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, 37–41], NPC [42, 43], IM [13, 44], EBV infection in HIV-infected individuals [8, 13, 45], BL  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 . 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 . One study found much higher EBV DNA loads (up to 107 copies/ml) , 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 [11–13, 26, 38, 56–59]. However, DNA loads as high as 5.5 × 105 copies/ml of whole blood and a positivity ratio of 72% have been reported . Differences in the results may be attributable to more sensitive methods associated with nested PCR and dual-labelled probes . 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) .
In the population sample the EBV VCA IgG antibody detection rate was consistent with levels of EBV sero-positivity in Western societies . One study previously showed a correlation between EBV VCA IgG antibody titres and EBV viral load (detectable versus non-detectable) . 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 , it is possible that sampling occurred late in the convalescent phase where low EBV DNA positivity ratios of 44% have been previously reported . 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 , unfractionated EDTA whole blood was used for DNA quantification where possible. The dynamic changes of EBV DNA are better reflected in circulating whole blood , 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, 62–64]. 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 . 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 . 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 . 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 . 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 . Ryan et al, found assay sensitivity was dependent on the specific gene segment and that different targets had varying lower limits of detection . 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 . To avoid overestimation in this study, we chose to use the next most sensitive EBV gene; EBNA-1 , 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 . Alternatively, selective drop out may have been due to low viral load and/or sampling error . 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 .
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 . Although there appears to be a strong concordance between laboratories for qualitative EBV DNA estimates, there continues to be marked inconsistency in quantitative results . It has been suggested that the use of unfractionated whole blood  or an international calibration standard could be the first step towards standardisation . 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].