A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples

Abstract

BackgroundFollowing the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012–2013 on more than 2000 faecal samples.MethodsWe describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic.ResultsOf 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR.The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7 -100).ConclusionsThe MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.

Support for targeted sampling of red fox (Vulpes vulpes) feces in Sweden: a method to improve the probability of finding Echinococcus multilocularis

“…The difference is also statistically significant ( P  < 0.001, Fisher’s test) when compared only to the two regions with an unknown status at study start (20/189, 10.6%, 95% CI: 6.6–15.9%). The national screening employed a newly designed magnetic-capture PCR [43] diagnostic technique with a reported sensitivity of 88% [43, 44], while the combined egg isolation and PCR technique used in this study has a lower reported sensitivity of 50% [45]. Thus, the difference cannot be explained by the diagnostic methods used.…”

Echinococcus multilocularis infection in animals

“…Conventional-PCR or Multiplex-PCR withsieving procedure for egg isolation from faeces: 40-80 samples depending on taeniid prevalence (for procedures see Mathis et al, 1996;Trachsel et al, 2007); Nested-PCR for total DNA isolation from faeces: around 70 samples (for procedures see Monnier et al, 1996;Dinkel et al, 1998;Van der Giessen et al, 1999); Real Time-PCR for total DNA isolation from faeces: 70 samples (for procedures see Dinkel et al, 2011;Knapp et al, 2013); MC-PCR with manual DNA fishing from faeces: 70 samples (for procedures see ; MC-PCR with automated DNA fishing from faeces: 240 samples (for procedures see Isaksson et al, 2014).…”

An autochthonous case of cystic echinococcosis in Finland, 2015

“…Before treatment with praziquantel, faecal samples were collected from the dogs. Mitochondrial DNA was extracted directly from the faecal material [3] because the small size of the samples did not allow isolation of parasite eggs. A fragment of mitochondrial ribosomal DNA of the E. granulosus sensu lato (s.l.)…”