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

Abstract

Toxoplasma reactivation is a life-threatening complication of allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We developed 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 </=0.75 parasites. These three patients became PCR negative during trimethoprim-sulfamethoxazole therapy but never developed clinically apparent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment. Real-time quantitative PCR is useful for diagnosis and follow-up of toxoplasma reactivation.

FIG. 1

Real-time quantitative LC-PCR test using fluorescence energy transfer. (A) Duplicate amplification plots obtained for T. gondii DNA dilutions from 0.75 × 10⁶ (right) to 0.75 parasite in 10 μl (left). Each slope corresponds to a particular input target quantity. (B) Plot of the duplicate Cp against the input target quantity (common log scale) showing the linearity of the results. The computer-calculated correlation coefficient is 1.