LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools

AbstractBackgroundSix species of the genusEntamoeba, i.e.,E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmaniican be found in human stools. Among these, onlyE. histolyticais considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical toE. disparandE. moshkovskii. In general,E. polecki,E. coli, andE. hartmaniican be differentiated morphologically fromE. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate amongEntamoebaspecies has limited epidemiologic information onE histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation ofEntamoebaspecies.MethodsPCR amplification was performed with biotinylatedEntamoebasp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of theEntamoebaspp studied. Regions of this fragment that could differentiate amongE. histolytica,E. moshkovskii, E. dispar, E. hartmaniiandE. coliwere selected to design hybridization probes to link to Luminex beads. 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 amebas in their stools.ResultsUsing this approach we were able to correctly identifyE. histoltyica,E. dispar,E hartmanni,E. coliandE. moshkovskiiin all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification ofE. dispar, E. moshkovskii, E. hartmanni, E. histolytica,andE. coli.ConclusionThese results show that this method could be used in the diagnostic detection ofEntamoebaspp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data onEntamoebaspp.