Abstract
Oral uptake of infectious Echinococcus multilocularis eggs shed by canids with their faeces may lead to development of alveolar echinococcosis in humans, which is clinically similar to a malignant infiltrative tumor and may be fatal if left untreated. E. multilocularis is therefore regarded as one of the most important and neglected metazoan parasites in the Northern hemisphere. The diagnosis of this tapeworm in the final host plays a key role in the epidemiology of E. multilocularis.The diagnostic performance of a magnetic-capture (MC) DNA extraction protocol in combination with a minor groove-binder real time PCR (MC-MGBqPCR) for the detection of E. multilocularis eggs was determined relative to a highly sensitive variant of the Intestinal Scraping Technique (IST) using faecal samples of foxes. In addition, we compared results obtained by MC-MGBqPCR with those of a previously validated protocol (QIAamp Fast DNA Stool Mini Kit (QT) combined with a TaqMan qPCR). Furthermore, a workflow using the NucleoMagVet DNA extraction kit (NM) in combination with MGBqPCR and TaqMan-qPCR was also included in the comparisons.To estimate the analytical sensitivity, phosphate-buffered saline and fox faecal samples were spiked with different numbers of eggs and tested in defined combinations of DNA extraction and PCR protocols. To assess the diagnostic sensitivity of the different workflows, samples were used that had been collected from the ampulla recti or the rectum of 120 foxes hunted in Brandenburg, Germany. The samples represented five IST categories formed according to the E. multilocularis worm burden of the foxes. For DNA extraction by MC or using two other commercial extraction kits, the supernatants obtained from 3 g of bead-beaten faecal samples were used. The extracted DNAs were then processed in the respective PCR protocols.The MC-MGBqPCR showed the highest diagnostic sensitivity (93%; 95% Confidence Interval (CI): 86–97%) relative to IST. The QT extraction protocol in combination with TaqMan-qPCR had the second highest sensitivity (89%; 95% CI: 80–94%), followed by NM with MGBqPCR (86%; 95% CI: 77–93%) in comparison to IST. The lowest diagnostic sensitivity was found for the NM combined with the TaqMan-qPCR protocol (72%; 95% CI: 62–82%).In conclusion, the MC-MGBqPCR seems to represent a suitable alternative to IST. However, applied to 3 g faecal samples, the less costly QT-TaqMan-qPCR workflow yielded a similar diagnostic sensitivity relative to IST. However, differences between these two workflows were not statistically significant.
Highlights
Echinococcus multilocularis is regarded as one of the most dangerous zoonotic metazoan parasites in the northern hemisphere (Eckert et al, 2000)
We have shown that protocol C, which is based on a commercial extraction kit in combination with a TaqMan real time PCR and on processing the supernatants of homogenized samples, yielded a similar diagnostic sensitivity as the magnetic DNA capture protocol and may represent a less expensive and faster alternative as compared to protocol A
The results showed that magnetic capture (MC), and the NucleoMag Vet protocols (B and D) perform well and detect E. multilocularis DNA in spiked phosphate-buffered saline (PBS) samples, and in most of faecal samples spiked with varying defined numbers of eggs
Summary
Echinococcus multilocularis is regarded as one of the most dangerous zoonotic metazoan parasites in the northern hemisphere (Eckert et al, 2000). Cats can be regarded as potential definitive hosts of E. multilocularis, but there are indications that they play a minor role in the lifecycle of the parasite (Thompson et al, 2003; Kapel et al, 2006). Worm eggs represent the infectious stage for intermediate hosts, which include a wide spectrum of species, among which rodents, e.g. Arvicolidae, play a prominent role (Woolsey et al, 2015a; Woolsey et al, 2015b). Successful infection of an intermediate host leads to the development of larval stages (metacestodes) of E. multilocularis in different organs, but mainly in the liver, causing alveolar echinococcosis (AE) (Matsumoto et al, 2010). Humans accidentally infected with E. multilocularis by oral uptake of infectious eggs serve as an aberrant (dead-end) intermediate hosts. While AE is still a rare disease in humans in Europe (Gottstein et al, 2015), an increasing prevalence has been reported (Schweiger et al, 2007)
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