Evaluation of genetic diversity of serine-rich histidine protease in symptomatic patients with Amoebic dysentery

Abstract

Amoebiasis is an intestinal infection in humans caused by the protozoan Entamoeba histolytica (the causative agent of amoebic dysentery). In 90% of cases, it is asymptomatic, and in 10% of cases, a range of clinical syndromes from dysentery to severe colitis, liver abscess and even peritonitis are observed.Trophozoite surface antigens of Entamoeba histolytica, which are serine-rich proteins, are the main factors in the occurrence of invasive amoebiasis, and their recombinant proteins are used in ELISA diagnostic kits for the diagnosis of amoebiasis and the differential diagnosis of Entamoeba histolytica from other species.Therefore, the present study was conducted with the aim of investigating the genetic diversity of serine-rich histidine protease of Entamoeba histolytica in patients with amoebiasis in Iran

Materials and methods: In this cross-sectional study, after obtaining the code of conduct and consent from the participants, 500 stool samples were collected from patients with digestive disorders from Saqez city. Entamoeba histolytica species were identified by using microscopic direct smears, trichrome staining and by targeting the 18s RNA gene. Next, in order to determine the serine-rich histidine protease gene type of Entamoeba histolytica, Nested-PCR technique was performed and then they were subjected to enzymatic digestion by PCR-RFLP method using ALU1 enzyme. Next, the products of the second stage of Nested-PCR were directly sequenced to check the genetic diversity.

Results: In the present study, the largest age group of 10 cases with Entamoeba histolytica ranged from 11 to 20 years old, and the frequency of Entamoeba histolytica parasite was identified with 67% in men and 33% in women, and the prevalence was 2%. According to AMOVA (Analysis of Molecular Variance) relatively high values of haplotypic diversity (genetic diversity) (Hd: 0.762) of Entamoeba histolytica including 2 new haplotypes were detected using DNAsp software. Alignment results of edited sequences showed that significant codon displacement occurred in dysentery samples (SREHP4-7*).While in the samples of mucoid diarrhea and watery diarrhea (SREHP1-3*), no significant codon displacement was observed. In this study, all the isolates were cut into two pieces of 152 and 300 base pairs, which is a definite confirmation of type 5.