Pathogenic and non-pathogenic zymodemes of Entamoeba histolytica in a rural area of Mexico. Concordance with serology.

OBJECTIVES: a) to describe the frequency of pathogenic and nonpathogenic zymodemes of E. histolytica in asymptomatic carriers in rural Mexico, b) to identify the isoenzymatic pattern of the zymodemes and c) to measure the concordance of pathogenic zymodemes and positive serology. DESIGN: Descriptive cross-sectional survey. Reference population: rural communities of the mexican highlands. STUDY UNITS: 2048 individuals from 341 families of 5 rural communities. METHODS: From the local census of each community, 70 to 100 families were selected by random sampling. All members of each family were studied. After obtaining the consent the following activities were done: a) filling of a questionnaire about housing and sanitary condition and structure of the family; age, sex, literacy and digestive symptoms and illness during the previous 4 weeks, in each individual; b) a blood sample for serology (CEIP and IHA) and c) a stool sample for coproparasitoscopic analysis (CPS) and zymodemes identification, in each individual. Statistical evaluation was done by kappa index and receiving operating curves (ROC). MAIN RESULTS: there were 122/1730 (7.1%) CPS studies positive to E histolytica cysts, 100/1730 (5.8%) zymodemes isolated and 137/1886 (7.3%) positive serologic studies. Of the 100 zymodemes 30 had a pathogenic pattern, three had a mixture (pathogenic/nonpathogenic) and 67 had a non-pathogenic patterns. There were two (XVII, XVIII) patterns described for the first time in Mexico. Concordance between pathogenic zymodemes and positive serology (IHA greater than 1:128) was very low, Kappa = 0.05. ROC curves for IHA in pathogenic and non-pathogenic zymodemes showed little relationship between positive serology and pathogenic pattern. CONCLUSIONS: Although the frequency of positive serology, coproparasitoscopic studies and zymodemes identification was similar, the concordance between serology and the coprologic studies was very low. This disagrees with other reports, and deserves further investigation using methodologic standards in design.

Efficacy of zymodemes of E. histolytica technique in an epidemiologic study and report of new zymodemes in Mexico.

OBJECTIVES: To determine the efficacy of zymodemes of E. histolytica technique in an epidemiologic study. To report the zymodemes for the first time described in Mexico. To report the main difficulties in the correct classification of zymodemes in this study. To describe the zymodemes identified in two evaluations done in 100 healthy individuals. DESIGN: A cross-sectional survey and a cohort of healthy persons. METHODS: We analyze the results of 1730 Robinson cultures performed in a cross-sectional survey done in the Municipality of Cadereyta, Queretaro, Mexico from October 1986 to August 1987. From positive cultures properly growth a Iysate was done and preserved in liquid nitrogen until the electrophoretic determination of 4 isoenzymes was done: EC 5.3.1.9. glucophosphate isomerase (GPI); EC 1.1.1.40 L-malate: NADP oxidoreductase (oxaloacetate decarboxylating) (ME); Ec 2.7.5.1 phosphoglucomutase (PGM); and EC 2.7.1.1 hexokinase (HK). RESULTS: Of 1730 cultures 289 (16.7%) developed trophozoites, in 100 (34.6%) was possible to make lysate and zymodeme. There were 67 ZNP, 3 mixtures (ZP and ZNP) and 30 ZP. There were two patterns for the first time described in Mexico (XVII and XVIII) and 30 strains had a pattern different from the 20 already described by Sargeaunt. Results observed in 100 individuals measured twice. [table: see text] There were 9 subjects in which zymodemes were isolated twice: at the first time 5 PZ and 4 NPZ, at the second time 1 PZ and 8 NPZ. At first time all 9 had negative serology and the second time one carrier of NPZ became positive. COMMENTS: In our study one tirth of Robinson cultures positive to trophozoites reached lysate. Efficacy of zymodemes and CPS is similar. We report for the first time in Mexico two non-pathogenic zymodemes (XVII and XVIII). We also report 30 zymodemes non-classified according to the standard proposed by Sargeaunt, one explanation for this feature is the possibility of zymodemes variability or new patterns. In endemic populations zymodeme pattern varies with time in asymptomatic carriers.

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)

[In vitro sensitivity of Entamoeba histolytica to metronidazole]. / Sensibilidad in vitro de Entamoeba histolytica a Metronidazol.

The chemotherapy of amebiasis includes different substances with intra and extraintestinal action. Nowadays, metronidazole is the election drug by its high efficacy and low toxicity. Some therapeutic failures with metronidazole in patients with invader amebiasis and some reports of resistance to it, of certain strains of Trichomonas vaginalis and species of Bacteroides have caused an increased interest to do new in vitro evaluation of this compound and to search of new antiamebic drugs. The object of this work was to standardize with our conditions a microtechnique to evaluate the sensibility to metronidazole, experimented by trophozoites of three amebic strains of different virulence (HK9:NIH, HM1:IMSS, HM3:IMSS) and to compare the effect exerted by the drug on them. We modified the technique proposed by Cedeño and Krogstad in 1983. First we read the results in a half of the time employed by them (24h) and after, we fixed the trophozoites with 0.25% glutaraldehyde before of make the counts of them. We compared the number of cells in microcultures of 3.2 x 10(4) trophozoites incubated during 24h with metronidazole (from 0.008 to 1 micrograms/ml) with the obtained in control microcultures. Each one of the strains showed to have a characteristic sensibility to the drug (p less than 0.001), which was directly proportional to the concentration. The middle effective dose of HK9 and HM1 strains was 0.3 microgram/ml and of 1 microgram/ml for HM3 strain. This technique seems to be reproducible and could be useful to quantify the in vitro activity of antiamebic agents.

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)