Untargeted metabolomics to discriminate liver and lung hydatid cysts: Importance of metabolites involved in the immune response.

The Echinococcus granulosus sensu lato species complex is responsible for the neglected zoonotic disease known as cystic echinococcosis (CE). Humans and livestock are infected via fecal-oral transmission. CE remains prevalent in Western China, Central Asia, South America, Eastern Africa, and the Mediterranean. Approximately one million individuals worldwide are affected, influencing veterinary and public health, as well as social and economic matters. The infection causes slow-growing cysts, predominantly in the liver and lungs, but can also develop in other organs. The exact progression of these cysts is uncertain. This study aimed to understand the survival mechanisms of liver and lung CE cysts from cattle by determining their metabolite profiles through metabolomics and multivariate statistical analyses. Non-targeted metabolomic approaches were conducted using quadrupole-time-of-flight liquid chromatography/mass spectrometry (LC-QTOF-MS) to distinguish between liver and lung CE cysts. Data processing to extract the peaks on complex chromatograms was performed using XCMS. PCA and OPLS-DA plots obtained through multiple statistical analyses showed interactions of metabolites within and between groups. Metabolites such as glutathione, prostaglandin, folic acid, and cortisol that cause different immunological reactions have been identified both in liver and lung hydatid cysts, but in different ratios. Considering the differences in the metabolomic profiles of the liver and lung cysts determined in the present study will contribute research to enlighten the nature of the cyst and develop specific therapeutic strategies.

Assessing the Potential Apoptotic Effects of Different Hydatid Cyst Fluids on Human Healthy Hepatocytes and Hepatocellular Carcinoma Cells.

Cystic Echinococcosis (CE) is a zoonotic infection caused by the larval form of Echinococcus granulosus in humans. Emerging evidence suggests an intriguing inverse association between E. granulosus infection and the occurrence of cancer. This study aimed to investigate the influence of diverse host-derived hydatid cyst fluids (HCF) with distinct genotypes on human liver hepatocytes (HC) and hepatocellular carcinoma cells (HepG2). Specifically, we examined their effects on cell proliferation, apoptosis sensitivity (BAX/BCL-2), apoptosis-related p53 expression, and the expression of cancer-related microRNA (hsa-miR-181b-3p). Cell proliferation assays, real-time PCR, and ELISA studies were conducted to evaluate potential anti-cancer properties. The findings revealed that animal-origin HCF (G1(A)) induced direct cell death by augmenting the susceptibility of HepG2 cells to apoptosis. Treatment with both G1(A) and G1(H) HCF sensitized HepG2 and HC cell lines to apoptosis by modulating the BAX/BCL-2 ratio, accompanied by upregulation of the p53 gene. Additionally, G1(A) HCF and human-derived HCFs (G1(H), G7(H)) reduced the expression of miR-181b-3p in HepG2 cells. Consequently, this study demonstrates the potential anti-cancer effect of HCF in HepG2 cells and provides the first comparative assessment of HCFs from human and animal sources with diverse genotypes, offering novel insights into this field.

First molecular detection of Entamoeba gingivalis subtypes in individuals from Turkey.

Entamoeba gingivalis is a parasitic protozoan that colonizes the human oral cavity and there are two subtypes (ST1 and ST2) that have been identified to date. However, there are no reports on the molecular detection or characterization of E. gingivalis in Turkey. The objective of this study was to detect the presence of E. gingivalis in Turkish healthy individuals and those with periodontal disease and to subtype the isolates using molecular techniques. Samples from the oral cavity of 94 individuals were taken and the presence of E. gingivalis was determined by PCR using primers for SsrRNA and the amplicons were then confirmed by DNA sequencing. Each participant completed a questionnaire that included demographic data, habits and lifestyle, as well as health status. The presence of E. gingivalis was detected in a total of 19 samples (11 patients and eight healthy individuals). Molecular characterization determined that 12 samples belonged to ST1 and seven samples belonged to ST2. The presence of E. gingivalis was higher in patients with periodontal disease than in healthy individuals, and this association was statistically significant (P < .05). This study constitutes the first report of molecular detection and subtyping of E. gingivalis in Turkey.

Molecular Characterization of Clarithromycin Resistance in Helicobacter pylori Strains.

BACKGROUND: The purpose of this study was to determine the antimicrobial status of stocked clinical Helicobacter pylori isolates by using antibiotic gradient test and subsequently identify the mutations that cause clarithromycin resistance by DNA sequencing. Turkey is a transition zone between Europe and Asia; therefore, we also aimed to show both continents' mutations in Turkish isolates. METHODS: One hundred forty-seven H. pylori isolates that had been stocked at -80°C between 1998 and 2008 were randomly selected and included in the study. Antibiotic susceptibility tests were performed using antibiotic gradient test for clarithromycin, amoxicillin, tetracycline, metronidazole, and levofloxacin. A polymerase chain reaction targeting the region of 23S rRNA gene domain V of H. pylori was performed and the mutations responsible for resistance against clarithromycin were defined by sequencing. RESULTS: All of the tested isolates were found susceptible to amoxicillin and tetracycline. However, clarithromycin, metronidazole, and levofloxacin resistance were detected in 28.5% (42/147), 44.8% (66/147), and 23.1% (34/147) of the isolates, respectively. Point mutations were detected in 46 isolates (46/147, 31.2%). The majority of mutations were defined as A2143G (19/46, 41.3%), A2142G (14/46, 30.4%), and A2142C (7/46, 15.2%), respectively. T2188C, T2182C, G1949A, G1940A, and C1944T mutations were also identified in the isolates. CONCLUSION: In conclusion, the most common mutations associated with clarithromycin resistance in H. pylori have been identified as A2143G, A2142G, and A2142C which are the most frequently detected mutations in European countries. Same mutations and other mutations like T2182C have also been detected frequently in north-eastern countries and China. Since Turkey is a transition zone between Europe and Asia, Turkey might have strains that carry mutations found in both continents.

Possible microRNA-based mechanism underlying relationship between chronic spontaneous urticaria and Blastocystis.

BACKGROUND: Blastocystis spp. has been proposed as a possible cause of extraintestinal clinical signs such as urticaria pathogenesis. OBJECTIVES: The aim of this study was to investigate the differences between microRNA (miRNA) expression profiles of Chronic spontaneous urticaria (CSU) patients in the presence or absence of Blastocystis spp. as well as healthy controls. Additionally, cellular pathways which are affected in the presence of Blastocystis spp. were identified. METHODS: Twenty patients diagnosed with CSU were enrolled in the study and divided into equally two groups according to the presence of Blastocystis spp. Besides, six healthy individuals were included in the study. The expression profiles of 372 human-derived miRNAs have been investigated in serum samples from CSU patients and healthy controls with miScript miRNA PCR Array Human miRBase Profiler. RESULTS: Compared to Blastocystis-negative (BN)-CSU patients, expression of 3 miRNAs (hsa-miR-3183, hsa-miR-4469, hsa-miR-5191) were found to be downregulated by at least two-fold (p < 0.05) in Blastocystis-positive (BP)-CSU patients. Additionally, the miRNA expression profiles of six healthy individuals (n = 3 Blastocystis-positive, n = 3 Blastocystis-negative) were analyzed and it was determined that the expressions of 7 miRNAs (hsa-miR-4661-5p, hsa-miR-4666a-5p, hsa-miR-4803, hsa-miR-5587-5p, hsa-miR-4500, hsa-miR-5680, hsa-miR-382-3p) increased at least 3-fold in the serum of individuals with Blastocystis-positive compared to Blastocystis-negative subjects. Most down-regulated miRNAs, in BP-CSU patients, affect cell adhesion molecules (CAMs), and signaling pathways therefore, Blastocystis spp. presence may influence the clinical presentation of urticaria by leading to unbalanced immunity. In addition, Blastocystis spp. presence may be influenced TGF- ß signaling pathway through altered miRNAs and may be laying the groundwork for the development of CSU in healthy individuals. CONCLUSIONS: As a consequence, this is the first report to show that the miRNA expression profile is affected by the presence of Blastocystis spp. Further miRNA-based studies are needed in order to enlighten the exact underlying molecular mechanisms of the relationship between Blastocystis spp. and CSU.

Evaluation of Potential Inflammatory Markers for Cystic Echinococcosis: P-selectin and Resistin

Objective: Cystic echinococcosis (CE) is one of the most common zoonotic diseases worldwide. Diagnosis of CE is predominantly based on imaging techniques and serological tests are used in cases of non-characteristic imaging findings as diagnostic reference. However, serological test results cannot be completely reliable as they are affected by multi-factors. P-selectin and resistin are inflammatory markers that are altered during the acute stages of infection. In this purpose, inflammatory markers as P-selectin and resistin have been investigated for a potential diagnostic reference for CE diagnosis. Methods: A total of 60 patients who were diagnosed with CE and twenty-five healthy individuals were included in this study. Blood samples were obtained from all participants. Obtained sera were evaluated using the P-selectin and resistin ELISA kits for protein levels. Additionally, the relative expression of SELP (P-selectin) and RETN (resistin) genes were determined using the comparative CT (ΔΔCT) method between groups as CE patients with active and inactive cysts, CE patients and healthy controls. Results: SELP (13.9-fold change, p<0.05) and RETN (8.1-fold change, p<0.05) were differentially expressed in CE patients compared in the control group. Whereas resistin protein levels were significantly higher in CE patients than the healthy controls (p<0.001), the difference in P-selectin protein levels was not significant (p>0.05). There was no difference between active and inactive CE patients in terms of P-selectin and resistin in gene and protein levels (p>0.05). Conclusion: Although there was no difference between the active and inactive CE patients, the good differentiation between the healthy controls and the CE patients suggested that resistin is a potential inflammatory diagnostic reference.

Can parasite-derived microRNAs differentiate active and inactive cystic echinococcosis patients?

Cystic Echinococcosis (CE) is a neglected zoonotic disease caused by the metacestode form of Echinococcus granulosus sensu lato. Non-invasive imaging techniques, especially ultrasound, are primarily used for CE diagnosis. MicroRNAs (miRNAs) are small, non-coding RNA molecules that act as post-transcriptional regulators in various biological processes. After identification of parasite-derived miRNAs, these miRNAs are considered to be potential biomarkers for diagnosis and follow-up. The focus of this research is to compare the expression profiles of certain parasite-derived miRNAs in CE patients with active and inactive cysts as well as healthy controls. Parasite-derived miRNAs, egr-let-7-5p, egr-miR-71a-5p, and egr-miR-9-5p, of inactive CE patients were found to be differentially expressed with 3.74-, 2.72-, and 20.78-fold change (p < 0.05), respectively, when compared with active CE patients. In this study, we evaluated for the first time the expression profile of three parasite-derived miRNAs in the serum of CE patients to determine their potential to distinguish between active and inactive CE. It was concluded that serum levels of parasite-derived miRNAs, egr-let-7-5p and egr-miR-9-5p, could be promising new potential biomarkers for stage-specific diagnosis of CE. Further studies are needed with larger sample set to validate discriminating potential of these miRNAs.

Metabolomic profiling of active and inactive liver cystic echinococcosis.

Cystic Echinococcosis (CE) is one of the life-threatening diseases worldwide. It is a parasitic zoonosis caused by tapeworms of the species Echinococcus granulosus sensu lato (s.l). The treatment options of CE vary from simple "watch and wait" approach to invasive treatment, based on the type and especially the nature of the cyst (active/inactive). Serological tests are inadequate to distinguish between active and inactive CE. A diagnostic reference that can determine whether the cyst is active or inactive can easily guide the treatment strategy. We aimed to test whether gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadropole time of flight mass spectrometry (LC-qTOF-MS) based metabolomics can establish a plasma metabolic fingerprint of CE patients and identify a diagnostic reference to discriminate active and inactive CE cysts. Metabolite concentrations were measured in plasma samples of 36 active CE patients, 17 inactive CE patients and 31 healthy controls. Multivariate statistical analysis on 232 identified metabolites obtained from two analytical platforms was performed by using principle component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) methods. The PLS-DA scores plot of the combined data set demonstrated a good separation between the groups. Compared to the healthy control group, decreased levels of squalene and increased levels of glyceric acid, 3-phosphoglycerate, glutamic acid, palmitoleic acid and oleic acid were determined in the CE patients. However, decreased levels of 3-phosphoglycerate and increased levels of 4-hydroxyphenylacetylglutamine, docosahexanoic acid were determined in active CE patients compared to the inactive CE patients. Determination of differences in metabolites may provide detailed understandings of potential metabolic process associated with active and inactive CE patients, and altered specific metabolic changes may provide some clues to obtain diagnostic reference for CE. This study has certain limitations: a. various factors affecting results of metabolomic studies such as lifestyle and dietary habits of the patients could not be fully controlled b. other infectious or malignant diseases of the liver should also be included as a positive control to evaluate the specificity of the diagnostic references.

[Evaluation of the Effect of Hydatid Cyst Fluid on the Apoptosis Pathway in BEAS-2B and A549 Cell Lines]. / BEAS-2B ve A549 Hücre Hatlarinda Hidatik Kist Sivisinin Apoptoz Yolagi Üzerine Etkisinin Degerlendirilmesi.

Some of the pathogenic microorganisms have been associated with cancer due to the activation of cancer precursors in the host because of the inflammatory processes. Additionally, some other pathogens prevents the tumor formation by creating an anti-neoplastic immune response which has been reported to stop the development of cancer. Cystic echinococcosis (CE) or cyst hydatid disease (CHD) is a zoonotic infection caused by the larval form of Echinococcus granulosus sensu lato in humans. It has been reported that there is a negative correlation between E.granulosus infection and cancer and it has been suggested that direct and/or indirect E.granulosus infection may have an anti-cancer effect. However, the molecular mechanisms of this effect still remains unclear. The aim of this study was to evaluate the effect of hydatid cyst fluid administration on cell proliferation and expression of some apoptotic genes (BCL-2, p53 and BAX) in human healthy lung epithelial (BEAS-2B) and human lung adenocarcinoma (A549) cell lines and understanding the molecular mechanism underlying the possible anti-cancer action mechanism of hydatid cyst fluid. In order to evaluate the effect of hydatid cyst fluid on cell proliferation and apoptotic gene expression, cell proliferation assay (XTT) and real-time polymerase chain reaction (Rt-PCR) were performed, respectively. After the application of hydatid cyst fluid, there was no change in the cell proliferation. A statistically significant decrease in BCL-2 gene expression (> 90 fold) and an increase in p53 gene expression (> 1.2 fold) were found. No significant change in BAX gene expression was detected. In this study, it was found that the application of hydatid cyst fluid did not directly cause cell death but it has shown for the first time to sensitize the A549 cell line, which is resistant to apoptosisand shed light on the possible mechanism of hydatid cyst fluid in the apoptotic pathway.

Human and animal cystic echinococcosis in Konya, Turkey: molecular identification and the first report of E. equinus from human host in Turkey.

Cystic echinococcosis is a neglected, zoonotic disease in Turkey. The disease is commonly seen in rural areas where the local population is in close contact with livestock and dogs. This research aimed to molecularly identify of hydatid cysts in cattle and human isolates from Konya, Turkey. Following sample collection, direct microscopy was performed. After direct examination, total DNA was extracted, and positive PCR products of cox 1 mitochondrial gene (~ 875 bp) were sequenced. A total of 83 hydatid cysts (cattle n = 57 and human n = 26), 82 were identified as Echinococcus granulosus sensu stricto (G1-G3 genotypes), and one human isolate was characterized as Echinococcus equinus (G4 genotype). Fertility rates of cysts belonging to cattle for liver and lung cysts were 93.3% and 80%, respectively. Out of 26 human originated isolates, 18 (69.2%) of cysts were found to be fertile. To the best of our knowledge, this is the first report of E. equinus from human host in Turkey.

Investigation of the relationship between CE cyst characteristics and genetic diversity of Echinococcus granulosus sensu lato in humans from Turkey.

Cystic echinococcosis (CE) is one of the most common zoonotic diseases worldwide, particularly in rural areas. This study aimed at the identification of the genotype/species belonging to Echinococcus granulosus sensu lato (s.l.) specimens in retrieved percutaneously from the human host and to investigate their relationship with cyst characteristics. The genetic identification of cyst material was performed by mt-CO1 gene polymerase chain reaction, and confirmed via sequencing. A total of 110 CE cysts were identified as E. granulosus s.l. In detail, 104 belonged to E. granulosus sensu stricto (G1 and G3) and six isolates were in the E. canadensis cluster (G6/7). All clusters were tested for the relationship between demographics, cyst features and genetic diversity. The relationship between genetic variation and certain clinical characteristics such as cyst volume and location were statistically significant for G6/7 cluster. Further studies are required with a larger sample set to investigate the relationship between the genetic variability of E. granulosus s.l. and cyst features.

Low prevalence of hypervirulent Klebsiella pneumoniae in Anatolia, screened via phenotypic and genotypic testing.

Hypervirulent Klebsiella pneumoniae (hvKP) strains are associated with vigorous clinical presentation and relapses. Initially reported from Asia, these variants have spread globally and become an emerging agent of significant health threat. This study was carried out to identify hvKP strains in a previously uninvestigated region and to evaluate the impact of commonly-employed phenotypic and genotypic markers as diagnostic assays. A total of 111 blood culture isolates, collected at a tertiary care center was investigated. The hvKP strains were sought by a string test and the amplification of partial magA, rmpA, iucA and peg344. All products were characterized via sequencing. Evidence for hvKP was observed in 10.8% via iucA amplification (7.2%), string test (2.7%) and magA amplification (0.9%). Specific products were not produced by assays targeting rmpA and peg344 genes. Antibiotic susceptibility patterns compatible with possible extensive or pan-antimicrobial resistance was noted in 66.7% of the hvKP candidate strains. Capsule type in the magA positive strain was characterized as K5. We have detected hvKP in low prevalence at a region with no prior documentation. Targetting the aerobactin gene via iucA amplification provided the most accurate detection in this setting. The epidemiology of hvKP in Anatolia requires elucidation for effective control and management.

Epidemiological factors associated with human cystic echinococcosis: a semi-structured questionnaire from a large population-based ultrasound cross-sectional study in eastern Europe and Turkey.

BACKGROUND: Cystic echinococcosis (CE) is a neglected parasitic zoonosis prioritized by the WHO for control. Several studies have investigated potential risk factors for CE through questionnaires, mostly carried out on small samples, providing contrasting results. We present the analysis of risk factor questionnaires administered to participants to a large CE prevalence study conducted in Bulgaria, Romania and Turkey. METHODS: A semi-structured questionnaire was administered to 24,687 people from rural Bulgaria, Romania and Turkey. CE cases were defined as individuals with abdominal CE cysts detected by ultrasound. Variables associated with CE at P < 0.20 in bivariate analysis were included into a multivariable logistic model, with a random effect to account for clustering at village level. Adjusted odds ratios (AOR) with 95% CI were used to describe the strength of associations. Data were weighted to reflect the relative distribution of the rural population in the study area by country, age group and sex. RESULTS: Valid records from 22,027 people were analyzed. According to the main occupation in the past 20 years, "housewife" (AOR: 3.11; 95% CI: 1.51-6.41) and "retired" (AOR: 2.88; 95% CI: 1.09-7.65) showed significantly higher odds of being infected compared to non-agricultural workers. "Having relatives with CE" (AOR: 4.18; 95% CI: 1.77-9.88) was also associated with higher odds of infection. Interestingly, dog-related and food/water-related factors were not associated with infection. CONCLUSIONS: Our results point toward infection being acquired in a "domestic" rural environment and support the view that CE should be considered more a "soil-transmitted" than a "food-borne" infection. This result helps delineating the dynamics of infection transmission and has practical implications in the design of specific studies to shed light on actual sources of infection and inform control campaigns.

Prevalence of abdominal cystic echinococcosis in rural Bulgaria, Romania, and Turkey: a cross-sectional, ultrasound-based, population study from the HERACLES project.

BACKGROUND: Cystic echinococcosis is a neglected zoonotic infection that is distributed worldwide and prioritised by WHO for control efforts. The burden of human cystic echinococcosis is poorly understood in most endemic regions, including eastern Europe. We aimed to estimate the prevalence of abdominal cystic echinococcosis in rural areas of Bulgaria, Romania, and Turkey. METHODS: We did a cross-sectional ultrasound-based survey that recruited volunteers from 50 villages in rural areas of Bulgaria, Romania, and Turkey. These villages were in provinces with annual hospital incidence of cystic echinococcosis within the mid-range for the respective countries. All people who attended a session were allowed to participate if they agreed to be screened. Abdominal ultrasound screening sessions were hosted in public community structures such as community halls, primary health-care centres, schools, and mosques. Lesions were classified using an adapted WHO classification. We reported the prevalence of abdominal cystic echinococcosis adjusted by sex and age through direct standardisation, using the country's rural population as a reference. FINDINGS: From July 1, 2014, to Aug 3, 2015, 24 693 individuals presented to screening sessions and 24 687 underwent ultrasound screening. We excluded a further six indivduals due to missing data, leaving 24 681 people in our analysis. Abdominal cystic echinococcosis was detected in 31 of 8602 people screened in Bulgaria, 35 of 7461 screened in Romania, and 53 of 8618 screened in Turkey. The age and sex adjusted prevalence of abdominal cystic echinococcosis was 0·41% (95% CI 0·29-0·58) in Bulgaria, 0·41% (0·26-0·65) in Romania, and 0·59% (0·19-1·85) in Turkey. Active cysts were found in people of all ages, including children, and in all investigated provinces. INTERPRETATION: Our results provide population-based estimates of the prevalence of abdominal cystic echinococcosis. These findings should be useful to support the planning of cost-effective interventions, supporting the WHO roadmap for cystic echinococcosis control. FUNDING: European Union Seventh Framework Programme.

Human cystic echinococcosis in Turkey: a preliminary study on DNA polymorphisms of hydatid cysts removed from confirmed patients.

Cystic echinococcosis caused by the larval stages of Echinococcus granulosus sensu lato s.l is endemic in Turkey with a high public health impact particularly in rural areas. The aim of this study was to investigate the genetic variation and population structure of E. granulosus s.s using metacestode isolates removed from surgically confirmed patients originating from several regions in Turkey and to investigate the occurrence of autochthonous transmission. Using DNA extracted from a total of 46 human-derived CE isolates, we successfully analysed an 827-bp fragment within the cox1 mitochondrial gene and confirmed the causative agent of human cystic echinococcosis in patients included in this study to be Echinococcus granulosus s.s (G1 and G3 genotypes). The haplotype parsimony network consisted of 28 haplotypes arranged within three main clusters and the neutrality indices were both negative and significant indicating negative selection or population expansion. The assessment carried out in this study using GenBank nucleotide sequence data from Turkey for sheep and cattle hosts demonstrated the importance of autochthonous transmission with sheep, cattle and humans harbouring the same haplotypes. Further studies are required to investigate the biological significance, if any, of E. granulosus s.s haplotypes and the genetic variability of CE from human patients using longer nucleotide sequences and a larger sample set.

Two haplotype clusters of Echinococcus granulosus sensu stricto in northern Iraq (Kurdistan region) support the hypothesis of a parasite cradle in the Middle East.

Human cystic echinococcosis (CE) caused by Echinococcus granulosus s.s. is a major public health problem in Iraqi Kurdistan with a reported surgical incidence of 6.3 per 100,000 Arbil inhabitants. A total of 125 Echinococcus isolates retrieved from sheep, goats and cattle were used in this study. Our aim was to determine species/genotypes infecting livestock in Iraqi Kurdistan and examine intraspecific variation and population structure of Echinococcus granulosus s.s. in this region and relate it to that of other regions worldwide. Using nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox 1) we identified E. granulosus s.s. as the cause of hydatidosis in all examined animals. The haplotype network displayed a double-clustered topology with two main E. granulosus s.s. haplotypes, (KU05) and (KU33). The 'founder' haplotype (KU05) confirmed the presence of a common lineage of non-genetically differentiated populations as inferred by the low non-significant fixation index values. Overall diversity and neutrality indices indicated demographic expansion. We used E. granulosus s.s. nucleotide sequences from GenBank to draw haplotype networks for the Middle East (Iran, Jordan and Turkey), Europe (Albania, Greece, Italy, Romania and Spain), China, Mongolia, Russia, South America (Argentina, Brazil, Chile and Mexico) and Tunisia. Networks with two haplotype clusters like that reported here for Iraqi Kurdistan were seen for the Middle East, Europe, Mongolia, Russia and Tunisia using both 827bp and 1609bp cox1 nucleotide sequences, whereas a star-like network was observed for China and South America. We hypothesize that the double clustering seen at what is generally assumed to be the cradle of domestication may have emerged independently and dispersed from the Middle East to other regions and that haplotype (KU33) may be the main haplotype within a second cluster in the Middle East from where it has spread into Europe, Mongolia, Russia and North Africa. Further studies using metacestodes of human origin are required to investigate the biological importance of E. granulosus s.s. haplotypes/clusters and their association, if any with clinical manifestations of CE infection.

Co-circulation of West Nile virus and distinct insect-specific flaviviruses in Turkey.

BACKGROUND: Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates. RESULTS: A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s.l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s.l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as "Ochlerotatus caspius flavivirus Turkey", was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified. CONCLUSIONS: We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected.

A novel rhabdovirus, related to Merida virus, in field-collected mosquitoes from Anatolia and Thrace.

Next-generation sequencing technologies have significantly facilitated the discovery of novel viruses, and metagenomic surveillance of arthropods has enabled exploration of the diversity of novel or known viral agents. We have identified a novel rhabdovirus that is genetically related to the recently described Merida virus via next-generation sequencing in a mosquito pool from Thrace. The complete viral genome contains 11,798 nucleotides with 83% genome-wide nucleotide sequence similarity to Merida virus. Five major putative open reading frames that follow the canonical rhabdovirus genome organization were identified. A total of 1380 mosquitoes comprising 13 species, collected from Thrace and the Mediterranean and Aegean regions of Anatolia were screened for the novel virus using primers based on the N and L genes of the prototype genome. Eight positive pools (6.2%) exclusively comprised Culex pipiens sensu lato specimens originating from all study regions. Infections were observed in pools with female as well as male or mixed-sex individuals. The overall and Cx. pipiens-specific minimal infection rates were calculated to be 5.7 and 14.8, respectively. Sequencing of the PCR products revealed marked diversity within a portion of the N gene, with up to 4% divergence and distinct amino acid substitutions that were unrelated to the collection site. Phylogenetic analysis of the complete and partial viral polymerase (L gene) amino acid sequences placed the novel virus and Merida virus in a distinct group, indicating that these strains are closely related. The strain is tentatively named "Merida-like virus Turkey". Studies are underway to isolate and further explore the host range and distribution of this new strain.

Isolation and genomic characterization of Culex theileri flaviviruses in field-collected mosquitoes from Turkey.

Vector surveillance for the arthropod-borne infections has resulted in the isolation of a growing number of novel viruses, including several flavivirus strains that exclusively replicate in insects. This report describes the isolation and genomic characterization of four insect-specific flaviviruses from mosquitoes, previously collected from various locations in Turkey. C6/36 Aedes albopictus and Vero cell lines were inoculated with mosquito pools. On C6/36 cells, mild cytopathic effects, characterized as rounding and detachment, were observed in four pools that comprised female Culex theileri mosquitoes. Complete (3 isolates, 10,697 nucleotides) or near-complete (1 isolate, 10,452 nucleotides) genomic characterization was performed in these culture supernatants via next generation sequencing. All strains demonstrated high genetic similarities, with over 99% identity match on nucleotide and amino acid alignments, revealing them to be different isolates of the same virus. Sequence comparisons identified the closest relative to be the Culex theileri flavivirus (CTFV) strains, originally characterized in Portugal. Phylogenetic analyses demonstrated that the isolates remained distinct as a cluster but formed a monophyletic group with CTFV strains, and shared a common ancestor with Quang Binh or related Culex flaviviruses. The organization of the viral genome was consistent with the universal flavivirus structure and stem-loops; conserved motifs and imperfect tandem repeats were identified in the non-coding ends of the viral genomes. A potential ribosomal shifting site, resulting in the translation of an additional reading frame, was detected. The deduced viral polyprotein comprised 3357 amino acids and was highly-conserved. Amino acid variations, presumably associated with adaptive environmental pressures, were identified. These isolates comprise the first fully characterized insect-specific flaviviruses in Turkey. Their impact on West Nile virus circulation, which is also endemic in the study region, remains to be explored.

Phlebovirus and Leishmania detection in sandflies from eastern Thrace and northern Cyprus.

BACKGROUND: Phlebotomine sandflies are vectors of several pathogens with significant impact for public health. This study was conducted to investigate and characterize phlebovirus and Leishmania infections in vector sandflies collected in the eastern Thrace region in Turkey and Northern Cyprus, where previous data indicate activity of these agents. METHODS: Field sampling of sandflies was performed at 4 locations in Edirne and Tekirdag provinces of eastern Thrace and at 17 locations in Lefkosa, Girne, Magosa and Guzelyurt provinces of northern Cyprus. In sandfly pools, phlebovirus RNA and Leishmania DNA were screened via a generic polymerase chain reaction (PCR) and kinetoplast minicircle PCR, respectively. Selected sandfly specimens unsuitable for pathogen detection were identified to species level. Cytochrome oxidase 1 gene region was used for DNA barcoding of selected specimens and pathogen positive pools. Positive amplicons were cloned and characterized by sequencing. RESULTS: A total of 2690 sandflies, collected from Eastern Thrace (15.4%) and Northern Cyprus (84.6%) were evaluated. Morphological examination of 780 specimens from Cyprus exhibited Phlebotomus perfiliewi sensu lato (72.6%), Phlebotomus tobbi (19.7%), Phlebotomus papatasi (2.8%), Laroussius sp. (1.6%) and Sergentomyia azizi (1.6%), Sergentomyia sp. (0.9%), Sergentomyia minuta (0.5%) and Phleobotomus jacusieli (0.1%) species. Pathogen screening was performed in 1910 specimens distributed in 195 pools. In eight pools of P.tobbi sandflies collected in Cyprus, Leishmania infantum DNA was demonstrated. Toscana virus (TOSV) genotype A sequences were identified in two pools of P. perfiliewi s.l. and one pool of P.tobbi sandflies from Cyprus. Co-infection of TOSV and Leishmania infantum was characterized in a P.tobbi pool. Sequences belonging to novel phleboviruses are revealed in three P. perfiliewi s.l. pools. One sequence, provisionally named Edirne virus, identified in Edirne province in eastern Thrace, demonstrated the highest rate of genomic similarity to Adria and Salehabad viruses. Furthermore, Girne 1 and Girne 2 viruses, identified in Girne province, revealed similarities to TOSV and Sandfly Fever Sicilian virus and related strains, respectively. CONCLUSIONS: Activity of TOSV genotype A strains in Cyprus and co-infection of sandfly vectors with L. infantum was documented for the first time. Novel phlebovirus strains of unknown medical significance was identified in sampling regions.