Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis.

Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens.

Assessment of pets (cats and dogs) in homes using electrostatic dust collectors and QPCR: new tools to evaluate exposure and risk of allergies.

Contradictory results are found in the literature concerning fungi, bacteria, and pet exposure and the risk of developing asthma. All these allergens have been thoroughly studied separately in cohort studies, and a variety of sampling and analytical methods are used. It is already possible to characterize fungi, mites, and bacteria by QPCR. The aim of our study is to evaluate QPCR systems to quantify the presence of cats and dogs in homes. Twenty-four houses were sampled with an Electrostatic Dust Collector which was analyzed by QPCR. Questionnaires on the presence of pets in homes were completed. The results from QPCR were correlated for real presence of cats and dogs, and highlighted indirect exposure. This study provides a useful screening tool that will be used in future large cohort studies, such as the ELFE cohort study.

Increased incidence and characteristics of alveolar echinococcosis in patients with immunosuppression-associated conditions.

BACKGROUND: An increased incidence of alveolar echinococcosis (AE) in patients with immunosuppression (IS) has been observed; our aim was to study this association and its characteristics. METHODS: Fifty AE cases with IS-associated conditions (ISCs) before or at AE diagnosis were collected from the French AE registry (1982-2012, 509 cases). There were 30 cancers, 9 malignant hematological disorders, 14 chronic inflammatory diseases, 5 transplants, and 1 case of AIDS; 9 patients had ≥2 ISCs. Characteristics of the 42 IS/AE cases and the 187 non-IS/AE cases diagnosed during the period 2002-2012 were statistically compared. RESULTS: There was a significant increase in IS/AE cases over time. Risk factors did not differ between IS/AE and non-IS/AE patients. However, AE was more frequently an incidental finding (78% vs 42%) and was diagnosed at earlier stages (41% vs 23%) in IS/AE than in non-IS/AE patients. Serology was more often negative (14% vs 1%) and treatment efficacy was better (51% regression after 1-year treatment vs 27%) in IS/AE patients. All IS/AE patients but 7 took IS drugs; 7 received biotherapeutic agents. When not concomitant, AE occurred in IS patients within a 48-month median time period. Atypical presentation and abscess-, hemangioma-, and metastasis-like images delayed AE diagnosis in 50% of IS/AE patients, resulting in inappropriate treatment. Liver images obtained for 15 patients 1-5 years before diagnosis showed no AE lesions. Albendazole efficacy was good, but 19 of 48 treated patients experienced side effects. CONCLUSIONS: Patients with immunosuppression are at increased risk for occurrence, delayed diagnosis, and progression of AE.

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.

Challenging the phylogenetic relationships among Echinococcus multilocularis isolates from main endemic areas.

Alveolar echinococcosis (AE) is a rare but severe disease that affects more than 18,000 people worldwide per year. The complete sequencing of the mitochondrial genome of Echinococcus multilocularis has made it possible to study the genetic diversity of the parasite and its spatial and temporal evolution. We amplified the whole mitochondrial genome by PCR, using one uniplex and two multiplex reactions to cover the 13,738 bp of the mitogenome, and then sequenced the amplicons with Illumina technology. In total, 113 samples from Europe, Asia, the Arctic and North America were analyzed. Three major haplogroups were found: HG1, which clustered samples from Alaska (including Saint-Lawrence Island), Yakutia (Russia) and Svalbard; HG2, with samples from Asia, North America and Europe; and HG3, subdivided into three micro-haplogroups. HG3a included samples from North America and Europe, whereas HG3b and HG3c only include samples from Europe. In France, HG3a included samples from patients more recently diagnosed in a region outside the historical endemic area. A fourth putative haplogroup, HG4, was represented by only one isolate from Olkhon Island (Russia). The increased discriminatory power of the complete sequencing of the E. multilocularis mitogenome has made it possible to highlight four distinct geographical clusters, one being divided into three micro-haplogroups in France.

Populations at risk for alveolar echinococcosis, France.

During 1982-2007, alveolar echinococcosis (AE) was diagnosed in 407 patients in France, a country previously known to register half of all European patients. To better define high-risk groups in France, we conducted a national registry-based study to identify areas where persons were at risk and spatial clusters of cases. We interviewed 180 AE patients about their way of life and compared responses to those of 517 controls. We found that almost all AE patients lived in 22 départements in eastern and central France (relative risk 78.63, 95% CI 52.84-117.02). Classification and regression tree analysis showed that the main risk factor was living in AE-endemic areas. There, most at-risk populations lived in rural settings (odds ratio [OR] 66.67, 95% CI 6.21-464.51 for farmers and OR 6.98, 95% CI 2.88-18.25 for other persons) or gardened in nonrural settings (OR 4.30, 95% CI 1.82-10.91). These findings can help sensitization campaigns focus on specific groups.

Detecting nested clusters of human alveolar echinococcosis.

Recent changes in the epidemiology of alveolar echinococcosis (AE) in Eurasia have led to increasing concerns about the risk of human AE and the need for a thorough evaluation of the epidemiological situation. The aim of this study was to explore the use of a National Register to detect complex distribution patterns on several scales. The data were human AE cases from the FrancEchino register, diagnosed in France from 1982 to 2011. We used the Kulldorff spatial scan analysis to detect non-random locations of cases. We proposed an exploratory method that was based on the successive detection of nested clusters inside each of the statistically significant larger clusters. This method revealed at least 4 levels of disease clusters during the study period. The spatial variations of cluster location over time were also shown. We conclude that National Human AE registers, although not exempted from epidemiological biases, are currently the best way to achieve an accurate representation of human AE distribution on various scales. Finally, we confirm the multi-scale clustered distribution of human AE, and we hypothesize that our study may be a reasonable starting point from which to conduct additional research and explore the processes that underlie such distributions.

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.

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.

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.

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.

Clinical features and evolution of alveolar echinococcosis in France from 1982 to 2007: results of a survey in 387 patients.

BACKGROUND & AIMS: Alveolar echinococcosis (AE) is a rare disease in humans, caused by the larval stage of the fox tapeworm Echinococcus multilocularis. METHODS: We present here 387 detailed AE cases diagnosed in France from 1982 to 2007 actively identified by a retrospective survey performed in 1997-1998 and prospectively thereafter. RESULTS: Male:female ratio was 1.03 and mean age 57.8 years at time of diagnosis. Among the 362 complete files (including 347 non dead-out and 15 dead-out lesions), 73% of the patients were symptomatic at first admittance. Among them, 83% presented with clinical patterns evocative either of a digestive or a hepatic disorder. Other symptomatic patients presented with erratic clinical pictures, generally due to metastasis or extra-hepatic location of the parasite. Except for a few patients with particularly severe AE who died shortly after the diagnosis, most patients were treated using benzimidazoles. Their mortality tends to merge with that of the general French population, matched by sex, age, and calendar year. This study also highlights an unexpectedly high frequency of blood-tied family cases (13% of patients submitted to a specific questionnaire). CONCLUSIONS: Even though the broad set of clinical features provoked by E. multilocularis makes AE a potential diagnostic trap for many physicians, our study revealed an improvement of its prognosis. However, as shown by our findings about the frequency of family cases, there is still a need for studies aimed at better describing this uncommon parasitic disease.

Should possible recurrence of disease contraindicate liver transplantation in patients with end-stage alveolar echinococcosis? A 20-year follow-up study.

Liver transplantation (LT) is currently contraindicated in patients with residual or metastatic alveolar echinococcosis (AE) lesions. We evaluated the long-term course of such patients who underwent LT and were subsequently treated with benzimidazoles. Clinical, imaging, serological, and therapeutic data were collected from 5 patients with residual/recurrent AE lesions who survived for more than 15 years. Since 2004, [(18) F]-2-fluoro-2-deoxyglucose (FDG)-positron emission tomography (PET) images were available, and the levels of serum antibodies (Abs) against Echinococcus multilocularis-recombinant antigens were evaluated. Median survival time after LT was 21 years. These patients were from a prospective cohort of 23 patients with AE who underwent LT: 5 of 8 patients with residual/recurrent AE and 4 of 9 patients without residual/recurrent AE were alive in September 2009. High doses of immunosuppressive drugs, the late introduction of therapy with benzimidazoles, its withdrawal due to side effects, and nonadherence to this therapy adversely affected the prognosis. Anti-Em2(plus) and anti-rEm18 Ab levels and standard FDG-PET enabled the efficacy of therapy on the growth of EA lesions to be assessed. However, meaningful variations in Ab levels were observed below diagnostic cutoff values; and in monitoring AE lesions, images of FDG uptake taken 3 hours after its injection were more sensitive than images obtained 1 hour after its injection. In conclusion, benzimidazoles can control residual/recurrent AE lesions after LT. Using anti-rEm18 or anti-Em2(plus) Ab levels and the delayed acquisition of FDG-PET images can improve the functional assessment of disease activity. The potential recurrence of disease, especially in patients with residual or metastatic AE lesions, should not be regarded as a contraindication to LT when AE is considered to be lethal in the short term.

Phylogenetic relationships within Echinococcus and Taenia tapeworms (Cestoda: Taeniidae): an inference from nuclear protein-coding genes.

The family Taeniidae of tapeworms is composed of two genera, Echinococcus and Taenia, which obligately parasitize mammals including humans. Inferring phylogeny via molecular markers is the only way to trace back their evolutionary histories. However, molecular dating approaches are lacking so far. Here we established new markers from nuclear protein-coding genes for RNA polymerase II second largest subunit (rpb2), phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold). Bayesian inference and maximum likelihood analyses of the concatenated gene sequences allowed us to reconstruct phylogenetic trees for taeniid parasites. The tree topologies clearly demonstrated that Taenia is paraphyletic and that the clade of Echinococcus oligarthrus and Echinococcusvogeli is sister to all other members of Echinococcus. Both species are endemic in Central and South America, and their definitive hosts originated from carnivores that immigrated from North America after the formation of the Panamanian land bridge about 3 million years ago (Ma). A time-calibrated phylogeny was estimated by a Bayesian relaxed-clock method based on the assumption that the most recent common ancestor of E. oligarthrus and E. vogeli existed during the late Pliocene (3.0 Ma). The results suggest that a clade of Taenia including human-pathogenic species diversified primarily in the late Miocene (11.2 Ma), whereas Echinococcus started to diversify later, in the end of the Miocene (5.8 Ma). Close genetic relationships among the members of Echinococcus imply that the genus is a young group in which speciation and global radiation occurred rapidly.

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.

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.

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.