Amoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.

The protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis – an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis.

To address the molecular diversity and occurrence of pathogenic species of the genus Entamoeba spp. in wild non-human primates (NHP) we conducted molecular-phylogenetic analyses on Entamoeba from wild chimpanzees living in the Issa Valley, Tanzania. We compared the sensitivity of molecular [using a genus-specific polymerase chain reaction (PCR)] and coproscopic detection (merthiolate-iodine-formaldehyde concentration) of Entamoeba spp. We identified Entamoeba spp. in 72 chimpanzee fecal samples (79%) subjected to species-specific PCRs for six Entamoeba species/groups (Entamoeba histolytica, Entamoeba nuttalli, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli and Entamoeba polecki ST2). We recorded three Entamoeba species: E. coli (47%), E. dispar (16%), Entamoeba hartmanni (51%). Coproscopically, we could only distinguish the cysts of complex E. histolytica/dispar/moshkovskii/nuttalli and E. coli. Molecular prevalence of entamoebas was higher than the prevalence based on the coproscopic examination. Our molecular phylogenies showed that sequences of E. dispar and E. coli from Issa chimpanzees are closely related to sequences from humans and other NHP from GenBank. The results showed that wild chimpanzees harbour Entamoeba species similar to those occurring in humans; however, no pathogenic species were detected. Molecular-phylogenetic methods are critical to improve diagnostics of entamoebas in wild NHP and for determining an accurate prevalence of Entamoeba species.

The world health organization currently recognizes diarrhoeal diseases as a significant cause of death in children globally. Protozoan parasites such as Giardia and Entamoeba that thrive in the oxygen-deprived environment of the human gut are common etiological agents of diarrhoea. In the urogenital tract of humans, the anaerobic protozoan parasite Trichomonas vaginalis is notorious as the most common non-viral, sexually transmitted pathogen. Even with high medical impact, our understanding of anaerobic parasite physiology is scarce and as a result, treatment choices are limited. Fluorescent proteins (FPs) are invaluable tools as genetically encoded protein tags for advancing knowledge of cellular function. These FP tags emit fluorescent colours and once attached to a protein of interest, allow tracking of parasite proteins in the dynamic cellular space. Application of green FPs-like FPs in anaerobic protozoans is hindered by their oxygen dependency. In this review, we examine aspects of anaerobic parasite biology that clash with physio-chemical properties of FPs and limit their use as live-parasite protein tags. We expose novel FPs, such as miniSOG that do not require oxygen for signal production. The potential use of novel FPs has the opportunity to leverage the anaerobe parasitologist toolkit to that of aerobe parasitologist.

This study developed a fast and high throughput dot-blot technique to evaluate the presence of Entamoeba in stool samples (n = 643) followed by a PCR-based method to validate and differentiate the two species E. histolytica and E. dispar. The prevalence rate of the parasite has been detected in a cross-sectional study carried out in the population of the Eastern and Northern parts of India. Of the various demographic features, prevalence was highest in the monsoon season (P = 0·017), in the <15 years age group (P = 0·015). In HIV-positive individuals, the prevalence rate was significantly high (P = 0·008) in patients with a CD4 cell count <200 as well as in patients without antiretroviral therapy (ART) (P = 0·011). Our analysis further confirmed that risk factors such as toilet facilities, living conditions, hygienic practices, drinking water source, occupation and level of education are important predictors as they were found to contribute significantly in the prevalence of the parasite.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0·27 μm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0·74 and 0·22 mm, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

Parasitic diseases are endemic in parts of the tropics, but there is no convincing evidence that their prevalence or incidence is increasing due to the HIV epidemic. Available scientific data on parasitic infections in patients with the Acquired Immunodeficiency Syndrome (AIDS) suggests a predominance of Pneumocystis carinii, Toxoplasma gondii and Cryptosporidium spp. For reasons which are unclear, parasitic infections such as Plasmodium falciparum, Strongyloides stercoralis and Entamoeba histolytica, where cell-mediated immune responses are also thought to be significant, do not appear to be opportunists of importance. It is being increasingly recognized that chemotherapy for parasitic diseases has a host-dependent component, although scientific data on this subject remain scanty. The management of opportunistic parasitic infections in patients infected with HIV is dogged by failures and relapses, aptly illustrating the notion of the relationship between chemotherapy and the immune response. This review discusses the immunity and chemotherapy of opportunistic parasite infections in patients infected with the Human Immunodeficiency Virus (HIV).

In this study, the proteome of axenically grown Entamoeba histolytica parasites was explored by two-dimensional gel electrophoresis (2-DE), employing a practical and effective procedure for the solubilization of E. histolytica proteins. Approximately 900 protein species in the pH range between 4 and 7 were detected by Coomassie Blue staining. Ninety-five spots were excised, trypsinated and subjected to mass spectrometry. The resultant data from peptide mass fingerprints were compared with those available in the E. histolytica genome and the (non-redundant) National Center for Biotechnology Information (NCBI) databases for the identification and categorization of proteins. Sixty-three of the proteins identified were predicted to relate to the cytoskeleton, surface, glycolysis, RNA/DNA metabolism, the ubiquitin-proteasome system, vesicular trafficking and signal transduction. The present study demonstrates, for the first time, that corresponding genes are indeed expressed in E. histolytica and provides a foundation for further proteomic studies of this parasite.

The protozoan parasite Entamoeba histolytica is the causative agent of amoebic dysentery. It is prevalent in developing countries that cannot prevent its fecal–oral spread and ranks second in worldwide causes of morbidity by parasitic infection. Improvements in sanitation would help curb disease spread. However, a lack of significant progress in this area has resulted in the need for a better understanding of the molecular and cellular biology of pathogenesis in order to design novel methods of disease treatment and prevention. Recent insight into the cellular mechanisms regulating virulence of E. histolytica has indicated that processes such as endocytosis, secretion, host cell adhesion and encystation play major roles in the infectious process. This review focuses on components of the molecular machinery that govern these cellular processes and their role in virulence, and discusses how an understanding of this might reveal opportunities to interfere with E. histolytica infection.

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.