Indirect haemagglutination (IHA) test in the serodiagnosis of amoebiasis

Among 72 patients clinically suspected of Entamoeba histolytica (E. histolytica) infections, 39 positive cases (54%) were detected serologically by the indirect hemagglutination (IHA) test. Parasitologically, microscopic examination of three consecutive stool specimens from all these patients indicated positivity for E. histolytica cysts and or trophozoites in 10 of the patients with IHA antibody titers greater than or equal to 1:128, which is of clinical significance. Another 2 patients were parasitologically positive but showed low IHA antibody titres (1:32-1:64); follow up indicated response to treatment with metronidazole. The highest serological positivity (100%) were detected in patients with liver abscess, all were clinically proven cases of extra-intestinal amoebiasis. IHA antibody levels of clinical significance were seen in all four patients with chronic dysentery with parasitological evidence of E. histolytica in their stools. In a group of patients with abdominal pain nine positives were detected serologically, four of which were positively diagnosed concurrently by parasitology; the remaining five patient's sera showed high IHA antibody titres with absence of cysts or trophozoites in stools, indicative possibly of persistence of antibodies from past infection. The serologic determination of E. histolytica IHA antibodies in a control group consisting of normal healthy school children and adults of both sexes without any clinical evidence of amoebiasis showed the absence of any positive titres of clinical significance; low titres (1:32-1:64) were detected in 5.2% of 232 sera tested. Parasitological examination of three consecutive stool specimens from all individuals in the control group showed the presence of cysts of E. histolytica in just two among 232 tested (0.9%).     

Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia

It is generally accepted that a majority of individuals infected by Entamoeba histolytica do not develop symptomatic disease. However, the parasite and the host factors contributing to the development of the disease, remain undetermined. It is also unclear why certain individuals develop extra-intestinal amebiasis without exhibiting apparent intestinal symptoms. An outbreak of amebic liver abscess in Tbilisi, Georgia in 1998-1999 suggested that the causative E. histolytica strain had an unusual propensity for extra-intestinal spread. To correlate the genetic differences with pathogenic potential of the parasite, we have examined the SREHP gene polymorphisms among Georgian E. histolytica isolates. Comparison of polymorphic patterns revealed the presence of several different genotypes of E. histolytica, thus preventing an association of a single genotype with hepatic disease, but supporting the previous finding of extensive genetic diversity among E. histolytica isolates from the same geographic origin.     

Tissue invasion by Entamoeba histolytica: evidence of genetic selection and/or DNA reorganization events in organ tropism

Entamoeba histolytica infection may have various clinical manifestations. Nine out of ten E. histolytica infections remain asymptomatic, while the remainder become invasive and cause disease. The most common form of invasive infection is amebic diarrhea and colitis, whereas the most common extra-intestinal disease is amebic liver abscess. The underlying reasons for the different outcomes are unclear, but a recent study has shown that the parasite genotype is a contributor. To investigate this link further we have examined the genotypes of E. histolytica in stool- and liver abscess-derived samples from the same patients. Analysis of all 18 paired samples (16 from Bangladesh, one from the United States of America, and one from Italy) revealed that the intestinal and liver abscess amebae are genetically distinct. The results suggest either that E. histolytica subpopulations in the same infection show varying organ tropism, or that a DNA reorganization event takes place prior to or during metastasis from intestine to liver.     

Partial characterization of a 36-kDa antigen of Entamoeba histolytica and its recognition by sera from patients with amoebiasis

A 36-kDa antigen of axenically grown pathogenic Entamoeba histolytica (HM1-IMSS) was eluted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved crude amoebic extract antigens. The immunoreactivity of this partially purified 36-kDa antigen with monoclonal antibody (MoAb) 3D(10) altered significantly (P<0.01) after heat and trypsin treatment but remained unaltered after treatment with sodium metaperiodate (P0.5), thereby indicating the protein nature of the epitope recognized by MoAb 3D(10). The epitope was found to be localized on the surface as well as in the cytoplasm of the E. histolytica trophozoites with the majority of it in the cytoplasm. In addition, this epitope was also found to be present on the cyst form of the parasite. The 36-kDa molecule was recognized by the sera from 29 (85%) of the 34 patients with amoebic liver abscess and five (83%) of the six patients with amoebic colitis. No serum samples from asymptomatic cyst passers, from patients with non-amoebic hepatic or intestinal disorders and apparently healthy subjects had antibodies that reacted with this 36-kDa molecule. The immune responses in man to this 36-kDa amoebic molecule indicate a potential specific role for this molecule in invasive amoebiasis.     

Detection of Entamoeba histolytica antigen in stool samples in Mersin, Turkey

Entameoba histolytica, 1 of the 2 Entamoeba species with similar morphology that infect humans, causes invasive intestinal and extraintestinal diseases, whereas Entamoeba dispar is found commensally and is noninvasive. Because of their morphologic similarity, E. histolytica and E. dispar cannot be differentiated microscopically. The antigens of E. histolytica and E. dispar, however, may be detected by the ELISA method. Previous studies have found that the detection of antigens in the stool is as sensitive and specific as cultures and isoenzyme analyses. Stool samples from 272 patients with diarrhea in the province of Mersin, Turkey, were examined for the presence of Entamoeba species microscopically and for Entamoeba (E. histolytica/E. dispar) antigens using the ELISA method. An E. histolytica-specific ELISA test was used to examine 29 E. histolytica/E. disparpositive samples. Twenty-four (8.82%) of the samples tested positive for E. histolytica/E. dispar by trichrome staining, and 29 (10.6%) of the samples tested positive for E. histolytica/E. dispar by the Entamoeba screening test. Entamoeba histolytica was positive in 21 (7.72%) and E. dispar positive in 8 (2.94%) samples. The detection of true E. histolytica infection is possible with the use of E. histolytica-specific antigen ELISA tests. Thus, real cases of amoebiasis can be detected and treated, and overtreatment of the patients with E. dispar, which is the nonpathogenic species, will be prevented.     

Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica.

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.     

Development of simple and stable ELISA for detection of copro antigen in intestinal amoebiasis

A simple, sensitive and stable ELISA (enzyme linked immunosorbent assay) was developed using rabbit antibody to fractionated Entamoeba histolytica antigen for the detection of copro antigen in the faeces of individuals with intestinal amoebiasis. In this test none of the healthy individuals, all trophozoite positive, 40% cyst passers and 6% individuals living in endemic area showed the presence of copro antigen. ELISA using polyclonal rabbit antibody could detect 1-5 trophozoites/well and 20-50 ng protein per well of NIH-200 strain of E. histolytica and the sensitivity of the test was comparable with that using monoclonal antibody. Cross reaction was observed only with E. invadens when faeces having other parasites were screened. The reagents of ELISA were stabilized and found to be stable for more than 6 months when stored at 4 degrees C.     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

Epidemiology of clinically relevant Entamoeba spp. (E. histolytica/dispar/moshkovskii/bangladeshi): A cross sectional study from North India

BackgroundEntamoeba infections have major impact on millions of the people worldwide. Entamoeba histolytica has long been accepted as the only pathogenic species. However, recent reports of other Entamoeba spp. in symptomatic cases have raised questions on their pathogenicity.Methodology/Principal findingsTotal 474 stool samples and 125 liver aspirates from patients with intestinal and extra intestinal manifestations and from community were included. Sewage samples from the hospital and the city were also included. Microscopic examination and molecular detection were performed to detect presence of E. histolytica/ dispar/ moshkovskii/ bangladeshi. The associated demographic and socioeconomic factors were statistically analyzed with the presence of Entamoeba. Microscopy detected Entamoeba spp. in 5.4% stool and 6.4% liver aspirate samples. Through nested multiplex PCR, prevalence of Entamoeba spp. in intestinal and extra-intestinal cases was 6.6% (20/301) and 86.4% (108/125) respectively and in asymptomatic population was 10.5% (13/123). Sewage samples did not show presence of any Entamoeba spp. Uneducated subjects, low economic conditions, untreated drinking water, consumption of raw vegetables and habit of not washing hands before meals were significantly associated with presence of Entamoeba spp.ConclusionsE. histolytica still remains the only Entamoeba spp. in invasive extra intestinal infections. E. dispar was detected in both asymptomatic and symptomatic intestinal infections. Routine identification of Entamoeba spp. should incorporate PCR based detection methods.     

Investigation of the prevalence of amoebiasis in Izmir province and determination of Entamoeba spp. using PCR and enzyme immunoassay

Amoebiasis is a common and life-threatening disease. The discrimination of the pathogenic Entamoeba histolytica from the non-pathogenic Entamoeba dispar could be done by advanced methods such as enzyme immunoassay (EIA) and PCR. The aim of this study was to investigate the prevalence of amoebiasis in Izmir province, and differentiate the Entamoeba species by PCR and EIA. Stool samples of 2,047 individuals were examined by direct microscopy, formalin ethyl acetate concentration, trichrome staining and culture, and those found to be positive for E. histolytica/dispar by any of these methods were further analyzed by PCR and EIA for species identification. Fifty-nine of 2,047 (2.9%) stool samples were found to be positive for E. histolytica/dispar with microscopy and/or culture. Among these positive samples, E. histolytica was detected in 14 (23.7%) and 5 (8.5%) samples with PCR and antigen-specific ELISA (EIA), respectively. E. dispar was diagnosed in 31 (52.5%) and 52 (88.1%) of 59 samples with species-specific PCR and EIA, respectively. Risk factors related to infection with Entamoeba spp. and other intestinal parasites included living in shanty houses (p < 0.01), a history of recent immigration to Izmir (p < 0.01), having no social security (p < 0.05) and living with a crowded family (p < 0.01). The results demonstrated the significance of amoebiasis as a public health problem among people with low socio-economic status in Izmir province.     

Autoimmunity in amoebiasis.

Amoebic liver abscess (ALA) and symptomatic intestinal amoebiasis cases were assessed by indirect haemagglutination assay for auto-reactive IgG and IgA class of antibodies in response to healthy human serum IgG and IgA. The present results indicated the presence of autoreactive IgG and IgA class of antibodies in ALA and intestinal amoebiasis respectively.     

Serodiagnosis of amoebiasis using a recombinant protein fragment of the 29 kDa surface antigen of Entamoeba histolytica

To develop an improved serodiagnostic test for amoebiasis, we performed a detailed analysis of the immunodominant epitopes of the 29 kDa surface antigen and evaluated its sensitivity and specificity. Enzyme-linked immunosorbent assay (ELISA) based on the fragment containing the immunodominant epitope was evaluated further and compared with full-length recombinant 29 kDa protein. Specificity and sensitivity of the two ELISAs were assessed using 55 human sera of parasitic protozoa infection cases (25 amoebiasis, 20 giardiasis and 10 toxoplasmosis sera) and 10 healthy control sera. The immunodominant epitope of the 29 kDa antigen is localised only in the N-terminus 14–54 amino acid residues. The sensitivities of the two ELISAs were very high, 92 and 96%, respectively. The specificity of the fragment was 100%, whereas the specificity of the full-length 29 kDa protein was 86.6%. These results indicate that the fragment containing the immunodominant epitope of the 29 kDa protein can be used to accurately serodiagnose amoebiasis without cross-reactivity from other parasites.     

Pathophysiology of amoebiasis

Few organisms are more aptly named than Entamoeba histolytica, an intestinal protozoan parasite that can lyse and destroy human tissue. Within the past four years, new models of E. histolytica infection have begun to illuminate how amoebic trophozoites cause intestinal disease and liver abscess, and have expanded our understanding of the remarkable killing ability of this parasite. Here, I summarize recent work on the interactions between E. histolytica and human intestine, and between E. histolytica and hepatocytes, and discuss what these studies tell us about the role of inflammation and programmed cell death in the pathogenesis of amoebiasis.     

Differentiation of Entamoeba histolytica and Entamoeba dispar by PCR: a preliminary study in Izmir, Turkey

The causative agent of amoebiasis is currently attributed to two distinct species (E. histolytica and E. dispar). The aim of this study was to differentiate these species by PCR in stool samples. Isolated genomic DNA was amplified by PCR and band products of 101 bp (E. dispar) were obtained. All seven stool samples were found to be E. dispar, not E. histolytica. Our results demonstrated the significance of E. histolytica/dispar differentiation in the diagnosis of amoebiasis. This study is preliminary to our current research project entitled "Investigation of the prevalence of amoebiasis and Entamoeba species in Izmir and its hinterland".     

Virulence of entamoeba histolytica strains from Bombay (India) to rats

The results of intracaecal inoculation experiments on young albino rats with different strains of E. histolytica isolated in cultures from patients with different varieties of clinical amoebiasis, such as acute amoebic dysentery, amoebic liver abscess and chronic amoebic colitis, as well as from asymptomatic infections, have been presented and discussed. Caecal scoring was performed with regard to the condition of the wall of the caecum and the contents of its lumen. Successful infection with virulent amoebae was associated with the presence of considerable ulceration of the caecum, whereas, in infections with avirulent amoebae, the changes were apparently only slight or absent. The virulence and pathogenicity were considerably different for clinical case and symptomless carrier strains of E. histolytica. In the former group, high caecal scores and pathogenicity indices with ulceration of the caecum were noted, whilst in the latter, the amoebae were either virulent or avirulent. Results similar to those for the former group were obtained with two carrier strains only.     

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS).

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.     

A rapid and sensitive screening kit for the detection of anti-Toxocara larval ES antibodies

We developed a new screening kit for the detection of anti-Toxocara larval excretory-secretory antibodies. The test can be performed within 3 min for one sample and does not require a high-priced supplemental instrument. Moreover, results are easily and directly observed. Using this test kit, 22 sera taken from healthy subjects were negative for anti-Toxocara larval excretory-secretory antibodies at the serum dilution of 1:20. Of 14 proven cases of parasitic infections other than toxocariasis, one (gnathostomiasis) showed a positive result, but the others were negative. In serologically diagnosed toxocariasis, the test kit showed good correlation with ELISA, immunoblot and double gel diffusion tests. We designated this test kit as ToxocaraCHEK.     

Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern India

ABSTRACT: BACKGROUND: Amebic dysentery is caused by the protozoan parasite Entamoeba histolytica and the ingestion of quadrinucleate cyst of E. histolytica from fecally contaminated food or water initiates infection. Excystation occurs in the lumen of small intestine, where motile and potentially invasive trophozoites germinate from cysts. The ability of trophozoites to interact and digest gut bacteria is apparently important for multiplication of the parasite and its pathogenicity; however the contribution of resident bacterial flora is not well understood. We quantified the population of Bacteroides, Bifidobacterium, Ruminococcus, Lactobacillus, Clostridium leptum subgroup, Clostridium coccoides subgroup, Eubacterium, Campylobacter, Methanobrevibacter smithii and Sulphur reducing bacteria using genus specific primers in healthy (N = 22) vs amebic patients (E. histolytica positive, N = 17) stool samples by Real-time PCR. RESULTS: Absolute quantification of Bacteroides (p = .001), Closrtridium coccoides subgroup (p = 0.002), Clostridium leptum subgroup (p = 0.0001), Lactobacillus (p = 0.037), Campylobacter (p = 0.0014) and Eubacterium (p = 0.038) show significant drop in their population however, significant increase in Bifdobacterium (p = 0.009) was observed where as the population of Ruminococcus (p = 0.33) remained unaltered in healthy vs amebic patients (E. histolytica positive). We also report high prevalence of nimE gene in stool samples of both healthy volunteers and amebic patients. No significant decrease in nimE gene copy no. was observed before and after the treatment with antiamebic drug. CONCLUSIONS: Our results show significant alteration in predominant gut bacteria in E. histolytica infected individuals. The frequent episodes of intestinal amoebic dysentery thus result in depletion of few predominant genera in gut that may lead to poor digestion and absorption of food in intestine. It further disturbs the homeostasis between gut epithelium and bacterial flora. The decrease in beneficial bacterial population gives way to dysbiosis of gut bacteria which may contribute to final outcome of the disease. Increase in the copy number of nimE gene harboring bacteria in our population reflects possible decrease in the availability of metronidazole drug during treatment of amoebiasis.     

Molecular basis of defence against oxidative stress in Entamoeba histolytica and Giardia lamblia

Gastrointestinal protozoa, viz. Entamoeba histolytica and Giardia, are able to survive in a microaerobic environment. The role of parasitic factors, particularly cysteine, cysteine-rich proteins, superoxide dismutase, and certain alternative mechanisms, has been described in the defence against oxidative stress. The role of the host-derived factors, particularly the phagocytosis of bacteria or host erythrocytes (intact or their enzymatic/non enzymatic components), in the detoxification of reactive oxygen metabolites in E. histolytica may provide novel approaches for the chemotherapy of invasive amoebiasis.