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.

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.

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.

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.

Large-scale countrywide screening for tick-borne pathogens in field-collected ticks in Latvia during 2017-2019.

BACKGROUND: Tick-borne diseases are of substantial concern worldwide in both humans and animals. Several hard tick species are of medical and veterinary interest in Europe, and changes in the range of tick species can affect the spread of zoonotic pathogens. The aim of the present study was to map the current prevalence and distribution pattern of ticks and related tick-borne pathogens in Latvia, a Baltic state in northern Europe. METHODS: Nearly 4600 Ixodes ricinus, I. persulcatus and Dermacentor reticulatus tick samples were collected in all regions of Latvia during 2017-2019 and were screened by molecular methods to reveal the prevalence and distribution pattern of a wide spectrum of tick-borne pathogens. RESULTS: New localities of D. reticulatus occurrence were found in western and central Latvia, including the Riga region, indicating that the northern border of D. reticulatus in Europe has moved farther to the north. Among the analyzed ticks, 33.42% carried at least one tick-borne pathogen, and 5.55% of tick samples were positive for two or three pathogens. A higher overall prevalence of tick-borne pathogens was observed in I. ricinus (34.92%) and I. persulcatus (31.65%) than in D. reticulatus (24.2%). The molecular analysis revealed the presence of tick-borne encephalitis virus, Babesia spp., Borrelia spp., Anaplasma phagocytophilum and Rickettsia spp. Overall, 15 and 7 tick-borne pathogen species were detected in Ixodes spp. and D. reticulatus ticks, respectively. This is the first report of Borrelia miyamotoi in Latvian field-collected ticks. CONCLUSIONS: This large-scale countrywide study provides a snapshot of the current distribution patterns of Ixodes and Dermacentor ticks in Latvia and gives us a reliable overview of tick-borne pathogens in Latvian field-collected ticks.

Babesiosis in Latvian domestic dogs, 2016-2019.

Canine babesiosis is tick-borne infection that represents a major veterinary issue in Central and Eastern Europe with a tendency to expand northwards. The first published report in Latvia about autochthonous cases of babesiosis in domestic dogs with no travel history was in 2013, and to the best of our knowledge, no other studies on this issue have been published to date. The aim of this study was to analyze the occurrence and clinical manifestations of babesiosis in Latvian domestic dogs with a history of tick exposure to determine the extent to which Babesia sp. causes the disease and to map outbreaks in Latvia. From 2016 to 2019, blood samples from dogs were collected, and molecular testing was performed by nested PCR using Babesia sp.-specific primers. In total, 43 of 262 samples were Babesia canis-positive. A seasonal pattern was observed for the outbreaks, as the majority of B. canis-positive samples (98%) were submitted between April and June, and there was a single canine babesiosis case recorded in October. Nearly half of the cases (46.5%) were recorded in the capital, Riga, and other cases were recorded in southern and western parts of Latvia. Clinical signs were consistent with typical manifestations of acute canine babesiosis; most common hematological changes were thrombocytopenia (89%) and normocytic normochromic anemia (69%). Blood smear microscopy was positive for 79% of cases. Two B. canis genotypes were distinguished on the basis of two nucleotide (GA → AG) substitutions in the 18S rRNA gene at positions 610/611; however, no relationship between the genotypes and the severity of the disease was found. In conclusion, canine babesiosis has become an endemic disease in the southern and western regions of Latvia and is caused solely by the large babesia species B. canis. Awareness among veterinarians and pet owners regarding the disease should be increased.

Tick-borne pathogens in ticks collected from dogs, Latvia, 2011-2016.

BACKGROUND: Different tick species are able to transmit different pathogens, and tick-borne diseases are of substantial concern worldwide for both humans and animals. Environmental changes and changes in the range of tick species, including Dermacentor reticulatus in Europe, can affect the spread of zoonotic pathogens. The aim of this study was to investigate the prevalence of the tick-borne pathogens in ticks removed from dogs in Latvia, and to explore possible changes between years 2011 and 2016. RESULTS: In 2011, only Ixodes ticks (221 Ixodes ricinus and 22 Ixodes persulcatus) were collected from dogs, while in 2016 tick samples belonged to Ixodes ricinus (360), Ixodes persulcatus (2) and Dermacentor reticulatus (27) species. In total, 35.8 and 40.0% of adult ticks were pathogen-positive in 2011 and 2016, respectively; the difference was not statistically significant (P > 0.05). The molecular analysis indicated the presence of 13 tick-borne microorganisms; the most prevalent pathogen was Rickettsia, followed by Borrelia burgdorferi sensu lato group spirochetes, Anaplasma phagocytophilum and Babesia species. Borrelia miyamotoi was also present. A co-infection with two and three tick-borne pathogens was detected in 7.9 and 7.4% of Ixodes ricinus and Dermacentor reticulatus, respectively. The results of this study confirmed that the spread of novel vectors could bring additional risk of exposure to novel emerging pathogens to pets and their owners, as both Babesia canis and Rickettsia raoultii were shown to be highly associated with Dermacentor reticulatus ticks in Latvia. CONCLUSIONS: This study demonstrates the potential danger from the inadvertent introduction of novel disease pathogens and vectors. Awareness of co-infections and Dermacentor reticulatus-related pathogens needs to be increased.

The prevalence of mitochondrial mutations associated with aminoglycoside-induced deafness in ethnic Latvian population: the appraisal of the evidence.

Aminoglycosides are potent antibiotics which are used to treat severe gram-negative infections, neonatal sepsis, and multidrug-resistant tuberculosis. Ototoxicity is a well-known side effect of aminoglycosides, and a rapid, profound, and irreversible hearing loss can occur in predisposed individuals. MT-RNR1 gene encoding the mitochondrial ribosomal 12S subunit is a hot spot for aminoglycoside-induced hearing loss mutations, however, a variability in the nature and frequency of genetic changes in different populations exists. The objective of this study was to analyze MT-RNR1 gene mutations in a Baltic-speaking Latvian population, and to estimate the prevalence of such genetic changes in the population-specific mitochondrial haplogroups. In the cohort of 191 ethnic non-related Latvians, the presence of two deafness-associated mutations, m.1555A>G and m.827A>G, three potentially pathogenic variations, m.961insC(n), m.961T>G and m.951G>A, and one unknown substitution, m961T>A was detected, and the aggregate frequency of all variants was 7.3%. All genetic changes were detected in samples belonged to the haplogroups H, U, T, and J. The presence of several aminoglycoside ototoxicity-related MT-RNR1 gene mutations in Baltic-speaking Latvian population indicates the necessity to include ototoxicity-related mutation analysis in the future studies in order to determine the feasibility of DNA screening for patients before administration of aminoglycoside therapy.

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.

Fatal Babesia canis canis infection in a splenectomized Estonian dog.

A previously splenectomized dog from Estonia was presented with a sudden lack of appetite and discoloration of the urine. Despite supportive therapy, its condition deteriorated dramatically during 1 day. Severe thrombocytopenia and high numbers of protozoan hemoparasites were evident in blood smears, and the hematocrit dropped from 46 to 33 %. The dog was euthanized before specific antibabesial treatment was initiated. Blood samples from the dog and from two other dogs in the same household tested positive for Babesia using molecular methods, and the sequences of partial 18S rRNA gene confirmed the causative species as Babesia canis canis. The risk of severe, rapidly progressing babesiosis in splenectomized dogs merits awareness.

Prevalence and phylogenetic analysis of Babesia spp. in Ixodes ricinus and Ixodes persulcatus ticks in Latvia.

Babesia spp. are tick-borne protozoan parasites that have been reported in many European countries and are considered to be emerging pathogens. Several Babesia spp. have been identified in ticks in Latvia. Recently, canine babesiosis cases were diagnosed for the first time in Latvia; therefore, continued studies on the prevalence and occurrence of new species are warranted. In the present study, questing tick samples collected in 2005-2007 were screened for the presence of Babesia spp.; in total, 432 Ixodes ricinus and 693 Ixodes persulcatus ticks were analyzed. Babesia spp. were detected in 1.4% of the I. ricinus ticks and in 1.9% of I. persulcatus ticks. Sequencing revealed that ixodid ticks in Latvia contained Babesia microti, Babesia capreoli, and Babesia venatorum. Babesia microti was the most prevalent species, accounting for 58% of all positive samples; moreover, two distinct B. microti genotypes were identified. Phylogenetic analysis of the full-length 18S rRNA gene of two B. capreoli/B. divergens isolates indicated a closer relationship to the B. capreoli clade than B. divergens. This is the first report of B. venatorum in I. persulcatus ticks in Latvia. Our results suggest that both I. ricinus and I. persulcatus ticks play important roles in the epidemiology of these zoonotic pathogens in Latvia.

Detection of tick-borne encephalitis virus in I. ricinus ticks collected from autumn migratory birds in Latvia.

Birds have a potential of spreading ticks via bird migration routes. In this study, we screened 170 ticks removed during autumn 2010 from 55 birds belonging to 10 species for the presence of tick-borne encephalitis virus (TBEV). In total, TBEV RNA was detected in 14% of I. ricinus tick samples obtained from different birds species. The results of this study indicate the possible role of migrating birds in the dispersal of TBEV-infected ticks along the southward migration route.

Prevalence of tick-borne pathogens in ticks collected from migratory birds in Latvia.

Migratory birds act as hosts and long-distance vectors for several tick-borne infectious agents. Here, feeding Ixodes ticks were collected from migratory birds during the autumn migration period in Latvia and screened for the presence of epidemiologically important non-viral pathogens. A total of 93 DNA samples of ticks (37 larvae and 56 nymphs) removed from 41 birds (order Passeriformes, 9 species) was tested for Lyme borreliosis spirochaetes, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp. Borrelia burgdorferi DNA was detected in 18% of the tick samples, and a majority of infected ticks were from thrush (Turdus spp.) birds. Among the infected ticks, Borrelia valaisiana was detected in 41% of cases, Borrelia garinii in 35%, and mixed Bo. valaisiana and Bo. garinii infection in 24%. Anaplasma phagocytophilum DNA was detected in 2% of ticks, R. helvetica in 12%, and Babesia spp. pathogens in 4% of ticks. Among these samples, 3 Babesia species were identified: Ba. divergens, Ba. microti, and Ba. venatorum. Coinfection with different pathogens that included mixed infections with different Borrelia genospecies was found in 20% of nymphal and 3% of larval Ixodes ticks. These results suggest that migratory birds may support the circulation and spread of medically significant zoonoses in Europe.

Autochthonous canine babesiosis caused by Babesia canis canis in Latvia.

This is the first report of confirmed canine babesiosis in Latvia supporting the observed geographical expansion of this disease. Between 2009 and 2011 three dogs which have not traveled outside of Latvia were diagnosed with babesiosis. Hematological analysis and serological tests for granulocytic anaplasmosis, ehrlichiosis and borreliosis were negative (Idexx SNAP 4Dx test). Peripheral blood erythrocytes of the three dogs contained large Babesia that were identified as Babesia canis canis by PCR. Sequences of partial 18S rRNA gene were 98-100% similar to the sequences of B. canis canis isolated from dogs in other European countries. We conclude that these are the first autochthonous canine babesiosis cases reported from Latvia.

Association between Anaplasma phagocytophilum seroprevalence in dogs and distribution of Ixodes ricinus and Ixodes persulcatus ticks in Latvia.

Anaplasma phagocytophilum has been detected in ticks in Latvia; however, this is the first study to investigate this pathogen in dogs in Latvia. The aims of this study were: (i) to determine A. phagocytophilum seroprevalence in dogs, (ii) to correlate A. phagocytophilum seroprevalence in dogs with the geographic distribution of the tick species Ixodes ricinus and Ixodes persulcatus, and (iii) to determine if seroprevalence for A. phagocytophilum is higher in dogs with clinical signs suggestive of canine granulocytic anaplasmosis (CGA). Peripheral venous blood samples were collected from 3 dog groups: (i) clinically healthy dogs (HD, n=400), (ii) clinically healthy hunting dogs (HHD, n=41), and (iii) dogs with a clinical suspicion of anaplasmosis (SD, n=29). Sampling was carried out in regions inhabited by I. ricinus (IR), I. persulcatus (IP), and in regions where both tick species were present (M). SNAP 4Dx test (IDEXX) was used to detect antibodies against A. phagocytophilum in the blood of all dogs; nested PCR was performed in selected dogs of the SD group. Seroprevalence for A. phagocytophilum was calculated and correlated with the prevalent tick species in the region. A. phagocytophilum seroprevalence was 11.0% in HD, 12% in HHD, and 17% in SD with no significant differences among groups. In the IR region, seroprevalence was 12.5% (34/272) while seroprevalence in the M region was 17% (13/76), and both were significantly higher than the seroprevalence of 2% in the IP region (2/93; p<0.0005). One CGA case was diagnosed. We conclude that A. phagocytophilum seroprevalence in Latvia is within the range reported from other European countries. CGA should be included in the differential list in Latvian dogs with appropriate clinical signs and laboratory abnormalities, especially in I. ricinus habitat areas.