Parasite Echinococcus Multilocularis Research Articles

A300 ADVANCED HEPATIC ECHINOCOCCOSIS: A CASE REPORT

Abstract Background Alveolar echinococcosis (AE) is a rare and potentially fatal disease caused by the parasite echinococcus multilocularis (EM). Humans are intermediate hosts who become infected through contact with definitive hosts (foxes, wolves, coyotes, dogs and cats) and ingestion of contaminated food and water. AE primarily affects the liver but has the potential for local or metastatic spread. It has a clinical latency time of 5-15 years and resembles a malignancy in appearance and growth, posing a challenge in diagnosis and management. Treatment includes benzimidazole therapy and surgical resection, however, removal of the entire parasitic mass is not always possible. Aims Present a case of hepatic AE involving the gallbladder, biliary tree, and spleen. Methods Case report. Results A 49-year-old farmer was admitted to a Saskatchewan hospital for a 2-week history of abdominal pain, jaundice, and progressive weight loss. Computed tomography (CT) of the abdomen and pelvis showed a necrotic mass in the gallbladder compromising the liver, splenic lesions and involvement of the biliary tree. The patient was given a preliminary diagnosis of metastatic cholangiocarcinoma and underwent a gallbladder biopsy which returned as echinococcus, confirmed with two liver biopsies and a positive EM nucleic acid test. He was then diagnosed with advanced hepatic AE with metastasis to the gallbladder, biliary tree, and spleen. The patient required a long-term hepatic drain and developed a hepatocutaneous fistula. While surgery is curative, his disease was too extensive for resection. He was started on Albendazole and received into our care for a liver transplant assessment 10 months later. Currently, there is limited literature on liver transplantation in advanced AE as it may not be curative. Additionally, subsequent immunosuppression may expedite residual parasite growth leading to aggressive disease recurrence. In this case, liver transplant would also require a splenectomy and excision of the hepatocutaneous fistula for complete parasite removal. Repeat CT of the abdomen and pelvis at thirteen months after the patient's initial hospitalization did not show disease progression. Given his stability on Albendazole, the added surgical complexity, and possibility of aggressive disease recurrence post liver-transplant, the multidisciplinary committee decided against liver transplant at this time. He continues to do well on Albendazole 15 months after initial presentation. Conclusions The reported mortality rate of AE is ampersand:003E 90% within 10-15 years of diagnosis if untreated or inadequately treated. This case highlights the complexities in management of advanced hepatic AE and the importance of early diagnosis and treatment with radical surgery. Although not curative, benzimidazole therapy is effective in slowing disease progression in unresectable cases. Further studies are needed to assess the role of liver transplantation in advanced hepatic AE. Funding Agencies None

Infection of sheep by Echinococcus multilocularis in Gansu, China: evidence from mitochondrial and nuclear DNA analysis

BackgroundIn the normal life cycle of the parasite (Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China.MethodsFrom 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed.ResultsThe isolates displayed ≥ 98.87% similarity to E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis.ConclusionsAs a result of the discovery of E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area.Graphical

Investigation of the mechanism of action of mefloquine and derivatives against the parasite Echinococcus multilocularis

Alveolar echinococcosis (AE) is caused by infection with the fox tapeworm E. multilocularis. The disease affects humans, dogs, captive monkeys, and other mammals, and it is caused by the metacestode stage of the parasite growing invasively in the liver. The current drug treatment is based on non-parasiticidal benzimidazoles. Thus, they are only limitedly curative and can cause severe side effects. Therefore, novel and improved treatment options for AE are needed.Mefloquine (MEF), an antimalarial agent, was previously shown to be effective against E. multilocularis in vitro and in experimentally infected mice. However, MEF is not parasiticidal and needs improvement for successful treatment of patients, and it can induce strong neuropsychiatric side-effects. In this study, the structure-activity relationship and mode of action of MEF was investigated by comparative analysis of 14 MEF derivatives. None of them showed higher activity against E. multilocularis metacestodes compared to MEF, but four compounds caused limited damage. In order to identify molecular targets of MEF and effective derivatives, differential affinity chromatography combined with mass spectrometry was performed with two effective compounds (MEF, MEF-3) and two ineffective compounds (MEF-13, MEF-22). 1′681 proteins were identified that bound specifically to MEF or derivatives. 216 proteins were identified as binding only to MEF and MEF-3. GO term enrichment analysis of these proteins and functional grouping of the 25 most abundant MEF and MEF-3 specific binding proteins revealed the key processes energy metabolism and cellular transport and structure, as well as stress responses and nucleic acid binding to be involved. The previously described ferritin was confirmed as an exclusively MEF-binding protein that could be relevant for its efficacy against E. multilocularis. The here identified potential targets of MEF will be further investigated in the future for a clear understanding of the pleiotropic effects of MEF, and improved therapeutic options against AE.

Dual inhibition of the Echinococcus multilocularis energy metabolism.

Alveolar echinococcosis is caused by the metacestode stage of the zoonotic parasite Echinococcus multilocularis. Current chemotherapeutic treatment options rely on benzimidazoles, which have limited curative capabilities and can cause severe side effects. Thus, novel treatment options are urgently needed. In search for novel targetable pathways we focused on the mitochondrial energy metabolism of E. multilocularis. The parasite relies hereby on two pathways: The classical oxidative phosphorylation including the electron transfer chain (ETC), and the anaerobic malate dismutation (MD). We screened 13 endochin-like quinolones (ELQs) in vitro for their activities against two isolates of E. multilocularis metacestodes and isolated germinal layer cells by the phosphoglucose isomerase (PGI) assay and the CellTiter Glo assay. For the five most active ELQs (ELQ-121, ELQ-136, ELQ-271, ELQ-400, and ELQ-437), EC50 values against metacestodes were assessed by PGI assay, and IC50 values against mammalian cells were measured by Alamar Blue assay. Further, the gene sequence of the proposed target, the mitochondrial cytochrome b, was analyzed. This allowed for a limited structure activity relationship study of ELQs against E. multilocularis, including analyses of the inhibition of the two functional sites of the cytochrome b. By applying the Seahorse XFp Extracellular Flux Analyzer, oxygen consumption assays showed that ELQ-400 inhibits the E. multilocularis cytochrome bc1 complex under normoxic conditions. When tested under anaerobic conditions, ELQ-400 was hardly active against E. multilocularis metacestodes. These results were confirmed by transmission electron microscopy. ELQ-400 treatment increased levels of parasite-released succinate, the final electron acceptor of the MD. This suggests that the parasite switched to MD for energy generation. Therefore, MD was inhibited with quinazoline, which did not induce damage to metacestodes under anaerobic conditions. However, it reduced the production of succinate compared to control treated parasites (i.e., inhibited the MD). The combination treatment with quinazoline strongly improved the activity of the bc1 inhibitor ELQ-400 against E. multilocularis metacestodes under anaerobic conditions. We conclude that simultaneous targeting of the ETC and the MD of E. multilocularis is a possible novel treatment approach for alveolar echinococcosis, and possibly also other foodborne diseases inflicted by platyhelminths, which cause substantial economic losses in livestock industry.

Dog Ownership and Risk for Alveolar Echinococcosis, Germany.

Human alveolar echinococcosis is caused by the parasite Echinococcus multilocularis, and dog ownership has been identified as a risk factor. We sought to specify the factors of dog ownership underlying this risk by conducting a case–control study among dog owners in Germany. The analysis revealed an increased odds ratio of ≈7-fold for dog owners whose dogs roam unattended in fields, 13-fold for dog owners who feed their dogs organic waste daily, 4-fold for dog owners who take their dog to a veterinarian only in case of illness, and 10-fold for dog owners who have never been informed by a veterinarian about the risk for infection. The results highlight the risk for infection associated with various factors of dog ownership and the value of veterinarians informing owners about prevention.

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.

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.

Tapeworms in Rodents from the Lower Anabar River Basin, with a Review of Species Diversity of Cestodes in Yakutia, Russia.

We aimed to explore the first data on the fauna of cestodes in rodents from the Lower Anabar River Basin situated in extreme north-western Yakutia, Russia and to develop a biological and biogeographical framework for examination of the species diversity of cestodes from Yakutia, which is an important faunal transition zone in the eastern Palaearctic. Field inventory of cestodes from rodents was conducted for the first time in the region of north-western Yakutia. Species diversity, intensity and prevalence of infection were assessed. It was noted a rather high (up to 82.5%) total cestode prevalence in rodents in the extreme north-western area of Yakutia. In Arvicolinae hosts, we collected specimens of six cestode species of four genera and three families. Arostrilepis microtis and Douthittia nordenskioeldi were first collected in voles Lasiopodomys gregalis from Yakutia (new host and geographical records). The zoonotic parasite Echinococcus multilocularis was found in the liver of L. gregalis. The list of cestodes from rodents in Yakutia based on review of previous publications and the material obtained in the present study includes 24 species of 16 genera and five families. Out of these, 17 parasitise rodents as adults stage and seven as larvae. The fauna of rodent tapeworms from the south subarctic tundra and pre-tundra larch woodlands of the north-western Yakutia is characterised by relatively impoverished species diversity compared to the fauna of cestodes from the taiga zone of the central Yakutia. The decrease in the species diversity of tapeworms does not affect the total infection prevalence of the definitive hosts, which is relatively high in this region.

Expression profiling of Echinococcus multilocularis miRNAs throughout metacestode development in vitro.

The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite Echinococcus multilocularis. MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro, determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E. multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E. multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E. multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE.

An altered microbiome in urban coyotes mediates relationships between anthropogenic diet and poor health

Generalist species able to exploit anthropogenic food sources are becoming increasingly common in urban environments. Coyotes (Canis latrans) are one such urban generalist that now resides in cities across North America, where diseased or unhealthy coyotes are frequently reported in cases of human-wildlife conflict. Coyote health and fitness may be related to habitat use and diet via the gut microbiome, which has far-reaching effects on animal nutrition and physiology. In this study, we used stomach contents, stable isotope analysis, 16S rRNA gene amplicon sequencing, and measures of body condition to identify relationships among habitat use, diet, fecal microbiome composition, and health in urban and rural coyotes. Three distinct relationships emerged: (1) Urban coyotes consumed more anthropogenic food, which was associated with increased microbiome diversity, higher abundances of Streptococcus and Enterococcus, and poorer average body condition. (2) Conversely, rural coyotes harbored microbiomes rich in Fusobacteria, Sutterella, and Anaerobiospirillum, which were associated with protein-rich diets and improved body condition. (3) Diets rich in anthropogenic food were associated with increased abundances of Erysipelotrichiaceae, Lachnospiraceae, and Coriobacteriaceae, which correlated with larger spleens in urban coyotes. Urban coyotes also had an increased prevalence of the zoonotic parasite Echinococcus multilocularis, but there were no detectable connections between parasite infection and microbiome composition. Our results demonstrate how the consumption of carbohydrate-rich anthropogenic food by urban coyotes alters the microbiome to negatively affect body condition, with potential relationships to parasite susceptibility and conflict-prone behavior.

An Update on the Epidemiological Situation of Alveolar Echinococcosis: Recording and Reporting Structures in Austria, Switzerland and Germany

Alveolar echinococcosis (AE) is a rare disease in Austria, Switzerland and Germany (DACh) caused by an infection with the parasite Echinococcus multilocularis. The aim of the study was to describe differences in the detection and reporting systems of alveolar echinococcosis in Austria, Switzerland and Germany and to describe epidemiological trends. As part of an epidemiological update on 6th September 2019 in Ulm, Germany, experts and representatives discussed differences in the reporting and recording systems as well as the current epidemiological situation. Since 2004, Austria has had an obligation to report suspected cases, diseases and deaths of alveolar echinococcosis by name in accordance with §1 Para. 1 of the Epidemiegesetz 1950 (EpidemieG) and the Ordinance on Notifiable Communicable Diseases. According to §7 Para. 3 of the German Infection Protection Act (IfSG), Germany has also been subject to a reporting obligation since 2001, but not by name. In addition, national registers are available in both countries, which can be used to answer scientific questions. In Switzerland, there is no obligation to report human alveolar echinococcosis since 1997. Efforts are currently being made to implement a national register for alveolar echinococcosis in Switzerland. Despite different reporting and recording systems, a similar epidemiological trend can be observed for DACh. In Austria, Switzerland and Germany there is a slightly increasing trend of human cases with alveolar echinococcosis. The direct comparability is limited due to different reporting obligations. The structures often do not allow a joint answering of scientific questions concerning diagnostics, treatment and care.

Surgery versus conservative drug therapy in alveolar echinococcosis patients in Germany – A health-related quality of life comparison

Alveolar echinococcosis (AE) is a rare zoonosis caused by the parasite Echinococcus multilocularis. Nothing is known about the health-related quality of life (HRQoL) in patients with AE receiving different types of therapy. Therefore, the aim of the study was to compare HRQoL in patients with AE in Germany depending on their therapeutic regimen namely conservative drug treatment with long-term benzimidazoles versus surgical therapy by resection of the parasitic liver lesions. The 36-Item Short Form Health Survey (SF-36) questionnaire, including other echinococcosis-related questions, was used to measure HRQoL. The SF-36 scales were evaluated according to the algorithms provided by the authors. The statistical analysis was performed with SAS version 9.2. The significance level was set at α = 0.05, p < 0.05 corresponds to statistical significance. The investigated group consisted of conservatively drug treated (n = 30) and patients with curative surgery (n = 25) with confirmed AE. The study was performed at an infectious disease outpatient department from April 2018 to October 2018. The conservatively drug treated patient group consisted of 15 men (50.0%) and 15 women (50.0%) with an average age of 55.7 ± 16.7 years (Median: 59). The surgery group consisted of nine men (36.0%) and 16 women (64.3%) with an average age of 53.3 ± 31.9 years (Median: 54). The physical quality of life of the conservatively drug treated patients did not show any significant differences to the surgical treated group (45.2 ± 11.4 vs. 47.6 ± 9.9; p = 0.4079). There was also no significant difference regarding the mental quality of life between the conservatively drug treated patients, and those treated with curative surgery (45.5 ± 10.6 vs. 47.3 ± 10.8; 0.5206). Nevertheless, there was a slight advantage in the physical and mental scores of the patients treated with surgery. Furthermore, for 13 of the 25 surgically treated patients, some aspects of the HRQoL improved significantly after surgery.The evaluation showed no statistically significant differences in HRQoL in patients with AE dependent on the applied treatment strategy (conservative drug versus curative surgical therapy).

Health-related quality of life in patients with alveolar echinococcosis: a cross-sectional study

The Alveolar echinococcosis (AE) is a rare zoonosis caused by the parasite Echinococcus multilocularis. To date, nothing is known about the health-related quality of life (HRQoL) in patients with AE. The aim of the study was to evaluate the HRQoL in patients with AE in comparison of the healthy population. We used the 36-Item Short Form Health Survey (SF-36) questionnaire to evaluate the HRQoL. The SF-36 scales have a range of 0-100 (0 represents the worst and 100 the most favourable state of health). SAS Version 9.2 was used for the statistical analysis of AE-cases (n = 30) and the healthy control group (n = 35). The analysis showed that the HRQoL in people with AE is reduced in comparison with the control population. The study group consisted of 15 (50.0%) men and 15 (50.0%) women; the control group of 16 (45.7%) men and 19 (54.3%) women. The mean age was 55.73 ± 16.65 years, while that of the control group was 54.57 ± 15.34years. The physical quality of life in patients with AE (45.21 ± 11.42) was not significantly less than that of the control group (50.54 ± 10.52); p = 0.0568. Nevertheless, AE-patients show lower SF-36 scores for the physical quality of life. For the mental quality of life, patients with AE had a significantly lower score (45.46 ± 10.57) than the control group (51.57 ± 9.04); p = 0.0154. The HRQoL in people with AE is reduced in comparison with a control population. Assessment of the physical and mental quality of life in patients with AE may help to evaluate the patient outcome.

Alveolar echinococcosis in Germany, 1992-2016. An update based on the newly established national AE database.

Alveolar echinococcosis is a rare disease caused by the parasite Echinococcus multilocularis. The newly established national database should enable us to determine high-risk areas and evaluate the endemic levels. The national database was implemented using SQL Workbench. We used EpiInfo™ software to display the prevalence of disease. Moran's I geodata analysis according to cluster and high risk areas was performed with GeoDa™. SAS Version 9.2 was used for the statistical analysis of the cases (n=523). The analysis showed a concentration of cases in Baden-Württemberg and Bavaria. Moran's I showed a heterogeneous case distribution throughout Germany (I=0.208815, Z=32.6175, p<0.001). In the period from 1992 to 2016, the prevalence was 0.64/100,000 inhabitants in the whole of Germany, 2.18/100,000 inhabitants in Baden-Württemberg and 1.48/100,000 inhabitants in Bavaria. The analysis also revealed a difference between men and women, with a prevalence of 0.58/100,000 and 0.69/100,000, respectively. The analysis of spatial autocorrelation and possible risk areas showed that the southeast regions of Baden-Württemberg (I=0.188514, Z=11.3197, p<0.001) and the southwest part of Bavaria (I=0.176953, Z=13.5144, p<0.001) constitute the high risk areas. The prevalence of disease is noticeably high in the Swabian Jura, the Bavarian Alps and the Alpine foothills. Raising awareness and educating doctors in high risk areas may prevent new cases and assist in earlier diagnosis.

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.

Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts

A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts.

MicroRNA discovery in the human parasite Echinococcus multilocularis from genome-wide data

The cestode parasite Echinococcus multilocularis is the aetiological agent of alveolar echinococcosis, responsible for considerable human morbidity and mortality. This disease is a worldwide zoonosis of major public health concern and is considered a neglected disease by the World Health Organization. The complete genome of E. multilocularis has been recently sequenced and assembled in a collaborative effort between the Wellcome Trust Sanger Institute and our group, with the main aim of analyzing protein-coding genes. These analyses suggested that approximately 10% of E. multilocularis genome is composed of protein-coding regions. This shows there is still a vast proportion of the genome that needs to be explored, including non-coding RNAs such as small RNAs (sRNAs). Within this class of small regulatory RNAs, microRNAs (miRNAs) can be found, which have been identified in many different organisms ranging from viruses to higher eukaryotes. MiRNAs are a key regulation mechanism of gene expression at post-transcriptional level and play important roles in biological processes such as development, proliferation, cell differentiation and metabolism in animals and plants. In spite of this, identification of miRNAs directly from genome-wide data only is still a very challenging task. There are many miRNAs that remain unidentified due to the lack of either sequence information of particular phylums or appropriate algorithms to identify novel miRNAs. The motivation for this work is the discovery of new miRNAs in E. multilocularis based on non-target genomic data only, in order to obtain useful information from the currently available unexplored data. In this work, we present the discovery of new pre-miRNAs in the E. multilocularis genome through a novel approach based on machine learning. We have extracted the most commonly used structural features from the folded sequences of the parasite genome: triplets, minimum free energy and sequence length. These features have been used to train a novel deep architecture of self-organizing maps (SOMs). This model can be trained with a high class imbalance and without the artificial definition of a negative class. We discovered 886 pre-miRNA candidates within the E. multilocularis genome-wide data. After that, experimental validation by small RNA-seq analysis clearly showed 23 pre-miRNA candidates with a pattern compatible with miRNA biogenesis, indicating them as high confidence miRNAs. We discovered new pre-miRNA candidates in E. multilocularis using non-target genomic data only. Predictions were meaningful using only sequence data, with no need of RNA-seq data or target analysis for prediction. Furthermore, the methodology employed can be easily adapted and applied on any draft genomes, which are actually the most interesting ones since most non-model organisms have this kind of status and carry real biological and sanitary relevance.AvailabilityWeb demo: http://fich.unl.edu.ar/sinc/web-demo/mirna-som/Source code: http://sourceforge.net/projects/sourcesinc/files/mirnasom/

Synthesis and Antigenicity against Human Sera of a Biotin-Labeled Oligosaccharide Portion of a Glycosphingolipid from the Parasite Echinococcus multilocularis.

Synthesis of a biotinylated analog of the carbohydrate portion of a glycosphingolipid from the parasite Echinococcus multilocularis has been achieved. We synthesized β-D-Galp-(1→6)-β-D-Galp-(1→6)-[α-L-Fucp-(1→3)]-β-D-Galp-(1→R: biotin probe) (1) and compared the antigenicity by an enzyme linked immunosorbent assay (ELISA) with biotinylated trisaccharide α-D-Galp-(1→4)-β-D-Galp-(1→3)-α-D-Galp-(1→R: biotin probe) (F), which has been shown to have significant antigenicity. Both of the oligosaccharides reacted with sera of alveolar echinococcosis (AE) patients, but showed different reactivity. Among the 60 sera of AE patients, more sera reacted with the linear sequence Galα1→4Galβ1→3GalNAcα1→R of oligosaccharide (F) than for branched compound 1. Some sera showed high specificity to one of the compound, indicating that the antibodies in the sera of AE patients differ in their specificity to recognize carbohydrate sequences of glycosphingolipids. Our results demonstrate that both of the biotinylated oligosaccharides 1 and F have good serodiagnostic potential and are complementary to detect infections caused by the parasite Echinococcus multilocularis.

E. multilocularis infection in animals

E. multilocularis infection in animals

Echinococcus multilocularis in dogs from two French endemic areas: No evidence of infection but hazardous deworming practices

The life cycle of the zoonotic parasite Echinococcus multilocularis is predominantly sylvatic, involving foxes as definitive hosts infected by predation of rodents, the intermediate hosts. The North-Eastern French departments of Meuse and Haute-Saône are highly endemic, with an estimated fox prevalence of 41% and 36% respectively. Although most of the parasites’ biomass occurs in foxes, domestic dogs can also be infected, leading to a major risk of human infection due to the close proximity of dogs and owners. In the present study, dog faeces were collected after praziquantel treatment provided by veterinarians. In all, 860 faecal samples were collected throughout Meuse (n=493) and Haute-Saône (n=367). Intestinal helminth eggs were isolated from the faeces using a flotation technique and observed by microscopy. Parasite species were identified in samples positive for taeniid eggs by sequence analysis after PCR amplification. To study the factors associated with infestation, each sample was linked to a questionnaire filled in by the dog owners.Taeniid eggs were observed in seven faecal samples (0.8%) but none of them were positive for E. multilocularis. Thus, the apparent prevalence of E. multilocularis in dog populations is lower than 1.00% for Haute-Saône and lower than 0.75% for Meuse. In Haute-Saône, a high proportion of dogs observed suspected preying on rodents were not dewormed monthly. In endemic areas, these dogs must be considered at risk of transmitting E. multilocularis to humans.