Epstein-Barr virus (EBV) causes infectious mononucleosis, an acute but self-limiting disease affecting children and young adults. After primary infection, the virus persists indefinitely in B-lymphocytes 1 , only to reactivate when cellular immunity is impaired. In immunocompromised individuals, EBV-related disorders following virus reactivation are associated with significant morbidity and mortality 2 . Up to 15% of transplant recipients develop post-transplant lymphoproliferative disease (PTLD), a heterogeneous group of disorders characterised by EBV transformation of lymphocytes 3 4 . Although uncommon, PTLD is aggressive and coupled with high mortality rates of 50-80% 4 . Also related to other diseases in immunosuppressed individuals, including chronic active EBV, fatal infectious mononucleosis (IM) and EBV-associated haemophagocytic syndrome (EBVAHS) 5 6 7 , EBV is linked to several malignancies such as nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma (BL) 5 . In HIV-infected individuals, EBV is associated with diseases such as oral hairy leukoplakia and AIDS-related non-Hodgkin's lymphoma 5 8 .

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 . Nevertheless, although 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 . Increasing virus burden is also believed to be a rapid indicator of immunopathological changes preceding and/or underlying the B-lymphocyte driven changes caused by EBV 16 . Therefore, determining EBV DNA loads in EBV-related disorders in immunocompromised populations is an important step towards disease diagnosis, management and treatment 17 .

Several methods for quantifying absolute DNA load have been developed since its first application to EBV diagnostics in 1999 18 19 20 . These include semi-quantitative, quantitative competitive and real-time PCR methods 21 , with each using different means for amplicon detection; visualisation on agarose gel, Southern blot analysis and enzyme immunoassay 21 . Real-time PCR quantification is generally preferred for its wider dynamic range, speed, ease of handling, sensitivity and specificity 2 22 23 24 25 . Although commercial assays incorporating probe-based chemistries are available 26 27 , in-house methods employing high saturating dyes such as SYBR Green I are more cost-effective and just as sensitive as the widely used TaqMan PCR 21 28 29 30 .

Here, in an effort to ascertain the relationship between EBV DNA load and disease, two real-time quantitative PCR (QPCR) assays using SYBR Green I dye and a single quantification standard incorporating two separate EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed. EBV DNA was measured in a range of clinical samples, including unfractionated whole blood, plasma, PBMC and CSF from patients with EBV-associated disorders or immune dysfunctions. EBV sero-status was also determined for individuals in a population sample to assess the correlation between DNA load and antibody titres.

With increasing availability of nucleic acid testing (NAT) methods, measuring EBV DNA in blood has proven valuable in diagnosing and monitoring PTLD 16 21 22 37 38 39 40 41 , NPC 42 43 , IM 13 44 , EBV infection in HIV-infected individuals 8 13 45 , BL 13 and chronic active EBV infection 18 46 . In this study, we successfully developed two in-house QPCR methods incorporating a novel single quantification standard containing two EBV DNA targets for measuring viral load on the Rotor-Gene 6000™. Substituting SYBR Green I dye as a fluorescent marker for product accumulation over fluorogenic probes, this method proved useful for quantifying EBV DNA concentrations in clinical samples from individuals with a variety of EBV-associated disorders or immune dysfunctions and in a healthy population sample.

Previous studies in PTLD have found that EBV DNA loads increased with disease progression and decreased with remission of lymphoproliferation 47 48 . This pattern was observed in Group 1, where EBV DNA loads appeared to be correlated with disease status. 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 . EBV DNA was also detected in CSF at concentrations comparable to plasma, however detectable CSF EBV DNA has been previously reported only in association with acquired immunodeficiency syndrome (AIDS)-related brain lymphoma 51 . The significance of EBV DNA in CSF of PTLD remains to be elucidated.

EBV DNA loads in IM patients were also similar to those reported in the literature 13 22 26 44 52 , although some authors described loads as high as 10⁶ and 10⁷ copies/ml 12 46 53 . In Group 3, EBV DNA loads were consistent with acute phase EBVAHS 46 54 , and correlated with the deterioration of the patient's disease condition. Elazary et al also found that a viral load ranging from 10⁴-10⁵ copies/ml was associated with poor patient outcome 54 . One study found much higher EBV DNA loads (up to 10⁷ copies/ml) 55 , but this may have been due to differences in sample type and detection methods. In Group 4, EBV DNA was detected in 33% of samples (22% of plasma, 67% of PBMC), compared to 34% to 76% positivity reported in other studies 8 26 . Notably however, these studies used whole blood for quantifying EBV DNA load, which could have increased the probability of viral DNA detection. As none of the Group 4 patients were known to have EBV-related disease, low positivity ratios and viral loads were expected.

Similar to our findings, the literature describes EBV DNA detectable from 10² to 10⁴ copies/ml and positivity ratios up to 29% in whole blood of healthy individuals 11 12 13 26 38 56 57 58 59 . However, DNA loads as high as 5.5 × 10⁵ copies/ml of whole blood and a positivity ratio of 72% have been reported 58 . Differences in the results may be attributable to more sensitive methods associated with nested PCR and dual-labelled probes 58 . Interestingly, another study showed 100% EBV DNA positivity in whole blood, although DNA loads were all below the detection limit of the assay (2.0 × 10³ copies/ml) 38 .

In the population sample the EBV VCA IgG antibody detection rate was consistent with levels of EBV sero-positivity in Western societies 2 . One study previously showed a correlation between EBV VCA IgG antibody titres and EBV viral load (detectable versus non-detectable) 60 . We similarly found a modest correlation with quantitative BHRF-1 DNA loads, and a weaker (not statistically significant) correlation with EBNA-1 DNA load (see Table 4).

We noted some discrepancies in our measures of EBV positivity. In one PTLD patient (Patient D), plasma was qualitative EBV PCR negative whilst simultaneously reporting an EBV DNA load of 1.3 × 10⁸ copies/ml in whole blood. However, a growing number of studies have shown that cell-associated EBV is detectable before plasma EBV DNA and can persist without accompanying plasma DNA loads 21 48 . In Group 2, Patient G, despite being EBV VCA IgM antibody positive, was EBV QPCR negative. As EBV DNA loads can change rapidly from being undetectable to being very high in a short period of time 38 , it is possible that sampling occurred late in the convalescent phase where low EBV DNA positivity ratios of 44% have been previously reported 46 . Other factors contributing to DNA load variation include differences in sample type, method of extraction or NAT, and target chosen for PCR amplification.

As specimen type is known to influence DNA loads and impact on assay performance 36 , unfractionated EDTA whole blood was used for DNA quantification where possible. The dynamic changes of EBV DNA are better reflected in circulating whole blood 38 , which also contains all the compartments that may harbour virus 13 21 61 . However, despite reports of greater test sensitivity with whole blood 12 36 , EBV DNA load has also been quantified in PBMC 14 16 62 63 64 . Although infection is typically associated with cell compartments 8 12 13 , EBV DNA is also found in cell-free blood partitions such as plasma or serum, usually in fragmented, cell-derived form 12 . In this study, 2 of 9 plasma samples from HIV-infected patients had detectable EBV DNA, compared to 2 out of 3 PBMC samples. As we did not have simultaneous plasma and PBMC samples from the same individuals, we were unable to assess the differences in viral load between these compartments. Further studies comparing suitability of different sample types in various EBV-related diseases and immune disorders are required.

The method of DNA purification is known to affect viral load measurements. One study showed yield from manually extracted DNA was 57% higher than that of robotic systems 65 . Therefore, to improve DNA recovery and maximise PCR sensitivity, samples here were purified using a commercial silica-based column method 61 66 . For optimal quantitation results, an earlier study showed that DNA should be subjected to PCR within one to two weeks post-extraction 67 . Here, delay between extraction and testing could have contributed to low DNA loads and positivity ratios in clinical samples. Furthermore, DNA from blood samples that had undergone more than four freeze-thaw cycles were found to be partially degraded 68 . Since the clinical samples used here were tested retrospectively, monitoring these conditions were not possible.

EBV DNA loads also vary according to type and size of gene target 69 . Ryan et al, found assay sensitivity was dependent on the specific gene segment and that different targets had varying lower limits of detection 15 . For EBV, BamHI-W is reportedly 10 times more sensitive than other targets for PCR, allowing for detection of viral DNA at trace amounts 8 13 15 . However, precise quantification of viral genomes is complicated by the number of reiterated BamHI-W sequences among EBV strains, which typically ranges between 7 and 11 repeats per genome 15 . To avoid overestimation in this study, we chose to use the next most sensitive EBV gene; EBNA-1 15 , and an abundantly expressed gene, BHRF-1, for QPCR.

Despite targeting highly conserved EBV regions, selective drop out of amplifiable EBV DNA at the EBNA-1 and BHRF-1 loci was observed in Group 4 (Patients N and X), and in 25 of 218 (11.5%) whole bloods from the population sample. Instead of amplifying both EBV DNA genes, only one target was detected, 93% of which had viral loads less than 2.0 × 10³ copies/ml. As beta-globin was detected in all samples, PCR inhibitors and/or defective nucleic acid purification methods were excluded 70 . Alternatively, selective drop out may have been due to low viral load and/or sampling error 71 . Since load determination is reliant on the amount of EBV genomes pipetted into a reaction and assumes viral homogeneity, QPCR results, particularly at low viral load levels are prone to random sampling error. This phenomenon is well documented in DNA quantification and results in less reliable viral load measurements 70 71 . Therefore, samples reporting low levels of target nucleic acid may not be reproducible in repeated assays from the same or different specimens 72 .

Currently, there are no standardised methods for measuring EBV DNA, complicating inter-laboratory comparisons in multicentre studies of EBV-related diseases. Standardisation is difficult as PCR assay conditions vary between laboratories, leading to variations in the accuracy and reproducibility of viral load quantification 21 . Although there appears to be a strong concordance between laboratories for qualitative EBV DNA estimates, there continues to be marked inconsistency in quantitative results 73 . It has been suggested that the use of unfractionated whole blood 26 or an international calibration standard could be the first step towards standardisation 73 . However, instrumentation, chemistries, gene targets and other test-related aspects remain diverse. One solution for enabling inter-laboratory comparisons is the distribution of proficiency panels such as QCMD. Such programs have already been used for assessing methods for the detection and quantification of EBV and other viruses 27 74 75 .

This is the first reported study that uses the SYBR Green I dye on the Rotor-gene 6000™ with a novel quantification standard containing two EBV targets for measuring EBV DNA load. The assays proved successful in the quantification of EBV genomes in clinical cases and should be considered as a cost effective and sensitive PCR alternative to probe-based assays. This approach can be modified to detect and quantify other latent herpesviruses such as HHV6, CMV, and VZV. This procedure is suitable for robotics and automation, and would be a useful addition in larger laboratories.

The authors declare that they have no competing interests.

MLL developed the assays, carried out all of the DNA work, assisted in the data analysis and result interpretation, and writing of the manuscript. On behalf of the Ausimmune Investigator group, RML supplied the whole blood and serum from the population sample, and was involved in the data analysis. VMR aided in primer design and JT performed the serology testing. MLL, DED, VMR, RML and ALP were involved in the design and conception of the study. All authors have read, reviewed and approved the final manuscript.