Molecular Technology Differentiates Five Entamoeba Species

histolytica is considered to be pathogenic, causing intestinal and extraintestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii.

Scientists at The Federal University of Rio de Janeiro (Brazil) extracted 74 DNA samples from human stools and nine of these DNA extracts were from stools that were positive for Entamoeba species by microscopy. Polymerase chain reaction (PCR) amplification was performed with biotinylated Entamoeba spp 18S ribosomal ribonucleic acid (rRNA) gene primers, designed to amplify a fragment ranging from 382 to 429 base pairs (bp) of the Entamoeba species studied.

All parasitological positive samples were tested using real-time PCR and nine stools samples were also analyzed by DNA sequencing. In addition regions of the fragments that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads (Austin, TX, USA). The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebae in their stools.

The investigators were able to correctly identify the five different species in all of the specimens studied. From the 24 samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, there was 100% concordance with the PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli. They were able to identify samples with more than one species of Entamoeba by performing a Luminex assay and all fifty parasitologically negative stool samples were negative in the assay.

The authors concluded that that the method could be used in the diagnostic detection of Entamoeba species in fecal samples. This diagnostic test was useful to clearly distinguish the pathogenic E. histolytica from the other species and also to strengthen epidemiologic data on Entamoeba species. The study was published on March 15, 2013, in the journal Parasites & Vectors.

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The Federal University of Rio de Janeiro

Luminex