Molecular differentiation by PCR of the Entamoeba dispar/histolytica/moshkovskii complex in the population of Coyaima, Tolima, Colombia

The Entamoeba histolytica/dispar/moshkovskii complex comprises morphologically indistinguishable species, making accurate diagnosis difficult and leading to overestimations in amebiasis prevalence. This study aimed to establish a sensitive and specific molecular method to differentiate these species in human fecal samples from Coyaima, Tolima, Colombia. A polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA (18S rRNA) gene was developed and optimized under various amplification conditions. The nested PCR showed higher sensitivity and specificity than the multiplex approach, achieving a detection limit of 160 picograms of E. histolytica DNA. Cross-reactivity tests confirmed the method specificity, as no amplification was observed with other intestinal protozoa or helminths. Epidemiological analysis revealed a prevalence of 24.54% for E. dispar, 1.04% for E. histolytica, and 0.78% for mixed E. histolytica/dispar infections, while E. moshkovskii was not detected in the study area. These findings demonstrate the effectiveness of the optimized protocol as a reliable molecular tool for differential diagnosis and epidemiological surveillance of the E. histolytica/dispar/moshkovskii complex, contributing to improved understanding of its distribution and transmission dynamics in endemic Colombian populations.