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

Abstract

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.

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References

  1. Al-Sabi’ MNS, Kapel CMO, Deplazes P, Mathis A (2007) Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes. Parasitol Res 101:731–736 https://doi.org/10.1007/s00436-007-0537-4

    Article  PubMed  Google Scholar 

  2. Catalano S, Lejeune M, Liccioli S, Verocai GG, Gesy KM, Jenkins EJ, Kutz SJ, Fuentealba C, Duignan PJ, Massolo A (2012) Echinococcus multilocularis in urban coyotes, Alberta, Canada. Emerg Infect Dis 18:1625–1628 https://doi.org/10.3201/eid1810.120119

    Article  PubMed  PubMed Central  Google Scholar 

  3. Conraths FJ, Deplazes P (2015) Echinococcus multilocularis: epidemiology, surveillance and state-of-the-art diagnostics from a veterinary public health perspective. Vet Parasitol 213:149–161 https://doi.org/10.1016/j.vetpar.2015.07.027

    Article  PubMed  Google Scholar 

  4. Davidson RK, Lavikainen A, Konyaev S, Schurer J, Miller AL, Oksanen A, Skírnisson K, Jenkins E (2016) Echinococcus across the north: current knowledge, future challenges. Food Waterborne Parasitol 4:39–53 https://doi.org/10.1016/j.fawpar.2016.08.001

    Article  Google Scholar 

  5. Deer DM, Lampel KA, González-Escalona N (2010) A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays. Lett Appl Microbiol 50:366–372 https://doi.org/10.1111/j.1472-765X.2010.02804.x

    Article  CAS  PubMed  Google Scholar 

  6. Deplazes P et al (2017) Global distribution of alveolar and cystic echinococcosis. Adv Parasitol 95:315–493 https://doi.org/10.1016/bs.apar.2016.11.001

    Article  CAS  PubMed  Google Scholar 

  7. Eckert J (2003) Predictive values and quality control of techniques for the diagnosis of Echinococcus multilocularis in definitive hosts. Acta Trop 85(2):157–163 https://doi.org/10.1016/S0001-706X(02)00216-4

    Article  CAS  PubMed  Google Scholar 

  8. 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 286

    Google Scholar 

  9. 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 302

    Google Scholar 

  10. Gesy K, Pawlik M, Kapronczai L, Wagner B, Elkin B, Schwantje H, Jenkins E (2013) An improved method for the extraction and quantification of adult Echinococcus from wildlife definitive hosts. Parasitol Res 112:2075–2078 https://doi.org/10.1007/s00436-013-3371-x

    Article  PubMed  Google Scholar 

  11. Holmes JC (1961) The importance of coyotes (Canis latrans) in the maintenance of sylvatic echinococcosis: preliminary observations. J Parasitol 47(Suppl):55

    Google Scholar 

  12. 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):583

    Article  CAS  Google Scholar 

  13. Jones A, Pybus MJ (2001) Taeniasis and echinococcosis. In: Samuel WM, Pybus MJ, Kocan AA (eds) Parasitic diseases of wild mammals. Iowa State University Press, Ames, pp 150–192

    Google Scholar 

  14. Karamon J, Sroka J, Cencek T (2010) Limit of detection of sedimentation and counting technique (SCT) for Echinococcus multilocularis diagnosis, estimated under experimental conditions. Exp Parasitol 124:244–246 https://doi.org/10.1016/j.exppara.2009.09.007

    Article  PubMed  Google Scholar 

  15. Klein C, Liccioli S, Massolo A (2014) Egg intensity and freeze-thawing of fecal samples affect sensitivity of Echinococcus multilocularis detection by PCR. Parasitol Res 113:3867–3873 https://doi.org/10.1007/s00436-014-4055-x

    Article  CAS  PubMed  Google Scholar 

  16. 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

    Article  CAS  PubMed  Google Scholar 

  17. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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

    Article  CAS  PubMed  Google Scholar 

  19. Massolo A, Liccioli S, Budke CM, Klein C (2014) Echinococcus multilocularis in North America: the great unknown. Parasite 21:73 https://doi.org/10.1051/parasite/2014069

    Article  PubMed  PubMed Central  Google Scholar 

  20. 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 57

    Google Scholar 

  21. Mercaldo ND, Lau KF, Zhou XH (2007) Confidence intervals for predictive values with an emphasis to case-control studies. Stat Med 26:2170–2183. https://doi.org/10.1002/sim.2677

    Article  PubMed  Google Scholar 

  22. Opel KL, Chung D, McCord BR (2010) A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 55:25–33. https://doi.org/10.1111/j.1556-4029.2009.01245.x

    Article  CAS  Google Scholar 

  23. 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

    Article  PubMed  PubMed Central  Google Scholar 

  24. Peregrine AS, Jenkins EJ, Barnes B, Johnson S, Polley L, Barker IK, de Wolf B, Gottstein B (2012) Alveolar hydatid disease (Echinococcus multilocularis) in the liver of a Canadian dog in British Columbia, a newly endemic region. Can Vet J 53(8):870–874

    PubMed  PubMed Central  Google Scholar 

  25. 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

    Article  PubMed  PubMed Central  Google Scholar 

  26. Schurer JM, Gesy KM, Elkin BT, Jenkins EJ (2013) Echinococcus multilocularis and Echinococcus canadensis in wolves from western Canada. Parasitol 141:159–163. https://doi.org/10.1017/S0031182013001716

    Article  Google Scholar 

  27. Skelding A, Brooks A, Stalker M, Mercer N, de Villa E, Gottstein B, Peregrine AS (2014) Hepatic alveolar hydatid disease (Echinococcus multilocularis) in a boxer dog from southern Ontario. Can Vet J 55:551–553

    PubMed  PubMed Central  Google Scholar 

  28. Thompson RCA (2015) Neglected zoonotic helminths: Hymenolepis nana, Echinococcus canadensis and Ancylostoma ceylanicum. Clin Microbiol Infect 21:426–432. https://doi.org/10.1016/j.cmi.2015.01.004

    Article  CAS  PubMed  Google Scholar 

  29. Vollset SE (1993) Confidence intervals for a binomial proportion. Stat Med 12:809–824 https://doi.org/10.1002/sim.4780120902

    Article  CAS  Google Scholar 

  30. Wahlström H, Comin A, Isaksson M, Deplazes P (2016) Detection of Echinococcus multilocularis by MC-PCR: evaluation of diagnostic sensitivity and specificity without gold standard. Infect Ecol Epidemiol 6:30173 https://doi.org/10.3402/iee.v6.30173

    Article  PubMed  Google Scholar 

  31. Waits LP, Paetkau D (2005) Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. J Wildl Manag 69(4):1419–1433 https://doi.org/10.2193/0022-541X(2005)69[1419:NGSTFW]2.0.CO;2

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Acknowledgements

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).

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Correspondence to Alessandro Massolo.

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Santa, M.A., Pastran, S., Klein, C. et al. 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. Parasitol Res 118, 119–125 (2019). https://doi.org/10.1007/s00436-018-6125-y

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Keywords

  • Echinococcus multilocularis
  • Echinococcus canadensis
  • Coyote
  • Real-time PCR
  • Coprodiagnosis
  • Magnetic beads