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.     

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.     

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.     

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.     

Plasma Epstein-Barr viral load predicting response after chemotherapy for post-transplant lymphoproliferative disease

Transplant patients are particularly at risk of developing B post-transplant lymphoproliferative disease (PTLD) related to intensive immunosuppressive treatment to prevent graft rejection. In EBV-positive PTLDs, EBV-DNA can be found in the patients' peripheral blood. Several methods have been described to assess peripheral blood EBV viral load. We report a case of a 13-year-old child who developed EBV-positive PTLD after renal transplantation. We assessed EBV plasma viral load by quantitative PCR and we found that the clearance of EBV-DNA correlated well with response to treatment.     

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

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

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.     

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.     

Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA.

A quantitative PCR assay for the detection of HIV-1 nucleic acids is described. The assay is based on a competitive internal standard nucleic acid which can be discriminated from target sequences by the presence of a new restriction enzyme site. The method was used to quantitate plasmid molecules containing HIV-1 sequences, HIV-1 DNA and HIV-1 RNA purified from HIV-1-infected tissue culture cells as well as HIV-1 DNA present in the peripheral blood mononuclear cells of an AIDS patient. The assay will be valuable for assessing viral load in AIDS patients and for the study of viral gene expression.     

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.     

Comparison of various blood compartments and reporting units for the detection and quantification of Epstein-Barr virus in peripheral blood

Epstein-Barr virus (EBV) infection is associated with a broad spectrum of disease. While quantification of EBV nucleic acid in the peripheral blood has been demonstrated to be useful for diagnosis and patient care, the optimal sample type and reporting format for such testing remain uncertain. Using quantitative real-time PCR (QRT-PCR), we evaluated EBV in whole blood (WB), peripheral blood mononuclear cells (PBMC), and plasma in 249 samples from 122 patients. In WB and PBMC, results were reported both in viral copies/ml and in copies/microg of total DNA. Trendings of quantitative values over time among the different sample types were compared. The sensitivities of QRT-PCR using WB and that using PBMC did not differ significantly (P = 0.33), and both were more sensitive than plasma alone (P < 0.0001). EBV viral load results from WB and PBMC paired sample types also showed a significant correlation (P < 0.05), as did results reported in copies/ml and copies/microg DNA for both WB and PBMC (R2 > 0.93). EBV viral loads detected using WB and PBMC trended very closely for the few patients who had multiple positive samples available for analysis. WB and PBMC show comparable sensitivities and a close quantitative correlation when assayed for EBV by QRT-PCR. The close correlation between copies/ml and copies/microg DNA also suggests that normalization to cell number or genomic DNA in cellular specimens may not be necessary.     

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.     

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.     

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.     

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.     

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.     

Epstein–Barr virus-associated leukemic lymphoma after allogeneic stem cell transplantation

Leukemic Epstein–Barr virus (EBV)-associated post-transplant lymphoproliferative diseases (PTLD) following allogeneic hematopoietic stem cell transplantation are extremely rare. We can successfully treat an EBV-associated leukemic lymphoma patient with rituximab, cidofovir, and donor lymphocyte infusion (DLI). In the present case, EBV-specific T cells that were present in the peripheral blood before rituximab administration treatment rapidly increased after DLI in association with a decrease in the EBV-DNA load.     

Case report: Kinetics of Epstein-Barr virus load in a bone marrow transplant patient with no sign of lymphoproliferative disease

The kinetics of Epstein-Barr virus (EBV) load was measured in peripheral blood mononuclear cells of a severely immunocompromised allogeneic bone marrow recipient child, in conditions not associated with lymphoproliferative disease. The viral doubling time was 46.4 hr. The study permitted monitoring EBV clearance from blood, when the anti-rejection therapy was interrupted. Likely, this is the first accurate kinetic assessment of EBV load increasing phase in a clinical context marked by the absence of an overt post-transplant lymphoproliferative disease. According to these data gamma-herpesviruses behave like beta-herpesviruses in being capable of rapid growth.     

Comparison of three methods for extraction of viral nucleic acids from blood cultures.

Reliable nucleic acid extraction techniques for blood specimens are required for the sensitive detection of viral DNA. Standardized procedures for processing blood specimens for the molecular detection of herpesviruses (cytomegalovirus [CMV], herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus [EBV]) have not been established. Three methods were used to extract DNA from blood specimens from healthy donors and asymptomatic immunocompromised patients: (i) IsoQuick treatment of whole blood, (ii) extraction of the peripheral blood leukocytes by lysis (lysis buffer and proteinase K), and (iii) extraction of peripheral blood leukocytes with phenol-chloroform (sodium docecyl sulfate solution and proteinase K). All blood specimens from 25 healthy blood donors were negative for CMV, herpes simplex virus and varicella-zoster virus nucleic acid sequences, regardless of the extraction method, while three samples (12%) extracted by the lysis technique were positive for EBV DNA. Of 25 blood samples from asymptomatic immunocompromised patients, CMV and EBV each were detected in nine specimens by lysis extraction, four each by IsoQuick and four (CMV) and six (EBV) by the phenol-chloroform method. Our results indicate that the lysis method is optimal for the detection of CMV and EBV DNA sequences by PCR from the leukocytic fraction of blood specimens. DNA of these viruses is frequently present in blood specimens from asymptomatic immunocompromised patients and occasionally from healthy donors.     

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.