Detection of EhCRT Gene Expression in Entamoeba histolytica-Infected Children and its Correlation with Interleukin 25 and Tumor Necrosis Factor Alpha

Abstract

Objectives: Entamoeba histolytica is a human enteric protozoan, which is the causative agent of amebiasis. The host activates a series of immunological responses to protect against the parasite after contact with the ameba and further invasion of the gut epithelium layer. As a result, the ameba has developed a variety of evasion mechanisms to hold out the immune response and continue to survive and cause disease. The calreticulin (EhCRT) is one of the immunogenic molecules of E. histolytica that induces an immune response in the human host. Increase in the expression of the EhCRT gene could provide control mechanism that allows the parasite to adapt and survive in host tissues. Aim of the Study: This study was designed to detect the EhCRT gene of E. histolytica by real-time polymerase chain reaction (PCR) in stool samples of children with amebiasis and its roles in host–parasite relationship via measuring the concentration of tumor necrosis factor alpha (TNFα) and interleukin 25 (IL25) by enzyme-linked immunoassay (ELISA) technique in their serum. Materials and Methods: A total of 86 diarrheal fecal samples were collected from children in age <1 year to 13 years suspected to be infected with E. histolytica during the period from December 30, 2020, to September 1, 2021. Microscopically positive samples were the subject to conventional PCR and real-time PCR for the detection of E. histolytica HM1:IMSS strain using (Psp) gene sequences and detection of calreticulin (EhCRT) expression. Blood was withdrawn from each child included in the study for ELISA test to measure the level of IL25 and TNFα. Results: Fecal samples for microscopic examination revealed that 71 (82.6%) children had amebic colitis, E. histolytica gene was detected in 44 samples (71%) using conventional PCR, and the immunogene EhCRT was expressed in 36 stool samples using real-time PCR. The results of the recent study showed highly significant elevation in the level of TNFα and IL25 in the amebic group (Eh+ve PCR). The majority of amebic children were in the age group of 1–4 years, had mucoid, acute, and with primary episodes of diarrhea. Conclusion: E. histolytica is a protozoan parasite highly prevalent among diarrheal children and is responsible for gastrointestinal amebiasis in the human host. The PCR is a useful tool in the diagnosis of E. histolytica infection. It is clear that the expression of the calreticulin gene (EhCRT) concedes with the duration of diarrhea a virulence factor that plays a role in host pathogenic pathways. The findings of this study showed that the level of TNFα in the serum of children infected with amebic colitis (Eh gene + ve) is significantly increased during the course of infection and the cytokine IL25 exhibits a significant drops in the same children.