A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020)

Background Surveillance of E. multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence and burden in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. Aim We reassessed the prevalence of E. multilocularis at the southern border of its distribution in Alto Adige (Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. Methods We compared results from the diagnostic test currently implemented for surveillance (based on Coproscopy+Multiplex PCR - CMPCR), against a real-time quantitative PCR (qPCR) for 235 fox faeces collected in 2019-2020. The performances of the two tests were estimated using a scraping technique (SFCT) as the gold standard applied to the small intestines of a subsample (n=123) of the same hosts. True prevalence was calculated and sample size required by each faecal test for the detection of the parasite was then estimated. Results True prevalence of E. multilocularis in foxes (14.3%) was definitely higher than reported in the last decade (never >5% from 2012 to 2018). The qPCR also had a higher sensitivity (83%) compared to CMPCR (21%). Agreement with the gold standard was far higher for qPCR (0.816) than CMPCR (0.298) as well, determining a smaller sample size required to detect the disease. Conclusions Alto Adige should be considered a highly endemic area. Surveillance at the edges of E. multilocularis distribution should adopt qPCR diagnostics on definitive hosts on a small geographic scale. AUTHOR SUMMARY Echinococcus multilocularis is an intestinal flatworm, whose adult stage in Europe is harboured mainly by the red fox (Vulpes vulpes), which spreads parasite’s eggs by faeces. This parasite is the agent of a severe and potentially lethal zoonosis, the alveolar echinococcosis, affecting humans after accidental ingestion of parasite’s eggs. In the Italian Alpine area, which represents the southernmost border of E. multilocularis European range, surveillance is hindered by a fragmented distributional pattern, where presence in foxes has been consistently reported only in few isolated foci in Alto Adige (Bolzano province – Italy) of low prevalence. In order to improve the efficiency of monitoring efforts, we tested the performances of two diagnostic protocols on fox faeces (sedimentation, filtration, counting technique followed by standard PCR, and whole stool real-time PCR) against a benchmark technique on fox intestines (scraping, filtration, counting technique - considered as the gold standard). This allowed not only to determine qPCR as a far more sensitive and sample-efficient diagnostic tool for E. multilocularis detection in marginally affected areas, but also to re-assess its prevalence in Alto Adige, which should be considered a highly endemic area. Consequent actions in the field and modifications in the surveillance strategy should be therefore considered.