Abstract
BackgroundReactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.ResultsEBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 102 to 1.3 × 108 copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 103 to 2.0 × 105 copies/ml in infectious mononucleosis (n = 7), 7.5 × 104 to 1.1 × 105 copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 102 to 5.6 × 103 copies/ml in HIV-infected patients (n = 12), and 2.0 × 102 to 9.1 × 104 copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).ConclusionTwo sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.
Highlights
Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals
EBV detection and load in the population sample Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1) DNA were detected in 11.0% and 21.6% of Group 5, respectively; 22.5% of samples were positive for at least one EBV DNA target (Table 4)
We found similar EBV DNA loads to those previously reported, with most studies showing EBV DNA concentrations ranging from 5.0 × 102 to 2.0 × 107 copies/ml in whole blood, plasma and serum [37,49,50]
Summary
Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV-related disorders following virus reactivation are associated with significant morbidity. Though sometimes detectable in the immunocompetent [9], EBV DNA is found in greater concentrations in immunosuppressed populations [10,11,12,13]. The presence of circulating EBV DNA does not always correlate with symptomatic infection, nor does it predict clinical disease in immunocompetent or immunosuppressed individuals [2,9]. The correlation between EBV burden and disease status is incompletely understood, several studies have shown an association between symptomatic infection and elevated DNA loads in clinical samples [14,15]. Determining EBV DNA loads in EBV-related disorders in immunocompromised populations is an important step towards disease diagnosis, management and treatment [17]
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