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.

The abundant polyadenylated transcript 2 DNA sequence of the pathogenic protozoan parasite Entamoeba histolytica represents a nonautonomous non-long-terminal-repeat retrotransposon-like element which is absent in the closely related nonpathogenic species Entamoeba dispar.

While comparing gene expression in the pathogenic organism Entamoeba histolytica and the closely related but nonpathogenic species Entamoeba dispar, we discovered that the E. histolytica abundant polyadenylated transcript 2 (ehapt2) and corresponding genomic copies are absent in E. dispar. Although polyadenylated, ehapt2 does not contain any overt open reading frame. Southern blot and sequence analyses revealed that about 500 copies of ehapt2 genomic elements were present in each cell and that the copies were distributed throughout the ameba genome. The various ehapt2 elements are regularly located in the vicinity of protein-encoding genes, downstream of pyrimidine-rich sequence stretches (40 to 125 bp; CT content, 79.2 to 85.5%), and are flanked by duplicated target sites of variable length. Target site duplications were obviously generated during integration of ehapt2 into the E. histolytica genome as one copy of the flanking repeat and the complete ehapt2 element are specifically absent in orthologous E. dispar genomic sequences. ehapt2 shares 3' sequences with EhRLE, a recently identified non-long-terminal-repeat (non-LTR) retrotransposon-like element of E. histolytica, which contains a conceptual open reading frame for reverse transcriptase. Thus, ehapt2 has all of the properties of nonautonomous non-LTR retrotransposons. A comparison of various E. histolytica isolates suggested that transposition of ehapt2 takes place at a very low frequency as the genomic localization of ehapt2 elements was found to be well conserved. A mobile element such as ehapt2 could be a suitable mechanism to explain the infrequent and late transition of E. histolytica from a harmless gut commensal to an invasive pathogen.

Epidemiology of amebiasis in a region of high incidence of amebic liver abscess in central Vietnam.

The recent identification of Entamoeba dispar as a separate species, which is nonpathogenic for humans but morphologically indistinguishable from Entamoeba histolytica, has prompted the World Health Organization to recommend reinforced efforts for reassessment of the epidemiology of amebiasis and, in particular, of E. histolytica. In this regard, the distribution of amebic liver abscess (ALA) cases were analyzed in the province of Thua Thien Hué (TT Hué) in central Vietnam, a region known for its high incidence of invasive amebiasis. In addition, in a particular area of Hué City, a parasitologic and seroepidemiologic survey was performed to identify possible risk factors for transmission of E. histolytica. Based on the analysis of hospital charts from April 1990 to April 1998, 2,031 cases of ALA were identified, indicating an ALA incidence of at least 21 per 100,000 inhabitants per year. Incidence varied substantially between the various districts of TT Hué and directly correlated with population density. The risk for ALA was significantly higher in summer and was age and sex dependent because 95% of the cases were adults, of which more than 80% were males. There was no clustering of cases within households and recurrent cases of ALA occured more frequently than predicted in the study population. Despite the higher incidence of ALA in males, the parasitologic and seroepidemiologic survey revealed a significant higher infection rate for intestinal protozoon parasites, including E. histolytica in females. Besides level of education and access to a toilet or tapwater, use of river water was identified as an important risk factor for E. histolytica infection.