Epstein-Barr Virus Load Research Articles

Long term treatment with abatacept or tocilizumab does not increase Epstein-Barr virus load in patients with rheumatoid arthritis - A three years retrospective study.

BackgroundEpstein-Barr Virus (EBV) is a widely disseminated lymphotropic herpes virus implicated in benign and malignant disorders. In transplant patients, immunosuppressive drugs (cyclosporine) diminish control of EBV replication, potentially leading to lymphoproliferative disorders (LPD). Rheumatoid arthritis (RA) patients have impaired control of EBV infection and have EBV load ten times higher than controls. As post transplant patients, patients with RA have increased risk of developing lymphomas. Immunosuppressive drugs used to treat RA (conventional disease modifying drugs cDMARDs or biologics bDMARDs) could enhance the risk of developing LPD in RA patients. We have previously shown that long term treatment with Methotrexate and/or TNF alpha antagonists does not increase EBV load in RA. Our objective was to monitor the Epstein-Barr Virus load in RA patients treated with Abatacept (CTLA4 Ig), a T cell coactivation inhibitor, and Tocilizumab, an anti IL6 receptor antibody.MethodsEBV load in the peripheral blood mononuclear cells (PBMCs) of 55 patients under Abatacept (in 34% associated with Methotrexate) and 35 patients under Tocilizumab (in 37% associated with Methotrexate) was monitored for durations ranging from 6 months to 3 years by real time PCR. The influences of treatment duration and disease activity score 28 (DAS28) index on EBV load were analyzed.ResultsAbatacept did not significantly modify EBV load over time. Tocilizumab significantly diminished EBV load over time. No patient (of 90) developed EBV associated lymphoma.ConclusionLong term treatment with Abatacept or Tocilizumab does not increase EBV load in the PBMNCs of patients with RA.

Comparison of eMAG™ versus NucliSENS® EasyMAG® performance on clinical specimens

BackgroundeMAG™ (bioMerieux) is a new nucleic acid extraction platform based on magnetic silica technology, like its predecessor, NucliSENS® easyMAG® (bioMerieux). Using the same reagents and disposables, eMAG™ adds further automation, allowing simultaneous extraction of 48 samples directly from primary tubes, and distribution of nucleic acid extracts on PCR strips or in tubes at the end of the extraction process. ObjectiveTo compare the performance of eMAG™ and easyMAG® on various clinical specimens. Study designRespiratory (n=199), whole blood (n=50), plasma (n=25) and urine (n=25) specimens were extracted in parallel on both platforms. Both qualitative (respiratory virus, cell control, CMV, EBV, HHV6 and BKV detection) and quantitative (respiratory virus and cell control cycle thresolds, and CMV, EBV, HHV6 and BKV viral loads) results were compared. ResultsDetection of qualitative targets showed good agreement, ranging from 84.6% for whole blood to 95.9% for respiratory specimens. Correlations between quantitative results were good, with R2 ranging from 0.802 to 0.995. Quantitative results showed average overall differences below 0.10 log10 copies/mL between eMAG™ and easyMAG®. ConclusionsThe two platforms showed comparable performance on the types of clinical specimen tested. With higher automation and throughput than easyMAG®, the eMAG™ platform is likely to be advantageous for laboratories performing a large number of molecular analyses.

Correlation between Epstein-Barr Virus Infection and Disease Activity of Systemic Lupus Erythematosus: a Cross-Sectional Study

Background:Systemic lupus erythematosus (SLE) is an autoimmune disease for whose pathogenesis viral infections are important. The Epstein-Barr virus (EBV) is the main infectious etiological agent. This study aimed to quantitative evaluation of EBV in SLE patients.Materials and Methods:In this cross-sectional study, 40 patients with SLE diagnosed based on American College of Rheumatology criteria were selected using purposive sampling. All were included in the study after obtaining informed consent for participation. Whole blood samples were taken and buffy coat preparations were isolated to determine viral load using the real-time polymerase chain reaction method and assessment with the SLE disease activity index (SLE-DAI).Results:From a total of 40 patients, 37 cases (92.5%) were women. The EBV test was positive in 67.5% and mean viral load was 5396 ± 1891.9 copy/ml. Twenty of forty patients had active and 50% inactive disease, mean EBV viral loads being 6798 and 28.25 copy/ml, respectively (P-value = 0.003). In terms of the severity of disease activity, 17.5 % of female patients had mild to moderate activity, whilst 32.5% of them had severe activity, with respective viral loads of 5,803.3 and 29.73 copy/ml (P-value = 0.003).Conclusion:The Epstein-Barr viral load in SLE patients with active disease was found to be markedly higher than in inactive cases. Thus, EBV may have an important role in the pathogenesis and activity of SLE.

Epstein–Barr Viral Load Monitoring for Diagnosing Posttransplant Lymphoproliferative Disorder in pediatric Liver and Heart Transplant Recipients

Epstein–Barr Viral Load Monitoring for Diagnosing Posttransplant Lymphoproliferative Disorder in pediatric Liver and Heart Transplant Recipients

Regulatory T Cells in Endemic Burkitt Lymphoma Patients Are Associated with Poor Outcomes: A Prospective, Longitudinal Study.

Deficiencies in Epstein-Barr virus (EBV)-specific T cell immunosurveillance appear to precede the development of endemic Burkitt lymphoma (eBL), a malaria-associated pediatric cancer common in sub-Saharan Africa. However, T cell contributions to eBL disease progression and survival have not been characterized. Our objective was to investigate regulatory and inflammatory T cell responses in eBL patients associated with clinical outcomes. By multi-parameter flow cytometry, we examined peripheral blood mononuclear cells from 38 eBL patients enrolled in a prospective cohort study in Kisumu, Kenya from 2008–2010, and 14 healthy age-matched Kenyan controls. Children diagnosed with eBL were prospectively followed and outcomes categorized as 2-year event-free survivors, cases of relapses, or those who died. At the time of diagnosis, eBL children with higher CD25+Foxp3+ regulatory T (Treg) cell frequencies were less likely to survive than patients with lower Treg frequencies (p = 0·0194). Non-survivors also had higher absolute counts of CD45RA+Foxp3lo naïve and CD45RA-Foxp3hi effector Treg subsets compared to survivors and healthy controls. Once patients went into clinical remission, Treg frequencies remained low in event-free survivors. Patients who relapsed, however, showed elevated Treg frequencies months prior to their adverse event. Neither concurrent peripheral blood EBV load nor malaria infection could explain higher Treg cell frequencies. CD8+ T cell PD-1 expression was elevated in all eBL patients at time of diagnosis, but relapse patients tended to have persistently high PD-1 expression compared to long-term survivors. Non-survivors produced more CD4+ T-cell IL-10 in response to both Epstein-Barr Nuclear Antigen-1 (EBNA-1) (p = 0·026) and the malaria antigen Plasmodium falciparum Schizont Egress Antigen-1 (p = 0·0158) compared to survivors, and were concurrently deficient in (EBNA-1)-specific CD8+ T-cell derived IFN-γ production (p = 0·002). In addition, we identified the presence of Foxp3-IL10+ regulatory Type 1 cells responding to EBNA-1 in contrast to the malaria antigen tested. These novel findings suggest that poor outcomes in eBL patients are associated with a predominantly immuno-regulatory environment. Therefore, Treg frequencies could be a predictive biomarker of disease progression and manipulation of Treg activity has potential as a therapeutic target to improve eBL survival.

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis.

Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection of EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains, which may poorly reflect primary viral envelope diversity. We report some of the first primary gp350 sequences, noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development.

AMC-053: Pilot Study of an Oncolytic Viral Strategy Using Bortezomib with ICE +/- Rituximab for Relapsed/Refractory HIV+ Lymphomas

Introduction:Both HIV+ Hodgkin and non-Hodgkin lymphomas have higher rates of latent infection by the gamma-herpesviruses (GHVs), Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpes virus (KSHV), than corresponding lymphomas in the HIV-seronegative population. Bortezomib, a proteasome inhibitor, induces lytic activation of both EBV and KSHV. Lytic activation of GHVs latently infecting lymphoma cells is hypothesized to beneficial both for direct tumor cell lysis as well as increased cytotoxic immune response due to viral lytic gene products. Furthermore, preclinical studies found proteasome inhibition impairs HIV infectivity via preservation of human anti-retroviral APOBEC3G, suggesting a novel therapeutic strategy to control HIV. Given that therapy of relapsed or refractory HIV-associated lymphoma (R/R-HAL) results in modest rates of remission, we sought to capitalize on the high viral association within HAL using an oncolytic strategy with bortezomib.Objectives:The primary objective of this study was to evaluate safety and overall response rate (ORR) of R/R-HAL to bortezomib combined with ifosfamide, carboplatin, etoposide +/- rituximab (ICE/R).The secondary objectives of this study were to estimate the impact of bortezomib on lytic activation of EBV and KSHV, using peripheral blood mononuclear cell (PBMC) viral loads, and on HIV using single copy plasma viral loads; to report overall survival at 1 year (1yr-OS); and to correlate EBV and KSHV viral load changes with lymphoma response.Methods:A 3+3 dose escalation design with a 7-day lead-in period of bortezomib alone prior to bortezomib + ICE/R allowed for assessment of early effects of bortezomib on viral loads. Bortezomib was given intravenously on day 1 and 8 of each cycle at one of 4 dose levels: 0.7, 1, 1.3 or 1.5 mg/m2. Standard dose ICE +/- R began day 8 of cycle 1 (28-day cycle); ICE +/- R began day 1 of all subsequent cycles (21-day cycle). Rituximab was included in the regimen only for CD20+ lymphoma.Binomial proportions were used to estimate ORR. The product-limit (Kaplan-Meier) method was used to estimate 1yr-OS. The Wilcoxon signed rank test was used to evaluate changes in viral loads.Results:Twenty-three subjects were enrolled from 7 sites within the AIDS Malignancy Consortium (AMC). More than 90% of enrolled subjects were men and half were minorities; at baseline, 20/23 were on antiretroviral therapy, median CD4 count was315/µL and median HIV viral load was undetectable. Mean age was 50 years. Over half of subjects had stage IV HAL; the majority had diffuse large B-cell lymphoma (DLBCL) (n=15), 2 had primary effusion (PEL), 3 had plasmablastic and 2 had Hodgkin lymphoma.Figure 1 summarizes grade 3-4 toxicities of the 22 subjects evaluable for adverse events during the dose-limiting toxicity period (cycles 1+2). The maximum tolerated dose was not reached at the highest dose cohort studied (bortezomib 1.5mg/m2).Responses occurred in 14/22 subjects initiating protocol therapy: 5 complete and 9 partial responses (PR). Of the 20 subjects who completed 2 or more cycles, the ORR was 80%. Nine of the responders underwent auto-hematopoietic stem cell transplant after protocol therapy. 1yr-OS was 55%.After bortezomib alone, median values of EBV PBMC viral load measured on day 8 were 2x greater than baseline. However, paired analysis did not confirm significant change in this small sample (n=16 evaluable), and there was no correlation found between change in EBV viral load and response. The 2 subjects with known KSHV+ lymphoma (PEL) each had more than a 1-log increase in day 8 KSHV viral load compared with baseline. Both of these subjects attained a PR from protocol therapy.Conclusions:Addition of bortezomib to ICE/R in R/R HAL is feasible with ORR (80%) and 1yr-OS (55%) comparing very favorably with a prior AMC retrospective report of ICE/R in R/R HAL (n=31, ORR 32%, 1yr-OS 38%, Bayraktar 2012). Evaluation of data collected from individual subjects suggests GHV lytic activation may occur with bortezomib alone. However, the lower bortezomib dose levels used to treat the bulk of study subjects and the limited study sample size limit our power to confirm this conclusion. We plan to further explore the effects of proteasome inhibition on GHVs. Evaluation of the impact of bortezomib on HIV replication is pending and will be presented at the meeting. [Display omitted] DisclosuresReid:Millennium: Research Funding. Sparano:Takeda: Research Funding.

Rituximab, etoposide, methylprednisolone, high-dose cytarabine, and cisplatin in the treatment of secondary hemophagocytic lymphohistiocytosis with classical Hodgkin lymphoma: a case report and review of the literature

BackgroundHemophagocytic lymphohistiocytosis is becoming an increasingly recognized disorder in adults. Classical Hodgkin lymphoma is a relatively uncommon etiology of hemophagocytic lymphohistiocytosis and may complicate treatment options. Rituximab, etoposide, methylprednisolone, high-dose cytarabine, and cisplatin are discussed here as a treatment regimen. Case presentationA 66-year-old Hispanic man previously in good health presented with a 1-month history of recurrent fevers, chills, and night sweats and a 3-week history of new onset jaundice. A bone marrow biopsy revealed a normocellular bone marrow with increased histiocytes with areas of hemophagocytic activity. He met five out of eight criteria for hemophagocytic lymphohistiocytosis diagnosis including fevers, pancytopenia, hemophagocytosis, ferritin of 23,292 ng/mL (>500 ng/mL), and soluble-CD25 of 15,330 pg/mL (>1033 pg/mL). A right cervical lymph node biopsy revealed CD15, CD30, MUM-1, and Epstein–Barr virus-encoded small ribonucleic acid-positive cells with morphologic findings of classical Hodgkin lymphoma, lymphocyte-rich subtype. He completed 2 weeks of hemophagocytic lymphohistiocytosis-directed therapy with etoposide and dexamethasone, but then was switched to rituximab, etoposide, methylprednisolone, high-dose cytarabine, and cisplatin due to minimal improvement in his pancytopenia and hepatic impairment. He completed one full cycle of rituximab, etoposide, methylprednisolone, high-dose cytarabine, and cisplatin with notable improvement in serial hepatic function panels and had an undetectable Epstein–Barr virus viral load. However, he eventually died due to complications of Enterococcus faecalis bacteremia and colonic microperforation in the setting of persistent pancytopenia.ConclusionsThis case discusses the challenges facing treatment of adult malignancy-associated hemophagocytic lymphohistiocytosis. Rituximab, etoposide, methylprednisolone, high-dose cytarabine, and cisplatin may be a viable option for patients with secondary hemophagocytic lymphohistiocytosis and Hodgkin lymphoma who cannot tolerate standard therapies due to hepatic impairment. Targeted therapy and immunotherapy are promising new areas of developing treatments.

The Epstein-Barr virus DNA load in the peripheral blood of transplant recipients does not accurately reflect the burden of infected cells.

Transplant recipients frequently exhibit an increased Epstein-Barr virus (EBV) load in the peripheral blood. Here, we quantitated the EBV-infected cells in the peripheral blood of these patients and defined the mode of viral infection, latent or lytic. These data indicated that there is no strong correlation between the number of infected cells and the EBV load (EBVL). This can be explained by a highly variable number of EBV copies per infected cell and by lytic replication in some cells. The plasma of these patients did not contain any free infectious viruses, but contained nevertheless EBV DNA, sometimes in large amounts, that probably originates from cell debris and contributed to the total EBVL. Some of the investigated samples carried a highly variable number of infected cells in active latency, characterized by an expression of the Epstein-Barr nuclear antigens (EBNA2) protein. However, a third of the samples expressed neither EBNA2 nor lytic proteins. Patients with an increased EBVL represent a heterogeneous group of patients whose infection cannot be characterized by this method alone. Precise characterization of the origin of an increased EBVL, in particular, in terms of the number of EBV-infected cells, requires additional investigations including the number of EBV-encoded small RNA-positive cells.

Prognostic significance of signal transducer and activator of transcription 5 and 5b expression in Epstein-Barr virus-positive patients with chronic lymphocytic leukemia.

Signal transducer and activator of transcription (STAT) proteins have been intensively studied in hematologic malignancies, and the efficacy of agents against STATs in lymphomas is already under research. We investigated the expression of total STAT5 and STAT5b in peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) in correlation with the presence of Epstein–Barr Virus (EBV) and its major oncoprotein (latent membrane protein 1, LMP1). The EBV load was measured in the peripheral blood by real‐time PCR for the BXLF1 gene and the levels of LMP1 by PCR and ELISA. Western blotting was performed for total STAT5 and STAT5b in protein extracts. STAT5b was only expressed in patients (not in healthy subjects) and STAT5 but particularly STAT5b expression was correlated with the presence of the virus (77.3% vs. 51.2%, P = 0.006 for STAT5b) and to the expression of LMP1 (58.3% vs. 21.6%, P = 0.011 for STAT5b). Moreover, the expression of STAT5b and the presence of EBV and LMP1 were strongly negatively correlated with the overall survival of the patients (log‐rank test P = 0.011, 0.015, 0.006, respectively). Double positive (for EBV and STAT5b) patients had the lowest overall survival (log‐rank test P = 0.013). This is the first report of a survival disadvantage of EBV+ patients with CLL, and the first time that STAT5b expression is correlated with survival. The correlation of STAT5 expression with the presence of the virus, along with our survival correlations defines a subgroup of patients with CLL that may benefit from anti‐STAT agents.

Factors influencing survival among Kenyan children diagnosed with endemic Burkitt lymphoma between 2003 and 2011: A historical cohort study.

Discovering how to improve survival and establishing clinical reference points for children diagnosed with endemic Burkitt lymphoma (eBL) in resource-constrained settings has recaptured international attention. Using multivariate analyses, we evaluated 428 children with eBL in Kenya for age, gender, tumor stage, nutritional status, hemoglobin, lactate dehydrogenase (LDH), Epstein-Barr virus (EBV) and Plasmodium falciparum prior to induction of chemotherapy (cyclophosphamide, vincristine, methotrexate and doxorubicin) to identify predictive and prognostic biomarkers of survival. During this 10 year prospective study period, 22% died in-hospital and 78% completed six-courses of chemotherapy. Of those, 16% relapsed or died later; 31% achieved event-free-survival; and 31% were lost to follow-up; the overall one-year survival was 45%. After adjusting for covariates, low hemoglobin (<8 g/dL) and high LDH (>400 mU/ml) were associated with increased risk of death (adjusted Hazard Ratio (aHR) = 1.57 [0.97-2.41]) and aHR = 1.84, [0.91-3.69], respectively). Anemic children with malaria were 3.55 times more likely to die [1.10-11.44] compared to patients without anemia or malarial infection. EBV load did not differ by tumor stage nor was it associated with survival. System-level factors can also contribute to poor outcomes. Children were more likely to die when inadvertently overdosed by more than 115% of the correct dose of cyclophosphamide (a HR = 1.43 [0.84-2.43]) or doxorubicin (a HR = 1.25, [0.66-2.35]), compared with those receiving accurate doses of the respective agent in this setting. This study codifies risk factors associated with poor outcomes for eBL patients in Africa and provides a benchmark by which to assess improvements in survival for new chemotherapeutic approaches.

Monitoring of Epstein–Barr virus load and killer T cells in patients with juvenile idiopathic arthritis treated with methotrexate or tocilizumab

Objectives: Methotrexate (MTX) is used for the treatment of polyarticular juvenile idiopathic arthritis (JIA), and an anti-interleukin-6 receptor monoclonal antibody (tocilizumab: TCZ) is also used and added for the treatment of intractable JIA. It has been reported that MTX might induce Epstein–Barr virus (EBV)-associated lymphoma, but the discussion about the effect of MTX and/or TCZ against reactivation of EBV in pediatric patients has been incomplete.Methods: The EBV loads in four polyarticular JIA and three systemic arthritis JIA patients treated with MTX and/or TCZ, and the percentage of EBV-specific killer T cells (EBV-CTLs) in some patients were prospectively monitored.Results: No patients had EBV-associated symptoms during the observation period. EBV loads in all patients were not significantly increased, and the levels of EBV loads were the same as EBV-seropositive healthy children following the administration of MTX and/or TCZ. EBV-CTLs were detectable during the observation period, but some patients had slightly low levels of EBV-CTLs.Conclusion: Treatment with MTX and/or TCZ did not severely affect EBV load and prevent induction of EBV-CTLs in JIA patients.

Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens

Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P=0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays.

Automated quantification of Epstein–Barr Virus in whole blood of hematopoietic stem cell transplant patients using the Abbott m2000 system

BackgroundAccurate quantification of Epstein-Barr virus (EBV) load in blood is essential for the management of post-transplant lymphoproliferative disorders. The automation of DNA extraction and amplification may improve accuracy and reproducibility. We evaluated the EBV PCR Kit V1 with fully automated DNA extraction and amplification on the m2000 system (Abbott assay). MethodologyConversion factor between copies and international units (IU), lower limit of quantification, imprecision and linearity were determined in a whole blood (WB) matrix. Results from 339 clinical WB specimens were compared with a home-brew real-time PCR assay used in our laboratory (in-house assay). ResultsThe conversion factor between copies and IU was 3.22 copies/IU. The lower limit of quantification (LLQ) was 1000 copies/mL. Intra- and inter-assay coefficients of variation were 3.1% and 7.9% respectively for samples with EBV load higher than the LLQ. The comparison between Abbott assay and in-house assay showed a good concordance (kappa = 0.77). Loads were higher with the Abbott assay (mean difference = 0.62 log10 copies/mL). SignificanceThe EBV PCR Kit V1 assay on the m2000 system provides a reliable and easy-to-use method for quantification of EBV DNA in WB.

Both high and low levels of cellular Epstein-Barr virus DNA in blood identify failure after hematologic stem cell transplantation in conjunction with acute GVHD and type of conditioning.

The level of Epstein-Barr virus DNA in blood has proven to be a biomarker with some predictive value in allogeneic hematopoietic stem cell transplantation patients (HSCT). We evaluated the impact of EBV load on survival of 51 patients (32M/19F, median age: 32 years, from < 1 to 68 years old), who had received HSCT for different types of malignancies (49 cases) or non-malignancies (2 cases). The overall survival [1]was compared between patients with extreme and moderate cell bound EBV DNA levels. Different sources of stem-cells (peripheral blood stem, n = 39; bone marrow, n = 9; or umbilical cord blood, n = 3) were used. Twenty patients received reduced-intensity conditioning regimen while the other 31 received myeloablative conditioning. Patients with high or very low level of cell bound EBV-DNA levels had a shorter OS than those with moderate EBV load: OS at 5 years was 67% vs 90% (p < 0.03). There was a conspicuous relationship between EBV load and the reconstitution dynamics of total and EBV-specific T cells, CD4+ and CD4- CD8- (double negative) T cells in the few patients where it was analyzed. This was not statistically significant. Two other factors were associated to early mortality in addition to high or low EBV load: acute GVHD II-IV (p < 0.02) and pre-transplant conditioning with total body irradiation (TBI) ≥6 Gy, (p < 0.03). All the patients meeting all three criteria died within two years after transplantation. This points to a subgroup of HSCT patients which deserve special attention with improvement of future, personalized treatment.

Epstein-Barr Virus.

This review covers relevant clinical and laboratory information relating to Epstein-Barr virus (EBV) infections in immunocompromised hosts. It describes the epidemiology and clinical manifestations with a primary focus on disease in solid organ and stem cell transplant recipients. The review pays particular attention to diagnostic approaches, including serologic testing and imaging, with an expanded discussion on the role of measuring the EBV load in peripheral blood, identifying both strengths and limitations of this assay. Additional attention is paid to potential additional strategies of immunologic monitoring that may enhance the performance of EBV load monitoring.

Epstein–Barr virus load in transplant patients: Early detection of post-transplant lymphoproliferative disorders

High levels of circulating EBV load are used as a marker of post-transplant lymphoproliferative disorders (PTLD). There is no consensus regarding the threshold level indicative of an increase in peripheral EBV DNA. The aim of the study was to clinically validate a developed EBV quantification assay for early PTLD detection.Transversal study: paired peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal lymphoid tissue (OLT) from children undergoing a solid organ transplant with (n=58) and without (n=47) PTLD. Retrospective follow-up: 71 paired PBMC and plasma from recipients with (n=6) and without (n=6) PTLD history. EBV load was determined by real-time PCR. The diagnostic ability to detect all PTLD (categories 1–4), advanced PTLD (categories 2–4) or neoplastic PTLD (categories 3 and 4) was estimated by analyzing the test performance at different cut-off values or with a load variation greater than 0.5log units.The higher diagnostic performance for identifying all, advanced or neoplastic PTLD, was achieved with cut-off values of 1.08; 1.60 and 2.47log EBVgEq/105 PBMC or 2.30; 2.60; 4.47loggEq/105 OLT cells, respectively. EBV DNA detection in plasma showed high specificity but low (all categories) or high (advanced/neoplastic categories) sensitivity for PTLD identification. Diagnostic performance was greater when: (1) a load variation in PBMC or plasma was identified; (2) combining the measure of EBV load in PBMC and plasma.The best diagnostic ability to identify early PTLD stages was achieved by monitoring EBV load in PBMC and plasma simultaneously; an algorithm was proposed.

(969) - Evaluation of Epstein-Barr Viral Loads as a Predictor of Development of Post-Transplant Lymphoproliferative Disorder

(969) - Evaluation of Epstein-Barr Viral Loads as a Predictor of Development of Post-Transplant Lymphoproliferative Disorder

Methylation of Epstein-Barr virus Rta promoter in EBV primary infection, reactivation and lymphoproliferation.

During Epstein-Barr virus (EBV) latency, the EBV genome is largely silenced by methylation. This silencing is overturned during the switch to the lytic cycle. A key event is the production of the viral protein Zta which binds to three Zta-response elements (ZRE) from the Rta promoter (Rp), two of which (ZRE2 and ZRE3) include three CpG motifs methylated in the latent genome. The bisulphite pyrosequencing reaction was used to quantify the methylation of ZRE2, ZRE3a, and ZRE3b in EBV-positive cell lines and in ex vivo samples of EBV-related diseases, in order to assess whether the level of methylation in these ZREs could provide additional information to viral DNA load and serology in the characterization of EBV-associated diseases. In PBMC from two patients with infectious mononucleosis, over time Rp became increasingly methylated whereas EBV load decreased. In tonsil from patients with chronic tonsillitis, the methylation was less than in EBV-associated tumors, regardless of the viral load. This was even more striking when only the ZRE3a and ZRE3b were considered since some samples presented unbalanced profiles on ZRE2. EBV reactivation in cell culture showed that the reduction in the overall level of methylation was closely related to the production of unmethylated virions. Thus, an assessment of the level of methylation may help to better characterize EBV replication in PBMC and in biopsies with high EBV load, during infectious mononucleosis and EBV-associated cancers. J. Med. Virol. 88:1814-1820, 2016. © 2016 Wiley Periodicals, Inc.

P753. Immunosuppression for inflammatory bowel disease does not affect Epstein–Barr viral load in the short-term: preliminary results

P753. Immunosuppression for inflammatory bowel disease does not affect Epstein–Barr viral load in the short-term: preliminary results