Assessing the role of individual foxes in environmental contamination with Echinococcus multilocularis through faecal samples.

Key parasite transmission parameters are difficult to obtain from elusive wild animals. For Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE), the red fox is responsible for most of the environmental contamination in Europe. The identification of individual spreaders of E. multilocularis environmental contamination is crucial to improving our understanding of the ecology of parasite transmission in areas of high endemicity and optimising the effectiveness of prevention and control measures in the field. Genetic faecal sampling appears to be a feasible method to gain information about the faecal deposition of individual animals. We conducted a 4 year faecal sampling study in a village that is highly endemic for E. multilocularis, to assess the feasibility of individual identification and sexing of foxes to describe individual infection patterns. Individual fox identification from faecal samples was performed by obtaining reliable genotypes from 14 microsatellites and one sex locus, coupled with the detection of E. multilocularis DNA, first using captive foxes and then by environmental sampling. From a collection of 386 fox stools collected between 2017 and 2020, tested for the presence of E. multilocularis DNA, 180 were selected and 124 samples were successfully genotyped (68.9%). In total, 45 unique individual foxes were identified and 26 associated with at least one sample which tested positive for E. multilocularis (Em(+)). Estimation of the population size showed the fox population to be between 29 and 34 individuals for a given year and 67 individuals over 4 years. One-third of infected individuals (9/26 Em(+) foxes) deposited 2/3 of the faeces which tested positive for E. multilocularis (36/60 Em(+) stools). Genetic investigation showed a significantly higher average number of multiple stools for females than males, suggesting that the two sexes potentially defecated unequally in the studied area. Three partially overlapping clusters of fox faeces were found, with one cluster concentrating 2/3 of the total E. multilocularis-positive faeces. Based on these findings, we estimated that 12.5 million E. multilocularis eggs were produced during the study period, emphasizing the high contamination level of the environment and the risk of exposure faced by the parasite hosts.

Genetic diversity of Echinococcus multilocularis specimens isolated from Belgian patients with alveolar echinococcosis using EmsB microsatellites analysis.

The genetic diversity of Echinococcus multilocularis (E. multilocularis) specimens isolated from patients with alveolar echinococcosis (AE), is a major field of investigation to correlate with sources of infection, clinical manifestations and prognosis of the disease. Molecular markers able to distinguish samples are commonly used worldwide, including the EmsB microsatellite. Here, we report the use of the EmsB microsatellite polymorphism data mining for the retrospective typing of Belgian specimens of E. multilocularis infecting humans. A total of 18 samples from 16 AE patients treated between 2006 and 2021 were analyzed through the EmsB polymorphism. Classification of specimens was performed through a dendrogram construction in order to compare the similarity among Belgian samples, some human referenced specimens on the EWET database (EmsB Website for the Echinococcus Typing) and previously published EmsB profiles from red foxes circulating in/near Belgium. According to a comparison with human European specimens previously genotyped in profiles, the 18 Belgian ones were classified into three EmsB profiles. Four specimens could not be assigned to an already known profile but some are near to EWET referenced samples. This study also highlights that some specimens share the same EmsB profile with profiles characterized in red foxes from north Belgium, the Netherlands, Luxembourg and French department near to the Belgian border. Furthermore, Belgian specimens present a genetic diversity and include one profile that don't share similarities with the ones referenced in the EWET database. However, at this geographical scale, there is no clear correlation between EmsB profiles and geographical location. Further studies including additional clinical samples and isolates from foxes and rodents of south Belgium are necessary to better understand the spatial and temporal circumstances of human infections but also a potential correlation between EmsB profiles and parasite virulence.

Complete mitochondrial exploration of Echinococcus multilocularis from French alveolar echinococcosis patients.

Alveolar echinococcosis (AE) is a parasitosis that is expanding worldwide, including in Europe. The development of genotypic markers is essential to follow its spatiotemporal evolution. Sequencing of the commonly used mitochondrial genes cob, cox1, and nad2 shows low discriminatory power, and analysis of the microsatellite marker EmsB does not allow nucleotide sequence analysis. We aimed to develop a new method for the genotyping of Echinococcus multilocularis based on whole mitochondrial genome (mitogenome) sequencing, to determine the genetic diversity among 30 human visceral samples from French patients, and compare this method with those currently in use. Sequencing of the whole mitochondrial genome was carried out after amplification by PCR, using one uniplex and two multiplex reactions to cover the 13,738 bp of the mitogenome, combined with Illumina technology. Thirty complete mitogenome sequences were obtained from AE lesions. One showed strong identity with Asian genotypes (99.98% identity) in a patient who had travelled to China. The other 29 mitogenomes could be differentiated into 13 haplotypes, showing higher haplotype and nucleotide diversity than when using the cob, cox1, and nad2 gene sequences alone. The mitochondrial genotyping data and EmsB profiles did not overlap, probably because one method uses the mitochondrial genome and the other the nuclear genome. The pairwise fixation index (Fst) value between individuals living inside and those living outside the endemic area was high (Fst = 0.222, P = 0.002). This is consistent with the hypothesis of an expansion from historical endemic areas to peripheral regions.

Real-time multiplex PCR for human echinococcosis and differential diagnosis.

Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the Echinococcus genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four Echinococcus species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi, and E. canadensis), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for Echinococcus spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (Taenia solium), and toxocariasis (Toxocara cati and T. canis)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10-4 ng/µL (seven PCR assays positive), corresponding to 37-42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of Echinococcosis species.

Title: PCR multiplex en temps-réel pour le diagnostic de l'échinococcose humaine et diagnostic différentiel. Abstract: L'identification moléculaire des pathogènes infectieux humains rares semble être l'une des méthodes actuelles les plus pertinentes pour un diagnostic et une prise en charge rapides des patients. Les techniques de PCR, en particulier la PCR quantitative en temps réel, sont bien adaptées à la détection d'ADN de pathogènes, même pour de faibles concentrations. Les infections à échinocoque sont dues à des helminthes du genre Echinococcus, des espèces étroitement apparentées, impliquées dans des lésions parasitaires affectant les animaux et accidentellement l'homme. Une qPCR multiplex (MLX qPCR), permettant la détection de quatre espèces d'Echinococcus impliquées en Europe dans l'échinococcose alvéolaire (EA) et kystique (EK) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi et E. canadensis), basée sur de courtes cibles mitochondriales a été développée ici. Une collection a été constituée de 81 tissus frais ou fixés en paraffine (FFPE) de lésions d'EA et EK. Les essais de qPCR ont été réalisées en triplex pour la détection d'Echinococcus spp., associés à une qPCR de contrôle d'inhibition. Une PCR duplex a été développée pour le diagnostic de deux autres helminthiases en impasse chez l'Homme (cysticercose (Taenia solium), et toxocarose (Toxocara cati et T. canis). La sensibilité de la qPCR a été évaluée et s'échelonne de 1 à 5 × 10−4 ng/µl (sept essais de qPCR positifs), correspondant à 37 à 42 cycles pour l'ADN quantifiable. La spécificité était de 100 % pour toutes les cibles. Cette qPCR multiplex, adaptée à de faibles quantités d'ADN peut être mise en œuvre au laboratoire pour un diagnostic moléculaire rapide des espèces d'Echinococcus.

Molecular diagnosis of alveolar echinococcosis in patients based on frozen and formalin-fixed paraffin-embedded tissue samples.

Confirmed diagnosis of alveolar echinococcosis (AE) is based on pathological criteria and molecular evidence. This parasite-borne disease, caused by the cestode Echinococcus multilocularis, sparingly involves humans as a dead-end host. In humans, the parasite mainly colonizes the liver but can colonize any organ and cause atypical forms, often difficult to characterize clinically. Moreover, molecular methods may be suitable to make the diagnosis of AE in cases of atypical forms, extra-hepatic localizations, or immunosuppressed patients. The aim of this study was to determine the most relevant published PCR techniques, for diagnosis of AE in patients and adopt the best strategy for molecular diagnosis depending on the nature of the tested sample. In this study, we evaluated nine end-point PCR assays and one real-time PCR assay (qPCR), targeting mitochondrial genes, using a total of 89 frozen or formalin-fixed paraffin-embedded (FFPE) samples from either 48 AE or 9 cystic echinococcosis patients. Targeted fragment-genes ranged from 84 to 529 bp. Six PCR assays were able to amplify the DNA of 100% of the frozen AE-samples and for one PCR, 69.8% of the FFPE AE-samples. The 16S rrnL PCR (84 bp) was positive in PCR for 77% of the AE samples and in qPCR for 86.5%. The sensitivity of the PCR assays was higher for fresh samples and FFPE samples stored for less than 5 years. The qPCR assay further increased sensitivity for the tested samples, confirming the need for the development of an Echinococcus spp. qPCR to improve the molecular diagnosis of echinococcoses.

TITLE: Diagnostic moléculaire de l'échinococcose alvéolaire chez les patients à partir d'échantillons de tissus congelés et fixés au formol et inclus en paraffine. ABSTRACT: La confirmation diagnostique de l'échinococcose alvéolaire (EA) est basée sur des critères anatomo-pathologiques et moléculaires. Cette maladie d'origine parasitaire, causée par le cestode Echinococcus multilocularis, implique sporadiquement l'homme, impasse parasitaire. Chez l'homme, le parasite colonise principalement le foie mais peut coloniser tout organe et causer des formes atypiques, souvent difficiles à caractériser cliniquement. En outre, les méthodes moléculaires permettent de réaliser le diagnostic de l'EA dans les formes atypiques, les localisations extra-hépatiques ou chez les patients immunodéprimés. Le but de cette étude était de déterminer les techniques PCR publiées les plus pertinentes, pour le diagnostic de l'EA chez les patients et adopter la meilleure stratégie par diagnostic moléculaire en fonction de la nature de l'échantillon testé. Dans cette étude nous avons évalué neuf PCR en point-final et une PCR-temps-réel (qPCR), ciblant des gènes mitochondriaux, utilisant 89 échantillons congelés ou fixés en paraffine (FFPE) de patients EA (n = 48) ou présentant une échinococcose kystique (n = 9). Les fragments de gènes ciblés allaient de 84 à 529 pb. Six tests PCR ont permis d'amplifier l'ADN de 100 % des échantillons EA congelés, et pour une PCR, 69,8 % des échantillons EA-FFPE. La PCR 16S rrnL (84 pb) était positive en PCR pour 77 % des échantillons EA et en qPCR pour 86,5 %. La sensibilité des tests PCR était plus importante pour les échantillons congelés et les FFPE stockés moins de 5 ans. Le test qPCR a permis d'augmenter la sensibilité pour les échantillons testés, confirmant le besoin de développement d'une qPCR Echinococcus spp. pour améliorer le diagnostic moléculaire des échinococcoses.

Soil contamination by Echinococcus multilocularis in rural and urban vegetable gardens in relation to fox, cat and dog faecal deposits.

Echinococcus multilocularis eggs are deposited on the ground with the faeces of the carnivore definitive hosts. A reliable assessment of the spatial distribution of E. multilocularis eggs in environments used by humans is crucial for the prevention of alveolar echinococcosis (AE). This study was conducted in 192 rural and 71 urban vegetable gardens in AE endemic areas of north-eastern France. Its objective was to explore the relationship between the spatial distribution of E. multilocularis estimated from the collection and molecular analysis of two types of samples: faeces and soil. A total of 1024 carnivore faeces and 463 soil samples were collected and analysed by real-time PCR. No fox droppings and no positive soil samples were collected from the urban gardens. Positive soil samples, positive carnivore faeces, or both, were found in 42%, 24% and 6% of the sampled rural gardens, respectively. No significant association was found between the detection of E. multilocularis in soil samples collected from 50 gardens during a single sampling session and the extent and frequency of deposits of fox and cat faeces collected during repeated sampling sessions conducted in the previous months. In 19/50 gardens, E. multilocularis was detected in the soil while no positive faeces had been collected in the previous 12 months. Conversely, in 8/50 gardens, no soil samples were positive although positive faeces had been collected in the previous months. Collecting and analysing faeces provide information on soil contamination at a given time, while analysing soil samples provides an overview of long-term contamination.

TITLE: Contamination du sol par Echinococcus multilocularis dans des jardins potagers ruraux et urbains en relation avec les dépôts fécaux de renards, de chats et de chiens. ABSTRACT: Les œufs d'Echinococcus multilocularis sont déposés sur le sol avec les fèces des carnivores hôtes définitifs. Une évaluation fiable de la distribution spatiale des œufs d'E. multilocularis dans les environnements utilisés par l'homme est cruciale pour la prévention de l'échinococcose alvéolaire (EA). La présente étude a été conduite dans 192 jardins potagers ruraux et 71 jardins potagers urbains des zones endémiques d'EA du nord-est de la France. Son objectif était d'explorer la relation entre la distribution spatiale d'E. multilocularis estimée à partir de la collecte et de l'analyse moléculaire de deux types d'échantillons : des fèces et du sol. Au total, 1024 fèces et 463 échantillons de sol ont été collectés et analysés par PCR en temps réel. Aucun excrément de renard et aucun échantillon de sol positif n'a été collecté dans les jardins urbains. Des échantillons de sol positifs, des fèces de carnivores positives ou les deux ont été trouvés dans 42 %, 24 % et 6 % des jardins ruraux échantillonnés. Aucune association significative n'a été trouvée entre la détection d'E. multilocularis dans les échantillons de sol collectés dans 50 potagers lors d'une unique session d'échantillonnage et l'importance et la fréquence des dépôts de fèces de renards et de chats collectées lors d'échantillonnages répétés conduits au cours des mois précédents. Dans 19/50 potagers, E. multilocularis a été détecté dans le sol alors qu'aucun excrément positif n'avait été collectés dans les 12 mois précédents. A l'inverse, dans 8/50 potagers aucun échantillon de sol n'était positif alors que des fèces positives avait été collectées dans les mois précédents. La collecte et l'analyse de fèces renseignent sur la contamination du sol à un instant donné, alors que l'analyse d'échantillons de sol fournissent un aperçu de la contamination à long terme.

Assessment of the Genetic Diversity of Echinococcus multilocularis from Copro-Isolated Eggs.

The genetic diversity of the parasite Echinococcus multilocularis, the infectious agent of alveolar echinococcosis, is generally assessed on adult worms after fox necropsy. We aimed to investigate E. multilocularis polymorphism through the microsatellite EmsB marker using a noninvasive approach. We tested batches of isolated eggs (1, 5, and 10) from 19 carnivore fecal samples collected in a rural town located in a highly endemic area in France to determine the best strategy to adopt using a minimal quantity of parasite DNA while avoiding genetic profile overlapping in the analysis. Several molecular controls were performed to formally identify the Taeniidae eggs. In total, 112 egg batches were isolated and 102 EmsB electrophoregrams were obtained in duplicate. Quality sorting was performed through the Pearson correlation coefficient (r) between each EmsB duplicate. Forty-nine batches with r > 0.9 remained in the analysis, mainly 5- or 10-egg batches. Three EmsB profiles were emphasized by hierarchical clustering and matched those from human lesions and adult worms previously genotyped and collected in the same area. We show that the genetic diversity of the parasite can be assessed from isolated E. multilocularis eggs in a spatiotemporal context using a noninvasive approach.

Red foxes harbor two genetically distinct, spatially separated Echinococcus multilocularis clusters in Brandenburg, Germany.

BACKGROUND: Alveolar echinococcosis (AE) is a clinically serious zoonosis caused by the fox tapeworm Echinococcus multilocularis. We studied the diversity and the distribution of genotypes of E. multilocularis isolated from foxes in Brandenburg, Germany, and in comparison to a hunting ground in North Rhine-Westphalia. METHODS: Echinococcus multilocularis specimens from 101 foxes, 91 derived from Brandenburg and 10 derived from North Rhine-Westphalia, were examined. To detect potential mixed infections with different genotypes of E. multilocularis, five worms per fox were analyzed. For genotyping, three mitochondrial markers, namely cytochrome c oxidase subunit 1 (Cox1), NADH dehydrogenase subunit 1 (Nad1), and ATP synthase subunit 6 (ATP6), and the nuclear microsatellite marker EmsB were used. To identify nucleotide polymorphisms, the mitochondrial markers were sequenced and the data were compared, including with published sequences from other regions. EmsB fragment length profiles were determined and confirmed by Kohonen network analysis and grouping of Sammon's nonlinear mapping with k-means clustering. The spatial distribution of genotypes was analyzed by SaTScan for the EmsB profiles found in Brandenburg. RESULTS: With both the mitochondrial makers and the EmsB microsatellite fragment length profile analyses, mixed infections with different E. multilocularis genotypes were detected in foxes from Brandenburg and North Rhine-Westphalia. Genotyping using the mitochondrial markers showed that the examined parasite specimens belong to the European haplotype of E. multilocularis, but a detailed spatial analysis was not possible due to the limited heterogeneity of these markers in the parasite population. Four (D, E, G, and H) out of the five EmsB profiles described in Europe so far were detected in the samples from Brandenburg and North Rhine-Westphalia. The EmsB profile G was the most common. A spatial cluster of the E. multilocularis genotype with the EmsB profile G was found in northeastern Brandenburg, and a cluster of profile D was found in southern parts of this state. CONCLUSIONS: Genotyping of E. multilocularis showed that individual foxes may harbor different genotypes of the parasite. EmsB profiles allowed the identification of spatial clusters, which may help in understanding the distribution and spread of the infection in wildlife, and in relatively small endemic areas.

Echinococcus multilocularis genetic diversity in Swiss domestic pigs assessed by EmsB microsatellite analyzes.

Assessing the genetic diversity of the parasite Echinococcus multilocularis provides key information about the temporal and spatial strain flow in a given area. Previous studies indicated that a historical endemic area conventionally presents a relatively high genetic diversity, whereas peripheral or newly endemic areas exhibit a more restricted variability of the parasite. The Swiss plateau region is part of the European historically endemic area, and the genetic diversity has already been investigated by assessing either human metacestode isolates or adult worms from foxes. To date, there have been no studies covering the whole geographical area affected by the parasite. The aim of the present study was to make use of the domestic pig to investigate the genetic diversity of E. multilocularis in relation to spatial distribution. A total of 55 E. multilocularis-induced hepatic lesions from slaughtered pigs from Switzerland were studied using EmsB microsatellite analyzes, and findings were compared to already published data (originating from human, primate, foxes, and rodent samples). A total of 12 EmsB profiles were described among the domestic pigs, some of them presenting a clear spatial organization in the Swiss plateau, with three of the main profiles geographically separated. One of the 12 EmsB profiles has been newly identified for Switzerland in this study, while the other 11 profiles had been previously described in other Swiss E. multilocularis isolates from other hosts. Overall, a total of 18 EmsB profiles have so far been described within the Swiss endemic area. Six profiles appeared only among human, primate, rodent, and fox samples. Based on a richness and diversity accumulation analysis, the sampling efficiency for the whole studied area has now been improved considerably by compilation of 178 E. multilocularis specimens obtained from four different intermediate and one definitive host species in Switzerland.

Unravelling the genetic diversity and relatedness of Echinococcus multilocularis isolates in Eurasia using the EmsB microsatellite nuclear marker.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe helminthic zoonotic disease distributed in the Northern Hemisphere. The lifecycle of the parasite is mainly sylvatic, involving canid and rodent hosts. The absence of genetic data from most eastern European countries is a major knowledge gap, affecting the study of associations with parasite populations in Western Europe. In this study, EmsB microsatellite genotyping of E. multilocularis was performed to describe the genetic diversity and relatedness of 785 E. multilocularis isolates from four western and nine eastern European countries, as well as from Armenia and the Asian parts of Russia and Turkey. The presence of the same E. multilocularis populations in the Benelux resulting from expansion from the historical Alpine focus can be deduced from the main profiles shared between these countries. All 33 EmsB profiles obtained from 528 samples from the nine eastern European countries belonged to the European clade, except one Asian profile form Ryazan Oblast, Russia. The expansion of E. multilocularis seems to have progressed from the historical Alpine focus through Hungary, Slovakia, the Czech Republic and southern Poland towards Latvia and Estonia. Most of the samples from Asia belong to the Asian clade, with one EmsB profile shared between Armenia and Turkey, and two between Turkey and Russia. However, two European profiles were described from two foxes in Turkey, including one harboring worms from both European and Asian clades. Three EmsB profiles from three Russian samples were associated with the Arctic clade. Two E. multilocularis profiles from rodents from Lake Baikal belonged to the Mongolian clade, described for the first time here using EmsB. Further worldwide studies on the genetic diversity of E. multilocularis using both mitochondrial sequencing and EmsB genotyping are needed to understand the distribution and expansion of the various clades.

Assessment of the exposure to Echinococcus multilocularis associated with carnivore faeces using real-time quantitative PCR and flotation technique assays.

The eggs of Echinococcus multilocularis, the infectious stage, are spread into the environment through wild and domestic carnivore faeces. The spatial location of the faeces containing infective E. multilocularis eggs is a key parameter for studying areas of exposure and understanding the transmission processes to the intermediate hosts and humans. Echinococcus multilocularis faecal prevalence is often assessed by detecting E. multilocularis DNA, not necessarily eggs. This work aimed to determine the percentage of faeces containing E. multilocularis eggs in a rural town and its surroundings and whether this level of precision is relevant in assessing exposure to E. multilocularis. For this purpose, we developed a combined molecular and microscopic approach to investigate the E. multilocularis exposure of potential hosts in the environment from field-collected carnivore faeces. Carnivore defecation patterns were then spatialized to study the spatial distribution of E. multilocularis. Faeces were screened for E. multilocularis DNA using a specific real-time quantitative PCR (qPCR). Echinococcus multilocularis eggs were morphologically identified from E. multilocularis-specific qPCR-positive faeces after sucrose flotation and individually confirmed through specific PCR and sequencing. The spatial distribution of E. multilocularis was studied using Kulldorff statistics. Echinococcus multilocularis eggs were identified mostly in fox faeces positive for E. multilocularis DNA by qPCR (n = 27/70) and only from 1 of 15 copro-samples from dogs and 1 of 5 from cats. The faecal prevalence of E. multilocularis DNA and eggs was overdispersed, with the same geographical patterns. These data suggest that E. multilocularis DNA and/or egg detection in carnivore faeces, mainly that of foxes, is appropriate in ecological studies of E. multilocularis transmission.

Genotyping Echinococcus multilocularis in Human Alveolar Echinococcosis Patients: An EmsB Microsatellite Analysis.

For clinical epidemiology specialists, connecting the genetic diversity of Echinococcus multilocularis to sources of infection or particular sites has become somewhat of a holy grail. It is very difficult to trace the infection history of alveolar echinococcosis (AE) patients as there may be an incubation period of five to 15 years before reliable diagnosis. Moreover, the variability of parasitic manifestations in human patients raises the possibility of genetically different isolates of E. multilocularis having different levels of pathogenicity. Thus, the exposure of human patients to different strains or genotypes circulating in geographically different environments may lead to different disease outcomes. Molecular tools, such as the microsatellite marker EmsB, were required to investigate these aspects. This genetic marker was previously tested on a collection of 1211 European field samples predominantly of animal origin, referenced on a publicly available database. In this study, we investigated a panel of 66 metacestode samples (between 1981 and 2019) recovered surgically from 63 patients diagnosed with alveolar echinococcosis originating from four European countries (France, Switzerland, Germany, Belgium). In this study, we identified nine EmsB profiles, five of which were found in patients located in the same areas of France and Switzerland. One profile was detected on both sides of the French-Swiss border, whereas most patients from non-endemic regions clustered together in another profile. EmsB profiles appeared to remain stable over time because similar profiles were detected in patients who underwent surgery recently and patients who underwent surgery some time ago. This study sheds light on possible pathways of contamination in humans, including proximity contamination in some cases, and the dominant contamination profiles in Europe, particularly for extrahepatic lesions.

Asian Admixture in European Echinococcus multilocularis Populations: New Data From Poland Comparing EmsB Microsatellite Analyses and Mitochondrial Sequencing.

The cestode Echinococcus multilocularis is the causative agent of a severe zoonotic disease: alveolar echinococcosis (AE). The parasite is distributed over a vast area in northern Eurasia and North America, but the impact of AE on human health is highly uneven between different regions. One hypothetical reason for this difference in virulence may be the genetic structure of E. multilocularis which-based on mitochondrial sequences and EmsB microsatellite profiles-forms four distinct clades. These clades correspond approximately to their continents of origin: Asia, Europe, and North America, with a fourth clade apparently restricted to Mongolia and neighboring regions, even though this clade has not yet been described by EmsB genotyping. However, there are various records of genetic variants from the "wrong" region, e.g., "European" haplotypes in Western Canada, which may be the result of introduction or natural migration of host animals. One such example, prompting this study, is the recent record of an "Asian" mitochondrial haplotype in worms from foxes in Poland. At the time, this could not be confirmed by EmsB microsatellite analysis, a method that has proven to possess greater discriminatory power with the E. multilocularis nuclear genome than sequencing of mitochondrial markers. Therefore, worms collected from foxes in Poland were examined both by EmsB analysis and sequencing of the full mitochondrial cox1 gene in order to allocate the samples to the European or Asian cluster. Based on EmsB analyses of 349 worms from 97 Polish red foxes, 92% of the worms clearly showed "European-type" EmsB profiles, but 27 worms (8%) from seven foxes showed profiles that clustered with samples of Asian origin. According to cox1 sequences, a total of 18 worms from 8 foxes belonged to the Asian cluster of haplotypes. The two methods did not fully agree: only 13 worms from three foxes belonged to Asian clusters by both EmsB and cox1, whereas 18 worms from nine foxes belonged to different clusters, according to each marker. Cross-fertilization between worms of Asian origin and those from the European Polish population may explain these conflicting results. The presence of clearly Asian elements in the Polish E. multilocularis population could be the result of introduction of E. multilocularis with host animals (e.g., domestic dogs), or the migration of foxes. In the absence of genetic data from eastern European countries, especially those bordering Poland, it cannot be concluded whether this Asian admixture is typical for a larger area toward central/eastern Europe, or the Polish parasite population is the western extreme of a gradient where both European and Asian elements mingle. Further studies are needed on this subject, preferably using both mitochondrial sequencing and EmsB microsatellite analysis.

Long-term retrospective assessment of a transmission hotspot for human alveolar echinococcosis in mid-west China.

BACKGROUND: Human alveolar echinococcosis caused by infection with Echinococcus multilocularis is one of the most potentially pathogenic helminthic zoonoses. Transmission occurs involving wildlife cycles typically between fox and small mammal intermediate hosts. In the late 1980s/early 1990s a large focus of human AE was identified in poor upland agricultural communities in south Gansu Province, China. More detailed investigations in 1994-97 expanded community screening and identified key risk factors of dog ownership and landscape type around villages that could support susceptible rodent populations. A crash of the dog population (susceptible domestic definitive host) in the early 1990s appeared to stop transmission. METHODOLOGY/FINDINGS: We subsequently undertook follow-up eco-epidemiological studies based on human population screening and dog survey, in 2005/6 and in 2014/15. Our observations show a decrease in human AE prevalence, especially marked in the 11-30 year old age category. In 2015, although the dog population had recovered and in addition, forest protection and the reforestation of some areas may have favoured red fox (wild definitive host) population growth, there was no evidence of infection in owned dogs. CONCLUSIONS/SIGNIFICANCE: Those observations suggest that over decades socio-ecological changes resulted in a cascade of factors that exacerbated and then interrupted parasite emergence, with probable elimination of peri-domestic transmission of E. multilocularis in this area, despite the relative proximity of large active transmission foci on the eastern Tibetan Plateau. This study case exemplifies how anthropogenic land use and behavioural changes can modify emergence events and the transmission of endemic zoonotic parasite infections, and subsequently the importance of considering processes over the long-term in a systems approach in order to understand pathogen and disease distribution.

Genetic diversity of Echinococcus multilocularis in red foxes from two Scandinavian countries: Denmark and Sweden.

Echinococcus multilocularis is an endemic parasite of red foxes in several European countries. This parasite has been present for decades in central Europe i.e. Switzerland, Eastern France, Southern Germany and Austria, which constitute the core endemic area of Europe. In the Scandinavian countries Sweden and Denmark, several recent findings were made in foxes. To better understand the dynamics and geographic spread of E. multilocularis in Europe, genetic studies have been undertaken using the DNA microsatellite marker EmsB. In Europe, the parasite spread in hitherto non-endemic areas was suspected to take place after founder events, in which the core endemic area presents a wider genetic diversity in comparison to newly endemic areas. However, identical parasite profiles can be shared between them, highlighting the parasite spreading in a mainland-island system. In this study, Swedish (27 adult worms from seven red foxes) and Danish (38 adult worms from nine red foxes) isolates were examined using fragment size analyses of the tandemly repeated microsatellite EmsB in order to compare the genetic profiles of the Scandinavian worms with a reference collection of European worm isolates from seven countries. Six EmsB profiles were detected in the Scandinavian panel. Three profiles were described in Denmark and four in Sweden. Only one of these profiles was detected in both countries. All profiles identified in the present study have previously been found in other European countries, suggesting an epidemiological link. Due to the relatively low number of Scandinavian E. multilocularis isolates analysed so far, firm conclusions cannot be made regarding the true genetic diversity. Nevertheless, the low genetic variation detected in Sweden and Denmark in this study is similar to the values obtained from peripheral areas of the main European endemic focus, which were more recently colonized by E. multilocularis; and continuous surveillance of this parasite is warranted to provide further insight into its epidemiology in Scandinavia.

First Case of Human Primary Vertebral Cystic Echinococcosis Due to Echinococcus Ortleppi.

Cystic echinococcosis (CE) is a cosmopolitan parasitic zoonosis affecting more than one million people worldwide. In humans, primary bone CE is rare and involvement of E. ortleppi is very uncommon. We report here the first case of primary vertebral cystic echinococcosis due to E. ortleppi in Burgundy, France.

Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure.

In zoonotic infections, the relationships between animals and humans lead to parasitic disease with severity that ranges from mild symptoms to life-threatening conditions. In cities and their surrounding areas, this statement is truer with the overcrowding of the protagonists of the parasites' life cycle. The present study aims to investigate the distribution of a parasite, Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis, using copro-sampling in historically endemic rural settlements of the eastern part of France and in newly endemic areas including urban parks and settlements surrounding Paris. Based on 2741 morphologically identified and geolocalized copro-samples, the density of fox faeces was generally higher in the surrounding settlements, except for one rural area where the faeces were at larger density downtown in the winter. Fox faeces are rare but present in urban parks. Dog faeces are concentrated in the park entrances and in the centre of the settlements. DNA was extracted for 1530 samples that were collected and identified from fox, dog, cat, stone marten and badger carnivore hosts. Echinococcus multilocularis diagnosis and host faecal tests were performed using real-time PCR. We failed to detect the parasite in the surroundings of Paris, but the parasite was found in the foxes, dogs and cats in the rural settlements and their surroundings in the historically endemic area. A spatial structuring of the carnivore stool distribution was highlighted in the present study with high densities of carnivore stools among human occupied areas within some potentially high-risk locations.

EWET: Data collection and interface for the genetic analysis of Echinococcus multilocularis based on EmsB microsatellite.

Evolution and dispersion history on Earth of organisms can best be studied through biological markers in molecular epidemiological studies. The biological diversity of the cestode Echinococcus multilocularis was investigated in different cladistic approaches. First the morphological aspects were explored in connection with its ecology. More recently, molecular aspects were investigated to better understand the nature of the variations observed among isolates. The study of the tandemly repeated multilocus microsatellite EmsB allowed us to attain a high genetic diversity level where other classic markers have failed. Since 2006, EmsB data have been collected on specimens from various endemic foci of the parasite in Europe (in historic and newly endemic areas), Asia (China, Japan and Kyrgyzstan), and North America (Canada and Alaska). Biological data on the isolates and metadata were also recorded (e.g. host, geographical location, EmsB analysis, citation in the literature). In order to make available the data set of 1,166 isolates from classic and aberrant domestic and wild animal hosts (larval lesions and adult worms) and from human origin, an open web access interface, developed in PHP, and connected to a PostgreSQL database, was developed in the EmsB Website for the Echinococcus Typing (EWET) project. It allows researchers to access data collection, perform genetic analyses online (e.g. defining the genetic distance between their own samples and the samples in the database), consult distribution maps of EmsB profiles, and record and share their new EmsB genotyping data. In order to standardize the EmsB analyses performed in the different laboratories throughout the world, a calibrator was developed. The final aim of this project was to gather and arrange available data to permit to better understand the dispersion and transmission patterns of the parasite among definitive and intermediate hosts, in order to organize control strategies on the ground.

Screening of antigenic vesicular fluid proteins of Echinococcus multilocularis as potential viability biomarkers to monitor drug response in alveolar echinococcosis patients.

PURPOSE: The only drugs available to treat alveolar echinococcosis (AE) are mostly parasitostatic and in many cases prescribed for life. Decision criteria for discontinuation rely on the absence of parasitic viability. The aim of the present study is to search for candidate proteins that may exhibit good potential as biomarkers for viability. EXPERIMENTAL DESIGN: Sixteen serum samples (five healthy controls, 11 patients with AE), are used. AE-patients are classified into three groups "Cured" (n = 2), "ABZ-responders" (n = 4) and "ABZ-nonresponders" (n = 5). Immunoreactive proteins from vesicular fluid (VF) are identified and quantified by LC-MS/MS analysis after immunoprecipitation (IP) using all 16 serum samples. RESULTS: Shotgun analysis of VF lead to the identification of 107 E. multilocularis proteins. Comparative proteomics reveal nine proteins more abundant in IP eluates from ABZ-nonresponder patients (cathepsin b, prosaposin a preprotein, actin modulator protein, fucosidase alpha L1 tissue, gluthatione-S-tranferase, beta galactosidase, elongation factor 2, H17g protein tegumental antigen, and NiemannPick C2 protein). CONCLUSIONS AND CLINICAL RELEVANCE: Detection of antibodies against these proteins by ELISA could be helpful to monitor the course of alveolar echinococcosis under albendazole (ABZ) treatment.

A step forward in the understanding of the presence and expansion of Echinococcus multilocularis in Eastern Europe using microsatellite EmsB genotyping in Poland.

Alveolar echinococcosis is a severe zoonotic disease caused by the parasite Echinococcus multilocularis. In Europe, the lifecycle of this cestode is mainly sylvatic based on a prey-predator interaction between the red fox and small rodents as definitive and intermediate hosts, respectively. National surveillance of E. multilocularis in red foxes in Poland has reported a clear distinction between low endemic areas (from 2 to 5.7%) in the western half and high endemic areas (11.8 to 50.0%) in the eastern half of the country. A drastic increase of prevalence has been observed in the eastern half of Poland since the 2000's. Microsatellite EmsB genotyping was performed on 301 E. multilocularis worms from 87 foxes sampled throughout Poland, leading to identification of 29 EmsB profiles. The main profile, Pol19, was identified across the country and accounted for 44.9% of the worms collected. The conformity of 18 Polish profiles was established by comparison with previous profiles identified in Europe, but none corresponded to the most common European profiles. Poland was confirmed as a peripheral area of the main European focus, with more recent colonization by the parasite. The sharing of common profiles mainly by neighboring provinces was confirmed by a clustering analysis identifying four main groups. Expansion of the parasite in Poland in these four groups appears to be influenced by the situation in neighboring countries. Acquiring EmsB genotyping data from eastern European countries, for which very few data are reported, is necessary to understand the expansion of the parasite in the whole of Europe.