Duplex realtime PCR method for Epstein–Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection

• Main Authors: María Dolores Fellner, Karina Durand, Marcelo Rodriguez, Lucía Irazu, Virginia Alonio, María Alejandra Picconi
• Format: Article
• Language: English
• Published: Elsevier 2014-05-01
• Series: Brazilian Journal of Infectious Diseases
• Online Access: http://www.sciencedirect.com/science/article/pii/S141386701300281X

Introduction: The quantification of circulating Epstein–Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. Objective: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. Methods: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. Results: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase – GAPDH reaction). Linear ranges comprised 107–10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000–32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p < 0.05). The EBV load in PBMC from the groups of BD, Non-Tx, Tx and Tx-PTLD exhibited increasing levels (p < 0.05). In BD, PBMC and plasma, EBV loads were undetectable. Conclusions: The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays. Keywords: EBV, real-time PCR, viral load, PTLD