Rapid diagnosis of alveolar echinococcosis from lung puncture sample using metagenomic next-generation sequencing: a case report.

INTRODUCTION: Alveolar echinococcosis (AE), caused by the larval forms of Echinococcus multilocularis, is a zoonotic disease affecting the liver, lungs, lymph nodes, kidneys, brain, bones, thyroid, and other organs. Diagnosing AE in a non-endemic area is usually challenging. With the rapid development and increasing application of sequencing techniques in recent years, metagenomic next-generation sequencing (mNGS) has become a powerful tool for diagnosing rare infectious diseases. CASE PRESENTATION: A 45-year-old woman was admitted to the hospital for the presence of pulmonary shadows for more than 3 months. The lung computed tomography (CT) at a local hospital revealed scattered solid and quasi-circular nodules in the left upper lobe, left lower lobe, right middle lobe, and right lower lobe. The largest nodule was located in the dorsal part of the right lung, measuring 2.0 × 1.7 × 1.5 cm. Moreover, abdominal CT revealed one space-occupying lesion each in the left and right lobes. The pathological analysis of the lung biopsy specimen revealed infiltration of lymphocytes, plasma cells, and eosinophils in the alveolar wall and interstitial area. No pathogenic bacteria were observed in the sputum smear and culture tests. There were no parasite eggs in the stool. The mNGS of the lung puncture tissue revealed 6156 sequence reads matching E. multilocularis; thus, the condition was diagnosed as AE. Albendazole 400 mg was administered twice daily, and the patient was stable during follow-up. CONCLUSION: This case emphasizes the role of mNGS in diagnosing AE. As a novel, sensitive, and accurate diagnostic method, mNGS could be an attractive approach for facilitating early diagnosis and prompt treatment of infectious diseases, especially when the infection was caused by rare pathogens.

Molecular analyses of exosome-derived miRNAs revealed reduced expression of miR-184-3p and decreased exosome concentration in patients with alveolar echinococcosis.

Both E. multilocularis and host-derived exosomes are involved in the pathogenic process of alveolar echinococcosis (AE). Exosomes secrete miRNAs that have regulatory roles in host-pathogen interactions in multiple ways. In the present study, we collected and purified supernatants of E. multilocularis cultures, as well as human plasma exosomes. High-throughput sequencing showed the identities of 45 exosomal miRNAs in E. multilocularis. The lengths of these miRNAs ranged from 19 to 25 nucleotides (nt), with the majority (n = 18) measuring 22 nt. Notably, emu-let-7-5p emerged as the most abundant among these miRNAs, with a detected count of 33,097 and also length of 22 nt. Nanoparticle tracking analysis (NTA) showed that the concentration of exosomes in the plasma of AE patients was lower compared to that in the healthy individuals. This result suggested that the concentration of plasma exosomes was able to distinguish AE patients from healthy individuals. Using qRT-PCR to assess the relative expression of 10 miRNAs of E. multilocularis, we showed that the expression of miR-184-3p was downregulated significantly in the exosomes of plasma from AE patients compared to that in the control group. In summary, this study indicates that AE induces a reduction in the concentration of human plasma exosomes, as well as downregulating miR-184-3p in infected individuals.

It's a small world for parasites: evidence supporting the North American invasion of European Echinococcus multilocularis.

Echinococcus multilocularis (Em), the causative agent of human alveolar echinococcosis (AE), is present in the Holarctic region, and several genetic variants deem to have differential infectivity and pathogenicity. An unprecedented outbreak of human AE cases in Western Canada infected with a European-like strain circulating in wild hosts warranted assessment of whether this strain was derived from a recent invasion or was endemic but undetected. Using nuclear and mitochondrial markers, we investigated the genetic diversity of Em in wild coyotes and red foxes from Western Canada, compared the genetic variants identified to global isolates and assessed their spatial distribution to infer possible invasion dynamics. Genetic variants from Western Canada were closely related to the original European clade, with lesser genetic diversity than that expected for a long-established strain and spatial genetic discontinuities within the study area, supporting the hypothesis of a relatively recent invasion with various founder events.

Global analysis of neuropeptides in cestodes identifies Attachin, a SIFamide homolog, as a stimulant of parasite motility and attachment.

Many anthelmintics target the neuromuscular system, in particular by interfering with signaling mediated by classical neurotransmitters. Although peptidergic signaling has been proposed as a novel target for anthelmintics, current knowledge of the neuropeptide complement of many helminth groups is still limited, especially for parasitic flatworms (cestodes, trematodes, and monogeneans). In this work, we have characterized the neuropeptide complement of the model cestode Hymenolepis microstoma. Peptidomic characterization of adults of H. microstoma validated many of the neuropeptide precursor (npp) genes previously predicted in silico, and identified novel neuropeptides that are conserved in parasitic flatworms. Most neuropeptides from parasitic flatworms lack significant similarity to those from other animals, confirming the uniqueness of their peptidergic signaling. Analysis of gene expression of ten npp genes by in situ hybridization confirmed that all of them are expressed in the nervous system and identified cryptic features, including the first evidence of dorsoventral asymmetry, as well as a new population of peripheral peptidergic cells that appears to be conserved in the trematode Schistosoma mansoni. Finally, we characterized in greater detail Attachin, an SIFamide homolog. Although its expression is largely restricted to the longitudinal nerve cords and cerebral commissure in H. microstoma, it shows widespread localization in the larval nervous system of Echinococcus multilocularis and Mesocestoides corti. Exogenous addition of a peptide corresponding to the highly conserved C-terminus of Attachin stimulated motility and attachment of M. corti larvae. Altogether, this work provides a robust experimental foothold for the characterization of peptidergic signaling in parasitic flatworms. Cover Image for this issue: https://doi.org/10.1111/jnc.15418.

Transcriptional effects of electroporation on Echinococcus multilocularis primary cell culture.

Echinococcus multilocularis is the etiological agent of alveolar echinococcosis (AE), a serious parasitic disease in the Northern Hemisphere. The E. multilocularis primary cell cultivation system, together with E. multilocularis genome data and a range of pioneering molecular-based tools have advanced the research on this and other cestodes. RNA interference (RNAi) and microRNA knock-down have recently contributed to the study of the cellular and molecular basis of tapeworm development and host-parasite interaction. These, as well as other techniques, normally involve an electroporation step for the delivery of RNA, DNA, peptides, and small molecules into cells. Using transcriptome data and bioinformatic analyses, we herein report a genome-wide comparison between primary cells of E. multilocularis and primary cells under electroporated conditions after 48 h of culture. We observed that ~ 15% of genes showed a significant variation in expression level, including highly upregulated genes in electroporated cells, putatively involved in detoxification and membrane remodeling. Furthermore, we found genes related to carbohydrate metabolism, proteolysis, calcium ion binding and microtubule processing significantly altered, which could explain the cellular dispersion and the reduced formation of cellular aggregates observed during the first 48 h after electroporation.

Echinococcus multilocularis infection in a red fox (Vulpes vulpes) on Prince Edward Island, Canada.

Objective: Molecular identification of small cestodes, morphologically consistent with Echinococcus multilocularis, recovered at necropsy from the gastrointestinal tract contents of a red fox, was accomplished by PCR using published species-specific n ad1 primers and methods. Animal: Red fox (Vulpes vulpes). Procedure: Small cestodes recovered from intestinal contents of a red fox trapped on Prince Edward Island in December 2020 (frozen at -20°C before being processed for parasite recovery in June 2021) were morphologically identified. Species identity confirmation and haplotyping of the cestodes were done via PCR and DNA sequencing of the n ad1, nad2, and cob genes. Results: Small cestodes morphologically consistent with E. multilocularis were detected in the gastrointestinal tract contents of a red fox trapped near Montague, PEI. The species identity was confirmed via PCR. Haplotyping revealed that they were of the European E1 haplotype. Conclusion: In Canada, E. multilocularis has been reported as far east as Québec, with most reports being in central and western provinces and territories. This is the first report of E. multilocularis infection in a canid host east of Ontario, Canada and illustrates the need for regular wildlife disease surveillance to enhance our understanding of emerging pathogens of veterinary and medical importance. Clinical Relevance: Echinococcus multilocularis is a highly pathogenic zoonotic cestode from the family Taeniidae that can cause alveolar echinococcosis (AE) when rodents, dogs, horses, pigs, non-human primates, or humans ingest its eggs. Alveolar echinococcosis is challenging to treat, and survival rates for untreated individuals are low.

Objectif: L'identification moléculaire de petits cestodes, morphologiquement compatibles avec Echinococcus multilocularis, récupérés à l'autopsie du contenu du tractus gastro-intestinal d'un renard roux, a été réalisée par PCR à l'aide d'amorces nad1 spécifiques à l'espèce et de méthodes publiées. Animal: Renard roux (Vulpes vulpes). Procédure: De petits cestodes récupérés du contenu intestinal d'un renard roux piégé à l'Île-du-Prince-Édouard en décembre 2020 (congelés à −20 °C avant d'être traités pour la récupération des parasites en juin 2021) ont été morphologiquement identifiés. La confirmation de l'identité des espèces et l'haplotypage des cestodes ont été effectués par PCR et séquençage de l'ADN des gènes nad1, nad2 et cob. Résultats: De petits cestodes morphologiquement compatibles avec E. multilocularis ont été détectés dans le contenu du tractus gastro-intestinal d'un renard roux piégé près de Montague, Î.-P.-É. L'identité de l'espèce a été confirmée par PCR. L'haplotypage a révélé qu'ils étaient de l'haplotype européen E1. Conclusion: Au Canada, E. multilocularis a été signalé aussi loin à l'est que le Québec, la plupart des signalements ayant été rapportés dans les provinces et territoires du centre et de l'ouest. Il s'agit du premier rapport d'infection à E. multilocularis chez un canidé hôte à l'est de l'Ontario, au Canada, et illustre la nécessité d'une surveillance régulière des maladies de la faune pour améliorer notre compréhension des agents pathogènes émergents d'importance vétérinaire et médicale. Pertinence clinique: Echinococcus multilocularis est un cestode zoonotique hautement pathogène de la famille des Taeniidae qui peut provoquer une échinococcose alvéolaire (EA) lorsque des rongeurs, des chiens, des chevaux, des porcs, des primates non humains ou des humains ingèrent ses oeufs. L'échinococcose alvéolaire est difficile à traiter et les taux de survie des personnes non traitées sont faibles.(Traduit par Dr Serge Messier).

Molecular Evidence for Local Acquisition of Human Alveolar Echinococcosis in Saskatchewan, Canada.

Alveolar echinococcosis (AE) is a life-threatening parasitic disease caused by the zoonotic cestode Echinococcus multilocularis. Our goals were to confirm infection, identify species, and analyze biogeographical origin of metacestode tissues from a suspected human AE case in Saskatchewan, Canada. We conducted polymerase chain reaction (PCR) targeting the nad1 mitochondrial gene for E. multilocularis and the rrnS ribosomal RNA gene for E. granulosus and conducted haplotype analysis at the nad2 locus. Our analysis confirmed AE and indicated that sequences matched infected Saskatchewan coyotes and European E3/E4 haplotypes. The patient had no travel history outside North America. This suggests autochthonous transmission of a European-type strain.

Parasite Circulating Cell-free DNA in the Blood of Alveolar Echinococcosis Patients as a Diagnostic and Treatment-Status Indicator.

BACKGROUND: Alveolar echinococcosis (AE) is a serious parasitic disease caused by the larvae of Echinococcus multilocularis. It is the less common but substantially more deadly of the 2 major echinococcosis diseases that can occur globally but are concentrated in central Asia. METHODS: We analyzed parasite circulating cell-free DNA (cfDNA) in 149 plasma samples using a DNA sequencing-based method (105 AE, 16 cystic echinococcosis, 4 liver cancer, 4 gallstones, and 20 healthy volunteers). After identifying the Echinococcus-specific cfDNA (Em-cfDNA) sequences in the samples, we determined whether Em-cfDNA could be used for AE diagnosis and as a potential indicator of the effectiveness of surgical treatment. We also examined potential associations between Em-cfDNA levels and clinical features of AE patients. RESULTS: Our work demonstrates that varying reads of Em-cfDNA were detectable in the plasma of 100% of preoperative AE patients and that all of the non-AE patients and healthy volunteers were negative. Em-cfDNA has good sensitivity and specificity for the diagnosis of AE. We also found that Em-cfDNA levels apparently have reference value for evaluating the therapeutic efficacy of surgery interventions for AE lesions. Finally, our analysis revealed that Em-cfDNA levels can reflect meaningful information about lesion size in preoperative AE patients. CONCLUSIONS: We demonstrate that sequencing-based monitoring of Em-cfDNA can be used in the clinic as a powerful diagnostic indicator for AE. We also note that there is a strong potential for use of this liquid-biopsy method to monitor ongoing disease status in postintervention AE patients.

First European Haplotype of Echinococcus multilocularis Identified in the United States: An Emerging Disease?

BACKGROUND: Echinococcus multilocularis is one of the most severe and lethal parasitic diseases of humans, most often reported in Europe and Asia. Only 1 previous case has been documented in the contiguous United States from Minnesota in 1977. European haplotypes have been identified in carnivores and domestic dogs as well as recently in patients in western and central Canada. METHODS: We used immunohistochemical testing with the monoclonal antibody Em2G11 and a species-specific enzyme-linked immunosorbent assay affinity-purified antigen Em2, as well as COX1 gene sequencing. RESULTS: Using pathology, immunohistochemical staining, specific immunodiagnostic testing, and COX1 gene sequencing, we were able to definitively identify E. multilocularis as the causative agent of our patient's liver and lung lesions, which clustered most closely with the European haplotype. CONCLUSIONS: We have identified the first case of a European haplotype E. multilocularis in the United States and the first case of this parasitic infection east of the Mississippi River. Given the identification of this haplotype in Canada, this appears to be an emerging infectious disease in North America.

microRNA-125b-5p is a promising novel plasma biomarker for alveolar echinococcosis in patients from the southern province of Qinghai.

BACKGROUND: Alveolar echinococcosis (AE) is caused by parasitic infection by Echinococcus multilocularis. Its diagnosis is usually based on clinical symptoms, ultrasound, and other imaging methods. MicroRNAs (miRNAs) play important roles in disease processes and can exist in a highly stable cell-free form in body fluids. It is important to identify specific, sensitive diagnostic markers for early diagnosis and evaluation of AE. In this study, we examined hsa-miR-125b-5p as a potential plasma biomarker of E. multilocularis infection. METHODS: Plasma samples from patients with AE and healthy individuals were screened for the presence of five miRNAs using miRNA chips. We used quantitative polymerase chain reaction to measure miRNA expression levels in plasma and liver tissue samples from patients with AE. RESULTS: hsa-miR-125b-5p was stably upregulated in the plasma and liver tissue samples from patients with AE. CONCLUSIONS: The results suggest that hsa-miR-125b-5p may be a promising biomarker for early, non-invasive diagnosis of AE.

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Investigation of Echinococcus multilocularis in foxes and dogs in Pakistan by detection of copro-DNA.

Alveolar echinococcosis (AE) is a zoonosis caused by Echinococcus multilocularis, a heteroxenous parasite belonging to Cestoda class. AE is currently considered an important public health issue, but epidemiological and notably molecular data from several endemic countries, including Pakistan, are sparse. Here we report the first detection of Echinococcus multilocularis in wildlife from Pakistan after real-time PCR and sequencing confirmation in the faecal samples of three foxes from northern Kaghan and Siran regions. The occurrence is estimated at 4.4% (95% CI 0.9-12.4). In order to go further in the epidemiological investigations on E. multilocularis and due to the potential presence of other Echinococcus species, we suggest the need for further epidemiological surveys targeting E. multilocularis and E. granulosus sensu lato isolates from humans and intermediate hosts as well as definitive hosts from wildlife in Pakistan.

Rodent control programmes can integrate Echinococcus multilocularis surveillance by facilitating parasite genotyping: the case of Arvicola terrestris voles screening in France.

The tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, the most serious parasitic disease for humans in Europe. In Europe, the E. multilocularis lifecycle is based on a prey-predator relationship between the red fox and small rodents. Over the last three decades, the surveillance of E. multilocularis infection in red foxes has led to the description of a wider distribution pattern across Europe. France constitutes the current European western border, but only the north-eastern half of the country is considered endemic. The red fox is the host mainly targeted in E. multilocularis surveillance programmes, but surveys targeting small rodents may be useful for obtaining molecular data, especially when the time-consuming trapping is already carried out in dedicated pest-control programmes. Here, we screened for parasitic lesions in the livers of 1238 Arvicola terrestris voles originating from the historical, but neglected focal area located in central France (Auvergne region) and from Hautes-Alpes, a recently identified endemic department in south-eastern France. This screening identified six voles infected with E. multilocularis in Hautes-Alpes and none in Puy-de-Dôme (Auvergne region) after molecular confirmation. The absence of infected rodents from Puy-de-Dôme can be mainly explained by the generally low prevalence reported in intermediate hosts. The infected Hautes-Alpes samples come all from the same trapping site situated at around 5 km from one of the three fox faecal samples with E. multilocularis DNA collected 15 years prior, thereby confirming the existence and persistence of the E. multilocularis lifecycle in the area. All the rodent E. multilocularis samples from Hautes-Alpes showed the same EmsB microsatellite marker profile. This profile has previously been described in Europe only in the Jura department (central eastern France), located at least 180 km further north. Successive migrations of infected foxes from the historical focal area, including from Jura, to Hautes-Alpes may explain the detection of the parasite in A. terrestris in Hautes-Alpes. Existing trapping efforts in areas where farmers trap A. terrestris for surveillance and pest control can be an effective complement to sampling foxes or fox faeces to obtain E. multilocularis molecular profiles.

Relationship between hepatic grayish-white solid nodules in horses imported from Canada and larval Echinococcus multilocularis infection.

Histopathological and genetic examinations were conducted on grayish-white solid hepatic nodules in 150 horses imported from Canada, in order to investigate larval Echinococcus multilocularis infection. Ten of the 150 horses (6.7%) were diagnosed with alveolar hydatid disease. The sequences of the mitochondrial cytochrome b genes obtained from all 10 polymerase chain reaction positive samples had 99 to 100% identity with the European haplotype E1 of E. multilocularis. Therefore, we concluded that the infections likely originated in Canada.

Relation entre les nodules hépatiques solides blanc-grisâtre trouvés chez des chevaux importés du Canada et l'infection larvaire à Echinococcus multilocularis . Des examens histopathologiques et génétiques ont été effectués sur des nodules hépatiques solides blanc-grisâtre observés chez 150 chevaux importés du Canada afin d'étudier l'infection larvaire à Echinococcus multilocularis. Dix des 150 chevaux (6,7 %) ont reçu un diagnostic de maladie hydatique alvéolaire. Les séquences des gènes mitochondriaux du cytochrome b obtenus à partir des 10 échantillons positifs par réaction d'amplification en chaîne par la polymérase ont montré une identité de 99 à 100 % avec l'haplotype européen E1 d'E. multilocularis. L'haplotype d'E. multilocularis obtenu à partir de cette étude suggère que les infections sont probablement originaires du Canada.(Traduit par Dr Serge Messier).

Metformin Suppresses Development of the Echinococcus multilocularis Larval Stage by Targeting the TOR Pathway.

Alveolar echinococcosis (AE) is a severe disease caused by the larval stage of the tapeworm Echinococcus multilocularis Current chemotherapeutic treatment options based on benzimidazoles are of limited effectiveness, which underlines the need to find new antiechinococcosis drugs. Metformin is an antihyperglycemic and antiproliferative agent that shows activity against the related parasite Echinococcus granulosus Hence, we assessed the in vitro and in vivo effects of the drug on E. multilocularis Metformin exerted significant dose-dependent killing effects on in vitro cultured parasite stem cells and protoscoleces and significantly reduced the dedifferentiation of protoscoleces into metacestodes. Likewise, oral administration of metformin (50 mg/kg of body weight/day for 8 weeks) was effective in achieving a significant reduction of parasite weight in a secondary murine AE model. Our results revealed mitochondrial membrane depolarization, activation of Em-AMPK, suppression of Em-TOR, and overexpression of Em-Atg8 in the germinal layer of metformin-treated metacestode vesicles. The opposite effects on the level of active Em-TOR in response to exogenous insulin and rapamycin suggest that Em-TOR is part of the parasite's insulin signaling pathway. Finally, the presence of the key lysosomal pathway components, through which metformin reportedly acts, was confirmed in the parasite by in silico assays. Taken together, these results introduce metformin as a promising candidate for AE treatment. Although our study highlights the importance of those direct mechanisms by which metformin reduces parasite viability, it does not necessarily preclude any additional systemic effects of the drug that might reduce parasite growth in vivo.

Molecular characterization of human Echinococcus isolates and the first report of E. canadensis (G6/G7) and E. multilocularis from the Punjab Province of Pakistan using sequence analysis.

BACKGROUND: Echinococcosis is a zoonotic parasitic disease causing serious health problems in both humans and animals in different endemic regions across the world. There are two different forms of human echinococcosis: Cystic Echinococcosis (CE) and Alveolar Echinococcosis (AE). CE is caused by the larval stage of Echinococcus granulosus sensu lato and AE by the larval stage of Echinococcus multilocularis. Geographically, CE is universally distributed, while AE is prevalent in the northern hemisphere. Although the disease is endemic in neighboring countries (China, Iran and India) of Pakistan, there are limited reports from that country. Besides, there are no comprehensive data on the genotyping of Echinococcus species in humans based on sequence analysis. This study aimed to detect the presence of human CE and to identify Echinococcus spp. in human isolates through genetic characterization of hydatid cysts in the Punjab Province of Pakistan. METHODS: Genetic analysis was performed on 38 human hydatid cyst samples collected from patients with echinococcosis using mitochondrial cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb) and NADH subunit 1 (nad1). Patient data including age, epidemiological history, sex, and location were obtained from hospital records. RESULTS: According to the sequence analysis we detected E. granulosus sensu stricto (n = 35), E. canadensis (G6/G7) (n = 2), and E. multilocularis (n = 1). Thus, the majority of the patients (92.1%, 35/38) were infected with E. granulosus s.s. This is the first molecular confirmation of E. canadensis (G6/G7) and E. multilocularis in human subjects from Pakistan. CONCLUSIONS: These findings suggested that E. granulosus s.s. is the dominant species in humans in Pakistan. In addition, E. canadensis (G6/G7) and E. multilocularis are circulating in the country. Further studies are required to explore the genetic diversity in both humans and livestock.

Fatty acid-binding proteins in Echinococcus spp.: the family has grown.

Fatty acid-binding proteins (FABPs) are small intracellular proteins that reversibly bind fatty acids and other hydrophobic ligands. In cestodes, due to their inability to synthesise fatty acids de novo, FABPs have been proposed as essential proteins, and thus, as possible drug targets and/or carriers against these parasites. We performed data mining in Echinococcus multilocularis and Echinococcus granulosus genomes in order to test whether this family of proteins is more complex than previously reported. By exploring the genomes of E. multilocularis and E. granulosus, six genes coding for FABPs were found in each organism. In the case of E. granulosus, all of them have different coding sequences, whereas in E. multilocularis, two of the genes code for the same protein. Remarkably, one of the genes (in both cestodes) encodes a FABP with a C-terminal extension unusual for this family of proteins. The newly described genes present variations in their structure in comparison with previously described FABP genes in Echinococcus spp. The coding sequences for E. multilocularis were validated by cloning and sequencing. Moreover, differential expression patterns of FABPs were observed at different stages of the life cycle of E. multilocularis by exploring transcriptomic data from several sources. In summary, FABP family in cestodes is far more complex than previously thought and includes new members that seem to be only present in flatworms.

miRNAs and lncRNAs in Echinococcus and Echinococcosis.

Echinococcosis are considered to be potentially lethal zoonotic diseases that cause serious damage to hosts. The metacestode of Echinococcus multilocularis and E. granulosus can result in causing the alveolar and cystic echinococcoses, respectively. Recent studies have shown that non-coding RNAs are widely expressed in Echinococcus spp. and hosts. In this review, the two main types of non-coding RNAs-long non-coding RNAs (lncRNAs) and microRNAs (miRNAs)-and the wide-scale involvement of these molecules in these parasites and their hosts were discussed. The expression pattern of miRNAs in Echinococcus spp. is species- and developmental stage-specific. Furthermore, common miRNAs were detected in three Echinococcus spp. and their intermediate hosts. Here, we primarily focus on recent insights from transcriptome studies, the expression patterns of miRNAs and lncRNAs, and miRNA-related databases and techniques that are used to investigate miRNAs in Echinococcus and echinococcosis. This review provides new avenues for screening therapeutic and diagnostic markers.

The fox tapeworm, Echinococcus multilocularis, in grey wolves and dogs in Slovakia: epidemiology and genetic analysis.

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is an important emerging parasite in the northern hemisphere. In epidemiological studies, the highest attention is being paid to foxes as the main reservoir hosts responsible for geographic expansion from multiple focal populations and the invasion of urban habitats, but little information is available on the parasite distribution in other carnivores. Hence, the study was designed to obtain updated information about the occurrence and genetic diversity of E. multilocularis in grey wolves and dogs in Slovakia. Faecal samples of wolves were collected from three locations under a certain level of environmental protection in the central and eastern parts of the country, and the presence of the parasite DNA was detected in 35.7% of 112 samples, with the highest rate (51.2%) recorded in the Poloniny National Park in north-eastern Slovakia. Among 110 faecal dog samples, E. multilocularis was detected in three faeces from segregated Roma settlements in the eastern part of the country, which accounted for an overall positivity of 2.7%. Sequence analysis of two mitochondrial genes, 12S rRNA and NADH dehydrogenase subunit 1, revealed four haplotypes in 13 isolates from wolves and dogs originating from four sites in eastern and central Slovakia, with all samples bearing a European-type pattern of E. multilocularis. The more than one-third positivity rate of E. multilocularis in wolf faecal samples dispersed over a large part of the country has corroborated the extensive circulation of the parasite in wildlife and confirmed the need to improve intervention control strategies.