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R. N. Belfort, S. Di Cesare, C. Miyamoto, D. Faingold, R. Belfort, Jr., M. N. Burnier, Jr.; Evaluating Different Methods of Toxoplasma gondii Detection in Peripheral Blood. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2493.
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Toxoplasmosis is the most common cause of infectious posterior uveitis worldwide. The mechanisms involved in the severity and recurrences of this particular disease are poorly understood. New diagnostic methods, such as identifying circulating parasites, are necessary to diagnose atypical cases, as well as to determine if recurrence is a local or systemic event. We have compared three methods to identify Toxoplasma gondii in peripheral blood.
RH strain of T. gondii was cultured on fibroblast monolayer for 5 weeks. A hemocitometer was used to determine the parasitic concentration and serial dilutions were made: 106, 105, 104, 103 and 102 parasites per milliliter. Cytospins and smears were performed from samples of 3 mL of blood, spiked with different parasitic concentrations. Smears were performed from whole blood. Cytospins were done from the white blood cell layer previously separated using Ficoll. Immunohistochemistry on smears and cytospins were performed on an automated Ventana machine using antibodies for T. gondii. 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. The primers used were: Forward 5’- CTA GTA TCG GTG CGG CAA TGT -3’and Reverse 5’- GGC AGC GTC TCT TCC TCT TTT -3’.
The cytospins were positive for both concentrations of 106 and 105 toxo/ml while the smears were only positive for 106 toxo/mL. The qPCR method was able to detect parasites in concentrations as low as 102 toxo/mL and the amplification product resulted in uniform melting curves.
To the best of our knowledge, this is the first side-by-side comparison of these three methods to detect T. gondii in peripheral blood. All three methods were able to identify T. gondii in peripheral blood. However, the qPCR method proved to be much more sensitive than cytospins or blood smears. These results indicate that qPCR is likely the most appropriate method to identify T. gondii in the peripheral blood of ocular Toxoplasmosis patients.
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