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 Dientamoeba fragilis , Giardia duodenalis , and Cryptosporidium spp. The metabarcoding assay detected Blastocystis (subtypes 1, 2, and 3) and archamoebid species and subtypes ( Entamoeba dispar , Entamoeba hartmanni , Entamoeba coli RL1 and RL2, Endolimax nana , Iodamoeba bütschlii 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 Giardia -specific reads were found in any of the five Giardia -positive samples included, and Dientamoeba -specific reads were observed only in 3/13 D. fragilis -positive samples. None of the samples were positive for Cryptosporidium 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.
Competing Interests: The authors declare that they have no competing interests.
(© 2022 The Authors.)