title: Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma.
abstract: BACKGROUND
  Epstein-Barr virus (EBV) DNA load monitoring in blood has been shown to be essential for the diagnosis of EBV-associated diseases. However, the methods currently used to assess EBV DNA load are often time-consuming and require prior blood separation.
  
  
  OBJECTIVE
  The aim of this study was to evaluate the relative diagnostic value of EBV DNA load monitoring in whole blood, peripheral blood mononuclear cells (PBMCs) and plasma after automated DNA extraction using the MagNA Pure extractor followed by LightCycler real-time quantitative PCR (LC-PCR).
  
  
  METHODS
  First, EBV DNA load was assessed retrospectively after automated or manual extraction on 104 PBMC specimens. Second, EBV DNA load was determined prospectively with the automated extraction procedure in the whole blood, PBMCs and plasma of 100 samples from patients with EBV-related diseases (group 1, n = 20), HIV-seropositive individuals (group 2, n = 66), and healthy EBV carriers (group 3, n = 14).
  
  
  RESULTS
  A good correlation was observed between automated and manual extraction on 104 PBMC specimens (r = 0.956; P < 0.0001). In the prospective study, 67 samples were positive in both whole blood and PBMCs, with a good correlation between EBV DNA loads in whole blood and PBMCs (r = 0.936; P < 0.0001). Only 18/100 samples were positive in plasma. Higher viral loads were regularly observed in the three blood compartments from group 1 than from groups 2 and 3.
  
  
  CONCLUSIONS
  This study demonstrated that an automated extraction of EBV DNA is easier to perform in whole blood or plasma than in PBMCs and facilitates the standardisation of EBV DNA measurement by real-time quantitative PCR. The quantitative detection of EBV DNA load in whole blood appeared more sensitive than in plasma for infectious mononucleosis in immunocompetent patients, probably because of a rapid loss of plasmatic EBV DNA. In transplant patients, EBV DNA load monitoring in whole blood and in plasma turned out to be equivalent in terms of feasibility and accuracy for the early diagnosis of post-transplant lymphoproliferative diseases (PTLDs).
authors.affiliation: Laboratoire de Virologie Moléculaire et Structurale, Faculté de Médecine, Université Joseph Fourier, Centre Hospitalier Universitaire, Michallon BP 217, Grenoble 38043, France. sfafi@chu-grenoble.fr
authors.forename: Samira
authors.initials: S
authors.lastname: Fafi-Kremer
authors.forename: Karen
authors.initials: K
authors.lastname: Brengel-Pesce
authors.forename: Gérard
authors.initials: G
authors.lastname: Barguès
authors.forename: Marie-Josette
authors.initials: MJ
authors.lastname: Bourgeat
authors.forename: Odile
authors.initials: O
authors.lastname: Genoulaz
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Seigneurin
authors.forename: Patrice
authors.initials: P
authors.lastname: Morand
mesh_terms: D017088:AIDS-Related Opportunistic Infections / Q000097:blood / Q000175:diagnosis / Q000821:virology; D001331:Automation / Q000379:methods; D004279:DNA, Viral / Q000097:blood / Q000302:isolation & purification*; D020031:Epstein-Barr Virus Infections / Q000097:blood / Q000175:diagnosis; D004854:Herpesvirus 4, Human / Q000302:isolation & purification*; D006801:Humans; D007244:Infectious Mononucleosis / Q000097:blood / Q000821:virology; D007963:Leukocytes, Mononuclear / Q000821:virology; D008232:Lymphoproliferative Disorders / Q000097:blood / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D014180:Transplantation / Q000009:adverse effects
chemical_list: D004279:DNA, Viral