Serodiagnosis of Acute Toxoplasmosis

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 28, No. 8 Use of Acute-Stage-Specific Antigens of Toxoplasma gondii for Serodiagnosis of Acute Toxoplasmosis YASUHIRO SUZUKI,'t PHILIPPE THULLIEZ,2 AND JACK S. REMINGTONl13* Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, California ; Laboratoire de Serologie Neonatale et de Recherche sur la Toxoplasmose, Institut de Puericulture de Paris, Paris, France2; and Division of Infectious Diseases, Department of Medicine, Stanford University School of Medicine, Stanford, California Received 20 February 1990/Accepted 15 May 1990 Antisera to acetone-fixed tachyzoites were prepared by immunizing rabbits intravenously with the fixed organisms. Immunoblots in which purified immunoglobulin G (IgG) of the antisera was used recognized four antigens of a supernatant (after centrifugation at 10,000 x g for 30 min) fraction of sonicated tachyzoites. A purified toxoplasma antigen fraction, referred to as acute-stage-specific (AC) antigen, that contained mainly three antigens was obtained by affinity chromatography by using the purified IgG. One antigen had a molecular weight of approximately 52,000, and two antigens had molecular weights of approximately 6,000. An enzymelinked immunosorbent assay (AC-ELISA) in which this purified antigen fraction, obtained by affinity chromatography, was used for detection of IgG antibody detected early acute infection in sera of patients with toxoplasmic lymphadenopathy. Ninety-two percent of sera obtained within 2 months after onset of lymphadenopathy were positive in the AC-ELISA, whereas only 9% of sera obtained later than 5 months after onset of the clinical illness were positive. The AC-ELISA appears highly sensitive and specific for diagnosis of acute toxoplasmosis. The most commonly recognized clinical manifestation of acute acquired infection with Toxoplasma gondii in immunocompetent adults is lymphadenopathy (3). Although criteria for histopathologic diagnosis of toxoplasmic lymphadenopathy have been described (2), serologic diagnosis is preferable to having the patient undergo a biopsy (1). However, the high prevalence of toxoplasma antibodies in otherwise normal individuals and the fact that titers may remain elevated for years following the acute infection have complicated interpretation of serologic test results obtained in individuals suspected of having acute toxoplasmosis. In 1986, Thulliez et al. (13) reported that a direct agglutination test for detection of immunoglobulin G (IgG) antibodies in which Formalin-fixed tachyzoites and acetone-fixed tachyzoites were used could, in a significant percentage of cases, differentiate between recently acquired and chronic (latent) infections with a single serum sample. Their results suggest that tachyzoite antigens recognized by IgG antibodies formed during the acute infection have specificities different from those formed later in the infection. We recently reported that acetone-treated tachyzoites have only tachyzoite-specific antigens on their surface, that these antigens are not present on the surface of bradyzoites (12), and that these antigens are important in distinguishing the stage of infection with T. gondii (12) (referred to as stage-specific antigen[s]). In the present study, we purified the tachyzoitespecific antigens, which showed three major bands on polyacrylamide gel electrophoresis, and used the preparation in an enzyme-linked immunosorbent assay (AC-ELISA) to determine whether it could detect acute-phase IgG antibodies for diagnosis of toxoplasmic lymphadenopathy. * Corresponding author. t Permanent address: Department of Parasitology, Jikei University School of Medicine, Tokyo 105, Japan MATERIALS AND METHODS Human sera. Sera were from 18 adult patients who had acute lymphadenopathic toxoplasmosis diagnosed by serology and histopathology (1, 15). These are the same patients with whom our previous study was performed (12). Control sera were from 21 chronically infected individuals who were healthy and had known stable toxoplasma IgG antibody titers (dye test titers ranged from 1:16 to 1:256) for more than 1 year. Toxoplasma antigens. Tachyzoites of the RH strain were harvested from the peritoneal fluids of mice infected 2 days earlier and purified as described previously (16). For preparation of acetone-fixed organisms, tachyzoites were treated with 30% acetone in phosphate-buffered saline (PBS) containing 0.01 M NaH2PO4, 0.01 M Na2HPO4, and M NaCi (ph 7.2) at 4 C for 72 h (13). Formalin-fixed tachyzoites for an agglutination test were prepared as described previously (13). Toxoplasma lysate was prepared by sonication of tachyzoites in PBS (9). Lysate supernatant antigens were obtained after centrifugation of the lysate at 10,000 x g for 30 min at 4 C. Gel electrophoresis and immunoblotting. Electrophoresis was performed in 5 to 15% polyacrylamide slab gels with the discontinuous sodium dodecyl sulfate buffer system described by Laemmli (4). Fresh tachyzoites were solubilized in the sample buffer as described earlier (12). Lysate supernatant antigens were also used for the electrophoresis. Molecular weight standards were myosin, P-galactosidase, phosphorylase b, bovine serum albumin, egg albumin, and carbonic anhydrase (Sigma Chemical Co., St. Louis, Mo.). Silver staining of the gels after electrophoresis was performed with a commercial kit (Bio-Rad Laboratories, Richmond, Calif.). For immunoblots, proteins separated by electrophoresis were transferred to nitrocellulose paper as described by Towbin et al. (14). Blots were first soaked in 5% nonfat dried milk in PBS and then incubated with sera from rabbits immunized with acetone-fixed tachyzoites at a

2 VOL. 28, 1990 TOXOPLASMA ELISA WITH ACUTE-STAGE-SPECIFIC ANTIGENS 1735 m- tle - a- 4- L*çA4u NU Antlsrm -AnUbWo.- 1/100 dilution as described previously (12). Thereafter, the nitrocellulose sheets were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (Caltag, South San Francisco, Calif.) at a dilution previously determined to be optimum (12). The color development was with a solution containing 0.1 mg of diaminobenzidine per ml of 0.1% H202 in PBS (12). Preparation of antisera against acetone-fixed tachyzoites. Female white rabbits weighing 2.5 kg obtained from Simonsen Laboratories (Gilroy, Calif.) were immunized intravenously twice at a 4-week interval with 5 x 107 acetone-fixed tachyzoites in 0.5 ml of borate buffer (ph 8.7). Sera were obtained 1 week after the second immunization. Purification of acute-stage-specific (AC) antigens. IgG antibodies were purified from rabbit antisera prepared against acetone-fixed tachyzoites by affinity chromatography by using protein A-Sepharose (Pharmacia, Uppsala, Sweden). The purified IgG antibodies were bound to Affi-gel 10 beads (Bio-Rad) according to the directions of the company. Lysate supernatant antigens were applied to the Affi-gel column, and the bound antigens were eluted with 0.2 M glycine buffer (ph 9.0) after thorough washing of the column with PBS to remove unbound antigens. The eluted fraction was dialyzed against PBS and thereafter concentrated by ultrafiltration by using a membrane with a molecular weight exclusion of >1,000. AC-ELISA. The purified AC antigens were diluted in 0.1 M carbonate buffer (ph 9.8) at concentrations of 25 to ,ug of protein per ml. Wells of polystyrene Immulon I microtiter plates (Dynatech Laboratories, Inc., Chantilly, Va.) were coated with 100,u of the purified AC antigens. After incubation at 4 C for 16 to 60 h, the plates were washed three times for S min each time with PBS containing 0.05% Tween 20. The wells were postcoated with 250,l of PBS containing 5% bovine serum albumin (Sigma Chemical Co.) at 37 C for 2 h. Thereafter, the plates were washed three times in PBS-Tween 20 and the wells were incubated at 37 C for 1 h with 100,ul of a 1:100 dilution (in PBS) of the sera to be tested. After washing three times in PBS-Tween 20 the wells were incubated at 37 C for 1 h with 100,tl of rabbit anti-human IgG antibody conjugated to horseradish peroxidase (Cooper Biochemical, West Chester, Pa.) in PBS containing 3% bovine serum albumin. After the plates were again washed three times in PBS-Tween 20, 100,ul of substrate solution (500 ptg of ortho-phenylenediamine per ml and 0.1% of H202 in 0.1 M citrate buffer [ph 4.5]) was added. The reaction of the enzyme substrate was allowed to continue for 10 min at 37 C and then read with a microplate photometer (Dynatech Laboratories, Inc.). Negative control wells for each serum sample were coated with PBS containing 5% bovine serum albumin without precoating with the purified AC antigens. Each serum sample was tested in triplicate. The results were expressed by using the formula: AC-ELISA value = (optical density value of experimental wells - optical density value of negative control wells) x 1,000. Serology. Toxoplasma IgG antibodies were measured in the Sabin-Feldman dye test (8), and IgM antibodies were measured in the double-sandwich IgM ELISA (DS-IgM- ELISA) (7). Titers in the DS-IgM-ELISA were calculated as described by Siegel and Remington (10). The agglutination tests with acetone- and Formalin-fixed tachyzoites were performed as described previously (14). Statistics. Levels of significance for comparisons between groups of patients were determined by using the x2 test. il.- m- os e- 2.- â-.4 FIG. 1. Immunoblot analysis of T. gondii antigens recognized by rabbit IgG antibodies against acetone-fixed tachyzoites. NRS, Normal rabbit serum. Two toxoplasma antigen preparations, lysate supernatant and whole antigens, were used for electrophoresis. Numbers represent molecular weight standards (103). RESULTS Antigens recognized by IgG antibodies of rabbits immunized with acetone-fixed tachyzoites. The purified IgG antibodies of the immunized rabbits recognized 16 antigens of whole tachyzoites in immunoblots (Fig. 1). The normal (prebleed) rabbit serum recognized 6 of the 16 antigens (Fig. 1). There were five major antigens (most intensely stained) recognized by the IgG antibodies that were not recognized by the control IgG. They had molecular weights of approximately 61,000, 54,000, 48,000, 30,000, and 6,000, respectively (arrows in Fig. 1). When lysate supernatant antigens were used for immunoblotting, IgG antibodies of the immunized rabbits reacted with four antigens with molecular weights of 54,000, 52,000, 30,000, and 6,000 (arrows in Fig. 1). None of the lysate supernatant antigens was recognized by the control IgG. Purification of AC antigens by affinity chromatography. The results of silver staining of the gel in which the purified AC antigens obtained from the affinity column were electrophoresed are shown in Fig. 2. Three major bands were observed (lanes 1 and 2 in Fig. 2). One of the antigens had a molecular weight of 52,000, and the other two bands were present in the area of the gel that corresponded to an approximate molecular weight of 6,000. The bands were not observed in the lane to which only sample buffer was applied (lane 3). Determination of optimum condition for coating microtiter plate wells with the purified AC antigens. The wells of microtiter plates were coated with either 2.5, 0.5, 0.25, 0.125, or ,ug of the purified AC antigens per ml for 60 h, and their reaction with IgG antibodies in sera of patients with acute lymphadenopathic toxoplasmosis (acute sera) and in control sera of chronically infected individuals (chronic

3 1736 SUZUKI ET AL. J. CLIN. MICROBIOL. >100 r , i [. w 40 FIG. 2. Silver staining of the purified AC antigens after electrophoresis: lanes 1 and 2, purified AC antigens (0.1,ug of protein); lane 3, sodium dodecyl sulfate sample buffer only; lane 4, lysate supernatant antigens (20,ug of protein). Numbers represent molecular weight standards (103). sera) was examined. At concentrations of AC antigens of to 0.125,ug/ml, no difference was detected in binding of IgG antibodies to the wells between acute and chronic sera. At a concentration of >0.25,ug of AC antigens per ml, higher AC-ELISA values were observed in the acute than the chronic sera; the greatest difference in AC-ELISA values was obtained at a concentration of 0.5,ug of AC antigens per ml. At 2.5 ptg of AC antigens per ml, AC-ELISA values in the chronic sera increased and the difference in values between the acute and chronic sera decreased. The wells of microtiter plates were incubated with either 2.5 or 0.5,ug of AC antigens per ml for 16 or 60 h to define an appropriate incubation time for coating the wells with the purified AC antigens. Thereafter, the wells were incubated with acute and chronic sera. In wells coated with 2.5,ug of the purified AC antigens per ml, the difference in binding of IgG antibodies between the acute and chronic sera was the same regardless of the incubation time of the wells with the purified antigens. In contrast, in wells coated with 0.5,ug of AC antigens per ml, the difference in binding of IgG antibodies between the acute and chronic sera was observed only in wells coated with the antigens for 60 h. The greatest difference in ELISA value between acute and chronic sera was obtained in wells coated with 0.5,ug of AC antigens per ml for 60 h. On the basis of these results, for the experiments described below, we coated the wells with 0.5 tg of AC antigens per ml for 60 h. AC-ELISA values in sera of patients with chronic (latent) infection. Serum specimens from 21 patients with chronic infection who had stable toxoplasma IgG titers and no clinical signs of infection were tested in the AC-ELISA. The AC-ELISA values were low. The mean plus 2 standard deviations of the AC-ELISA values was 9.9 (Fig. 3). We 30 F O a 0 * * J, I e 8 T.~ 3-4 0,5_ --, -* -< Months aitt onet of lymphadwbopth y CChroni control FIG. 3. Distribution of AC-ELISA values following onset of clinical illness in patients with toxoplasmic lymphadenopathy. The horizontal line indicates the mean plus 2 standard deviations of values in the control group of chronically infected individuals. therefore defined AC-ELISA values of.10 as suggestive of recent acute infection with T. gondii. AC-ELISA values in sera of patients with acute lymphadenopathic toxoplasmosis. The AC-ELISA values in sera of patients with acute lymphadenopathic toxoplasmosis are shown in Fig. 3. Of the serum specimens obtained from 13 patients within 2 months after onset of their lymphadenopathy (group 1), 92% (12 of 13) had positive AC-ELISA values (.10). In contrast, at 3 to 4 months after onset of lymphadenopathy (group 2), only 38% (3 of 8) of the patients had positive values. In sera drawn later than 5 months after onset of lymphadenopathy (group 3), the prevalence of positive values was only 9% (1 of 11). The percentages of positive patients in the AC-ELISA were significantly different between groups 1 and 2 (P < 0.05) and between groups 1 and 3 (P < 0.01). Comparison between results of AC-ELISA and other serologic tests. The percentages of patients who had serologic test titers suggestive of acute toxoplasma infection as a cause for their lymphadenopathy are shown in Table 1. During the first 2 months of lymphadenopathy, a high percentage of patients had such titers in each of the serologic tests; the highest percentage (92%) was obtained in the AC-ELISA. In contrast, at 3 to 4 months after the onset of lymphadenopathy, only the AC-ELISA showed a significant decrease in prevalence of positivity (P < 0.05) when compared with the prevalence of positives in the first 2 months. At greater than 5 months after the onset of lymphadenopathy, a high percentage of the patients was positive only in the dye test. The lowest prevalence of positive results in these latter patients was in the AC-ELISA (9%). DISCUSSION The results described above reveal that an ELISA in which purified toxoplasma AC antigens are used for detec-

4 VOL. 28, 1990 TOXOPLASMA ELISA WITH ACUTE-STAGE-SPECIFIC ANTIGENS 1737 TABLE 1. Results suggesting acute toxoplasma infection in four different serologic tests performed on sera collected at various time intervals following onset of toxoplasmic lymphadenopathy Serologic test % Patients with titer at following mo after onset of lymphadenopathy (no. with titer/no. tested) s AC-ELISA (10) 92 (12/13) 38 (3/8) 9 (1/11) Dye test (.1:1,024)a 85 (11/13) 88 (7/8) 73 (8/11) DS-IgM-ELISA (22)a 77 (10/13) 100 (8/8) 27 (3/11) DS-IgM-ELISA (23) 77 (10/13) 100 (8/8) 27 (3/11) DS-IGM-ELISA (24) 54 (7/13) 88 (7/8) 27 (3/11) Agglutination test' 77 (10/13) 88 (7/8) 36 (4/11) a These cutoff values are based on the results of the study by Brooks et al. (1) ḃ Interpretation of results of the agglutination test was based on the report by Thulliez et al. (13). tion of acute-stage-specific IgG antibodies was valuable for diagnosis of acute lymphadenopathic toxoplasmosis. We previously reported that tachyzoite-specific antigens, which detect acute-stage-specific IgG antibodies, are useful for differentiating between the acute and chronic stages of the infection and that acetone-fixed tachyzoites appear to have only the specific antigens on their surface (12). The affinitypurified AC-antigen fraction contained three main antigens: one with a molecular weight of approximately 52,000 and two with molecular weights of approximately 6,000. The molecular weights of the antigens used for the AC-ELISA are consistent with those of tachyzoite-specific antigens observed in our previous study (12). It is noteworthy that differentiation between acute and chronic sera in the AC- ELISA was dependent on the concentration of AC antigens used for coating the plates. The results obtained in the AC-ELISA appeared to correlate with duration of lymphadenopathy in the patients with acute lymphadenopathic toxoplasmosis. When compared with the results in the dye test, DS-IgM-ELISA, and agglutination test, only the AC-ELISA revealed a significant decrease in titer value by 4 months after onset of lymphadenopathy in patients with serologic test results considered consistent with a diagnosis of the acute infection. Only 9% of the patients had a positive reaction in the AC-ELISA when tested more than 5 months after clinical onset of their disease. These results indicate that the AC-ELISA may be more suitable than the dye test, DS-IgM-ELISA, and agglutination test as a serologic test for determining the acuteness of infection in patients with toxoplasmic lymphadenopathy. In contrast to the AC-ELISA, a high percentage (73%) of patients still had high titers in the dye test more than 5 months after onset of lymphadenopathy. This observation is consistent with previous studies which noted that high titers in the dye test are not specific for the acute infection (7, 16). In regard to the DS-IgM-ELISA, Brooks et al. (1) reported that 50% of patients tested 7 to 12 months after onset of lymphadenopathy still had a titer of -2. Our results in the present study were similar. Thus, in cases of toxoplasmic lymphadenopathy the IgM-ELISA titers decrease slowly and in many patients do not become negative for more than a year. In regard to the differential agglutination test in which Formalin-fixed and acetone-fixed antigens were used (13), the results in the present study are consistent with those of a study of a large number of non-acquired immune deficiency syndrome patients by Dannemann et al. (submitted for publication). In that study it was difficult to precisely estimate from the pattern of the results of the agglutination test with Formalin-fixed antigens and acetone-fixed antigens when the patient acquired the infection. There was a wide range in months from onset of the acute pattern in the results of the agglutination test using Formalin- and acetone-fixed antigens to the time the results converted to a chronic pattern. Whether our data can be extrapolated to immunocompetent adults with suspect toxoplasma infection but without lymphadenopathy is not known. It has been reported that patients who exhibit lymphadenopathy following acute infection with T. gondii differ in their immune response to T. gondii from those with other manifestations of toxoplasma infection (5, 11). However, in a preliminary study, each of the serum specimens from six mothers of babies with congenital toxoplasma infection showed positive values (mean value of the six patients = 17) in the AC-ELISA (unpublished observations). ACKNOWLEDGMENTS We thank Yun S. Park, Dorothy Gibbons, and Pamela Stepick- Biek for their technical assistance. This work was supported in part by grants from the Ohyama Health Foundation and the Ministry of Education, Science, and Culture of Japan, by Public Health Service grant A from the National Institutes of Health, and by a MacArthur Foundation grant in molecular parasitology. LITERATURE CITED 1. Brooks, R. G., R. E. McCabe, and J. S. Remington Role of serology in the diagnosis of toxoplasmic lymphadenopathy. Rev. Infect. Dis. 9: Dorfman, R. F., and J. S. Remington Value of lymphnode biopsy in the diagnosis of acute acquired toxoplasmosis. N. Engl. J. Med. 289: Krick, J. A., and J. S. Remington Toxoplasmosis in the adult-an overview. N. Engl. J. Med. 298: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Luft, B. J., G. Kansas, E. G. Engleman, and J. S. Remington Functional and quantitative alternations in T lymphocyte subpopulations in acute toxoplasmosis. J. Infect. Dis. 150: Naot, Y., and J. S. 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5 1738 SUZUKI ET AL. J. CLIN. MICROBIOL. Antigen(s) responsible for immunoglobulin G responses specific for the acute stage of Toxoplasma infection in humans. J. Clin. Microbiol. 26: Thulliez, P., J. S. Remington, F. Santoro, G. Ovlaque, S. D. Sharma, and G. Desmonts Une nouvelle reaction d'agglutination pour le diagnostic stade evolutif de la toxoplasmose acquise. Pathol. Biol. 34: Towbin, H. T., T. Staehelin, and J. Gordon Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: Welch, P. C., H. Masur, T. C. Jones, and J. S. Remington Serologic diagnosis of acute lymphadenopathic toxoplasmosis. J. Infect. Dis. 142: Wilson, C. B., V. Tsai, and J. S. Remington Failure to trigger the oxidative metabolic burst by normal macrophages: possible mechanism for survival of intracellular pathogens. J. Exp. Med. 151: