November 2010
Volume 51, Issue 11
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Clinical and Epidemiologic Research  |   November 2010
Contribution of anti-Hsp70.1 IgG Antibody Levels to the Diagnostic Certainty of Clinically Suspected Ocular Toxoplasmosis
Author Affiliations & Notes
  • Bernabé F. F. Chumpitazi
    From the Laboratory of Parasitology and Mycology,
  • Laurence Bouillet
    the Departments of Internal Medicine and
    INSERM (Institut National de la Santé et de la Recherche Médicale) Unit 882, CEA (French Atomic Energy and Alternative Energies Commission)-Grenoble, and
  • Hélène Fricker-Hidalgo
    From the Laboratory of Parasitology and Mycology,
  • Tiffany Lacharme
    Ophthalmology,
  • Jean-Paul Romanet
    Ophthalmology,
  • Christian Massot
    the Departments of Internal Medicine and
  • Christophe Chiquet
    Ophthalmology,
  • Hervé Pelloux
    From the Laboratory of Parasitology and Mycology,
    the CNRS (Centre National de la Recherche Scientifique) UMR (Unité Mixte de Recherche) 5163, Joseph Fourier University (Grenoble 1), Grenoble, France.
  • Corresponding author: Bernabe F. F. Chumpitazi, Laboratoire de Parasitologie-Mycologie, DAI, Pôle Biologie, CHU de Grenoble, Hôpital Michallon, BP 217, F-38043, Grenoble, France; bchumpitazi@chu-grenoble.fr
Investigative Ophthalmology & Visual Science November 2010, Vol.51, 5530-5536. doi:10.1167/iovs.10-5244
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      Bernabé F. F. Chumpitazi, Laurence Bouillet, Hélène Fricker-Hidalgo, Tiffany Lacharme, Jean-Paul Romanet, Christian Massot, Christophe Chiquet, Hervé Pelloux; Contribution of anti-Hsp70.1 IgG Antibody Levels to the Diagnostic Certainty of Clinically Suspected Ocular Toxoplasmosis. Invest. Ophthalmol. Vis. Sci. 2010;51(11):5530-5536. doi: 10.1167/iovs.10-5244.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Laboratory diagnosis of ocular toxoplasmosis, the major cause of posterior uveitis worldwide, can be improved. Heat shock protein (Hsp) 70 is involved in cellular infection by Toxoplasma gondii but also in the immune response to this parasite. The authors postulate that infected patients may exhibit serum IgG anti-Hsp70.1 antibodies and that determining the presence of these antibodies could improve the diagnosis of suspected ocular toxoplasmosis.

Methods.: This retrospective case–control study included 26 laboratory-confirmed cases of ocular toxoplasmosis (group A), 41 clinically suspected cases (group B), and 67 currently healthy blood donors who were chronically infected with T. gondii (group C). Laboratory and clinical data were analyzed according to the ocular presentation and Goldmann-Witmer's coefficient. Serum and aqueous humor were sampled at the time of uveitis. Serum anti-Hsp70.1 antibody levels were obtained by ELISA. The probability of ocular toxoplasmosis was estimated by a logistic regression analysis that combined data from serum IgG anti-Hsp70.1 and aqueous-humor IgG anti-T. gondii antibody levels.

Results.: Serum IgG anti-Hsp70.1 antibody levels were significantly increased in groups A and B when compared to the levels in control group C (P ≤ 0.0034). These levels correlated with the retinal lesion size (r = 0.301; P < 0.0349). Logistic probability and anti-Hsp70.1 antibodies in sera confirmed that 10 of 23 cases in group B were true ocular toxoplasmosis.

Conclusions.: Anti-Hsp70 may play a role in the immunopathogenesis of ocular Toxoplasma infection. This study showed that the anti-Hsp70.1 antibody and the logistic probability test can confirm clinically suspected ocular toxoplasmosis.

Toxoplasma gondii is the microorganism most frequently responsible for posterior uveitis, which usually presents as an active creamy-white focal retinitis, with or without hyperpigmented retinochoroidal scarring. 1,2 Ocular toxoplasmosis (OT) may lead to a poor visual prognosis, especially when the macula is impaired or when complications occur. 3 Atypical eye lesions may include punctuate outer retinal toxoplasmosis, retinal vasculitis, a unilateral pigmentary retinopathy mimicking retinitis pigmentosa, neuroretinitis, papillitis, and pseudomultiple retinochoroiditis. 4 For the diagnosis of OT, the sensitivity of the Desmonts' coefficient also called the Goldmann-Witmer coefficient (GWC) is 74%, with a specificity of 100%. 5,6 In cases of atypical OT, GWC sensitivity may decrease to 45% and specificity to 93%. 7 Despite improvement in diagnostic tests such as polymerase chain reaction (PCR) and quantification of anti-Toxoplasma antibodies, 8 10 the sensitivity of the laboratory diagnosis remains insufficient at the present time. 
During inflammation, the eye's immune tolerance is disrupted, but the repercussions on systemic immunity are not well understood. For example, local antibody production is usually observed during infection, although MHC class II expression is low and limits antigen presentation. 11 A localized breakdown of the blood–retinal barrier (BRB) or migration of T. gondii-infected leukocytes through the BRB, allowing retinal infection by active tachyzoites of T. gondii and subsequently by encysted bradyzoites, may induce failure of the eye's tolerance and the development of localized inflammation. The encysted bradyzoites are resistant to treatment and to the immune system. 12 These events may explain why retinal lesions and relapses appear many years after the initial infection by T. gondii. 13  
Heat shock protein 70 (Hsp70) is upregulated during cellular stress and is involved in protein-folding as well as in intracellular antigen processing and in the inflammatory process. 14 16 Increased expression of bag1/Hsp30 and Hsp70 has been associated with bradyzoite development and also with the conversion of bradyzoites to tachyzoites during Toxoplasma reactivation. 17,18 The expression of T. gondii Hsp70 is associated with the virulent strains and is increased just before the death of the host cell. 19,20 Hsp70 is released from cells undergoing necrosis and/or through physiological secretion mechanisms. It enters the bloodstream and therefore can act at distant sites in the body. 21 Moreover, vaccination with the T. gondii Hsp70 gene induces significant protection against Toxoplasma infection in B6 and BALB/c mice. 22 Anti-Hsp70 IgG autoantibody, produced by B-1 cells of C57BL/6 mice, has been observed after T. gondii infection. 23,24 To our knowledge, the presence of human anti-Hsp70.1 antibody has not yet been investigated in patients with clinically suspected OT. In this study, we hypothesized that anti-Hsp70 antibodies are present in the sera of patients with clinically suspected OT. The objective was to assess the potential value of serum IgG anti-Hsp70.1 as a diagnostic tool for the laboratory diagnosis of OT in patients in whom the diagnosis cannot be confirmed by using available techniques. 
Materials and Methods
Patient and Control Groups
Our research adhered to the tenets of the Declaration of Helsinki. This retrospective case–control study included 26 patients with clinically suspected and laboratory confirmed OT (group A) and 41 patients with a clinically suspected OT but lacking laboratory confirmation (group B) from 2002 to 2007 and managed by the Grenoble University Hospital (Table 1). Briefly, the clinical diagnosis of OT required at least one active creamy-white focal retinal lesion in either eye combined or not with hyperpigmented retinochoroidal scars in either eye 2 , defined as:
  1.  
    Primary OT presenting an active retinal lesion without retinochoroidal scarring. 25
  2.  
    Recurrent OT presenting an active retinal lesion with pigmented retinochoroidal scars in either eye.
Table 1.
 
Characteristics of the Patients at the Beginning of the Study
Table 1.
 
Characteristics of the Patients at the Beginning of the Study
Parameter Group A Group B Group C
Ocular toxoplasmosis Confirmed Suspected Absent
Patients, n 26 41 67
Age, y (mean ± SD) 41.0 ± 19.3 40.2 ± 18.8 43.6 ± 12.2
    Range 18–80 10–73 18–64
Male/female 12/14 15/26 34/33
GWC ≥2 (n = 23)* ≤2 (n = 38)† Not done
DNA T. gondii PCR Positive (n = 3) Negative (n = 3) Not done
Serum anti-Toxoplasma IgG, IU/mL, median (range) 92 (15–300) 80 (17–300) 61 (25–175)
Aqueous humor anti-Toxoplasma IgG, IU/mL, median (range) 2.7 (0.4–27.7) 0.1 (0–1.6) Not done
Probably acute/acquired toxoplasmosis, n 1/24 5/32 2/65
Affected eyes, n 27 43 0
For each patient, we collected the following clinical data: date of examination, presence and location of cicatricial lesions, any active inflammation related to posterior and anterior segments, treatment of each new episode, and follow-up (median, 10.4 months). Underlying diseases, immunosuppression, and other causes of uveitis were also determined from patient files. Laboratory analyses included the GWC and T. gondii DNA detection by PCR. 6,26,27 The patients were then grouped according to clinical and laboratory criteria (Table 1). 
Sixty-seven healthy blood donors who were positive for IgG anti-Toxoplasma antibodies constituted the control group C (Table 1). This group, mainly chronically infected by T. gondii, was considered to be a nonuveitis group. 
Anti-Toxoplasma Therapy
For patients with active toxoplasmic chorioretinitis, the anti-Toxoplasma treatment was pyrimethamine (1 mg/kg/d), sulfadiazine (100 mg/kg/d), and folinic acid (50 mg/wk) for at least 4 weeks. A combination of antiparasite drugs with corticosteroids was usually administered for periods limited to 1 or 2 months for active toxoplasmic lesions near the macula or optic nerve or in cases of severe intraocular inflammatory response. 
Anti-Toxoplasma Antibodies
To determine the patients' specific serologic characteristics, all three groups were screened for serum IgG and IgM anti-Toxoplasma antibodies (Toxo IgG, IgM; BioMérieux, Marcy l'Etoile, France). 
Anti-Hsp70.1 Antibodies
Anti-Hsp70.1 levels were assayed by ELISA, as described recently, 28 but with some modifications: (1) a coating for the laboratory plates was prepared containing 100 μL of human recombinant Hsp70 (NSP-555; Stressgen- Tebu, Saint Quentin-Yvelines, France) per well at 2 μg protein/milliliter; (2) each assay was repeated three times; (3) the wells were incubated with 100 μL of sera diluted to 1:200 for 1 hour at 37°C; and (4) the enzymatic activity was developed with 0.75 mg/mL o-phenylene-diamine dihydrochloride and 0.03% sodium perborate in 0.05 M citrate phosphate buffer [pH 5], for 10 minutes at room temperature in the dark. The reaction was stopped with 200 μL of 2 N H2SO4, and the absorbance was read at 492 nm. Reproducibility for intra- and interassays was higher than 95%. The validation of the test was confirmed by inhibition of the antibody response with human recombinant Hsp70 (5 μg/mL). 28  
Determination of Cutoffs
The most appropriate cutoff level of IgG anti-Hsp70.1 antibodies was determined as previously described. 28 Briefly, the cutoff was selected by the best addition of Youden's index and Yule's Q coefficient and specificity ≥83%. The cutoffs were calculated for serum IgG anti-Hsp70.1, aqueous humor (AH) IgG anti-Toxoplasma antibodies, and the logistic probability test. 
Statistical Analysis
For data with normal distribution, the mean, standard deviation, and the range are given. Otherwise the median and the 10th and 90th percentiles are reported. Nonparametric tests were used. The correlations were established with Z correlation coefficients. The differences between data sets were evaluated, first by the Kruskal-Wallis test (three groups) and then by the Mann-Whitney U test (two groups). 
Logistic Test
A logistic regression analysis was performed, as described by Fried et al. 29 The included variables were the levels of serum IgG anti-Hsp70.1 and AH IgG anti-Toxoplasma antibodies. The GWC values were not included in this analysis, because they were used to define groups A and B. The number of analyzed cases was 24 for group A and 38 for group B, since paired corresponding data were required for IgG anti-Hsp70.1 and AH IgG anti-Toxoplasma antibodies. Group C was not included in this analysis. 
Results
No Significant Difference in Serum IgG or IgM Anti–T. gondii Antibody Levels in the Three Study Groups
The serum anti-Toxoplasma IgG or IgM antibody levels were examined in relation to OT (Fig. 1). The control group of healthy donors (group C) as well as the suspected (group B) and confirmed (group A) toxoplasmic uveitis groups did not show any statistically significant difference in the levels of IgG and IgM anti-Toxoplasma antibodies (Kruskal-Wallis test for the three groups; P = 0.29 and P = 0.54, respectively). 
Figure 1.
 
Serum antibody anti–Toxoplasma gondii obtained by enzyme-linked fluorescence assay (ELFA) in control, suspected, and ocular toxoplasmosis patients. IgG (A) and IgM (B) antibody anti–Toxoplasma gondii levels observed by ELFA in sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B) and control (C) patients. All data represent one sample per patient. The box-and-whisker plots show the median of 26 (A), 40 (B), and 67 (C) measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and the 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. The P-values for data from (A) and (B) were 0.29 and 0.54 (Kruskal-Wallis' test for the three groups), respectively. No significant differences were observed between the groups.
Figure 1.
 
Serum antibody anti–Toxoplasma gondii obtained by enzyme-linked fluorescence assay (ELFA) in control, suspected, and ocular toxoplasmosis patients. IgG (A) and IgM (B) antibody anti–Toxoplasma gondii levels observed by ELFA in sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B) and control (C) patients. All data represent one sample per patient. The box-and-whisker plots show the median of 26 (A), 40 (B), and 67 (C) measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and the 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. The P-values for data from (A) and (B) were 0.29 and 0.54 (Kruskal-Wallis' test for the three groups), respectively. No significant differences were observed between the groups.
Significant Difference in the Levels of Serum Anti-Hsp70.1 IgG Antibodies among the Three Groups
A significant difference (P < 0.0001) was found in the levels of serum IgG anti-Hsp70.1 antibodies in the three groups (Fig. 2). A further two-by-two analysis of groups was performed that showed a significant difference in the levels of serum IgG anti-Hsp70.1 antibodies between group B (median, 0.353) and group C (median, 0.214; P < 0.0001; Fig. 2). A significant difference was also identified in the levels of serum IgG anti-Hsp70.1 antibodies between groups A (median, 0.446) and C (median, 0.214; P = 0.0034; Fig. 2). 
Figure 2.
 
Serum levels of anti-Hsp70.1 antibodies in blood donors and in patients with suspected toxoplasmic uveitis. IgG antibody anti-Hsp70.1 levels observed by ELISA in the sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B), and control (C) patients. The box-and-whisker plots show the median of 26 (A), 41 (B), and 67 (C) averages of three measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. All data represents one sample per patient.
Figure 2.
 
Serum levels of anti-Hsp70.1 antibodies in blood donors and in patients with suspected toxoplasmic uveitis. IgG antibody anti-Hsp70.1 levels observed by ELISA in the sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B), and control (C) patients. The box-and-whisker plots show the median of 26 (A), 41 (B), and 67 (C) averages of three measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. All data represents one sample per patient.
Kinetics of Serum Anti-Hsp70.1 IgG Antibodies in Clinically Suspected Ocular Toxoplasmosis
We analyzed the anti-Hsp70.1-antibody kinetics in two patients: one with laboratory confirmed OT (Fig. 3A) and one classified as having true clinically suspected OT (Fig. 3B). 
Figure 3.
 
Kinetics of serum IgG anti-Hsp70.1 antibodies in patients with suspected toxoplasmic uveitis. (A) A patient with confirmed ocular toxoplasmosis. A significant decrease (16.7%) in the marker was observed (from point 2 to 3). Point 1 served as the reference background for this patient. (B) A patient with suspected ocular toxoplasmosis. Within a week, a significant increase (50.9%) in the marker was observed (from point 1 to 2).
Figure 3.
 
Kinetics of serum IgG anti-Hsp70.1 antibodies in patients with suspected toxoplasmic uveitis. (A) A patient with confirmed ocular toxoplasmosis. A significant decrease (16.7%) in the marker was observed (from point 2 to 3). Point 1 served as the reference background for this patient. (B) A patient with suspected ocular toxoplasmosis. Within a week, a significant increase (50.9%) in the marker was observed (from point 1 to 2).
A sample collected before the uveitis episode was used as the patient's reference background signal (0.952 ± 0.014 for patient 42 and 0.377 ± 0.013 for patient 19). A significant increase (50.9%) in the marker was observed in the next week, from points 1 to 2 (0.569 ± 0.002; Fig. 3B), and a significant decrease (28.8%) in the marker was seen in the 3 months after the episode, from points 3 to 4 (0.385 ± 0.018; Fig. 3B). Thus, serum IgG anti-Hsp70.1 antibodies increased during the toxoplasmic uveitis episode. 
Value of Serum Anti-Hsp70.1 Antibodies for Confirmation of Clinically Suspected Cases of OT
After taking into account the sensitivities of the tests used and the number of cases in group A, the number of biologically confirmed OT cases was nine. 
After an update of the clinical records of group B, the OT clinical diagnosis was confirmed for 23 of 41 patients of group B. Among the initially suspected OT cases that were finally discarded we found:
  1.  
    Ten patients who were negative for anti-Hsp70.1 antibodies or the logistic probability test: Three cases of viral uveitis were confirmed by PCR (CMV, herpes simplex virus, and Varicella zoster virus), two of Behçet's disease, one of postinfection encephalitis due to T. gondii, one of neuroretinitis, one of iridocyclitis, one of idiopathic intermediate uveitis, and one of idiopathic retinitis and papillitis.
  2.  
    Five patients were positive for anti-Hsp70.1 antibodies: two with idiopathic panuveitis, one with sarcoid-panuveitis, one with encephalitis, and one with Candida chorioretinitis.
  3.  
    Three patients were negative for anti-Hsp70.1 antibodies, with no value for the logistic probability test (lack of AH anti-T. gondii antibody data): one had CMV retinitis, one idiopathic uveitis, and one acute neuroretinitis.
After performing a logistic regression analysis, we obtained the equation   where P(x) is the logistic probability of having OT, x 1 is a dichotomous variable representing the IgG anti-Hsp70.1 antibody obtained with a cutoff of 0.462, and x 2 is the IgG anti-Toxoplasma antibody in AH (IU/mL). The odds ratio for x 1 was 2.52 (95% CI, 0.73–8.67) and for x 2 was 1.34 (95% CI, 1.04–1.72). 
The characterizations of the serum anti-Hsp70.1 IgG antibody, the AH anti-Toxoplasma IgG antibody, and the logistic test are shown in Table 2. The results of the logistic test improved the distinction between groups A and B (Table 2). In group A, 16 of the 24 patients (two immunocompromised patients did not have AH anti-Toxoplasma IgG records) were reconfirmed by the logistic test. Five patients in group B with a positive logistic test result were reclassified to the group of confirmed OT and are listed in Table 3. None of these patients had a viral etiology (negative viral PCR, data not shown). Given the clinical data, which are essential, and the logistic test results, four of five patients were confirmed as having OT (Table 3). The logistic test associated with GWC data improved the diagnosis of the OT cases. 
Table 2.
 
Discrimination between Groups with Confirmed and Clinically Suspected OT According to the Logistic Test
Table 2.
 
Discrimination between Groups with Confirmed and Clinically Suspected OT According to the Logistic Test
Parameter Antibody Logistic Test P(x) *
Serum anti-Hsp70.1 IgG AH IgG anti-Toxoplasma
Youden's index 0.335 0.350 0.535
Yule's Q coefficient 0.670 0.687 0.859
Cutoffs 0.462 1.1 IU/mL 0.395
Confirmed OT group (A; n = 26), % (95% CI) 50 (32–68)† 51 (36–67)† 67 (47–82)†
Clinically suspected OT group (B; n = 41), % (95% CI) 24 (14–39)† 16 (9–28)† 13 (6–27)†
Nonuveitis control group (C; n = 67), % (95% CI) 12 (6–22)† Not done Not done
Sensitivity (%) 50 51 67
Specificity (%) 83 84 87
Positive predictive value (%) 42 68 76
Negative predictive value (%) 87 72 80
Table 3.
 
Patients with Clinically Suspected OT (Group B) with a Positive Logistic Test Result
Table 3.
 
Patients with Clinically Suspected OT (Group B) with a Positive Logistic Test Result
Parameter Patient
1 2 3 4 5
Age, y/sex 41/Female 71/Male 55/Female 58/Female 44/Female
Clinical signs Panuveitis Panuveitis, macular edema Panuveitis Posterior uveitis Panuveitis
Chorioretinal lesions, n Right 1 Right 1 0 Right 1 Left 1
Lesion size in papillary diameter 1.5 3 / 1 2
Lesion localization Peripapillary Macula and peripheral / Peripheral Nasal and superior juxta-papillary
Therapy Pyrimethamine and sulfadiazine TMP-SMX and triamcinolone No Pyrimethamine and sulfadiazine Pyrimethamine and sulfadiazine
Immunocompromised Yes No No No No
Underlying disease AIDS Polyangiitis Sarcoidosis None None
GWC 1.45 1.32 0.2 1.52 0.2
Logistic probability of having OT, P(x) * 0.821 0.628 0.474 0.474 0.482
Final clinical diagnosis Acquired OT Retinal detachment and OT Sarcoidosis Recurrent OT Acquired OT
Treatment outcome Negative Favorable Negative Favorable Favorable
In group B, six patients with anti-Hsp70.1 antibody in sera and with a negative logistic test result were analyzed (Table 4). Five had clinical OT (primary parameter) and were thus considered to be confirmed cases, and one had Candida chorioretinitis. 
Table 4.
 
Patients with Clinically Suspected OT (Group B) with Positive anti-Hsp70.1 Antibody Serology and a Negative Logistic Test Result
Table 4.
 
Patients with Clinically Suspected OT (Group B) with Positive anti-Hsp70.1 Antibody Serology and a Negative Logistic Test Result
Parameter Patient
6 7 8 9 10 11
Age, y/sex 20/Male 18/Female 50/Female 38/Female 63/Female 73/Female
Clinical signs Panuveitis Panuveitis Posterior uveitis Panuveitis Panuveitis Panuveitis
Lesions, n Right 3 Right 1 Right 1 Right 1 Left 1 Right 2/left 1
Lesion size in papillary diameter 1 1 0.5 1 0.5 3
Lesion localization Macula and temporal inferior retina Temporal inferior retina Peripapillary Peripapillary retina Inferior papillary retina Temporal inferior retina
Anti-Toxoplasma therapy Pyrimethamine and sulfadiazine Pyrimethamine and sulfadiazine Pyrimethamine and sulfadiazine No Pyrimethamine and sulfadiazine No
Immunocompromised No No No No Yes No
Underlying disease None None Autoimmune thyroiditis None Type II diabetes Cholongio-carcinoma
GWC 1.61 0.36 0 0.2 0.13 0
Anti-Hsp70.1* 0.633 0.634 0.543 0.752 0.667 1.531
Diagnosis OT OT OT OT OT Ocular candidiasis
When the logistic test results, the presence of anti-Hsp70.1 antibodies, and the clinical data were taken into account, nine patients in group B were classified in the group of confirmed OT. An additional patient (data not shown) in group B with anti-Hsp70.1 antibody in his serum was also classified in the group of confirmed OT. 
In immunocompromised patients in groups A and B with confirmed clinical evidence of OT, the logistic test was more sensitive (4/6; 66.7%) than the serum anti-Hsp70 test alone (1/8; 12.5%) for the confirmation of OT. The association of both tests improved this sensitivity (5/8; 62.5%). 
Relationships between OT Severity and Local and Systemic Immunity
The severity of OT was evaluated by the length of the major axis of the chorioretinal lesion (papillary diameter, PD). Local immunity was assessed by the level of AH IgG anti-Toxoplasma antibodies, systemic immunity by serum IgG anti-Hsp70.1 antibodies, and the combination of both immunities by the logistic probability P(x). The results are shown in Table 5. The Z correlation (r) results were statistically significant (P < 0.05). 
Table 5.
 
Z Coefficient Correlations of Chorioretinal Lesion Size, with Local, Systemic Immunity and Logistic Probability
Table 5.
 
Z Coefficient Correlations of Chorioretinal Lesion Size, with Local, Systemic Immunity and Logistic Probability
r P
AH IgG anti-Toxoplasma antibody (n = 47) 0.497 0.0003
Serum IgG anti-Hsp70.1 antibody (n = 49) 0.301 0.0349
Logistic probability, P(x), (n = 47) 0.485 0.0004
Discussion
Our results show the presence of anti-Hsp70.1 antibodies in the sera of patients with laboratory-confirmed OT and clinically suspected OT. These findings are consistent with the production of anti-Hsp-70 autoantibodies by resistant BALB/c and susceptible C57BL/6 mice. 23 Anti-Hsp70 autoantibodies may be induced by a cross-reaction mechanism with T. gondii Hsp70, since the Hsp70 N-terminal ATP-binding domain is well conserved between species. 24,30 The production of these antibodies is transient in mice 31 and probably also in humans, as observed in two cases in the present study. On the other hand, antibody transport across the BRB is infrequent. 32 The hypothesis of Chen et al. 23 was that T. gondii Hsp70 could pass the BRB. Calderwood et al. 21 suggested that Hsp70 can enter the bloodstream after release of intracellular Hsp70 by active secretion mechanisms or after necrosis. T. gondii Hsp70, present in the anterior chamber during intraocular T. gondii infection, may be captured by antigen-presenting cells via their Hsp70 receptors and then carried through the trabecular meshwork via the blood vessels, thus making contact with the systemic immune system. 33 This proposed route may explain why we observed an antibody response to Hsp70.1 in the sera of OT patients. The relationship between intraocular toxoplasmic immunity and systemic immunity appears to be supported by the correlation between the levels of IgG anti-Toxoplasma antibodies in AH and in sera (data not shown) and by a positive correlation between these antibodies and the retinal lesion size, as has been described with the serum anti-glycoinositolphospholipid IgA antibody. 34  
The gold standard for the diagnosis of OT remains the ophthalmic examination; however, atypical lesions may be observed in elderly persons or immunocompromised patients that may complicate the ophthalmic diagnosis. 4,9,35,36 Laboratory tests are then generally necessary to confirm the clinical diagnosis or suspicion of OT with typical and/or atypical lesions. The sensitivity of GWC for the diagnosis of OT is 74% and 81%, with a specificity of 100%. 5,8 In atypical OT cases, GWC sensitivity can decrease to 45%, 7 in which case complementary laboratory tests (e.g., PCR, immunoblot analysis) have been used to increase sensitivity and to confirm clinical OT diagnosis. 7 9 In the present study, three of our cases were confirmed by PCR and ten by the logistic test and anti-Hsp70 antibodies in sera. To confirm clinically suspected OT cases, we recommend the following algorithm: Perform GWC and/or PCR; if GWC or PCR are not informative, perform complementary analysis with the anti-Hsp70 antibody test; if this test is positive, the clinically suspected case is confirmed. The logistic test, based on the presence of anti-Hsp70 antibodies in sera and in anti-Toxoplasma antibodies in AH, may enhance the sensitivity of the confirmation of toxoplasmic uveitis, as seen in the present study. However, it is essential to note that Hsp70 may be expressed as a result of other diseases, and thus the ophthalmic data should always be considered when diagnosing OT. Therefore, the kinetics of the evolution of anti-Hsp70 antibody levels must be determined in each patient before, during, and after the episode of OT, and ophthalmic clinical data must be obtained. 
The mechanism of toxoplasmic pathogenesis is complex, because parasite and host specificities are interrelated. The host immune response may be subjected to the parasite's influence and induce several pro- and anti-inflammatory proteins. 37,38 Hsp70 acts as a molecular chaperone during the cell cycle in prokaryote and eukaryote cells. The importance of Hsp70 during T. gondii growth and differentiation has been reported. 17 19 Also, a relationship between the virulence of T. gondii infection and the expression of Hsp70 has been observed. 39 The increased level of Hsp70 just before the death of the host cell has suggested an immunomodulated protective role for tachyzoites against host immunologic responses during the invasion of the host cells. 19,39 This protective role may be associated with host NF-κB expression and nitric oxide downregulation. 39 In some OT patients, such as those who are immunocompetent, serum anti-Hsp70 antibodies may be a marker of OT. The data on immunocompromised patients suggest that the logistic test performs better than the anti-Hsp70 test alone for OT confirmation. A prospective study is thus necessary to confirm our findings in both immunocompromised and immunocompetent OT patients. 
Footnotes
 Disclosure: B.F.F. Chumpitazi, None; L. Bouillet, None; H. Fricker-Hidalgo, None; T. Lacharme, None; J.-P. Romanet, None; C. Massot, None; C. Chiquet, None; H. Pelloux, None
The authors thank Monique Baccard (Laboratory of Virology, Grenoble University Hospital) for providing access to the serum library facilities; Ali Rahim (Laboratory of Parasitology-Mycology, Grenoble University Hospital) for extraction of the laboratory OT data; and Jean-Louis Quesada and Alison Foote (Clinical Research Centre, Grenoble University Hospital) for helpful statistical discussions and English editing, respectively. 
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Figure 1.
 
Serum antibody anti–Toxoplasma gondii obtained by enzyme-linked fluorescence assay (ELFA) in control, suspected, and ocular toxoplasmosis patients. IgG (A) and IgM (B) antibody anti–Toxoplasma gondii levels observed by ELFA in sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B) and control (C) patients. All data represent one sample per patient. The box-and-whisker plots show the median of 26 (A), 40 (B), and 67 (C) measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and the 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. The P-values for data from (A) and (B) were 0.29 and 0.54 (Kruskal-Wallis' test for the three groups), respectively. No significant differences were observed between the groups.
Figure 1.
 
Serum antibody anti–Toxoplasma gondii obtained by enzyme-linked fluorescence assay (ELFA) in control, suspected, and ocular toxoplasmosis patients. IgG (A) and IgM (B) antibody anti–Toxoplasma gondii levels observed by ELFA in sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B) and control (C) patients. All data represent one sample per patient. The box-and-whisker plots show the median of 26 (A), 40 (B), and 67 (C) measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and the 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. The P-values for data from (A) and (B) were 0.29 and 0.54 (Kruskal-Wallis' test for the three groups), respectively. No significant differences were observed between the groups.
Figure 2.
 
Serum levels of anti-Hsp70.1 antibodies in blood donors and in patients with suspected toxoplasmic uveitis. IgG antibody anti-Hsp70.1 levels observed by ELISA in the sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B), and control (C) patients. The box-and-whisker plots show the median of 26 (A), 41 (B), and 67 (C) averages of three measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. All data represents one sample per patient.
Figure 2.
 
Serum levels of anti-Hsp70.1 antibodies in blood donors and in patients with suspected toxoplasmic uveitis. IgG antibody anti-Hsp70.1 levels observed by ELISA in the sera of confirmed ocular toxoplasmosis (A), clinically suspected ocular toxoplasmosis (B), and control (C) patients. The box-and-whisker plots show the median of 26 (A), 41 (B), and 67 (C) averages of three measurements (horizontal bars within box), respectively; the lower and upper quartiles (box) and 10th to 90th percentiles (vertical bars). Points above or below these levels are shown separately. All data represents one sample per patient.
Figure 3.
 
Kinetics of serum IgG anti-Hsp70.1 antibodies in patients with suspected toxoplasmic uveitis. (A) A patient with confirmed ocular toxoplasmosis. A significant decrease (16.7%) in the marker was observed (from point 2 to 3). Point 1 served as the reference background for this patient. (B) A patient with suspected ocular toxoplasmosis. Within a week, a significant increase (50.9%) in the marker was observed (from point 1 to 2).
Figure 3.
 
Kinetics of serum IgG anti-Hsp70.1 antibodies in patients with suspected toxoplasmic uveitis. (A) A patient with confirmed ocular toxoplasmosis. A significant decrease (16.7%) in the marker was observed (from point 2 to 3). Point 1 served as the reference background for this patient. (B) A patient with suspected ocular toxoplasmosis. Within a week, a significant increase (50.9%) in the marker was observed (from point 1 to 2).
Table 1.
 
Characteristics of the Patients at the Beginning of the Study
Table 1.
 
Characteristics of the Patients at the Beginning of the Study
Parameter Group A Group B Group C
Ocular toxoplasmosis Confirmed Suspected Absent
Patients, n 26 41 67
Age, y (mean ± SD) 41.0 ± 19.3 40.2 ± 18.8 43.6 ± 12.2
    Range 18–80 10–73 18–64
Male/female 12/14 15/26 34/33
GWC ≥2 (n = 23)* ≤2 (n = 38)† Not done
DNA T. gondii PCR Positive (n = 3) Negative (n = 3) Not done
Serum anti-Toxoplasma IgG, IU/mL, median (range) 92 (15–300) 80 (17–300) 61 (25–175)
Aqueous humor anti-Toxoplasma IgG, IU/mL, median (range) 2.7 (0.4–27.7) 0.1 (0–1.6) Not done
Probably acute/acquired toxoplasmosis, n 1/24 5/32 2/65
Affected eyes, n 27 43 0
Table 2.
 
Discrimination between Groups with Confirmed and Clinically Suspected OT According to the Logistic Test
Table 2.
 
Discrimination between Groups with Confirmed and Clinically Suspected OT According to the Logistic Test
Parameter Antibody Logistic Test P(x) *
Serum anti-Hsp70.1 IgG AH IgG anti-Toxoplasma
Youden's index 0.335 0.350 0.535
Yule's Q coefficient 0.670 0.687 0.859
Cutoffs 0.462 1.1 IU/mL 0.395
Confirmed OT group (A; n = 26), % (95% CI) 50 (32–68)† 51 (36–67)† 67 (47–82)†
Clinically suspected OT group (B; n = 41), % (95% CI) 24 (14–39)† 16 (9–28)† 13 (6–27)†
Nonuveitis control group (C; n = 67), % (95% CI) 12 (6–22)† Not done Not done
Sensitivity (%) 50 51 67
Specificity (%) 83 84 87
Positive predictive value (%) 42 68 76
Negative predictive value (%) 87 72 80
Table 3.
 
Patients with Clinically Suspected OT (Group B) with a Positive Logistic Test Result
Table 3.
 
Patients with Clinically Suspected OT (Group B) with a Positive Logistic Test Result
Parameter Patient
1 2 3 4 5
Age, y/sex 41/Female 71/Male 55/Female 58/Female 44/Female
Clinical signs Panuveitis Panuveitis, macular edema Panuveitis Posterior uveitis Panuveitis
Chorioretinal lesions, n Right 1 Right 1 0 Right 1 Left 1
Lesion size in papillary diameter 1.5 3 / 1 2
Lesion localization Peripapillary Macula and peripheral / Peripheral Nasal and superior juxta-papillary
Therapy Pyrimethamine and sulfadiazine TMP-SMX and triamcinolone No Pyrimethamine and sulfadiazine Pyrimethamine and sulfadiazine
Immunocompromised Yes No No No No
Underlying disease AIDS Polyangiitis Sarcoidosis None None
GWC 1.45 1.32 0.2 1.52 0.2
Logistic probability of having OT, P(x) * 0.821 0.628 0.474 0.474 0.482
Final clinical diagnosis Acquired OT Retinal detachment and OT Sarcoidosis Recurrent OT Acquired OT
Treatment outcome Negative Favorable Negative Favorable Favorable
Table 4.
 
Patients with Clinically Suspected OT (Group B) with Positive anti-Hsp70.1 Antibody Serology and a Negative Logistic Test Result
Table 4.
 
Patients with Clinically Suspected OT (Group B) with Positive anti-Hsp70.1 Antibody Serology and a Negative Logistic Test Result
Parameter Patient
6 7 8 9 10 11
Age, y/sex 20/Male 18/Female 50/Female 38/Female 63/Female 73/Female
Clinical signs Panuveitis Panuveitis Posterior uveitis Panuveitis Panuveitis Panuveitis
Lesions, n Right 3 Right 1 Right 1 Right 1 Left 1 Right 2/left 1
Lesion size in papillary diameter 1 1 0.5 1 0.5 3
Lesion localization Macula and temporal inferior retina Temporal inferior retina Peripapillary Peripapillary retina Inferior papillary retina Temporal inferior retina
Anti-Toxoplasma therapy Pyrimethamine and sulfadiazine Pyrimethamine and sulfadiazine Pyrimethamine and sulfadiazine No Pyrimethamine and sulfadiazine No
Immunocompromised No No No No Yes No
Underlying disease None None Autoimmune thyroiditis None Type II diabetes Cholongio-carcinoma
GWC 1.61 0.36 0 0.2 0.13 0
Anti-Hsp70.1* 0.633 0.634 0.543 0.752 0.667 1.531
Diagnosis OT OT OT OT OT Ocular candidiasis
Table 5.
 
Z Coefficient Correlations of Chorioretinal Lesion Size, with Local, Systemic Immunity and Logistic Probability
Table 5.
 
Z Coefficient Correlations of Chorioretinal Lesion Size, with Local, Systemic Immunity and Logistic Probability
r P
AH IgG anti-Toxoplasma antibody (n = 47) 0.497 0.0003
Serum IgG anti-Hsp70.1 antibody (n = 49) 0.301 0.0349
Logistic probability, P(x), (n = 47) 0.485 0.0004
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