A high-throughput sequencing assay to comprehensively detect and characterize unicellular eukaryotes and helminths...

Several of the most devastating human diseases are caused by eukaryotic parasites transmitted by arthropod vectors or through food and water contamination. These pathogens only represent a fraction of all unicellular eukaryotes and helminths that are present in the environment and many uncharacterized organisms might have subtle but pervasive effects on health, including by modifying the microbiome where they reside. Unfortunately, while we have modern molecular tools to characterize bacterial and, to a lesser extent, fungal communities, we lack suitable methods to comprehensively investigate and characterize most unicellular eukaryotes and helminths: the detection of these organisms often relies on microscopy that cannot differentiate related organisms, while molecular assays can only detect the pathogens specifically tested.

Here, we describe a novel sequencing-based assay, akin to bacterial 16S rRNA sequencing, that enables high-throughput detection and characterization of a wide range of unicellular eukaryotes and helminths, including those from taxonomical groups containing all common human parasites. We designed and evaluated taxon-specific PCR primer pairs that selectively amplify all species from eight taxonomical groups (Apicomplexa, Amoeba, Diplomonadida, Kinetoplastida, Parabasalia, Nematoda, Platyhelminthes, and Microsporidia). We then used these primers to screen DNA extracted from clinical, biological, and environmental samples, and after next-generation sequencing, identified both known and previously undescribed organisms from most taxa targeted.

This novel high-throughput assay enables comprehensive detection and identification of eukaryotic parasites and related organisms, from a wide range of complex biological and environmental samples. This approach can be easily deployed to many settings and will efficiently complement existing methods and provide a holistic perspective on the microbiome.