Use of fluorescence resonance energy transfer hybridization probes to evaluate quantitative real-time PCR for diagnosis of ocular toxoplasmosis.

Toxoplasma gondii infection is an important cause of chorioretinitis in Europe and the United States. Ophthalmological examination and a good clinical response to adequate therapy mainly support ocular toxoplasmosis diagnosis. However, clinical diagnostic may be difficult in some atypical cases. In these cases, laboratory confirmation, based on detection of local specific antibodies and parasite DNA by conventional PCR, is therefore important to confirm the disease etiology. More recently, real-time PCR has been developed to improve prenatal congenital toxoplasmosis diagnosis. We therefore examined the diagnostic value of quantitative real-time PCR for the detection of T. gondii in aqueous humor samples, associated with quantification of human beta-globin to control sample quantitative quality, by using a double fluorescence resonance energy transfer hybridization probes system with a double fluorescence reading. Of the 23 the clinically toxoplasmosis suspect patients, 22 showed serological evidence of exposure to Toxoplasma; one had a serological profile indicative of active infection. The analysis of paired aqueous humor and serum samples revealed an intraocular antibody production in 9 of 23 cases (39.1%). The quantitative real-time PCR revealed positive and high parasite numbers and high Toxoplasma/human genome ratios in three cases. Furthermore, PCR was the only positive confirmatory test in two cases (11.1%). None of the patients included in the control group (n = 7) had evidence of either local specific antibody production or T. gondii DNA detection, suggesting a good relative assay specificity. On the whole, quantitative real-time PCR appears to be useful for diagnosing atypical ocular toxoplasmosis presentations.