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Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador

Manuel CalvopiñaOne Health Research Group, Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador;

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Carlos Bastidas-CaldesOne Health Research Group, Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador;
Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador;
Programa de Doctorado en Salud Pública y Animal, Universidad de Extremadura, Extremadura, Caceres, Spain;

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Francisco RomeroIngeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador;

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Irina Villacrés-GrandaPrograma de Doctorado Interuniversitario en Ciencias de La Salud, Universidad de Sevilla, Sevilla, Spain;

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Jean-Pierre PointierPSL Research University, USR 3278 CNRS–EPHE, CRIOBE Université de Perpignan, Perpignan, France;

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Hidekazu TakagiDepartment of Microbiology and Immunology, Aichi Medical University School of Medicine, Aichi, Japan;

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Hiromu SugiyamaDepartment of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan

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ABSTRACT.

Here, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis, Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata, Biomphalaria cousini, and Aplexa marmorata, isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus, but not to C. sinensis, Fasciola hepatica, or Paragonimus mexicanus. The sequence of amplified DNA product matched that of human-isolated Amphimerus. This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2–gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest.

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Author Notes

Address correspondence to Manuel Calvopiña, One Health Research Group, Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador. E-mail: manuelcalvopina@gmail.com

Financial support: This research was supported by a grant from the Direccion General de Investigacion y Vinculacion (DGIV) de la Universidad de las Américas (UDLA), Quito-Ecuador (BIO.CBC.20.02) and by the Japan Agency for Medical Research and Development (AMED 21fk0108136j0102) to HS.

Authors’ addresses: Manuel Calvopiña, One Health Research Group, Facultad de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador, E-mail: manuelcalvopina@gmail.com. Carlos Bastidas-Caldes, One Health Research Group, Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador, Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador, and Programa de Doctorado en Salud Pública y Animal, Universidad de Extremadura, Extremadura, Caceres, Spain, E-mail: cabastidasc@gmail.com. Francisco Romero, Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador, E-mail: francisco.romero@udla.edu.ec. Irina Villacrés-Granda, Programa de Doctorado Interuniversitario en Ciencias de La Salud, Universidad de Sevilla, Sevilla, Spain, E-mail: irinamaribel@gmail.com. Jean-Pierre Pointier, PSL Research University, USR 3278 CNRS–EPHE, CRIOBE Université de Perpignan, Perpignan, France, E-mail: pointier@univ-perp.fr. Hidekazu Takagi, Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Aichi, Japan, E-mail: htakagi@aichi-med-u.ac.jp. Hiromu Sugiyama, Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan, E-mail: hsugi@nih.go.jp.

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