Fig 1.
Map of the localities studied in Pedro Pablo Gómez (PPG), Manabí, Ecuador.
Rural communities involved in the PPG region (left panel, red) are located at ~493 (A, latitude: -80.551933°, longitude: -1.70995°), ~483 (B, latitude: -80.562917°, longitude: -1.703733°), and ~588 (C, latitude: -80.55195°, longitude: -1.742683°) meters above sea level. Amphimerus sp. adult flukes were described for the first time infecting humans in the province of Esmeraldas (right panel, blue). Maps were developed using shapefiles from https://www.naturalearthdata.com/.
Table 1.
Human population showing small trematode eggs in Pedro Pablo Gómez communities.
Participants were divided by age, gender, and communities to assess associations with small trematode egg presence, although none was statistically significant. Percentages are calculated with the totals of each row.
Fig 2.
Characteristics of Amphimerus sp. in different morphological stages from Pedro Pablo Gómez, Manabí, Ecuador.
(A) Adult Amphimerus sp. fluke isolated from the liver of a naturally infected dog. Note the anterior-posterior division of the ungrouped vitelline glands (arrow) at the level of the ovary, which extend beyond the testes reaching the posterior end of the body. (B) Egg isolated from a human fecal sample resembling characteristic opisthorchiid and heterophyid eggs: pyriform shape, anterior operculum, and an apparent posterior spine (knob). (C) Metacercaria of Amphimerus sp. isolated from fish collected in the communities of Pedro Pablo Gómez, showing an oval shape within a cyst wall; the larva inside features a round and prominent ventral sucker (VS) and a black, round to oval, excretory bladder (EB).
Fig 3.
Freshwater fish incriminated as potential second intermediate hosts of Amphimerus sp., collected in Pedro Pablo Gómez, Manabí, Ecuador.
All fish species were positive for Amphimerus sp. metacercariae. (A) Rhoadsia altipinna, known locally as ‘ancha’. (B) Bryconamericus bucay, known locally as ‘engorda’. (C) Andinoacara rivulatus, known locally as ‘vieja’. (D) Piabucina aureoguttata, known locally as ‘guaija’. Pictures by Daniel Romero-Alvarez.
Fig 4.
Molecular diagnosis of Amphimerus sp. metacercariae based on PCR-RFLP.
(A) Multiple metacercariae were isolated from fish collected at each community. (B) Different metacercariae identified based on PCR-RFLP as shown in panel D. (C) First round of PCR amplification using universal trematode primers (green arrow). (D) Digested (L1-L6) and undigested (L7) PCR products by the restriction enzyme Hind III. Lanes 1–5 corresponds to (L1) adult Amphimerus sp. flukes from a human case at Esmeraldas, followed by adult parasites from a human (L2), a dog (L3), and a cat (L4), and (L5) Amphimerus sp. metacercariae from freshwater fish (i.e., Bryconamericus bucay) from Pedro Pablo Gómez; the two fragments correspond to 374 and 140 base pairs (red arrows). Lane 6 corresponds to the metacercariae of Haplorchis pumilio with fragments of 440 and 86 base pairs (blue arrows). Lane 7 corresponds to unidentified metacercariae from fish in Pedro Pablo Gómez. L: 100 base pairs DNA Ladder (New England Biolabs, Ipswich, Massachusetts, United States).
Table 2.
Metacercariae identified by molecular diagnosis.
Metacercariae isolated from fish of the three communities studied were tested by PCR-RFLP and DNA sequencing to identify trematode species in the area. Percentages are calculated with the totals of each row.
Fig 5.
Schematic life cycle of Amphimerus sp. in Manabí province, Ecuador.
(A) All opisthorchiids use two intermediate hosts (prosobranch mollusc and fish for first and second, respectively); we lack information on the snail species involved in Amphimerus sp. life cycle in Ecuador. Opossums play a role in the sylvatic maintenance of A. neotropicalis and A. pseudofelineus in Latin America [42] and might be incriminated in the life cycle of Amphimerus sp. in Ecuador as well. (B) Landscape of rural community B within Pedro Pablo Gómez (PPG) parish, Manabí province, Ecuador. (C) Local dish being prepared in one of PPG studied communities from fish collected in surrounding streams.