title: 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: BACKGROUND
  Following 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  Of 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).
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Virology Immunobiology and Parasitology, National Veterinary Institute, Uppsala, Sweden. mats.isaksson@sva.se.
authors.forename: Mats
authors.initials: M
authors.lastname: Isaksson
authors.affiliation: Department of Virology Immunobiology and Parasitology, National Veterinary Institute, Uppsala, Sweden. asa.hagstrom@sva.se.
authors.forename: Åsa
authors.initials: Å
authors.lastname: Hagström
authors.affiliation: Institute of Parasitology, Vetsuisse and Medical Faculty, University of Zurich, Zurich, Switzerland. mtarmua@vetparas.uzh.ch.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Armua-Fernandez
authors.affiliation: Department of Epidemiology, National Veterinary Institute, Uppsala, Sweden. helene.wahlstrom@sva.se.
authors.forename: Helene
authors.initials: H
authors.lastname: Wahlström
authors.affiliation: Department of Pathology and Wildlife Diseases, National Veterinary Institute, Uppsala, Sweden. erik.agren@sva.se.
authors.forename: Erik Olof
authors.initials: EO
authors.lastname: Ågren
authors.affiliation: Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden. andrea.miller@slu.se.
authors.forename: Andrea
authors.initials: A
authors.lastname: Miller
authors.affiliation: Precision System Science, Stockholm, Sweden. anders.holmberg@pssbio.com.
authors.forename: Anders
authors.initials: A
authors.lastname: Holmberg
authors.affiliation: Precision System Science, Stockholm, Sweden. morten.lukacs@pssbio.com.
authors.forename: Morten
authors.initials: M
authors.lastname: Lukacs
authors.affiliation: Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy. adriano.casulli@iss.it.
authors.forename: Adriano
authors.initials: A
authors.lastname: Casulli
authors.affiliation: Institute of Parasitology, Vetsuisse and Medical Faculty, University of Zurich, Zurich, Switzerland. deplazesp@access.uzh.ch.
authors.forename: Peter
authors.initials: P
authors.lastname: Deplazes
authors.affiliation: Department of Virology Immunobiology and Parasitology, National Veterinary Institute, Uppsala, Sweden. mikael.juremalm@sva.se.
authors.forename: Mikael
authors.initials: M
authors.lastname: Juremalm
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000235:genetics* / Q000302:isolation & purification; D004443:Echinococcosis / Q000453:epidemiology / Q000469:parasitology*; D048210:Echinococcus multilocularis / Q000235:genetics / Q000302:isolation & purification*; D062665:Epidemiological Monitoring; D005589:Foxes / Q000469:parasitology*; D008280:Magnetics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013548:Sweden / Q000453:epidemiology
chemical_list: D018509:DNA, Helminth