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R. N. Belfort, R. Belfort, Jr., J. Isenberg, B. F. Fernandes, A. Romano, M. N. Burnier; Evaluating the Presence of Toxoplasma Gondii in Peripheral Blood of Patients With Uveitis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5868.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanisms involved in the severity and recurrences of ocular toxoplasmosis are poorly understood. New diagnostic methods, such as identifying circulating parasites, are necessary to diagnose atypical cases and to better understand recurrences. We have developed a sensitive method to identify Toxoplasma gondii in peripheral blood of patients with uveitis.
YFP RH strain of T. gondii was cultured on fibroblast monolayer for 5 weeks. Fluorescence was used to determine the parasitic concentration and serial dilutions were made: 10^6 to 10^0 parasites per milliliter. Cytospins and whole blood smears from spiked blood were stained using monoclonal antibodies on an automated Ventana. For qPCR, different concentrations of T. gondii were added to 1mL of blood. qPCR was performed using the DNA extracted from spiked blood and targeting a 62bp fragment of the B1 gene or the 529bp fragment. The primers used were: B1 Forward 5’- CTA GTA TCG GTG CGG CAA TGT -3’and Reverse 5’- GGC AGC GTC TCT TCC TCT TTT -3’ e 529 CGC TGC AGG GAG GAA GAC GAA AGT TG- 3’ and reverse 5'- CGC TGC AGA CAC AGT GCA TCT GAA TT- 3’. Twenty milliliters of blood from patients presenting with different etiology of uveitis had DNA extracted up to six hours after blood was collected, to prevent loss of DNA. The genetic material was preserved in special FTA paper plates or frozen until testes by qPCR for T. gondii using the methodology developed by us was performed. Tests were run in triplicate.
The qPCR method using the 529 bp fragment as target was able to detect parasites in concentrations as low as 1 toxo/mL, better than cytospins or blood smears. Out of 32 patients with uveitis tested, only two presented positive PCR for T. gondii in peripheral blood.
qPCR method using the 529bp target proved to be more sensitive than qPCR using the B1 gene target, cytospins or blood smears. Despite the high sensitivity of our method, only immunosuppressed patients presented circulating parasites in peripheral blood.
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