Epstein-Barr Virus Viral Load Research Articles

Experience with Epstein barr virus and Cytomegalovirus infection in pediatric liver transplant recipients: A 2014–2017 study

IntroductionThis study presents our experience with Epstein Barr virus (EBV) and Cytomegalovirus (CMV) infections in the pediatric liver transplant population and their potential association with the development of the post-transplant lymphoproliferative syndrome (PTLD). Patients and methodsThis retrospective descriptive study covers the period from 2014 to 2017 and includes pediatric liver transplanted recipients who underwent viral load monitoring for EVB and CMV during the first-year post-transplant. ResultsA total of 89 patients were included in the study, with a median age of 0.68 years (RIQ 0.31–0.96) (10.8 months RIQ 8.4–25.2). The most common underlying pathology leading to transplantation was biliary atresia, observed in 55 (61.6 %) cases. Regarding EBV viral loads, values exceeding 10,000 copies/ml were observed in 9 (8 %) patients at 3 months, 33 (29.3 %) at 6 months, 31 (27.6 %) at 9 months and 25 (22.3 %) at 12 months post-transplantation. The probability of developing EBV infection within one-year post-transplantation was 81.3 %, while the probability of CMV infection was 29 %. A total of 8 (8.9 %) biopsy-confirmed graft rejections occurred, only 1 was EBV and CMV-negative. The likelihood of graft rejection in patients with EBV infection was 21.5 %, and for CMV it was 20.8 %. Importantly, only one case of PTLD was documented during 12 months follow-up. ConclusionCharacterizing this pediatric liver transplant population and monitoring EBV and CMV viral loads enables timely interventions, potentially reducing the risk of PTLD and graft rejection.

Disease-Modifying Treatments for Multiple Sclerosis Affect Measures of Cellular Immune Responses to EBNA-1 Peptides.

Epstein-Barr virus (EBV) has been strongly implicated in the pathogenesis of multiple sclerosis (MS). Despite this, there are no routinely used tests to measure cellular response to EBV. In this study, we analyzed the cellular response to EBV nuclear antigen-1 (EBNA-1) in people with MS (pwMS) using a whole blood assay. This cross-sectional study took place in a dedicated MS clinic in a university hospital. We recruited healthy controls, people with epilepsy (PWE), and pwMS taking a range of disease-modifying treatments (DMTs) including natalizumab, anti-CD20 monoclonal antibodies (mAbs), dimethyl fumarate (DMF), and also treatment naïve. Whole blood samples were stimulated with commercially available PepTivator EBNA1 peptides and a control virus-cytomegalovirus (CMV) peptide. We recorded the cellular response to stimulation with both interferon gamma (IFN-γ) and interleukin-2 (IL-2). We also compared the cellular responses to EBNA1 with IgG responses to EBNA1, viral capsid antigen (VCA), and EBV viral load. We recruited 86 pwMS, with relapsing remitting MS, in this group, and we observed a higher level of cellular response recorded with IFN-γ (0.79 IU/mL ± 1.36) vs healthy controls (0.29 IU/mL ± 0.90, p = 0.0048) and PWE (0.17 IU/mL ± 0.33, p = 0.0088). Treatment with either anti-CD20 mAbs (0.28 IU/mL ± 0.57) or DMF (0.07 IU/mL ± 0.15) resulted in a cellular response equivalent to control levels or in PWE (p = 0.26). The results of recording IL-2 response were concordant with IFN-γ: with suppression also seen with anti-CD20 mAbs and DMF. By contrast, we did not record any differential effect of DMTs on the levels of IgG to either EBNA-1 or VCA. Nor did we observe differences in cellular response to cytomegalovirus between groups. This study demonstrates how testing and recording the cellular response to EBNA-1 in pwMS may be beneficial. EBNA-1 stimulation of whole blood samples produced higher levels of IFN-γ and IL-2 in pwMS compared with controls and PWE. In addition, we show a differential effect of currently available DMTs on this response. The functional assay deployed uses whole blood samples with minimal preprocessing suggesting that employment as a treatment response measure in clinical trials targeting EBV may be possible.

Epstein-Barr virus-associated post-transplant lymphoproliferative disorders in pediatric transplantation: A prospective multicenter study in the United States.

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLD) is the most common malignancy in children after transplant; however, difficulties for early detection may worsen the prognosis. The prospective, multicenter, study enrolled 944 children (≤21 years of age). Of these, 872 received liver, heart, kidney, intestinal, or multivisceral transplants in seven US centers between 2014 and 2019 (NCT02182986). In total, 34 pediatric EBV+ PTLD (3.9%) were identified by biopsy. Variables included sex, age, race, ethnicity, transplanted organ, EBV viral load, pre-transplant EBV serology, immunosuppression, response to chemotherapy and rituximab, and histopathological diagnosis. The uni-/multivariable competing risk analyses revealed the combination of EBV-seropositive donor and EBV-naïve recipient (D+R-) was a significant risk factor for PTLD development (sub-hazard ratio: 2.79 [1.34-5.78], p = .006) and EBV DNAemia (2.65 [1.72-4.09], p < .001). Patients with D+R- were significantly more associated with monomorphic/polymorphic PTLD than those with the other combinations (p = .02). Patients with monomorphic/polymorphic PTLD (n = 21) had significantly more EBV DNAemia than non-PTLD patients (p < .001) and an earlier clinical presentation of PTLD than patients with hyperplasias (p < .001), within 6-month post-transplant. Among non-liver transplant recipients, monomorphic/polymorphic PTLD were significantly more frequent than hyperplasias in patients ≥5 years of age at transplant (p = .01). D+R- is a risk factor for PTLD and EBV DNAemia and associated with the incidence of monomorphic/polymorphic PTLD. Intensive follow-up of EBV viral load within 6-month post-transplant, especially for patients with D+R- and/or non-liver transplant recipients ≥5 years of age at transplant, may help detect monomorphic/polymorphic PTLD early in pediatric transplant.

Analytical and clinical evaluation of the cobas Epstein–Barr virus test at a tertiary care cancer hospital

BackgroundEpstein–Barr Virus (EBV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Cobas EBV test (Roche Molecular, Pleasanton, CA) has recently been FDA-cleared for the monitoring of EBV viral loads in plasma samples of transplant patients. In this study, we compared the viral loads obtained by a laboratory-developed test (EBV LDT) using Altona Analyte specific reagents (ASR) to those obtained on the Cobas EBV test. MethodsThe analytical performance of the assay was established using the EBV verification panel from Exact Diagnostics and the EBV ATCC strain B95-8. The clinical evaluation was performed using 343 plasma samples initially tested on the EBV LDT. ResultsThe analytical sensitivity (<18.8 IU/mL), precision (SD < 0.17 log) and linear range (35.0 IU/mL to 1E + 08 IU/mL) of the Cobas EBV assay established by the manufacturers were confirmed. The strength of the qualitative agreement was substantial between the cobas EBV and the EBV LDT (85.6 %; κ = 0.71) and almost perfect when discordant results were resolved (96.4 %; κ = 0.93). The quantitative agreement was moderate (82.9 %; κ = 0.53) with the viral load obtained on the Cobas EBV test being lower across the linear range of the two tests (mean log difference of 1.0). While the absolute values of the viral loads were markedly different, the overall trends observed in patients with multiple consecutive results were similar between the two tests. ConclusionsThe Cobas EBV test provides an accurate and valid, in vitro diagnostic (IVD) option for monitoring of EBV viral loads in transplant patients and should provide an opportunity for increased standardization and commutability of tests results across laboratories.

Analytical and Clinical Performance of the NeuMoDx™ Platform for Cytomegalovirus and Epstein-Barr Virus Viral Load Testing.

DNA assays for viral load (VL) monitoring are key tools in the management of immunocompromised patients with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. In this study, the analytical and clinical performances of the NeuMoDx™ CMV and EBV Quant Assays were compared with artus CMV and EBV QS-RGQ Kits in a primary hospital testing laboratory. Patient plasma samples previously tested using artus kits were randomly selected for testing by NeuMoDx assays. The NeuMoDx CMV Quant Assay and artus CMV QS-RGQ Kit limits of detection (LoDs) are 20.0 IU/mL and 69.7 IU/mL, respectively; 33/75 (44.0%) samples had CMV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.9503; 20 samples (60.6%) had lower NeuMoDx CMV quantification values versus the artus kit. The LoD of the NeuMoDx EBV Quant Assay and artus EBV QS-RGQ Kit are 200 IU/mL and 22.29 IU/mL, respectively; 16/75 (21.3%) samples had EBV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.8990. EBV quantification values with the NeuMoDx assay were higher versus the artus kit in 15 samples (93.8%). In conclusion, NeuMoDx CMV and EBV Quant Assays are sensitive and accurate tools for CMV and EBV DNA VL quantification.

EBV and multiple sclerosis: expression of LMP2A in MS patients.

Several evidences, including increased serum titers of Epstein-Barr virus (EBV)-specific antibodies and the presence of EBV DNA in brain of patients suggest a possible role of this virus in the pathogenesis of Multiple Sclerosis (MS), a chronic neurodegenerative disease with an unknown etiopathology. Aim of the present study is to verify if the expression of LMP2A and EBNA-1, two EBV genes, is altered in MS patients. EBV viral load, LMP2A and EBNA-1 gene expression and EBNA-1 antibodies titers were evaluated in blood of EBV-seropositive MS patients (n = 57; 31 relapsing remitting -RRMS- and 26 progressive -PMS-patients) and age- and sex-matched healthy controls (HC, n = 49). Results showed that EBNA-1 and VCA antibodies titers are significantly augmented in MS patients compared to HC (p < 0.05 for both antibodies); detection of EBV DNA was more frequent as well in MS patients compared to HC, although without reaching statistical significance. Regarding viral gene expression, LMP2A was significantly more frequently detected and more expressed in MS patients compared to HC (p < 0.005) whereas no differences were observed for EBNA-1. Considering patients alone, EBNA-1 was significantly more frequent in PMS compared to RRMS (p < 0.05), whereas no differences were observed for LMP2A. Increased expression of the LMP2A latency-associated gene in MS patients supports the hypothesis that EBV plays a role in disease etiopathology.

Investigating the role of Epstein-Barr virus and human papillomavirus types 16 and 18 co-infections in cervical disease of Iranian women.

High-risk human papillomaviruses (HR-HPVs) are known to contribute to cervical cancer (CC), but the role of Epstein-Barr virus (EBV) in this process remains unclear, despite EBV's widespread detection in premalignant and malignant cervical tissues. In this cross-sectional study of 258 cervical samples, including both formalin-fixed paraffin-embedded (FFPE) and fresh cervical tissues, the presence and viral load of HR-HPVs (HPV-16 and HPV-18) and EBV were evaluated in Iranian women with cervical intraepithelial neoplasia (CIN), squamous cell carcinoma (SCC), and a cervicitis control group using real-time PCR. The study revealed a significant correlation between disease severity and both increased HPV-16 positivity and HPV-16 and HPV-18 co-infection (p<0.001). Interestingly, the control group had a higher frequency of EBV-positive cases than SCC/CIN groups (p<0.001). HPV-16 DNA load increased with disease severity (P<0.001), while HPV-18 showed no significant difference (P=0.058). The control group had a higher EBV DNA load compared to SCC/CIN groups (P=0.033). HPV-16 increased the risk of CIN II, CIN III, and SCC, while HPV-18 increased the risk of CIN II and CIN III. Notably, EBV was associated with a lower risk of CIN groups and SCC. No significant difference in EBV co-infection with HPV-16/18 was found, failing to support the hypothesis that EBV is a cofactor in CC. However, high EBV viral load in the control group suggests a potential "hit and run hypothesis" role in CC progression. This hypothesis suggests that EBV may contribute briefly to the initiation of CC with an initial impact but then becomes less actively involved in its ongoing progression.

Epstein‐Barr virus in tonsillar tissue of Iranian children with tonsillar hypertrophy: Quantitative measurement by real‐time PCR

AbstractBackground and ObjectivesEpstein‐Barr virus (EBV) infection is ubiquitous all around the world. Tonsils seem to be candidate replication sites for EBV, and these tissues can be infected acutely or chronically. Some studies reported an association between EBV infection and tonsillar hypertrophy. In this study, we aimed to evaluate the presence and copy number of the EBV genome in tonsil tissue specimens of patients with tonsillar hypertrophy.MethodsA cross‐sectional study was performed on 50 fresh tonsil tissue samples from children, who underwent tonsillectomy because of tonsillar hypertrophy. Patients' tonsil tissues were evaluated using real‐time polymerase chain reaction for EBV genome and viral load. Finally, the results were analyzed using SPSS software.ResultsEBV genome was detected in 58% (29/50) of tonsillar tissues. The relationship between EBV genome detection rate and age groups was in the statistical significance range (P = 0.051). Among 29 positive cases, the average EBV viral load was (3.1 × 105) copy/g ± (0.5 × 105) copy/g. No significant difference was observed among different sex and age groups for EBV viral load.ConclusionHerein, EBV genome detection could support the colonization of EBV in the tonsils, which may have a direct or indirect association with the pathogenesis of tonsillar hypertrophy.

Clinical characteristics and outcomes of Epstein-Barr virus viral load after allogeneic hematopoietic stem cell transplantation

Epstein-Barr virus (EBV) reactivation can occur following allogenic hematopoietic stem cell transplantation (allo-HSCT). However, the clinical characteristics and outcomes of EBV-viral load are not well known. Thus, we retrospectively analyzed the clinical features and prognostic impact of the EBV viral load in 121 allo-HSCT recipients from our hospital. EBV DNA quantification was performed in whole blood after transplantation. Patients were grouped based on whether EBV DNA quantification reached > 1000 copies/mL during follow-up (N = 50) or not (N = 71). Patients with EBV > 1000 EBV copies/mL were relatively more common in the groups with graft versus host disease (GVHD) prophylaxis including ATG, haploidentical donor type, peripheral blood as a donor source, and acute GVHD II–IV. The 20-month OS and DFS were not significantly different between patients with < 1000 EBV copies/mL and patients with > 1000 EBV copies/mL (20-month OS, 56.0% vs. 60.6%; p = 0.503, 20-month DFS, 50.0% vs. 57.7%; p = 0.179). Immunosuppressant (ISS) dose reduction was achieved after the maximum increase in EBV in 41/50 (82%) patients. Additionally, 30/50 (60%) patients achieved a 50% dose reduction or no restarting of ISS within 3 months of the maximum EBV increase. Among cases wherein EBV DNA quantification reached > 1000 copies/mL, those that achieved rapid dose reduction of ISS tended to have longer overall survival (“not reached” vs 5.4 months, p < 0.001) and disease-free survival (88.4 months vs 5.3 months, p < 0.001) than those in patients who did not. Our data highlight the importance of rapid ISS reduction in post-transplant EBV reactivation.

Post-transplant lymphoproliferative disorder in pediatric liver transplant recipients: Experience from a South African transplant center.

Post-transplant lymphoproliferative disorder (PTLD) is a clinically heterogeneous potentially fatal complication of pediatric liver transplantation (PLT). We determined the prevalence, complications, and associated factors for PTLD in PLT recipients from Wits Donald Gordon Medical Centre, South Africa from January 2012 to August 2019. We performed a retrospective record review of 150 PLT recipients. Histologically proven PTLD occurred in 17/150 PLT recipients (11.3%). Children with PTLD were significantly younger at transplant (17.9 vs. 32.7 months, p = 0.001) with a significantly higher prevalence of obstructive etiology (17/17 vs. 81/133, p = 0.001). Fifteen (88.2%) children with PTLD were Epstein-Barr virus (EBV) seronegative at transplant. High post-transplant EBV viral load at a threshold value of 4.8 log10 DNA copies/mL (sensitivity: 80.0% [95% confidence interval {CI}, 46.7%-100.0%]; specificity: 73.1% [95% CI 42.3%-93.3%; area under the curve {AUC} 75.8%]) and low post-transplant albumin levels at a threshold value of 21.5g/L (sensitivity: 70.6% [95% CI, 41.2%-94.1%]; specificity: 85.7% [95% CI, 60.4%-94.5%; {AUC} 74.8%]) were associated with PTLD. The prevalence of cytomegalovirus (CMV) disease was significantly higher in children who developed PTLD versus non-PTLD (12/17 vs. 18/133; p < 0.001). CMV disease and the combination of post-transplant high EBV viral load and low albumin were independently associated with an increased risk of developing PTLD. Four (23.5%) children with PTLD died, however, survival was equivalent to non-PTLD PLT (p = 0.580). The prevalence of PTLD in our cohort mirrors international cohorts, with mortality similar to non-PTLD PLT recipients.

Systemic EBV-Positive T-cell Lymphoma of Childhood: A Case Report and Comprehensive Literature Review

Abstract Introduction/Objective Systemic EBV-positive T-cell lymphoma of childhood (SETLC) is an extremely rare disease in which most cases have been reported in Eastern Asian countries. It is characterized by a clonal proliferation of EBV- infected cytotoxic T cells that most commonly affect immunocompetent children and young adults. Patients present with systemic disease with rapid disease progression and multi-organ failure leading to death. Here, we report a case of SETLC in an 18 year old male, and review clinicopathologic features of the entity from 12 reported cases in the literature. Methods/Case Report A previously healthy 18 year old male presented with fever, abdominal pain, and vomiting. Clinical workup revealed elevated serum EBV viral load, hepatosplenomegaly, lymphadenopathy, pancytopenia, DIC, and multi-organ failure. Peripheral blood smear and bone marrow biopsy showed increased atypical lymphocytes with CD3+, CD8+, CD56-, TIA-1+ phenotype. In addition, prominent hemophagocytosis was noted in the bone marrow. T- cell receptor beta gene rearrangement was positive by PCR. A diagnosis of SETLC was established. Despite chemotherapy, he deteriorated rapidly and passed. Results (if a Case Study enter NA) NA Conclusion SETLC is an extremely rare disease in which most cases have been reported in Eastern Asian countries. Common clinical manifestations include fever, hepatosplenomegaly, lymphadenopathy, cytopenia, elevated LFT, coagulopathy, hyperferritinemia, hemophagocytic lymphohistiocytosis, and high serum EBV viral loads. It is a systemic disease with multiple organs involvement by EBV positive T cells. Comprehensive literature review of 12 reported cases along with our case demonstrated these neoplastic T cells were positive for CD2, CD3, CD8, TIA1 or granzyme, and EBER in situ hybridization; negative for CD56 in most cases. Hemophagocytosis was a common finding, and T cell receptor gene rearrangement confirmed clonality of the neoplastic T cells in all reported cases. Currently, there is no standard treatment. Most patient died from the disease weeks to months after disease onset.

Combination of Mitoxantrone Hydrochloride Liposome with Tislelizumab in Patients with Relapsed or Refractory NK/T Cell Lymphoma: A Phase Ib/Ⅱ Clinical Trial

Background : Extranodal NK/T cell lymphoma (ENKTL) is a highly aggressive malignancy associated with Epstein-Barr virus (EBV) infection, and remains a high unmet clinical need for improving prognosis. Mitoxantrone hydrochloride liposome (PLM60) is a nano-drug that has been approved as the first treatment option for relapsed/refractory (r/r) PTCL, and has shown certain efficacy and safety in a pivotal phase Ⅱ study. Tislelizumab is a humanized immunoglobulin G4 variant monoclonal antibody against PD-1. This trial aims to investigate the safety and efficacy of combining PLM60 with tislelizumab in patients (pts) with r/r ENKTL. Methods: Pts with r/r ENKTL were recruited in this ongoing, prospective, multicenter, open-label phase Ib/Ⅱ study (NCT05464433). Phase Ib was 3+3 design with two dose levels of PLM60 (d1, 16 mg/m 2 and 20 mg/m 2) plus tislelizumab 200 mg (d1, Q4W) induction therapy for up to 6 cycles, then tislelizumab 200 mg (Q3W) to continue maintenance therapy until disease progression, discontinuation, withdrawal or one year of therapy (including induction therapy period). Phase II was a dose expansion at the recommended phase II dose (RP2D). The primary endpoints were to assess the tolerability (i.e. dose-limiting toxicity (DLT)) and explore RP2D of PLM60 in phase Ib, and the objective response rate (ORR) of phase II. Secondary endpoints were complete response (CR) rate, ORR, disease control rate (DCR) and safety of phase Ib, and DCR, progression-free survival (PFS), overall survival (OS) and safety of phase II. Results: At data cut-off on 11 July 2023, 13 eligible pts were enrolled (phase Ib, n=6 and phase II, n=7). 6 pts of phase Ib were evaluable for response with a median age of 29.5 (range, 22-47) years, and 5 pts (83.3%) were ECOG 1. 3 pts (50.0%) had advanced stage III or IV (Ann Arbor). No DLT occurred at 16 mg/m 2 and 20 mg/m 2in phase Ib study. RP2D was PLM60 20 mg/m 2 plus tislelizumab 200 mg. The CR rate, ORR and DCR were 33.3% (2/6, 95% CI 4.3%-77.7%), 100.0% (6/6, 95% CI 54.1%-100.0%) and 100.0% (6/6, 95% CI 54.1%-100.0%), respectively. At data cutoff, the phase II study is being enrolled. Overall, the 11 pts evaluated in phase Ib and phase II had an ORR of 81.8% (9/11) (Figure 1). Furthermore, a decrease in EBV viral load was observed during treatment with PLM60 and tislelizumab except for one pt who experienced viral load rebounded and disease progression after 3 cycles of therapy (Figure 2). With a median follow-up of 2.1 (range, 1.0-5.6) months currently, the median PFS and OS will be reported after long-term follow-up. Treatment-related adverse events (TRAEs) of any grade occurred in all 13 enrolled pts, in which 7 (53.8%) pts were grade 3/4. The common grade 3/4 TRAEs were leucopenia (38.5%), neutropenia (30.8%), lymphocyte count decreased (30.8%), thrombocytopenia (7.7%), alanine aminotransferase increased (7.7%) and diarrhea (7.7%). Three patients had treatment delayed because of AEs. Moreover, no adverse cardiac events occurred during this study. Conclusions: PLM60 plus tislelizumab proves an encouraging efficacy in r/r ENKTL pts with manageable safety profiles.

Flowrna in the Diagnosis of EBV-Related Lymphoproliferative Disease

Introduction Epstein-Barr (EBV) viraemia is frequently encountered in the context of haemophagocytic lymphohistiocytosis (HLH), systemic inflammation and lymphoproliferative disease (LPD). Although EBV typically infects B lymphocytes triggering both polyclonal proliferation and lymphoma, EBV can also infect T and NK cells. Understanding the phenotype of EBV-infected cells can help identify the underlying diagnosis and guide the use of appropriate targeted therapy. We have developed an EBV FlowRNA assay combining the detection of EBV-encoded small RNA (EBER) with multicolour flow cytometry (Collins, Blood, 2020). Here we present a larger case series illustrating how this assay can be used in diagnostic workup. Methods Anticoagulated blood samples were received from patients with EBV viraemia and either HLH or confirmed or suspected LPD within 36 hours of collection. Mononuclear cells were isolated by density centrifugation and one to three million cells were stained with a fixable viability marker and conjugated antibodies to cell surface markers for lymphocyte immunophenotyping. FlowRNA staining was then performed as per manufacturers' instructions (PrimeFlow RNA, Thermo Fisher), using target probe sets (Thermo Fisher) for EBER 1/2 and Beta-2-microglobulin (B2M). The sample was then acquired on a LSRFortessa X-20 cytometer and subsequent analysis performed in FlowJo version 10. Results Between February 2021 and June 2023, we received 23 adequate samples from 19 patients. A definitive EBV+ population was identified in single lymphocyte subsets from 15 samples. Significant EBV-infected subsets were identified; CD19+ B cells from 4 patients, CD56+ NK cells from 6 patients, CD4+ T cells from 1 patient and CD8+ T cells from 4 patients. All patients with EBV-infected T/NK cells had an underlying lymphoma. The frequency of infected cells detected ranged from 0.017% in a case of nodal CD8+ T cell lymphoma (figure A) to 22% in a patient with relapsed extra-nodal NK T lymphoma (ENKTL) with heavy marrow infiltration and an EBV viral load of 1x10 6 copies per ml (figure B). In 5 samples no EBER+ cells were identified despite EBV DNAemia of up 2x10 6 copies per ml. Although a small number of EBER+ events could be detected in a further two samples, the very low frequency of EBV+ cells made it impossible to confirm this was a genuine population. Discussion FlowRNA can be used to give useful clinical information with a short turnaround time. In two patients with suspected relapse of ENKTL, CD56+EBER+ cells could be detected in the peripheral blood despite no lesions being visible with imaging. The diagnosis was only later confirmed by bone marrow biopsy. Importantly, FlowRNA enables the distinction between EBV positivity in circulating tumour cells from bystander B cell reactivation. In one patient with known T cell lymphoma and EBV DNAemia, the B cells were EBER+ but tumour cells were negative confirming bystander infection. Conversely, in another patient with CD8+ nodal lymphoma, EBER+ T cells were identifiable pre and post-treatment. This technique can therefore be used to monitor therapy and has the potential to identify measurable residual disease (MRD). Clonality of EBER+ B cells can be confirmed by Kappa/Lambda staining; in two cases of EBV-related HLH, EBER detection in polyclonal B lymphocytes helped confirm EBV-related lymphoproliferation rather than underlying lymphoma. The assay can be highly sensitive, detecting very small lymphocyte populations. However, to achieve this at least 100,000 lymphocytes must be analysed. Measuring sufficient cell numbers can be challenging in the context of post-treatment lymphopenia, although using up to 50 ml of blood can increase the cell yield. Additionally, this technique can only detect EBER+ cells when present in the peripheral blood. We have noted negative FlowRNA with high EBV viral load in conditions previously associated with cell-free viral DNA including ENKTL and Hodgkin lymphoma, which represented 3/5 negative samples. FlowRNA has several significant strengths, it is highly sensitive, reproducible, has a short turnaround time and by its nature as a blood test, samples can be easily obtained. The ability to monitor low-frequency tumour cells in the blood gives potential for MRD monitoring. We are now working to validate this test for routine diagnostic use.

Clinical, laboratory and virological aspects in infectious mononucleosis of EBV- and HHV-6 etiology in children

Purpose: To characterize clinical and laboratory features of infectious mononucleosis (IM) of EBV and HHV-6 etiology among children. Material and methods. A single-center prospective study was performed at Pediatric Research and Clinical Center for Infectious Diseases in 2021 — 2022, which included 84 patients with IM EBV (n = 40), HHV-6 (n = 11), EBV + HHV-6 (n = 33). Methods of laboratory diagnostics were clinical, biochemical blood tests, qualitative and quantitative PCR of blood (whole blood, plasma) to herpesvirus 4—6 types. Results. Patients with EBV IM were significantly older than children with HHV-6 IM and EBV + + HHV-6 IM (р ≤ 0,004). The main complaint of patients was fever (median 39,0°С, Q1—Q3 38,5—39,3°С). Nasal stuffiness (93%), tonsillitis (86%), peripheral cervical lymphadenopathy (100%) dominated among clinical manifestations in all groups. Splenomegaly was noted more rarely than hepatomegaly in children. There were no significant differences in the level of leukocytes among patients (р &gt; 0,05). Increased values of lymphocytes, atypical mononuclear cells, hepatic cytolysis syndrome were characteristic of EBV-monoinfection (р ≤ 0,007). On the contrary, level of neutrophils was significantly higher in the HHV-6 IM group. Genotype HHV-6B was detected in 100% of cases. Assay of EBV viral load showed following: this indicator in plasma (р = 0,0008) and whole blood (р = 0,028) in EBV patients was significantly higher than among EBV + HHV-6 IM. A direct moderate correlation was found between viral loads in different human fluids (r = 0,413, р &lt; 0,05). Conclusions. The results emphasize the importance of clinical and etiological diagnosis, including HHV-6 determination, for the well-timed registration of children with IM and their coverage with regular medical check-up.

Extranodal nasal-type NK/T lymphoma treated with chemotherapy and radiotherapy: case series from aEuropean tertiary referral center and review of the literature.

Extranodal nasal-type NK/T-cell lymphoma (ENKTL) is very rare in western countries and few data are available regarding the prognosis and the outcome of patients treated for this disease. We aimed to evaluate the prognosis, the pattern and risk factors of disease failure after combined therapy and also performed areview of the literature. We retrospectively analyzed 20patients with (ENKTL) who underwent L‑Asparaginase based chemotherapy followed by (chemo-) radiotherapy between 2010 and 2020 in our center. Data on clinical characteristics and irradiation were collected. Failure patterns were recorded as local (tumor site), regional (regional lymph nodes) or distant failure (metastasis and/or nonregional lymph nodes). During amedian follow-up period of 46months, disease failure was observed in 8patients (40%). The 3‑year progression-free survival (PFS) and overall survival (OS) rates were 62.5 and 83.0%, respectively. The failure patterns were local (n = 6, 30%), regional (n = 3, 15%) and distant (n = 4, 20%). Among patients with local failure, all failures occurred within the radiation fields (100%). Univariate analysis showed that bilateral regional lymph node involvement (p = 0.0002), initial circulating EBV viral load ≥ 3.5 log (p = 0.03) and no negativation of EBV PCR after induction CT (p = 0.0497) were independent predictors of PFS. Patients with bilateral lymph node involvement and/or high EBV viral load have asignificant recurrence rate despite multimodal therapy. These results need to be confirmed by larger studies. Given the high rate of local recurrence within radiotherapy fields, the value of dose escalation should be considered. Patients at risk of relapse should be included in dedicated trials.

Pre-Emptive Use of Rituximab in Epstein-Barr Virus Reactivation: Incidence, Predictive Factors, Monitoring, and Outcomes.

Post-transplant lymphoproliferative disease (PTLD) is a fatal complication of hematopoietic cell transplantation (HCT) associated with the Epstein-Barr virus (EBV). Multiple factors such as transplant type, graft-versus-host disease (GVHD), human leukocyte antigens (HLA) mismatch, patient age, and T-lymphocyte-depleting treatments increase the risk of PTLD. EBV reactivation in hematopoietic cell transplant recipients is monitored through periodic quantitative polymerase chain reaction (Q-PCR) tests. However, substantial uncertainty persists regarding the clinically significant EBV levels for these patients. Guidelines recommend initiating EBV monitoring no later than four weeks post-HCT and conducting it weekly. Pre-emptive therapies, such as the reduction of immunosuppressive therapy and the administration of rituximab to treat EBV viral loads are also suggested. In this study, we investigated the occurrence of EBV-PTLD in 546 HCT recipients, focusing on the clinical manifestations and risk factors associated with the disease. We managed to identify 67,150 viral genomic copies/mL as the cutoff point for predicting PTLD, with 80% sensitivity and specificity. Among our cohort, only 1% of the patients presented PTLD. Anti-thymocyte globulin (ATG) and GVHD were independently associated with lower survival rates and higher treatment-related mortality. According to our findings, prophylactic measures including regular monitoring, pre-emptive therapy, and supportive treatment against infections can be effective in preventing EBV-related complications. This study also recommends conducting EBV monitoring at regular intervals, initiating pre-emptive therapy when viral load increases, and identifying factors that increase the risk of PTLD. Our study stresses the importance of frequent and careful follow-ups of post-transplant complications and early intervention in order to improve survival rates and reduce mortality.

Genotype characterization of Epstein-Barr virus among adults living with human immunodeficiency virus in Ethiopia.

Epstein-Barr virus (EBV) is a human lymphotropic herpesvirus with a causative agent in cancer. There are two genotypes of EBV (EBV genotype 1 and EBV genotype 2) that have been shown to infect humans. This study aimed to characterize the EBV genotype among people with human immunodeficiency virus (PWH) and HIV-negative individuals in Ethiopia. DNA was extracted from peripheral blood mononuclear cells (PBMCs). Conventional polymerase chain reaction (cPCR) targeting EBNA3C genes was performed for genotyping. A quantitative real-time PCR (q-PCR) assay for EBV DNA (EBNA1 ORF) detection and viral load quantification was performed. Statistical significance was determined at a value of p < 0.05. In this study, 155 EBV-seropositive individuals were enrolled, including 128 PWH and 27 HIV-negative individuals. Among PWH, EBV genotype 1 was the most prevalent (105/128, 82.0%) genotype, followed by EBV genotype 2 (17/128, 13.3%), and mixed infection (6/128, 4.7%). In PWH, the median log10 of EBV viral load was 4.23 copies/ml [interquartile range (IQR): 3.76-4.46], whereas it was 3.84 copies/ml (IQR: 3.74-4.02) in the HIV-negative group. The EBV viral load in PWH was significantly higher than that in HIV-negative individuals (value of p = 0.004). In PWH, the median log10 of EBV viral load was 4.25 copies/ml (IQR: 3.83-4.47) in EBV genotype 1 and higher than EBV genotype 2 and mixed infection (p = 0.032). In Ethiopia, EBV genotype 1 was found to be the most predominant genotype, followed by EBV genotype 2. Understanding the genotype characterization of EBV in PWH is essential for developing new and innovative strategies for preventing and treating EBV-related complications in this population.

Detection and Quantification of the Epstein-Barr Virus in Lymphoma Patients from Ethiopia: Molecular and Serological Approaches.

The Epstein-Barr virus (EBV) is a known oncogenic virus associated with various lymphoma subtypes throughout the world. However, there is a lack of information regarding EBV prevalence in lymphoma patients, specifically in Ethiopia. This study aimed to investigate the presence of the EBV and determine its viral load in lymphoma patients from Ethiopia using molecular and serological approaches. Lymphoma patient samples were collected from the Ethiopian population. DNA and serum samples were extracted and subjected to molecular detection methods, including quantitative polymerase chain reaction (qPCR) analysis targeting the EBNA1 gene. Serological analyses were performed using an enzyme-linked immunosorbent assay (ELISA) to detect EBV viral capsid antigen IgG antibodies. EBV DNA was detected in 99% of lymphoma patients using qPCR, and serological analyses showed EBV presence in 96% of cases. A high EBV viral load (>10,000 EBV copies/mL) was observed in 56.3% of patients. The presence of high EBV viral loads was observed in 59.3% of HL patients and 54.8% of NHL patients. This study provides important insights into the prevalence and viral load of the EBV among lymphoma patients in Ethiopia. The findings contribute to the limited knowledge in this area and can serve as a foundation for future research.

Screening and Management of PTLD.

Posttransplant lymphoproliferative disorder (PTLD) represents a heterogeneous group of lymphoproliferative diseases occurring in the setting of immunosuppression following hematopoietic stem cells transplant and solid organ transplantation. Despite its overall low incidence, PTLD is a serious complication following transplantation, with a mortality rate as high as 50% in transplant recipients. Therefore, it is important to establish for each transplant recipient a personalized risk evaluation for the development of PTLD based on the determination of Epstein-Barr virus serostatus and viral load following the initiation of immunosuppression. Due to the dynamic progression of PTLD, reflected in the diverse pathological features, different therapeutic approaches have been used to treat this disorder. Moreover, new therapeutic strategies based on the administration of virus-specific cytotoxic T cells have been developed. In this review, we summarize the available data on screening and treatment to suggest a strategy to identify transplant recipients at a higher risk for PTLD development and to review the current therapeutic options for PTLD.

Host Genetic Variation Has a Profound Impact on Immune Responses Mediating Control of Viral Load in Chronic Gammaherpesvirus Infection.

Chronic infection with the gammaherpesvirus EBV is a risk factor for several autoimmune diseases, and poor control of EBV viral load and enhanced anti-EBV responses elevate this risk further. However, the role of host genetic variation in the regulation of immune responses to chronic gammaherpesvirus infection and control of viral replication remains unclear. To address this question, we infected C57BL/6J (B6) and genetically divergent wild-derived inbred PWD/PhJ (PWD) mice with murine gammaherpesvirus-68 (MHV-68), a gammaherpesvirus similar to EBV, and determined the effect of latent gammaherpesvirus infection on the CD4 T cell transcriptome. Chronic MHV-68 infection of B6 mice resulted in a dramatic upregulation of genes characteristic of a cytotoxic Th cell phenotype, including Gzmb, Cx3cr1, Klrg1, and Nkg7, a response that was highly muted in PWD mice. Flow cytometric analyses revealed an expansion of CX3CR1+KLRG1+ cytotoxic Th cell-like cells in B6 but not PWD mice. Analysis of MHV-68 replication demonstrated that in spite of muted adaptive responses, PWD mice had superior control of viral load in lymphoid tissue, despite an absence of a defect in MHV-68 invitro replication in PWD macrophages. Depletion of NK cells in PWD mice, but not B6 mice, resulted in elevated viral load, suggesting genotype-dependent NK cell involvement in MHV-68 control. Taken together, our findings demonstrate that host genetic variation can regulate control of gammaherpesvirus replication through disparate immunological mechanisms, resulting in divergent long-term immunological sequelae during chronic infection.