title: Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia.
abstract: BACKGROUND
  Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method.
  
  
  METHODS
  In this study, a total of 334 human faecal samples were collected from different Orang Asli settlements. Faecal samples were processed by direct wet smear and formalin ethyl acetate concentration methods followed by iodine staining and was microscopically examined for Entamoeba species and other intestinal parasites. Microscopically positive samples were then subject to nested PCR and real-time PCR.
  
  
  RESULTS
  The overall prevalence of Entamoeba infection was 19.5% (65/334). SK Posh Piah recorded highest Entamoeba prevalence (63.3%) while Kampung Kemensah had the lowest prevalence (3.7%) of Entamoeba. Microscopically positive samples were then tested by real-time PCR and nested PCR for the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infection. Real-time PCR showed higher Entamoeba detection (86.2%) compared to nested PCR (80%), although the McNemar test value showed no significant difference between the two methods (p = 0.221).
  
  
  CONCLUSIONS
  This study is the first in Malaysia to report the use of real-time PCR in identifying and differentiating the three Entamoeba infections. It is also proven to be more effective compared to the conventional nested PCR molecular method.
authors.affiliation: Tropical Infectious Disease Research and Education Center (TIDREC), Department of Parasitology, Faculty of Medicine, University Malaya, 50603 Kuala Lumpur, Malaysia. lauyeeling@um.edu.my.
authors.forename: Yee Ling
authors.initials: YL
authors.lastname: Lau
authors.forename: Claudia
authors.initials: C
authors.lastname: Anthony
authors.forename: Siti Aminah
authors.initials: SA
authors.lastname: Fakhrurrazi
authors.forename: Jamaiah
authors.initials: J
authors.lastname: Ibrahim
authors.forename: Init
authors.initials: I
authors.lastname: Ithoi
authors.forename: Rohela
authors.initials: R
authors.lastname: Mahmud
mesh_terms: D000293:Adolescent; D000818:Animals; D002648:Child; D002675:Child, Preschool; D004747:Entamoeba / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D004749:Entamoebiasis / Q000175:diagnosis* / Q000469:parasitology*; D005243:Feces / Q000469:parasitology; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D007223:Infant; D008296:Malaysia; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D055815:Young Adult