Routine use of real‐time quantitative PCR for laboratory diagnosis of Epstein‐Barr virus infections

Abstract

Real-time quantitative polymerase chain reaction (PCR) on the LightCycler instrument (LC-PCR) was developed to measure the Epstein-Barr virus (EBV) load in clinical samples. LC-PCR detected two copies of the EBV genome per 500 ng of DNA. Its specificity was confirmed by assays in EBV-negative cell lines, other human herpesviruses and EBV-seronegative individuals. Excellent inter-assay reproducibility of LC-PCR was obtained in 43 samples (r = 0.983). LC-PCR results were compared with a routinely used ELISA-PCR of 150 samples and a good correlation was found (r = 0.956). A total of 88 individuals were studied, including healthy EBV-seropositive adults (n = 32), patients with EBV-associated disease (n = 34), and HIV-infected patients (n = 22); 37.5% of PBMC samples from healthy individuals contained EBV DNA, while no serum sample was positive. The viral load was significantly higher in PBMCS and saliva specimens in patients recently infected with HIV (19 and 39,400 copies/microg DNA, respectively), as well as in AIDS patients (122 and 331,130 copies/microg DNA) than in the control population (0 and 35 copies/microg DNA). This study confirmed that EBV load measurement with LC-PCR is helpful in the management of EBV-related post-transplantation lymphoproliferative disorders and probably of EBV-associated primary central nervous system B-cell lymphoma.

Evaluation of a commercial real-time PCR assay for quantitation of Epstein-Barr virus DNA in different groups of patients

“…Similar results were obtained when peripheral blood mononuclear cells were tested for EBV DNA (Brengel-Pesce et al, 2002). Positive results for EBV DNA may be caused indirectly by immunosuppression through either retroviral infection or immunosuppressive therapy.…”

“…Some of these methods lack inclusion of an internal control and they usually are not designed for accurate quantitation of EBV DNA. Recently, molecular assays for detection of EBV DNA based on real-time PCR have been reported (Brengel-Pesce et al, 2002;Jebbink et al, 2003;Kimura et al, 1999;Niesters., 2000;Stevens et al, 2002a). Compared to conventional PCR-based assays, real-time PCR offers several important advantages.…”

A prospective study of Epstein–Barr virus load in 85 hematopoietic stem cell transplants

“…The weak positive predictive value did not seem to be entirely related to the semiquantitative as compared with more accurate quantitative or real time PCR, [37][38][39][40] since in another study we demonstrated a good correlation between the semi-quantitative method and real time quantitative PCR. 41 In fact, from our study and others published, it seems that a closer followup of the viral burden between day +30 and day +90 and the demonstration of a fast increase in viral burden is probably a better predictor of disease onset than is an elevated absolute value of viral burden. Recently, Stevens et al, 42 using a weekly quantitative PCR in unfractionated whole blood from patients with lung transplants, found that 78% of the samples from patients with EBV-LPD vs 3.4% of the samples from patients without EBV-LPD were superior to a cut-off value similar to our cut-off value.…”

Monitoring of Epstein‐Barr virus load after hematopoietic stem cell transplantation for early intervention in post‐transplant lymphoproliferative disease

“…For protection they were given immunoglobulins after transplantation resulting in high IgG antibody titers against virus capsid antigen and nuclear antigen of EBV. [Kenagy et al, 1995;Martinez et al, 1995;Bai et al, 1997;Rowe et al, 1997;Brengel-Pesce et al, 2002;Gärtner et al, 2002;Sirvent-von Bueltzingsloewen et al, 2002]. High amounts of EBV in peripheral blood mononuclear cells were detected in these patients [Savoie et al, 1994;Rooney et al, 1995;Lucas et al, 1998].…”