Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.     

Detection of Epstein-Barr virus genomes in peripheral blood B cells from solid-organ transplant recipients by fluorescence in situ hybridization

Resolution of Epstein-Barr Virus (EBV) infection in pediatric solid-organ transplant recipients often leads to an asymptomatic carrier state characterized by a persistently elevated circulating EBV load that is 2 to 4 orders of magnitude greater than the load typical of healthy latently infected individuals. Elevated EBV loads in immunosuppressed individuals are associated with an increased risk for development of posttransplant lymphoproliferative disease. We have performed fluorescence in situ hybridization (FISH) studies with peripheral blood B cells from carriers of persistent EBV loads in order to directly quantitate the number of EBV genomes per infected cell. Patients were assigned to two groups on the basis of the level of the persistent load (low-load carriers, 8 to 200 genomes/10(5) peripheral blood lymphocytes; high-load carriers, >200 genomes/10(5) peripheral blood lymphocytes). FISH analysis revealed that the low-load carriers predominantly had circulating virus-infected cells harboring one or two genome copies/cell. High-load carriers also had cells harboring one or two genome copies/cell; in addition, however, they carried a distinct population of cells with high numbers of viral genome copies. The increased viral loads correlated with an increase in the frequency of cells containing high numbers of viral genomes. We conclude that low-load carriers possess EBV-infected cells that are in a state similar to normal latency, whereas high-load carriers possess two populations of virus-positive B cells, one of which carries an increased number of viral genomes per cell and is not typical of normal latency.     

Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders.

A quantitative competitive PCR (QC-PCR) assay for Epstein-Barr virus (EBV) has been developed to provide accurate measurement of EBV genome load in pediatric transplant recipients at risk for developing posttransplant lymphoproliferative disorder (PTLD). The assay quantifies between 8 and 5,000 copies of the EBV genome in 10(5) lymphocytes after a 30-cycle amplification reaction. For 14 pediatric patients diagnosed with PTLD, the median EBV genome load was 4,000, and 13 of the 14 patients had values of >500 copies per 10(5) lymphocytes. Only 3 of 12 control transplant recipients not diagnosed with PTLD had detectable viral genome loads (median value, 40). This median was calculated by using the highest value obtained by PCR testing on each of these patients posttransplantation. PCR values of >500 copies per 10(5) lymphocytes appear to correlate with a diagnosis of PTLD. By a modified protocol, the EBV genome copy number in latently infected adults was estimated to be <0.1 copy per 10(5) lymphocytes.     

Quantification of Epstein-Barr virus load in Argentinean transplant recipients using real-time PCR.

BACKGROUND: Post-transplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplant recipients that is caused by suppression of T-cell function. OBJECTIVE: Evaluation of the diagnostic value of EBV DNA load in non-fractionated whole blood samples (n = 297) from 110 pediatric transplant patients by real-time PCR. RESULTS AND CONCLUSIONS: Patients with PTLD had a median viral load of 1.08 x 10(5) copies/ml blood (n = 24), which was significantly higher compared with patients without PTLD (median: 50 copies/ml blood, n = 273, P < 0.0001). From receiver operating characteristic (ROC) curve analysis we obtained a cut-off value of 6215 copies/ml blood with a sensitivity of 95.8%, specificity of 71.4%, negative predictive value (NPV) of 99.5% and positive predictive value (PPV) of 22.8%. Thus, real-time PCR proved to be more useful in ruling out than in indicating the presence of PTLD. Further analysis showed that patients without PTLD but developing a post-transplant EBV-primary infection had associated high viral loads that were indistinguishable from those of the PTLD group (statistically not significant). Similarly, the presence of clinical symptoms of disease in patients without PTLD was associated with higher viral loads than in patients that were asymptomatic (P < 0.0001), but the difference was much less significant when compared with the PTLD group of patients (P = 0.0391). These patients who had a high viral load may benefit from a close follow-up of the viral burden.     

A semiquantitative PCR method (SQ-PCR) to measure Epstein-Barr virus (EBV) load: its application in transplant patients.

BACKGROUND: High Epstein-Barr virus load has been related to an increased risk of Posttransplant Lymphoproliferative Disorders (PTLD) in transplant recipients. OBJECTIVES: Development of a method to quantitate EBV DNA levels in peripheral blood mononuclear cells (PBMC) and evaluate its usefulness in transplant patients. STUDY DESIGN: We designed a semiquantitative nested PCR based on a limiting dilution analysis to detect high viral loads in PBMC. This method was applied to 25 healthy carriers, and 85 solid organ transplant recipients as follows: (A) 53 asymptomatic patients; (B) 24 symptomatic patients; (C) eight patients with PTLD. RESULTS: In healthy carriers the reciprocal of the limiting dilution (RLD) ranged between non-detected (ND) and 1, the median RLD was ND, which is equivalent to a viral load of <1 copy per 10(5) PBMC. In the transplant population the medians RLD (range) were: (A) asymptomatic group: ND (ND-64), median equivalent to a viral load of <1 copy per 10(5) PBMC; (B) symptomatic group: 4 (ND-256), median equivalent to a range of viral load of 4-64 copies per 10(5) PBMC. (C) PTLD group: 256 (16-16384), median equivalent to a range of viral load of 256-4096 copies per 10(5) PBMC. Statistically significant differences were found between all groups: A+B vs. C (P<0.0001); A vs. B (P<0.0001); A vs. C (P<0.0001), B vs. C (P<0.0001). We also observed a good correlation between viral loads and clinical findings in four follow-up patients. Considering the RLD=256 as a cutoff point to detect transplant patients with PTLD, resulted in sensitivity 75%, specificity 96.7%, positive predictive value 60%, negative predictive value 98.3%. CONCLUSION: This SQ-PCR method enables us to differentiate between transplant patients with and without PTLD; therefore, it could be applied as a marker for early detection of this pathology.     

Epstein-Barr virus infection in paediatric liver transplant recipients: detection of the virus in post-transplant tonsillectomy specimens.

AIMS: Post-transplant lymphoproliferative disease (PTLD) is an important and serious complication in transplant patients. Recent studies have suggested that quantitative assessment of Epstein-Barr virus (EBV) infection in transplant patients might help to identify those at risk of developing PTLD. Therefore, tonsils from paediatric liver transplant recipients were studied for evidence of EBV infection. METHODS: Tonsils were studied by in situ hybridisation for the detection of the small EBV encoded nuclear RNAs (EBERs). The phenotype of EBV infected cells was determined by double labelling in situ hybridisation and immunohistochemistry. The expression of viral latent and lytic antigens was determined by immunohistochemistry. Tonsils from patients without known immune defects were studied as controls. RESULTS: Tonsils from transplant patients showed pronounced follicular hyperplasia and minor paracortical hyperplasia. In situ hybridisation revealed variable numbers of EBV infected B cells in the tonsils from transplant patients (range, 2-1000/0.5 cm(2); mean, 434/0.5 cm(2); median, 105/0.5 cm(2)). Lower numbers were detected in the control tonsils (range, 1-200/0.5 cm(2); mean, 47/0.5 cm(2); median, 9/0.5 cm(2)). The latent membrane protein 1 (LMP1) of EBV was not detected and there were only rare cells in two cases showing expression of the EBV encoded nuclear antigen 2 (EBNA2). There was no evidence of lytic infection. None of the patients developed PTLD within a follow up period of up to five years. CONCLUSIONS: These data indicate that tonsillar enlargement in paediatric liver transplant patients does not necessarily imply a diagnosis of PTLD. Furthermore, the presence of increased numbers of EBV infected cells in tonsils from liver transplant recipients by itself does not indicate an increased risk of developing PTLD.     

Using Epstein-Barr Viral Load Assays To Diagnose,Monitor, and Prevent Posttransplant Lymphoproliferative Disorder

Summary: Epstein-Barr virus (EBV) DNA measurement is being incorporated into routine medical practice to help diagnose, monitor, and predict posttransplant lymphoproliferative disorder (PTLD) in immunocompromised graft recipients. PTLD is an aggressive neoplasm that almost always harbors EBV DNA within the neoplastic lymphocytes, and it is often fatal if not recognized and treated promptly. Validated protocols, commercial reagents, and automated instruments facilitate implementation of EBV load assays by real-time PCR. When applied to either whole blood or plasma, EBV DNA levels reflect clinical status with respect to EBV-related neoplasia. While many healthy transplant recipients have low viral loads, high EBV loads are strongly associated with current or impending PTLD. Complementary laboratory assays as well as histopathologic examination of lesional tissue help in interpreting modest elevations in viral load. Circulating EBV levels in serial samples reflect changes in tumor burden and represent an effective, noninvasive tool for monitoring the efficacy of therapy. In high-risk patients, serial testing permits early clinical intervention to prevent progression toward frank PTLD. Restoring T cell immunity against EBV is a major strategy for overcoming PTLD, and novel EBV-directed therapies are being explored to thwart virus-driven neoplasia.     

Posttransplant lymphoproliferative disorders in heart-lung transplant recipients: primary presentation in the allograft

Heart-lung transplant recipients are predisposed to acute rejection episodes, bronchiolitis obliterans, and opportunistic infections. In 9.4% of recipients at the University of Pittsburgh, a posttransplant lymphoproliferative disorder (PTLD) developed, and in 60% of cases, it presented in the allografted lungs and was associated with primary infection by Epstein-Barr virus (EBV). The PTLD is histologically indistinguishable from a primary pulmonary lymphoma and consists of a mixed population of large lymphoid cells, immunoblasts, and plasma cells. Two cases of PTLD were monoclonal with immunohistochemical and Southern blot analysis. Despite this, there was clinical recovery with reduced immunosuppression and acyclovir. We discuss the role of EBV in the development of PTLD and the pathogenesis of primary presentation in the allograft.     

Detection of Epstein-Barr virus DNA in sera from transplant recipients with lymphoproliferative disorders

Early diagnosis of Epstein-Barr Virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is important because many patients respond to reduction in immunosuppression, especially if PTLD is detected at an early stage. Previous studies have found elevated EBV DNA levels in blood from patients with PTLD, but these assays required isolation of cellular blood fractions and quantitation. We evaluated the presence of cell-free EBV DNA in serum from solid-organ transplant recipients as a marker for PTLD. Five of 6 transplant recipients with histopathologically documented PTLD had EBV DNA detected in serum at the time of diagnosis (sensitivity = 83%), compared with 0 of 16 matched transplant recipients without PTLD (specificity = 100%) (P < 0.001 [Fisher's exact test]). Furthermore, EBV DNA was detected in serum 8 and 52 months prior to the diagnosis of PTLD in two of three patients for whom stored sera were analyzed. Detection of EBV DNA in serum appears to be a useful marker for the early detection of PTLD in solid-organ transplant recipients. Further studies to define the role of such assays in evaluating solid-organ transplant patients at risk for PTLD are warranted.     

Expression of Epstein-Barr virus-encoded small RNA (by the EBER-1 gene) in liver specimens from transplant recipients with post-transplantation lymphoproliferative disease.

BACKGROUND. Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative disease (PTLD) develops in 1 to 10 percent of transplant recipients, in whom it can be treated by a reduction in the level of immunosuppression. We postulated that the tissue expression of the small RNA transcribed by the EBER-1 gene during latent EBV infection would identify patients at risk for PTLD. METHODS. We studied EBER-1 gene expression in liver specimens obtained from 24 patients 2 days to 22 months before the development of PTLD, using in situ hybridization with an oligonucleotide probe. Control specimens were obtained from 20 recipients of allografts with signs of injury due to organ retrieval, acute graft rejection, or viral hepatitis in whom PTLD had not developed 9 to 71 months after the biopsy. RESULTS. Of the 24 patients with PTLD, 17 (71 percent) had specimens in which 1 to 40 percent of mononuclear cells were positive for the EBER-1 gene. In addition, 10 of these 17 patients (59 percent) had specimens with histopathological changes suggestive of EBV hepatitis. In every case, EBER-1-positive cells were found within the lymphoproliferative lesions identified at autopsy. Only 2 of the 20 controls (10 percent) had specimens with EBER-1-positive cells (P < 0.001), and such cells were rare. CONCLUSIONS. EBER-1 gene expression in liver tissue precedes the occurrence of clinical and histologic PTLD. The possibility of identifying patients at risk by the method we describe here and preventing the occurrence of PTLD by a timely reduction of immunosuppression needs to be addressed by future prospective studies.     

Semiquantitative PCR analysis of Epstein-Barr virus DNA in clinical samples of patients with EBV-associated diseases

The laboratory diagnosis of primary and reactivated Epstein-Barr virus (EBV) infection is based on serologic methods in immunocompetent patients. However, in immunocompromised patients, serologic data are difficult to interpret and do not often correlate with clinical data. In order to find a useful and practical marker for diagnosis of EBV-related diseases, a polymerase chain reaction (PCR) assay was established for semiquantitative detection of EBV sequences. The method was based on a nested PCR, using primers of the virus capsid antigen p23 region and an endpoint dilution. This method was carried out on 68 plasma samples, 68 samples of peripheral blood mononuclear cells and 5 cerebrospinal fluid samples of 39 patients with various diseases to evaluate the EBV-genome copy number. Samples from patients suffering from infectious mononucleosis served as positive controls for active EBV infection. In 5 patients with infectious mononucleosis, high copy numbers of EBV genomes in peripheral blood mononuclear cells were detected within a range of 1,000-40,000 copies in 10(5) peripheral blood mononuclear cells. In contrast, samples from 19 latently infected persons either showed low copy numbers (10-100 in 10(5) peripheral blood mononuclear cells) or were EBV PCR negative. Comparable results were observed in seven renal transplant patients without any symptoms. The practical value of the semiquantitative detection of EBV DNA was demonstrated in three bone marrow transplant recipients. Two developed a lymphoproliferative disease associated with extremely high amounts of EBV DNA in plasma (16,000 and 50,000 copies/ml, respectively) and peripheral blood mononuclear cells (100,000 and 6.5 million copies in 10(5) peripheral blood mononuclear cells, respectively). The high EBV load in plasma and peripheral blood mononuclear cells was reduced dramatically after successful antiviral therapy in one case. The third bone marrow transplant recipient developed an EBV-induced transverse myelitis with an increased number of EBV-genome copies in peripheral blood mononuclear cells and EBV-positive cerebrospinal fluid samples. After combined antiviral and immune therapy, the EBV-genome copy numbers decreased and the patient recovered completely. These data demonstrate a good correlation between semiquantitative detection of EBV genomes and clinical findings. The method is recommended for the diagnosis of EBV-associated diseases in patients after transplantation, as well as for monitoring the response to therapy.     

In situ hybridization for Epstein-Barr virus NotI repeats in posttransplant lymphoproliferative disorder.

Epstein-Barr Virus-associated posttransplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. In PTLD, B-cell expansions range from reactive hyperplasias to large cell lymphomas and are often associated with active Epstein-Barr virus (EBV) infection. The lymphoproliferations may infiltrate transplant allografts and therefore may need to be distinguished from acute cellular rejection (ACR). A total of 36 tissue specimens from 11 transplant patients (six kidney, two heart, three liver) with PTLD were studied for EBV content by automated in situ hybridization (ISH) on formalin-fixed or Bouin's-fixed, paraffin-embedded tissue using a synthetic 3' terminally biotin-labeled oligonucleotide DNA probe from the EBV NotI tandem repeat region. The NotI repeat is abundantly transcribed during productive EBV infection and may encode an EBV early antigen. EBV serologies from the 11 patients showed seven primary acute infections and one acute reactivation. Two serologic studies indicated infection of indeterminant onset, and serology was not performed on one patient. Histologically, seven patients presented with polymorphous infiltrates in transplant allograft biopsies, three of which progressed to disseminated monomorphous cell populations and death within 3 to 6 wk. Tissues examined by ISH from all 11 patients showed nuclear staining for EBV in the atypical lymphoid infiltrates (34/36 specimens). The nuclear signal ranged from a stippled pattern of positivity to homogeneous nuclear staining and was localized predominantly in follicular center cells and immunoblasts, although some smaller lymphocytes also contained the viral genome. ISHs performed on 31 allograft biopsies with ACR from 24 transplant patients (six kidney, five heart, 13 liver) without clinical evidence of PTLD and with serological evidence of past EBV infection were negative for the virus. Cell lines containing EBV in the productive state (EB3 and P3HR1) were positive with ISH for NotI, while a latently infected cell line (Raji) was negative. These data indicate that ISH with the NotI probe identifies amplified genome in EBV infections and is useful in discriminating the atypical infiltrate of EBV-associated PTLD from that seen in ACR.     

Pediatric solid-organ transplant recipients carry chronic loads of Epstein-Barr virus exclusively in the immunoglobulin D-negative B-cell compartment

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 transplant recipients who had become chronic viral load carriers (>8 copies/10(5) lymphocytes for >2 months). A total of 19 patients with viral loads ranging from 20 to 5,000 viral genome copies/10(5) 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 CD19(hi) 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.     

EBV viral load in tumor tissue is an important prognostic indicator for nasal NK/T-cell lymphoma

We retrospectively studied 19 cases of nasal NK/T-cell lymphoma for various potential prognostic factors and performed real-time quantitative polymerase chain reaction for Epstein-Barr virus (EBV) viral load in tumor tissue. Patients with a low EBV viral load (<1 copy per cell) more frequently survived for more than 2 years compared with patients with a high EBV viral load (>/=1 copies/cell) (7/7 vs 3/9; P = .014; Fisher exact test). Furthermore, the patients with low EBV viral loads had a better overall survival than patients with high viral loads (50% accumulative survival: not reached vs 4-5 months; Kaplan-Meier survival analysis; P = .049). In contrast, the overall survival of the patients did not correlate with the extent of lesion, age, stage, necrosis, histologic subtypes, CD56 expression, or angiocentric or angiodestructive growth pattern. Our findings suggest that the EBV viral load in tumor tissues is a useful indicator for predicting outcome of nasal NK/T-cell lymphoma.     

The systemic distribution of Epstein-Barr virus genomes in fatal post-transplantation lymphoproliferative disorders. An in situ hybridization study.

The systemic distribution of Epstein-Barr virus (EBV) genomes was studied in paraffin-embedded tissues from 12 fatal cases of Post-transplantation lymphoproliferative disease (PTLD), using an in situ hybridization technique employing an alpha-35S-dCTP-radiolabeled BamHI-W fragment of EBV DNA. The presence of EBV was documented in various PTLD-involved organs. The hybridization signal for the virus localized predominantly in the abnormal lymphoid cells, but signals also were detected in hepatocytes and/or adrenal cortical cells in five cases. The distribution of autoradiographic label within the lymphoid cells was focal and its intensity varied from field to field suggesting a nonuniformity of the viral genomic load in the infected tissues. Recruitment of EBV genome-bearing cells was not observed into inflammatory mononuclear infiltrates found in organs without histopathologic evidence of PTLD.     

Evaluation of use of Epstein-Barr viral load in patients after allogeneic stem cell transplantation to diagnose and monitor posttransplant lymphoproliferative disease

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 10(6) versus 4 x 10(4) copies/microg of DNA; P < 0.0001). A threshold value of 10(5) copies/microg 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 10(4) copies/microg 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.     

Monitoring of EBV-DNAemia by quantitative real-time PCR after adult liver transplantation.

BACKGROUND: Post-transplant lymphoproliferative disease (PTLD) causes significant morbidity and mortality in transplantation. The clinical significance of Epstein-Barr virus (EBV) in the development of PTLD is clear, but not all EBV-reactivations cause PTLD. OBJECTIVES: We retrospectively analyzed EBV-DNAemia in liver transplant patients by a quantitative TaqMan-based real-time plasma PCR. STUDY DESIGN: Altogether 1284 specimens, obtained from 105 patients for frequent monitoring of cytomegalovirus (CMV) and human herpesvirus-6 and -7 (HHV-6, HHV-7) during the post-transplant year, were retrospectively tested for EBV-DNA. RESULTS: Altogether, 14/105 (13%) patients showed EBV-DNAemia, which usually occurred within 3 months after transplantation and subsided within a few weeks. EBV-DNAemia occurred concurrently with CMV in 10/14, with HHV-6 in 11/14, and with all three betaherpesviruses in 4/14 cases. The peak viral loads were relatively low (median 2100 EBV-DNA copies/ml, range 568-6600), except in one patient who first had low-level EBV-DNA (562-3022 copies/ml) in the early post-transplant period, but on day 175 after transplantation developed high-level DNAemia (9851-86,975copies/ml) which continued for 6 months and developed into PTLD at 6 months after transplantation. CONCLUSION: Low-level EBV-DNAemia is common after liver transplantation, often occurring together with betaherpesviruses, but seldom leads to high viral loads or PTLD. However, monitoring of EBV-DNA levels in the patients can be useful.     

High levels of Epstein-Barr virus DNA in blood of solid-organ transplant recipients and their value in predicting posttransplant lymphoproliferative disorders

Epstein-Barr virus (EBV) DNA was quantitated in peripheral blood mononuclear cells (PBMC) from 25 healthy subjects, 105 asymptomatic solid-organ transplant (SOT) recipients, and 15 SOT recipients with symptomatic EBV infections by using a newly developed quantitative-PCR technique. Patients with symptomatic EBV infections had significantly higher (P < 0.001) median EBV DNA levels than asymptomatic SOT recipients and immunocompetent individuals. In SOT recipients, the positive predictive value of EBV DNA levels of >1, 000 genome equivalents (GE)/0.5 microg of total PBMC DNA was 64.7% for symptomatic EBV infection, while the negative predictive value was 96.1%. In 19 of 32 (59.3%) asymptomatic SOT recipients, EBV DNA levels were consistently below 1,000 GE for as long as 18 months, while 10 of 32 (31.2%) patients had 1,000 to 5,000 EBV GE at least once during follow-up. In a minority of patients (3 of 32; 9.3%), >/=5,000 GE could be detected at least once during follow-up. Reduction of immunosuppressive treatment decreased EBV DNA levels by >/=1 log(10) unit in patients with symptomatic EBV infections. Quantification of EBV DNA is valuable for the diagnosis and monitoring of symptomatic EBV infections in SOT recipients.     

Effect of herpesvirus infections on T-lymphocyte subpopulations and blastogenic responses in renal transplant recipients receiving cyclosporine

The immunosuppressive effects of three herpesviruses--cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV)--were assessed in 29 renal transplant recipients treated with cyclosporine and prednisone. The ratios of Leu 3-positive ("helper-inducer") to Leu 2-positive ("suppressor-cytotoxic") T lymphocytes in peripheral blood were only moderately and transiently decreased during primary CMV infection, with or without concurrent reactivated EBV and HSV infections. This effect was due to an increase in absolute numbers of Leu 2-phenotypic and decrease in Leu 3-phenotypic T cells and was associated with symptomatic viral illness. Reactivated CMV infection alone or together with reactivated EBV and HSV infections resulted in less significant alterations in T-cell subsets than did primary CMV infection. Lymphocyte blastogenesis was not significantly altered during the herpesvirus infections. The data suggest that cyclosporine treatment inhibits the activation of suppressor cells and depression of cellular immune function that have been associated with herpesvirus infections in renal transplant recipients undergoing conventional immunotherapy.     

Lytic and latent EBV gene expression in transplant recipients with and without post-transplant lymphoproliferative disorder

Background

Epstein–Barr virus (EBV) is associated with post-transplant lymphoproliferative disorder (PTLD), which has significant morbidity and mortality in transplant recipients. To devise prophylactic measures, we need predictors of PTLD and a better understanding of the physiopathogenesis of the disease.

Objectives

To identify a molecular pattern of EBV gene products in blood that is specific to PTLD and can be used for the diagnosis of this disease.

Study design

We evaluated the ratio between latent and replicating EBV nucleic acids in individuals with PTLD by comparison with transplant recipients without PTLD and immunocompetent hosts with EBV DNA-emia. Subjects were prospectively identified between July 2009 and October 2010 at the University of Colorado Hospital. EBV DNA, LMP-2A Latency III and BZLF1 Lytic genes mRNA were quantified using real-time PCR.

Results

We found that PTLD subjects (N = 7) had significantly higher EBV DNA-emia compared with non-transplant immunocompetent subjects (N = 69; p < 0.0001), and transplant recipients without PTLD (N = 105; p < 0.0001). The ratios between LMP-2A and BZLF1 mRNA in transplant recipients were significantly lower than in non-transplant subjects (p = 0.04). However, PTLD and non-PTLD transplant recipients displayed similar ratios.

Conclusions

These results suggest that EBV replication makes a larger contribution to the circulating EBV DNA in transplant recipients compared with immunocompetent hosts. Transplant recipients seem to lose control over EBV replication, which may contribute to the development of PTLD.