Mitigating the global impact of fasciolosis: developing diagnostics for triclabendazole resistance in Fasciola species

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Summary:Fasciolosis and one health Fasciola spp infect livestock and, as a zoonotic parasite, this leads to infection in humans. Estimates of infection suggest between 2.4 and 17 million people are infected worldwide, typically in LMIC endemic regions. WHO recognises fasciolosis as a neglected tropical disease. Triclabendazole is the drug of choice for treating infection in livestock as it targets all life cycle stages and it is the only drug licenced for humans. Triclabendazole resistance in Fasciola species was initially reported in livestock species due to intensive treatment regimes, inevitably this led to infection of humans with triclabendazole resistant parasites and failure to effectively treat infections. The ability to detect drug resistant parasites in livestock and humans is key to effective control; better control in livestock reduces the risk of transmission to humans and if humans get infected, testing for resistance can inform on the off-licence use of alternative therapeutics. We need a molecular diagnostic for triclabendazole resistance in Fasciola spp that can be used on both livestock and humans in LMIC settings.Project aims and objectivesFasciolosis is caused by two parasites, Fasciola hepatica and F. gigantica. Fasciola spp have a complex demography, they are either present as species-specific infections (e.g Fasciola hepatica in Peru) or as hybrid forms in regions where their distributions overlap (e.g in India and Pakistan) and introgression of genetic loci between the species has been reported. We have made substantial inroads into identifying a genomic locus that confers triclabendazole resistance in F. hepatica and have localised it to a cluster of seven genes; amongst which are two candidate genes, an ADP ribosylation factor (ARF) and ABC genes (ABCB1), both of which have non-synonymous SNPs that are under selection by triclabendazole. In livestock detection of infection is by coproantigen/presence of eggs in faeces or by antibody detection in serum. Similarly, human faecal samples can be used to detect eggs or Fasciola spp DNA. This project aims to build on our existing understanding of triclabendazole resistance in livestock to mitigate its impact on human health. We will: 1. Conduct whole genome re-sequencing from multiple isolates from both livestock and humans from countries representing different demographics (Peru, India, Pakistan, Cameroon and Senegal)2. Carry out comparative genomics to localise genetic exchange within F. gigantica, F. hepatica and hybrids/introgressed species, with a particular focus on triclabendazole-resistance genes.3. Determine if triclabendazole selection is placed on SNPs within ARF and ABCB1 genes in multiple F. hepatica and F. gigantica isolates of known resistance status from both livestock and humans. 4. Optimise diagnostic molecular methods for species-specific detection of Fasciola spp in faecal samples and duodenal/bile aspirates in humans, in collaboration with clinicians in India and Peru.5. Establish proof-of-principal methodology for molecular diagnostic detection e.g Lec-lamp, to detect SNPs in triclabendazole resistance genes in F. hepatica and F. gigantica isolates for point-of-care use in LMIC settings.The goal of this project is to generate novel genomic sequence data, mapping this to current reference genomes and interrogate gene annotations from multiple liver fluke isolates in order to determine whether resistance genes are conserved across the different species and hybrid forms of Fasciola. Candidate gene sequence variation between known drug resistant and susceptible isolates will be interrogated in order to identify gene changes linked to resistance (resistance markers). Finally, a bespoke molecular diagnostic tool will be developed to detect these resistance markers in samples from livestock and humans. The project aims to combine discovery science with applied diagnostics to positively impact on livestock and human heal