Comparison of Immunoblotting, Calculation of the Goldmann-Witmer Coefficient, and Real-Time PCR Using Aqueous Humor Samples for Diagnosis of Ocular Toxoplasmosis

Abstract

We compared three biological methods for the diagnosis of ocular toxoplasmosis (OT). Paired aqueous humor and serum samples from 34 patients with OT and from 76 patients with other ocular disorders were analyzed by three methods: immunoblotting or Western blotting (WB), the calculation of the GoldmannWitmer coefficient (GWC), and PCR. WB and GWC each revealed the intraocular production of specific anti-Toxoplasma immunoglobulin G in 81% of samples (30 of 37). PCR detected toxoplasmic DNA in 38% of samples (13 of 34). Nine of the 13 PCR-positive patients were immunocompetent. Combining the techniques significantly improved the diagnostic sensitivity, to 92% for the GWC-WB combination, 90% for the WB-PCR combination, and 93% for the GWC-PCR combination. The combination of all three techniques improved the sensitivity to 97%.Ocular toxoplasmosis (OT) is the main cause of posterior uveitis worldwide and is a frequent cause of vision loss (14,15,21). The current gold standard for the diagnosis of OT is ophthalmologic examination, but the findings may be equivocal for patients with atypical lesions. In particular, toxoplasmic retinochoroiditis can mimic acute retinal necrosis syndrome (1). Therefore, laboratory methods often are necessary to confirm the diagnosis of OT. The most reliable sample type is that of aqueous humor, which can be tested for local specific antibody (Ab) production or for Toxoplasma gondii DNA by PCR.Local Ab production can be detected with an immunoblotting method and quantified by calculating the GoldmannWitmer coefficient (GWC) (3). In both cases, the specific Ab profiles of serum and aqueous humor samples are compared. Specific Abs can be detected by enzyme-linked immunosorbent assay (ELISA) and/or by immunofluorescence (IF) assay.The aim of this study was to compare the sensitivities and specificities of these three biological methods for the diagnosis of OT. The design of this study is that of a prospective case series. MATERIALS AND METHODS Patients and methods.We analyzed data from a series of 110 patients diagnosed with various ocular disorders during a 15-month period (December 2004 to February 2006 at the Department of Ophthalmology, Pitié-Salpêtrière Hospital, Paris, France. In most cases, the clinical findings were suggestive of atypical Toxoplasma gondii retinochoroiditis but were inconclusive. In order to confirm the diagnosis, anterior-chamber paracentesis was performed, and aqueous humor was sampled (vitreous humor for 18 patients). Blood was sampled simultaneously. Some patients were tested two or three times during the study, yielding a total of 120 samples. Clinical findings suggestive of T. gondii retinochoroiditis (i.e., focal retinal necrosis and choroidal edema with possible old scars) associated with successful outcomes of specific treatments were considered the gold standard. Considering these findings, a final diagnosis of OT was made for 34 patients (39 samples). The controls consisted of nontoxoplasmic ocular infection (see Table S1 in the supplemental material) and...

Molecular Detection and Genotyping of Toxoplasma gondii from Clinical Samples

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