

Comparison of telomere length between population-specific mitochondrial haplogroups among different age groups in a Latvian population.

Population studies have demonstrated that telomere length (TL) displays great diversity among different populations. Previously described controversial findings associated longevity with specific mitochondrial DNA haplogroups (hgs) (e.g., J and U). These observations may be influenced by population diversity, geographic location, and/or specific historic background. The aims of this study were to identify a specific hg which correlates with aging in a Latvian populating and to evaluate the possible association of TL variability with specific mitochondrial hgs. The results show no significant correlation between TL, mitochondrial DNA hgs and longevity. A slight increase in frequency was observed among centenarians of hg H; however, these findings were not statistically significant. TL did not show any statically significant difference, only hg W had slightly longer telomeres among others. An insignificant increase in TL was observed in the 55-89 age group of hg W but in the <90 age group for hg J which also had the longest TL in the 20-45 age group. In conclusion this study indicates that specific mitochondrial DNA hgs do not have a significant, if any, influence on the variation of TL in Latvians.

Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages.

Point mutations associated with Leber hereditary optic neuropathy in a Latvian population.

PURPOSE: To study mutations associated with Leber hereditary optic neuropathy (LHON) in patients suspected of having this mitochondrial disorder in a Latvian population. Additional aims were to determine the heteroplasmy status of all non-synonymous polymorphisms identified in the current study and to identify the mitochondrial haplogroups of the studied participants because these factors may contribute to the manifestation of LHON. METHODS: Twelve patients, including patients in two families, were enrolled in the current study. LHON was suspected based on the findings of ophthalmologic examinations. In clinically affected individuals, the presence of all previously reported LHON-associated mutations was assessed with sequencing analysis. Additionally, the SURVEYOR endonuclease assay was used to detect heteroplasmy. The mitochondrial haplogroups were identified with restriction analysis and the sequencing of hypervariable segment 1. RESULTS: In one family (mother and son), there was one primary LHON-associated mutation, G11778A. In addition, one rare previously reported LHON-associated polymorphism, A13637G, was detected in two unrelated patients. A non-synonymous polymorphism at T6253C was found in one individual. This mutation was reported in the background of the 3460 mutation among LHON patients in a Chinese population. No non-synonymous point mutations in mitochondrial DNA were found in five of the study participants. CONCLUSIONS: Molecular analysis of 12 patients with suspected LHON confirmed the diagnosis in four patients and allowed the use of appropriate prophylactic measures and treatment. Further investigations and additional studies of different populations are necessary to confirm the role of the non-synonymous polymorphisms A13637G and T6253C in the manifestation of LHON and the associations of these polymorphisms with mitochondrial haplogroups and heteroplasmy.