High Prevalence of Human Liver Infection by Amphimerus spp. Flukes, Ecuador

Amphimerus spp. flukes are known to infect mammals, but human infections have not been confirmed. Microscopy of fecal samples from 397 persons from Ecuador revealed Opisthorchiidae eggs in 71 (24%) persons. Light microscopy of adult worms and scanning electron microscopy of eggs were compatible with descriptions of Amphimerus spp. This pathogen was only observed in communities that consumed undercooked fish.


T he Study
In June 2009, during a routine fecal examination for the parent study, 4 samples tested positive for eggs of the Opisthorchiidae family in 3 indigenous Chachi communities along the Cayapas River in the northern coastal rainforest of Ecuador. In January 2010, a followup survey was conducted in the same 3 communities (total population 589); all villagers, whether symptomatic or not, were asked to provide a fecal sample. Specifi cally, a community meeting was held in each village, study objectives were explained, and villagers were asked for their voluntary participation. Flasks were distributed to all villagers and collected the next day in the school and by going house to house. The Chachis, the predominant group in these 3 communities, represent 13% of the 24,000 inhabitants in the region. Afro-Ecuadorians and mestizos also reside in this region (10,11).
A total of 297 (50.4%) community members 3-77 years of age provided samples. To each person providing a sample, a questionnaire was administered regarding types of food eaten and cooking practices. Samples were preserved in 10% formalin, transported to a laboratory in Quito, and stored at 4°C until examination by light microscopy. Eggs were concentrated by using the formalin-ether technique. In addition, 120 fecal samples from Afro-Ecuadorian and mestizo persons were examined. The villagers were informed of the study in their local Chapalache language by community health community workers. The ethical committee of the Central University approved this study.
Duodendoscopy was performed in 4 patients by a gastroenterology specialist to examine the biliary liquid; the microscopy of this liquid showed eggs identical to those found in their feces. These patients received praziquantel (75 mg/kg in 3 doses/3 d), and were purged with 10 mg of bisacodilo. Fecal samples were collected and examined for worms as previously described (12). Recovered worms were fi xed with 10% formalin, stained with Diff-Quik fi xative (Sysmex, Kobe, Japan), and identifi ed by comparing their morphologic features to known adult Clonorchis and Opisthorchis spp. worms. Community health workers collected and examined the livers of 3 cats and 3 dogs from 1 of the 3 communities. All 6 livers had eggs and high numbers of adult parasites in the bile ducts. Adult parasites were stained, and microscopic studies showed them to be identical to those in the human specimens.
A total of 71 (24%) of the 297 fecal samples from the indigenous Chachi were positive for Opisthorchiidae eggs (Table). In contrast all 120 samples from Afro-Ecuadorian and mestizo persons were negative. Eggs were yellow-brown and measured 28-33 μm ×12-15 μm (n = 20). The operculum and the shoulders, however, were not prominent as they are in Clonorchis and Opisthorchis eggs. Occasionally, a small knob, but most frequently a curved spine, was seen on the abopercular end. Although, by light microscopy, the shape and size of the eggs resembled that of the other liver fl ukes, the patterns of the eggshell surface were distinct when viewed with SEM ( Figure 1). This observation is corroborated with published photographs (3). After participants were treated with praziquantel, a total of 8 worms were recovered from 4 human participants and dozens from cat and dog livers; all were placed in saline. The worms were delicate, leaf-shaped, elongated, and red-pink and measured 8-13.6 mm long (average 10.2 mm) × 0.5-1.1 mm wide (n = 15). After a few minutes, the worms coiled in an S shape and became transparent or whitish. Once stained, the following features were observed: 1) the vitellaria divided into an anterior and posterior group with the posterior group extending the level of the posterior testis; 2) a ventral sucker larger than oral sucker; and 3) 2 rounded testes ( Figure 2). On the basis of these morphologic characteristics of the adults and the SEM fi ndings of the eggs, the parasite was identifi ed as Amphimerus spp.

Conclusions
Our study demonstrates that the liver fl uke of the genus Amphimerus can infect humans. We found a high prevalence (15.5%-34.1%) of infection with Amphimerus spp. trematodes in the surveyed communities (Table). Samples from the Afro-Ecuadorian and mestizo population were all negative for Opisthorchiidae eggs. Amphimerus spp. trematodes are believed to be transmitted, as are the other members of the Opisthorchiidae family, by ingestion of raw or undercooked fi sh (2). In our survey, most Chachis reported eating smoked fi sh caught in the rivers. Food sharing is more common among Chachi than Afro-Ecuadorians and mestizo families (13). Notably, the most remote village (120 km inland from the coast) had the highest prevalence. Our results suggest that Amphimerus spp. fl ukes are zoonotic pathogens of domestic animals living with humans.
Amphimeriasis should be considered an endemic liver fl uke infection among residents of this Chachi population in Ecuador. Further studies are needed to determine the complete epidemiology and geographic distribution of infection in this region, as well as in other provinces of Ecuador where freshwater fi sh is eaten undercooked or where the same tropical ecology is found. For example, the Amazonian region has indigenous groups where other foodborne trematodiasis-like paragonimiasis are endemic (14). Amphimerus spp. fl ukes infecting domestic and wild animals have been reported from Ecuador's neighboring countries as well as from Central and North America. The existence of undiscovered foci of human infections is possible.  In 1971, Yamaguti (1) suggested that a parasite previously reported in Ecuador (8) as O. guayaquilensis might in fact be Amphimerus spp. Subsequently, publications referred to this parasite as A. guayaquilensis (5,7); however, the accuracy of this reclassifi cation is unclear. Molecular analysis could help clarify the ambiguities in genus/species identifi cation of O. guayaquilensis parasites and the conspecifi c species of Amphimerus (15).
We have much to learn about the pathology and epidemiology of Amphimerus spp. fl ukes. For example, nothing is known about the clinical and pathologic signifi cance of infections with this parasite. Praziquantel eliminated the parasites in these patients, but whether the dose and treatment time were adequate are unknown. Additionally, little is known about epidemiologic factors responsible for the differences in the number of infections among the different population groups. Future studies can help determine the direct and indirect public health implications of this new foodborne zoonosis.