title: Duplex realtime PCR method for Epstein-Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection.
abstract: BACKGROUND
  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 10(7)-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.
authors.affiliation: Oncogenic Viruses Service, Virology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina. Electronic address: oncovir1@anlis.gov.ar.
authors.forename: María Dolores
authors.initials: MD
authors.lastname: Fellner
authors.affiliation: Oncogenic Viruses Service, Virology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina.
authors.forename: Karina
authors.initials: K
authors.lastname: Durand
authors.affiliation: Operational Team Quality Management, Parasitology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Rodriguez
authors.affiliation: Operational Team Quality Management, Parasitology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina.
authors.forename: Lucía
authors.initials: L
authors.lastname: Irazu
authors.affiliation: Oncogenic Viruses Service, Virology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina.
authors.forename: Virginia
authors.initials: V
authors.lastname: Alonio
authors.affiliation: Oncogenic Viruses Service, Virology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina.
authors.forename: María Alejandra
authors.initials: MA
authors.lastname: Picconi
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000097:blood*; D020031:Epstein-Barr Virus Infections / Q000175:diagnosis*; D005260:Female; D016027:Heart Transplantation / Q000009:adverse effects*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007223:Infant; D016030:Kidney Transplantation / Q000009:adverse effects*; D016031:Liver Transplantation / Q000009:adverse effects*; D008232:Lymphoproliferative Disorders / Q000175:diagnosis* / Q000821:virology; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019562:Viral Load; D055815:Young Adult
chemical_list: D004279:DNA, Viral
keywords: EBV; PTLD; real-time PCR; viral load