Identification of Entamoeba histolytica by real-time PCR method

The intestinal protozoan parasite Entamoeba histolytica is endemic in large parts of the world and is considered responsible for millions of cases of dysentery and liver abscess each year. Prior to invasive disease, E. histolytica may reside in the human gut for months or even years as symptomatic infection. The World Health Organization has recommended that Entamoeba histolytica `should be specifically identified and if present should be treated??. Classic microscopic examination of the parasite E. histolytica in stool can not differentiate it from the nonpathogenic but identically appearing parasites Entamoeba dispar and Entamoeba moshkovskii.A number of assays have been developed during recent years, such as protein and DNA detection systems, which are able to distinguish E. histolytica from E. dispar. Amplification of ameba DNA fragments by PCR has been proven to constitute a sensitivite and specific method to detect E. histolytica or E. dispar from feces. Recently developed closed-tube, real-time PCR methods can circumvent these problems. These methods allow specific detection of the amplicon by binding to one or two fluorescence-labeled the time needed to obtain results. In addition, the closed reaction tube minimizes the potential for cross-contamination, and the assay output is numerical rather than qualitative, allowing appropriate diagnostic statistics to be applied.In this study, stool samples were tested by microscopy, antigen detection assay, traditional PCR and RT-PCR methods for identification of Entamoeba histolytica and Entamoeba dispar. A total of 74 stool 13 (17,6%) of which were positive by the E. Histolytica-specific antigen detection test, while the other 61 (82,4%) stool were negative by the antigen detection test. DNA was extracted from the stool by the QIAGEN method and the small-subunit rRNA gene of E. histolytica and then amplified by traditional and real-time PCR. A total of 74 stool, 19 were positive by real-time PCR assay and 16 were positive by the traditional PCR test. Compared to the real time PCR assay, the antigen detection test was found 79% sensitive and 96% specific. When the traditional PCR test results were compared to the real-time PCR assay, the sensitivity of traditional PCR was 84,2% and the specificity was 100%. In conclusion, real-time PCR being the more sensitive and specific the other methods.KEW WORDS: Entamoeba histolytica, Coproantigen ELISA, PCR, Real-Time PCR