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

Abstract

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.

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

“…Real-time quantitative PCR is a fast and reliable method to measure EBV DNA load in clinical specimens but there is a need to standardise the measurement of EBV load in the clinical setting, and the best-suited blood compartment to use is still a matter of debate (Dehee et al, 2001;Hoshino et al, 2001;Jabs et al, 2001;Jebbink et al, 2003;Kimura et al, 1999;Leung et al, 2002; In this study, we assessed the feasibility of combining the MagNA Pure ® automated extraction with a real-time LightCycler PCR for EBV DNA quantification in whole blood, PBMCs and plasma in order to simplify the DNA extraction step and to compare the clinical value of the different blood compartments.…”

New Highly Sensitive Real-Time PCR Assay for HIV-2 Group A and Group B DNA Quantification

“…To normalize the HIV-2 DNA quantification, the amount of total DNA in extracts was determined by spectrophotometry (Nanodrop, Thermo Scientific, Wilmington, NC, USA) (labs A and B) or by quantification of the albumin gene (lab C) using the LightCycler FastStart DNA Master Hybprobe kit (Roche, Mannheim, Germany) and serial dilutions of human genomic DNA (Roche) as the standard (29,30).…”

Assessment of immunovirological features in HIV related non-Hodgkin lymphoma patients and their impact on outcome

“…4 The impact of the tumour-cell EBV status on the prognosis of patients with lymphoma has been studied with controversial results, 5,6 whereas the absolute EBV DNA load had poor diagnostic and prognostic values for AIDS-NHL, particularly because of its lack of specificity and its high fluctuation over time. 7,8 However, a recent study indicated EBV quantification in HIV patients treated for NHL as a surrogate marker of clinical outcome of AIDS-NHL. 9 Recently, a broader relationship between HHV8 infection and ARL has been proposed, beyond that one known for clinically distinctive primary effusion lymphoma.…”