Diagnosis of invasive amebiasis by enzyme-linked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies.

Saliva from subjects with amebic liver abscess (ALA), acute amebic colitis, asymptomatic infection with Entamoeba histolytica or Entamoeba dispar, and uninfected controls was tested by enzyme-linked immunosorbent assay (ELISA) for the presence of E. histolytica galactose-inhibitable lectin antigen and salivary immunoglobulin (IgG) antibodies to a recombinant cysteine-rich lectin-derived protein (LC3). Salivary lectin antigen was found in 65.8% of subjects with acute colitis, compared to 22.2% of those convalescent from ALA, 10.0% with asymptomatic E. histolytica infection, 9.8% with E. dispar infection, and 2.6% of controls (subjects from the United States and study patients with nonamebic diarrhea) (P < 0.001 for each compared to values for subjects with colitis). Salivary anti-LC3 IgG antibodies were found in 92% of ALA patients regardless of duration of illness and in 83.3% of colitis patients who were symptomatic for at least 7 days (P < 0.001 compared to other study groups). Serum anti-LC3 IgG antibodies were detected in 56.3% of subjects with acute colitis, 100% of subjects with ALA or prolonged colitis, 45% of subjects with asymptomatic E. histolytica infection, 32.3% of subjects with E. dispar infection, and 23.4% of diarrhea controls. In comparison to ELISA for serum anti-LC3 IgG antibodies, the salivary lectin antigen assay is a more sensitive and specific test for acute amebic colitis. Detection of salivary anti-LC3 IgG antibodies by ELISA is an effective means for the diagnosis of ALA and prolonged cases of amebic colitis.

Infection and immunity mediated by the carbohydrate recognition domain of the Entamoeba histolytica Gal/GalNAc lectin.

Entamoeba histolytica causes invasive amebiasis, a major parasitic disease of the developing world, whose primary symptoms are liver abscess and colitis. All strains of E. histolytica express a 260-kDa surface Gal/GalNAc lectin that is antigenically conserved and immunogenic. The lectin is required for adherence to human intestinal epithelial cells and contact-dependent killing of immune effector cells. By expression cloning, the carbohydrate recognition domain (CRD) was identified within the lectin heavy-subunit cysteine-rich region. Of interest for a hepatic parasite, the CRD had sequence identity to the receptor-binding domain of hepatocyte growth factor (HGF) and competed with HGF for binding to the c-Met HGF receptor. In an animal model of invasive disease, immunization with the CRD inhibited liver-abscess formation, yet in humans, a naturally acquired immune response against the CRD did not persist.

A comparison of mebendazole and albendazole in treating children with Trichuris trichiura infection in Durban, South Africa.

OBJECTIVE: To compare the efficacy of mebendazole 500 mg and albendazole 400 mg single-dose treatments of Trichuris trichiura infection in children in the Durban area of KwaZuluNatal, South Africa. DESIGN: A single-blind randomised trial in children with a documented moderate infection of T. trichiura. Ova were counted in stool specimens before and 10 days after treatment by the formal-ether concentration method. SETTING: Two shelters for abandoned and orphaned children in Durban. PARTICIPANTS: Ninety-six children aged between 2 and 12 years. OUTCOME MEASURES: The number of children who showed reduced T. trichiura ova counts after the treatments, and reductions in ova counts, both expressed as percentages. Statistical analysis using the Wilcoxon 2-sample test and the chi-square test. RESULTS: Eighty-two children completed the trial; 42 received mebendazole and 40 albendazole. Of the mebendazole group 85% showed a reduction in T. trichiura ova count, compared with 75% of children who received albendazole. Mebendazole treatment was associated with a median percentage reduction in ova count of 72.2%, which significantly exceeded the 44.1% reduction after albendazole (P = 0.024). CONCLUSION: The mebendazole 500 mg single-dose therapy was more efficacious than the albendazole 400 mg single-dose therapy in treating T. trichiura infection in these children.

Longitudinal study of the antibody response to recombinant Entamoeba histolytica antigens in patients with amebic liver abscess.

Serology is a critical component in the diagnosis of amebic liver abscess. However, in areas endemic for amebiasis there is a high background level of seropositivity for amebiasis (owing to previous infection with Entamoeba histolytica), which may complicate the interpretation of a positive serologic test result. Recently, we reported that serologic tests based on recombinant E. histolytica antigens might offer improved diagnosis of current invasive amebiasis because they apparently differentiated active infection from past exposure to the parasite. To confirm this finding, we have performed a longitudinal study on 20 patients with amebic liver abscess by examining their seroreactivity over time with recombinant versions of two major E. histolytica proteins, the serine rich E. histolytica protein (SREHP), and the 170-kD subunit of the galactose-specific adhesin. We found that more than 50% of the patients examined had become seronegative by one or both recombinant tests within 180 days of their diagnosis of amebic liver abscess. In the case of the recombinant SREHP-based tests, 12 patients had become seronegative 90 days after presentation. In contrast, all patients remained seropositive by a standard conventional test, an indirect hemagglutination test, at more than six months after presentation. Our study shows that patients lose seroreactivity with the recombinant SREHP or 170-kD antigen-based tests more rapidly than with a conventional serologic test; this may make them useful for the serologic diagnosis of amebiasis in endemic areas.

Entamoeba histolytica and Entamoeba dispar are distinct species; clinical, epidemiological and serological evidence.

The name of the causative organism of invasive amoebiasis, Entamoeba histolytica, was first introduced in 1903, even though this intestinal amoeba had been recognised since 1875. The marked disparity between the number of infected individuals and those with invasive amoebiasis resulted in a number of explanatory hypotheses being proposed. Although none of these were universally accepted, Brumpt's concept of two morphologically identical species gained increasing acceptance 50-60 years later when technology became available to investigate this anomaly. Sargeaunt spear-headed this drive by establishing the value of isoenzyme electrophoresis for studying the host-parasite relationship. From this foundation, incorporation of clinical, epidemiological and serological parameters to studies of the parasite resulted in the conclusion that a species complex comprising two morphologically identical amoebae was implicated with the disease. The two organisms have been named E. histolytica and Entamoeba dispar. The former is a pathogen and is responsible for invasive amoebiasis, while the latter is a gut commensal. Demonstration of the existence of this species complex has subsequently been confirmed by studies on the nucleic acids from several independent laboratories. The acceptance of E. histolytica and E. dispar as distinct species has had a major impact on our understanding of amoebiasis and its clinical management.

Differentiation of Entamoeba histolytica and Entamoeba dispar infections.

In this article, Terry jackson and Jonathan Ravdin briefly review the latest information on monoclonal antibody-based ELISAs that use antigen capture as a tool in the differential detection of human infection with Entamoeba histolytica and E. dispar. Current technology of culture and isoenzyme analysis is not widely available, is cumbersome and too time-consuming. A further potential benefit of antigen detection tests is that they can be used to monitor the efficacy of therapy; this is a shortcoming of serological tests owing to the persistence of the antibody response after successful treatment.

Protection against amebic liver abscess formation in the severe combined immunodeficient mouse by human anti-amebic antibodies.

We have used serum from patients with amebic liver abscess to investigate the role of antibody in the prevention of invasive amebiasis using the severe combined immunodeficient (SCID) mouse model of Entamoeba histolytica infection. The SCID mice were passively immunized with serum or purified antibody from patients with amebic liver abscess 24 hr prior to the direct intrahepatic challenge with 10(6) virulent E. histolytica trophozoites. This treatment reduced the mean abscess size in these animals from 24.5% to 3.5% (P < 0.0001). These results demonstrate that human anti-amebic antibodies are capable of exerting a protective effect in an animal model of amebic liver abscess formation.

Evaluation of three serological tests for the detection of antiamebic antibodies applied to sera of patients from an area endemic for amebiasis.

Two enzyme immuno assays based on a single recombinant Entamoeba histolytica antigen (P1-EIA) or soluble E. histolytica extract (SA-EIA) as well as a latex agglutination test using an E. histolytica membrane fraction (M-LA) were evaluated for its use to detect anti-amebic serum antibodies in patients from Durban, South Africa, an area endemic for amebiasis. In a previous study, all three test systems were found to be reliable in terms of sensitivity and specificity when applied to sera of European individuals. By analysing a total of 167 serum samples of patients from the Durban area, suffering from invasive amebiasis (n = 76) or miscellaneous diseases unrelated to E. histolytica infection (n = 91), the present study revealed sensitivity for the detection of anti-amebic antibodies of 97.4% for SA-EIA, 86.8% for P1-EIA and 96.1% for M-LA, respectively. Specificity was high for P1-EIA (96.7%) and M-LA (92.3%) but substantially lower for SA-EIA (62.6%). In addition, antibody responses to the recombinant P1 antigen were analysed in 16 patients with amebic liver abscess before and after anti-amebic treatment. The results indicated that most of the patients lost their specific antibody response within 7 month of follow up. Therefore, P1-EIA seems to be a valuable test for distinguishing between present and past E. histolytica infections.

A recombinant cysteine-rich section of the Entamoeba histolytica galactose-inhibitable lectin is efficacious as a subunit vaccine in the gerbil model of amebic liver abscess.

The 170-kDa subunit of the galactose-inhibitable adherence lectin of Entamoeba histolytica mediates attachment to colonic mucins and host cells. The DNA fragment encoding the 170-kDa subunit was produced by polymerase chain reaction (PCR) and divided into four sections by restriction endonucleases. The third section (designated LC3, base pairs 2273-3397) encodes a cysteine-rich fusion protein that was recognized by adherence-inhibitory anti-lectin monoclonal antibodies and serum antibodies from 95% of subjects with amebic liver abscess. Immunization of gerbils with purified recombinant LC3-encoded protein (10 micrograms) with Titermax adjuvant elicited a high-titer serum anti-LC3 IgG antibody response and protective immunity against intrahepatic challenge with 0.5 x 10(6) virulent axenic trophozoites (strain HM1:IMSS; 71% vaccine efficacy, P < .01). In summary, a recombinant cysteine-rich portion of the 170-kDa lectin subunit was highly antigenic, immunogenic, and effective as a subunit vaccine in an experimental animal model of amebic liver abscess.

Protection of gerbils from amebic liver abscess by immunization with recombinant Entamoeba histolytica 29-kilodalton antigen.

The goal of our study was to obtain a highly conserved Entamoeba histolytica recombinant antigen for study as a subunit amebiasis vaccine. We screened a Uni-Zap cDNA library of E. histolytica (strain HM1:IMSS) with human immune sera and isolated a dominant 804-bp cDNA clone. A 33-kDa fusion protein expressed from the cDNA clone was determined by monoclonal antibody binding, DNA hybridization, and nucleotide sequence to be the complete E. histolytica 29-kDa antigen. Serum antibodies to the recombinant protein were detected by enzyme-linked immunosorbent assay in 80% of subjects from Egypt and South Africa with amebic liver abscess. Similar results were found with the native 29-kDa protein. Native and recombinant 29-kDa antigens induced proliferation of lymphocytes harvested from patients with amebic liver abscess (P < 0.01 compared with controls). Intraperitoneal immunization of gerbils with the recombinant fusion protein (10 micrograms) with Titermax adjuvant elicited an antigen-specific serum immunoglobulin G antibody response and was partially protective (54%) against intrahepatic challenge with 5 x 10(5) virulent axenic trophozoites (strain HM1:IMSS). In summary, the recombinant form of the E. histolytica 29-kDa antigen demonstrated serologic specificity for amebic liver abscess, exhibited conserved T-cell epitopes, and was effective as a subunit vaccine in an experimental animal model of amebic liver abscess.

Control of an amoebiasis outbreak in the Philippi area near Cape Town.

Previous studies in Durban have shown that serological investigations, in combination with iso-enzyme electrophoresis, are invaluable for monitoring the endemicity of pathogenic strains of Entamoeba histolytica. We therefore proposed that antibody profiles could be used to detect epidemic situations. An outbreak of amoebiasis in the normally non-endemic Philippi area near Cape Town provided an opportunity for testing this hypothesis. Seven of 9 patients presenting at a district hospital with invasive amoebiasis originated from a single farm in Philippi. Iso-enzyme electrophoresis and serological investigations were used to monitor the endemicity of amoebiasis on 16 of the 49 farms in this district. In an attempt to contain disease transmission all inhabitants on farms from which patients came (including those where cyst-passers were identified) and all seropositive subjects were treated. The antibody profiles proved invaluable for confirming that the farm from which the hospitalised patients originated was the central focus of the outbreak, and also identified subjects infected with pathogenic zymodemes of E. histolytica on the adjacent 4 farms. On all 5 of these farms, 62.5-100% of seropositive subjects were strongly positive. In contrast weak to negative serological responses occurred on the remaining 11 farms. In addition the success of treatment was indicated by a notable drop in strong seropositive responses on the affected 5 farms to 11.5% within 9 months. The infection pathways implied that the pathogenic strain of E. histolytica was introduced into this non-endemic area by a foreigner from an endemic area; this suggests that the pathogenicity of E. histolytica is an immutable stable feature.

Invasive amoebiasis is associated with the development of anti-neutrophil cytoplasmic antibody.

Features of tissue damage in invasive amoebiasis, in particular polymorphonuclear neutrophil (PMN) degranulation and vasculitis, bear resemblance to that seen in Wegener's granulomatosis, the latter being associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We therefore tested sera from patients with confirmed amoebic liver abscess (ALA) for the presence of ANCA by means of an indirect fluorescent antibody test using pure neutrophils as substrate. ANCA was detected in 97.4% of amoebic sera; the pattern of staining was cytoplasmic, homogeneous, without central accentuation (C-ANCA). A proteinase 3 (PR3) ELISA demonstrated PR3 specificity in 75% of C-ANCA-positive ALA sera. Possible explanations are (i) a cross-reacting antibody to a component of Entamoeba histolytica, or (ii) an antibody to PMN components released, and possibly modified, by the action of E. histolytica on PMN. It is possible that this antibody contributes to the pathogenesis of invasive amoebiasis.

Differentiation of pathogenic Entamoeba histolytica infections from nonpathogenic infections by detection of galactose-inhibitable adherence protein antigen in sera and feces.

We determined whether epitope-specific monoclonal antibodies to the galactose-inhibitable adherence protein (GIAP) of Entamoeba histolytica could be used in an enzyme-linked immunosorbent assay (ELISA) to detect antigen in serum and feces and differentiate between nonpathogenic zymodemes and the potentially invasive pathogenic organisms that require treatment. Overall, 57% of subjects from Cairo, Egypt, with symptomatic intestinal amebiasis and 42% with asymptomatic infection possessed GIAP antigen in their sera, whereas 4% of uninfected controls or subjects with other parasitic infections possessed GIAP antigen in their sera (P < 0.001). In subjects from Durban, South Africa, only 6% of uninfected controls or those with nonpathogenic E. histolytica infection were positive for GIAP in serum, whereas 3 of 4 with asymptomatic pathogenic intestinal infection and 75% with amebic liver abscess were positive for GIAP in serum. Fifteen stool samples from patients with intestinal amebiasis were available for study; all had a positive ELISA result for fecal GIAP antigen. Epitope-specific monoclonal antibodies identified 8 of 15 subjects with fecal antigen from pathogenic strains. Seven of those eight subjects had adherence protein antigen in their sera, whereas none of seven with apparent nonpathogenic E. histolytica infection had adherence protein antigen in their sera. In summary, we were able to detect E. histolytica adherence protein antigen directly in serum and fecal samples by ELISA. The presence of amebic antigen in serum demonstrated 94% specificity for pathogenic E. histolytica infection, and amebic antigen is present during asymptomatic intestinal infection. In conjunction with antibody detection, this method should be very useful in the diagnosis and management of intestinal amebiasis.

Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based on monoclonal antibodies to the galactose-specific adhesin.

Monoclonal antibodies (MAbs) directed against pathogen-specific epitopes of the galactose adhesin of Entamoeba histolytica were used in an ELISA to detect antigen from pathogenic E. histolytica. Single stool specimens from 74 patients in Bangladesh were used. The ELISA for pathogenic E. histolytica was positive in all 12 stool specimens with pathogenic amebae subsequently cultured, in no stool specimens with nonpathogenic E. histolytica and in 2 of 40 stools with other or no intestinal parasites detected. Specificity and sensitivity of the assay for pathogenic E. histolytica were 97% and 100%, respectively. These preliminary data offer promise for an ELISA using MAbs to the galactose adhesin as a rapid and sensitive means to detect the presence of pathogenic E. histolytica infection in stool specimens.

Immunological similarity between the 170 kD amoebic adherence glycoprotein and human beta 2 integrins.

Amoebiasis is an important cause of morbidity and mortality in developing countries. Virulence of Entamoeba histolytica depends on the trophozoite's being able to bind to colonic epithelium; binding is mediated by the 170 kD subunit of a 260 kD lectin. We found, by microcytofluorometry and western blotting studies, that this 170 kD subunit protein shares an epitope with human beta 2 integrins--a subfamily of cell-surface glycoproteins expressed exclusively on leucocytes and involved in many adhesive and signalling interactions. The immunological similarity between these proteins may explain the trophozoite's ability to bind to and invade colonic epithelium.

Use of a recombinant 170-kilodalton surface antigen of Entamoeba histolytica for serodiagnosis of amebiasis and identification of immunodominant domains of the native molecule.

We expressed the gene that encodes one of the major surface antigens of Entamoeba histolytica, the 170-kDa protein (1,270 amino acids), as a glutathione S-transferase fusion protein containing amino acids 1 to 1202 (lacking the putative transmembrane and cytoplasmic regions) and as separate fusion proteins containing each of three major domains of the 170-kDa molecule. Lysates from bacteria induced to express one of these proteins were used as the target antigens in a Western blot (immunoblot) analysis to determine whether a recombinant 170-kDa antigen could serve as the basis for a serologic test used to detect invasive amebiasis and whether there are differences in humoral immunogenicity among the three major domains of the 170-kDa antigen. Among patients with invasive amebiasis from three major areas where the disease is endemic and two sites in the United States, 54 (90%) of 60 had antibodies to the recombinant 170-kDa protein. Among 37 patients from regions where the disease is endemic and 20 patients from the United States without amebic disease, 1 (2%) of 57 had antibodies to the recombinant 170-kDa protein. We found significant differences in seroreactivity to each of three major domains of the molecule among patients seropositive for the complete construct, ranging from 100% seroreactivity with the fusion protein containing the domain designated cysteine rich and 89% seropositivity with the fusion protein incorporating a portion of the region designated cysteine poor to only 9% seropositivity for the fusion protein containing the pseudorepeat domain. Our study indicates that a serologic test based on the recombinant 170-kDA antigen could serve as a highly sensitive and specific test for acute invasive amebiasis.

Asymptomatic intestinal colonization by pathogenic Entamoeba histolytica in amebic liver abscess: prevalence, response to therapy, and pathogenic potential.

Since the application of isoenzyme electrophoresis to the study of Entamoeba histolytica, the prevalence and natural history of asymptomatic intestinal colonization in patients with amebic liver abscess (ALA) has not been addressed. We prospectively evaluated this enteric phase in 50 patients with ALA, using two dosage regimens of metronidazole. The overall prevalence of asymptomatic colonization was 72% (36/50). All these isolates, without exception, proved to express pathogenic zymodemes. Despite a 100% clinical response of the hepatic lesions, failure to eradicate the organism from the bowel occurred in 20 of these 36 subjects. During longitudinal posttreatment surveillance, three carriers returned with second bouts of invasive disease: one with dysentery and two with liver abscesses. Thus, in patients with ALA, there is a high prevalence of intestinal colonization with exclusively pathogenic strains, and treatment with metronidazole frequently results in a continued carrier state. These carriers have a propensity for developing recurrent invasive disease and constitute a public health hazard.

Serodiagnosis of invasive amebiasis using a recombinant Entamoeba histolytica antigen-based ELISA.

Amebic serology remains a critical component in the diagnosis of invasive amebiasis. A recombinant serine rich-Entamoeba histolytica-protein/maltose binding protein (SREHP/MBP) fusion protein was evaluated as the target antigen in an ELISA test to detect acute invasive amebiasis. Retrospective analysis of 65 serum samples from patients with amebic liver abscess and 40 asymptomatic control patients showed the SREHP/MBP ELISA test had a sensitivity of 74% and specificity of only 55% for the diagnosis of amebic liver abscess. This study was repeated under identical conditions using a purified recombinant SREHP cleaved from the MBP component. The purified recombinant SREHP-based ELISA had a sensitivity of 79% and specificity of 87%. Our data suggest a purified recombinant SREHP-based ELISA could prove useful in the serodiagnosis of acute invasive amebiasis.