Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabi province, Ecuador

Author summary Among neglected tropical diseases, foodborne trematodiases rank among the least studied, especially in the Americas. In Ecuador, a parasite of the genusAmphimerus, family Opisthorchiidae, has been found in the biliary ducts of humans, cats, and dogs, in the northern province of Esmeraldas. Reports as old as 1949 document opisthorchiid infections in the coastal province of Manabi. Given a lack of studies elucidating the infective larval stage of these flukes in Ecuador, we decided to conduct parasitological and molecular experiments to characterize the presence of these human liver infections in the area. We found adultAmphimerussp. flukes in humans and other domestic animals, and its metacercariae in four edible freshwater fish species:Rhoadsia altipinna,Bryconamericus bucay,Andinoacara rivulatus, andPiabucina aureoguttata. Moreover, we found a prevalence of 35.8% of small trematode eggs in humans. Finally, we also found the metacercariae of the intestinal flukeHaplorchis pumilioin all fish species examined. Our findings confirm the area of Pedro Pablo Gomez as an endemic region for opisthorchiid liver infections due toAmphimerussp., and expand the human differential diagnosis of small trematode eggs to liver and intestinal flukes. Consumption of undercooked freshwater fish of the species identified here could lead toAmphimerussp. infections. Surveillance in other areas known for raw fish consumption would likely unveil new cases of foodborne trematodiases in Ecuador and other regions. Amphimerussp. is a fluke that dwells in the biliary tracts of vertebrate definitive hosts including humans, domestic, and wild mammals in Latin America. Opisthorchiid liver infections are rarely studied in the Americas confirming its status as a neglected tropical disease. In Ecuador, small trematode eggs were reported in human cases from the province of Manabi in 1949, and recently,Amphimerussp. adults were recovered from human and reservoir hosts in the province of Esmeraldas. Due to the lack of research on the infectious sources ofAmphimerussp. in the continent, we have developed a series of epidemiological studies with parasitological and molecular techniques to elucidate the endemicity of opisthorchiid fluke infections in Manabi, Ecuador. We developed a cross-sectional study in three communities at Pedro Pablo Gomez parish in the province of Manabi, Ecuador. We examined a total of 176 fecal samples to detect opisthorchiid eggs, and four fish species to find opisthorchiid metacercariae. To study adult worms, we treated and purged seven patients in a family and dissected the livers of a dog and a cat infected. We observed morphological features of adults and metacercariae and used polymerase chain reaction with restricted fragment length polymorphism (PCR-RFLP) and DNA sequencing of a section of the ITS2 gene for identification. Small trematode eggs were detected in 63 (35.8%) out of 176 fecal samples of residents in the three study sites. Adult opisthorchiid flukes were recovered from human patients, a dog and a cat, and they were morphologically and molecularly identified asAmphimerussp. Opisthorchiid metacercariae were also identified molecularly asAmphimerussp. in four fish species, i.e.,Rhoadsia altipinna,Bryconamericus bucay,Andinoacara rivulatus, andPiabucina aureoguttata. Metacercariae of the heterophyidHaplorchis pumiliowere also found in the four fish species examined. This is the first study to confirm the current endemicity ofAmphimerussp. in Pedro Pablo Gomez, Manabi, Ecuador. The adult worms isolated here shared morphological characteristics with previousAmphimerussp. descriptions and were molecularly similar toAmphimerussp. described in the neighboring province of Esmeraldas. Moreover, this study is the first to document four fish species as infection sources ofAmphimerussp. detected via a molecular protocol targeting the metacercariae of the parasite. Fish species identified here should be targeted for public health campaigns to avoid further human liver-fluke infections byAmphimerussp. or potential intestinal-fluke infections byH.pumilioor others.