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Clinical guidelines and recent studies of EBV infection focus predominantly on incidence and implications of viremia in the first posttransplant year . Screening for EBV DNA during the first year is recommended, particularly for high-risk EBV donor seropositive recipient seronegative patients. There is little reporting on prevalence, patterns and outcomes of EBV infection beyond the first year. Such information is important because most PTLD in this population presents late, beyond the first year, and over 50% of late cases are associated with EBV infection [8, 9]. In addition, there are conflicting analyses relating to the associations of EBV DNAemia with PTLD development, graft dysfunction and loss, acute rejection, opportunistic infection, adverse events, risk of death and overall burden of immunosuppression [6, 7, 12, 14-17].
Bamoulid et al, Holman et al and Holmes et al reported EBV DNAemia rates from 40% to 56% in adult renal transplant recipients during the first posttransplant year [6, 13, 18]. We report a lower rate of EBV DNAemia (>1000 copies/mL) at recruitment of 16% and 24% during follow-up in individuals within the first posttransplant year, while 31% of the study population at recruitment and 46% of individuals overall, had one or more samples with detectable DNA during the study year. However, Bamoulid et al  reported a similar prevalence (19%) of DNAemia levels >1000 copies/mL. Further, Holmes et al  reported 7% DNAemia >10 000 copies/mL, which compares to our prevalence of 7% chronic HVL (>10 000 copies/mL) in the first year posttransplant.
In this cross-sectional analysis, DNAemia prevalence and persistence appear to increase, rather than fall, with time from transplant, perhaps as a consequence of prolonged exposure to immunosuppression and/or immunosenescence, although we could not confirm an effect of age on DNAemia in analyses adjusted for time. However, studies such as this may suffer from selection effects, with prevalence rates being conditional on survival. This is a limitation of our study and a cohort study would be required to further investigate factors associated with late EBV DNAemia from time of transplant. Attention should be given to co-morbidities such as history of diabetes, cardiovascular disease and autoimmune disease, while analysis of markers of under-immunosuppression such as donor-specific antibody detection, proteinuria and rate of graft decline as well as over-immunosuppression such as viral, bacterial infection or skin cancer may also be of interest.
High DNAemia rates during the first year might be expected as immunosuppression levels are high and seronegative recipients experience primary infection. Falling rates of detection with increasing time from transplant have been reported in heart and lung recipients . In our series, low rates in the first year may be due to sample size, but may reflect immunosuppressive practice, including MMF and minimal use of T cell depleting antibody.
In analyses adjusted for time from transplant, MMF was the only agent with a significant association with DNAemia rates. Reduced incidence of DNAemia in those receiving MMF has been noted in other studies [6, 18, 20]. It has been suggested that this may be due to an anti-B cell effect, reducing the EBV-carrying B lymphocyte population . Treatment with rituximab is associated with transient depletion of B cell numbers with corresponding falls in EBV viral loads, and studies have also reported subsequent rises in viral loads as B cell numbers are later reconstituted [6, 21, 22]. Individuals on MMF or azathioprine in our study had lower total lymphocyte counts than those not on anti-metabolite/proliferatives, but only MMF was significantly associated with DNAemia. Analysis of B cell numbers in relation to EBV viral loads in larger numbers of patients on MMF may be helpful. These observations raise the following questions. Does the observed lower risk of EBV DNAemia in MMF-treated patients correspond to a reduced risk of EBV positive PTLD? Should we consider changing asymptomatic individuals with high-level EBV DNAemia to MMF, and should low-dose MMF with steroids be the maintenance agents of choice in new cases of EBV positive PTLD, rather than low-dose calcineurin inhibitors? PTLD risk registry analyses suggest that MMF-treated individuals overall may have a reduced incidence and risk of PTLD development [23-26], although recent analyses report a high prevalence of MMF use in primary central nervous system PTLD [27, 28]. To date there is no clear evidence to guide immunosuppression prescribing in the late posttransplant period as treatment or prevention of EBV-related disease or PTLD.
In the event of detection of EBV DNAemia in stable patients, the likelihood of persistence of DNAemia over at least the following year of follow-up was increased if the recipient was EBV seronegative at transplant [6, 14, 18], and the higher the viral load at recruitment with 88% of those with initially undetectable levels remaining free of EBV detection.
The implications of EBV DNAemia in otherwise stable transplant recipients in the late posttransplant period are unclear. EBV is a latent herpes virus, and after primary infection, it is anticipated that a steady state will be reached, where numbers of EBV-infected B cells are controlled by effective EBV-specific T cell responses . EBV DNA in blood is found in healthy members of the general population, although typically at significantly lower levels than following transplantation [18, 30]. While detection of CMV and BK virus DNA in blood, particularly if >1000 copies/mL, is typically associated with clinical symptoms and end-organ damage, detection of late EBV DNAemia may not identify those with a clinical illness requiring treatment, and/or those who are over-immunosuppressed. In our study those with DNAemia did not have significantly poorer survival during follow-up or poorer graft-related outcomes, nor did they have greater rates of specific symptoms, hematological or biochemical abnormalities. Further, increased DNAemia prevalence was not associated with previous ATG use, higher calcineurin inhibitor trough concentration, or greater numbers of maintenance immunosuppressive agents. In support of an “over-immunosuppressed” argument, we show, in univariate analysis, an association of DNAemia with a history of nonmelanoma skin cancer, warts, extra-pulmonary tuberculosis (albeit small numbers), time from transplant and longer duration of immunosuppression.
Recent studies have reported associations between EBV DNAemia in the first year after transplant and opportunistic infections, adverse events, and in some studies, greater rates of graft loss and graft dysfunction [6, 14, 20, 31, 32]. In the Bamoulid et al report, there is also concern raised that preemptive reduction of immunosuppression for EBV DNAemia persistently >104 log/mL copies may influence subsequent graft loss .
Risk of PTLD has been associated with EBV donor positive/recipient negative individuals, those with higher levels of EBV DNA detection, chronic HVL, detectable DNA in plasma and those receiving T cell depleting antibodies [7, 8, 23, 33-36]. Preemptive strategies to reduce PTLD incidence include reduction of immunosuppression, and more recently, the use of rituximab in transplant patients with persistent high-level DNAemia. Reduction in incidence of PTLD is reported in liver transplant and pediatric populations with such strategies [5-7, 37-42]. We calculated an increased risk of PTLD during follow-up for those with DNAemia at recruitment compared to those without detectable DNAemia. Should screening for EBV DNA be performed in the late posttransplant period, how frequently and in whom should immunosuppression be reduced preemptively? We show late EBV DNAemia is common, 46% ≥1 sample, and 16% with persistent viral loads >1000 copies/mL, but is not associated with poorer graft function or specific symptoms. However, it is of note that two of six PTLD cases (33%) occurred in seronegative recipients (high risk). Further, an EBV tissue histology-negative patient had persistent EBV DNAemia during the sampling period, yet, at the time of development of PTLD, had undetectable EBV DNA in blood. EBV seronegative recipients also had a greater incidence of DNAemia at recruitment and of chronic HVL. While only 10% of the study population, they accounted for at least 33% of PTLD cases during the follow-up period.
Screening EBV seronegative recipients for DNAemia, symptoms and lymphadenopathy for the lifetime of their graft is logistically and clinically sensible. This is a small group with a greater risk of PTLD than seropositive recipients, persisting into the late posttransplant period. However, there is no evidence from our study to support the use of EBV DNAemia detection in the low-risk seropositive group to prompt a change of immunosuppression or investigation for PTLD. Attention should perhaps focus as much on screening for symptoms and regular clinical examinations as on EBV DNAemia screening, particularly as EBV negative histology PTLD accounts for up to 50% of late PTLD cases in adult renal transplant recipients.