We determined the prevalence of Entamoeba (E.) histolytica, E. dispar and E. moshkovskii in patients with chronic diarrhoea associated with abdominal pain or discomfort mimicking irritable bowel syndrome. Stool samples were collected from 161 patients with chronic diarrhoea and from 157 healthy controls. Stool microscopy with modified trichrome stain, culture and polymerase chain reaction (PCR) for Entamoeba spp. differentiation was performed. Microscopy demonstrated Entamoeba cysts in 44% (57/129) of patients with diarrhoea compared to 29% (44/151) of controls (P=0·009). In patients with diarrhoea, PCR for E. histolytica was positive in 9% (11/129) (P=0·008), E. dispar in 19% (24/129) (P=0·117) and E. moshkovskii in 19% (24/129) (P<0·001). E. histolytica and E. moshkovskii were significantly associated with diarrhoea while E. dispar was found equally in both groups.

Since it is known that Entamoeba dispar is non-pathogenic and morphologically similar to E. histolytica, there are many targets used in PCR for differentiating these species. However, obtaining high quality DNA from fecal samples is fundamental for PCR. Most methods are laborious or use kits that make diagnosis expensive. In the present work, a new simple, fast and cheap technique of DNA extraction from fecal samples was combined with a PCR for an episomal target in order to identify E. histolytica and E. dispar in feces.

Since 1993, strains of Entamoeba histolytica sensn lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

We have recently identified 2 surface proteins in Entamoeba histolytica as intermediate subunits of galactose- and N-acetyl-D-galactosamine-inhibitable lectin (EhIgl1 and EhIgl2); these proteins both contain multiple CXXC motifs. Here, we report the molecular characterization of the corresponding proteins in Entamoeba dispar, which is neither pathogenic nor invasive. Two Igl genes encoding 1110 and 1106 amino acids (EdIgl1 and EdIgl2) were cloned from 2 strains of E. dispar. The amino acid sequence identities were 79% between EdIgl1 and EdIgl2, 75–76% between EdIgl1 and EhIgl1, and 73–74% between EdIgl2 and EhIgl2. However, all the CXXC motifs were conserved in the EdIgl proteins, suggesting that the fold conferred by this motif is important for function. Comparison of the expression level of the Igl genes by real-time RT-PCR showed 3–5 times higher expression of EdIgl1 compared to EdIgl2. Most EdIgl1 and EdIgl2 proteins were co-localized on the surface and in the cytoplasm of trophozoites, based on confocal microscopy. However, a different localization of EdIgl1 and EdIgl2 in intracellular vacuoles and a different level of phenotypic expression of the two Igls were also observed. These results demonstrate that Igls are important proteins even in non-pathogenic amoeba and that Igl1 and Igl2 may possess different functions.

The virulence of Entamoeba histolytica is governed by adhesion/colonization in the gut which is mediated by a galactose specific lectin. Two morphologically identical but distinct species i.e. pathogenic E. histolytica and non-pathogenic E. dispar, can be differentiated by distinct epitopes in the lectin. Both species bind to colonic epithelial cells, but only E. histolytica infection induces an inflammatory response and subsequent pathogenesis. Thus, comparing the responses of the intestinal cells to pathogenic and non-pathogenic lectins is a point of interest. The pathogenic lectin causes cytolysis of epithelial and immune-competent cells. Our data (both qualitative and mRNA quantitation) indicate that the epithelial cells responded to E. histolytica lectin with an increased expression of pro-inflammatory IL-2, IL-6, IL-8, MIP-1α, MCP-1, RANTES, GROα and GMCSF as compared to E. dispar lectin. The pathogenic LCM induced a significant increase in intracellular calcium concentration, proliferative response and chemotaxis of lymphocytes from ALA patients as compared to non-pathogenic LCM. High RANTES and IL-6 were induced in patients' lymphocytes by pathogenic LCM, along with their receptors CCR5 and CD126 as compared to NP-LCM. The local release of such a complex network of cytokines/chemokines could explain the histopathology of E. histolytica infection. The comparative low levels of these chemokines/pro-inflammatory cytokines and high levels of anti-inflammatory IL-10 in response to non-pathogenic E. dispar could explain the absence of an acute inflammatory response and the disease process. The cytokines and chemokines may provide a mechanism for initiation, amplification or containment of inflammation during disease state.

Infection with Entamoeba histolytica, the protozoan parasite that causes amoebic colitis and liver abscess, results in 34 million to 50 million symptomatic cases of amoebiasis (all illnesses caused by E. histolytica, including amoebic dysentery) worldwide each year, causing 40 thousand to 100 thousand deaths annually. As a result of accruing biochemical, genetic and immunological data, E. histolytica was re-defined in 1993 to recognise the existence of two morphologically identical but genetically distinct human parasites: E. histolytica, the aetiological agent of invasive intestinal and extraintestinal amoebiasis, and Entamoeba dispar, a non-pathogenic intestinal parasite. Because microscopy is unable to distinguish between these two organisms, it should no longer be relied upon to diagnose amoebiasis. Sensitive and specific molecular techniques that are able to distinguish E. histolytica from E. dispar have been developed recently; they include (1) the detection of an E. histolytica antigen using an enzyme-linked immunosorbent assay (ELISA), (2) the use of the polymerase chain reaction (PCR) to amplify amoebic DNA, and (3) the culture of stool samples followed by isoenzyme analysis. Of these three test methods, only antigen detection using ELISA can be performed rapidly and easily, making it the diagnostic test method of choice for clinical use in the developing world, where the morbidity and mortality caused by E. histolytica are greatest. However, the PCR method is a powerful tool for the genetic typing of different amoebic strains. Together these two methods should result in both improved clinical diagnosis and treatment of amoebiasis, and a greater understanding of the epidemiology of E. histolytica. Such knowledge will not only assist public health efforts to control amoebiasis, but also facilitate the careful testing of the anti-amoebic vaccines that are currently being developed.

Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl–Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species.

Virulent strains of Entamoeba histolytica have been reported to produce a mixture of phosphoglycoconjugates that share some structural features with the lipophosphoglycans (LPGs) of Leishmania. Purification of these glycoconjugates is essential to their precise structural characterization. In this study we have extracted ‘LPG-like’ molecules from various virulent E. histolytica strains and purified on the basis of charge differences, 2 apparently related glycoconjugates a ‘LPG’ and a ‘lipophosphopeptidoglycan (LPPG)’. In marked contrast to the abundance of these ‘LPG’ and ‘LPPG’ molecules in the virulent strains, avirulent E. histolytica and E. dispar strains produce either very low, or no detectable levels of LPG, and either low levels or modified forms of ‘LPPG’. Monospecific polyclonal antibodies prepared against that ‘LPG’ of the virulent strain HM-1: IMSS cl6 identified epitopes shared between both the ‘LPG’ and the ‘LPPG’ of this and other virulent strains, using Western blot analysis. Flow cytometric analysis of a range of strains using these antibodies identified a surface distribution of these molecules and confirmed a correlation between surface exposure of epitopes bound by these antibodies and parasite virulence.