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

Amphimerus sp. 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 Manabí in 1949, and recently, Amphimerus sp. adults were recovered from human and reservoir hosts in the province of Esmeraldas. Due to the lack of research on the infectious sources of Amphimerus sp. in the continent, we have developed a series of epidemiological studies with parasitological and molecular techniques to elucidate the endemicity of opisthorchiid fluke infections. We developed a cross-sectional study in three communities at Pedro Pablo Gómez parish in the province of Manabí, 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 as Amphimerus sp. Opisthorchiid metacercariae were also identified molecularly as Amphimerus sp. in four fish species, i.e., Rhoadsia altipinna, Bryconamericus bucay, Andinoacara rivulatus, and Piabucina aureoguttata. Metacercariae of the heterophyid Haplorchis pumilio were also found in the four fish species examined. This is the first study to confirm the current endemicity of Amphimerus sp. in Pedro Pablo Gómez, Manabí, Ecuador. The adult worms isolated here shared morphological characteristics with previous Amphimerus sp. descriptions and were molecularly similar to Amphimerus sp. described in the province of Esmeraldas. Moreover, this study is the first to document four fish species as infection sources of Amphimerus sp. 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 by Amphimerus sp. or potential intestinal-fluke infections by H. pumilio or others.

Laboratory propagation of freshwater cyclopoid copepods as an intermediate host for parasitic helminths.

Among the freshwater cyclopoids, small and medium-sized species can be propagated in the laboratory with a flagellate Chilomonas as the food. By contrast, large species need to be given, in addition to the flagellate, a larger food organism. For this, although a metazoan food is preferable, a ciliate Stentor may in part serve as a substitute. Methods for infection experiment involving copepods are given, and photographs of some parasite larvae in the copepod host are shown. Usage of carbonated water is helpful to immobilize cyclopoids and thus recommended for infection experiments.

First confirmed human case of Diphyllobothrium stemmacephalum infection and molecular verification of the synonymy of Diphyllobothrium yonagoense with D. stemmacephalum (Cestoda: Diphyllobothriidea).

Several tapeworm species in the genus Diphyllobothrium Cobbold, 1858 have uncertain taxonomic positions, leading to taxonomic confusion as well as misdiagnosis of infections. Taxonomic revision based on DNA sequence analysis is considered necessary to resolve the taxonomy of several cases, including that between Diphyllobothrium stemmacephalum, the type species of the genus, and Diphyllobothrium yonagoense. Diphyllobothrium yonagoense was synonymized with D. stemmacephalum based on morphological observations by Andersen (1987), however no molecular studies have been undertaken to verify the validity of this synonymization. In the present study, the first human case confirmed molecularly as D. stemmacephalum infection is reported, and the validity of the synonymization of D. yonagoense with D. stemmacephalum was assessed based on molecular phylogenetics. Diphyllobothrium stemmacephalum and D. yonagoense grouped into the same clades with high bootstrap confidence values for both cox1 and nad3. Genetic distances between the two taxa were very small (0.000-0.012 and 0.000-0.017 for cox1 and nad3, respectively) and were considered to fall within the range of intraspecific variation. Using these molecular analyses, this study verified molecularly that D. yonagoense is a junior synonym of D. stemmacephalum. Further, the closer phylogenetic relationship between D. stemmacephalum and Diplogonoporus species rather than other diphyllobothriids, including Diphyllobothrium nihonkaiense and Diphyllobothrium latum, was corroborated. The genus name for D. nihonkaiense and D. latum is also discussed.

High prevalence of the liver fluke Amphimerus sp. in domestic cats and dogs in an area for human amphimeriasis in Ecuador.

BACKGROUND: Amphimerus sp. is a liver fluke which recently has been shown to have a high prevalence of infection among an indigenous group, Chachi, who reside in a tropical rainforest in the northwestern region of Ecuador. Since it is unknown which animals can act as a reservoir and/or definitive hosts for Amphimerus sp. in this endemic area, a study was done to determine the prevalence of infection in domestic cats and dogs. This information is important to understand the epidemiology, life cycle and control of this parasite. METHODOLOGY/FINDINGS: In July 2012, three Chachi communities located on Rio Cayapas, province of Esmeraldas, were surveyed. A total of 89 of the 109 registered households participated in the study. Of the 27 cats and 43 dogs found residing in the communities, stool samples were collected from 14 cats and 31 dogs (total of 45 animals) and examined microscopically for the presence of Amphimerus eggs. The prevalence of infection was 71.4% in cats and 38.7% in dogs, with similar rates of infection in all three communities. Significantly more cats were infected than dogs (p = 0.042). CONCLUSIONS/SIGNIFICANCE: The data show a high rate of Amphimerus sp. infection in domestic cats and dogs residing in Chachi communities. It can be concluded that these animals act as definitive and reservoir hosts for this liver fluke and that amphimeriasis is a zoonotic disease. These findings provide important epidemiological data which will aid in the development and implementation of control strategies against the transmission of Amphimerus.

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.

[Molecular identification of human Diphyllobothrium nihonkaiense using mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequence].

Identification of Diphyllobothrium species has been carried out based on their morphology, especially sexual organs. In addition to these criteria, PCR-based identification methods have been developed recently. A 20 year-old Japanese living in Kochi Prefecture passed tapeworm. He was successfully treated with single dose of gastrografin. We examined the morphologic features of the proglottids and eggs using histology and scanning electron microscope. We also analyzed mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the proglottids. The causative tapeworm species was identified as D. nihonkaiense based on the results of morphologic features and genetic analysis. We discussed the advantage of PCR-based identification methods of Diphyllobothrium species using cox1 sequence in the clinical laboratory.

Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina.

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.