Detection of Echinococcus multilocularis in the Definitive Host: Coprodiagnosis by PCR as an Alternative to Necropsy

Abstract

Recently, extensions of the range of Echinococcus multilocularis in Europe and North America and drastic increases in fox populations in Europe put an increasing proportion of the human population at risk of alveolar echinococcosis. To obtain data on the local infection pressure, studies of the prevalence of the parasite in the animals that transmit the parasite, foxes, dogs, and cats, are urgently required. Such investigations, however, have been hampered by the need for necropsy of the host animal to specifically diagnose infection with the parasite. In this study, a nested PCR and an improved method for DNA extraction were developed to allow the sensitive and specific diagnosis of E. multilocularisinfections directly from diluted fecal samples from foxes. The target sequence for amplification is part of the E. multilocularismitochondrial 12S rRNA gene. The specificity of the method was 100% when it was tested against 18 isolates (metacestodes and adult worms) of 11 cestode species, including E. granulosus. The sensitivity of the method was evaluated by adding egg suspensions and individual eggs to samples of diluted feces from uninfected foxes. The presence of one egg was sufficient to give a specific signal. To confirm the PCR results, an internal probe which hybridized only withE. multilocularis amplification products but not with the DNA of other cestodes was constructed. In order to investigate the applicability of this method for epidemiological studies, 250 wild foxes from a area in southern Germany where echinococcosis is highly endemic were examined by both necropsy and PCR of rectal contents. The sensitivity correlated with the parasites’ number and stage of maturity. It ranged from 100% (>1,000 gravid worms) to 70% (<10 nongravid worms). On the basis of positive PCR results for 165 foxes, the sensitivity of the traditional and widely used necropsy method was found to be not higher than 76%. We therefore present this PCR system as an alternative method for the routine diagnosis of E. multilocularis in carnivores.

Annual assessment of Echinococcus multilocularis surveillance reports submitted in 2019 in the context of Commission Delegated Regulation (EU) 2018/772

“…The choice is sound as no risk factors were reported to justify the identification of subareas within the Irish territory Appendix C -United Kingdom. Assessment tables of the surveillance report (Mathis et al, 1996;Dinkel et al, 1998) • Method based on the concentration of helminth eggs by a combination of sequential sieving of faecal samples and flotation of the eggs in zinc chloride solution • DNA of the taeniid eggs retained in the 20 microns sieve was obtained after alkaline lysis • Nested PCR was performed using E. multilocularis species-specific primers against the mitochondrial 12S rRNA gene…”

Assessment of Echinococcus multilocularis surveillance reports submitted in 2017 in the context of Commission Regulation (EU) No 1152/2011

“…Type of test: Both methods used for detection of E. multilocularis in the UK were well described. GB selected a PCR Cest1-Cest2 NAD1 test (Mathis et al, 1996;Dinkel et al, 1998) for detection of E. multilocularis in rectal content. In NI, the SCT test (Eckert, 2003), considered as the reference standard for detection of E. multilocularis eggs from individual intestinal content, was used.…”

“…www.efsa.europa.eu/efsajournal 57 EFSA Journal 2017;15(11):5051 Appendix D -Assessment tables for the surveillance report of the United Kingdom (Mathis et al, 1996;Dinkel et al, 1998) • Method based on the concentration of helminth eggs by a combination of sequential sieving of faecal samples and flotation of the eggs in zinc chloride solution • The uneven geographical distribution of the population means sampling of animals also uneven. The sampling activity targeted the regions with higher fox density…”

Assessment of Echinococcus multilocularis surveillance reports submitted in 2016 in the context of Commission Regulation (EU) No 1152/2011

“…Population fraction The estimation of the proportion of the population allocated in each category of each risk indicator must be reported Proportions of the population allocated in each category of each risk indicator were reported and justified See Appendix D -Assessment tables for the surveillance report of United Kingdom (Mathis et al, 1996;Dinkel et al, 1998). The method is based on the concentration of helminth eggs by a combination of sequential sieving of faecal samples and flotation of the eggs in zinc chloride solution.…”