Monitoring Of Epstein-Barr Virus Load Research Articles

CRISPR Cas12a-mediated amplification-free digital DNA assay improves the diagnosis and surveillance of Nasopharyngeal carcinoma

Sensitive and accurate cell-free plasma Epstein-Barr virus (EBV) DNA measurement is essential in the routine diagnosis, monitoring and treatment of Nasopharyngeal Carcinoma (NPC). This measurement in commercial and in-house assay are commonly based on real-time quantitative PCR (qPCR) method, which requires reference materials for standardization and lack quantitative precision due to amplification bias or cross-contamination. To address these issues, we developed a CRISPR/Cas12a-mediated amplification-free digital DNA assay, which targets the repetitive sequences of EBV DNA and utilizes the cis-cleavage activity of CRISPR-Cas12a prior to droplet generation. By this mean, more activated Cas12a-crRNA duplexes could be produced for subsequent target detection and counting, thus improving the performance in detecting low EBV DNA load. We demonstrated that it was more robust than conventional qPCR for detecting plasma EBV DNA in a case-control study of 208 participants, especially when the target concentrations were around the diagnostic cut-off value for NPC. More importantly, this assay allowed a more accurate diagnosis of early-stage NPC, with an area under the curve (AUC) of 0.9883 (versus 0.7682 for qPCR). Furthermore, its absolute quantification capability enabled dynamic monitoring of EBV load in NPC patients during initial diagnosis, treatment, and recurrence, thereby potentially improving disease management and prognosis. Taken together, our results demonstrate that this amplification-free digital assay has the potential to be a robust tool to improve the diagnosis and surveillance of NPC.

Dynamic monitoring of plasma Epstein-Barr Virus DNA load can predict the occurrence of lymphoproliferative disorders after haploidentical hematopoietic stem cell transplantation

Objective: To determine the optimal cutoff value of Epstein-Barr virus (EBV) DNA load that can assist in the diagnosis of post-transplant lymphoproliferative disease (PTLD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT) . Methods: The data of patients with EBV infection after haplo-HSCT from January to December 2016 were retrospectively analyzed. Through constructing the receiver operating characteristic (ROC) curve and calculating the Youden index to determine the cutoff value of EBV-DNA load and its duration of diagnostic significance for PTLD. Results: A total of 94 patients were included, of whom 20 (21.3% ) developed PTLD, with a median onset time of 56 (40-309) d after transplantation. The median EBV value at the time of diagnosis of PTLD was 70,400 (1,710-1,370,000) copies/ml, and the median duration of EBV viremia was 23.5 (4-490) d. Binary logistic regression was used to analyze the peak EBV-DNA load (the EBV-DNA load at the time of diagnosis in the PTLD group) and duration of EBV viremia between the PTLD and non-PTLD groups. The results showed that the difference between the two groups was statistically significant (P=0.018 and P=0.001) . The ROC curve was constructed to calculate the Youden index, and it was concluded that the EBV-DNA load ≥ 41 850 copies/ml after allogeneic hematopoietic stem cell transplantation had diagnostic significance for PTLD (AUC=0.847) , and the sensitivity and specificity were 0.611 and 0.932, respectively. The duration of EBV viremia of ≥20.5 d had diagnostic significance for PTLD (AUC=0.833) , with a sensitivity and specificity of 0.778 and 0.795, respectively. Conclusion: Dynamic monitoring of EBV load in high-risk patients with PTLD after haplo-HSCT and attention to its duration have important clinical significance, which can help clinically predict the occurrence of PTLD in advance and take early intervention measures.

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.

Unexpected Higher Tacrolimus Levels at the Onset of Epstein-Barr Virus Associated Post-Transplantation Lymphoproliferative Disorder in Living Donor Liver Transplant Infants

Introduction: Post-transplantation lymphoproliferative disorder (PTLD) is a life threatening disease, which occurs as high as 20% of liver transplanted children. Especially, liver transplanted infants are high risk group of Epstein-Barr virus (EBV) associated PTLD because of their immature immunity and primary EBV transmission from EBV positive graft implantation. In this study, we investigated relationship between changes of tacrolimus (Tac) trough levels and symptomatic infectious episodes associated with EBV. Patients and methods: From July 1991 to June 2011, 85 pediatric living donor liver transplantations (LDLT) were performed at Tohoku University Hospital. Of these patients, there were 32 infants, who received Tac and steroids as a primary induction therapy, with an age of less than 2 years old at LDLT. In this study, a retrospective review of the transplant records of these 32 patients was conducted. The indication for LDLT included biliary atresia (n=31) and fulminant hepatitis (n=1). 32 infants were all survived for over 1-year. The definition of PTLD was infectious episodes with peripheral-blood mononuclear cells and/or serum EBV DNA positivity (cut-off value>102.5 copies/μg DNA) and with persisting abnormal nodal swelling with/without extranodal findings confirmed by contrast enhanced computed tomography images. Results: 21 (66%) of 32 patients experienced symptomatic viral infection. 8 (25%) of these patients were EBV seronegative at the onset of viral infectious episode. Of the remaining 13 patients, 8 (25%) experienced infectious episodes with sudden positive change of blood EBV DNA load without the development of PTLD at a median of 358 (161-580) posttransplant days (non-PTLD group). 5 (16%) patients developed PTLD at a median of 198 (112-447) posttransplant days (PTLD group). All 13 patients resolved their symptoms and signs associated with EBV disorders and all have maintained good allograft function during a median follow-up time of 2264 (157-4024) days after the development of symptomatic EBV infection or PTLD. The clinical characteristics of these patients were summarized in Table 1. Although the averages of Tac trough levels for 2 months before the onset of infectious episodes were similar in both patients, those levels at the onset in PTLD group patients were significantly higher than those of non-PTLD group. Moreover, most of the patients developing PTLD showed more than twice trough levels of Tac before infectious episodes.Conclusions: Among infants with an age of less than 2 years old at LDLT, an unexpected increase of Tac trough level at the onset of infectious episodes with sudden positive change of blood EBV DNA load may predict the development of PTLD. In such clinical settings, we need to perform frequent EBV load monitoring and imaging studies.

TNFα antagonist therapy does not increase the Epstein-Barr virus burden in patients with rheumatoid arthritis or ankylosing spondylitis

ObjectiveThe risk of non-Hodgkin lymphoma is increased in rheumatoid arthritis (RA) but not in ankylosing spondylitis (AS). In RA, the degree of inflammation is closely associated with the lymphoma risk. Whether immunosuppressants such as methotrexate and TNFα antagonists affect the lymphoma risk in RA is unclear. The Epstein-Barr virus (EBV) may contribute to the pathogenesis of RA and may be involved in the development of lymphoma in patients taking methotrexate and/or TNFα antagonists, although these points remain debated. EBV load monitoring during immunosuppressive treatment may predict the occurrence of EBV-related lymphoma. Here, our objective was to prospectively measure the EBV load in patients receiving TNFα antagonists for RA or AS. MethodsWe prospectively studied patients with RA or AS before and after TNFα antagonist therapy initiation. The EBV load was measured in blood samples using the EBV R-gene Quantification Kit. Disease activity at the time of blood sampling was evaluated by determining the Disease Activity Score 28 (DAS28) in RA patients and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) in AS patients. ResultsWe included 46 patients with RA (82.6% women; mean age, 52.7±11.3 years) and 27 with AS (men, 81.5%; mean age, 45.1±12.7 years). In the RA group, the EBV load was measured at baseline and 9.72±5.7 months later. The baseline EBV load was undetectable in 33 (70.2%) patients; mean EBV load in the 13 remaining patients was 9389copies/ml (3.47 log10±0.45). Baseline EBV load did not correlate with disease activity (DAS28). At the follow-up assay, the EBV load became positive in five patients and increased significantly in one patient (four patients on etanercept, one on adalimumab, and one on infliximab); it became negative in six patients (five on adalimumab and one on etanercept) and showed non-significant changes in six patients. Mean EBV load in patients positive at follow-up was 3.63±0.52 log10 copies/ml. Mean DAS28 was 4.78±1.1 at baseline and 2.94±1.24 at follow-up. At follow-up, a good EULAR response was noted in 33 (71.7%) patients and a moderate EULAR response in seven (15.2%) patients. In the AS group, the baseline EBV load measurement occurred after 12.9±10.6 months. Baseline EBV load was undetectable in 25 (92.6%) patients; mean load in the remaining two patients was 4.15±0.46 log10 copies/ml. At follow-up, the EBV load became positive in two patients (one on adalimumab and one on infliximab) and became negative in one patient (on adalimumab). Mean load in positive patients was 3.33±0.24 log10 copies/ml. Mean BASDAI was 55.1±16.2 at baseline and 17.88±18.62 at follow-up. A positive EBV load was significantly more common in the RA group than in the AS group (P=0.039). EBV load changes did not differ significantly between the RA and AS groups or across TNFα antagonists. No cases of lymphoma were recorded. ConclusionIntroducing TNFα antagonist therapy does not affect the EBV load in patients with RA or AS. EBV load monitoring is probably unnecessary in patients given TNFα antagonists for RA or AS.

Posttransplant Lymphoproliferative Disorder in Pediatric Liver Transplantation

The success rate of pediatric liver transplantation has improved in recent years. Advances in immunosuppression have reduced the risk of rejection, but have enhanced the risk of posttransplant lymphoproliferative disorder (PTLD). Since 1994, we have performed 197 orthotopic liver transplantations in 157 recipients younger than 15 years. Herein we have performed a retrospective study to review the incidence and clinical characteristics, along with the treatment and outcomes of PTLD diagnosed over this 14-year experience. We documented 8 cases of PTLD (5%), half of which occurred during the first 2 years posttransplantation; 5 presented with abdominal involvement and 2 with thoracic masses. The histological findings showed lymphoma in 6 cases. All were treated with reduction of immunosuppression and 2 received Rituximab. Three patients died, a mortality rate of 37.5%. One subject experienced rejection, and the others responded to treatment. PTLD is a life-threatening condition that requires a high index of suspicion, appropriate imaging, biopsy diagnosis, and prompt treatment to achieve positive results. Quantitative monitoring of Epstein-Barr virus load may be useful to detect a high-risk population.

Monitoring of Epstein-Barr virus load and killer T cells in pediatric renal transplant recipients

The aim of this study is to establish a monitoring method to prevent Epstein-Barr virus (EBV)-associated symptoms including post-transplant lymphoproliferative disorder (PTLD) that occur after pediatric renal transplantation. Circulating EBV loads were quantified by real-time PCR every 1 - 3 months after grafting in 22 pediatric recipients (13 EBV-seronegative [R(-)] and 9 EBV-seropositive [R(+)] recipients before grafting). The peripheral blood cell populations of non-specific activated killer cells (CD8+HLA-DR+ phenotype) in 13 R(-) recipients and EBV-specific cytotoxic T cells (CTLs) reactive with a tetramer expressing HLA-A24-restricted EBV-specific antigens in 8 of 13 R(-) recipients were determined by flow cytometry. EBV-associated symptoms including PTLD (2 cases) were found in 4 R(-) and none of the R(+) recipients. The maximum of EBV load in the R(-) group was significantly higher that in the R(+) group. In R(-) recipients, 4 symptomatic cases had significantly more EBV genome than asymptomatic cases. EBV-specific CTLs were detected in 6 of the 8 R(-) recipients, but these CTLs could not be detected in 1 of the 2 cases at onset of PTLD. The percentage of CD8+HLA-DR+ cells was significantly higher in asymptomatic recipients than in recipients with EBV-associated symptoms whose EBV loads were over 400 copies/microg DNA. Monitoring of killer T cells and EBV loads may allow assessment of the risk of EBV-associated symptoms, and high EBV loads and low EBV-specific and/or non-specific CTL responses may be predictive for development of EBV-associated symptoms such as PTLD.

Monitoring of Epstein–Barr virus load and antibody in pediatric renal transplant patients

Epstein-Barr virus (EBV) infection can lead to life-threatening post-transplant lymphoproliferative disorder (PTLD). The aim of the present study was to establish EBV monitoring methods to prevent PTLD. EBV-DNA load was investigated, using real-time polymerase chain reaction (PCR) and anti-EBV antibody titers, in peripheral blood mononuclear cells of 21 renal transplant patients (seven recipients who were EBV-seronegative, R[-]; 14 who were EBV-seropositive, R[+]) before grafting. The mean age at entry and the mean follow-up period was 7.8 years of age (range, 3.3-12.0 years) and 1.8 years (range, 0.4-4.0 years), respectively, in the R(-) group, and 12.5 years of age (range, 3.9-17.7 years) and 3.8 years (range, 0.8-8.2 years) in the R(+) group, respectively. The mean maximum load of the EBV genome was 1071 copies/microg DNA (range, 106-20700 copies/microg DNA) in the R(-) group, and 61 copies/microg DNA (range, <50-552 copies/microg DNA) in the R(+) group. During follow up no patient in the R(+) group had any noticeable symptoms that could be related to EBV, but three recipients in the R(-) group developed EBV-related symptoms including adenoid hypertrophy, cervical lymphadenopathy, and PTLD (B cell lymphoma), in one patient each. In the R(-) group the first leukocyte-associated viremia was detected at 30-180 days, and seroconversion at 43-266 days after transplantation. Viral DNA detection using PCR is a useful tool for EBV surveillance, but the maximum EBV load was not markedly elevated (2474 copies/microg DNA) in a patient with PTLD. Therefore, EBV surveillance using only monitoring of EBV load in peripheral leukocyte may be insufficient. Histology may therefore be necessary to accurately diagnose PTLD.

Persistent detection of Epstein-Barr virus DNA after pediatric liver transplantation: Unclear risks and uncertain responses

Persistent detection of Epstein-Barr virus DNA after pediatric liver transplantation: Unclear risks and uncertain responses

Monitoring of Epstein‐Barr virus load after hematopoietic stem cell transplantation for early intervention in post‐transplant lymphoproliferative disease

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease is a life-threatening complication following hematopoietic stem cell transplantation. A quantitative polymerase chain reaction to evaluate EBV-genome copy numbers based on a nested polymerase chain reaction and an end-point dilution was used. Applying this assay EBV load was prospectively screened weekly in 123 patients after transplantation. The results demonstrate that EBV reactivations with more than 1,000 EBV-genome copies measured in 10(5) peripheral blood mononuclear cells were observed in 31 patients (25.2%). Three patients developed lymphoproliferative disease with extremely high EBV-genome copies in peripheral blood mononuclear cells (>100,000 copies/10(5) cells) and plasma. After combined antiviral and immune therapy two of three patients showed a dramatic decrease of EBV load and survived, while the third patient died of lymphoma. A subclinical EBV reactivation was observed in 24 cases (19.5%) with EBV-genome copies in 10(5) peripheral blood mononuclear cells ranging between 2,500 and mostly 10,000. After reduction of immunosuppression the EBV levels normalized. In four patients, the high copy number of > or =80,000 copies/10(5) peripheral blood mononuclear cells and plasma positivity prompted us to start pre-emptive therapy with rituximab and cidofovir for prevention of lymphoproliferative disease. After drug administration the high EBV load was reduced remarkably. Ninety-two patients (74.8%) who had < or =1,000 copies/10(5) peripheral blood mononuclear cells did not develop EBV-associated lymphoproliferative disease. In conclusion, monitoring of EBV load is a sensitive and useful parameter in the surveillance of EBV reactivation for early intervention in EBV-associated lymphoproliferative disease as well as for follow-up of the efficacy of therapy.

Post-transplant lymphoproliferative disease and other Epstein-Barr virus diseases in allogeneic haematopoietic stem cell transplantation after introduction of monitoring of viral load by polymerase chain reaction

The clinical value of monitoring of Epstein-Barr virus (EBV) viraemia by quantitative polymerase chain reaction during 1 y was evaluated. 39 recipients of allogeneic hematopoietic stem cell transplantation (SCT) were followed. More than 100 EBV genome equivalents (gEq)/ml in blood or plasma were found in 16/39 patients (41%) at 34 d (range 1-139) post-transplant. Seven of these 16 patients developed EBV disease; 3 post-transplant lymphoproliferative disease (PTLD), 1 myelitis, 1 encephalitis and 2 reactivations with fever. EBV diseases were only found in the high-risk group among recipients of mismatched related or unrelated donor grafts or in patients who underwent reduced-intensity conditioning. In this group, 3/20 (15%) developed PTLD. Conditioning with antithymocyte globulin was significantly associated with EBV disease (p<0.01). EBV load in plasma was more strongly associated with EBV disease than viral load in blood. A cut-off level of 1000 gEq/ml plasma distinguished EBV disease from asymptomatic viraemia, but not PTLD from other EBV diseases. Weekly monitoring of EBV load in plasma in high-risk patients in the first 3 months following SCT seems to be of value for prediction of EBV disease. Therapy for PTLD including rituximab was evaluated during 2 y and showed response in 4/6 cases.

Recent developments in the prevention and treatment of Epstein?Barr virus-associated lymphoproliferative diseases

Epstein–Barr virus (EBV) infection and its relevance in post-transplant lymphoproliferative diseases (PTLDs) is an increasing area of concern. PTLD can differ clinically from a mononucleosis-like syndrome to malignant lymphoma. The incidence varies between < 1 and > 20% depending on different risk factors and the kind of transplant. Despite several treatment regimens, including reduction of immunosuppression, antiviral drugs, adoptive immunotherapy and administration of anti-CD20 monoclonal antibodies, the mortality rate is still high. Novel therapeutic strategies for managing PTLD use pharmacological induction of the viral thymidine kinase gene in tumour cells, the target of antivirals based on nucleoside analogues, followed by treatment with ganciclovir. Further treatment modalities include the development of vaccines and targeting of the latent EBV episomes. Prevention of PTLD by pre-emptive therapy based on molecular monitoring of EBV load will represent the main aim to reduce occurrence. This review examines patents and literature for the treatment of EBV-associated lymphoproliferative diseases.

Prospective monitoring of the Epstein-Barr virus DNA by a real-time quantitative polymerase chain reaction after allogenic stem cell transplantation.

Epstein-Barr virus (EBV)-related lymphoproliferative disorder (LPD) is a serious complication of haematopoietic stem cell transplantation (HSCT). To clarify the frequency, natural course and risk factors for LPD, we prospectively monitored 38 allogeneic (allo)-HSCT patients, focusing on the use of anti-thymocyte globulin (ATG). We used a recently developed real-time polymerase chain reaction assay to monitor EBV genome load. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. Of the 19 patients given ATG, 47.4% (nine patients) had a significant increase in EBV genome load (10(2.5) copies/microg DNA). Of these nine patients, two developed LPD. Therefore, 10.5% of the patients receiving allo-HSCT with ATG developed LPD. In contrast, none of the 19 patients without ATG had a significantly increased EBV load. The increases in viral load were observed in the second or third month after HSCT. We found that the peak viral loads of LPD patients were > 10(4.0 ) copies/microg DNA. On the other hand, the viral loads of most patients with no symptoms were < 10(2.5) copies/microg DNA. In conclusion, routine monitoring of EBV load during the second and third months after transplantation may benefit patients undergoing HSCT with ATG. We propose that an EBV load > 10(2.5) copies/microg DNA is the reactivation of EBV, and that an EBV load > 10(4.0) copies/microg DNA is indicative of developing LPD.

Epstein-Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease.

The Epstein-Barr virus load in the peripheral blood at the time of diagnosis of post-transplant lymphoproliferative disease (PTLD) is elevated 1000- to 10,000-fold compared to the level detected in normal latency. With the use of quantitative polymerase chain reaction (PCR), changes in the viral load over time can be measured with a two- to fourfold accuracy. This has allowed early detection of first-time infections and reactivations that may lead to PTLD and has provided an opportunity to intervene before symptomatic disease has occurred. Viral load monitoring has also been used to follow patients with PTLD and, along with other parameters, provided an assessment of the effectiveness of therapeutic protocols. Viral load monitoring has led to the discovery that at least two-thirds of transplant recipients become persistent viral load carriers. While the persistent load appears to be largely carried in latently infected memory B cells, more work is needed to clearly define this type of persistent infection and determine the risks associated with it. New diagnostic tests need to be developed to distinguish the persistent latent viral loads from viral loads that are likely to become symptomatic PTLD.