Evaluation of cysteine proteinase gene polymorphism of Entamba histolytica in dysentery patients in Iran by sequencing method

Abstract

Amebiasis in humans is caused by Entamba histolytica , which is also one of the global causes of acute bloody diarrhea. 3.6% of all human infections with E.histolytica are detected by microscopical examination, trichrome, and molecular methods. The treatment is carried out based on the location and the clinical symptoms. Despite the documented dysentery cases in Saqqez, no research has been done to find the gene responsible for the pathogenicity of E.histolytica amoeba. In this study, single step PCR method is used to sequence the E.histolytica amoeba strains that were previously discovered by multiplex PCR to assess cysteine proteinase gene polymorphism.

Materials and methods: In this cross-sectional study, 500 stool samples from patients with intestinal disorders were analyzed for the kind of amoebas using microscopic trichrome and molecular methods after receiving consent inform. After DNA extraction from positive samples of Entamba histolytica (trophozoite and cyst), the cysteine proteinase 4 gene was amplified and sequenced to evaluate the polymorphism.

Results: In this investigation, Amoeba E.histolytica was found in 10 cases (2%) out of 500 stool samples utilizing microscopic and molecular and staining techniques. In terms of stool appearance, 20% of stools had a watery diarrheal appearance, and 20% of stools had a diarrheal appearance. Mucoidi, and bloody diarrhea represented 60% of stool cases (dysentery). The most affected age range is between 11 and 20 years, and 67% of males and 33% of women were found to have E.histolytica. E.histolytica (Hd: 0.689), which includes 3 novel haplotypes, displayed significantly high genetic diversity in dysentery sample sequencing data. According to the alignment results of the modified sequences, the amino acids in the dysentery samples were changed in codons No. 16 (Valine instead of Glutamate) and 115 (Glutamine instead of Leucine) (E.h 2-E.h 6). Phenylalanine rather than leucine at codon position 179 was found in samples with mucoid diarrhea and watery diarrhea (E.h 7–E.h 9).