Molecular typing of Belgian Echinococcus multilocularis specimens from alveolar echinococcosis human lesions using EmsB microsatellites analysis

Background. The genetic diversity of Echinococcus multilocularis (Em) is a major field of investigations to correlate with sources of infection or variable clinical manifestations of the alveolar echinococcosis (AE). Molecular markers able to distinguish strains are already used such as EmsB microsatellites. This marker is present in about 40 copies in the Em genome. Here, we report the use of EmsB microsatellite polymorphism for the typing of Belgian specimens isolated from patients with AE between 2019 and 2020.

Material and methods. Total genomic DNA was isolated from liver, pleural fluid and bile samples using a DNA extraction kit for tissue (Qiagen). The PCR was performed according to Knapp et al, 2020. The EmsB A primer was 5’-labeled with FAM-fluorochrome. Fragment size analysis was performed on an ABI3500 automatic sequencer (ThermoFisher). The fluorescence signal was detected by colorimetric reading. Correspondences were established to assess the size of the amplified fragments using Gene mapper (ThermoFisher). “R studio” was used to generate a distance matrix, calculate the Euclidian distance and obtain a UPGMA method dendrogram in order to assess the similarity among samples. The profiles obtained were compared with those included in the EWET data collection.

Results
Seven specimens have been successfully analyzed. According to a comparison with European samples previously characterized (Knapp et al., 2020), 3 Belgian specimens shared the same P5 genomic profile while one strain had a P8 profile. These P5 and P8 strains were included into European profiles with strains from France, Switzerland and Germany. The three other isolates could not be classified into existing profiles but were placed between P6 and P7 profiles. Five strains originated from patients living in Wallonia, the Southern part of Belgium (Namur, Hainaut and Luxembourg) while the two others originated from neighboring provinces (Walloon Brabant and Bruxelles).

Conclusions
The EmsB microsatellites analysis allowed to genotypically characterize Em clinical specimens isolated in Belgium for the first time. This study highlights that some samples share the same genotypic profile but that heterogenetic diversity exist in Belgium. Some profiles are unique and differ from other European profiles. Further studies including more clinical samples are ongoing.