Development of a PCR test for the diagnosis of Toxoplasma gondii infections
AbstractIntroduction.Toxoplasmosis is a paucisymptomatic and self-limiting disease in the normoergic patient, but it becomes potentially dangerous in immunocompromised patients (transplanted or HIV-infected patients) and during pregnancy, because if transmitted from mother to fetus can cause serious malformations to the future child and consequences to the newborn. Recent studies evidenced that a proper early administrated treatment can reduce the seriousness of symptoms in newborns and immunosuppressed patients. Because the serological diagnosis in these categories of patients is often insufficient, the search of DNA of Toxoplasma gondii by Polymerase Chain Reaction (PCR) is progressively used. Although PCR is a specific and sensitive technique, today a fully standardization is still lacking. The aim of this study was the development of a new test based on PCR for the detection of the Toxoplasma gondii parasite in different biological samples. Methods. In this study we proved the performances of a new PCR system, with ready-to-use reagents, where the highly repetitive RE region of T. gondii (GenBank accession number AF146527) is the target gene. As reference, we used the control panel of the European Molecular Biology QCMD 2008 Toxoplasma gondii (TGDNA08) EQA Program. EQA panel for the identification of T. gondii consists of eight samples containing different concentrations of the parasite and two negative samples (seven amniotic fluid and three plasma samples). Results. All negative samples were confirmed, as strong positive too. Only one sample with low concentration (5 parasites/ml) provided positive results in 50% of the ten repetitions of the intra-assay test, defining the threshold of sensitivity of the test. Conclusions.This study, although preliminary, demonstrates the specificity and sensitivity of the new test on different biological samples.
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Copyright (c) 2010 Alessia Moiana, Maurizio Gramegna, Francesca Genco, Valeria Meroni
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