Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.

Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and pron...

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Main Authors: Nichola Eliza Davies Calvani, Peter Andrew Windsor, Russell David Bush, Jan Šlapeta
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2017-09-01
Series:PLoS Neglected Tropical Diseases
Online Access:http://europepmc.org/articles/PMC5617325?pdf=render
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spelling doaj-d5fa4a2149584e42bea01193458a16b32020-11-25T02:33:24ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352017-09-01119e000593110.1371/journal.pntd.0005931Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.Nichola Eliza Davies CalvaniPeter Andrew WindsorRussell David BushJan ŠlapetaFasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples.A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74-0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65-88%), compared to the sensitivity (91-100%) of the new molecular diagnostic workflow.Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.http://europepmc.org/articles/PMC5617325?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Nichola Eliza Davies Calvani
Peter Andrew Windsor
Russell David Bush
Jan Šlapeta
spellingShingle Nichola Eliza Davies Calvani
Peter Andrew Windsor
Russell David Bush
Jan Šlapeta
Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.
PLoS Neglected Tropical Diseases
author_facet Nichola Eliza Davies Calvani
Peter Andrew Windsor
Russell David Bush
Jan Šlapeta
author_sort Nichola Eliza Davies Calvani
title Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.
title_short Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.
title_full Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.
title_fullStr Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.
title_full_unstemmed Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.
title_sort scrambled eggs: a highly sensitive molecular diagnostic workflow for fasciola species specific detection from faecal samples.
publisher Public Library of Science (PLoS)
series PLoS Neglected Tropical Diseases
issn 1935-2727
1935-2735
publishDate 2017-09-01
description Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples.A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74-0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65-88%), compared to the sensitivity (91-100%) of the new molecular diagnostic workflow.Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.
url http://europepmc.org/articles/PMC5617325?pdf=render
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