Genetic diversity and phylogenetic positioning of Entamoeba histolytica isolates from stool samples of patients from few Hospitals at Diyala governorate

Entamoeba histolytica is a protozoan parasite that causes amoebiasis, an infection primarily affecting the intestines. The study aimed to determine the phylogenetic placement of six isolates of E. histolytica that were amplified from three genetic loci, rRNA, cysteine proteinase, and amoebapore C to evaluate the genetic variation pattern of each sequence. DNA extracted from cultured and frozen stool samples using PrestoTM Stool DNA Extraction Kit and Geneaid kits was analyzed for E. histolytica diversity in amoebiasis patients at Baquba General Hospital from March-June 2023. Polymerase chaing kreaction (PCR) and sequencing of virulence genes (rRNA, cysteine proteinase, amoebapore C) followed by phylogenetic analysis revealed genetic variation and potential implications for pathogenicity. The conducted sequencing reactions confirmed the precise identification of the examined samples, which were determined to be attributed to variable isolates of E. histolytica. Aligning the rRNA sequences of samples A1 and A2 with the most closely related rRNA sequences of E. histolytica (GenBank acc. no. OP925909.1) revealed the presence of two nucleic acid variants (212C>T in A1, and 427G>C in A1 and A2) not present in the reference sequences. Aligning the cysteine proteinase sequences of samples B1 and B2 with the most closely related genomic DNA sequences of E. histolytica (GenBank acc. no. X91642.1) revealed the presence of four nucleic acid variants (95A>T and 96A>T in B1 and B2, and 246A>T and 247A>T in B2) not present in the reference sequences. Translation of nucleic acid sequences to amino acid residues showed that both 95A>T and 96A>T variants caused a missense effect of p.46E>V, while both 246A>T and 247A>T variants showed another missense effect of p.97T>S. Aligning the amoebapore C sequences of samples C1 and C2 with the most closely related genomic sequences of E. histolytica (GenBank acc. no. X76903.1) revealed their entire similarity. It was inferred from the tree that the investigated protozoan samples occupied distinct phylogenetic positions and were suitable for the vicinity of various clinical isolates that were isolated from several locations worldwide. However, the rRNA-based tree provided ample genetic diversity with more effective discrimination compared with the other used loci.