# Identification of Paragonimus mexicanus and P. caliensis in freshwater crabs from Costa Rica: evidence of zoonotic lung fluke diversity in Central America

## TL;DR

This study identifies two species of lung flukes in Costa Rican freshwater crabs, revealing new host species and geographic clusters of infection, which highlights public health risks from undercooked crabs.

## Contribution

The first molecular confirmation of infected female crabs and the identification of three new host species for Paragonimus in Costa Rica.

## Key findings

- Six crab species were found to host Paragonimus, including three new hosts.
- Male crabs had significantly higher infection rates than females (p = 0.0059).
- Infections were most common in the Caribbean and Northern regions of Costa Rica.

## Abstract

Paragonimus spp. are foodborne trematodes with complex life cycles involving freshwater snails and decapod crustaceans. In Central America, Paragonimus mexicanus is a known zoonotic agent, whereas the public health significance of P. caliensis remains unclear. Between 2015 and 2016, we surveyed freshwater crabs across 51 sites in 15 river basins spanning three major climatic regions in Costa Rica. A total of 419 crabs (10 species) were examined for metacercariae; male crabs were identified morphologically, and infected females were identified to species by 16S rRNA and COI sequencing. Metacercariae were detected in six crab species, three of which represent new hosts of Paragonimus. The highest prevalence in male crabs occurred in Ptychophallus uncinatus (55.6%, 25/45). Molecular analyses confirmed the presence of two Paragonimus species in crabs: P. mexicanus and P. caliensis. A binomial GLM showed that males had higher odds of infection than females (p = 0.0059). Most infections occurred in areas along the Caribbean coast and in the Northern Region, which drains into Lake Nicaragua and Río San Juan. A binomial GLM revealed that the probability of Paragonimus infection varied significantly across climatic regions. Crabs from the Caribbean slope had the highest odds of infection, followed by the Northern Region. In contrast, crabs from the Pacific slope were significantly less likely to be infected. These patterns support the existence of geographic clusters of transmission within Costa Rica. We provide the first species-level, molecular confirmation of infected female crabs in Costa Rica and identify three freshwater crab species as newly recognized second intermediate hosts. These findings expand the known host range and distribution of Paragonimus in Costa Rica and highlight the need for public health education about the risks of consuming undercooked freshwater crabs.

Paragonimiasis is a foodborne parasitic infection caused by lung flukes of the genus Paragonimus, typically acquired through the consumption of raw or undercooked freshwater crabs harboring infective metacercariae. Although Paragonimus spp. have been reported in various crab species across Latin America, their distribution and host range in Costa Rica remain poorly understood. To address this gap, between 2015 and 2016, we collected a total of 419 freshwater crabs from 51 sites across 24 river basins in Costa Rica. Crab specimens were identified morphologically and examined for Paragonimus metacercariae using both microscopic and molecular techniques. Infection was detected in six crab species, three of which represent new hosts for Paragonimus. Male Ptychophallus uncinatus showed the highest infection prevalence (55.6%). Male crabs had significantly higher odds of infection than females (p = 0.0059), with infections most frequently detected in river basins draining into the Caribbean Sea and Lake Nicaragua. Molecular analyses identified two Paragonimus species: Paragonimus mexicanus, a known zoonotic agent, and P. caliensis, whose pathogenicity to humans remains uncertain. Notably, this study provides the first molecular identification of infected female freshwater crab hosts in the region. Our findings demonstrate that Paragonimus spp. in Costa Rica have a broader geographic and host range than previously recognized, underscoring the need for greater public health awareness regarding the consumption of freshwater crabs and for continued surveillance and research on the transmission dynamics of this neglected zoonotic parasite.