Amplicon-based next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection of single-celled parasites in human faecal samples.

Comprehensive detection and differentiation of intestinal protists mostly rely on DNA-based methods. Here, we evaluated next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection and differentiation of intestinal eukaryotic protists in the stool of healthy Tunisian individuals. Thirty-six faecal DNA samples previously evaluated by microscopy and ameboid species-specific PCRs were tested. The hypervariable regions V3-V4 and V3-V5 of the 18S rRNA gene were amplified using three universal eukaryotic primer sets and sequenced using Illumina®MiSeq sequencing. In addition, real-time PCR assays were used to detect , , and spp. The metabarcoding assay detected (subtypes 1, 2, and 3) and archamoebid species and subtypes (, , RL1 and RL2, , RL1) in 27 (75%) and 22 (61%) of the 36 stool samples, respectively. Meanwhile, the assay had limited sensitivity for flagellates as evidenced by the fact that no -specific reads were found in any of the five -positive samples included, and -specific reads were observed only in 3/13 -positive samples. None of the samples were positive for by any of the methods. In conclusion, a large variety of intestinal eukaryotic protists were detected and differentiated at species and subtype level; however, limited sensitivity for common flagellates was observed.