Real-Time Polymerase Melting Curve Analysis for Genotyping of Entamoeba histolytica from diarrheal patients in Al-Diwaniyah Teaching Hospital of Iraq

Entamoeba histolytica is a significant cause of amoebic colitis and liver abscess in developing countries, including Iraq. Genetic diversity among E. histolytica isolates may influence disease manifestation and transmission. This study aimed to genotype Entamoeba histolytica isolates from diarrheal patients in Al-Diwaniyah Teaching Hospital, Iraq, using Real-Time PCR with SYBR Green I and melting curve analysis. A total of 55 stool specimens from patients with diarrhea were collected between January and December 2025. All the specimens were tested positive to E. histolytica on ssrRNA gene by PCR. Real-Time PCR was done in a nested manner on the SREHP gene using the nested Genotyping technique and then the melting curve was analyzed. Melting curve analysis showed the existence of four different genotypes with different melting temperatures: 84 o C (Genotype-I), 83 o C (Genotype-II), 82 o C (Genotype-III), and 81 o C (Genotype-IV). Genotype-II was the most prevalent (47.3%), followed by Genotype-I (23.6%), Genotype-III (18.2%), and Genotype-IV (10.9%). This population did not have any Genotype-V (79C). Inference: SYBR Green I and melting curve analysis of Real-Time PCR is a quick and dependable technique to genotype E. histolytica . Genotype-II predominates implying that it may be involved in symptomatic disease in this area. This approach can be used in epidemiological and large-scale screening in areas where the disease is endemic such as Iraq.