Valutazione analitica e applicazione clinica di un metodo Real Time PCR per il dosaggio della carica virale di Epstein-Barr virus

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Maria Teresa Bortolin *
Chiara Pratesi
Rosamaria Tedeschi
Stefania Zanussi
Ettore Bidoli
Monica D’Andrea
Carlo Costanzo
Antonella Selva
Paolo De Paoli
(*) Corresponding Author:
Maria Teresa Bortolin | micro@cro.it

Abstract

We assessed the performance of a Real Time PCR assay to be used for EBV viremia evaluation in clinical specimens. Sensitivity and intra-/interassay reproducibility were evaluated by using DNA serial dilutions from the Namalwa cell line. EBV DNA was analyzed in serum samples from 39 patients (pts) with undifferentiated type nasopharyngeal carcinoma (UCNT), from 5 infectious mononucleosis (IM) pts and from 18 healthy donors. Results obtained by Real Time PCR were compared with those obtained by quantitative competitive (QC)-PCR assay.We thereafter measured the dynamics of EBV DNA load in 5 HIV-seropositive (HIV+) and 9 HIV-seronegative (HIV-, as controls) pts with lymphoma, treated with high-dose chemotherapy (HCT) followed by autologus stem-cell transplantation (ASCT). We found a sensitivity of 100% at 10 EBV copies. The Spearman correlation for both the intra- and the interassay reproducibility was statistically significant (r=0.99; p20 copies/reaction and >30% for EBV viral loads <20 copies/reaction. No EBV DNA was detected in healthy donors. Higher EBV DNA loads were found by Real Time PCR (range 1173-46328 copies/ml) than by QC-PCR (range 450-5000 copies/ml) (p<0.05). 54% of UCNT and 100% of IM pts were EBV DNA positive. Two HIV+(40%) and 2 HIV-(22%) pts with lymphoma had detectable EBV viremia during the follow-up. The Real Time PCR is a suitable technique for high-throughput screening and frequent monitoring of patients at risk for developing EBV-associated diseases.

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