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

Santa, Maria A; Pastran, Sonya; Klein, Claudia; Ruckstuhl, Kathreen E.; Massolo, Alessandro

doi:10.1007/s00436-018-6125-y

Cited by 10 publications

(9 citation statements)

References 25 publications

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“…qPCR following Knapp et al 2016 [ 21 ], with minor modifications (at the Animal, Environmental and Antique DNA and Sequencing Platforms of the Fondazione E. Mach): in brief, whole DNA was extracted from 200 mg of faecal sample as described in Santa et al 2019 [ 22 ], using the automated magnetic bead based extraction kit Mag-Bind Stool DNA 96 kit (Omega Bio-Tek, USA) after a freeze-thawing step to facilitate DNA release from eggs. The extraction step was followed by qPCR amplification (Viia 7 Real-time PCR System, ThermoFisher Scientific, Waltham, MA) targeting the mitochondrial DNA marker rrnL, using 10 pmol of species-specific primers for E .…”

Section: Methodsmentioning

confidence: 99%

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019–2020)

et al. 2022

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Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

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Section: Methodsmentioning

confidence: 99%

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019–2020)

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“…One methodological limitation in the study of these parasites is the sampling adequacy problem in characterizing the Echinococcus species and strains distribution across space and hosts. Genetic analyses of E. multilocularis are usually conducted on only a few parasites from each definitive host, whereas the worm burden per definitive host can go as high as hundreds of thousands of worms [17,18]. Therefore, this could lead to an underestimation of the genetic diversity and prevalence of less common genetic variants [19].…”

Section: Introductionmentioning

confidence: 99%

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta, Canada

et al. 2021

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Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.

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“…Multiplex PCR amplification and Sanger sequencing were then performed on harvested eggs. Knapp et al 2016 [19], with minor modifications (at the Animal Genetics laboratories and Sequencing Platform of the Fondazione E. Mach): in brief, whole DNA was extracted from 50 mg of faecal sample as described in Santa et al 2019 [20], followed by qPCR amplification (Viia 7 Real-time PCR System, ThermoFisher Scientific, Waltham, MA) targeting the mitochondrial DNA marker rrnL, using 10 pmol of species-specific primers for E. multilocularis (202 bp) and 0.2 pmol of hydrolysis probe. The qPCR-positive samples were amplified with the same primer pair and sequenced with the dideoxy chain-termination protocol on an ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems) using the BigDye Terminator cycle sequencing kit (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020)

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et al. 2021

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Background: Surveillance of E. multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence and burden in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. Aim: We reassessed the prevalence of E. multilocularis at the southern border of its distribution in Alto Adige (Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. Methods: We compared results from the diagnostic test currently implemented for surveillance (based on Coproscopy+Multiplex PCR - CMPCR), against a real-time quantitative PCR (qPCR) for 235 fox faeces collected in 2019-2020. The performances of the two tests were estimated using a scraping technique (SFCT) as the gold standard applied to the small intestines of a subsample (n=123) of the same hosts. True prevalence was calculated and sample size required by each faecal test for the detection of the parasite was then estimated. Results: True prevalence of E. multilocularis in foxes (14.3%) was definitely higher than reported in the last decade (never >5% from 2012 to 2018). The qPCR also had a higher sensitivity (83%) compared to CMPCR (21%). Agreement with the gold standard was far higher for qPCR (0.816) than CMPCR (0.298) as well, determining a smaller sample size required to detect the disease. Conclusions: Alto Adige should be considered a highly endemic area. Surveillance at the edges of E. multilocularis distribution should adopt qPCR diagnostics on definitive hosts on a small geographic scale.

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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

Cited by 10 publications

References 25 publications

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019–2020)

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019–2020)

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta, Canada

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020)

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