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.

Comparison of antigen-capture ELISA to stool-culture methods for the detection of asymptomatic Entamoeba species infection in Kafer Daoud, Egypt.

We performed a prospective field study in the village of Kafer Daoud in Menofia, Egypt to compare the fecal culture method with enzyme linked immuno assay (ELISA) for detection of 170 kDa lectin antigen in feces for diagnosis of asymptomatic Entamoeba histolytica and Entamoeba dispar infection. All subjects with E. histolytica or E. dispar infection detected by culture also had positive ELISA for amebic antigen in their feces and an additional 57 Entameoba infections missed by culture were detected by ELISA (P < 0.001 compared to culture). The presence of fecal anti-lectin IgA antibodies and serum anti-LC3 (recombinant cysteine-rich lectin protein) IgG antibodies were positive predictors for E. histolytica infection (P < 0.03). Of interest, infection with Trichomonas hominis but not Blastocystis hominis was positively associated with E. histolytica infection (P < 0.05). In conclusion, ELISA for detection of fecal lectin antigen is a more sensitive method than fecal culture for detecting asymptomatic E. histolytica infection.

The cysteine-rich region of the Entamoeba histolytica adherence lectin (170-kilodalton subunit) is sufficient for high-affinity Gal/GalNAc-specific binding in vitro.

Adherence of Entamoeba histolytica trophozoites to colonic mucin, epithelium, and other target cells is mediated by the amebic Gal/GalNAc lectin. We constructed in vitro expression vectors containing full-length (residues 1 to 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin, hgl2. In vitro transcription followed by translation using a nuclease-treated rabbit reticulocyte lysate system was carried out. Immunoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipitation with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which recognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hgl2 enhanced immunoreactivity (P < 0.05) with the conformationally dependent MAb 3F4. Binding of PDI-refolded full-length (P < 0.001) and cysteine-rich (P = 0.005) Hgl2 to CHO cells was galactose dependent and competitively inhibited by native hololectin (50% inhibitory concentration of 39.6 ng/ml). The cysteine-poor region (1 to 353) did not bind CHO cells. Both full-length (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjugate GalNAc(19)BSA in a GalNAc-specific manner. The smaller cysteine-rich fragment (480 to 900) also exhibited GalNAc-specific binding but to a lesser extent (P < 0.05) than residues 1 to 1280 and 356 to 1143. Neither the cysteine-poor fragment (1 to 480), luciferase (protein control), nor control translation reactions (without hgl2 lectin mRNA) bound GalNAc(19)BSA. Binding to GalNAc(19)BSA was shown to be dependent on the concentration of GalNAc(19)BSA coated in each well or (35)S-lectin added (K(D) = 0.85 +/- 0.37 pM). Binding was competitively inhibited by the terminal GalNAc-containing glycoprotein asialofetuin (P < 0.005). Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subunit contains one or more carbohydrate-binding domains.

Serum IgM antibody response to the galactose-inhibitable adherence lectin of Entameoba histolytica.

An ELISA for detection of serum IgM antibodies to the galactose-inhibitable adherence lectin of Entamoeba histolytica revealed that 2.8% of uninfected controls, 0.0% of controls infected with other parasites, 13.4% of asymptomatic amebic infections, 55% of colitis patients, and 77% of amebic liver abscess patients from Cairo, Egypt and Durban, South Africa had serum anti-lectin IgM antibodies. Of acute amebic colitis patients with symptoms for less than one week, only 6% possessed serum IgG anti-lectin antibodies, yet 45% had serum IgM antibodies to the amebic lectin. This compares with 65% of sera in acute colitis patients positive for lectin antigen as determined by ELISA with anti-lectin monoclonal antibodies. In conclusion, an ELISA for serum anti-lectin IgM antibodies appears to have greater clinical utility in the setting of acute amebic colitis than an ELISA for anti-lectin IgG antibodies, but is no more sensitive than an ELISA for detection of lectin antigen in sera.

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.

Humoral and mucosal IgA antibody response to a recombinant 52-kDa cysteine-rich portion of the Entamoeba histolytica galactose-inhibitable lectin correlates with detection of native 170-kDa lectin antigen in serum of patients with amebic colitis.

Humoral and mucosal IgA responses to a recombinant cysteine-rich portion (designated LC3) of the Entamoeba histolytica galactose-inhibitable lectin's 170-kDa subunit were determined in patients with amebic colitis. All patients had 170-kDa amebic antigen in serum, compared with 1 of 50 cyst passers and 1 of 31 controls (P < .01). Seven days after treatment, serum and fecal 170-kDa antigen became undetectable in 12 of the 13 patients (P < .001). Serum anti-LC3 IgA was found in 83.8% of colitis patients, compared with 2% of controls and 12% of asymptomatic cyst passers (P < .001). Salivary and fecal anti-LC3 IgA levels were higher in patients than in cyst passers (P < .001). In conclusion, in amebic colitis, development of humoral and mucosal IgA responses to the recombinant LC3-encoded protein correlates with detection of amebic 170-kDa antigen in serum and feces.

Oral immunization with a recombinant cysteine-rich section of the Entamoeba histolytica galactose-inhibitable lectin elicits an intestinal secretory immunoglobulin A response that has in vitro adherence inhibition activity.

The LC3-encoded 52-kDa recombinant protein includes amino acids 758 to 1134 of the 170-kDa subunit of the galactose-inhibitable lectin. Oral immunization of BALB/c mice with the LC3-encoded protein and cholera holotoxin induced an intestinal secretory immunoglobulin A (IgA) response (P < 0.01 compared with the control). There was a negative correlation (P = 0.001) between intestinal anti-LC3 IgA and serum IgA and IgG antibody responses. Intestinal secretions from immunized mice completely inhibited the galactose-specific adherence of axenic trophozoites ot Chinese hamster ovary cells (P < 0.01).

Development of amebicidal cell-mediated immunity in gerbils (Meriones unguiculatus) immunized with the galactose-inhibitable adherence lectin of Entamoeba histolytica.

The galactose-inhibitable adherence lectin of Entamoeba histolytica is a protective antigen in the gerbil model of amebic liver abscess. To further understand the mechanisms of vaccine efficacy, we studied the cell-mediated immune response to the lectin in gerbils. Splenocytes harvested from immunized gerbils demonstrated in vitro proliferation and production of interleukin-2 and gamma-interferon in response to purified adherence lectin (P < 0.05 for each compared to control splenocytes). Splenocytes from immunized gerbils developed direct amebicidal activity (P = 0.014) following in vitro stimulation with the adherence lectin. Splenocytes harvested from immunized gerbils following intrahepatic challenge with viable E. histolytica adherence lectin. Splenocytes harvested from immunized gerbils following intrahepatic challenge with viable E. histolytica trophozoites demonstrated proliferative and amebicidal responses (P < 0.05). In conclusion, immunization with the E. histolytica galactose-inhibitable adherence lectin elicits an amebicidal cell-mediated response that is enhanced by parasite challenge.

Interdisciplinary integration for quality improvement: the Cleveland Veterans Affairs Medical Center Firm System.

BACKGROUND: Many of the characteristics of Firm Systems lend themselves to the application of principles of continuous quality improvement (CQI). A Firm System is defined as two or more parallel practices organized on the principle of continuity of relationships between patients and an interdisciplinary group of health care professionals and trainees. Firm Systems are organized around the care of the patient or customer and emphasize access, continuity, and quality of care. CASE STUDY: The Firm System was implemented at the Cleveland Veterans Affairs Medical Center (VAMC) not as a CQI initiative per se, but as an effort to coordinate the processes involved in the delivery of patient care. The primary goals of this implementation were to improve the quality of patient care, medical education, and health care research. The main strategy to deal with problems caused by uncoordinated care were to move from a departmental approach to an integrated interdisciplinary approach. This approach represented a paradigm shift within the organization that extended to planning, documentation, and the general work environment. Most important, the institution had leaders who were committed to the Firm System and willing to authorize resources to ensure its success. CONCLUSION: VA hospitals are ideal settings for Firm Systems because they provide longitudinal, comprehensive care with a centralized, prepaid payment mechanism, and they have well-developed information systems that allow the random assignment of patients to Firms. Recommendations to others interested in implementing Firm Systems include creation of a written plan that can gain general support; identification of resources needed for successful implementation; remembering that the patient is the most important customer, as well as that complex systems have many customers; monitoring of performance; and the importance of randomizing patients and providers.

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.

Immunization of rats with the 260-kilodalton Entamoeba histolytica galactose-inhibitable lectin elicits an intestinal secretory immunoglobulin A response that has in vitro adherence-inhibitory activity.

The 260-kDa galactose-inhibitable lectin of Entamoeba histolytica mediates binding of amebic trophozoites to purified colonic mucins and intestinal epithelial cells. Parenteral immunization of Lewis rats with immuno-affinity-purified lectin with Freund's adjuvant and then intra-Peyer's patch inoculation of lectin with cholera toxin B subunit as adjuvant elicited a significant anti-lectin secretory immunoglobulin A response in the bowel lumen. Purified intestinal secretory immunoglobulin A from three or four immunized animals studied possessed inhibitory activity (P < 0.02) in an in vitro assay of trophozoite galactose-specific adherence to Chinese hamster ovary cells.

Secretory immunoglobulin A antibodies to the galactose-inhibitable adherence protein in the saliva of patients with amebic liver disease.

Monoclonal antibodies directed against the 260-kD galactose-inhibitable adherence protein (GIAP) of Entamoeba histolytica inhibit binding of amebic trophozoites to purified colonic mucins, suggesting that anti-GIAP secretory immunoglobulin A (sIgA) may have a role in host defense in invasive amebiasis. We determined by enzyme-linked immunosorbent assay (ELISA) whether a salivary anti-GIAP sIgA response was present in patients from the Republic of South Africa with invasive E. histolytica infection. In 13 patients with amebic liver abscess (ALA), salivary anti-GIAP sIgA was significantly higher (mean +/- SD optical density [OD] = 0.448 +/- 0.258) than that determined for seven South African adult patients hospitalized with nonamebic illness (0.084 +/- 0.072; P = 0.002), seven healthy South African Adults (0.194 +/- 0.119: P = 0.025), and seven healthy adults from Charlottesville, Virginia (0.036 +/- 0.023; P = 0.004). Of the patients with ALA, nine had acute disease, and four had been cured of amebiasis 2-8 months previously. There was no significant difference between these two groups in the anti-GIAP sIgA levels. All ALA patients had a high titer serum anti-amebic antibody response, and there was no direct correlation between the level of anti-GIAP salivary IgA and anti-GIAP serum antibodies (R = 0.187). These findings demonstrate that the E. histolytica GIAP is a mucosal antigen in naturally occurring invasive E. histolytica infection.

Role of mucosal secretory immunity in the development of an amebiasis vaccine.

Invasive colonic infection by the enteric protozoan Entamoeba histolytica elicits a mucosal anti-amebic IgA antibody response. The E. histolytica galactose-inhibitable adherence protein (GIAP) mediates parasite binding to colonic mucins and epithelial cells. Anti-GIAP secretory IgA antibodies are found during invasive amebiasis and can be elicited by immunization of rats with the native protein. Entamoeba histolytica contains potent IgA degradative activities due to its cysteine-specific proteases. Research is needed to further understand the role of secretory antibodies in natural or vaccine-induced immunity to E. histolytica.