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

Abstract

BackgroundFasciolosis, 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.Methodology/Principal findingsA 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.Conclusions/SignificanceSpecies-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.

Do triclabendazole medicated molasses blocks have a role in control of Fasciola gigantica in smallholder cattle production in Lao PDR?

“…Thus these results should be interpreted with caution as they may significantly underestimate the level of F. gigantica infection in animals with chronic fasciolosis. Research is currently in progress to apply a potentially more sensitive molecular diagnostic test for F. gigantica infection in cattle in Laos, with preliminary results suggesting improved diagnostic acumen that may assist future planned studies on liver fluke epidemiology and control in the region (Calvani et al 2017). The baseline data (Day 1 data collection) identified a high prevalence of F. gigantica infestation in all the study groups, confirming the widespread distribution of this parasite at potentially pathogenic levels in Laos (Copeman and Copland 2008;Rast et al 2013).…”

<p>Human Fascioliasis: Current Epidemiological Status and Strategies for Diagnosis, Treatment, and Control</p>

“…74 These inconsistencies between studies may be related to differences in DNA extraction protocols which in the case of the hardy Fasciola eggs is a critical step. 75 The lack of training and laboratory capacity in endemic areas where molecular testing is most needed is a significant barrier for implementation. In these settings, the ideal diagnostic method should be easily deployable to areas without much laboratory capacity, require minimal training of personnel, and have a high accuracy.…”

“… 74 These inconsistencies between studies may be related to differences in DNA extraction protocols which in the case of the hardy Fasciola eggs is a critical step. 75 …”

Immunodiagnosis of cattle fascioliasis using a 27 kDa Fasciola gigantica antigen

“…The most reliable diagnostic method is the presence of eggs in the stool of infected individuals. However, despite its reliability, an overwhelming consensus exists that this method is not completely reliable [6]. Disadvantages of this method include that eggs are not detected until a late stage of infection when liver damage has occurred.…”