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Open Access Research

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

Helena Lúcia Carneiro Santos12, Kakali Bandyopadhyay3, Rebecca Bandea3, Regina Helena Saramago Peralta4, José Mauro Peralta1* and Alexandre Januário Da Silva3

Author Affiliations

1 Instituto de Microbiologia da Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

2 LAPSA, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

3 Center for Global Health, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA, USA

4 Departamento de Patologia, Universidade Federal Fluminense, Niterói, RJ, Brazil

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Parasites & Vectors 2013, 6:69  doi:10.1186/1756-3305-6-69

Published: 15 March 2013

Abstract

Background

Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species.

Methods

PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment 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. 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.

Results

Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in 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 of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli.

Conclusion

These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.

Keywords:
Entamoeba; 18SrRNA; PCR; Molecular diagnostics; Multiplex assay