Evaluating different methods of Toxoplasma gondii detection in peripheral blood

Abstract

Introduction: The mechanisms involved in the virulence and recurrences of ocular toxoplasmosis are poorly understood. New detection methods are necessary to diagnose atypical cases and to better understand recurrences. A sensitive method to identify Toxoplasma gondii in peripheral blood of patients is necessary. Purpose: To detect the presence of T. gondii in peripheral human blood using immunohistochemistry and PCR. Methods: Yellow fluorescent protein RH strain of T. gondii was cultured on fibroblast monolayer for 5 weeks. Cytospins and whole blood smears from spiked blood were stained using monoclonal antibodies on an automated Ventana. For real time PCR, different concentrations of T. gondii were added to human blood and DNA was extracted. PCR was performed 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’. For the 529 bp fragment primers used were 5’- CGC TGC AGG GAG GAA GAC GAA AGT TG- 3’ and reverse 5´- CGC TGC AGA CAC AGT GCA TCT GAA TT- 3’. All assays were run in triplicate. Results: The qPCR was able to detect fewer parasites than immunohistochemistry. Moreover, qPCR targeting the 529 bp fragment was better than the B1 gene, being able to detect parasites in concentrations as low as 1 toxo/mL. Conclusions: The qPCR method using the 529bp target proved to be more sensitive than qPCR using the B1 gene target and cytospins or blood smears using immunohistochemistry.