Roadmap for development of diagnostic tests for helminths

What are we trying to achieve and why? What is the problem we are trying to solve?

The increasing levels of anthelmintic resistance (AR) in many helminths is leading to an urgent need to move towards evidence-based anthelmintic use and have better anthelmintic stewardship. This requires quick, accurate, sensitive and cheap molecular tests in order to:

• Quantify the DNA or RNA from different helminth species present in fecal samples before and after drug treatments.
• Detect AR mutations for each of the major anthelmintic classes in each of the parasite species of interest using parasite DNA isolated from faeces.
• Provide farmers and veterinarians with an assessment as to the clinical consequences/production impacts of parasite infections and anthelmintic resistance status present in the helminth population on their farm.

What are the scientific and technological challenges (knowledge gaps needing to be addressed)?

• Levels of parasite DNA (or RNA) in fecal samples may not reflect adult helminth burdens and may not detect pre-patent/immature parasite stages.
• High volumes of feces may need to be processed to obtain required sensitivity and mitigate effects of parasite aggregeation in fecal samples.
• Presence of parasite stages or “free DNA” ort RNA in feces is variable. It will be determined by the physical location of the parasite, eg for Fasciola, immature stages are in the liver parenchyma so parasite DNA unlikely to be present in faeces.
• Molecular genetic basis of resistance to different classes of drug in each different species is incomplete and molecular markers still lacking.
• Accurate quantitation of the frequency of resistance mutations (and the different species) in helminth populations needed (rather than simple presence or absence)
• Diagnostic platforms, either lab-based or penside, need to be sufficiently rapid, cheap and scalable to provide meaningful and sufficiently timely information for field use.

What approaches could/should be taken to address the research question?

• Laboratory based tests or penside tests both have potential roles in providing timely information at the herd level for surveillance, monitoring, anthelmintic stewardship and herd/flock health planning.
• Further development and validation of molecular markers and Next-generation sequencing approaches for species differentiation of eggs and larvae in fecal samples.
• More work to identify the major genetic mutations (markers) underlying AR for the different drug classes for the major parasite species of interest: includes genetic , mapping, population genomics and functional genomics approaches.
• More field based research piloting molecular diagnostic approaches to establish proof of concept.

What else needs to be done before we can solve this need?

Existing knowledge including successes and failures

• Accurate, quick and simple quantitive diagnosis of specific helminth species and AR mutations would improve surveillence, monitoring and anthelmintic stewardship. Several PCRs have been described but they have not transitioned to diagnostic use and are only available in specific laboratories because specialized equipment needs and costs. There are also problems in reproducibility between laboratories, with published methods often not working in other diagnostic laboratories.
• There has been recent progress in applying next -generation amplicon sequencing technologies for quantitation of helminth species in fecal samples (eg. Nemabiome ITS-2 rDNA sequencing) and of benzimidazole resistance mutations in cattle and sheep nematodes but these require more validation in the field.
• Good understanding of the most important mutations underlying benzimidazole resistance in cattle and sheep trichostrongylid nematode species. Recent progress in genetically mapping mutations underlying ivermectin resistance, levamisole resistance and monepantel resistance in Haemonchus controtus. This should allow
  more proof of concept field diagnostic studies to be performed