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Braz J Infect Dis. 2014 May-Jun;18(3):271-80. doi: 10.1016/j.bjid.2013.07.011. Epub 2014 Jan 2.

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

Author information

1
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.
2
Oncogenic Viruses Service, Virology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina.
3
Operational Team Quality Management, Parasitology Department, National Institute of Infectious Diseases "Carlos G. Malbrán", Av. Vélez Sársfield 563, C1282AFF Buenos Aires, Argentina.

Abstract

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 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.

KEYWORDS:

EBV; PTLD; real-time PCR; viral load

PMID:
24389276
DOI:
10.1016/j.bjid.2013.07.011
[Indexed for MEDLINE]
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