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 × 106 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.

Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

“…Another study used the assay in serum for diagnosis and follow-up of four stem cell transplant patients (82). They compared their results with a conventional PCR but also were able to quantitate parasitemia using real-time PCR by correlating extracted DNA assay crossing points with corresponding tachyzoite counts of cultured Toxoplasma gondii.…”

Challenging loop—mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii

“…In the present study, the sensitivity and specificity of LAMP and nested-PCR assays targeting the 529 bp RE and B1 gene were evaluated for the detection of T. gondii DNA. Due to the different studies reported a good sensitivity and specificity for the molecular markers targeting the repeated region in the T. gondii genome for detecting the parasite [6,38,39,44,45], these genomic targets were selected.…”

Real-time PCR in the microbiology laboratory

“…Likewise, the study of new and emerging viruses has been ideally complemented by the use of homogeneous real-time PCR assays as tools to demonstrate and strengthen epidemiological links between unique viral sequences and the [177,234,240,253,254,266,267,280,284,292,294] Solid tissues [172,198,238,[256][257][258]271,288,295] Cerebrospinal fluid [136,190,192,194,248] Peripheral blood mononuclear cells [183] Bone marrow [120] Whole blood [179,291] Plasma [118] Serum [171,172,180,279,287] Swabs [192,259,296] Bronchoalveolar lavage [243,246] Amniotic fluid [286] Saliva and sputum [158,233] Faeces [264,285] Urine [12,…”