title: LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Instituto de Microbiologia da Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Helena Lúcia Carneiro
authors.initials: HL
authors.lastname: Santos
authors.forename: Kakali
authors.initials: K
authors.lastname: Bandyopadhyay
authors.forename: Rebecca
authors.initials: R
authors.lastname: Bandea
authors.forename: Regina Helena Saramago
authors.initials: RH
authors.lastname: Peralta
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Peralta
authors.forename: Alexandre Januário
authors.initials: AJ
authors.lastname: Da Silva
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics; D004747:Entamoeba / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004749:Entamoebiasis / Q000175:diagnosis* / Q000469:parasitology; D005243:Feces / Q000469:parasitology*; D018732:Forensic Anthropology; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D010274:Parasitology / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics
chemical_list: D016054:DNA, Protozoan; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S