Redefining the Entamoeba landscape with next-generation sequencing: Implications for diagnosis and epidemiology

Entamoeba is a genus of anaerobic protists that infect humans and a wide range of animal hosts worldwide, and that are also detected in environmental reservoirs. Of particular importance is Entamoeba histolytica, the causative agent of amoebiasis, which accounts for more than 55,000 deaths each year and remains a major public health concern. Given this substantial burden, and the fact that pathogenic and non-pathogenic species of Entamoeba cannot be distinguished based on morphology alone, molecular approaches have become essential for accurate diagnosis, taxonomy, and epidemiological surveillance. In this Chapter, we systematically review the impact of DNA sequencing technologies on Entamoeba research from 1987 to 2025. A bibliographic search was conducted using PubMed and Google Scholar, combining the names of all recognized Entamoeba species with keywords related to sequencing technologies, spanning from first- to fourth-generation platforms. Original research articles employing DNA sequencing for molecular identification, phylogenetic analyses, and genomic investigations were included. The literature illustrates a clear transition from morphology-based classification to Sanger sequencing of the small subunit of the ribosomal RNA (SSU rRNA) gene, which established the molecular taxonomic framework of the genus and enabled the recognition of numerous new species and ribosomal lineages. The expansion of diversity revealed by high-throughput sequencing is also addressed, together with the contribution of long-read technologies to resolving highly AT-rich and repetitive genomes. Despite these advances, challenges remain in species delineation and marker-associated biases, highlighting the ongoing need for integrative genomic approaches in Entamoeba research.