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

Abstract

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance

“…Given the overall limited performances of traditional and partly modified copromicroscopic techniques (e.g., FT and CMPCR), the adoption of different approaches for the analysis of fecal material, such as the use of molecular test alone for all collected samples, should be at least considered. The use of real-time PCR has led indeed to a further important increase in the sensitivity of the copromicroscopic test for the detection of E. multilocularis ( 46 – 50 ) allowing for the detection of low-intensity infections.…”

Assessing Red Fox (Vulpes vulpes) Demographics to Monitor Wildlife Diseases: A Spotlight on Echinococcus multilocularis

“…According to parasitological investigations, two fecal samples from Alto Isarco-Wipptal, corresponding to two individual foxes (6.45%), tested positive for E. multilocularis . Based on test performances according to Obber et al [ 33 ], true prevalence was estimated at 31% (CI 50%: 12–43.5).…”

“…Parasitological analyses on collected samples were performed at the Parasitology laboratory of Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) using the copromicroscopy and multiplex PCR technique described in Citterio et al [ 21 ] and referred to as CMPCR in Obber et al [ 33 ]. Briefly, floatation, filtration, and sieving of 2 g of feces were used to isolate Taenidae eggs, which were then processed for DNA extraction, PCR amplification, and Sanger sequencing.…”

“…Briefly, floatation, filtration, and sieving of 2 g of feces were used to isolate Taenidae eggs, which were then processed for DNA extraction, PCR amplification, and Sanger sequencing. True prevalence based on test performances [ 33 ] was then calculated using the Rogan–Gladen formula as in Lang and Reiczigel [ 43 ].…”

“…In Italy, the distribution of E. multilocularis has long been limited to the Alps, corresponding to the southern margin of its central European range. In particular, since 2002, it has consistently been reported in the Alto Isarco-Wipptal district and in Val Pusteria-Pustertal Valley, both in the province of Bolzano-Bozen (northeastern Italy) [ 21 , 28 , 29 , 30 , 31 , 32 , 33 ], although recent findings in the Ligurian Alps suggest the emergence of a novel endemic area, possibly as the result of an expansion of its French range towards the southeast [ 34 ].…”