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

• DOI: 10.1016/j.parepi.2022.e00242
• Publication Date: 2022-01-30T01:04:48Z
• Authors: Amal Chihi; Lee O'Brien Andersen; Karim Aoun; Aïda Bouratbine; Christen Rune Ste...
• Keywords: Entamoeba; Blastocystis; Giardia; Amplicon

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