Copyright  © 2000 Medicina On line - Revista Virtual de Medicina
Volume 1- Número 1- Ano I (Jan/Fev/Mar de 1998)
Analysis of aqueous humor in ocular toxoplasmosis: Detection of low avidity IgG specific to Toxoplasma gondii.
Vinhal, F.A.1,
Pena, J.D.O.1,
Katina, J.H.2,
Brandao, E.O.1,
Silva, D.A.O.1,
Orefice, F.2
and Mineo, J.R.1*
From the Laboratory of Immunology,
Federal University of Uberlândia - MG, Brazil1,
Uveitis Center, Faculty of Medicine,
Federal University of Minas Gerais2
SUMMARYUsing a modified enzyme-linked immunosorbent assay that included dissociation of antigen antibody complexes with 6M urea solution, we analyzed the avidity of  toxoplasma-specific IgG in aqueous humor and serum samples from 24 patients with toxoplasmic chorioretinitis. As a control, we studied aqueous humor and serum samples from 14 cataract patients without history of uveitis and serum samples from 10 patients with recent primary systemic toxoplasmic infection without ocular lesion.
IgG avidity was markedly lower in aqueous humor samples from patients with toxoplasmic chorioretinitis than in serum samples, despite those samples presenting higher levels of  toxoplasma-specific IgG than in serum samples.
The detection of the low-avidity toxoplasma-specific antibodies can offer a valuable aid to make a specific etiologic diagnosis and perhaps contribute to understand the pathogenic mechanisms of ocular toxoplasmosis.
INTRODUCTION
Human toxoplasmosis is caused by the intracellular protozoan parasite Toxoplasma gondii   that infects virtually every eucariotic cell. Because this microorganism is ubiquitous in nature, it produces one the most common infections of humans with approximately 500 million people throughout the world having antibodies to this parasite (Kean 1972).  Also, it is the most common cause of retinochoroiditis worldwide in man with figures ranging from 28 to 55% of all cases of posterior uveitis  (Newman et al. 1982;  O'Connor  & Hogan 1967).
The diagnosis of toxoplasmic retinochoroiditis is based mainly on the typical clinical picture and a positive serological test for toxoplasmosis.  However, other inflammatory processes such as tuberculosis, syphilis and candidiasis, and even noninflammatory conditions may mimic this entity, thus emphasizing the need for an accurate differential diagnosis (Turunen et al. 1983).  In AIDS patients, any opportunistic infection could conceivably also cause retinitis or chorioretinitis, so it will tax the diagnostic skills of the ophthalmologist to focus the differential diagnosis on the possibility of toxoplasmic retinochoroiditis (Holland et al. 1988; Pavesio et al. 1992;  Smith 1988).
Conventional serology is of little value in ocular toxoplasmosis because significant changes of toxoplasma antibody levels in serum are  rarely observed.  In cases where the clinical picture is atypical, or the vitreous involvement is so marked that it obstructs the view of the fundus, or even in cases in which other infectious diseases must be considered, studies of the  aqueous humor may be helpful. Analysis of the aqueous humor may provide evidence of specific antibody formation by cells of the uveal tract, especially if more antibody per unit of immunoglobulin can be detected in the aqueous humor than in the serum (Kijlstra 1986;  McCabe & Remington 1983;  O'Connor 1957;  O'Connor 1974).  However, the determination of this coefficient has not been measured by standardized techniques by different laboratories and this fact makes  the interpretation of the results difficult.
An enzyme-linked immunosorbent assay (Avidity-ELISA) which measures the antigen binding avidity of toxoplasma specific IgG antibodies using urea to elute low avidity IgG was reported recently (Camargo et al. 1991; Hedman et al. 1989; Joynson et al. 1990).  It was shown that serum samples from patients with acute toxoplasmosis had low avidity IgG whereas sera from patients with chronic infection had high avidity IgG.
In the present study we reported the results that were obtained by using this assay for avidity measurement of the toxoplasma specific IgG antibodies in  aqueous humor and serum samples from material diagnosed to be of toxoplasmic chorioretinitis origin by clinical and laboratory evidence.
SUBJECTS AND METHODS
PATIENTS AND SAMPLES:
Forty-eight serum samples and thirty-six aqueous humor samples from forty- eight patients were studied. Each patient had toxoplasma-specific IgG antibody as measured by an indirect toxoplasma IgG-ELISA, indirect IgG-immunofluorescence and passive hemagglutination tests with titers equal or higher than 1:16. These patients were divided into three groups based on the clinical picture as following:
  • Group I: This group consisted of twenty four patients with acute posterior uveitis. All of these patients presented typical lesions of recurrent active toxoplasmic chorioretinitis and were studied from 10 days to 12 weeks after the onset of ocular symptoms. This group consisted of thirteen males and eleven females, ranging in age from  13 to 58 years with the mean being 26 years. One serum sample from each patient was obtained and at the same time aqueous humor samples from twenty three patients were obtained as described. (18)    The amount  of  aqueous  humor  obtained was approximately 200 ul per patient.
  • Group II: Fourteen cataract patients without clinical history of uveitis were studied as controls. This group consisted of six males and eight females ranging in age from 21 to 79 years with the mean being 46 years. One serum sample from each patient,  and aqueous humor samples from thirteen patients were obtained as described above.
  • Group III: Ten patients with recent primary  systemic toxoplasma infection without clinical history of ocular lesions were also studied. This group was included  as positive control of avidity assay and only serum samples were collected from these patients.
All the serum and aqueous humor samples were collected and preserved  at -20°C until tested.
TOXOPLASMA ANTIGEN:
Toxoplasma gondii  (RH strain) was grown intraperitoneally in Swiss mice for 48-72 hours (17)    The peritoneal exudate was obtained and the parasites were purified as described. (17)   Organisms were pelleted by centrifugation and the pellet was suspended in distilled water and sonicated.   Solution of 1.7M NaCl was added at the same volume and the lysate was centrifugated for 30 min at 10,000 g. The supernatant was collected for use as toxoplasma antigen in avidity-ELISA.
AVIDITY-ELISA:
An immunoenzymatic assay was performed to isolate low avidity IgG from immobilized parasite antigen. (2,4)     In this assay,  low avidity IgG antibody is dissociated from antigen, whereas high avidity antibodies remain bound. For this, the toxoplasma antigen was diluted with 0.01 M Carbonate pH 9.6 and used at a concentration of 2.5 ?g protein/ml for coating ELISA PVC microtiter plates (Hemobag, SP, Brazil). After treatment with the antigen for 18 Hs at 4°C, the plates were washed three times with phosphate-buffered saline containing 0.05% Tween 20  (PBST).  The serum and aqueous humor samples were diluted at 1:10 and 1:1,000 respectively and they were applied in duplicate to the microtiter wells. After incubation for 60 min (serum samples) or 90 min (aqueous humor samples) at 37°C, one well of each doublet was washed (three times, 5 min each) with PBST  The other well was washed one time ( 5 min) with 6M urea dissolved in PBST and two times (5 min each) with PBST only. The residual antigen-bound IgG was detected with antibody to human IgG conjugated to peroxidase (type VI, Sigma Co., St.Louis, MO) according method described by Wilson and Nakane. (19)   The conjugate was applied for 60 min at 37°C, washed with PBST, and revealed by substrate solution  consisting of 0.4 mg of o-phenylenediamine per ml diluted in 0.1M citrate-Na2HPO4  buffer (pH 5.5) activated by 0.005% (vol/vol) H202 . After incubation for 15 min at room temperature, the reaction was stopped with 2N H2SO4. The intensity of the color reaction was measured with a microwell
reader system (model 210, Organon Teknika, Belgium) at a wavelength of 492 nm.
STATISTICS:
Two values were obtained for each group of serum or aqueous humor samples, one for the toxoplasma antibody washed with urea (urea+) and one for the antibody washed without urea (urea-).  The results were expressed as  the mean (+/- standard deviation) absorbance value observed for each group. The urea-treatment ratio (U.T.R.) was calculated and expressed in percentage [A492 (urea+) / A492 (urea-)] x 100.  The  Student t  test was applied to paired or unpaired samples to compare  experiments among the groups. Differences were considered significant when p < 0.05.
RESULTS
Figure 1 shows the results obtained in the avidity ELISA of serum samples. When the toxoplasma-bound antibodies were washed with PBS without 6M urea, the mean  (+/- standard deviation)  absorbances for the groups I, II and III were .386 (+/-134), .332 (+/-.170) and .527 (.+/-262), respectively.  After wash with 6M urea-PBS, the results became .360 (+/-.152), .294 (+/-.104) and .373 (+/-.227). The comparison revealed no statistically significant differences (p > 0.05).
Figure 2 shows the results obtained in the avidity ELISA with aqueous humor samples. When total toxoplasma IgG was measured,  the mean (+/- s.d.)  absorbance values were .676 (+/-.262) and .172 (+/-.021) for the groups I and II, respectively. The values for the remaining toxoplasma-bound antibodies were .509 (+/-.180) and .164 (+/-.040).  Comparison of the results of these groups revealed significant differences between the total antibodies and  the antibodies bound to toxoplasma after washing with 6M-urea-PBS (p < 0.0001).
Figure 3 shows the urea-treatment ratios in the avidity ELISA for serum and aqueous humor samples.
For serum samples, the UTR values of groups I, II and III were  6.7%,  11.4%   and 29.2% respectively.  Comparison of the results of groups I and II showed no statistically significant difference (p = 0.41). However, the results obtained with both groups were significantly different when compared with group III   ( p = 0.001 and 0.012,  respectively).
For aqueous humor samples, the UTR values of groups I and II were 24.7% and 4.6%, respectively. These results showed a significant difference   (p = 0.002). The comparison between UTR values for aqueous humor and serum samples of group I also revealed a significant difference (p = 0.001).  On the other hand, the difference between the sera and aqueous humor values of group II  wasn't  statistically significant (p = 0.49).
 Fig. 1. Avidity of toxoplasma-IgG in serum samples from patients with toxoplasmic chorioretinitis (Group I), cataract patients (Group II) and patients with recent primary systemic toxoplasma infection (Group III). Antigen-bound antibodies were washed with 6M urea solution or without urea and the antibody levels were determined by enzyme-labelled antibody. The intensity of the reaction was measured at 492 nm.
Fig. 2. Avidity of toxoplasma-IgG in aqueous humor samples from patients with toxoplasmic chorioretinitis (Group I) and cataract patients (Group II).  Antigen-bound antibodies were washed with 6M urea solution or without urea and the antibody levels were determined by enzyme-labelled antibody. The intensity of the reaction was measured at 492 nm.
Fig. 3. Avidity of toxoplasma-IgG in aqueous humor and serum samples from patients with toxoplasmic chorioretinitis (Group I), cataract patients (Group II)  and serum samples from patients with recent primary systemic toxoplasma infection  (Group III). Antigen-bound antibodies were washed either with 6M urea (urea+) or without urea (urea-) and the residual antigen-bound IgG was detected by enzyme-labelled antibody. U.T.R. = ratio (urea+:urea-) of the respective intensity of the reaction measured at 492 nm.
DISCUSSION

Only a few laboratory tests are currently useful to the ophthalmologist in the examination of patients with uveitis. 1    In cases of severe uveitis in which clinical examination and laboratory testing have not provided any answers concerning the cause, the assessment of local intraocular antibody production may be useful. Aqueous humor analysis is now used in cases of severe uveitis in various centers. 8      Analysis of vitreous humor samples for local intraocular antibody production, however, has not been widely reported probably because obtaining  enough volume of undiluted vitreous humor samples is not always easy and not always without hazards. 1
By using the dye test, detailed studies about the synthesis of intraocular toxoplasma antibodies have been carried out. 6   The dye test has been widely replaced by immunoassays in the serological diagnosis of toxoplasmosis because of the inherent technical problems of the dye test.6   To study the intraocular synthesis of toxoplasma antibodies during ocular toxoplasmosis,  a detailed study was conducted by using a modified enzyme immunoassay. 6  The results indicated that IgG-class anti-toxoplasma antibodies are produced locally within the eye in cases of toxoplasmic chorioretinitis and it was concluded that the detection of these antibodies can offer a valuable aid to make a specific etiologic diagnosis of ocular toxoplasmosis.  However, the quantification of the levels of antibodies to unrelated antigens offers indirect evidence that the increased toxoplasma specific IgG antibody levels in the aqueous humor were not a result of increased capillary permeability or polyclonal antibody stimulation.
In our study we demonstrated directly that IgG antibodies in aqueous humor samples from patients with toxoplasmic chorioretinitis are different from antibodies present in their serum samples not only in terms of level but also in terms of the avidity to toxoplasma antigen. Despite the higher level of the toxoplasma specific IgG antibodies in aqueous humor samples, these molecules present lower avidity to Toxoplasma gondii.   The avidity of toxoplasma specific IgG antibodies was measured as a function of hydrogen bond dissociation. The hydrogen-bond disrupting agent, urea, was used to elute IgG from the immobilized antigen. As a result, IgG antibodies of low avidity were dissociated in conditions where high-avidity antibodies mostly have remained antigen-bound. Recently, it was demonstrated by using sodium thiocyanate to dissociate the antigen-antibody complexes that low-avidity antibody to retinal S-antigen in serum samples was more prevalent in patients with acute retinal vasculitis than in healthy subjects. 5     It was discussed that probably the association of low-avidity antibody with normal levels of circulating immune complexes may suggest defective regulation of antiretinal autoimmunity and has important pathogenic implications. Nevertheless, in that study that included subjects with ocular toxoplasmosis, it was not measured the avidity of the IgG antibody against Toxoplasma gondii  neither in the serum nor  in the aqueous humor samples. It was described in another study that patients with ocular toxoplasmosis present lymphocyte proliferative responses to both parasite and retinal antigens. 11  In this study, 16 (40%) of 40 patients had positive in vitro response to the retinal S-antigen. The proliferative responses to the p22 toxoplasma membrane antigen approached that of a crude antigen preparation of Toxoplasma gondii.  On the other hand,  the response to the p30, the immunodominant membrane antigen, was considerably less striking. They postulated that the immune response to the p22 toxoplasma antigen may increase the risk of developing  ocular disease or a sub strain of toxoplasma with p22 as its immunodominant membrane antigen, p30, may have a greater affinity for the retina.
Our results imply a major difference between serum and aqueous humor samples from patients with toxoplasmic chorioretinitis in comparison with samples from subjects without clinical history of uveitis. Those patients present high levels of toxoplasma-specific IgG antibodies in aqueous humor samples and these molecules present low avidity to toxoplasma antigen.
Beyond its utility as a diagnostic tool for specific etiologic diagnosis of toxoplasmic chorioretinitis, the measurement of IgG-avidity in the aqueous humor may also contribute to understand the pathogenic mechanisms of ocular toxoplasmosis.

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