Jenny Knapp

State-of-the-art Echinococcus and Taenia: phylogenetic taxonomy of human-pathogenic tapeworms and its application to molecular diagnosis.

The taxonomy of tapeworms belonging to the family Taeniidae has been controversial because of the paucity of adult phenotypic characters and the great plasticity of larvae in intermediate hosts. The family consists of the medically important two genera Echinococcus and Taenia, which are closely related to each other. Cladistic approaches using the molecular data of DNA and the numerical data of morphologic characters are clarifying phylogenetic relationships among the members of these genera. The nucleotide data of worldwide taeniid parasites accumulated in public DNA databases may provide a basis for the development of molecular diagnostic tools, and make it possible to identify the parasites, at least the human Taenia spp. by non-morphologists. Furthermore, the detection of intraspecific genetic variations prompts evolutionary and ecological studies to address fundamental questions of parasite distributional patterns. Here, we introduce the recent advances of taeniid phylogeny and its application to molecular diagnosis.

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.

Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries

The taxonomic and phylogenetic status of Echinococcus granulosus strains are still controversial and under discussion. In the present study, we investigated the genetic polymorphism of E. granulosus isolates originating from three countries of Africa, including a region of Algeria, where the common G1 sheep and the camel G6 strains coexist sympatrically. Seventy-one hydatid cysts were collected from sheep, cattle, camels, and humans. Two mitochondrial markers (cox1 and nad1) were used for strain identification. Two nuclear markers (actII and hbx2) were used to study the possible occurrence of cross-fertilization. Despite the heterogeneity observed among the G1 isolates, they were all localized within one robust cluster. A second strong cluster was also observed containing all of the G6 isolates. Both strains appeared as two distinct groups, and no cases of interbreeding were found. Thus, the attribution of a species rank can be suggested. We also found the Tasmanian sheep G2 strain for the first time in Africa. Because of the slight variations observed between the common sheep and the Tasmanian sheep strains, further studies should be carried out to elucidate the epidemiological relevance of this genetic discrimination.

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.

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, [18F]‐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‐Em2plus 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‐Em2plus 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. Liver Transpl 17:855‐865, 2011.

Genetic diversity of the cestode Echinococcus multilocularis in red foxes at a continental scale in Europe

Background Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level. Methodology/Principal Findings The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas. Conclusions/Significance The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a “mainland–island” system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.

Assessment of Use of Microsatellite Polymorphism Analysis for Improving Spatial Distribution Tracking of Echinococcus multilocularis

ABSTRACT Alveolar echinococcosis (AE)—caused by the cestode Echinococcus multilocularis—is a severe zoonotic disease found in temperate and arctic regions of the northern hemisphere. Even though the transmission patterns observed in different geographical areas are heterogeneous, the nuclear and mitochondrial targets usually used for the genotyping of E. multilocularis have shown only a marked genetic homogeneity in this species. We used microsatellite sequences, because of their high typing resolution, to explore the genetic diversity of E. multilocularis. Four microsatellite targets (EmsJ, EmsK, and EmsB, which were designed in our laboratory, and NAK1, selected from the literature) were tested on a panel of 76 E. multilocularis samples (larval and adult stages) obtained from Alaska, Canada, Europe, and Asia. Genetic diversity for each target was assessed by size polymorphism analysis. With the EmsJ and EmsK targets, two alleles were found for each locus, yielding two and three genotypes, respectively, discriminating European isolates from the other groups. With NAK1, five alleles were found, yielding seven genotypes, including those specific to Tibetan and Alaskan isolates. The EmsB target, a tandem repeated multilocus microsatellite, found 17 alleles showing a complex pattern. Hierarchical clustering analyses were performed with the EmsB findings, and 29 genotypes were identified. Due to its higher genetic polymorphism, EmsB exhibited a higher discriminatory power than the other targets. The complex EmsB pattern was able to discriminate isolates on a regional and sectoral level, while avoiding overdistinction. EmsB will be used to assess the putative emergence of E. multilocularis in Europe.

Genetic diversity of Echinococcus multilocularis on a local scale

Echinococcusmultilocularis is the causative agent of human Alveolar Echinococcosis (AE), and it is one of the most lethal zoonotic infections in the Northern Hemisphere. In France, the eastern and central regions are endemic areas; Franche-Comté, Lorraine and Auvergne are particularly contaminated. Recently, several human cases were recorded in the French Ardennes area, a region adjacent to the western border of the E. multilocularis range in France. A previous study in this focus described a prevalence of over 50% of the parasite in red foxes. The present study investigated the genetic diversity of adult worms collected from foxes in a 900km(2) area in the Ardennes. Instead of a conventional mitochondrial target (ATP6), two microsatellite targets (EmsB and NAK1) were used. A total of 140 adult worms isolated from 25 red foxes were genotyped. After hierarchical clustering analyses, the EmsB target enabled us to distinguish two main assemblages, each divided into sub-groups, yielding the differentiation of six clusters or assemblage profiles. Thirteen foxes (52% of the foxes) each harbored worms from at least two different assemblage profiles, suggesting they had become infected by several sources. Using the NAK1 target, we identified 3 alleles, two found in association with the two EmsB assemblages. With the NAK1 target, we investigated the parasite breeding system and the possible causes of genetic diversification. Only one fox harbored hybrid worms, indicative of a possible (and rare) occurrence of recombination, although multiple infections have been observed in foxes. These results confirm the usefulness of microsatellite targets for assessing genetic polymorphism in a geographically restricted local range.

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.

Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools

The oncosphere stage of Echinococcus multilocularis in red fox stools can lead, after ingestion, to the development of alveolar echinococcosis in the intermediate hosts, commonly small mammals and occasionally humans. Monitoring animal infection and environmental contamination is a key issue in public health surveillance. We developed a quantitative real-time PCR technique (qPCR) to detect and quantify E. multilocularis DNA released in fox faeces. A qPCR technique using a hydrolysis probe targeting part of the mitochondrial gene rrnL was assessed on (i) a reference collection of stools from 57 necropsied foxes simultaneously investigated using the segmental sedimentation and counting technique (SSCT) (29 positive for E. multilocularis worms and 28 negative animals for the parasite); (ii) a collection of 114 fox stools sampled in the field: two sets of 50 samples from contrasted endemic regions in France and 14 from an E. multilocularis-free area (Greenland). Of the negative SSCT controls, 26/28 were qPCR-negative and two were weakly positive. Of the positive SSCT foxes, 25/29 samples were found to be positive by qPCR. Of the field samples, qPCR was positive in 21/50 (42%) and 5/48 (10.4%) stools (2 samples inhibited), originating respectively from high and low endemic areas. In faeces, averages of 0.1 pg/μl of DNA in the Jura area and 0.7 pg/μl in the Saône-et-Loire area were detected. All qPCR-positive samples were confirmed by sequencing. The qPCR technique developed here allowed us to quantify environmental E. multilocularis contamination by fox faeces by studying the infectious agent directly. No previous study had performed this test in a one-step reaction.