Rapid determination of Trypanosoma cruzi urinary antigens in human chronic chagas disease by agglutination test

EXPERIMENTAL PARASITOLOGY 68, 208-215 (1989)

Rapid Determination of TI-ypanosoma cruzi Urinary Antigens Human Chronic Chagas Disease by Agglutination Test A. M. KATZIN,* A. MARcIPAR,t

in

H. FREILJJ,$ R. CORRAL,+ AND J. F. YANovsKYt

*Catedra de Microbiologia, Parasitologia e Inmunologia, Facultad de Medicina, UBA, SLaboratorio Virologia Hospital de Nirios, and tPolychaco SAIC, Buenos Aires, Argentina

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KATZIN, A. M., MARCIPAR, A., FREILJJ, H., CORRAL, R., AND YANOVSKY, J. F. 1989. Rapid determination of Trypanosoma cruzi urinary antigens in human chronic Chagas disease by agglutination test. Experimental Parasitology, 68, 208-215. Detection of Trypanosoma cruzi in man becomes particularly difficult during the chronic stage of Chagas disease because of the low parasitemia. We were able to develop a simple and straightforward method for determining the concentration of T. cruzi antigens in urine using nitrocellulose micellar suspension (Nitrocell-Mr, Polychaco Argentina) and for their subsequent detection through a “latex” type agglutination test. The latex used was an esferocell nitrocellulose suspension (Esferocell-Mr, Polychaco). Specific antigens for T. cruzi were detected in 54 of 58 urine samples from chronic chagasic patients. The antigens characterized by affinity chromatography and SDS-PAGE were glycoproteins with apparent molecular weights (and pls) of 100 kDa (pZ 5 to SS), 80 kDa (pZ 6.0), and 50 kDa (PZ 6.5 to 7.0). This method is practical and fulfills the requirement of large-scale epidemiological studies. It is also helpful in cases of conflictive serology. o 1989Academic press, Inc. INDEX DESCRIPTORSAND ABBREVIATIONS: Chagas disease: Antigenuria; Trypanosoma cruzi; Diagnostic; Agglutination test; Phosphate-buffered saline (PBS); Cellulose nitrate suspension (CNS); Nitrocellulose micellar suspension (NMS); Urine final concentrate (UFC); Cellulose nitrate suspension with bound anti-T. cruzi IgG (CNSGTc); Urine T. cruzi antigens (UTcAg); Tris-buffered saline (TBS); Polyacrylamide gel electrophoresis (PAGE); Sodium dodecyl sulfate (SDS); Enzyme-linked immuno-adsorbent assay (ELBA); Direct agglutination (DA); Indirect immunofluorescence (IFF).

nosis and hemoculture. However, some isolates are difficult to adapt to laboratory hosts, to tissue, or to axenic cultures (Camargo and Takeda 1979). In order to improve diagnosis, various research groups are studying the detection of specific T. cruzi antigens in body fluids. The presence of free and antibody-bound antigens in serum from animals and human patients, revealed by an ELISA and contraimmunoelectrophoresis, has been reported (Araujo et al. 1981; Marcipar et al. 1982; Kahn et al. 1983). Urinary antigens have been described in mice, in dogs, and in acute pediatric and congenitally infected patients by means of double diffusion or ELISAs (Bongertz et al. 1981; Corral et al. 1984). In this paper we report a simple and straightforward method for concentration of T. cruzi antigens in urine of chronic cha-

INTRODUCTION

The detection of antigens in/or during infectious diseases caused by viruses (Walters et al. 1976), bacteria (Edwards 1971), fungi (Warren et al. 1977), and parasites (Kohanteb et al. 1987) has proved extremely useful in diagnosis. In protozoan systemic infections this may be the approach of choice for demonstrating the presence of parasites (Araujo and Remington 1980; Voller and Dosavigny 1981.). Detection of Trypanosoma cruzi, the causative agent of Chagas disease, is particularly difficult during the chronic stage of the disease because of the low parasitemia. The methods used at present are xenodiagTo whom correspondence

should be addressed:

A.M.K., Dt. Parasitologia, Instituto Clencias Biomedicas, Universidade de Sao Paula, Cidade Universitaria, CEP 05508, Sao Paulo, Brasil. 208 0014-4894189 $3.00 Copyright0 1989by Academic AU rights of reproduction

Press, Inc. in any form reserved.

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OF

gasic patients by using nitrocellulose micellar suspension. We demonstrate by a nitrocellulose esferocell agglutination test the presence of T. cruzi antigens in such concentrates. The antigens were characterized and identified by affinity chromatography and unidimensional and two-dimensional SDS-PAGE. MATERIAL

Human

AND

METHODS

Patients

Urine samples were collected from 58 patients with chronic Ttypanosoma cruzi infection, showing positive serology for Chagas disease (titers > l/32 by DA and IIF tests). Patients were adults, ages 25-52, of both sexes, living in a nonendemic area. Three patients presented positive hemocultures and xenodiagnosis and five had cardiopathy. The control population consisted of 30 healthy individuals from the same nonendemic area with negative serology for T. cruzi infection, 8 patients with schistosomiasis from Belo Horizonte, Brazil, and 2 patients with acute toxoplasmosis.

Anti-T.

Specific antiserum to T. cruzi was obtained from a horse immunized by subcutaneous inoculation of T. cruzi suspension (5 ml, Tulahuen strain, 10’ epimastigates/ml) previously washed in PBS, pH 7.2, with complete Freund’s adjuvant. The animal was bled on Day 30 postinoculation and the immunoglobulin fraction of serum was precipitated with ammonium sulfate. Anti-T. cruzi antibodies were purified by affinity chromatography with a total homogenate of epimastigotes coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia) (2 mg of protein/ml of gel). Two hundred milligrams of precipitated horse immunoglobulin was applied to the column (15 x 2 cm) and specific anti-T. cruzi IgG (titer I:8000 by hemagglutination test) was eluted in 0.1 M glycine-HCl buffer pH 2.9.

Preparation of Cellulose Nitrate Suspension with Anti-T. cruzi

ZgG

Puritled anti-T. cruzi IgG was dialyzed against 7 liters of PBS and then incubated with 2% CNS (Esferocell-Mr, Polychaco) in PBS (30 mg IgG/g cellulose nitrate suspension) for 30 mitt at 20 C. CNSGTc was washed and centrifuged three times in PBS (2OOOg, 10 min). The pellet was resuspended in the same buffer, adjusted to 2% (w/v), and stored at 4 C until used.

Urine

concentration

T. cruzi Antigens procedure.

CrUzi

A 300-ml volume of

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urine was collected from each patient and preserved with 1% acetic acid at 4 C until tested. Samples were centrifuged at 2OOOgfor 15 min. The supematant was concentrated by mixing with nitrocellulose micellar suspension (Nitrocell-Mr, Polychaco) to a final concentration of 2% (v/v) and incubated for 18 hr at 4 C or for 3 hr at 20 C. The suspension was centrifuged 15 min at 2OOOg and the pellet, containing the proteins attached to the nitrocellulose micellar suspension, was washed twice with 10 ml of PBS. The pellet was dissolved in 15 ml of cold acetone at -20 C and centrifuged at SOOOgfor 10 min in order to extract the nitrocellulose. The precipitate was dissolved in 0.2 ml PBS and centrifuged again as in the previous step. The supematant (UFC) was filtered through Whatrnan No. 1 paper and was tested for the presence of UTcAg. Agglutination assay. UFC (30 ~1) and CNSGTc (60 ~1) were mixed on a glass slide, rocked for 2 min, and examined either visually or microscopically (40X) for agglutination detection. Results were read within 2 min and any degree of agglutination was considered positive. Controls with cellulose nitrate suspension coated with normal horse IgG were performed in parallel.

Characterization

cruzi Antiserum

Determination of in Urine

T.

of UTcAg

Zodination of UFC. UFC (200 pl) was incubated with 1 ml PBS containing 200 @/ml Nai3’I in glass tubes precoated with 20 ug Iodogen (Fraker and Speck 1978). After 10 mitt at 4 C, the UFC was dialyzed against 8 liters of buffer containing 10 mikf Tris, 150 mM NaCl, pH 7.4 (TBS). Lectin-afJiity

chromatography.

UFC

was radioio-

dinated as described above, Nonidet-P40 was added to 2% final concentration and samples were applied directly to a concanavalin A-Sepharose column (Pharmacia, 5 x 0.25 cm) previously equilibrated in TBS containing 2% Nonidet-P40. The flow rate was adjusted to 1 drop/20 set until the total sample was in contact with the bed surface. The flow was then interrupted for 1 hr and restored with a rate of 1 drop/l0 sec. Fractions of 0.3 ml were collected. Nonadsorbed material was washed with one bed volume of the described buffer (peak 1). The column was subsequently washed until radioactivity decreased to background level (approx 35 vol). Bound material was eluted with the same buffer containing 0.1 M a-methyl-Dmannoside and 0.1 M a-methyl-D-glucoside (peak 2). Materials from both peaks (about 1.2 ml) were precipitated by addition of 3 vol of cold ethanol at - 20 C for 48 hr, centrifuged at 10,OOOgfor 30 mitt, and solubilized in 200 pl appropriate buffer (1% Nonidet-P40, 10 mM Tris, 100 mM NaCl, pH 7.4) for immunoprecipitation, prior to analysis by SDS-PAGE (Katzin et al. 1984). Zmmunoprecipitation. Immunoprecipitation was performed as described by Zingales and Colli (1984).

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All materials present in peaks 1 and 2 were incubated overnight with 50 pl of anti-T. cruzi IgG purified as described above (anti-T. cruzi IgG used with CNSGTc). The antigen-antibody complexes were mixed for 30 min at room temperature with 70 ul of a 10% suspension of heat-killed and formalin-fixed Stnphylococcus aureus Cowan 1 strain (Kessler 1975). The immune complexes were washed twice with a solution containing 0.05% (v/v) Nonidet-P40, 0.1% (v/v) SDS, 0.3 M NaCl, pH 8.6, and the precipitate was resuspended in 70 ul electrophoresis sample buffer (Zingales and Colli 1984) for unidimensional SDS-PAGE or in 60 ul electrophoresis sample buffer for twodimensional SDS-PAGE (O’Farrell 1975). SDS-PAGE. Unidimensional SDS-PAGE was performed as described by Laemmli (1970) using a 7-14% acrylamide gradient. Electrophoresis was carried out at 90 V for 4 hr. Two-dimensional PAGE was performed under the equilibrium conditions described by O’Farrell (1975). Ampholines in three different pH ranges (I%, pH 3.5 to 10.0; 0.7%, pH 4.0 to 6.0; 0.3%, pH 9.0 to 11.0) were combined to obtain a broad pH gradient. After isoelectric focusing (16 hr at 400 V), the pH gradient was measured directly on the firstdimension gel by means of a surface electrode. As commented by O’Farrell (1975), direct measurement of pH on urea gels may not give precise values. Thus, all references to isoelectric pH values found in this work are only approximate and have been used for descriptive purposes. The second-dimension slab gels were prepared as linear 7 to 14% gradients. The following molecular weight markers (daltons) were included in unidimensional and two-dimensional SDSPAGE: trypsinogen (24,000), ovoalbumin (45,000), bovine serum albumin (68,000), human transferrin (SO,OOO), and B-galactosidase (120,000). Gels were dried and exposed to Kodak X-Omat fdm with intensifying screens for 7 to 10 days at - 70 C.

Characterization of T. cruzi Antigens Detected by CNSGTc Trypomastigote forms of T. cruzi (5 x 10’ cells) ob-

ET AL.

tamed as previously described (Andrews and Colli 1982) were radioiodinated using 300 @i/ml Na13rI (Katzin and Colli 1983) and lysed in 0.5 ml lysis buffer containing 1% Nonidet-P40, 1 mM phenylmethylsulfonyl fluoride, 1 mM N-p-tosyl-L-lysylchloromethyl ketone, and 10 mMTris/Cl, pH 8.6, and then centrifuged at 10,OOOgfor 30 min. The soluble extract (about 200 ~1) was incubated for 2 hr at 20 C with 50 ~1 of CNSGTc. The immune complexes were washed twice with PBS, pH 7.2, and resuspended in 60 ul of electrophoresis sample buffer. Parallel samples of T. cruzi antigens were immunoprecipitated by Kessler’s method, described above, using anti-T. cruzi IgG (the same anti-T. cruzi IgG used with CNSGTc). The material was analyzed by SDS-PAGE and radioautography.

RESULTS

Agglutination

Assay of Urine Samples

Of 58 chronic chagasic patients, 54 (91%) were positive for UTcAg by the CNSGTc agglutination test. Three positive patients had positive hemoculture and xenodiagnosis, and five patients presented cardiopathy. Schistosomiotic and toxoplasmotic patients as well as noninfected controls were negative (Table I). Characterization

of the Assay

Both the sensitivity and the specificity of the CNSGTc agglutination assay for UTcAg detection were evaluated using T. cruzi and Toxoplasma gondii (prepared as described previously; Freilij et al. 1987) and Leishmania spp. (Behringwerke AG Mar-

TABLE1 Results Obtained by Agglutination Patients

Urine samples

Chagasic Toxoplasmotic Schistosomiotic Normal

58’ 2 8 30

Tests with Urine Samples Agg. CNSGTc” 54 2 8 30

positived negative negative negative

Agg. CNSG’ 58 negative 2 negative 8 negative 30 negative

0 Agglutination tests with cellulose nitrate suspension-bound anti-T. cruzi IgG. * Agglutination tests with cellulose nitrate suspension-bound normal IgG. c Number of patients. d Three patients had positive xenodiagnosis and hemocultures. Five patients presented cardiopathy.

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OF T.

burg Federal Republic of Germany) standard soluble antigens. The protein concentration of each of these antigens was measured by Lowry’s method (Lowry et al. 1951). The agglutination test used was capable of detecting at least 0.5 p,g of T. cruzi antigen. When the CNSGTc reagent was used with either Leishmania spp. or T. gondii standard antigens in final concentrations ranging from 10 to 100 pg/50 pl, no reactions were obtained. Electrophoretic analysis revealed several bands of T. cruzi trypomastigote antigenic surface proteins recognized by CNSGTc and also by the same anti-T. cruzi IgG bound to S. aureus Cowan 1 strain. Some of them (100, 80, and 55 kDa) were comparable to those UTcAg characterized above (Figs. 1 and 3).

Cruzi

ANTIGENS

211

1, 90% of total radioactivity of the original sample) and the second peak constituted the specific eluate (peak 2, 10% of total radioactivity). Protein bands were observed only from immunoprecipitates of peak 2 (Fig. I), with approximate molecular weights (and pls) of 100kDa (pZ 5.0 to 5.5), 80 kDa (pZ 6.0), and 55 kDa (pZ 6.5 to 7.0), as revealed by autoradiography (Fig. 2). DISCUSSION

Although it has been a long time since Carlos Chagas first described the disease subsequently known by his surname, an accurate diagnosis of this disease is still difficult. Current serological methods of diagnosis are relatively simple and sensitive but they present problems of cross-reactivity and other nonspecific reactions. In addition, low detectable antibody titers result Characterization of UTcAg in Urine of from the formation of immune complexes Chagasic Individuals or immunosuppression in patients undergoing treatment (WHO 1983; Freilij et al. Iodinated UFC samples chromoto1983;Corral et al. 1984). Direct methods of graphed through a Sepharoseconcanavalin A column yielded two peaks; the first peak parasitological diagnosis, with or without corresponded to nonbound material (peak blood concentration, are useful during the abcdef

g h

i

j

klmn

120

24 FIG. 1. Autoradiography. Urine samples labeled with I 13’ Na were chromatographed by Sepharoseconcanavalin A (peak 2) and immunoprecipitated with anti-l: cruzi antiserum using the method of Kessler (1975). Urine samples from chagasic patients: lanes a-h. Urine samples from normal patients: lanes i-n. Patients b and c have positive xenodiagnosis and hemoculture. Patient c presented chronic chagasic cardiopathy. kDa, kilodaltons.

212

KATZIN

l-2060,g 660 y

45-

24-

PHI

”

”

’ 1’

”

4.0 4.5 5.0 5.5 6.0 6.5 6.7 7.0 23 25

FIG. 2. Autoradiography of 1311-labeled T. cruzi antigens eliminated in the urine of patient e, with chronic chagasic disease. The antigens were isolated by aftinity chromatography (Sepharose-Con A), and peak 2 was inununoprecipitated with anti-T. cruzi antisera using the method of Kessler (1975) and analyzed by twodimensional SDS-PAGE. kDa, kilodaltons; pH, pH gradient.

acute stage of the disease, in congenital infections, and in immunocompromised patients (Freilij et al. 1983). These diagnostic techniques, however, are much less effective during the chronic phase of infection, when under optimal conditions, trypomastigotes can be detected only in 50% of the cases(Cancado et al. 1979; Chiari et al. 1979). Moreover, these methods require complex facilities (insect house, sterile area, skilled personnel) and results are obtained only after 30-90 days. The search for antigenic fractions of parasite molecules is a simple and more sensitive procedure, applicable to a variety of infectious diseases. The detection of free antigens and specific immune complexes in sera of infected animals and in individuals with acute and chronic T. cruzi infection has been described. Kahn et al. (1983) have reported 65% positivity for circulating immune complexes in chronic chagasic patients and 55% positivity in antigenic ELISA determinations. Araujo ef al. (1981) and Marcipar et al. (1982) have found by

ET AL.

similar techniques that antigenemia can be demonstrated during the chronic stage in 50 and 82% of the cases, respectively. The occurrence of T. cruzi antigens in urine was originally described by Bongertz et al. (1981). Antigenuria was detected by double diffusion tests in acutely infected experimental animals. The first report of urinary excretion of T. cruzi antigens in human Chagas disease yielded 100% positivity in ELISAs performed in children with acute and congenital infection (Corral et al. 1984). However, when we tested this technique with urine from chronic chagasic patients, only 32% of the casesgave positive results (unpublished data). The lower sensitivity might be related to the reduced sample volume and to the fact that the ethanol precipitation step used in this technique provides only a lofold concentration of antigens. The possibility of increasing the sample volume, together with the development of nitrocellulose micellar suspension adsorbent which provides an approx loo-fold concentration of antigen in urine, has enabled us to devise a test that revealed specific T. cruzi antigenuria in 54 of 58 chronic chagasic patients studied. Thus, it is possible to achieve a parasitologic immunodiagnosis despite low parasitemia, which is so characteristic at this stage of T. cruzi infection. The occurrence of antigenuria was detected in patients with or without cardiopathy, thus suggesting no relation to clinical forms. The method proved to be highly specific, as T. cruzi antigens could not be detected in the urine specimens of the control population of healthy individuals with negative serology for Chagas disease, S. mansoni-infected patients, and acute toxoplasmosis cases. Furthermore, the “latex” reagents did not react with total antigenic homogenates from other parasites such as Leishmania spp. and T. gondii. The T. cruzi antigens detected in patients’ urine were also present in the soluble extract of epimastigotes used as stan-

DETERMINATION

OF T. CrUZi

dard. Anti-T. cruzi serum prepared against epimastigotes reacted with trypomastigotes as described by different authors (Zingales and Colli 1985; Andrews et al., 1984; Lanar and Manning 1984). T. cruzi antigens found in urine were glycopoteins, detectable only in the specific eluate (peak 2) from the Sepharose-concanavalin A column. These glycoproteins eliminated in urine may correspond to several glycopeptides previously described for T. cruzi (Katzin and Colli 1983, Zingales and Colli 1985). Parasite antigens excreted in urine share epitopes with trypomastigote surface glycoproteins and have approximately the same molecular weight (Fig. 3). Similar glycoproteins were described previously by Andrews et al. (1984) and Lanar and Manning (1984). In urine of patients with acute Chagas disease, two glycoproteins with similar molecular weights and pZ values have been described (Freilij et al. 1987). The other bands detected in some urine samples were probably a result of antigen proteolysis; it is very difficult to control hydrolytic mechanims in urine samples because of bacterial contamination. The presence in urine of antigens of moa b

213

ANTIGENS

lecular weights above the limits acceptable for glomerular filtration mechanisms demonstrates that these mechanisms depend on the size and spatial configuration of the molecule (Berliner and Glebish 1981). Moreover, localization of parasites in the urinary tract must not be discarded either (Bite and Zeledon 1970; Postan et uf. 1983). On the other hand, Younes-Chennouti et al. (1988) described persistence of T. cruzi antigens in the inflammatory lesions of chronically infected mice. These antigens could be shed into the circulation and finally eliminated in the urine. Xenodiagnosis and parasitologic diagnosis by hemoculture may confirm only 20 to 50% of chronic patients. If any of these methods are repeated twice or more, or if a greater number of insects and cultures are used, a 90% positivity can be achieved (Albunquerque et al. 1972; Cancado et al. 1979). This improvement has two drawbacks: the need of special facilities and the long time necessary to obtain trustworthy results. The urine test simplifies diagnosis since results are obtained within 24 hr after collection of the samples. Moreover, the sensitivity of the assayis higher than that of the traditional parasitological methods conducted under standard conditions. In addition, this method is practical and may fulfill the requirements of large-scale epidemiological studies. It is also helpful in cases of doubtful serology. A possible correlation between the presence of these antigens in urine and active infection may offer a new tool for rapid evaluation of the disease in therapeutic studies. ACKNOWLEDGMENTS

24-

FIG. 3. Surface antigens of trypomastigotes immunoprecipitated with (a) CNSGTc and (b) the same antiT. cruzi IgG that is conjugated to CNSGTc (see Material and Methods) and immunoprecipitated with Sraphylococcus aureus Cowan 1 (Kessler’s methods).

The authors are deeply indebted to Dr. G. Gazinelli (Belo Horizonte, Brazil) for sending us urine from schistosomiotic patients. We especially thank L. S. Ozaki and R. V. Milder for their critical reading of the manuscript. A.M.K. is a research member of CONICET Argentina. REFERENCES ALBUNQUERQUE,

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ZINGALES,

Received 13 June 1988; accepted with revision 21 October 88