Date Published: February 14, 2018
Publisher: Public Library of Science
Author(s): William Cevallos, Pedro Fernández-Soto, Manuel Calvopiña, María Buendía-Sánchez, Julio López-Abán, Belén Vicente, Antonio Muro, Ana Paula Arez.
Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients’ stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas.
Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp. (within the family Opisthorchiidae), was recently reported as an endemic disease in the tropical Pacific side of Ecuador. Data showing high prevalence of infection among an indigenous group, the Chachis, and also domestic cats and dogs residing in the same communities have been noted and, actually, human amphimeriasis has been reported as a new emerging food-borne zoonotic disease. Parasites of the genus Amphimerus infect humans after ingestion of raw or undercooked freshwater fish containing viable metacercariae. Human disease is mostly asymptomatic, occasionally causing non-specific, generalised symptoms. However, histopathological studies in cats and a double-crested cormorant infected with Amphimerus spp. showed the presence of liver cirrhosis and pancreatitis [1,2]. Similarly, as occur in other human infections by parasites of the family Opisthorchiidae, affected individuals with Amphimerus spp. can suffer from suppurative cholangitis, cholelithiasis and cholangiocarcinoma [3–5]. Since the Chachi community habitually consumes smoked freshwater fish, an estimated 13% of the inhabitants living along the Rio Cayapas in the Province of Esmeraldas are a risk of acquiring amphimeriasis .
The indigenous Chachi communities, who live in remote villages along the Río Cayapas in the north-western coastal rainforest of Ecuador, have been shown to have a high prevalence of infection (15.5%-34.1%) with Amphimerus sp. . Infection in domestic cats and dogs residing in this endemic area has also been reported as high (71.4% and 38.7%, respectively) and these animals have been proposed to serve as definite hosts and reservoirs for the parasite . The prevalence data obtained in these studies were assessed according to eggs findings in both human and animal stool samples by classical parasitological methods.
In conclusion, to the best of our knowledge, we demonstrate for the first time that common filter paper is useful for long-term storage of human stool samples for later quality DNA extraction of human-parasitic trematode eggs. Additionally, this simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to be used as an effective molecular large-scale screening test for amphimeriasis-endemic areas. The system ‘air-dried stool sample on filter paper’-LAMP assay could also be very interesting and useful for molecular diagnosis of other human infectious parasitic diseases in remote areas with poor settings.