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.

Effect of NAT2, GSTM1 and CYP2E1 genetic polymorphisms on plasma concentration of isoniazid and its metabolites in patients with tuberculosis, and the assessment of exposure-response relationships.

Objectives: Isoniazid is a key drug in the chemotherapy of tuberculosis (TB), however, interindividual variability in pharmacokinetic parameters and drug plasma levels may affect drug responses including drug induced hepatotoxicity. The current study investigated the relationships between isoniazid exposure and isoniazid metabolism-related genetic factors in the context of occurrence of drug induced hepatotoxicity and TB treatment outcomes. Methods: Demographic characteristics and clinical information were collected in a prospective TB cohort study in Latvia (N = 34). Time to sputum culture conversion (tSCC) was used as a treatment response marker. Blood plasma concentrations of isoniazid (INH) and its metabolites acetylisoniazid (AcINH) and isonicotinic acid (INA) were determined at three time points (pre-dose (0 h), 2 h and 6 h after drug intake) using liquid chromatography-tandem mass spectrometry. Genetic variations of three key INH-metabolizing enzymes (NAT2, CYP2E1, and GSTM1) were investigated by application PCR- and Next-generation sequencing-based methods. Depending on variables, group comparisons were performed by Student's t-test, one-way ANOVA, Mann-Whitney-Wilcoxon, and Kruskal-Wallis tests. Pearson correlation coefficient was calculated for the pairs of normally distributed variables; model with rank transformations were used for non-normally distributed variables. Time-to-event analysis was performed to analyze the tSCC data. The cumulative probability of tSCC was obtained using Kaplan-Meier estimators. Cox proportional hazards models were fitted to estimate hazard rate ratios of successful tSCC. Results: High TB treatment success rate (94.1%) was achieved despite the variability in INH exposure. Clinical and demographic factors were not associated with either tSCC, hepatotoxicity, or INH pharmacokinetics parameters. Correlations between plasma concentrations of INH and its metabolites were NAT2 phenotype-dependent, while GSTM1 genetic variants did not showed any effects. CYP2E1*6 (T > A) allelic variant was associated with INH pharmacokinetic parameters. Decreased level of AcINH was associated with hepatotoxicity, while decreased values of INA/INH and AcINH/INH were associated with month two sputum culture positivity. Conclusion: Our findings suggest that CYP2E1, but not GSTM1, significantly affects the INH pharmacokinetics along with NAT2. AcINH plasma level could serve as a biomarker for INH-related hepatotoxicity, and the inclusion of INH metabolite screening in TB therapeutic drug monitoring could be beneficial in clinical studies for determination of optimal dosing strategies.

Exploring Variability in Rifampicin Plasma Exposure and Development of Anti-Tuberculosis Drug-Induced Liver Injury among Patients with Pulmonary Tuberculosis from the Pharmacogenetic Perspective.

Genetic polymorphisms can exert a considerable impact on drug pharmacokinetics (PK) and the development of adverse drug reactions (ADR). However, the effect of genetic polymorphisms on the anti-tuberculosis (anti-TB) drug, and particularly rifampicin (RIF), exposure or anti-TB drug-induced liver injury (DILI) remains uncertain. Here, we evaluated the relationship between single nucleotide polymorphisms (SNPs) detected in the RIF pharmacogenes (AADAC, SLCO1B1, SLCO1B3, ABCB1, and NR1I2) and RIF PK parameters, as well as anti-TB treatment-associated DILI. In total, the study enrolled 46 patients with drug-susceptible pulmonary TB. The RIF plasma concentration was measured using the LC-MS/MS method in the blood samples collected pre-dose and 2 and 6 h post-dose, whilst the DILI status was established using the results from blood biochemical analysis performed before and 10-12 days after treatment onset. The genotyping was conducted using a targeted NGS approach. After adjustment for confounders, the patients carrying the rs3732357 GA/AA genotype of the NR1I2 gene were found to have significantly lower RIF plasma AUC0-6 h in comparison to those with GG genotype, while the difference in RIF plasma Cmax was insignificant. None of the analyzed SNPs was related to DILI. Hence, we are the first to report NR1I2 intronic SNP rs3732357 as the genetic component of variability in RIF exposure. Regarding anti-TB treatment-associated DILI, the other preexisting factors promoting this ADR should be considered.

Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia.

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.

Comparative Microbiome Analysis of Three Epidemiologically Important Tick Species in Latvia.

(1) Background: Amplicon-based 16S rRNA profiling is widely used to study whole communities of prokaryotes in many niches. Here, we comparatively examined the microbial composition of three tick species, Ixodes ricinus, Ixodes persulcatus and Dermacentor reticulatus, which were field-collected in Latvia. (2) Methods: Tick DNA samples were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). (3) Results: The results showed significant differences in microbial species diversity and composition by tick species and life stage. A close similarity between microbiomes of I. ricinus and I. persulcatus ticks was observed, while the D. reticulatus microbiome composition appeared to be more distinct. Significant differences in alpha and beta microbial diversity were observed between Ixodes tick life stages and sexes, with lower taxa richness indexes obtained for female ticks. The Francisella genus was closely associated with D. reticulatus ticks, while endosymbionts Candidatus Midichlorii and Candidatus Lariskella were associated with I. ricinus and I. persulcatus females, respectively. In I. ricinus females, the endosymbiont load negatively correlated with the presence of the Rickettsia genus. (4) Conclusions: The results of this study revealed important associations between ticks and their microbial community and highlighted the microbiome features of three tick species in Latvia.

Assessment of Amikacin- and Capreomycin-Related Adverse Drug Reactions in Patients with Multidrug-Resistant Tuberculosis and Exploring the Role of Genetic Factors.

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.

Tracing microbial communities associated with archaeological human samples in Latvia, 7-11th centuries AD.

In the grave environment, microorganisms are major ecological participants in the successional decomposition of vertebrates and could infiltrate the skeleton/skeletal material during taphonomic processes. The diversity of archaeological skeleton-associated microbial assemblages and the impact of various factors are poorly understood. This study aimed to evaluate the taxonomic microbial composition of archaeological human bone and teeth samples from the 7th to 11th centuries AD from two burial sites in Latvia. Samples were analysed by a shotgun metagenomics-based approach. The results showed a strong presence of the environmental DNA in the samples, and variability in microbial community structure between individual samples. Differences in microbial composition were observed between bone and tooth samples, as well as between different burial sites. Furthermore, the presence of endogenous ancient DNA (aDNA) in tooth samples was detected. Overall, compositions of microbial communities associated with archaeological human remains in Latvia dated 7-11th century AD were influenced by the sample type and burial location. These findings indicate that, while the content of historical DNA in archaeological samples is low, the comparison of archaeological skeleton-associated microbial assemblages across time and space, along with aDNA damage profile analysis, is important and could help to reveal putative ancient microorganisms.

Oral microbiome variations related to ageing: possible implications beyond oral health.

The global population is getting older due to a combination of longer life expectancy and declining birth rates. Growing evidence suggests that the oral microbiota composition and distribution may have a profound effect on how well we age. The purpose of this study was to investigate age-related oral microbiome variations of supragingival plaque and buccal mucosa samples in the general population in Latvia. Our results indicated significant difference between supragingival plaque bacterial profiles of three age groups (20-40; 40-60; 60 + years). Within supragingival plaque samples, age group 20-40 showed the highest bacterial diversity with a decline during the 40-60 age period and uprise again after the age of 60. Among other differences, the important oral commensal Neisseria had declined after the age of 40. Additionally, prevalence of two well-documented opportunistic pathogens Streptococcus anginosus and Gemella sanguinis gradually rose with age within our samples. Furthermore, supragingival plaque and buccal mucosa samples significantly differed in overall bacterial composition.

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.

Application of natural sciences methodology in archaeological study of Iron Age burials in Latvia: pilot study.

Natural sciences provide several modern methodologies that could be successfully applied in archaeological studies. In this pilot study, archaeological human remains from two Iron Age cemeteries (7th-twelfth centuries AD), Lejasbiteni and Cunkani-Drengeri, which are located in different regions of Latvia, were studied. We applied ancient DNA (aDNA) and tooth enamel peptide analysis to determine the biological sex of the individuals. In addition, aDNA analysis was used to perform mtDNA haplogroup analysis. In most cases, the results of aDNA analysis regarding the biological sex of individuals coincided with the gender assigned based on grave orientation and grave goods. The results of sex determination using peptide analysis in all four individuals for whom data were available matched the possible gender. Of the 17 samples that had sufficient DNA for sequencing, seven samples had enough reads to perform mtDNA haplogroup analysis. The H2a2a, I4a1, H2a2a1, and H16c mtDNA haplogroups were identified in the individuals from the Lejasbiteni cemetery, while the T2b and K1a + 150 mtDNA haplogroups were identified in the individuals from the Cunkani-Drengeri cemetery. Overall, the obtained results demonstrated the feasibility of applying aDNA and tooth enamel peptide analysis for biological sex determination within archaeological studies. The availability of human aDNA data will be highly useful for investigating the demographic history and social structures in Iron Age Latvia.

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis.

Introduction: Pharmacogenetics studies provide clinically relevant information on the identified associations between genetic variants and individual variability in drug response, which, in turn, offers great promise for guiding personalized drug therapy and clinical trial design. However, there is a lack of information concerning the evidence-based clinical annotations of specific CYP2E1 genetic variants. Aim: To design and evaluate the next-generation sequencing-based method for full-length CYP2E1 gene polymorphism analysis. Materials and Methods: Seven gene-specific oligonucleotide primer pairs targeting overlapping CYP2E1 gene fragments spanning all nine gene exons with interleaving introns, untranslated (UTR) and intergenic regions were designed. Human DNA samples (n = 3) were used as a training set to check the primer performance and to optimize the PCR conditions. The effectiveness of the developed target amplification and sequencing protocol was evaluated using the test set comprising human DNA samples (n = 3) obtained from tuberculosis patients. Sequencing data analysis was performed on the Galaxy online-based platform. Results: The sequencing data quality was sufficient for the detection of genetic variants dispersed throughout the CYP2E1 gene with a high degree of confidence in fully covered regions achieving optimal reading depth of the targeted fragment with high base call accuracy. Conclusion: Developed protocol can be applied in subpopulation-level association studies to determine whether single nucleotide variants (SNVs) or variant combinations from multiple regions of the CYP2E1 gene are of clinical significance.

Babesiosis in Southeastern, Central and Northeastern Europe: An Emerging and Re-Emerging Tick-Borne Disease of Humans and Animals.

There is now considerable evidence that in Europe, babesiosis is an emerging infectious disease, with some of the causative species spreading as a consequence of the increasing range of their tick vector hosts. In this review, we summarize both the historic records and recent findings on the occurrence and incidence of babesiosis in 20 European countries located in southeastern Europe (Bosnia and Herzegovina, Croatia, and Serbia), central Europe (Austria, the Czech Republic, Germany, Hungary, Luxembourg, Poland, Slovakia, Slovenia, and Switzerland), and northern and northeastern Europe (Lithuania, Latvia, Estonia, Iceland, Denmark, Finland, Sweden, and Norway), identified in humans and selected species of domesticated animals (cats, dogs, horses, and cattle). Recorded cases of human babesiosis are still rare, but their number is expected to rise in the coming years. This is because of the widespread and longer seasonal activity of Ixodes ricinus as a result of climate change and because of the more extensive use of better molecular diagnostic methods. Bovine babesiosis has a re-emerging potential because of the likely loss of herd immunity, while canine babesiosis is rapidly expanding in central and northeastern Europe, its occurrence correlating with the rapid, successful expansion of the ornate dog tick (Dermacentor reticulatus) populations in Europe. Taken together, our analysis of the available reports shows clear evidence of an increasing annual incidence of babesiosis across Europe in both humans and animals that is changing in line with similar increases in the incidence of other tick-borne diseases. This situation is of major concern, and we recommend more extensive and frequent, standardized monitoring using a "One Health" approach.

Development of rapid antigen test prototype for detection of SARS-CoV-2 in saliva samples.

Background: The development of easy-to-perform diagnostic methods is highly important for detecting current coronavirus disease (COVID-19). This pilot study aimed at developing a lateral flow assay (LFA)-based test prototype to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva samples. Methods: Mice were immunized using the recombinant receptor-binding domain (rRBD) of SARS-CoV-2 virus spike protein. The combinations of the obtained mouse anti-receptor-binding domain (RBD) polyclonal antibodies (PAbs) and several commercial antibodies directed against the SARS-CoV-2 spike protein were used for enzyme-linked immunosorbent assay (ELISA) to select antibody pairs for LFA. The antibody pairs were tested in a LFA format using saliva samples from individuals with early SARS-CoV-2 infection (n = 9). The diagnostic performance of the developed LFA was evaluated using saliva samples from hospitalized COVID-19 patients (n = 111); the median time from the onset of symptoms to sample collection was 10 days (0-24 days, interquartile range (IQR): 7-13). The reverse transcription-polymerase chain reaction (rRT-PCR) was used as a reference method. Results: Based on ELISA and preliminary LFA results, a combination of mouse anti-RBD PAbs (capture antibody) and rabbit anti-spike PAbs (detection antibody) was chosen for clinical analysis of sample. When compared with rRT-PCR results, LFA exhibited 26.5% sensitivity, 58.1% specificity, 50.0% positive prediction value (PPV), 33.3% negative prediction value (NPV), and 38.7% diagnostic accuracy. However, there was a reasonable improvement in assay specificity (85.7%) and PPV (91.7%) when samples were stratified based on the sampling time. Conclusion: The developed LFA assay demonstrated a potential of SARS-CoV-2 detection in saliva samples. Further technical assay improvements should be made to enhance diagnostic performance followed by a validation study in a larger cohort of both asymptomatic and symptomatic patients in the early stage of infection.

Telomere length and mitochondrial DNA copy number in multidrug-resistant tuberculosis.

Multidrug resistant tuberculosis (MDR-TB) is a severe disease that requires prolonged chemotherapy and is associated with an increased probability of treatment failure and death. MDR-TB is a state of heightened oxidative stress and inflammation, which could be related to the aging-related processes and immunosenescence. We, therefore, tested the hypothesis that MDR-TB is associated with alterations in aging biomarkers in peripheral blood cells. We investigated 51 MDR-TB patients and 57 healthy individuals and carried out an analysis of covariance to assess the possible impact of different variables on biomarker perturbations. The results showed that MDR-TB patients had significantly reduced telomere length (TL) and increased mitochondrial DNA copy number (mtDNA CN) (P < 0.05) in comparison to the controls, and MDR-TB infection was the main influencing factor. Male sex and extrapulmonary TB strongly influenced mtDNA CN increment, and MDR-TB patients with normal weight had longer telomeres than those who were underweight (P < 0.05). In conclusion, the evidence for shorter telomeres and higher mtDNA CN in the peripheral blood cells of MDR-TB patients was obtained indicating the connection between MDR-TB and aging biomarkers. The observed associations highlight a complicated interplay between MDR-TB and immunosenescence, thus further studies are required to achieve full understanding.

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.

Haemotropic Mycoplasma species in pet cats in Latvia: a study, phylogenetic analysis and clinical case report.

OBJECTIVES: The aim of this study was to evaluate whether haemotropic Mycoplasma species are detected in pet cats in Latvia, to perform a phylogenetic analysis of the detected pathogens and to report a clinical case of feline infectious anaemia. METHODS: Peripheral blood samples (n = 125) from pet cats were submitted; 99 samples were adequate to test for the presence of Mycoplasma species DNA by nested PCR. A clinical case was added in the later stages of the study. Positive isolates were subjected to phylogenetic analysis. RESULTS: The prevalence of 'Candidatus Mycoplasma haemominutum' was 15% (n = 15/99), that of Mycoplasma haemofelis was 5% (5/99) and that of 'Candidatus Mycoplasma turicensis' was 2% (n = 2/99). Cases of coinfection included 'Candidatus M haemominutum' + M haemofelis (4%; n = 4/99) and 'Candidatus M haemominutum' + 'Candidatus M turicensis' (1%; n = 1/99). This is the first published report of M haemofelis infection in the Baltic states. Two different 'Candidatus M turicensis' isolates were discovered after phylogenetic analysis. CONCLUSIONS AND RELEVANCE: This report is the first of an autochthonous feline infectious anaemia case in the Baltic region. The prevalence of Mycoplasma species was similar to that in other northern European countries. Phylogenetic analysis revealed variability of the isolates; one of the 'Candidatus M turicensis' genotypes was detected for the first time in Europe.

Development of a real-time PCR method for rapid diagnosis of canine babesiosis and anaplasmosis.

BACKGROUND: Canine babesiosis and anaplasmosis, caused by Babesia canis and Anaplasma phagocytophilum, respectively, are significant tick-borne diseases in Baltic countries. Both diseases can be diagnosed on the basis of clinicopathological findings, by direct pathogen detection in blood smears or by indirect pathogen detection; however, because of high selectivity and specificity, molecular methods may be advantageous. The goal of this study was to develop a duplex real-time polymerase chain reaction (RT-PCR) method for the detection of B. canis and A. phagocytophilum in canine clinical samples. METHODS: Sequence-based polymorphism analysis of genes encoding B. canis-specific merozoite surface protein Bc28.1 (Bc28.1) and A. phagocytophilum malate dehydrogenase (mdh) was performed on pathogen isolates present in Latvian domestic dogs. The obtained results were used to design a species-specific duplex RT-PCR assay. RESULTS: The presence of three B. canis Bc28.1 gene sequence types was revealed in canine samples with a nonuniform geographical distribution, and two types of A. phagocytophilum mdh genes were detected. The novel duplex RT-PCR assay provided correct classification of samples positive and negative for B. canis and A. phagocytophilum. The analytical sensitivity of this assay was ten gene copies/ reaction for both pathogens. CONCLUSIONS: A novel duplex RT-PCR molecular method was developed for the detection of B. canis and A. phagocytophilum in canine clinical samples. Sequence variability of Bc28.1 and mdh genes indicated the genetic variability of B. canis and A. phagocytophilum isolates occurring in Latvian domestic dogs.

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.

The Postmedieval Latvian Oral Microbiome in the Context of Modern Dental Calculus and Modern Dental Plaque Microbial Profiles.

Recent advantages in paleomicrobiology have provided an opportunity to investigate the composition of ancient microbial ecologies. Here, using metagenome analysis, we investigated the microbial profiles of historic dental calculus retrieved from archaeological human remains from postmedieval Latvia dated 16-17th century AD and examined the associations of oral taxa and microbial diversity with specific characteristics. We evaluated the preservation of human oral microbiome patterns in historic samples and compared the microbial composition of historic dental calculus, modern human dental plaque, modern human dental calculus samples and burial soil microbiota. Overall, the results showed that the majority of microbial DNA in historic dental calculus originated from the oral microbiome with little impact of the burial environment. Good preservation of ancient DNA in historical dental calculus samples has provided reliable insight into the composition of the oral microbiome of postmedieval Latvian individuals. The relative stability of the classifiable oral microbiome composition was observed. Significant differences between the microbiome profiles of dental calculus and dental plaque samples were identified, suggesting microbial adaptation to a specific human body environment.