1. To determine critical steps in transmission of F. hepatica infection to humans analyzing environmental, temporal-spatial, and host infection distribution data using a One-Health approach.
2. To demonstrate the accuracy and efficiency of PCR and recombinase polymerase amplification tests to assess F. hepatica infection in multiple hosts and environmental samples.
3. To define the effect of chronic fascioliasis on innate and adaptive immune responses and their association with Fasciola reinfection, treatment failure, and susceptibility to other infections.

We hypothesize that F. hepatica transmission risk among humans is determined by crucial temporal and spatial associations between host distribution, intensity of infection, and susceptibility among humans. These crucial lifecycle steps can be targeted for surveillance and control interventions. To detect these associations, we will establish novel tools and models of transmission that can be replicated in other complex zoonotic diseases.

We will determine the epidemiology of Fasciola infection using a cross sectional study and prospective cohorts of human, livestock, and snails in two endemic regions of the Peruvian highlands. We will use a One-Health approach to collect georeferenced human, animal, snail, weather, and environmental data to feed Fasciola based transmission models. We will utilize quantitative real time PCR and qualitative RPA tests to assess alternative surveillance strategies (i.e., environmental DNA), precisely determine the infection burden, and assess the efficacy of treatment in low resource settings. We will evaluate the innate and T-cell responses in cohorts of human with chronic fascioliasis before and after treatment to define if memory immune responses predict susceptibility to reinfection and treatment failure. We will evaluate if these responses are Fasciola specific via assessing vaccine response.

In a significant expansion of our laboratory capacity, we have opened the new facility to house the Cusco Branch of the Tropical Medicine Institute of Universidad Peruana Cayetano Heredia. This is a 7 stories high 12,000 square facility including 6 BSL2 laboratories for microbiology and parasitology, dedicated cryobank space, and a data management center all fully backed up by uninterruptible power supply and a power generator. At Universidad Nacional de Cajamarca a new Tropical Medicine Research Center has been created that expands the scope of work in the school of veterinary medicine. In addition, a new collaboration with University of Galway in Ireland is allowing us to develop different serologic tests to diagnose fascioliasis in multiple hosts at the same time.

We have demonstrated that the risk for chronic fascioliasis decreases 4% for each year of age in Cajamarca and Cusco and the in the communities studied water played a significant role in transmission; with drinking water piped into the households decreased risk of infection by 40% and drinking water from artificial reservoirs increased the risk by 68%. Importantly, drinking from artificial reservoirs or irrigation channels while working on farms increased risk 56% to 132%. These findings have significant implications for control given that most water safety interventions are focused on households and providing safe water in farms might be less feasible. The prevalence of fascioliasis in livestock varied by species and location, but the infection was associated with poor body condition in all livestock species regardless of location. Modifiable farming practices were associated with species specific risk of infection suggesting that training farmers could impact transmission. Our drone based high resolution imaging was able to predict a spatial risk for infection among human and sheep at the community level.

We have developed novel diagnostics for chronic fascioliasis capable of detecting the infection in several types of specimens. Applying our concept of a single simple PCR test for all hosts, we have standardized a Fasciola hepatica PCR with a sensitivity of 80% and specificity of 92% in a single human stool sample and 73% sensitivity and 84% specificity in snails. Similarly, our eDNA PCR in water can amplify Fasciola DNA in 19% of man-made canals and 12% of natural creeks in the Cusco region. The detection of Fasciola cell-free DNA in urine has a sensitivity of 86% and specificity 44% to diagnose the infection, detecting Fasciola DNA in 35 samples negative by stool microscopy. Our mut-CL1 antigen ELISA showed 94% sensitivity and 93-100% specificity in initial standardization. In Cajamarca, it had 93% sensitivity and 85% specificity. The preliminary mut-CL1 antigen Western Blot detected antibodies in 83% of infected subjects and was negative in 84% of uninfected subjects.

A total of 277 PBMC samples were collected and exposed to Fasciola E/S antigens. The analysis of PBMC samples of subjects with chronic Fasciola infection showed that they produce significantly higher amounts of IL-5, IL-9, IL-13, IL-22, IL-4, and IL-17F compared to subjects without infection, indicating a Th2/Th9/Th22 biased immune environment. Higher levels of IL-2, IFN-gamma, and TNF alpha were also observed. No difference in baseline cytokine responses was found between subjects who cured or failed triclabendazole treatment.

We have not changed our big themes which focus on transmission, novel tools, and human immunology.