Complete mitochondrial exploration of Echinococcus multilocularis from French alveolar echinococcosis patients

Echinococcosis is a zoonotic disease caused by the cestode Echinococcus spp., one of the 17 neglected tropical diseases prioritized by the WHO, with a goal of control or even eradication by 2050 (World Health Organization et al., 2012). The most common forms in humans are alveolar echinococcosis (AE), caused by Echinococcus multilocularis, and cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato. AE is a severe parasitic disease caused by the larval stage (metacestode) of E. multilocularis. This cestode involves several mammals in its life cycle. In Europe, the definitive hosts are carnivores, especially foxes (Vulpes vulpes), and intermediate hosts include rodents, mainly those of the genera Arvicola, Myodes, and Microtus in Europe (Oksanen et al., 2016). There is also a domestic cycle involving dogs and possibly cats. These animals, due to their proximity to humans, present a significant risk of parasite transmission (Poulle et al., 2017, Torgerson et al., 2020). In humans, AE evolves mostly in the liver, infecting its host through escape mechanisms from the immune system (Brehm and Koziol, 2017). AE is more severe in immunocompromised patients (e.g., those with HIV or chronic inflammatory diseases, or undergoing immunosuppressive therapy or chemotherapy) (Chauchet et al., 2014). However, even without underlying immunosuppression, there is a high degree of inter-individual variability that may be related to host-dependent factors (Eiermann et al., 1998) and the immune response, as well as, potentially, the parasite and certain virulence factors (Vuitton, 2003).

The mortality rate 10 years after diagnosis has been estimated to be over 90% without treatment (Ammann and Eckert, 1996). Various clinical forms can be observed, with dissemination to neighboring organs or those more distant from the initial hepatic location. However, the parasitic lesion may also evolve into a calcified lesion, which then becomes inactive (Eckert and Deplazes, 2004, Wen et al., 2019).

In Europe and North America, this zoonosis is expanding. In North America, it is only recently that several cases of autochtone AE have been reported in Alberta, Canada, probably due to the involvement of European E. multilocularis strains (Massolo et al., 2019, Houston et al., 2021). In Europe, an increasing number of AE cases is being reported in national and international registers (Vuitton et al., 2015, Gottstein and Deplazes, 2021). A growing number of European nations are reporting AE, including the Baltic countries (Marcinkutė et al., 2015), Scandinavia (Wahlström et al., 2015), Belgium, and the Netherlands (van Dommelen et al., 2012, Cambier et al., 2018), which were previously free of the disease. Its reported expansion is partially due to better diagnostic methods and more exhaustive declaration of cases, but also to the growth and modification of the animal reservoirs (Vuitton et al., 2015).

Two current methods are useful for exploring the diversity of E. multilocularis to follow the genetic evolution of AE in time and space. Studies based on sequencing analysis of the mitochondrial genes cob, cox1, and nad2 (3,558 bp in total) have made it possible to trace back the evolutionary history of the parasite on a global scale and differentiate European, Asian, and North American clades (Nakao et al., 2009, Spotin et al., 2018). The highly polymorphic microsatellite marker EmsB (Echinococcus microsatellite Bart) (Bart et al., 2006, Valot et al., 2015) is a relevant genotyping tool due to its strong discriminatory power relative to other microsatellite markers (Knapp et al., 2007), but due to its nature, phylogenetic studies are complex.

The nuclear and mitochondrial genomes of E. multilocularis have been reported to show low genetic variability (Haag et al., 1997). The mitochondrial genome is easily amplifiable due to its presence in several copies in the targeted cells and its relatively short length (GenBank acc. No. AB018440.2, 13,738 bp). Recent studies analyzed the genetic diversity of E. granulosus using long mitochondrial genome sequences or the entire mitogenome (Kinkar et al., 2016, Kinkar et al., 2017, Laurimäe et al., 2016, 2018). These analyses have permitted the broad typing of parasites in Europe, South America, and Asia, showing high diversity, particularly related to livestock transport around the world. The amplification and analysis of the whole mitochondrial genome of E. multilocularis would likely make possible a finer analysis of genetic diversity.

In this context, we aimed to (i) develop a new method for the analysis of E. multilocularis polymorphism by sequencing the whole mitochondrial genome from PCR products, (ii) describe the genetic diversity of human AE surgical specimens collected from French patients using this new method, and (iii) compare the results with those obtained using two genotyping methods currently performed, such as sequencing of the mitochondrial genes cob, cox1, and nad2, and analysis of the microsatellite marker EmsB.

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