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.     

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.     

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.     

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.     

Hodgkin lymphoma in a renal transplant recipient associated with low peripheral blood Epstein-Barr virus genome copies.

Posttransplant lymphoproliferative disorders are often accompanied by >500 Epstein-Barr virus (EBV) genome copies/10(5) lymphocytes, and they occur shortly after transplantation. Hodgkin lymphoma occurs rarely after transplantation, appearing a mean of 4.2 years posttransplant, and although Hodgkin lymphoma has strong associations with EBV, no quantitative analysis of peripheral blood EBV genome copies has been reported. A mixed cellularity Hodgkin lymphoma developed in a 17-year-old boy 4 years after a renal transplant. Serial EBV genome copy numbers from blood by competitive polymerase chain reaction had been obtained to assess for lymphoproliferative disease. Epstein-Barr virus genome copy numbers peaked at 500 copies/10(5) lymphocytes 8 months prior to Hodgkin lymphoma diagnosis but fell to 8 copies/10(5) lymphocytes at diagnosis. Reliance on EBV levels greater than 500 copies may result in delay of biopsy and diagnosis of Hodgkin disease in the posttransplant setting.     

Prediction of cytomegalovirus (CMV) plasma load from evaluation of CMV whole-blood load in samples from renal transplant recipients

In a prospective cohort of 82 renal transplant recipients, we evaluated the capacity of the cytomegalovirus (CMV) load in whole blood (WB) to predict the plasma CMV load, aiming to identify active CMV infections by using WB samples only and to deduce a WB threshold. Using quantitative real-time PCR, a total of 1,474 WB samples were assayed, of which 279 were positive for CMV, and 140 out of the 276 paired plasma samples tested positive. Thirty (36.6%) patients presented with at least one positive plasma PCR result, and 21 infection episodes (19 patients) required curative treatment (median follow-up time, 12 months). When the plasma CMV load was >500 copies/ml (n = 70), more than 94% (95% confidence interval, 86.0%, 98.4%) of WB samples had >500 copies/ml. Two prediction models were built: log₁₀ plasma viral load (VL) was calculated as −0.3777 + 0.9342 × log₁₀ WB VL and as −0.3777 + 0.8563 × log₁₀ WB VL for patients with and without treatment, respectively. In the validation sample (578 routine samples), 77.2% of the observed and expected plasma viral loads were concordant (95% confidence intervals, 73.5 and 80.5%). According to the model, the plasma viral load was >500 copies/ml when the WB load was >3,170 or >4,000 copies/ml in patients with or without treatment, respectively. WB seems to be an appropriate candidate for routine CMV monitoring of transplant recipients by using a single assay.     

Use of real-time PCR to measure Epstein-Barr virus genomes in whole blood

The measurement of the Epstein–Barr viral load in peripheral blood has been recognised as an important way of monitoring the response to treatment in patients with Epstein–Barr virus (EBV)-related malignancies. In particular, EBV load in transplant recipients can be used as a predictive parameter for Post-transplant Lymphoproliferative Disorder (PTLD). The aim was to develop a rapid and reliable PCR protocol for the quantification of the cell-associated EBV genome. Real-time PCR using TaqMan methodology was established. This technique was applied to determine the EBV load in various study groups including healthy controls, transplant recipients, patients on haemodialysis, and patients with infectious mononucleosis. The baseline level of EBV genomes in the immunosuppressed renal transplant recipients was significantly different from that in the healthy controls.     

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.     

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.     

Quantitative Analysis of Epstein-Barr Virus Load by Using a Real-Time PCR Assay

To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 10⁷ copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 10^3.7 copies/μg of DNA in patients with EBV-related lymphoproliferative disorders, 10^4.1 copies/μg of DNA in patients with chronic active EBV infections, and 10^2.2 copies/μg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.     

Surface immunoglobulin-deficient Epstein-Barr virus-infected B cells in the peripheral blood of pediatric solid-organ transplant recipients

Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, normally causes an asymptomatic latent infection with very low levels of circulating virus in the peripheral blood of infected individuals. However, EBV does have pathogenic potential and has been linked to several diseases, including posttransplant lymphoproliferative disease (PTLD), which involves very high circulating viral loads. As a consequence of immunosuppression associated with transplantation, children in particular are at risk for PTLD. Even in the absence of symptoms of PTLD, very high viral loads are often observed in these patients. EBV-infected B cells in the circulations of 16 asymptomatic pediatric solid-organ transplant recipients from Children's Hospital of Pittsburgh were simultaneously characterized for their surface immunoglobulin (sIg) isotypes and EBV genome copy numbers. Patients were characterized as having high and low viral loads on the basis of their stable levels of circulating virus. Patients with high viral loads had both high- and low-copy-number cells. Cells with a high numbers of viral episomes (>20/cell) were predominantly Ig null, and cells with low numbers of episomes were predominantly sIgM positive. Patients with low viral loads carried the vast majority of their viral load in low-copy-number cells, which were predominantly IgM positive. The very rare high-copy-number cells detected in carriers with low viral loads were also predominantly Ig-null cells. This suggests that two distinct types of B-lineage cells contribute to the viral load in transplant recipients, with cells bearing high genome copy numbers having an aberrant Ig-null cellular phenotype.     

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.     

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.     

Epstein‐Barr virus related post‐transplant lymphoproliferative disorder prevention strategies in allogeneic hematopoietic stem cell transplantation

Epstein‐Barr virus associated post‐transplant lymphoproliferative disorders (EBV PTLD) are recognized as a significant cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). The number of patients at risk of developing EBV PTLD is increasing, partly as a result of highly immunosuppressive regimens, including the use of anti‐thymocyte globulin (ATG). Importantly, there is heterogeneity in PTLD management strategies between alloHSCT centers worldwide. This review summarizes the different EBV PTLD prevention strategies being utilized including the alloHSCT and T‐cell depletion regimes and the risk they confer; monitoring programs, including the timing and analytes used for EBV virus detection, as well as pre‐emptive thresholds and therapy with rituximab. In the absence of an institution‐specific policy, it is suggested that the optimal pre‐emptive strategy in HSCT recipients with T‐cell depleting treatments, acute graft vs host disease (GVHD) and a mismatched donor for PTLD prevention is (a) monitoring of EBV DNA post‐transplant weekly using plasma or WB as analyte and (b) pre‐emptively reducing immune suppression (if possible) at an EBV DNA threshold of >1000 copies/mL (plasma or WB), and treating with rituximab at a threshold of >1000 copies/mL (plasma) or >5000 copies/mL (WB). There is emerging evidence for prophylactic rituximab as a feasible and safe strategy for PTLD, particularly if pre‐emptive monitoring is problematic. Future management strategies such as prophylactic EBV specific CTLs have shown promising results and as this procedure becomes less expensive and more accessible, it may become the strategy of choice for EBV PTLD prevention.     

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.     

Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen

Epstein-Barr virus (EBV) DNA load monitoring in peripheral blood has been shown to be a useful tool for the diagnosis of aberrant EBV infections. In the present study we compared the relative diagnostic values of EBV DNA load monitoring in unfractionated whole blood and simultaneously obtained serum or plasma samples from Burkitt's lymphoma (BL) patients, transplant recipients, human immunodeficiency virus (HIV)-infected individuals, and infectious mononucleosis (IM) patients by a quantitative competitive PCR (Q-PCR). The EBV DNA load in BL patients was mainly situated in the cellular blood compartment (up to 4.5 x 10(6) copies/ml). EBV DNA loads in unfractionated whole blood and parallel serum samples showed no correlation. In transplant recipients, IM patients, and HIV-infected patients, the EBV burden in the circulation was almost exclusively restricted to the cellular blood compartment, because serum or plasma samples from these patients yielded negative results by Q-PCR, despite high viral loads in corresponding whole-blood samples. A 10-fold more sensitive but qualitative BamHI-W-repeat PCR occasionally revealed the presence of EBV at <2,000 copies of EBV DNA per ml of serum. Spiking of 100 copies of EBV DNA in samples with negative Q-PCR results excluded the presence of inhibitory factors in serum or plasma that influenced the Q-PCR result. Serum samples from all populations were often positive for beta-globin DNA, indicating cell damage in vivo or during serum preparation. We conclude that serum is an undesirable clinical specimen for EBV DNA load monitoring because it omits the presence of cell-associated virus and uncontrolled cell lysis may give irreproducible results or overestimation of the DNA load. Unfractionated whole blood is strongly preferred since it combines all blood compartments that may harbor EBV and it best reflects the absolute viral burden in the patient's circulation.     

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.     

Development of a real-time quantitative assay for detection of Epstein-Barr virus

With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10(7) copies of DNA per ml using two sample preparation methods based on absorption. A precision study yielded an average coefficient of variation for both methods of less than 12%, with a coefficient of regression for the standard curve of a minimum of 0. 98. We detected EBV DNA in 19.2% of plasma samples from immunosuppressed solid-organ transplant patients without symptoms of EBV infections with a mean load of 440 copies per ml. EBV DNA could be detected in all transplant patients diagnosed with posttransplant lymphoproliferative disorder, with a mean load of 544,570 copies per ml. No EBV DNA could be detected in healthy individuals in nonimmunosuppressed control groups and a mean of 6,400 copies per ml could be detected in patients with infectious mononucleosis. Further studies revealed that the inhibitory effect of heparinized plasma could be efficiently removed by use of an extraction method with Celite as the absorbent.     

Quantification of adenovirus DNA in unrelated donor hematopoietic stem cell transplant recipients

BACKGROUND: Adenovirus (AdV) infection is a life threatening condition in immunosuppressed patients. Quantitative AdV assays can improve the clinical management of these patients. OBJECTIVES: To evaluate quantitative measurement of AdV DNA with PCR in blood from hematopoietic stem cell transplant (HSCT) recipients. STUDY DESIGN: Quantitative PCR was used to measure viral DNA levels of AdV in consecutive blood samples from 40 HSCT recipients (27 adults and 13 children) during a 1-year post-engraftment period. All patients received grafts from unrelated donors and were given anti-T-cell antibodies in the conditioning regimen. RESULTS: In the group of 40 patients, six (15%) had detectable AdV DNA in blood for different lengths of time. None of these six patients suffered from severe graft-versus-host disease. In three of the patients a high AdV viral load (>10,000copies/mL) was detected, one of whom also had high viral load of EBV and CMV and one of EBV only. These three patients died within 2 months after detection of ADV viremia. A low AdV viral load (<500copies/mL) was detected in three surviving patients and they did not have concomitant high viral load of neither CMV nor EBV. CONCLUSIONS: AdV viremia was present in 15% of the HSCT recipients and a high AdV viral load was associated with fatal outcome. Screening for AdV DNA with quantitative PCR in blood may be of clinical importance in allogeneic HSCT recipients in order to prevent severe clinical virological complications.     

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.