title: Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples.
abstract: BACKGROUND
  Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount.
  
  
  METHODS
  A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination.
  
  
  RESULTS
  2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces.
  
  
  CONCLUSIONS
  The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.
authors.affiliation: Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Clos Chapelle-aux-Champs, 30 B1.30.24, 1200 Brussels, Belgium.
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Durant
authors.forename: Leonid M
authors.initials: LM
authors.lastname: Irenge
authors.forename: Renata
authors.initials: R
authors.lastname: Fogt-Wyrwas
authors.forename: Catherine
authors.initials: C
authors.lastname: Dumont
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Doucet
authors.forename: Bernard
authors.initials: B
authors.lastname: Mignon
authors.forename: Bertrand
authors.initials: B
authors.lastname: Losson
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Gala
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D005243:Feces / Q000469:parasitology*; D010063:Ovum / Q000145:classification*; D018510:RNA, Helminth / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D012987:Soil / Q000469:parasitology*; D013045:Species Specificity; D014119:Toxocara / Q000145:classification*; D014120:Toxocariasis / Q000175:diagnosis / Q000469:parasitology
chemical_list: D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer; D018510:RNA, Helminth; D012987:Soil