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

Abstract : 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 speciesspecific 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.
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https://hal-riip.archives-ouvertes.fr/pasteur-03597404
Contributor : Abdelhakim Ben Hassine Connect in order to contact the contributor
Submitted on : Friday, March 4, 2022 - 11:22:51 AM
Last modification on : Tuesday, March 8, 2022 - 3:06:04 AM

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Amal Chihi, Lee O'Brien Andersen, Karim Aoun, Aïda Bouratbine, Christen Rune Stensvold. Amplicon-based next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection of single-celled parasites in human faecal samples. Parasite Epidemiology and Control, Elsevier, 2022, 17, pp.e00242. ⟨10.1016/j.parepi.2022.e00242⟩. ⟨pasteur-03597404⟩

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