Real-time PCR for diagnosis and follow-up of toxoplasma reactivation afterallogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes

Toxoplasma reactivation is a life-threatening complication of allogeneic st em cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We d eveloped a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect and quantify Toxoplasma gondii DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from 0.75 x 10(6) to 0.75 parasites per PCR mixture. Serial samples from four patients with toxoplasma reactivation were evaluated. Three patients had several consecutive PCR-positive samples which corresponded to less tha n or equal to 0.75 parasites. These three patients became PCR negative duri ng trimethoprim-sulfamethoxazole therapy but never developed clinically app arent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The p arasite count decreased to 5 parasites in 3 days after pyrimethamine-clinda mycin treatment. Real-time quantitative PCR is useful for diagnosis and fol low-up of toxoplasma reactivation.