Needle in a Haystack: A Droplet Digital Polymerase Chain Reaction Assay to Detect Rare Helminth Parasites Infecting Natural Host Populations.

Helminths infect humans, livestock, and wildlife, yet remain understudied despite their significant impact on public health and agriculture. Because many of the most prevalent helminth-borne diseases are zoonotic, understanding helminth transmission among wildlife could improve predictions and management of infection risks across species. A key challenge to understanding helminth transmission dynamics in wildlife is accurately and quantitatively tracking parasite load across hosts and environments. Traditional methods, such as visual parasite identification from environmental samples or infected hosts, are time-consuming, while standard molecular techniques (e.g., PCR and qPCR) often lack the sensitivity to reliably detect lower parasite burdens. These limitations can underestimate the prevalence and severity of infection, hindering efforts to manage infectious diseases. Here, we developed a multiplexed droplet digital PCR (ddPCR) assay to quantify helminth loads in aquatic habitats using 18S rRNA target genes. Using Schistocephalus solidus and their copepod hosts as a case study, we demonstrate ddPCR's sensitivity and precision. The assay is highly reproducible, reliably detecting target genes at concentrations as low as 1 pg of DNA in lab standards and field samples (multi-species and eDNA). Thus, we provide a toolkit for quantifying parasite load in intermediate hosts and monitoring infection dynamics across spatio-temporal scales in multiple helminth systems of concern for public health, agriculture, and conservation biology.