A single-round PCR assay was developed for detection and differential diagnosis of the three Entamoeba species found in humans, Entamoeba moshkovskii, Entamoeba histolytica, and Entamoeba dispar, that are morphologically identical as both cysts and trophozoites. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. PCR generates a 166-bp product with E. histolytica DNA, a 752-bp product with E. dispar DNA, and a 580-bp product with E. moshkovskii DNA. Thirty clinical specimens were examined, and the species present were successfully detected and differentiated using this assay. It was possible to detect as little as 10 pg of E. moshkovskii and E. histolytica DNA, while for E. dispar the sensitivity was about 20 pg of DNA. Testing with DNA from different pathogens, including bacteria and other protozoa, confirmed the high specificity of the assay. We propose the use of this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three morphologically indistinguishable Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.Infections with Entamoeba spp. can result in either a harmless colonization of the intestine or an invasion of the colon wall and damage of other host tissues such as the liver, lung, and brain (amoebiasis). In most cases, a clinical diagnosis of amoebiasis can be confirmed and usually depends on the visualization of parasites by light microscopy of a wet smear or stained specimens. This procedure is inexpensive and simple, but it has several limitations, such as being incapable of distinguishing between the cysts and trophozoites of the diseasecausing species Entamoeba histolytica, the nonpathogenic species Entamoeba dispar, and the amphizoic amoeba Entamoeba moshkovskii, which occasionally infects humans. Multiple samples often have to be requested and examined, and the presence of cysts of different species of Entamoeba, Iodamoeba, or Endolimax can make the diagnosis even more difficult (4, 5). Furthermore, with the reports of sporadic cases of human infection with E. moshkovskii (3, 6) and the recent finding of a high prevalence and association of E. moshkovskii with E. histolytica and E. dispar in young children in Bangladesh (2), the differentiation of the three species in clinical samples by other means becomes of great importance both for diagnosis and for epidemiological studies.

Abstract. Multiplex real -time polymerase chain reaction (PCR) was developed for differential detection of Entamoeba histolytica , Entamoeba dispar , and Entamoeba moshkovskii . Specific primers were designed for all three species, and then differentiation of E. histolytica and E. dispar was achieved simultaneously using a hybridization probe and melting curve analysis, whereas E. moshkovskii was detected with a separate probe under the same condition. This assay detected as little as 0.2 pg of E. histolytica DNA and 2 pg each for E. dispar and E. moshkovskii DNA. Thirty-five clinical samples suspected to be E. histolytica infection by microscopy were tested. The results showed 32 positive samples; four samples were E . histolytica and 28 samples were E. dispar . Interestingly, one E. dispar positive sample showed a mixed infection with E. moshkovskii . This is the first report of E. moshkovskii infection from Thailand and this assay is currently the most rapid and sensitive method to differentiate these human amoebas.

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