title: Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.
abstract: BACKGROUND
  In endemic areas with very low infection prevalence, the frequency and intensity of Echinococcus multilocularis can be extremely low. This necessitates efficient, specific and sensitive molecular tools. We wanted to compare the existing molecular tools, used in the Norwegian national surveillance programme, and compare these with new techniques for detection of this zoonotic pathogen in fox faeces. Here we present the results of screening samples containing a known level of E. multilocularis eggs with two highly sensitive DNA isolation and extraction methods combined with one conventional PCR and three real-time PCR methods for detection.
  
  
  METHODS
  We performed a comparison of two extraction protocols; one based on sieving of faecal material and one using targeted DNA sampling. Four methods of molecular detection were tested on E. multilocularis-egg spiked fox faeces.
  
  
  RESULTS
  There were significant differences between the multiplex PCR/egg sieving DNA extraction methods compared to the new DNA fishing method and the three real-time PCR assays. Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low.
  
  
  CONCLUSIONS
  The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs. Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.
authors.affiliation: Norwegian Veterinary Institute, Post boks 750 Sentrum, 0106, Oslo, Norway. oivind.oines@vetinst.no.
authors.forename: Øivind
authors.initials: Ø
authors.lastname: Øines
authors.forename: Mats
authors.initials: M
authors.lastname: Isaksson
authors.forename: Åsa
authors.initials: Å
authors.lastname: Hagström
authors.forename: Saraya
authors.initials: S
authors.lastname: Tavornpanich
authors.forename: Rebecca K
authors.initials: RK
authors.lastname: Davidson
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000737:chemistry*; D018509:DNA, Helminth / Q000235:genetics; D004443:Echinococcosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D048210:Echinococcus multilocularis / Q000302:isolation & purification*; D005243:Feces / Q000737:chemistry / Q000469:parasitology*; D005589:Foxes; D010270:Parasite Egg Count / Q000379:methods / Q000662:veterinary; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity
chemical_list: D000947:Antigens, Helminth; D018509:DNA, Helminth