Duplex realtime PCR method for Epstein-Barr virus and human DNA quantification: its application for post-transplant lymphoproliferative disorders detection
Braz. j. infect. dis
;
18(3): 271-280, May-June/2014. tab, graf
Article
in English
| LILACS
| ID: lil-712960
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 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. .
Full text:
Available
Index:
LILACS (Americas)
Main subject:
DNA, Viral
/
Heart Transplantation
/
Kidney Transplantation
/
Liver Transplantation
/
Epstein-Barr Virus Infections
/
Lymphoproliferative Disorders
Type of study:
Diagnostic study
Limits:
Adolescent
/
Adult
/
Child
/
Child, preschool
/
Female
/
Humans
/
Infant
/
Male
Language:
English
Journal:
Braz. j. infect. dis
Journal subject:
Communicable Diseases
Year:
2014
Type:
Article
Affiliation country:
Argentina
Institution/Affiliation country:
National Institute of Infectious Diseases Carlos G. Malbrán/AR
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