Triclabendazole (TCBZ) resistance is an emerging problem in fascioliasis that is not well understood. Studies including small numbers of parasites fail to capture the complexity of susceptibility variations between and within Fasciolahepatica populations. As the first step to studying the complex resistant phenotype–genotype associations, we characterized a large sample of adult F. hepatica with diverging TCBZ susceptibility. We collected parasites from naturally infected livestock slaughtered in the Cusco and Cajamarca regions of Peru. These parasites were exposed to TCBZ sulfoxide (TCBZ.SO) in vitro to determine their susceptibility. We used a motility score to determine the parasite’s viability. We titrated drug concentrations and times to detect 20% non-viable (susceptible conditions) or 80% non-viable (resistant conditions) parasites. We exposed 3348 fully motile parasites to susceptible (n = 1565) or resistant (n = 1783) conditions. Three hundred and forty-one (21.8%) were classified as susceptible and 462 (25.9%) were classified as resistant. More resistant parasites were found in Cusco than in Cajamarca (p < 0.001). Resistant parasites varied by slaughterhouse (p < 0.001), month of the year (p = 0.008), fluke length (p = 0.016), and year of collection (p < 0.001). The in vitro susceptibility to TCBZ.SO in wildtype F. hepatica was associated with geography, season, and morphometry.

Fascioliasis is a parasitic infection caused by Fasciola spp., primarily affecting ruminant animals. These digenean flatworms cause severe liver damage in their hosts, resulting in substantial economic losses within the livestock industry. Human fascioliasis is an emerging public health concern, with an estimated global prevalence of 2.6 million cases. Infection in humans typically occurs through the ingestion of aquatic vegetation or water contaminated with metacercariae. Triclabendazole (TCBZ) remains the only drug recommended by WHO for the treatment of human fascioliasis and is widely used in livestock. However, the increasing prevalence of TCBZ resistance in livestock, along with reports of TCBZ-resistant human infections, poses a growing challenge to disease control. Although it has been suggested that resistant livestock infections may contribute to the emergence of resistance in human populations, this relationship has not been systematically investigated. In this study, we characterized the mitochondrial genomes of TCBZ-resistant and TCBZ-sensitive F. hepatica isolates from human infections and conducted a comparative haplotype analysis with F. hepatica samples obtained from cattle in the same region of Peru. Maximum-likelihood phylogenetic and haplotype network analyses of 304 animal and 11 human F. hepatica samples identified five distinct haplogroups. Mitochondrial haplotypes from human infections clustered into monophyletic groups alongside those from animal hosts, supporting the hypothesis of local zoonotic transmission from animal reservoirs. Additionally, a phylogeographic analysis of global ND1 sequence diversity provided insights into the demographic history of the parasite across pre- and post-domestication periods and revealed genetic signatures of global dissemination that have shaped its present-day distribution.