EBV is the etiologic agent of most post-transplant lymphoproliferative disorder (PTLD), which is an important cause of morbidity and mortality in both solid organ transplant recipients and HSCT patients. PTLD results from uncontrolled EBV-induced proliferation of B-cells in the immunocompromised setting. Quantitative EBV DNA PCR can be used to aid in the early diagnosis of PTLD, track the course of the disease, and monitor response to treatment.
Procedure
Extraction of EBV viral DNA from plasma, CSF, urine, other biological fluids, or tissues followed by amplification and detection using real-time, quantitative PCR. An internal control is added to ensure the extraction was performed correctly and the PCR reaction was not inhibited. ViraCor’s assay design includes multiple targets to account for viral mutations, which exponentially reduces the chance of false negative results.
Specimens
Whole Blood: 3 to 5 ml collected in EDTA (lavender top) tube. Do not freeze; ship ambient. Testing will be performed on plasma separated from the submitted whole blood specimen. Whole blood specimens are accepted as a matter of convenience for the originating laboratory. Special arrangements for testing of whole blood may be made by calling ViraCor prior to specimen shipment.
Plasma: 3 to 5 ml separated from whole blood collected in EDTA (lavender top) tube; ship ambient.
Bone Marrow: 2 ml minimum, collected in an EDTA (lavender top) tube. Do not freeze; ship ambient.
Bronchial Lavage/Bronchial Wash: 1 to 3 ml, collected in sterile, screw-cap tube; ship ambient.
CSF: 1 ml minimum, submitted in sterile, screw-cap tube; ship on dry ice.
Pleural Fluid: 1 ml submitted in a sterile, screw-top tube; ship ambient.
Throat Gargle: Instruct patient to gargle with 2 to 3ml sterile saline. Expectorate into sterile cup, then transfer contents to sterile, screw-cap tube for shipment; ship ambient.
Tissue: Place in a sterile, screw-cap tube, add a small amount of saline to keep moist. Prefer 1 mm x 1 mm specimen. Prefer fresh over formalin fixed for maximum sensitivity; ship ambient.
Upper respiratory aspirate (NP aspirate, nasal aspirate, tracheal aspirate, etc.): Instill 1 to 2 ml sterile saline into desired location and gently aspirate contents. Place collected fluid into sterile, screw-cap tube; ship ambient.
Upper respiratory swab (NP swab, throat swab): Swab desired location with sterile, flexible shaft swab, preferably a flocked swab. Place swab into 1 to 2 ml sterile saline, M4, or viral transport media in sterile, screw- cap tube. Do not use calcium alginate swab or wood shafted swab; ship ambient.
Other specimens may be accepted for testing; however the following comment will appear in the final report: "The clinical utility of this result has not yet been demonstrated in the peer reviewed literature and is therefore unknown." Call ViraCor for further information.
Causes for Rejection
Whole blood frozen. Call ViraCor at 800-305-5198 if specimen is greater than 96 hrs old.
Specificity
The primers and probes used in this assay are specific for known EBV strains based on similarity search algorithms. Additionally, no cross reactivity was detected when tested against adenoviruses, BKV, CMV, HSV-1, HSV-2, HHV-6 variant A, HHV-6 variant B, HHV-7, HHV-8, JCV, parvovirus B19, SV-40, and VZV.
Epstein-Barr Virus Assay
Range 100 copies/ml to 1 x 1010 copies/ml
Turnaround Time
Same day (within 8 to 12 hours of receiving specimen), Monday through Saturday
Shipping
Ship Monday through Friday. Friday shipments must be labeled for Saturday delivery. All specimens must be labeled with patient's name and collection date. Multiple tests can be run on one specimen. Ship specimens FedEx Priority Overnight® to:
ViraCor Laboratories, 1001 NW Technology Dr, Lee's Summit, MO 64086
The CPT codes provided are based on ViraCor’s interpretation of the American Medical Association’s Current Procedural Terminology (CPT) codes and are provided for informational purposes only. CPT coding is the sole responsibility of the billing party. Questions regarding coding should be addressed to your local Medicare carrier. ViraCor assumes no responsibility for billing errors due to reliance on the CPT codes illustrated in this material. PCR tests are performed pursuant to a license agreement with Roche Molecular Systems, Inc. This assay was developed and the performance characteristics were determined at ViraCor Laboratories. This test is performed in a CLIA certified laboratory. FDA approval is not required for the performance of this test.
AS04-0808
Pathogen Overview
ABOUT EPSTEIN-BARR VIRUS
Epstein-Barr virus (EBV) is an omnipresent member of the Herpesviridae family. It is a linear, double-stranded DNA virus with an icosahedral capsid. EBV is also known as HHV-4. EBV is a member of the Gammaherpesvirinae subfamily, along with HHV-8. The virus was discovered in 1964 by Drs. Epstein and Barr, after whom it was named. The virions which make up EBV are hexagonal nucleocapsids, with a complex surrounding envelope. EBV is most commonly found in B lymphocytes and also in epithelial cells of the nasopharynx. The virus has also been seen in epithelial cells, T lymphocytes, smooth muscle cells, and follicular dendritic cells. EBV enters the B lymphocyte and the viral DNA forms a self-replicating circular episome within the cell nucleus. EBV establishes latency in the B lymphocytes following primary infection. As B lymphocytes proliferate, the number of cells available to carry the virus increases. In an immunocompetent individual, specific cytotoxic T lymphocytes control the amount of B cell proliferation. In an immunocompromised patient, a latent EBV infection can reactivate and potentially cause lymphoproliferative disease, which can progress to a malignancy. This is due to a lack of T cells to control the EBV-driven proliferation of B cells.
EPSTEIN-BARR VIRUS CLINICAL MANIFESTATIONS
EBV is the causative agent of heterophile-positive infectious mononucleosis, which is most commonly seen in young adults or older adolescents. In transplant patients, EBV is the most common cause of post-transplant lymphoproliferative disorder (PTLD).
EBV antibodies are found in 90 to 95% of virtually all populations by the time they reach adulthood. In developed countries, approximately 50% of the population seroconverts before the age of 5. Childhood primary EBV infections are usually asymptomatic. Between the ages of 10 and 20, a second wave of seroconversion typically occurs. When adolescents or adults experience a primary EBV infection, it takes the form of infectious mononucleosis (IM), which most often manifests itself clinically in individuals who are not exposed to EBV until their second decade of life. IM typically presents with sore throat, fever, lymphadenopathy, and enlarged spleen. This disease is generally self-limiting and lasts approximately 2 to 4 weeks. Immunocompetent individuals usually do not experience additional complications, but some develop a chronic, persistent manifestation of the disease which can involve significant organ system dysfunction and last over 6 months.
Immunocompromised patients, such as hematopoietic stem cell transplant (HSCT) patients and solid organ transplant patients, are at risk for EBV-associated lymphoproliferation. EBV-induced proliferation may lead to PTLD. Transplant patients who are seronegative at the time of transplant are at highest risk for PTLD. If patients experience a primary EBV infection while being treated with immunosuppressive drugs, the risk of developing PTLD is dramatically increased. This is particularly common in the pediatric transplant population.
Additional risk factors for development of PTLD in the HSCT population include the intensity and duration of immunosuppressive therapy, unrelated donor, T-cell-depleted allografts, use of antithymocyte globulin, and immunosuppression to prevent graft-versus-host disease (GVHD). If lymphoproliferative disease develops in HSCT patients with GVHD, the prognosis is quite poor. The disease can manifest itself in forms ranging from benign polyclonal hyperplasia to malignant lymphoma. HIV patients sometimes develop nonmalignant lymphoproliferative diseases, such as follicular hypoplasia, primary lymphoid hyperplasia, and oral hairy leukoplasia, as well as lymphoid, muscular, and epithelial cell malignancies.
EPSTEIN-BARR VIRUS LABORATORY DIAGNOSIS
Heterophile antibodies are present in approximately 90% of the population presenting with IM in the second decade of life. However, in children younger than 5 years of age, the heterophile antibody will often be negative. Testing for these antibodies has been important to the diagnosis of IM. In some cases of IM and other EBV-associated illnesses, testing for EBV-specific antibodies has been shown to be useful. It is important, particularly in the pediatric population, to ascertain the EBV serostatus prior to transplantation, since seronegative patients are at significantly higher risk for PTLD when they acquire an EBV primary infection. Antibody testing is not useful for diagnosis or monitoring of PTLD. Molecular testing methods that facilitate early detection of EBV replication can facilitate early intervention, with the goal of restoring adequate function of the immune system to control EBV replication. For these reasons, real-time, quantitative polymerase chain reaction (PCR) is considered the best option for detecting and monitoring the overall EBV burden (viral load). Although both PBMCs and plasma are useful material for detection of EBV in immunocompromised patients, a higher positive predictive value for PTLD is achieved if plasma is used. Due to the highly sensitive nature of quantitative, real-time PCR, latent EBV can be detected when testing specimens containing PBMC, which is not useful for the physician.
EPSTEIN-BARR VIRUS TREATMENT
Most cases of IM require only supportive care, as over 95% of patients recover without therapy or complications. Each individual should limit his or her activity level to comfortably tolerable, and heavy lifting should be avoided to prevent spleen ruptures in patients with splenomegaly.
The usefulness of antiviral therapy to treat immunocompromised patients with EBV has not been supported by sufficient clinical evidence to become a frequently recommended option. Acyclovir has benefited some HIV patients with EBV-related diseases, but results in the literature are mixed. Additional agents have been shown to have antiviral activity against EBV, but only in vitro. Rituximab, a chimeric anti-CD20 monoclonal antibody developed for the treatment of B cell lymphoma, has been utilized with some success to curb the proliferation of EBV driven proliferating B cells. Currently, the best approach appears to be restoration of T cell function by reduction of immunosuppression, to as great of an extent as possible. Other experimental approaches that may prove promising include tumor-directed monoclonal antibodies and EBV-specific cytotoxic T cells.
Selected References
Knipe D, Howley P. Fields Virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2006.
Miller LE. The lab and Epstein-Barr virus infections. ADVANCE for Medical Laboratory Professionals. 2002:19-21. Available at
www.ACVANCEforMLP.com
Rose C, Green M, Webber S, Ellis D, Reyes J, et al. Pediatric solid-organ transplant recipients carry chronic loads of Epstein-Barr virus exclusively in the immunoglobulin D-negative B-cell compartment. J Clin Microbiol. 2001;(39):1407-1415.
Schooley RT. Epstein-Barr virus (infectious mononucleosis). In: Mandell GL, Bennett JE, Dolin, eds. Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases. Vol. 2. 4th ed. New York, NY: Churchill Livingstone; 1995:1364-1377.
van Esser JWJ, van der Holt B, Meijer E, Niesters HGM, Trenschel R, et al. Epstein-Barr virus (EBV) reactivation is a frequent event after allogenic stem cell transplantation (SCT) and quantitatively predicts EBV-lymphoproliferative disease following T-cell depleted SCT. Blood. 2001;(98):972-978.
Wagner HJ, Wessel M, Jabs W, et al. Patients at risk for development of post-transplant lymphoproliferative disorder: plasma versus peripheral blood mononuclear cells as material for quantification of Epstein-Barr viral load by using real-time quantitative polymerase chain reaction. Transplantation. 2001;(72):1012-1019.
PAO-04-0707 PCR tests are performed pursuant to a license agreement with Roche Molecular Systems, Inc.
Abstracts & Publications
Ahya Vivek, Douglas Lisa, Andreadis Charalambos, Arnoldi Sharon, Svoboda Jakub, Kotloff Robert, Hadjiliadis Denis, Sager Jeffrey, Woo Joseph, Pochettino Alberto, Schuster Stephen, Stadtmauer Edward, Tsai Donald. Association between elevated whole blood Epstein-Barr Virus (EBV)-encoded RNA EBV polymerase chain reaction and reduced incidence of acute lung allograft rejection. The Journal of Heart and Lung Transplantation. 2007;26(8):839-844.
Accurate functional assessment of patient immunosuppression after solid-organ transplantation remains elusive. Despite therapeutic serum immunosuppressive drug levels many lung transplant recipients still develop allograft rejection. We investigated the hypothesis that detection of latent Epstein-Barr virus (EBV) in peripheral blood may be a functional marker for the net effects of administered immunosuppression. METHODS: A retrospective analysis was performed on data obtained from a prospective trial investigating the ability of a novel EBV polymerase chain reaction (PCR) panel for LMP (latent membrane protein 1), EBNA (EBV nuclear antigen) and EBER (EBV-encoded RNA) to predict future development of post-transplant lymphoproliferative disorder (PTLD). Thirty-one lung transplant patients were followed for up to 2 years after transplantation with EBV PCR panels performed on plasma and whole blood. Patients were assessed for occurrences of Grade 2 or higher acute rejection and episodes of infection. RESULTS: Patients with whole blood EBER-positive PCR had a statistically significant lower incidence (45% vs 83%) of Grade 2 or higher acute allograft rejection than patients with no positive assays (odds ratio [OR] = 0.17, 95% confidence interval [CI] 0.021 to 1.2, p = 0.048). Positive whole blood EBER PCR did not correlate with increased risk for infectious complications (OR = 1.6, 95% CI 0.22 to 11, p = 0.69). CONCLUSIONS: These results suggest that whole blood EBER EBV PCR load may represent an important functional measure of immunosuppression in solid-organ transplant patients.
Berger C, Day P, Meier G, et al. Dynamics of Epstein-Barr virus DNA levels in serum during EBV-associated disease. J Med Virol. 2001;(64):505-512.
A real-time polymerase chain reaction assay for quantitation of Epstein-Barr virus (EBV) DNA in serum was developed. This assay detected EBV DNA in 24 (89%) of 27 sera from patients with infectious mononucleosis, but only in 9 (18%) of 51 sera from EBV carriers (P < 0.001) and in none of the sera from 32 EBV-seronegative individuals. EBV DNA levels were higher in sera from infectious mononucleosis (median 8,000, range 1833-150,069 copies/ml) than from carriers (median < 2, range < 2-2980; P < 0.001). In sera of 36 children with infectious mononucleosis followed prospectively, EBV DNA levels correlated inversely with the duration of symptoms. Among 18 children with tumors including Hodgkin's disease (n = 7), non-Hodgkin's lymphoma (n = 6), Burkitt's lymphoma (n = 1), lymphoproliferative disorder (n = 4), and osteosarcoma (n = 1), EBV DNA was detected in serum from those 9 (100%) expressing EBV in the tumor (Hodgkin's disease, 3; non-Hodgkin's lymphoma, 2; lymphoproliferative disorder, 4), the levels peaking at diagnosis and correlating with disease activity. Quantitation of EBV DNA in serum may offer a simple means of monitoring patients at risk of EBV-associated lymphoproliferation.
Campe H, Jaeger G, Abou-Ajram C, et al. Serial detection of Epstein-Barr virus DNA in sera and peripheral blood leukocyte samples of pediatric renal allograft recipients with persistent mononucleosis-like symptoms defines patients at risk to develop post-transplant lymphoproliferative disease. Pediatr Transplantation. 2003;(7):46-52.
We tested blood samples of 25 pediatric renal transplant recipients for Epstein–Barr virus (EBV) DNA load by quantitative polymerase chain reaction (PCR). Eleven of these transplant recipients showed clinical persistent mononucleosis-like symptoms years after transplantation (Tx). A quantitation of EBV DNA by PCR in peripheral blood lymphocyte (PBL) and serum samples revealed variable EBV DNA titers. The majority of EBV PCR results in samples of the 14 asymptomatic transplant recipients was repeatedly below detection limit. In contrast, patients with mononucleosis-like symptoms showed persistent EBV genome titers over a period of 6 months, ranging from 75 to 18 750 copies/10 000 PBL and from 680 to 335 000 copies/mL serum, respectively. One child suffering from this mononucleosis-like condition developed an EBV-associated Burkitt-like lymphoma 29 months after Tx. Whereas clinical and histological investigations did not indicate a post-transplant lymphoproliferative disorder (PTLD) until tumor detection, EBV titers in PBL and serum had been high for at least 8 months. We propose that pediatric transplant recipients who show both, recurrent mononucleosis-like symptoms and a sustained high EBV genome load, are at increased risk for severe EBV-related post-transplant complications.
Gärtner BC, Schäfer H, Marggraff K, et al. Evaluation of use of Epstein-Barr viral load in patients after allogeneic stem cell transplantation to diagnose and monitor posttransplant lymphoproliferative disease. J Clin Microbiol. 2002;40(2):351-358.
Epstein-Barr virus (EBV)-induced posttransplant lymphoproliferative disease (PTLD) continues to be a serious complication following transplantation. The aim of the present study was to evaluate the EBV load as a parameter for the prediction and monitoring of PTLD. The EBV load was analyzed by a quantitative competitive PCR with 417 whole-blood samples of 59 patients after allogeneic stem cell transplantation (SCT). The EBV load was positive for all 9 patients with PTLD and for 17 patients without PTLD. The viral loads of patients with manifest PTLD differed from the loads of those without PTLD (median loads, 1.4 x 106 versus 4 x 104 copies/µg of DNA; P < 0.0001). A threshold value of 105 copies/µg of DNA showed the best diagnostic efficacy (sensitivity, 87%; specificity, 91%). However, in patients with less than three major risk factors for PTLD, the positive predictive value of this threshold was rather low. One week prior to the manifestation of PTLD, the EBV load was as low in patients who developed PTLD as in patients without disease (median, 2.2 x 104 copies/µg of DNA; P was not significant). EBV DNA tested positive first at 20 to 71 days prior to the clinical manifestation of PTLD and occurred with the same delay after transplantation regardless of disease (median delay, 52 versus 63 days; P was not significant). EBV DNA was detected earlier in patients with primary infections than in those with reactivations (33 versus 79 days; P = 0.01), but the peak levels were similar in the two groups. EBV primary infection or EBV reactivation is frequent in patients after allogeneic SCT but results in PTLD only in a subgroup of patients. Although evaluation of the EBV load has limitations, the EBV load represents a valuable parameter to guide therapy.
Kullberg-Lindh C, Ascher H, Saalman R, Olausson M, Lindh M. Epstein-Barr viremia levels after pediatric liver transplantation as measured by real-time polymerase chain reaction. Pediatr Transplantation. 2006;(10):83-90.
Effective immunosuppression has improved the results following liver transplantation, but also increased the risk for opportunistic infections. Epstein–Barr virus (EBV) infection in transplant patients can cause various symptoms including the life-threatening premalignant condition, post-transplantation lymphoproliferative disorder (PTLD). Serum specimens from 24 consecutive children (mean 7.6 specimens/patient), who had undergone liver transplantation in Göteborg from January 1995 to May 2002, were analyzed retrospectively for EBV DNA by real-time TaqMan® polymerase chain reaction (PCR). The results were related to clinical picture, immunosuppression, graft rejection and infections with other agents. Eleven patients (46%) developed primary EBV infection at a mean time of 4.8 months after transplantation, and six (25%) reactivated EBV infection at a mean of 4.0 months after transplantation. Four of the 11 patients with primary infection had symptomatic EBV infection: two had PTLD and two hepatitis. One patient in the group with reactivated infection developed PTLD. EBV DNA levels were significantly higher in the group with primary symptomatic infection compared with the patients with primary asymptomatic infection (mean 65500 copies/mL; range 14200–194300 vs. 3700 copies/mL; range 100–9780). In patients with symptomatic infection EBV DNA levels did not differ between PTLD and hepatitis patients. The data suggest that quantitative analysis of EBV DNA in serum by real-time PCR is useful for identification of EBV-related disease.
Lei KIK, Chan LYS, Chan WY, Johnson PJ, Lo YMD. Quantitative analysis of circulating cell-free Epstein-Barr virus (EBV) DNA levels in patients with EBV-associated lymphoid malignancies. Br J Haematol. 2000;(111):239-246.
Cell-free Epstein-Barr virus (EBV) DNA has recently been detected in the plasma and serum of patients with Hodgkin[apos]s disease, post-transplant lymphoproliferative disease (PTLD) and acquired immunodeficiency syndrome-related lymphoma. However, no data are available on the temporal variation of plasma/serum EBV DNA levels in patients with EBV-associated lymphoid malignancies during the course of therapy. Using a real-time quantitative polymerase chain reaction assay, we studied the plasma EBV DNA levels in 13 patients with EBV-associated lymphoid malignancies (six patients with Hodgkin’s disease, four with nasal natural killer/T-cell lymphoma, two cases of PTLD and one patient with Burkitt’s lymphoma) at presentation and during therapy. Plasma EBV DNA was detected in 12 of the 13 patients (median 2266 copies/ml; interquartile range 181-8379 copies/ml), but not in any of 35 healthy control subjects (P < 0.0001). The EBV status in tumour cells was also examined in 12 of these patients using in situ hybridization for EBV-encoded small RNAs (EBERs). EBER positivity was observed in 11 patients, all of whom had EBV DNA detectable in plasma. The one patient who had no detectable plasma EBV DNA was also negative for EBERs in tumour tissue. Serial measurements of plasma EBV DNA levels were performed in nine of the patients during the course of therapy. All patients who responded to therapy demonstrated a significant reduction of plasma EBV DNA to low or undetectable levels, whereas in two patients with ineffective therapy, disease progression was associated with a rapid increase in plasma EBV DNA levels. We concluded that plasma EBV DNA is detectable in a wide range of EBV-associated lymphoid malignancies. As plasma EBV DNA levels correlate well with the therapeutic response, such analysis may be a valuable tool for monitoring clinical progress.
Rose C, Green M, Webber S, et al. Pediatric solid-organ transplant recipients carry chronic loads of Epstein-Barr virus exclusively in the immunoglobulin D-negative B-cell compartment. J Clin Microbiol. 2001;(39):1407-1415.
Solid-organ transplant recipients are at risk for development of lymphoproliferative diseases. The purpose of this study was to examine the distribution of Epstein-Barr virus (EBV) load in the peripheral blood of pediatric patients who had become chronic viral load carriers (>8 copies/105 lymphocytes for >2 months.) A total of 19 patients with viral loads ranging from 20 to 5,000 viral genome copies/105 lymphocytes were studied. Ten patients had no previous diagnosis of posttransplant lymphoproliferative disease (PT-LPD), while nine had recovered from a diagnosed case of PT-LPD. No portion of the peripheral blood viral load was detected in the cell-free plasma fraction. Viral DNA was found in a population of cells characterized as CD19hi and immunoglobulin D negative, a phenotype that is consistent with the virus being carried exclusively in the memory B-cell compartment of the peripheral blood. There was no difference in the compartmentalization based upon either the level of the viral load or the past diagnosis of an episode of PT-LPD. These results have implications for the design of tests to detect EBV infection and for the interpretation and use of positive EBV PCR assays in the management of transplant recipients.
Schooley RT. Epstein-Barr Virus (Infectious Mononucleosis). In Mandell GL, Bennett JE, Dolin, eds. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases. Vol 2. 4th ed. New York, NY: Churchill Livingstone; 1995:1364-1377.
This chapter appears in the 2nd volume of the definite textbook on infectious diseases. Chapter 118 discusses the history, description, epidemiology, pathogenesis, clinical manifestations, laboratory diagnosis treatment and prevention of Epstein-Barr virus.
Stevens SJ, Verschuuren EA, Verkuujlen SA, et al. Role of Epstein-Barr virus DNA load monitoring in prevention and early detection of post-transplant lymphoproliferative disease. Leuk Lymphom. 2002;43(4):831-840.
Posttransplant lymphoproliferative disease (PTLD) is a severe and life-threatening complication after stem cell or solid-organ transplantation, virtually always associated with presence of Epstein-Barr virus (EBV) in the proliferating cells. PTLD is probably caused by the iatrogenically impaired T-cell response allowing outgrowth of EBV-positive B-cells. Quantitative EBV DNA load monitoring is a minimally invasive technique increasingly recognized as a valuable tool in posttransplant patient management. In this review, we focus on the clinical utility of EBV DNA load monitoring in the peripheral blood of transplant recipients using PCR and we discuss the currently most-widely used techniques and their value and limitations in predicting and diagnosing PTLD. Options for EBV DNA load-guided pre-emptive therapy and application of monitoring EBV DNA load dynamics in the prediction of clinical response after therapy are described. Origins of elevated EBV DNA loads in immunosuppressed patients and recent insights in the EBV life cycle in the immuncompromised host are discussed. Finally, a standardization of methodology, clinical specimen type, and cut-off values is proposed. This is essential for comparisons between different institutes and more adequate patient management.
van Esser JWJ, van der Holt B, Meijer E, et al. Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBV-lymphproliferative disease following t-cell-depleted SCT. Blood. 2001;98(4):972-978.
Reactivation of the Epstein-Barr virus (EBV) after allogeneic stem cell transplantation (allo-SCT) may evoke a protective cellular immune response or may be complicated by the development of EBV-lymphoproliferative disease (EBV-LPD). So far, very little is known about the incidence, recurrence, and sequelae of EBV reactivation following allo-SCT. EBV reactivation was retrospectively monitored in 85 EBV-seropositive recipients of a T-cell-depleted (TCD) allo-SCT and 65 EBV-seropositive recipients of an unmanipulated allo-SCT. Viral reactivation (more than 50 EBV genome equivalents [gEq]/mL) was monitored frequently by quantitative real-time plasma polymerase chain reaction until day 180 after SCT. Probabilities of developing viral reactivation were high after both unmanipulated and TCD-allogeneic SCT (31% ± 6% versus 65% ± 7%, respectively). A high CD34+ cell number of the graft appeared as a novel significant predictor (P = .001) for EBV reactivation. Recurrent reactivation was observed more frequently in recipients of a TCD graft, and EBV-LPD occurred only after TCD-SCT. High-risk status, TCD, and use of antithymocyte globulin were predictive for developing EBV-LPD. Plasma EBV DNA quantitatively predicted EBV-LPD. The positive and negative predictive values of a viral load of 1000 gEq/mL were, respectively, 39% and 100% after TCD. Treatment-related mortality did not differ significantly between TCD and non-TCD transplants, but the incidence of chronic graft-versus-host disease was significantly less in TCD patients. It is concluded that EBV reactivation occurs frequently after TCD and unmanipulated allo-SCT, especially in recipients of grafts with high CD34+ cell counts. EBV-LPD, however, occurred only after TCD, and EBV load quantitatively predicted EBV-LPD in recipients of a TCD graft.
Wagner H, Fischer L, Jabs WJ, et al. Longitudinal analysis of Epstein-Barr viral load in plasma and peripheral blood mononuclear cells of transplanted patients by real-time polymerase chain reaction. Transplantation. 2002;74(5):656-664.
BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a significant cause of morbidity and mortality in transplant recipients and is caused by iatrogenic suppression of T cell function. Elevations in the Epstein-Barr viral (EBV) load in plasma (>1000 EBV copies/100 µL plasma) or peripheral blood mononuclear cells (PBMC) (>5000 EBV copies/µg PBMC DNA) as determined by real-time quantitative polymerase chain reaction (RQ-PCR) have been shown to be sensitive indicators for the development of PTLD in patients.
METHODS: The diagnostic value of frequent monitoring of EB viral load in peripheral blood from 46 patients after heart transplantation was investigated compared with 21 healthy controls in a prospective longitudinal study. EB viral load was detected in PBMC and plasma using real-time quantitative (RQ)- polymerase chain reaction (PCR)-based assays and compared with serological parameters of EBV infection or with the occurrence of CMV reactivations.
RESULTS: EB viral load was significantly increased in PBMC and in plasma from transplanted patients compared with healthy controls. Regarding levels and fluctuations of EB viral load in PBMC, patients were grouped in three distinct categories with high, intermediate, or low EB viral load. Although in one patient without PTLD, the EB viral load exceeded the threshold value for PTLD of 5000 EBV copies/µg PBMC DNA, all patients had an EB viral load in plasma of less than 1000 EBV copies/100 µL plasma. No correlation was found between the level of EB viral load and serological parameters of EBV reactivations in patients or in healthy control individuals. EBV and cytomegalovirus reactivations occurred independently in the majority of patients.
CONCLUSIONS: EB viral load measurements in plasma and PBMC of patients using RQ-PCR are superior to serology and are a powerful tool for monitoring transplanted patients.
Weinstock DM, Ambrossi GG, Brennan C, Kiehn TE, Jakubowski A. Preemptive diagnosis and treatment of Epstein-Barr virus-associated post transplant lymphoproliferative disorder after hematopoietic stem cell transplant: an approach in development. Bone Marrow Transplant. 2006;37(6):539-546.
Hematopoietic stem cell transplant (HSCT) recipients are at risk for Epstein–Barr virus (EBV)-associated, post transplant lymphoproliferative disorder (PTLD). Studies have suggested that early treatment may improve the outcome of patients with PTLD. Thus, significant attention has been focused on PCR-based approaches for preemptive (i.e., prior to clinical presentation) diagnosis. Reports from several transplant centers have demonstrated that HSCT recipients with PTLD generally have higher concentrations of EBV DNA in the peripheral blood than patients without PTLD. However, the PCR values of patients with PTLD typically span multiple orders of magnitude and overlap significantly with values from patients without PTLD. Thus, questions remain about the sensitivity and predictive value of these assays. Preemptive strategies using rituximab and/or EBV-specific cytotoxic T lymphocytes have been evaluated in patients with elevated EBV viral loads. We review the current literature, discuss our institutional experience and identify several areas of future research that could improve the diagnosis and treatment of this life-threatening disorder in HSCT recipients.
