Epstein-Barr Virus Serostatus Research Articles

Training Load, Immune Status, and Clinical Outcomes in Young Athletes: A Controlled, Prospective, Longitudinal Study.

Introduction: Beside positive effects on athlete's health, competitive sport can be linked with an increased risk of illness and injury. Because of high relative increases in training, additional physical and psychological strains, and an earlier specialization and professionalization, adolescent athletes needs an increased attention. Training can alter the immune system by inducing a temporary immunosuppression, finally developing infection symptoms. Previous studies identified Epstein Barr Virus (EBV) as potential indicator for the immune status. In addition to the identification of triggering risk factors for recurrent infections, the aim was to determine the interaction between training load, stress sense, immunological parameters, and clinical symptoms.Methods: A controlled, prospective, longitudinal study on young athletes (n = 274, mean age: 13.8 ± 1.5 yrs) was conducted between 2010 and 2014. Also 285 controls (students, who did not perform competitive sports, mean age: 14.5 ± 1.9 yrs) were recruited. Athletes were examined 3 times each year to determine the effects of stress factors (training load: training hours per week [Th/w]) on selected outcome parameters (clinical [susceptibility to infection, WURSS-21: 21-item Wisconsin Upper Respiratory Symptom Survey], immunological, psychological end points). As part of each visit, EBV serostatus and EBV-specific IgG tiers were studied longitudinally as potential immune markers.Results: Athletes (A) trained 14.9 ± 5.6 h weekly. Controls (C) showed no lower stress levels compared to athletes (p = 0.387). Twelve percent of athletes reported recurrent infections (C: 8.5%, p = 0.153), the presence of an upper respiratory tract infection (URTI) was achieved in 30.7%. EBV seroprevalence of athletes was 60.3% (C: 56.6%, p = 0.339). Mean EBV-specific IgG titer of athletes was 166 ± 115 U/ml (C: 137 ± 112 U/ml, p = 0.030). With increasing Th/w, higher stress levels were observed (p < 0.001). Analyzes of WURSS-21 data revealed no relationship to training load (p = 0.323). Also, training load had no relation to EBV serostatus (p = 0.057) or the level of EBV-specific IgG titers (p = 0.364).Discussion: Young elite athletes showed no increased sense of stress, no higher prevalence of recurrent infections, and no different EBV-specific serological parameters compared to controls. Also, no direct relationship between training loads, clinical complaints, and EBV-specific immune responses was found. With increasing training loads athletes felt more stressed, but significant associations to EBV-specific serological parameters were absent. In summary, EBV serostatus and EBV-specific IgG titers do not allow risk stratification for impaired health. Further investigations are needed to identify additional risk factors and immune markers, with the aim to avoid inappropriate strains by early detection and following intervention.

Risk factors for post-transplant lymphoproliferative disorder after Thymoglobulin-conditioned hematopoietic cell transplantation.

Epstein-Barr virus (EBV)-induced post-transplant lymphoproliferative disorder (PTLD) occurs frequently when rabbit antithymocyte globulin (ATG) is used in hematopoietic cell transplant (HCT) conditioning. We retrospectively studied 554 patients undergoing ATG-conditioned myeloablative HCT. Strategies used to minimize mortality due to PTLD were either therapy of biopsy-diagnosed PTLD in the absence of EBV DNAemia monitoring (n=266) or prompt therapy of presumed PTLD (based on clinical/radiologic signs and high EBV DNAemia, in the setting of weekly EBV DNAemia monitoring) (n=199). Both strategies resulted in similar mortality due to PTLD (0.7% vs 1% at 2years, P=.43) and similar overall survival (63% vs 67% at 2years, P=.23) even though there was a trend toward higher PTLD incidence with the prompt therapy. Donor positive with recipient negative EBV (D+R-) serostatus was a risk factor for developing PTLD. Older patient age, HLA-mismatched donor, and graft-versus-host disease were not associated with increased risk of PTLD. In summary, in ATG-conditioned HCT, D+R- serostatus, but not older age, mismatched donor or GVHD is a risk factor for developing PTLD. EBV DNAemia monitoring may be a weak risk factor for developing/diagnosing PTLD; the monitoring coupled with prompt therapy does not improve survival.

Impact of Donor Epstein-Barr Virus Serostatus on the Incidence of Graft-Versus-Host Disease in Patients With Acute Leukemia After Hematopoietic Stem-Cell Transplantation: A Study From the Acute Leukemia and Infectious Diseases Working Parties of the European Society for Blood and Marrow Transplantation.

We investigated the effect of Epstein-Barr virus (EBV) serostatus on the overall outcome of allogeneic hematopoietic stem-cell transplantation (allo-HSCT). The study included 11,364 patients who underwent allogeneic peripheral-blood or bone marrow transplantation for acute leukemia between 1997 and 2012. We analyzed the impact of donor and recipient EBV serologic status on overall survival, relapse-free survival, relapse incidence, nonrelapse mortality, and incidence of graft-versus-host disease (GVHD) after allo-HSCT. Patients receiving grafts from EBV-seropositive donors had the same overall survival as patients who received grafts from EBV-seronegative donors (hazard ratio [HR], 1.05; 95% CI, 0.97 to 1.12; P = .23). Seropositive donors also had no influence on relapse-free survival (HR, 1.04; 95% CI, 0.97 to 1.11; P = 0.31), relapse incidence (HR, 1.03; 95% CI, 0.94 to 1.12; P = .58), and nonrelapse mortality (HR, 1.05; 95% CI, 0.94 to 1.17; P = .37). However, in univariate analysis, recipients receiving grafts from seropositive donors had a higher risk of chronic GVHD than those with seronegative donors (40.8% v 31.0%, respectively; P < .001; HR, 1.42; 95% CI, 1.30 to 1.56). When adjusting for confounders, higher risk was identified for both acute and chronic GVHD. In seronegative patients with seropositive donors, the HR for chronic GVHD was 1.30 (95% CI, 1.06 to 1.59; P = .039). In seropositive patients with seropositive donors, the HR was 1.24 (95% CI, 1.07 to 1.45; P = .016) for acute GVHD and 1.43 (95% CI, 1.23 to 1.67; P < .001) for chronic GVHD. Seropositive patients with seronegative donors did not have an increased risk of GVHD. Our data suggest that donor EBV status significantly influences development of acute and chronic GVHD after allo-HSCT.

Clinical Characteristic and Prognosis of Post-Transplant Lymphoproliferative Disorder in Pediatric Heart Transplant Recipients in Japan

Purpose: Post-transplant lymphoproliferative disorder (PTLD) caused by Epstein-Barr virus (EBV) has recently has been recognized as a serious complication of various organ transplantations. In our institute, we have followed-up 34 pediatric patients after heart transplantation. We retrospectively investigated clinical features of PTLD in heart transplant recipients.Methods: The evaluation period was from July 2004 to June 2015. Six patients with PTLD (4 boys, 2 girls) were diagnosed by blood examination, lymph node histology and EBV viral load in peripheral blood and invasive organ.Results: Median age at onset of PTLD was 4.5 years old (range, 3.2-16.2 years). Median time from transplantation to onset was 18 months (range, 7-45 months). Bone marrow and central nervous system invasion were not detected in all cases. Four patients were EBV-seronegative recipients for EBV-seropositive donors. We performed the lymphoma-based chemotherapy combined with the anti-CD20 monoclonal antibody rituximab and achieved remission. As a result, five patients remained alive in remission, and one patient died of therapy-related toxicity. Although one patient relapsed after chemotherapy, she achieved second remission after multimodal chemotherapy combined with rituximab. Abdominal lymph node involvement was detected in four cases, and one patient experienced severe abdominal symptoms including intestinal perforation.Conclusion: EBV serostatus and the period from transplantation to onset of PTLD appear to represent important prognostic factors. Early detection followed by pre-emptive reduction of immunosuppressive agents is essential to the management of PTLD, but may increase the risk of fatal rejection. We administered chemotherapy combined with rituximab, and this treatment strategy might be effective. However, severe fatal toxicity developed in one patient, and severe abdominal symptoms due to tumor lysis were noted in many cases. These findings suggested the importance of carefully selecting the induction treatment and perform various supportive therapies. We should establish appropriate treatment strategies according to prognostic factors because of the distinctive characteristics of PTLD by accumulating more cases. DisclosuresNo relevant conflicts of interest to declare.

Current preventive strategies and management of Epstein–Barr virus-related post-transplant lymphoproliferative disease in solid organ transplantation in Europe. Results of the ESGICH Questionnaire-based Cross-sectional Survey

There is limited clinical evidence on the utility of the monitoring of Epstein–Barr virus (EBV) DNAemia in the pre-emptive management of post-transplant lymphoproliferative disease (PTLD) in solid organ transplant (SOT) recipients. We investigated current preventive measures against EBV-related PTLD through a web-based questionnaire sent to 669 SOT programmes in 35 European countries. This study was performed on behalf of the ESGICH study group from the European Society of Clinical Microbiology and Infectious Diseases. A total of 71 SOT programmes from 15 European countries participated in the study. EBV serostatus of the recipient is routinely obtained in 69/71 centres (97%) and 64 (90%) have access to EBV DNAemia assays. EBV monitoring is routinely used in 85.9% of the programmes and 77.4% reported performing pre-emptive treatment for patients with significant EBV DNAemia levels. Pre-emptive treatment for EBV DNAemia included reduction of immunosuppression in 50.9%, switch to mammalian target of rapamycin inhibitors in 30.9%, and use of rituximab in 14.5% of programmes. Imaging by whole-body 18-fluoro-deoxyglucose positron emission tomography (FDG-PET) is used in 60.9% of centres to rule out PTLD and complemented computer tomography is used in 50%. In 10.9% of centres, FDG-PET is included in the first-line diagnostic workup in patients with high-risk EBV DNAemia. Despite the lack of definitive evidence, EBV load measurements are frequently used in Europe to guide diagnostic workup and pre-emptive reduction of immunosuppression. We need prospective and controlled studies to define the impact of EBV monitoring in reducing the risk of PTLD in SOT recipients.

Risk of Early and Late Non-Hodgkin Lymphoma (NHL) in Australian Liver and Cardiothoracic Transplant Recipients.

Aims: To determine whether risk of NHL is increased in cardiothoracic compared to liver transplant recipients, after adjustment for extent of immunosuppression and competing risk of death. Methods: A retrospective cohort study in Australia using population-based liver (n=1924) and cardiothoracic (n=2631) transplant registries (1984-2006). NHL incidence was ascertained by probabilistic record linkage with the Australian Cancer Database. Adult and pediatric transplant unit medical records were retrospectively reviewed to ascertain putative risk factor data, including demographics, co-morbid conditions, immunosuppressive agents received at transplantation and at regular intervals during follow-up, and serum viral antibody status at transplantation. Multiple imputation was used to impute missing Epstein-Barr virus (EBV) data. Adjusted hazard ratios (HR) for early (n=31) and late-onset (n=69) NHL were estimated using the Fine and Gray proportional subdistribution hazards model. Results: After imputation there were 22 early (<1yr) and 45 late (≥1yr) NHL cases available for multivariate analysis. Compared to liver recipients, lung recipients were at increased risk of early NHL (HR 5.18, 95%CI 1.39-19.3) after adjustment for recipient EBV seronegativity at transplantation (HR 3.33, 95%CI 1.08-10.4), receipt of OKT3 antibody (HR 10.0, 95%CI 2.50-40.0), male gender (HR 3.47, 95%CI 1.44-8.37), age, calendar year of transplant, receipt of ATG/ALG, and current dose of immunosuppressive agents. For late NHL, heart recipients were at increased risk compared to liver recipients (HR 3.66, 95%CI 1.33-10.0) after adjusting for EBV serostatus, age, sex, calendar year of transplant, time since transplantation, receipt of OKT3 and ATG/ALG, and the current dose and number of immunosuppressive agents. Imputed and non-imputed results were similar. Conclusions: Differences in immunosuppressive regimen do not appear to explain the excess risk of NHL in heart and lung compared to liver transplant recipients. There is growing evidence of heterogeneity in the risk factors for early and late NHL after solid organ transplantation.

Epstein-Barr virus and risk of non-Hodgkin lymphoma in the cancer prevention study-II and a meta-analysis of serologic studies

Epstein-Barr virus (EBV) causes rare, malignant lymphomas. The role of EBV in other non-Hodgkin lymphomas (NHLs) remains unclear, but mildly reduced immune function could lead to reactivation of EBV and subsequent NHL. We examined the association between prospectively-collected plasma EBV antibodies and NHL risk in the Cancer Prevention Study-II (CPS-II) Nutrition Cohort and conducted a meta-analysis of our and published results. The CPS-II study included 225 NHL cases and 2:1 matched controls. No associations were observed between EBV serostatus or antibody levels and risk of NHL overall. However, when including only the three most common types of NHL (diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma), high compared to low early antigen (EA-D) diffuse and BZLF1-encoded replication activator antibodies were associated with approximately 60% higher risk of NHL. Odds ratios (ORs) for EBV nuclear antigen-1 and viral capsid antigen (VCA)-p18 were elevated but not statistically significant. In the meta-analysis, both EA (summary OR = 1.52, 95% confidence interval (CI): 1.16-2.00) and VCA (summary OR = 1.20, 95% CI: 1.00-1.44) were positively associated with NHL risk. These results suggest EBV may be associated with a wider spectrum of NHL subtypes, but further study is needed to confirm and fully understand these associations.

AB0525 T-Lymphocyte Subsets and CMV and EBV DNA in Blood of SLE Patients and Kidney Transplant Recipients

BackgroundInfections are important cause of morbidity in SLE. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can cause serious complications in immunocompromised subjects. The clinical impact of CMV and EBV infections in...

Cytomegalovirus &amp; Epstein Barr Virus serostatus as a predictor of the long-term outcome of kidney transplantation

Cytomegalovirus (CMV) and Epstein Barr Virus (EBV) disease and asymptomatic infection have been associated with poor outcomes in kidney transplantation. Recipients who acquire primary infection through transplantation from a seropositive donor may be at particular risk of complications. The primary aim of this study was to evaluate the relationship between donor/recipient (D/R) CMV and EBV serostatus pretransplant and allograft and patient survival in a large cohort of kidney transplant recipients. Data were obtained from the Australian and New Zealand Dialysis and Transplant Registry and the Australian and New Zealand Organ Donation Registry on 4516 first deceased donor kidney transplants performed between 1998 and 2010. Graft and patient survival were analysed using the Kaplan-Meier method. Pretransplant CMV D/R serostatus was available for the whole cohort, with EBV D/R serostatus available for 2566 transplants (56.8%). Serostatus for both viruses was significantly associated with donor and recipient age and recipient smoking status. For both viruses the majority of transplants were in a D+/R+ serostatus setting: 45.3% for CMV and 77.9% for EBV. D/R serostatus for either virus did not have a significant effect on graft or patient survival. We conclude that in the current era of viral prophylaxis and surveillance, long-term outcome for the kidney transplant population is unaffected by D/R CMV and EBV serostatus.

Influence of CMV/EBV serostatus on respiratory infection incidence during 4 months of winter training in a student cohort of endurance athletes

The purpose of this study was to examine the influence of previous infection with cytomegalovirus (CMV) or Epstein Barr virus (EBV) on the incidence, severity and duration of upper respiratory tract infections (URTIs) in endurance athletes during a 4-month winter training period. Blood samples were obtained from 236 subjects (166 males and 70 females, aged 18-35years) at the start of the study period. Plasma samples were analysed for CMV and EBV serostatus. Weekly training and daily illness logs were kept for the next 16weeks. With regard to CMV/EBV serostatus, the results indicated that athletes with previous CMV infection (n=58, 25% of cohort) had significantly fewer URTI symptom days (median 2 vs. 4days, p=0.033) during the study period than those with no previous infection (n=178, 75%), whereas positive EBV serostatus (n=197, 84%) had no influence on URTI episode incidence, severity or duration. Moreover, we found that athletes with prior infection of both CMV and EBV (n=50, 21%) had 50% fewer URTI episodes (p=0.04) and symptom days (median 2 vs 8days, p=0.01) than athletes who were seronegative for both CMV and EBV (n=31, 13%). Previous coinfection with CMV and EBV might promote protective immune surveillance to lower the risk of URTI. Further research is required to clarify why previous CMV and EBV infection reduces the incidence of URTI.

Pretransplant Epstein-Barr Virus Serostatus and Incidence of Posttransplant Lymphoproliferative Disorder in Pediatric Renal Transplants

Pediatric renal transplant recipients are at increased risk of Epstein-Barr virus infection which may be due to the high percentage of Epstein-Barr virus seronegative recipients at the time of transplant in the pediatric age group. We aimed to assess the Epstein-Barr virus serostatus of recipients and donors and the incidence of posttransplant lymphoproliferative disorder in pediatric renal transplant at the Labafinejad Hospital in Tehran, Iran. We reviewed the clinical records of 183 children who had a renal transplant at the Labafinejad Hospital in Tehran, Iran, between 2003 and 2011. Of all the patients, 61.2% were Epstein-Barr virus seropositive at the time of transplant. Graft survival rate and the rate of acute rejection episodes were not statistically different between the seropositive and seronegative recipients. Three patients (0.005%) had posttransplant lymphoproliferative disorder after transplant. We showed that the rate of seronegative recipients in our cohort is similar to other studies, but the rate of posttransplant lymphoproliferative disorder was low in our recipients.

Reliability of the Siemens Enzygnost and Novagnost Epstein–Barr Virus assays for routine laboratory diagnosis: agreement with clinical diagnosis and comparison with the Merifluor Epstein–Barr Virus immunofluorescence assay

BackgroundDiagnosis of Epstein–Barr virus (EBV) infection is routinely conducted by clinical laboratories, especially to diagnose infectious mononucleosis. At an estimated general population incidence of 1:200, this represents a potentially significant testing burden. We evaluated the reliability of the Siemens Novagnost® and Enzygnost® EBV microtiter assays measuring VCA IgM and IgG, and EBNA-1 IgG for clinical diagnosis of EBV-related infectious mononucleosis.MethodsRemnant sera from 537 patients tested for EBV infection were used to compare the Siemens assays to each other and to the Merifluor assay. The Siemens assays are qualitative/semiquantitative, automatable enzyme immunoassays. The Merifluor assays are manual, qualitative indirect immunofluorescent assays. Testing was conducted on the Siemens and Merifluor assays in parallel. All assays were conducted and interpreted according to each manufacturer’s specifications. Agreement of serostatus between each of the three assays was assessed. Discrepant results were resolved using a third method (Mikrogen recomLine).ResultsFinal EBV serostatus indicated 2.9% of the population had an acute infection, 89.6% had a past infection, and 7.5% were EBV naive. All three assays demonstrated 100% agreement with acute infection. Agreement with past-infection serostatus was 99.1% for Enzygnost, between 86% and 98.8% for Novagnost, and 98.1% for Merifluor. Seronegative agreement was 100% for Enzygnost, 89.7% for Novagnost, and 92.3% for Merifluor.ConclusionsThe Siemens Enzygnost and Novagnost EBV microtiter assays are suitable for clinical rule-in of acute EBV infection and for identifying EBV-naive individuals. Both assays also adequately identify remote EBV infections. Because these assays can be automated, they can improve speed and efficiency of EBV testing, especially in high-volume laboratories.

NKG2C+CD57+ CD56dim NK cells are enriched following Epstein-Barr virus transformed lymphoblastoid cell line co-culture with peripheral blood mononuclear cells from cytomegalovirus+ donors (P4359)

Abstract Cytomegalovirus (CMV) is a persistent herpesvirus that promotes NKG2C+ Natural Killer (NK) cell differentiation. Highly mature CD57+ NKG2C+ NK cells have been proposed as the equivalent of mouse memory-like NK cells, which have features as long-term persistence and enhanced recall responses. AIM: To examine if co-infection with another common herpesvirus, Epstein-Barr virus (EBV), affects the NKG2C+ CD56dim NK cell population. METHODS: PBMCs from 45 five-year old children with known EBV and CMV serostatus were phenotyped. Further, PBMCs from 6 CMV sero+ EBV sero- infants were used for co-culture with EBV+ lymphoblastoid cell-lines (LCLs) during 72 hours. EBV- LCLs (Ramos) served as controls. Anti CD3, CD14, CD19, CD56, CD57, NKG2C and ki-67 antibodies were used for flow cytometric analysis. RESULTS: EBV and CMV co-infection was related to higher proportions of NKG2C+ and NKG2C+CD57+ CD56dim NK cells compared to infection with CMV or EBV alone, or seronegativity. In contrast to co-culture with Ramos, PBMC co-culture with EBV+ LCLs generally led to enrichment of NKG2C+CD57+ NK cells (stimulation index median 1.23, range 0.87-1.30), which was not related with higher NK cell proliferation. CONCLUSIONS: EBV co-infection seems to promote the existence of higher proportions of memory-like NK cells. Our data herein indicate that this is not due to enhanced proliferation of these cells; rather it suggests that memory-like NK cells may be maintained by EBV co-infection.

Does CMV Mismatch Status Modify the Risk of PTLD Due to EBV Mismatch Status in Lung and Heart Transplant Recipients?

Purpose Epstein-Barr virus (EBV) mismatch is a known risk factor for posttransplant lymphoproliferative disorders (PTLD) in lung and heart transplant recipients (LTx and HTx). We evaluated the potential modifying effect of cytomegalovirus (CMV) mismatch on the relation between EBV mismatch and PTLD in LTx and HTx. Methods and Materials We performed a study of U.S. LTx and HTx recipients from 2000-2011 in the Scientific Registry of Transplant Recipient (SRTR). Adjustment for sextiles of the propensity score (based on the probability of being an EBV mismatch) was used to account for confounding in Cox proportional hazards models stratified for organ type. Results The study population included 5957 LTx (61 PTLDs; 1.02%) and 7870 HTx (39 PTLDs; 0.5%) with complete data on CMV and EBV serostatus. A total of 272 LTx and 457 HTx were concurrently CMV and EBV mismatches. After adjustment for sextiles of the propensity score, the hazard ratio (HR) for PTLD was 4.4 (95% CI: 2.9-6.7) and 1.6 (95% CI: 1.1-2.5) in EBV and CMV mismatch recipients, respectively. When compared to patients who were neither CMV nor EBV mismatch, the adjusted HR for PTLD in patients who were CMV mismatch only, EBV mismatch only, and both CMV/EBV mismatch were 1.8 (95% CI: 1.1-3.3), 4.8 (95% CI: 2.9-8.1), and 7.0 (95% CI: 3.9-12.7), respectively. However, the HRs for the association of EBV mismatches and PTLD in CMV mismatch and non-mismatch patients did not significantly differ (P value for interaction=0.6). [ figure 1 ] Conclusions EBV mismatch status is significantly associated with risk for PTLD in LTx and HTx. Concomitant CMV mismatch status in EBV mismatch patients does not significantly modify the relative hazard of PTLD.

Molecular pathogenesis of B-cell posttransplant lymphoproliferative disorder: what do we know so far?

Posttransplant lymphoproliferative disorder (PTLD) is a potentially fatal disease that arises in 2%–10% of solid organ and hematopoietic stem cell transplants and is most frequently of B-cell origin. This very heterogeneous disorder ranges from benign lymphoproliferations to malignant lymphomas, and despite the clear association with Epstein-Barr Virus (EBV) infection, its etiology is still obscure. Although a number of risk factors have been identified (EBV serostatus, graft type, and immunosuppressive regimen), it is currently not possible to predict which transplant patient will eventually develop PTLD. Genetic studies have linked translocations (involving C-MYC, IGH, BCL-2), various copy number variations, DNA mutations (PIM1, PAX5, C-MYC, RhoH/TTF), and polymorphisms in both the host (IFN-gamma, IL-10, TGF-beta, HLA) and the EBV genome to B-cell PTLD development. Furthermore, the tumor microenvironment seems to play an important role in the course of disease representing a local niche that can allow antitumor immune responses even in an immunocompromised host. Taken together, B-cell PTLD pathogenesis is very complex due to the interplay of many different (patient-dependent) factors and requires thorough molecular analysis for the development of novel tailored therapies. This review aims at giving a global overview of the currently known parameters that contribute to the development of B-cell PTLD.

The Seville Expert Workshop for Progress in Posttransplant Lymphoproliferative Disorders

: Posttransplant lymphoproliferative disorders (PTLDs) are associated with significant morbidity and mortality among solid-organ transplant patients, but approaches to diagnosis and management vary considerably. An international multidisciplinary panel evaluated current understanding of risk factors and classification systems and developed recommendations to aid in PTLD prevention. We considered evidence on PTLD risk factors including Epstein-Barr virus serostatus and immunosuppression and identified knowledge gaps for future research. Recommendations address prophylactic and preemptive strategies to minimize PTLD development, including modulation of immunosuppression and antiviral drug regimens. Finally, new classification criteria were outlined that may help facilitate standardized reporting and improve our understanding of PTLD.

Development of multiplex serological assays to detect oncoviral infections

Serological markers of infection (antibodies or antigens) of viruses that cause cancer are most often detected using ELISA-based methodologies. In many cases, multiple markers of infection must be assessed to determine a final sero-status. Volume requirements and costs of reagents for single analyte ELISAs are high and studies which include multiple viruses can require milliliters of plasma, often not available from archived cohorts. Thus, we sought to develop a Luminex® bead-based customizable panel initially including Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Epstein-Barr Virus (EBV) and often the concomitant infection, Human Immunodeficiency Virus (HIV) to reduce sample volume requirements, overall cost and increase flexibility. Peptides, antigens and antibodies were sourced from multiple manufacturers and tested for suitability in this platform. Where necessary, suitable reagents were designed and produced in-house. The HCV assay multiplexes four HCV peptides designed to detect antibodies raised to HCV Core (2), HCV NS4, HCV NS5 gene regions. The HBV assay multiplexes a HBV early antigen peptide, a recombinant HBV core protein and either a recombinant HBV surface antigen or an antibody specific for HBV surface antigen to assess HBV infection. The EBV assay multiplexes peptides specific to viral capsid antigen, EBNA-1 and early antigen (Cyto-Barr, Zuidhorn, The Netherlands). HIV-1 assay development is ongoing and the list of antigens to be included in the assay has not been finalized. These assays can be run singly or with any combination (multi-plex) of the above listed targets. Each target has been independently validated using samples of known molecular and serological status to determine specificity (false positive versus false negative) and re-evaluated under multiplex conditions to confirm assay performance. In addition, where possible, samples were assayed on commercial testing platforms as well as our multiplex assay to assess concordance (94-99%). Dependent on the panel selected and the expected antibody titers in a particular population, plasma or serum volumes in the range of 10 µL to 125 µL per subject would be required to determine the HBV, HCV, EBV and/or HIV serostatus of a subject. This assay platform is inherently flexible and the benefits include amenability to expansion to include other oncogenic viruses as well as screening large epidemiological cohorts or smaller subsets of samples in an economical and high throughput manner.

Associations Between EBV Serostatus and Organ Transplant Type in PTLD Risk: An Analysis of the SRTR National Registry Data in the United States

In a prior multiorgan transplant database study, recipient Epstein-Barr virus (EBV) seronegativity was not associated with increased risk for posttransplant lymphoproliferative disorders (PTLD) in liver transplants (LTX), at variance with prior single center reports and with data from kidney and heart transplants (KTX and HTX). The Scientific Registry of Transplant Recipients (SRTR) in the United States is the only other registry with data on the required variables for comparison.Our study set comprised 112 756 KTX (580 PTLDs; 0.51%), 13 937 HTX (140 PTLDs; 1.0%) and 40 437 LTX (383 PTLDs; 0.95%) performed January 2003 onward. The unadjusted hazard ratio (HR) for PTLD if recipient EBV seronegative was 5.005 for KTX, 6.528 for HTX and 2.615 for LTX (p < 0.001 for all). In models adjusted for multiple covariates, the adjusted HR was 3.583 (p < 0.001) for KTX, 4.037 (p < 0.001) for HTX, 1.479 (p = 0.03) for LTX. Interaction models using EBV seropositive KTX as reference group showed significantly higher risk for all other EBV seronegative organ transplant groups and also for EBV seropositive LTX (AHR 2.053, p < 0.0001).Recipient EBV seronegativity is still significantly associated with risk for PTLD in LTX, though less so because of higher baseline risk in the EBV seropositive LTX group.

Impact of Epstein-Barr virus donor and recipient serostatus on the incidence of post-transplant lymphoproliferative disorder in kidney transplant recipients

Post-transplant lymphoproliferative disorder (PTLD) is a serious complication of transplantation. Using the OPTN/UNOS database, primary kidney recipients (2000-2009) were stratified according to transplant type (deceased donor, DD or living donor, LD), donor (D) and recipient (R) Epstein-Barr virus (EBV) serostatus (R+; D+/R- and D-/R-) and recipient age. Incidence and adjusted risk of PTLD and death were compared. Of the 137 939 primary kidney recipients transplanted between 2000 and 2009, 913 subsequently developed PTLD. In 90 208 recipients with known EBV serostatus, we found a trend toward a decrease in PTLD incidence in years 2007-2009 when compared to 2000-2003. This was due to a significant decrease in PTLD incidence in EBV- recipients. Of those, 61 273 had a known donor serostatus and were further examined. In adults, PTLD incidence (in 1000 person-years) in DD and LD was 7.0 and 7.0 in D+/R-; 3.0 and 2.5 in D-/R- and 1.2 and 1.0 in R+, respectively. The hazard ratio (HR) for PTLD (R+ as reference) in D+/R- (6.2 in DD and 7.2 in LD) was double to thrice than for D-/R- transplants (2.4 in both DD and LD). In pediatric recipients, PTLD incidence in DD and LD was 15.9 and 17.3 in D+/R-; 12 and 18 in D-/R- and 1.2 and 2.2 in R+, respectively. The HR for PTLD was 17.4 and 6.9 in D+/R- and 15.9 and 7.6 in D-/R- in DD and LD, respectively. A D+/R-, compared with a D-/R- transplant, may contribute to an increase in PTLD incidence of 35 and 42% in adult DD and LD transplants, respectively.

Risk Factors for Post-Transplant Lymphoproliferative Disorder in Solid Organ Transplant Recipients

Abstract 4465 Introduction:Solid organ transplant recipients are profoundly immune suppressed and are at risk for post-transplant lymphoproliferative disorder (PTLD). PTLD is associated with substantial morbidity and mortality. With limited treatment options, earlier detection of disease and earlier initiation of therapy may be essential for optimizing therapeutic outcomes. Thus, identifying at risk groups is critical. Methods:We conducted an IRB-approved retrospective study of 1,496 solid organ transplant recipients treated at our institution to identify risk factors for the development of PTLD. We identified 104 cases of PTLD going back over the past 20 years, through 1991. A cross-sectional sample of solid organ transplants evaluated at our institution was used as controls (N = 1,392). Charts were reviewed for relevant demographic variables, reason for transplantation, Epstein-Barr virus (EBV) serostatus pre-transplant, and Human Leukocyte Antigen (HLA)-type. Information on outcomes, such as organ rejection over follow up and death was also recorded. In individuals with PTLD, date of diagnosis, tumor characteristics (i.e., EBV status), tumor pathology, and PTLD treatment was recorded. Survival analysis (Kaplan-Meier plots and Cox proportional hazards regression) was conducted on collected data, to determine risk factors for the development of PTLD. Results:In the cohort as a whole, 86/1,496 (5.8%) of individuals were EBV seronegative at baseline, and 20.9% (312/1496) did not have an available documented baseline EBV serology. EBV serostatus was the major risk factor for EBV-associated PTLD (N=74, or 71% of PTLD cases): EBV-seronegative individuals had a Hazard Ratio (HR) =7.70 [95% CI = 3.8 – 15.5, p < 0.001] for EBV-associated PTLD compared to seropositive individuals. Thus, further analyses were stratified by EBV serostatus, to examine the effect of HLA-type on risk of development of PTLD. In an exploratory data analysis, we found that HLA-B40 was a major risk factor for the development of EBV-associated PTLD in EBV-seronegative individuals, while HLA-B8 was a risk factor for the development of EBV-associated PTLD in EBV-seropositive individuals. In baseline seronegative individuals HLA-B40 was associated with a HR=5.99 [95% CI = 1.95 – 18.4, p=0.002] for PTLD (see figure below for Kaplan Meier curves). In baseline seropositive individuals HLA-B8 was associated with a HR = 3.65 [95% CI = 1.52 – 8.76, p= 0.004] for PTLD. In EBV seropositive individuals, receipt of either a liver, cardiac, lung or pancreas transplant (compared to a kidney transplant alone) was associated with an increased risk of PTLD as well, HR = 6.72 [95% CI = 2.78 – 16.24, p < 0.001. Conclusions:While the relation of HLA-type and EBV-associated PTLD seen in the study are novel, we recommend future studies to test these observations. These results demonstrate that risk factors for EBV-associated PTLD may verify substantially with baseline EBV-serostatus. We recommend future studies consider stratifying analyses by baseline EBV-serostatus, as EBV-associated PTLD resulting from primary EBV-infection may represent a distinct pathology than EBV-associated PTLD arising in individuals who are known to be EBV-seropositive. Disclosures:No relevant conflicts of interest to declare. [Display omitted]