Abstract
There are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.