Assay Detects Fish Trematode DNA in Human Stools

Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals.

The diagnosis of Amphimeriasis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes and no immunological or molecular methods have been developed to date.

Scientists at the Universidad Central del Ecuador (Quito, Ecuador) and their colleagues obtained human stool samples from indigenous communities during February 2016. The samples were processed for parasitological screening under light microscopy by direct examination, simple sedimentation, formalin-ether concentration and Kato-Katz techniques. After parasitological screening, a total of 44 stool samples were selected, including 25 (56.81%) that were positive for Amphimerus sp. eggs-by one or more parasitological methods-and 19 (43.18%) negative samples.

DNA extraction was performed using the Mini Stool DNA Extraction kit. To determine the specificity of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assays to amplify only Amphimerus sp. DNA, a total of 16 DNA samples from several helminths, including trematodes, cestodes, nematodes and protozoa were used. DNA was measured using a Nanodrop ND-100 spectrophotometer. The LAMP assay was called LAMPhimerus.

The team obtained from the total of 25 parasitologically positive stool samples, 9/25 (36%) and 18/25 (72%) positive results when they applied LAMPhimerus for 60 min and 120 min, respectively. Additionally, positive results included 5/19 (26.3%) samples that were negative in all previously applied parasitological tests. Of the 11 that were simultaneously positive on three parasitological tests 82% were also positive on the LAMP assay; only the two samples, with the same very low egg count, were negative on the LAMP assay.

The authors have developed, for the first time, a LAMP assay (namely, LAMPhimerus) for the sensitive and specific detection of Amphimerus sp. DNA in human stool samples. After further studies for validation, the method could be readily adapted for effective field diagnosis and disease surveillance in amphimeriasis-endemic areas. The study was published on June 19, 2017, in the journal Public Library of Science Neglected Tropical Diseases.

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Universidad Central del Ecuador