Duplex realtime PCR method for Epstein-Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
Fellner, María Dolores; Durand, Karina; Rodriguez, Marcelo; Irazu, Lucía; Alonio, Virginia; Picconi, María Alejandra.
Braz. j. infect. dis
; 18(3): 271-280, May-June/2014. tab, graf
Artículo
en Inglés
| LILACS | ID: lil-712960
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. .Asunto(s)
Adolescente Adulto Niño Preescolar Femenino Humanos Lactante Masculino Persona de Mediana Edad Adulto Joven ADN Viral/sangre Infecciones por Virus de Epstein-Barr/diagnóstico Trasplante de Corazón/efectos adversos /genética Trasplante de Riñón/efectos adversos Trasplante de Hígado/efectos adversos Trastornos Linfoproliferativos/diagnóstico Trastornos Linfoproliferativos/virología Reacción en Cadena en Tiempo Real de la Polimerasa Sensibilidad y Especificidad Carga Viral
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