Evaluation of an automated magnetic bead-based DNA extraction and real-time PCR in fecal samples as a pre-screening test for detection of Echinococcus multilocularis and Echinococcus canadensis in coyotes
- 16 Downloads
Efficient and sensitive diagnostic tools are essential for the study of the eco-epidemiology of Echinococcus species. We evaluated an automated magnetic bead-based DNA extraction commercial kit followed by qPCR (MB-qPCR), for the detection of Echinococcus multilocularis and Echinococcus canadensis in coyote (Canis latrans) fecal samples. The diagnostic sensitivity was determined by validating the method against the scraping, filtration, and counting technique (SFCT) for samples collected in Canada. From the 60 samples tested, 27 out of 31 SFCT positives samples for Echinococcus cestodes were positive in the MB-qPCR for E. multilocularis, with a sensitivity of 87.1% (95% CI 70.2 to 96.4%). Two samples were also positive for E. canadensis in the MB-qPCR and confirmed by morphological identification of adult worms. The agreement of the MB-qPCR and the SFCT was statistically significant with a kappa value of 0.67 (95% CI 0.48–0.85; p value < 0.001). The magnetic bead-based DNA extraction followed by qPCR proved to have a sensitivity comparable to the SFCT to detect E. multilocularis. Although the diagnostic sensitivity for E. canadensis was not estimated, MB-qPCR identified E. canadensis cases previously overlooked when using SFCT. We propose a combination of molecular and morphological identification using the MB-qPCR and the SFCT to detect both parasites, allowing for a more efficient large-scale surveillance, and detecting co-infections of Echinococcus species that can be difficult to identify when only based on morphology.
KeywordsEchinococcus multilocularis Echinococcus canadensis Coyote Real-time PCR Coprodiagnosis Magnetic beads
We thank the Alberta Trappers’ Association and the trappers who provided animal carcasses. All the undergrad students that helped with the processing of samples and Marion Wassermann from the University of Hohenheim who provided DNA material. This research was supported by the ACA (Alberta Conservation Association) (N030-00-90-247), and by MITACS Inc. through the MITACS Accelerate program (internal fund number: 10018836) matching funds provided by Animal Health, Veterinary Scientific Affairs of Bayer Inc. (internal fund number: 10017067).
Compliance with ethical standards
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
- Eckert J, Gemmell MA, Meslin FX, Pawlowski ZS (2001) WHO/OIE manual on echinococcosis in humans and animals: a public health problem of global concern. OIE/World Health Organization, Paris, p 286Google Scholar
- FAO/WHO (2014) Multicriteria-based ranking for risk management of food-borne parasites. Microbiological risk assessment series N°23. Food and Agriculture Organization of the United Nations/World Health Organization, Rome, p 302Google Scholar
- Holmes JC (1961) The importance of coyotes (Canis latrans) in the maintenance of sylvatic echinococcosis: preliminary observations. J Parasitol 47(Suppl):55Google Scholar
- Isaksson M, Hagström Å, Armua-Fernandez MT, Wahlström H, Ågren EO, Miller A, Holmberg A, Lukacs M, Casulli A, Deplazes P, Juremalm M (2014) 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. Parasite Vector 7(583 https://doi.org/10.1186/s13071-014-0583-6):583CrossRefGoogle Scholar
- Knapp J, Millon L, Mouzon L, Umhang G, Raoul F, Ali ZS, Combes B, Comte S, Gbaguidi-Haore H, Grenouillet F, Giraudoux P (2014) Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools. Vet Parasitol 201:40–47 https://doi.org/10.1016/j.vetpar.2013.12.023 CrossRefGoogle Scholar
- Knapp J, Umhang G, Poulle M-L, Millon L (2016) Development of a real-time PCR for a sensitive one-step Coprodiagnosis allowing both the identification of carnivore feces and the detection of Toxocara spp. and Echinococcus multilocularis. Appl Environ Microbiol 82:2950–2958 https://doi.org/10.1128/AEM.03467-15 CrossRefGoogle Scholar
- Maas M, van Roon A, Dam-Deisz C, Opsteegh M, Massolo A, Deksne G, Teunis P, van der Giessen J (2016) Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts. Vet Parasitol 230:20–24 https://doi.org/10.1016/j.vetpar.2016.10.016 CrossRefGoogle Scholar
- Massolo A, Preiksaitis J, Klein C, Sis B, Houston S, Kowalewska-Growchowska K (2015) Locally acquired alveolar echinococcosis in an immunocompromosed patient in Canada: clinical presentation and epidemiologic investigation. ArcticNet Annual Scientific Meeting, Vancouver, p 57Google Scholar
- Otero-Abad B, Armua-Fernandez MT, Deplazes P, Torgerson PR, Hartnack S (2017) Latent class models for Echinococcus multilocularis diagnosis in foxes in Switzerland in the absence of a gold standard. Parasit Vectors 10(612):612. https://doi.org/10.1186/s13071-017-2562-1 CrossRefPubMedPubMedCentralGoogle Scholar
- Santa MA, Pastran SA, Klein C, Duignan P, Ruckstuhl K, Romig T, Massolo A (2018) Detecting co-infections of Echinococcus multilocularis and Echinococcus canadensis in coyotes and red foxes in Alberta, Canada using real-time PCR. Int J Parasitol Parasites Wildl 7:111–115. https://doi.org/10.1016/j.ijppaw.2018.03.001 CrossRefPubMedPubMedCentralGoogle Scholar