Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.