Further insight into the genetic diversity of Entamoeba coli and Entamoeba hartmanni.

Despite the species' wide distribution, studies of the genetic diversity within Entamoeba coli and Entamoeba hartmanni remain limited. In the present study, we provide further insight into the genetic diversity of both species based on analysis of partial nuclear small subunit ribosomal DNA sequences generated from human fecal DNAs from samples collected in Africa, South America, and Europe. Reinforcing the previous recognition that E. coli is a species complex, our data confirm the existence of the two subtypes, ST1 and ST2, previously identified plus, potentially, a new subtype, ST3. While ST1 appears to be genetically quite homogenous, ST2 shows a substantial degree of intrasubtype diversity. ST2 was more common in samples collected outside Europe, whereas ST1 showed no geographical restriction. The potentially novel subtype is represented to date exclusively by sequences from South American and African samples. In contrast to previous reports, our new data also indicate substantial variation in E. hartmanni that could also support the establishment of subtypes within this species. Here, however, no links were identified between subtype and geographical origin.

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.

Development and evaluation of molecular tools for detecting and differentiating intestinal amoebae in healthy individuals.

Amoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.