Quantification of Epstein-Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR.

Epstein-Barr virus (EBV) reactivation is more likely to occur in immunocompromised patients with subsequent higher susceptibility to EBV-associated lymphoproliferations. In contrast to transplant recipients, limited data are available concerning the EBV load in HIV-infected patients, with or without AIDS-related non-Hodgkin's lymphomas. We developed a TaqMan real-time PCR assay, allowing both the EBV genome and a cellular gene to be quantified in order to obtain a reliable normalized measurement of the EBV load in peripheral blood mononuclear cells (PBMCs). With a wide 6-log(10) quantification range and inter-assay variations of less than 24%, this quantitative PCR was sufficiently accurate and reproducible for routine follow-up. The EBV load was determined in PBMCs from 113 HIV-infected patients, 11 patients with primary HIV infection and 24 HIV-seronegative healthy controls. The rates of EBV detection were similar in the three groups. However, EBV loads were higher in the HIV-infected group (P < 0.00001) except for the patients with primary HIV infection. Unexpectedly, EBV loads were not correlated with the clinical stages of HIV infection or HIV replication, and did not depend on the degree of immunodepression, as judged by CD4+ counts. This study contributes towards the definition of the baseline EBV load during HIV infection and stresses the broad inter-individual variability of the EBV load in HIV-infected patients. Real-time PCR provides a useful tool that can be used in further longitudinal studies to assess the relevance of the EBV load to identify HIV-infected patients with a high risk of EBV-associated lymphoproliferations.

Dehee A ，Asselot C ，Piolot T ，Jacomet C ，Rozenbaum W ，Vidaud M ，Garbarg-Chenon A ，Nicolas JC ... -  《JOURNAL OF MEDICAL VIROLOGY》

[Epstein Barr viral load monitoring in mononuclear lymphocytes and serum of renal transplant recipients using a quantitative PCR protocol].

Merlino C ，Giacchino F ，Bergallo M ，Bonello F ，Bollero C ，Segoloni GP ，Cavallo R ... -  《-》

Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma.

Fafi-Kremer S ，Brengel-Pesce K ，Barguès G ，Bourgeat MJ ，Genoulaz O ，Seigneurin JM ，Morand P ... -  《JOURNAL OF CLINICAL VIROLOGY》

Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR.

Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/microl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10-100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100-500 copies/10(5) PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/10(5) cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50-10 ng DNA.

Krumbholz A ，Meerbach A ，Zell R ，Gruhn B ，Henke A ，Birch-Hirschfeld E ，Wutzler P ... -  《JOURNAL OF MEDICAL VIROLOGY》

Real-time polymerase chain reaction (RQ-PCR) for the monitoring of Epstein-Barr virus (EBV) load in peripheral blood mononuclear cells.

Wagner HJ ，Jabs W ，Smets F ，Wessel M ，Fischer L ，Offner G ，Kirchner H ，Bucsky P ... -  《KLINISCHE PADIATRIE》