[Development of a real-time PCR assay for quantitative diagnosis of Toxoplasma gondii after allogeneic bone marrow transplantation].
OBJECTIVE
A sensitive, rapid and specific diagnostic method is essential for the diagnosis of toxoplasmosis in immunocompromised hosts or in congenital infection. We report the development of a real-time PCR assay for quantitative diagnosis of toxoplasmosis with competitive internal amplification control. This PCR was applied after allogeneic stem cell transplantation to estimate the frequency of reactivation.


METHODS
Primers and Taqman probe (FAM-BHQ1) were designed to amplify the 529 bp element of T. gondii. The internal amplification control was developed by cloning a fragment of Arabidopsis thaliana DNA flanked by sequences specific for T. gondii 529 bp element. A Taqman probe specific for the competitive internal control was designed and tested (YY-BHQ1). We determined the repeatability and reproducibility of the method. A prospective study was performed on adults who received an allogeneic hematopoietic stem cell transplantation. After transplantation, patients were monitored once per week during the first 100 days; they were then monitored once every 2 weeks until day 180 after transplantation (i.e., day +180).


RESULTS
A total of 451 samples from 40 patients were tested. Twenty-five patients had both positive toxoplasmosis serology and an adequate chemoprophylaxis. One sample from one patient was found positive. The rate of reactivation in the population of this study is 4%.


CONCLUSIONS
A monitoring by T. gondii PCR should be realized weekly for patients receiving an allogeneic hematopoietic stem cell transplantation, without an adequate chemoprophylaxis.
Daval
S
S
Laboratoire de Parasitologie-Mycologie, CHU de Clermont-Ferrand, 58 rue Montalembert, 63003 Clermont-Ferrand cedex 1, France.
Poirier
P
P
Armenaud
J
J
Cambon
M
M
Livrelli
V
V
D000328:Adult; D000368:Aged; D000818:Animals; D000981:Antiprotozoal Agents / Q000008:administration & dosage / Q000627:therapeutic use; D017360:Arabidopsis / Q000235:genetics; D001667:Binding, Competitive; D016026:Bone Marrow Transplantation / Q000009:adverse effects*; D003199:Computer Systems; D015342:DNA Probes; D018744:DNA, Plant / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D007165:Immunosuppression Therapy / Q000009:adverse effects*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011183:Postoperative Complications / Q000175:diagnosis / Q000469:parasitology*; D011292:Premedication; D011446:Prospective Studies; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014122:Toxoplasma / Q000302:isolation & purification* / Q000502:physiology; D014123:Toxoplasmosis / Q000175:diagnosis* / Q000209:etiology / Q000469:parasitology / Q000635:transmission; D014184:Transplantation, Homologous / Q000009:adverse effects*; D055815:Young Adult
D000981:Antiprotozoal Agents; D015342:DNA Probes; D018744:DNA, Plant; D016054:DNA, Protozoan; D019914:Taq Polymerase