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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 89(1): 47-51, jan./mar. 1994
Mara Rosenzvit, Sergio Angel*, Luis Alvarez*, Viviana Mellare*, Jorge Blanco*, Viviana Pszenny*, Juan Carlos Garberi*/+ Instituto de Investigaciones Medicas "Alfredo Lanari", Departamento de Sustancias Vasoactivas, Donato Alvarez 3150, Buenos Aires, Argentina *Centro de Investigaciones Medicas Albert Einstein (CIMAE), Luis Viale 2831 (1416), Buenos Aires, Argentina This work was supported by the grant of the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and by Fundacion Roemmers. +Corresponding author Received 17 May 1993, Accepted 19 November 1993
Key words: toxoplasmosis serodiagnosis - microELISA - epidemiology Toxoplasmosis is one of the most frequent and worldwide dispersed infections in humans (Frenkel 1971). Most of the time the absence of clear symptoms requires a serological assay for a correct diagnosis. The severe consequences that the acute stage of the disease produces on fetuses and neonates (Remington & Desmonts 1983) indicate a preventive screening test in pregnant women. Indirect hemagglutination (IHA) is the most utilized system in Argentina and other countries. Indirect immunofluorescence (IIF) is another system widely used. The first (IHA) is very simple but often leads to false positive results, consumes a great quantity of antigen and often recent infections are not detected (Tomasi et al. 1986). IIF is sensitive and rapid, but immunofluorescent microscopy and "trained eyes" are necessary, resulting in important difficulties for the assay automatization. Enzyme linked immunosorbent assay (ELISA) is the other alternative system for toxoplasmosis serodiagnosis (Ambroise-Thomas & Desgeorges 1980, Tomasi et al. 1986). It is very simple and has sensitivity levels similar to IIF. The aim of our work was to design a simple and economic test for immunodiagnosis of toxoplasmosis useful in clinical laboratories and that could be applied in epidemiological studies. Micro-SIA was found to be more sensitive and reliable than IIF and ELISA due to the virtual absence of background (Conway de Macario et al. 1983). On the other hand, it is very simple, rapid and requires small amounts of sera, antisera and reagents. In view of this advantages, we have designed a slide micro-immunoenzymatic assay (micro-SIA), with nonparticulate antigens for immunodiagnosis of toxoplasmosis. MATERIALS AND METHODS Antigen - Tachyzoites of T. gondii, RH strain, harvested from mouse peritoneal exudate, 72 hr after infection, were resuspended in phosphate-buffered saline (PBS), pH 7.4 with the addition of EDTA, aprotinin and phenylmethylsulfonyl fluoride (PMSF) at final concentrations of 2.5mM, 100 units/ml and 0.1 mM respectively, and frozen and thawed three times. The soluble extract (1.5 mg of proteins/ml) was diluted in PBS to make the desired antigen dilution and was used to sensitize the immunoassay slides. Sera - Control sera (classified by results of immunofluorescence and indirect hemagglutination) were used at appropriate dilutions. In addition, 448 serum samples were collected from Buenos Aires city and its suburbs people. Samples were decomplemented by heating 30 min at 56 C and stored at -20 C until use. For testing these sera, serial dilutions were made in PBS, pH 7.4, 1.5% non fat milk. A standard WHO serum was used to determine the sensitivity of micro-SIA and IIF tests. SIA procedure - The assay presented here is a modification of the one described by Conway de Macario et al. (1983). Briefly, slides with 24 circles (Cel Line Associates, Inc., New Jersey) were cleaned with 95% (v/v) ethanol. Five ul of the antigen dilution were loaded on each micro-SIA well and air-dried. Antigen coated slides were washed in distilled water, blocked, washed again in distilled water and air-dried. The airdrying procedure could be shortened by using a Titan micro hood (Helena Laboratories, Beaumont, TX). Then, 5 ul of serum dilution were placed on each circle and slides were kept at room temperature in a humid chamber, for 20 min. Circles were then washed three times in PBS and incubated with 5 ul of peroxidase conjugated rabbit immunoglobulins to human IgA, IgG and IgM from Dako, Denmark diluted 1:1000 for 20 min. Serum and second antibody were diluted in PBS, pH 7.4, 1.5% non fat milk. The wells were washed three times in PBS for 5 min each and once in distilled water for 1 min after each incubation. Slides were air-dried as described above prior to the addition of 10 ul of the substrate solution (o-phenylenediamine dihydrochloride in 0.1M citrate buffer, pH 4.5 containing 0.15% of 30% H202 (v/v), to obtain a 1mg/ml solution). After a further 15 min incubation at room temperature in the presence of substrate, 10 ul of 1N sulfuric acid were added and the absorbance read at 42Onm in a minireader MR590 (Dynatech Instruments, Alexandria, VA). Sera dilution cutoff value, determined as the most frequent titre for the general population, was 1:100. IIF procedure - Parasites for IIF test were obtained as described above and then were purifled by one cycle of centrifugation in a sucrose gradient (Garberi et al. 1990), washed three times in PBS, and fixed in 1% formalin during 30 min at room temperature. IIF test procedure, reactives and equipment were the same as those used by the "Instituto Nacional de Microbiologia Carlos G. Malbran". Briefly, about 50 fixed parasites per microscope field were loaded in each well. Then, slides were air-dried and washed in distilled water. Each well was incubated with 20 ul of serum dilution for 30 min. After washing twice in PBS, 20 ul of fluorescein conjugated anti-human IgG from Dako, Denmark diluted 1:200 in PBS were placed on each circle and incubated for 30 min. Slides were washed again in PBS and examinated under Carl Zeiss fluorescence microscopy with a low power (12 V) halogen lamp. The validity of this test was confirmed by comparison with Sabin-Feldman assay which was calibrated to IU/ml with reference to WHO standard. Regression analysis - To compare micro-SIA with IIF each human sera, in total 448, was titrated by both methods by successive 1:2 dilutions, starting from 1:12.5 for micro-SIA and from 1:4 for IIF assay. Regression analysis was performed considering the logarithm of microSIA and IIF titres as independent variables. Evaluation of epidemiological data - To determine the positivity or negativity of the reaction each human sera was assayed at a dilution correspondent to the cutoff value by both techniques. For micro-SIA assay the reaction was considered positive when the absorbance obtained was higher than 0.01. Contingency tables were constructed and the chi square test was performed in order to determine significant differences in toxoplasmosis prevalence among people harbouring physiological (pregnancy) or pathological (allergy, rheumatism, cancer, endocrinological anomalies, HIV and Trypanosoma cruzi infections) alterations, and a healthy control group. People selected for this analysis were between 30 to 55 years old. RESULTS In order to find the adequate antigen mass to perform Toxoplasma micro-SIA, different quantities of T. gondii antigen were loaded in the wells of the slide. For each antigen quantity, the assay was performed using different serum dilutions, i.e.: 1:100; 1:200 and 1:400 (Fig. 1). The largest differences between the absorbances of positive and negative control sera were obtained with antigens levels of 500 and 700 ng of T. gondii protein. We chose 500 ng of antigen mass because practically no absorbance was obtained for the negative control serum, even at 1:100 dilution. Experiments were performed with different washing and blocking conditions. It was found that Toxoplasma micro-SIA had better results when washings were carried out in PBS. The blocking agent chosen was non fat milk diluted in PBS in a final concentration of 1.5%. The optimum blocking time was found to be 40 min, because it produced the higher difference in absorbance between negative and positive control serum (Fig. 2). Fig. 1: effect of antigen quantity (expressed as Toxoplasma gondii protein mass) on absorbance readings obtained after performing micro-SIA assay with positive and negative control sera, at three sample dilutions. Fig. 2: effect of blocking time on absorbance readings (X+/- SD) obtained after performing micro-SIA assay with positive and negative control sera. Fig. 3: effect of serum incubation time on absorbance readings (X+/-SD) obtained after performing micro-SIA assay with positive and negative control sera. On the other hand, the best conditions for the incubation with serum were 20 min at room temperature (Fig. 3). Longer incubations resulted in higher absorbances, but the resolution observed between positive and negative sera was not as good as for 20 min. Shorter incubations had similar problems, accompained by a very low absorbance. Experiments designed to study the stability of antigen coated and blocked slides gave a shelf life at 4 C of at least one year (data not shown). Once the assay conditions were established, analyses were made to determine if micro-SIA is appropriate for toxoplasmosis diagnosis. Micro-SIA and IIF toxoplasmosis titres were determined in 448 sera obtained from Buenos Aires city and its suburbs (Argentina) people selected at random. Titre was defined as the inverse of the highest serum dilution giving an absorbance at 420 nm higher than 0.01. A good correlation was found between micro-SIA and IIF titres (r=0.855) (Fig. 4). The sensitivity of Toxoplasma micro-SIA, determined with a WHO control serum, resulted in 3 IU/ml at 1:100 serum dilutions (data not shown). At the same time the sensitivity of IIF test was 10 IU/ml at 1:32 sera dilution. Therefore, this test resulted more sensitive than IIF. In coincidence with this result, for all the sera tested micro-SIA titres were higher than IIF titres (Fig. 4). In addition, 206 sera (out of 448) which had low IIF titres (32 or 64) were analyzed by micro-SIA. Thirty-three percent of these sera gave titres of 200 or 400, 61.2% gave titres of 100 and only 5.8% gave titres of 50 (data not shown). Fig. 4: correlation between micro-SIA and IIF titres of 448 sera samples. Each point represents more than one serum sample, n > 36 (filled circles) or n < 36 (open circles). The calculated regression line is shown.
TABLE I Contingency table to determine independence in prevalence of toxoplasmosis positive titres among people harbouring physiological or pathological alterations and controls micro-SIA IIF People group No. of No. of No. of No. of positive negative positive negative sera sera sera sera Pregnancy 34(50.0%) 34(50.0%) 34(50.0%) 34(50.0%) Allergy 20(50.0%) 20(50.0%) 20(50.0%) 20(50.0%) Rheumatism 18(45.0%) 22(55.0%) 18(45.0%) 22(55.0%) Cancer 24(57.1%) 18(42.9%) 23(54.8%) 19(45.2%) End. Anom. 31(51.7%) 29(48.3%) 31(51.7%) 29(48.3%) HIV 24(50.0%) 20(50.0%) 22(45.8%) 26(54.2%) Chagas 14(43.8%) 18(56.2%) 14(43.8%) 18(56.2%) Control 34(56.7%) 26(43.3%) 32(53.3%) 28(46.7%) End. Anom.: endocrinological anomalies. Number between brackets indicates percentage of positive and negative sera for each group. To determine the reproducibility of the method, one control serum was assayed 20 times in the same slide and 20 times in different assays. The intra assay variation coefficient resuited 5.48% and the inter assay variation coefficient was 6.24%. The interference of the method with other physiological (pregnancy) or pathological (allergy, rheumatism, cancer, endocrinological anomalies and HIV or T. cruzi infections) was analyzed. Chi square test indicated that there are no significant differences in percentage of Toxoplasma micro-SIA positive results among physiological or pathological alterations above mentioned and healthy control people. The same results were obtained for IIF assay (Table I). Four hundred and forty eight sera were analyzed to determine the prevalence of toxoplasmosis in Buenos Aires city and its suburbs (Table II). Positive serology was 56.3% (micro-SIA) or 54% (IIF).
TABLE II Prevalence of toxoplasmosis in Buenos Aires and Buenos Aires suburbs (Argentina) determinated by micro-SIA or IIF assays Technique No. of positive No. of negative sera sera micro-SIA 252(56.3%) 196(43.7%) IIF 242(54.0%) 206(46.0%) Number between brackets indicates percentage of positive and negative sera for each group. DISCUSSION There are various immunodiagnosis systems to detect toxoplasmosis: Dye test, IIF, IHA, ELISA, and IgM specific tests (Sabin & Feldman 1949, Walton et al. 1966, Tomasi et al. 1986, Dully et al. 1989). Because of the complexity of the immune ,response, it is recommended to perform more than one assay. Sensitivity and simplicity of the tests are two important features required. We report here a micro-ELISA system (named slide micro-immunoenzymatic assay) for Toxoplasma serology. Micro-SIA system showed a high sensitivity, three times as much as IIF. Also, it resulted more sensitive than ELISA, for which a sensitivity of 8 IU/ml was reported (Joss et al.1989). It is also very simple, rapid and economic, because it required little amounts of samples and reagents (5-10 ul). No special laboratory equipment is required and results can be assesed by naked eye because of background absence. Antigen coated slides could be stored at 4 C for at least one year. This allowed the preparation in advance of a stock of slides and to perform several determinations very rapidly (about 1 hr). The correlation found between IIF and micro-SIA was very good (r= 0.855), indicating that this assay could be an alternative to the indirect immunofluorescent test which requires a fluorescence microscopy and technical training. Recently, recombinant T. gondii antigens useful for serodiagnosis were obtained (Gross et al. 1992, van Gelder et al. 1993). We consider that Toxoplasma micro-SIA may be used with these antigens. Furthermore, no interference or false positivity was found with sera obtained from people harbouring other physiological or pathological alterations. All the features stated above make Toxoplasma micro-SIA very suitable for epidemiological studies and allowed us to analyze the prevalence of toxoplasma infection in samples obtained at random from Buenos Aires city and its suburbs (these two places concentrate 40% of argentinian population). The high prevalence of toxoplasmosis seropositivity found in this study (55-56%) is in good agreement with reports from other countries around the world like France (55%), Holland (58%), Italy (60%) and Austria (62%), (Remington & Desmonts 1983) confirming the worldwide dissemination of the disease. ACKNOWLEDGMENTS To Dr Jose Burdman for his critical reading. To Marcelo Lamami and Ricardo Damnoti for graphical and photographical works. REFERENCES Ambroise-Thomas P, Desgeorges PT 1980. Las posibilidades del test ELISA en el diagndstico serologico de la toxoplasmosis. Acta Bioquim Clin Latino-americana ABCLDL 14: 169-177. Conway de Macario E, Macario AJL, JoveIl RT 1983. Quantitative slide micro-immunoenzymatic assay (micro-SIA) for antibodies to particulate and nonparticulate antigens J Immunol Meth 59: 39-47. Dully KT, Wharton PJ, Johnson JD, New L, Holliman RE 1989. Assessment of immunoglobulin M immunosorbent agglutination assay (ISAGA) for detecting toxoplasmic specific IgM. J Clin Pathol 42: 1291-1295. Frenkel JK 1971. Toxoplasmosis: Pathology of protozoal and helminthic diseases. p. 254-290. In RA Marcial Rojas, The Coccidia. Williams and Wilkins, Baltimore. Garberi JC, Blanco JC, Angel SD, Pszenny V, Arakelian MC, Pastini A 1990. Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites. Preliminary characterization of its genome. Mem Inst Oswaldo Cruz 85: 429-434. Gross U, Roos T, Appoldt D, Heeseman J 1992. 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Van Gelder P, Bosman F, De Meuter F, van Heuverswyn H, Herion P 1993. Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli. J Clin Microbiol 31: 9-15. Walton BC, Benchoff BM, Brooks WH 1966. Comparison of indirect fluorescent antibody test and methylene blue dye test for detection of antibodies to Toxoplasma gondii. Am J Trop Med Hyg 15: 149-152. Copyright 1994 Memorias do Instituto Oswaldo Cruz The following images related to this document are available:Line drawing images[oc94009b.gif] [oc94009d.gif] [oc94009c.gif] [oc94009a.gif] |
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