title: Evaluation of a Real-time PCR-based assay using the lightcycler system for detection of Toxoplasma gondii bradyzoite genes in blood specimens from patients with toxoplasmic retinochoroiditis.
abstract: PCR based methods have advantages over traditional methods for the diagnosis of toxoplasmosis, especially when serology fails and clinical symptoms are not evident. However, current PCR-based assays are often labour-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. We have developed and evaluated a highly sensitive Real-time PCR (Light-cycler, LC-PCR) to detect and quantify Toxoplasma gondii B1 and bradyzoite specific genes (SAG-4, MAG-1) in serum and peripheral blood mononuclear cells (PBMC) specimens, from five immunocompetent subjects with clinically suspected toxoplasmic retinochoroiditis (TRC) or without a suspected T. gondii infection. A standard curve for quantitation of parasitic load was generated using SYBR Green I fluorescent detection. The results were compared with those obtained with a nested PCR (n-PCR). In TRC patients, both PCR methods confirmed ophtalmoscopy and fluorangiographic findings. Among the TRC patients, the use of LC-PCR was more sensitive than n-PCR for detection and quantification of either B1 gene (P<0.001) or SAG-4/MAG-1 gene (P<0.05). LC-PCR has been shown particularly useful to accurately determine the parasite DNA load in follow-up specimens in whom the performance of either B1 or SAG-4 and MAG-1 in detecting T. gondii loads, varied with respect to specific antitoxoplasmic treatment.
authors.affiliation: Section of Infectious Diseases, Department Clinical and Experimental Medicine, via Fossato di Mortara, 23, 44100 Ferrara, Italy. cnc@dns.unife.it
authors.forename: Carlo
authors.initials: C
authors.lastname: Contini
authors.forename: Silva
authors.initials: S
authors.lastname: Seraceni
authors.forename: Rosario
authors.initials: R
authors.lastname: Cultrera
authors.forename: Carlo
authors.initials: C
authors.lastname: Incorvaia
authors.forename: Adolfo
authors.initials: A
authors.lastname: Sebastiani
authors.forename: Stephan
authors.initials: S
authors.lastname: Picot
mesh_terms: D000328:Adult; D000818:Animals; D002825:Chorioretinitis / Q000175:diagnosis* / Q000469:parasitology; D016054:DNA, Protozoan / Q000302:isolation & purification; D005260:Female; D017125:Genes, Protozoan; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics*; D014126:Toxoplasmosis, Ocular / Q000175:diagnosis*
chemical_list: D016054:DNA, Protozoan