Measurement of the Epstein-Barr Virus (EBV) load in the peripheral blood using DNA amplification techniques has become increasingly available for the management of organ transplant recipients (1). In this volume of the American Journal of Transplantation, Tsai and colleagues add to our knowledge of this test by exploring the use of EBV PCR for the diagnosis and management of post-transplant lymphoproliferative disorders (PTLD) in adult solid organ transplant (SOT) recipients (2). As most published experience to date has been derived from pediatric organ transplant or bone marrow transplant recipients, analysis of data derived from adult SOT is necessary to determine whether or not results obtained from this population will be similar to findings from children and marrow recipients. The findings that EBV viral loads obtained from adult SOT recipients undergoing evaluation for PTLD were highly specific but only marginally sensitive contrast with previously published work, which has typically found the EBV viral load to be extremely sensitive but to lack specificity for the presence of EBV-associated disease (1–3).
The differences between the current investigation and previously published studies may be explained by a number of reasons. One obvious explanation for these differences could be variations in PCR assay methods, units or cut-offs of positivity. Published experience has been based on determination of EBV viral load within peripheral blood lymphocytes (PBL), whole blood or plasma. Amplification of EBV genome has been performed using a wide variety of primer sets and target genes. Results of viral load detection using PCR have been reported as genome copies/105 PBL, genome copies/mL or genome copies/µg DNA. Definitions for ‘elevated’ loads, ‘high’ loads or ‘positive’ loads have been arbitrarily determined and have varied between laboratories and publications. Efforts to standardize methods, sites of sampling, and levels of load that denote risk have not been undertaken but are urgently needed.
Differences in patient population may also explain the variations in sensitivity and specificity observed between the current report and previously published studies. Obtaining results from a cohort of patients in whom PTLD is clinically suspected may lead to enhanced specificity for the presence of EBV disease compared with results obtained from patients undergoing surveillance or random sampling in the absence of a clinical illness. Specificity may also be affected by the EBV serostatus of the patient before transplantation. Experience from our center suggests that children developing primary EBV infection after transplantation are more likely to become chronic asymptomatic carriers of high EBV loads than those who are seropositive at the time of the transplant (4,5). As the majority of pediatric transplant recipients are seronegative before transplantation, high load carriers are seen frequently in this population. In contrast, adults are rarely EBV seronegative before transplantation, suggesting that high low carriers will be observed infrequently in these patients. Thus elevated loads in adults may be more likely to be associated with PTLD, possibly explaining the higher specificity reported in the current study compared with previous reports. Additional data are necessary to confirm these hypotheses.
Not only the specificity but also the sensitivity of the EBV viral load could be affected by the EBV serostatus at the time of the transplant. It is possible that EBV viral loads may be higher in patients with PTLD that develops in recipients who experience primary infection following their transplant compared with patients with PTLD who were EBV-seropositive before transplantation. ‘Low-load’ PTLD (where EBV viral loads at the time of diagnosis of PTLD fall below predicted cut-offs) may be seen more frequently in patients who are seropositive before transplantation than those who are seronegative. Because the vast majority of children are EBV-seronegative before transplantation, EBV viral loads at the time of PTLD may be higher in children resulting in a greater sensitivity compared with adults where seropositive patients may account for significant proportions of cases of PTLD. Again, this hypothesis has yet to be confirmed.
The availability of a noninvasive, accurate test that can predict risk for, or presence of, EBV-associated PTLD is a highly desirable development for the field of transplantation. Unfortunately, the technology to acquire a result currently exceeds our understanding of its meaning. While one or more differences in the test or patient populations may explain the contrast between the results of Tsai et al. and previous publications, neither this work nor previously published work clarifies this for the transplant clinician. What we do know is that the EBV viral load is elevated in most pediatric patients with PTLD. However, an elevated EBV PCR alone in these patients does not differentiate between asymptomatic infection, symptomatic EBV infection (not fulfilling the diagnostic criteria of PTLD) and PTLD. We do not know if elevated EBV viral loads will be detected less often in adults compared with pediatric organ transplant recipients with PTLD, though the current study suggests this may be the case. We know that development of a high EBV viral load occurs uncommonly in patients who are EBV-seropositive before transplantation. However, we do not know if pre-existing immunity against EBV before transplantation will affect the EBV viral load at the time of diagnosis of EBV-associated PTLD. We know that intermittent elevations in EBV viral load can be observed with treatment of rebound rejection but that these transient elevations are not typically associated with relapsing disease. We do not know the significance of the high-load carrier state nor whether the use of EBV viral load monitoring plays any role in identifying relapse of PTLD. Finally, we do not know whether additional laboratory tests can be identified that may add to the sensitivity or specificity of the EBV viral load in organ transplant recipients, though preliminary work characterizing viral gene expression in the load looks promising (4). Determining the answers to these questions about the EBV viral load for which we have no answers remains an important challenge to all of us who care for patients undergoing transplantation. Collaborative, prospective studies aimed at answering these questions must be designed and as importantly funded if we are to maximize the full potential of the EBV viral load to impact on the incidence and outcome of EBV disease in organ transplant recipients.