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.     

Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica

A monoclonal antibody, EH3015, prevents in vitro adherence of Entamoeba histolytica trophozoites to mammalian cells and inhibits amebic liver abscess formation in hamsters. By immunoaffinity chromatography with the monoclonal antibody, purified E. histolytica antigens with molecular masses of 150 and 170 kDa under non-reduced conditions were obtained. Hamsters were immunized with these antigens (group I) or with fractions further purified by polyacrylamide gel electrophoresis (group II). Pooled immune sera from the two groups inhibited in vitro amebic adherence to Chinese hamster ovary cells by 98% at 1:10 dilutions. The immunized hamsters were challenged by the intrahepatic injection of E. histolytica trophozoites. Complete protection from abscess formation was observed in 38% of hamsters in group I and 67% in group II, whereas all control hamsters inoculated only with adjuvant developed amebic liver abscesses. In the immunized hamsters, the abscesses in the two groups were significantly smaller than in the controls. These results demonstrate that the E. histolytica antigens are possible vaccine candidates for amebiasis. Received: 10 August 2000 / Accepted: 5 September 2000     

Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests

A noninvasive diagnostic test for amebic liver abscess is needed, because amebic and bacterial abscesses appear identical on ultrasound or computer tomography and because it is rarely possible to identify Entamoeba histolytica in stool specimens from patients with amebic liver abscess. Here we report a method of detection in serum of circulating E. histolytica Gal/GalNAc lectin to diagnose amebic liver abscess, which was used in patients from Dhaka, Bangladesh. The TechLab E. histolytica II test (which differentiates the true pathogen E. histolytica from Entamoeba dispar) detected Gal/GalNAc lectin in the sera of 22 of 23 (96%) amebic liver abscess patients tested prior to treatment with the antiamebic drug metronidazole and 0 of 70 (0%) controls. After 1 week of treatment with metronidazole, 9 of 11 (82%) patients became serum lectin antigen negative. The sensitivity of the E. histolytica II antigen detection test for intestinal infection was also evaluated. Antigen detection identified E. histolytica infection in 50 samples from 1, 164 asymptomatic preschool children aged 2 to 5 years, including 16 of 16 (100%) culture-positive specimens. PCR analysis of stool specimens was used to confirm that most antigen-positive but culture-negative specimens were true-positive: PCR identified parasite DNA in 27 of 34 (79%) of the antigen-positive, culture-negative stool specimens. Antigen detection was a more sensitive test for infection than antilectin antibodies, which were detected in only 76 of 98 (78%) amebic liver abscess patients and in 26 of 50 (52%) patients with intestinal infection. We conclude that the TechLab E. histolytica II kit is a sensitive means to diagnose hepatic and intestinal amebiasis prior to the institution of metronidazole treatment.     

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.     

Sensitivity and specificity of a new commercial enzyme-linked immunoassay kit for detecting Entamoeba histolyticaIgG antibodies in serum samples

The study presented here was conducted to evaluate the performance of the newly available RIDASCREEN Set (R-Biopharm AG, Darmstadt, Germany) for the detection of immunoglobulin G antibodies against Entamoeba histolytica. The sensitivity and specificity of this new enzyme-linked immunosorbent assay were evaluated using a panel of sera from 239 individuals. The assay was positive for 43 of 44 patients with invasive amebiasis, including all 18 patients with amebic liver abscess, while it was negative for 190 of 195 adult controls who were either healthy individuals or patients with other parasitic diseases. The kit was found to be highly specific (97.4%) and sensitive (97.7%) for detecting antibodies against E. histolytica in humans. Although antibody titers in patients with amebic liver abscess tend to be higher on average than in patients with invasive amebiasis, it is not possible to distinguish the two forms solely based on the results of this commercial test.     

Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites.     

Amebic lung abscess with coexisting lung adenocarcinoma: a unusual case of amebiasis

Amebic lung abscess with concurrent lung cancer, but without either a liver abscess or amebic colitis, is extremely uncommon. Here, we report a 70-year-old man presenting with pulmonary amebiasis and coexisting lung adenocarcinoma. During his first-time hospitalization, the diagnosis of lung amebiasis was confirmed by morphological observation and PCR in formalin-fixed and paraffin-embedded sediments of pleural effusion. Almost four months later, the patient was readmitted to hospital for similar complaints. On readmission, lung adenocarcinoma was diagnosed by liquid-based sputum cytology and thought to be delayed because coexisting amebic lung abscess. This case demonstrated that sediments of pleural effusion may be used for further pathological examination after routine cytology has shown negative results. At the same time, we concluded that lung cancer may easily go undetected in the patients with pulmonary amebiasis and repetitive evaluation by cytology and imaging follow-up are useful to find potential cancer.     

Serologic reactivity to purified recombinant and native 29-kilodalton peripheral membrane protein of pathogenic Entamoeba histolytica.

The 29-kDa peripheral membrane protein of Entamoeba histolytica has recently been demonstrated to have epitopes on pathogenic clinical isolates which were not detected by monoclonal antibodies on nonpathogenic isolates. To analyze the serological response to this protein, we tested 93 serum specimens (from 33 patients with amebic liver abscess, 7 patients with colitis, 2 patients with ameboma, 18 individuals harboring a nonpathogenic zymodeme strain, 10 healthy Mexican migrant workers, and 23 healthy controls) by enzyme-linked immunosorbent assay (ELISA) using immunoaffinity-purified native or recombinant protein. When tested by ELISA with the native antigen, 79% (26 of 33) of the serum specimens from patients with amebic liver abscess, 4 of 9 serum specimens from symptomatic patients with colitis or ameboma, and serum from one migrant worker were positive. None of the 18 subjects harboring a nonpathogenic strain or 23 control individuals were seropositive to the native antigen (sensitivity, 71%; specificity, 98%). Of 30 serum specimens from patients with amebic liver abscess tested with recombinant antigen, 27 were seropositive (90%). In addition, six patients with colitis or ameboma and two individuals who harbored a nonpathogenic strain were seropositive to the recombinant antigen. One healthy Mexican migrant worker tested positive by both ELISAs (sensitivity, 87%; specificity, 94%). Immunoblotting of 51 serum specimens to sodium dodecyl sulfate-denatured native 29-kDa protein was less sensitive (65%) than ELISA in detecting serum antibodies to the antigen. These results suggest a similar antibody response to native and recombinant antigens (r = 0.86) and support the potential utility of a quantitative assay with defined recombinant antigen for the serodiagnosis of invasive amebiasis in nonendemic areas in conjunction with other diagnostic tools.     

Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess.

Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis.     

Progress towards development of a vaccine for amebiasis.

The application of molecular biologic techniques over the past decade has seen a tremendous growth in our knowledge of the biology of Entamoeba histolytica, the causative agent of amebic dysentery and amebic liver abscess. This approach has also led to the identification and structural characterization of three amebic antigens, the serine-rich Entamoeba histolytica protein (SREHP), the 170-kDa subunit of the Gal/GalNAc binding lectin, and the 29-kDa cysteine-rich protein, which all show promise as recombinant antigen-based vaccines to prevent amebiasis. In recent studies, an immunogenic dodecapeptide derived from the SREHP molecule has been genetically fused to the B subunit of cholera toxin, to create a recombinant protein capable of inducing both antiamebic and anti-cholera toxin antibodies when administered by the oral route. Continued progress in this area will bring us closer to the goal of a cost-effective oral combination "enteric pathogen" vaccine, capable of inducing protective mucosal immune responses to several clinically important enteric diseases, including amebiasis.     

Host-pathogen interaction in amebiasis and progress in vaccine development

Entamoeba histolytica, the causative organism of invasive intestinal and extraintestinal amebiasis, infects approximately 50 million people each year, causing an estimated 40 to 100 thousand deaths annually. Because amebae only infect humans and some higher non-human primates, an anti-amebic vaccine could theoretically eradicate the organism. Uncontrolled epidemiologic studies indicate that acquired immunity to amebic infection probably occurs and that such a vaccine might be feasible. Application of molecular biologic techniques has led to rapid progress towards understanding howEntamoeba histolytica causes disease, and to the identification of several amebic proteins associated with virulence. These proteins are now being evaluated as potential vaccine components. Parenteral and oral vaccine preparations containing recombinant amebic proteins have been effective in preventing disease in a gerbil model of amebic liver abscess. Although systemic and mucosal cellular and humoral immunity both appear to play a role in protection againstEntamoeba histolytica, the relative importance of each in the human immune response remains unknown. No animal model of intestinal amebiasis currently exists, moreover, so it has been impossible to evaluate protection against colonization and colitis. Further investigation of the fundamental mechanisms by whichEntamoeba histolytica causes disease and of the human immune response to amebic infection is necessary to assess the true feasibility of an anti-amebic vaccine.     

Amebic lung abscess: an unexpected diagnosis

Pleuropulmonary amebiasis is a very rare complication of amebiasis infection and direct pulmonary involvement is exceptional. The clinical diagnosis is difficult without any intestinal or extraintestinal manifestations. A percutaneous drainage is necessary to aspirate pus. We report herein the case of a 56-year-old man presenting with an amebic lung abscess in his right lower lung and pleural effusion. The diagnosis was suspected by our laboratory from a serological assay (antiamebic antibodies reaching 320 by IHA) and established from a direct examination of the pus in which rare trophozoites of Entamoeba were detected. We pointed out the importance of the communication between the clinician and the biologist. Protozoan infection should be suspected in pleuropulmonary infection when antibiotics failed even in France. This patient left endemic area a long time ago and the way of his amebiasis contamination is unknown: recurrence of amebic infection is rare and prevalence in industrialized countries reaches only 1%. Several hypothesis are exposed.     

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1–7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1–2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host. Received: 26 September 1999 / Accepted: 24 November 1999     

Laboratory diagnosis of amebiasis

The detection of Entamoeba histolytica, the causative agent of amebiasis, is an important goal of the clinical microbiology laboratory. To assess the scope of E. histolytica infection, it is necessary to utilize accurate diagnostic tools. As more is discovered about the molecular and cell biology of E. histolytica, there is great potential for further understanding the pathogenesis of amebiasis. Molecular biology-based diagnosis may become the technique of choice in the future because establishment of these protozoa in culture is still not a routine clinical laboratory process. In all cases, combination of serologic tests with detection of the parasite (by antigen detection or PCR) offers the best approach to diagnosis, while PCR techniques remain impractical in many developing country settings. The detection of amebic markers in serum in patients with amebic colitis and liver abscess appears promising but is still only a research tool. On the other hand, stool antigen detection tests offer a practical, sensitive, and specific way for the clinical laboratory to detect intestinal E. histolytica. All the current tests suffer from the fact that the antigens detected are denatured by fixation of the stool specimen, limiting testing to fresh or frozen samples.     

Role of the Entamoeba histolytica cysteine proteinase in amebic liver abscess formation in severe combined immunodeficient mice.

Evidence from in vitro studies suggest that the Entamoeba histolytica cysteine proteinase plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. We used affinity-purified antibodies against a recombinant E. histolytica cysteine proteinase to demonstrate that the proteinase is present extracellularly in amebic liver abscesses in mice with severe combined immunodeficiency (SCID mice). Treatment of E. histolytica trophozoites with specific cysteine proteinase inhibitor E-64 blocked or greatly reduced liver abscess formation at 48 h in SCID mice. Our study suggests an important role for a functional cysteine proteinase in amebic liver abscess formation.     

HLA characterization in adult asymptomatic cyst passers of Entamoeba histolytica/E. dispar

The present work aimed at studying the possible association of HLA antigens with Entamoeba histolytica/E. dispar asymptomatic infection in a Mexican mestizo population. A case-control design was selected for evaluation of the role of genetic markers in parasite infection. For this purpose the HLA-A, HLA-B, and HLA-DR profiles of a population of asymptomatic E. histolytica/E. dispar adult cyst passers (cases) and a corresponding nonparasitized adult group (controls) followed for 12 months were identified. Entamoeba species were identified through zymodeme patterns and/or amplification of species-specific DNA sequences. A healthy, nonparasitized group of individuals was included as a control. Our results show that apparently, no specific HLA marker is associated with the asymptomatic cyst passers' condition. These findings have to be added to previous results in which, in contrast to a demonstrated association between HLA-DR3 and amebic liver abscess in Mexican mestizo adults and infants, no significant association with amebic rectocolitis was found. Received: 18 March 1999 / Accepted: 16 April 1999     

Entamoeba histolytica: Antibody responses and lymphoreticular changes in gerbils (Meriones unguiculatus) in response to experimental liver abscess and amebic extract injection

Antibody responses and histological changes in hepatic lymph nodes and spleen of gerbils (Meriones unguiculatus) during the course of experimental hepatic amebiasis (5–60 days), or in those injected with extracts ofEntamoeba histolytica, are described. Lymph node and spleen responses in infected animals paralleled the proliferation of the amebic liver abscess. However, spleen follicle responses were similar in animals that received low or high doses of the amebic extract and differed histologically from those with amebic liver abscess. Liver abscesses, up to 30 days postinfection (pi), doubled in weight between 10 and 15 and between 20 and 30 days pi. Early changes (10 days pi) in the lymphoreticular tissues were characterized by increased size and weight of the organs, hyperplastic follicles, and blastogenesis in the T-dependent areas. At 20 and 30 days pi, the size of spleen follicles increased and there was depletion of lymphocytes from the periarterial area (PAA), as well as gross extension of the red pulp, accompanied by extramedullary erythropoiesis and megakaryocytosis. The paracortical areas (PCA) of lymph nodes were depleted of lymphocytes and histocytosis throughout the organ, and there was intense plasma cell activity in the medulla. At 60 days pi, lymphocyte repopulation was noted in the PCA and PAA; germinal centers were depleted of blast cells and the spleen red pulp had contracted. Antiamebic antibody titers were low throughout the infection. Changes in the cellularity of the lymphoid organs are discussed in relation to the proliferation of the amebic liver abscesses in infected animals and in those which were injected with the amebic extract.     

Evaluation of recombinant fragments of Entamoeba histolytica Gal/GalNAc lectin intermediate subunit for serodiagnosis of amebiasis

We have recently identified a 150-kDa surface antigen of Entamoeba histolytica as an intermediate subunit (Igl) of galactose- and N-acetyl-D-galactosamine-inhibitable lectin, which is a cysteine-rich protein consisting of 1,101 amino acids (aa) and containing multiple CXXC motifs in amino acid sequences. In the present study, full-length Igl except for the signal sequences (aa 14 to 1088) and three fragments of Igl-the N-terminal part (aa 14 to 382), the middle part (aa 294 to 753), and the C-terminal part (aa 603 to 1088)-were prepared in Escherichia coli, and the reactivity of these recombinant proteins with sera from patients with amebiasis was examined by means of enzyme-linked immunosorbent assay (ELISA). Sera from 57 symptomatic patients with amebic liver abscess or amebic colitis, sera from 15 asymptomatic cyst passers, sera from 40 individuals with other protozoan infections, and sera from 50 healthy controls were used. The sensitivity and specificity of the recombinant full-length Igl in the ELISA were 90 and 94%, respectively. When three fragments were used as antigens in the ELISA, the sensitivities were 56% in the N terminus, 92% in the middle part, and 97% in the C terminus. The specificities of the three antigens were 96% in the N terminus and 99% in both the middle and C-terminal fragments. These results demonstrate that Igl is well recognized in not only symptomatic but also asymptomatic patients with E. histolytica infection and that the carboxyl terminus of Igl is an especially useful antigen for the serodiagnosis of amebiasis.     

Human amebic liver abscess: expression of intercellular adhesion molecules 1 and 2 and of von Willebrand factor in endothelial cells

Liver invasion by amebas with production of amebic liver abscess (ALA) is the most common extraintestinal lesion produced by the protozoan parasite Entamoeba histolytica. This hepatic damage is characterized by the presence of extensive tissue necrosis. However, little is known about the parasite and host factors involved in the process of tissue damage. During the early establishment of amebas in the liver parenchyma as well as during the extension of the tissue necrosis, parasites interact with sinusoidal endothelial cells. As a consequence of ameba-endothelial cell interactions, the latter can be activated and express proinflammatory factors that could be related to tissue destruction. We studied by immunohistochemistry the localization of antigenic molecules of E. histolytica trophozoites and of molecules such as intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and von willebrand factor in activated endothelial cells of human ALA, which could be related to the pathophysiological mechanisms of tissue destruction in amebiasis. Received: 11 November 1996 / Accepted: 18 December 1996     

Oral vaccination with recombinant Yersinia enterocolitica expressing hybrid type III proteins protects gerbils from amebic liver abscess

Protection against invasive amebiasis was achieved in the gerbil model for amebic liver abscess by oral immunization with live attenuated Yersinia enterocolitica expressing the Entamoeba histolytica galactose-inhibitable lectin that has been fused to the Yersinia outer protein E (YopE). Protection was dependent on the presence of the YopE translocation domain but was independent from the antibody response to the ameba lectin.