DEVELOPMENT OF NESTED PCR FOR IDENTIFICATION OF ENTAMOEBA COLI IN HUMAN FECAL SAMPLES

Abstract

Microscopic examination is the gold standard for detecting Entamoeba spp in stool specimens, although it is often not adequately sensitive or specific. Here, a nested PCR assay was developed for the detection of E. coli in fecal samples with a limit of detection of 1 pg/20 mg of sample and good specificity (no cross-amplification of other intestinal Entamoeba spp or protozoa). The nested PCR assay employs two set of primers, one set for amplifying Entamoeba genus DNA and the other specific for E. coli small subunit rDNA. Applying this technique to stored fecal samples (n = 55) from school children in western Thailand, 33% of the samples were positive for E. coli compared to 29% by microscopic examination, the latter method also showing 29% mixed infections, all among Entamoeba sp-positive samples. Sensitivity and specificity of the in-house nested PCR assay compared to microscopy was 100% and 95% respectively, with positive and negative predictive value of 0.9 and 1.0 respectively. Kappa analysis (κ = 0.9) indicated an excellent agreement of nested PCR assay and microscopy. This in-house nested PCR assay is suitable for both laboratory screening and epidemiology of E. coli infection in human population.