Prospective study of Entamoeba dispar infection in a cohort of mothers and their infants: relationship to serum antibody response.

The sera of cohorts of newborn infants and their mothers, characterized as cyst passers of Entamoeba with nonpathogenic zymodemes (E. dispar) and seropositive for amoebic antigens, were analyzed. Both cohorts were followed for a period of 12 months by microscopic examination of feces and determination of serum anti-amoebic antibody titers using the indirect hemagglutination assay. Control groups (noncyst passer mothers and their infants) were included and followed. To characterize antigens involved in the induction of IgG and IgA antibody responses, Western blots of serum from all participants were tested and immunoplots of the frequency of antigenic recognition were constructed. Results of clinical follow-up and microscopic examination of feces showed that during the 12-month period none of the cyst passer mothers had episodes of diarrhea attributable to E. histolytica invasion; five of 21 children of cyst passer mothers became infected during the study, five of five infected children developed serum antiamebic antibodies (titers 1:64-1:128); none of the cohort of children from cyst passer mothers had diarrhea due to E. histolytica. Western blot analysis showed that there are antigenic fractions that induce serum antibodies of the IgG and IgA classes against E. dispar very early in the host-parasite relationship. Our results suggest that mechanisms of antibody induction different from intestinal invasion may be operating in amebic infection. Intestinal absorption of antigen, systemic reflection of secretory antibody response, and priming of newborns by maternal anti-idiotypic antibody transfer are discussed.

Study of human Entamoeba histolytica infection by zymodeme, indirect hemagglutination and electroimmunotransfer blot assays.

Human amebiasis is a parasitic infection which involves asymptomatic as well as intestinal and extraintestinal symptomatic stages in individuals harbouring Entamoeba histolytica. Several factors have been proposed to explain the variability in the outcome of the disease. Among these are differences in virulence of E. histolytica strains and methods such as zymodeme analysis have been used to differentiate invasive from non invasive strains of this parasite (Sargeaunt and Williams, 1978). In order to establish a possible correlation between zymodeme analysis and serological data in human cases of amebiasis, a cross-sectional epidemiological study was carried out in the community area of Cadereyta, State of Queretaro, Mexico. In this study, fecal and serum samples from individuals were also tested by Indirect Hemagglutination assay (IHA) to determine antibody titre and by a standardized Electroimmunotransfer blot assay (EITB) for recognition of E. histolytica specific antigens. Zymodemes were determined in a total of 81 samples. Of these, 27 had a pathogen zymodeme (PZ) while 54 had a non pathogen zymodeme (NPZ). Values obtained by IHA varied from negative to titres up to 1:256 in serum samples tested. Reactivity to E. histolytica antigens determined by EITB was observed in all sera. Patterns of antigen recognition were complex and showed reactivity to several parasite molecules. Among these, components with M. Wt. of 165, 119, and doublets of 98-100 and 50-52 Kd were more frequently recognized by antibodies present in the sera tested. In general, antibody titres detected by IHA did not showed a direct correlation with zymodeme analysis. Samples with PZ or NPZ had similar variable levels of reactivity to E. histolytica antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of an immunoblot methodology for the detection of relevant Entamoeba histolytica antigens by antibodies induced in human amebiasis.

The complexity of the clinical spectrum in human amebiasis and the high variability in laboratory methods used to detect Entamoeba histolytica infections have impeded the collection and evaluation of reliable epidemiological data. Thus, more sensitive, specific and standardized methods are needed in order to accurately identify infections with this parasite. An important step in the development of serological diagnostic methods is the identification and isolation of specific parasite antigens which are immunogenic in the host. In this work, we have standardized an electroimmunotransfer blot technique to characterize E. histolytica antigens recognized by antibodies present during human amebic infections. An important aspect was an investigation of technical variations in the preparation of cell lysates including the use of different protease inhibitors and solubilizing agents. The highest yield of protein was achieved by homogenization of trophozoites in the presence of 10 mM p-hydroxymercuribenzoate (pHMB) as a protease inhibitor and by lysis using Triton X-100 and a mixture of protease inhibitors. Recovery of degraded vs non-degraded proteins in the cell extracts was evaluated by gradient polyacrylamide gel electrophoresis. Both quantitative and qualitative differences were noted between the different methods of preparing soluble cell extracts. A more complete set of antigenic components was obtained by homogenization and use of pHMB. Thus parasite extracts prepared by this method were selected for protein transfer. In this, the optimal protein concentration was of 120 micrograms of protein per cm of gel width and efficient transfer of proteins to nitrocellulose sheets was achieved at 100 V for 2 hrs and at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)