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

Abstract

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

Keywords: Alveolar echinococcosis; Echinococcus multilocularis; European endemic area; contamination event; genotyping; microsatellite EmsB.

Figure 1

Electrophoregrams for genotyping performed on four human alveolar echinococcosis (AE) samples for the four Multiplex mixture conditions (MLX-A to D) and the Platinum mixture (PL).

Figure 2

Electrophoregrams for the genotyping performed on 14 EchinoRisk samples from three foxes in Multiplex PCR master mix (Qiagen) conditions (MLX A to D) and Platinum Taq DNA polymerase mixture (Invitrogen) conditions (PL) at the Besançon Laboratory (NRC-E) and in AmpliTaq DNA polymerase mixture (Applied Biosystems) conditions at the Berne Laboratory (IPA). The arrows indicate the 2 bp shift observed between the results obtained with the ABI PRISM 3100 Genetic Analyzer and the Applied Biosystems 3130 Genetic Analyzer.

Figure 3

Dendrogram constructed from EmsB amplification data by hierarchical clustering analysis (Euclidean distance and unweighted pair group method). Approximately unbiased (au) p-values are indicated at tree nodes as percentages calculated by multiscale bootstrapping (B = 1000). E. granulosus sensu stricto (G1 strain, originating from an Algerian sheep), and three E. multilocularis from Alaska (ALK-SLI), Japan (JAP-I3J-R) and China (CHI-7PRC-r) were used as control outgroups. A genetic distance threshold of 0.1 was used to distinguish EmsB profiles. A representative EmsB electrophoregram is provided for each profile.

Figure 4

Distribution of the EmsB profiles described for human AE lesions in the countries studied (a), and French endemic Région profiles (b). TDB: Territoire de Belfort; Ct.: Canton; G. Ct.: Canton of Geneva.

Figure 5

Distribution over time, from 1986 to 2018, of the nine EmsB profiles (P1 to P9) for the 63 alveolar echinococcosis patients genotyped.

Figure 6

Distribution of the 66 AE human samples and the 1211 EWET (EmsB Website for Echinococcus Typing) reference data (a), and graphical representation of the EmsB profiles described for human AE lesions and similar EWET reference data selected on the basis of a Euclidean distance of less than 0.1 to the tested samples (b).