Characterization of the mitochondrial genome sequences of the liver fluke Amphimerus sp. (Trematoda: Opisthorchiidae) from Ecuador and phylogenetic implications.

Amphimerus Barker, 1911 is a liver fluke infecting several animal species and humans. Being a digenetic trematode of the Opisthorchiidae family, Amphimerus is closely related to the genera Metorchis, Clonorchis and Opisthorchis. Recently, a high prevalence of Amphimerus infection in humans, cats, and dogs had been demonstrated in a tropical Pacific region of Ecuador. Hence, we determined and characterized the entire mt genome sequences of adult liver flukes, morphologically identified as Amphimerus, collected in the endemic region of Ecuador, and examined its phylogenetic relationships with flukes in the Opisthorchiidae family using Bayesian inference (BI) based on the concatenated amino acid sequences and partial cox1 sequences. The complete mt genome sequence (15, 151 bp in length) of the Amphimerus sp. contains 35 genes, including 12 protein-coding genes (PCGs, without atp8), two rRNAs (rrnL and rrnS) and 21 tRNAs, lacking trnG. The gene content and arrangement of the Ecuadorian Amphimerus mt genome was similar to those of other trematodes in the Opisthorchiidae family. All genes in the circular mt genome of Amphimerus sp. are transcribed from the same strand in one direction, with the A + T content of 60.77%. Genetic distances between Amphimerus sp. and other genera in Opisthorchiidae were rather high, ranging from 26.86% to 28.75% at nucleotide level and 29.37%-31.12% at amino acid level. Phylogenetic analysis placed the Ecuadorian Amphimerus within the branch of Opisthorchiidae, but very distinct from Opisthorchis. Our results indicate that the liver fluke Amphimerus from Ecuador does not belong to the genus Opisthorchis, and that it should be assigned under the genus Amphimerus. The determination of the mt genome of the Ecuadorian Amphimerus provides a new genetic resource for future studies on taxonomy and molecular epidemiology of Opisthorchiidae trematodes.

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans

BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.