PROJECT SUMMARY/ABSTRACT Diseases attributed to parasites cause substantial morbidity and mortality globally. However, there is mounting evidence that the relationship between humans and symbionts (defined here as all host-dependent eukaryotic organisms, including parasites and commensals) is not always detrimental and, in fact, may sometimes confer important health advantages. Symbiont assemblages may aid in digestion, confer protective immunity, or, paradoxically, provide protection from pathogenic parasite infection and disease. We currently understand the biology and ecology of eukaryotic parasites that cause human disease, but we know very little about the overall structure and function of communities of protozoans and helminths that also colonize humans. This shortcoming hinders progress in the field of parasitology, compared to the fields of bacteriology and mycology, which have revolutionized our understanding of human health through the study of bacterial and fungal microbiota. Indeed, most current parasitological methods are designed for a different purpose: to target specific, pathogenic organisms. Although methods for unbiased community analysis have been validated and standardized for prokaryotes and fungi, no such methods currently exist for eukaryotic symbionts, including parasites. With no standardized methods in place, research into the beneficial effects of eukaryotic symbiont communities and their potential role in parasitic disease represents a critical knowledge gap. The ultimate objective of this work is to create a uniform, validated research tool for the study of human symbiont assemblages, comparable to what has been achieved for the bacterial and fungal microbiomes. To this end, we have developed a novel comprehensive (able to identify all organisms regardless of prior characterization, including novel organisms) assay for eukaryotic symbiont assemblage characterization that is based on newly-designed PCR primers, a novel noise reduction strategy, and a user- friendly bioinformatic pipeline. Our goals in developing this assay were to 1) create an “industry standard” for symbiont community assessment, analogous to other standardized microbiome assays, 2) develop a broadly- applicable research method useful for all organisms and all sample types, 3) contribute to our understanding of the basic biology and ecology of human eukaryotic symbiont communities and 4) inform future development of “universal” diagnostic tools and targeted anti-parasitic therapeutics. We will optimize the assay in silico using custom databases, in vitro using a “mock community”, and ex vivo using samples from experimentally-infected animals. We will then validate the pipeline and assess its performance by analyzing human samples from two different populations in which intestinal parasites have previously been characterized by microscopy and PCR. Through completion of this project, we hope to provide a critically-needed research tool that will improv...