Whole Blood Epstein-Barr Virus Research Articles

Disparities in the Levels of Whole-Blood Epstein-Barr Virus between the Cancer and Non-Cancer Populations in Zhejiang, China

ObjectiveThis study aimed to investigate the prevalence of Epstein-Barr virus (EBV) infection in patients with and without cancer. MethodsA total of 26,648 participants who underwent whole-blood EBV DNA (WBEBV) assays between January 1, 2020, and August 31, 2023, were included. The chi-square test was used for categorical data analysis, and R software was used to analyze the differences in EBV DNA load levels and the diagnostic capabilities of WBEBV. ResultsPositive rates were 10.2% and 25.4% for healthy controls (HC) and patients, respectively. The positivity rate for EBV-associated neoplasms (EN) was the highest at 7.53%, followed by leukemia (Le) at 5.49%. The subgroup analysis showed that the positivity rate for abnormal proliferation or hyperplasia (APH) was 31.9%, followed by 30.5% for Le. The WBEBV of patients with transplants (TP), especially living-related transplants (LT), was the highest among all subgroups. WBEBV at diagnosis was used to differentiate between infectious mononucleosis (IM) and chronic active Epstein-Barr virus (CAEBV), with a sensitivity of 67.4% (95% confidence interval [CI]: 57.6–75.8) and specificity of 72% (95% CI: 63.3–79.3). We conclude that the prevalence of EBV infection is low in the healthy population in this region and that a high EBV load at baseline is more common in LT, IM, and Lymphocyte Leukemia (LL). ConclusionThis study used a large-sample survey to characterize the prevalence of whole-blood EBV levels in various diseases, including the stages and subtypes. The EBV detection rate was higher in patients with malignant disease than in those with benign disease. Our study provides clinicians with baseline information regarding EBV-associated diseases.

The Clinical Significance and Prognostic Role of Whole-Blood Epstein-Barr Virus DNA in Lymphoma-Associated Hemophagocytic Lymphohistiocytosis.

To evaluate the role of circulating Epstein-Barr virus (EBV) DNA in lymphoma-associated hemophagocytic lymphohistiocytosis (HLH). We conducted a retrospective cohort study to explore the clinical and prognostic significance of EBV DNA in lymphoma-associated HLH. We included adult patients with combined diagnoses of lymphoma and HLH from January 2010 and November 2022 by retrieving the medical record system. A total of 281 patients with lymphoma-associated HLH were identified. Elevated whole-blood EBV DNA was observed in 54.4% (153/281) of patients, and the median copy number was significantly higher in the T/NK-cell malignancies (199,500, interquartile range, 30,000-1,390,000) than that in the B-cell non-Hodgkin lymphoma (5520, interquartile range, 1240-28,400, P < 0.001). The optimum cutoff for predicting survival was 16,100 copies/mL. Compared to the patients with EBV DNA ≤ 16,100 copies/mL, those with EBV DNA > 16,100 copies/mL were younger and had more T/NK-cell malignancies, lower levels of neutrophils and fibrinogen, and higher levels of hemoglobin, alanine aminotransferase, aspartate aminotransferase, lactic dehydrogenase, and β2-microglobulin. A higher load of EBV DNA (> 16,100 copies/mL), thrombocytopenia (< 100 × 109/L), neutropenia (< 1 × 109/L), hypofibrinogenemia (≤ 1.5g/L), and elevated levels of creatinine (> 133μmol/L) were independent adverse predictors of 60-day overall survival and overall survival. A prognostic index based on EBV DNA and the other four factors was established to categorize the patients into four groups with significantly different outcomes. Our study identified high EBV load as a risk factor for lymphoma-associated HLH and established a prognostic index to predict outcomes.

Prospective evaluation of blood Epstein-Barr virus DNA load and antibody profile in HIV-related non-Hodgkin lymphomas.

The value of Epstein-Barr virus (EBV) biomarkers on the prognosis of HIV-related non-Hodgkin's lymphoma (NHL) has been poorly explored in the combined antiretroviral therapy (cART) era. We evaluated EBV DNA load and EBV antibodies in HIV-NHL patients enrolled in the French ANRS-CO16 Lymphovir Cohort between 2008 and 2015. Whole blood and plasma EBV DNA load and serological profiles were analyzed in 76 HIV-infected patients at diagnosis of NHL and 6 months after the initiation of chemotherapy. Prechemotherapy whole blood (WB) and plasma EBV DNA loads were positive for 80 and 45% of HIV-NHL patients, respectively. Pretreatment WB EBV DNA positivity was associated with a positive plasma HIV-1 RNA load (relative risk (RR), 4.42 [1.33; 14.72]) and plasma EBV DNA positivity with EBV in situ detection (RR 10.62 [2.38; 47.49]). Following chemotherapy, the proportions of patients with positive WB or plasma EBV DNA declined from 81 to 23% (P < 0.0001) and from 43 to 8% (P < 0.0001), respectively. Estimated 2-year progression-free survival did not differ according to prechemotherapy WB positivity (82% versus 67%, P = 0.15) or plasma EBV DNA positivity (76% versus 81%, P = 0.52). The plasma EBV DNA load correlates with in situ EBV detection. The WB EBV DNA load correlates with HIV load. WB and plasma EBV DNA loads at NHL diagnosis do not constitute prognostic markers for HIV-NHL patients in the modern cART era.

Reappraisal of the prognostic value of Epstein-Barr virus status in monomorphic post-transplantation lymphoproliferative disorders\u2013diffuse large B-cell lymphoma

The role of the Epstein-Barr virus (EBV) status in the blood for predicting survival in post-transplantation lymphoproliferative disorders–diffuse large B-cell lymphoma (PTLD–DLBCL) is unknown. We evaluated the prognostic values of pre-treatment EBV-encoded small RNA (EBER) detected with in situ hybridization in tissues and EBV DNA in the whole blood (WB) and plasma in 58 patients with monomorphic PTLD–DLBCL after solid organ transplantation. There were no significant differences in the rates of overall response, complete response, and survival according to EBER EBV and WB EBV status. In contrast, patients with positive plasma EBV DNA had significantly lower rates of overall response (60.0% vs. 94.4%, P = 0.043) and complete response (40.0% vs. 88.9%, P = 0.019) as well as worse progression-free survival (PFS) (P = 0.035) and overall survival (OS) (P = 0.039) compared with patients with negative plasma EBV DNA. In multivariate analysis, plasma EBV DNA positivity was a significantly unfavorable prognostic factor for PFS [hazard ratio (HR) 4.92, 95% confidence interval (CI) 1.22–19.86, P = 0.025] and OS (HR 4.48, 95% CI 1.14–17.63, P = 0.032). Despite small number of 6 patients with plasma EBV positivity, plasma EBV DNA positivity might be more prognostic for survival than EBER or WB EBV DNA positivity in patients with monomorphic PTLD–DLBCL.

Automated quantification of Epstein-Barr virus in whole blood for post-transplant lymphoproliferative disorders monitoring

BackgroundStandardized and sensitive assays for Epstein Barr Virus (EBV) are needed to define universal cutoff for treatment initiation in allogeneic hematopoietic stem cells transplant recipients. In a context of accreditation and the availability of EBV international standard, we evaluated the Abbott RealTime EBV (RT) assay for EBV quantification in whole blood.MethodsThe RT assay was compared on 282 prospective clinical samples with the Artus EBV PCR Kit V1 assay (V1) and we analyzed the kinetics of EBV load in 11 patients receiving rituximab treatment.ResultsThe estimated limit of detection was 88 IU/mL. The assay was linear (r2 = 0.9974) in the range of all samples tested (100 to 1,000,000 IU/mL). Intra-assay coefficients of variation (CV) ranged between 0.35 and 1.35%, and inter-assay CV between 3.40 and 4.5%. On samples above the limit of quantification, the two assays were strongly correlated. EBV RT values were on average 0.30 log10 IU/mL lower than those measured with the V1 assay. In patients treated with rituximab, the RT assay remained positive in 5 patients at the time it dropped below undetectable levels with the V1 assay.ConclusionsIn conclusion, the RT assay is a reliable assay for EBV load in whole blood. Its sensitivity will enable to estimate the kinetics of EBV load and the impact of treatments to control EBV reactivations.

Superiority of Epstein-Barr Virus (EBV) DNA in the Plasma over Whole Blood in Prognostication of Extranodal NK/T Cell Lymphoma, Nasal Type (ENKTL)

Background Extranodal natural killer T cell lymphoma (ENKTL) is a rare subtype of non-Hodgkin lymphoma, of which pathogenesis is related to Epstein-Barr virus (EBV) infection. The presence of EBV-DNA in blood is a well-known prognosticator, which is also included in a prognostic model, PINK-E (Kim SJ et al, Lancet Oncol 2016). Furthermore, its presence after treatment is known to be a predictor of the treatment failure (Kim SJ et al, Lancet Haematol 2015). However, no consensus have been made on which blood sample (i.e, whole blood vs. plasma) is best to be used for the test. While EBV in plasma is known to have a higher specificity and sensitivity for EBV-related diseases as compared with EBV in peripheral blood mononuclear cells (Kanakry JA et al., Blood 2016), PINK-E model incorporated EBV status regardless of types of samples. We aimed to compare the prognostic performance of EBV DNA titers from whole blood (WB) and plasma. Method The EBV DNA viral load was measured in both WB and plasma samples by real-time quantitative PCR at the time of diagnosis, during and after completion of planned treatment. DNA was extracted using Artus® EBV RG PCR kit (Qiagen Inc.). The limit of detection level was 2.66 log copies/mL according to the manufacturer. Prognostic accuracy was compared by using time-dependent receiver operating characteristic (ROC) curves and corresponding area under the curve (AUC) values. Results A total of 60 patients with newly diagnosed ENKTL between September 2014 and September 2018 were included in this analysis. Twenty nine patients (48.3%) were in stage I, 6 (10.0%) in stage II, and 25 (41.7%) in stage IV. Forty six (76.7 %) patients had nasal involvement. Among them, 33 patients underwent concurrent chemoradiotherapy with weekly cisplatin and 53 received L-asparaginase containing chemotherapy (VIDL, n=46, 86.8%; SMILE, n=7, 13.2%) as definitive treatment or after chemoradiotherapy. Fifty two patients were evaluable for response: 33 (63.5%) complete response, 9 (17.3%) partial response, and 10 (19.2%) progressive disease. With a median follow-up duration of 34.1 months (range, 1.2-57.5 months), 2-year progression-free survival (PFS) and overall survival (OS) rates were 55.0% and 63.0%, respectively. EBV-DNA was detected in 37 (61.7%) in pretreatment WB and 23 patients (38.3%) in plasma samples respectively (Table 1). There was moderate agreement (ƙ=0.49) between the tests. Discordant results were noted in 16 (26.7%) patients, 15 of whom were WB (+) and plasma (-). Presence of EBV DNA in plasma was significantly correlated with unfavorable clinical characteristics than that in WB (Table 1). WB EBV positivity (2-year PFS rates, 42% vs 74%; HR 2.61, 95% CI, 1.04-6.52, p=0.041) and plasma EBV positivity (2-year PFS rates, 23% vs 75%; HR 4.87, 95% CI, 2.15-11.03, p&lt;0.0001) were both associated with poor PFS. Plasma EBV positivity at diagnosis was a highly significant prognosticator of OS (2-year OS rates of 27% vs. 85%; HR 8.38, 95% CI, 3.03-23.19, p&lt;0.0001). EBV status in the WB also tended to be associated with survival but without statistical significance (2-year OS rates, 47% vs 79%, HR 3.34, 95% CI, 1.12-9.96, P=0.071). Furthermore, WB EBV positivity in those with plasma EBV negativity was not prognostic for OS (2-year OS rates, 86% vs 84%, p=0.944) (Figure 1). On the other hand, plasma EBV status in those with WB EBV positivity was a significant predictor of OS (2yr OS rates, 84% vs. 29%, p&lt;0.001). Multivariable analysis showed plasma EBV positivity was significantly association with poor PFS (HR 4.22, 95% CI, 1.79-9.97, p=0.001) and OS (HR 8.39, 95% CI, 3.03-23.19, p=0.002) (Table 2). The prognostic accuracy assessed by the time dependent ROC curve analysis for 2-year OS rates also showed higher AUCs with plasma than WB (0.79, 95% CI, 0.63-0.92 for plasma; 0.66, 95% CI, 0.37-0.81 for WB). After completion of treatment, 22 patients were tested for EBV titer. Among those with plasma EBV positivity, WB EBV positivity was not associated with OS (2-year OS rates, 83% vs 62%, p=0.488). By contrast, plasma EBV positivity among those with WB EBV positivity was a significant predictor of OS (2yr OS rates, 62% vs. 17%, p=0.008) (Figure 2). In addition, post treatment plasma EBV DNA titer was a significant prognostic factors for PFS and OS by multivariable analysis (Table 3). Conclusion EBV DNA test in plasma is more accurate prognostic marker for ENKTL than that in WB as it can prognosticate regardless of EBV status in the WB. Disclosures No relevant conflicts of interest to declare.

Evaluation of four commercial extraction-quantification systems to monitor EBV or CMV viral load in whole blood

BackgroundMeasurement of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viral loads is commonly used to monitor posttransplant patients. Two new systems (eMAG/eSTREAM and Versant/kPCR) have been recently commercialized. ObjectivesTo evaluate the performance of four systems to quantify CMV and EBV in whole blood. Study designThree extraction and real-time PCR amplification systems: m2000SP/RT (M2000), eMAG/eSTREAM (EMAG), and Versant/kPCR (KPCR) were compared with our routine system Qiasymphony/RGQ (QS/RGQ). The 4 systems were tested using 3 dilutions in triplicate according to the WHO international standard (WHO-IS) for intra-assay reproducibility; 56 whole blood samples (24 patients, 4 follow-ups) for CMV and 45 samples (27 patients, 3 follow-ups) for EBV. ResultsFor CMV, the mean of the WHO-IS (expected value: 4.7 Log IU/ml) was: QS/RGQ=4.84, M2000=4.61, EMAG=4.33, and KPCR=4.79. One patient (10 samples) presented a major underquantification by QS/RGQ. Of the 46 remaining samples, 41 were quantified with QS/RGQ, 43 with M2000, 33 with EMAG and 24 with KPCR. For EBV, the mean of the WHO-IS was: QS/RGQ=4.70, M2000=4.61, EMAG=4.62, and KPCR=4.57. Among the 45 samples, 43 were quantified with QS/RGQ, 39 with M2000, 40 with EMAG and 32 with KPCR. ConclusionThe results obtained with the WHO-IS were very good. The results of patients' samples were well correlated with the announced sensitivity of each system. The elevated threshold of the KPCR CMV assay may be problematic for the follow-up of highly immunocompromised patients who require early introduction of treatment.

Evaluation of the new Abbott Real-Time EBV assay: fully automated quantification of EBV in whole blood by targeting BLLF1

The accurate measurement of the Epstein–Barr virus (EBV) DNA level in the blood is required for managing EBV-associated diseases. A new commercial Abbott Real-Time EBV assay, which targets the BLLF1 gene, was evaluated on 120 clinical whole blood samples and the Qnostics EBV analytical panel. The limit of detection of the assay was 48.9 IU/mL (95% confidence interval, 48.1–49.8 IU/mL). The assay was linear from 2 to 5 log10 IU/mL (R2 = 0.997). The within-run coefficients of variation (CVs) ranged from 1.68% to 4.75% and the between-run CVs ranged from 1.73% to 12.83% for samples with high, medium, and low viral loads. EBV DNA loads measured by Abbott EBV assay strongly correlated with the results quantified by another commercial Nanogen EBV Real-Time Alert Q-PCR assay (r = 0.879, P < 0.0001). The fully automated Abbott Real-Time EBV assay targeting BLLF1 reduced both hands-on time and turnaround time and demonstrated a reliable performance for EBV DNA quantification in whole blood.

Co-Infections by Double-Stranded DNA Viruses after Ex Vivo T Cell–Depleted, CD34+ Selected Hematopoietic Cell Transplantation

Recipients of ex vivo T cell–depleted (TCD) hematopoietic cell transplantation (HCT) are at risk of infection by double-stranded (ds) DNA viruses. We report rates of dsDNA viremia, end-organ disease (EOD), infection-related mortality, and overall survival (OS) in a contemporary cohort of adult TCD HCT recipients routinely monitored for cytomegalovirus (CMV), adenovirus (ADV), human herpesvirus 6 (HHV6), and Epstein-Barr virus (EBV). Healthcare utilization in the first 6 months post-HCT was compared between patients with dsDNA viremia versus no viremia. This was an observational study of adult patients with acute leukemia and myelodysplastic syndrome who received CD34+ selected, peripheral blood HCT at Memorial Sloan Kettering Cancer Center from March 2012 through December 2014. Patients were prospectively monitored by quantitative PCR assays for CMV, ADV, HHV6, and EBV in whole blood or plasma. The cumulative incidence of viremia(s) at day +180, EOD at 1 year, and OS at 1 year were estimated by the Kaplan-Meier method and compared by the log-rank test among patient with and without viremia/EOD. Standardized incidence ratios were used to compare overall length of hospital stay (LOS), number of readmissions after HCT, and length of readmissions through day +180. Of 156 patients, 96 (62%) were CMV recipient seropositive. Forty-two patients received grafts from matched related (27%), 86 from matched unrelated (55%), and 28 from mismatched (18%) donors. Overall, 132 patients (85%) had ≥1 viremia and 52 (33%) ≥2 viremias by day +180. The cumulative incidences for CMV, HHV6, ADV, and EBV viremia were 44%, 61%, 7%, and 16%, respectively, with median times of onset 28 days (interquartile range [IQR], 25 to 33), 33 days (IQR, 25 to 47), 60 days (IQR, 19 to 84), and 79 days (IQR, 54 to 106) post-HCT, respectively. Twenty-eight patients (18%) developed EOD by dsDNA viruses at 1 year post-HCT. Treatment for CMV accounted for 91% total antiviral treatment-days. Compared with patients with no viremia, patients with CMV viremia, HHV6 viremia, or ≥2 viremias experienced longer LOS (P < .001) and a higher number of readmissions (P < .001) by day +180. OS rate at 1 year was 79% and was similar between patients with or without dsDNA viremias. EOD was associated with lower 1-year OS rates (63.4%) versus without EOD (81.1%) (P = .02). Of 33 patients who died, 10 died due to infection, and 7 of these infection-related deaths were due to dsDNA viruses. Viremia by dsDNA viruses occurred in 85% of TCD HCT recipients by day +100 and 33% of patients experienced ≥2 viremias by day +180. CMV accounted for most antiviral use. CMV, HHV6, or ≥2 viremias were associated with more readmissions and longer LOS. One year OS rate was 78%. EOD by dsDNA viruses was associated with decreased 1-year OS. Infections by dsDNA viruses pose a substantial burden after TCD HCT.

Acute liver dysfunction not resulting from hepatitis virus in immunocompetent children.

The aim of the present study was to clarify the roles of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) in immunocompetent children with acute liver dysfunction not resulting from hepatitis virus. Sixty-eight children (median age, 3 years) hospitalized as a result of acute liver dysfunction were enrolled in this study. Hepatitis A, B, and C were excluded. The prevalence of CMV, EBV, and HHV-6 and viral DNA load in whole blood was prospectively evaluated on multiplex real-time polymerase chain reaction (PCR). Of the 68 children with acute liver dysfunction, multiplex real-time PCR was positive in 30 (44%). CMV, EBV, and HHV-6 DNA were detected in 13 (19%), 14 (21%), and seven (10%), respectively. Serum CMV immunoglobulin (Ig)G/IgM and EBV viral capsid antigen IgG/IgM were measured in 40 (CMV DNA positive, n = 10; negative, n = 30) and 45 (EBV DNA positive, n = 14; negative, n = 31) of the 68 children, respectively. Eighteen percent (CMV, 7/40) and 9% (EBV, 4/45) were positive for both PCR and viral-specific IgM. There was no significant difference in CMV and EBV viral load between IgM-positive and -negative children with viremia. CMV, EBV, and HHV-6 DNA were frequently detected in immunocompetent children with acute liver dysfunction, but primary CMV and EBV infection were confirmed in 10-20% of the children with acute liver dysfunction. The combination of PCR assay and serology is necessary to make a diagnosis of acute liver dysfunction due to primary CMV, EBV and/or HHV-6 infection in immunocompetent children.

Quantification of Epstein-Barr Virus DNA Is Helpful for Evaluation of Chronic Active Epstein-Barr Virus Infection

Chronic active Epstein-Barr virus infection (CAEBV) presents with chronic or recurrent infectious mononucleosis-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Immunological methods are useful for the diagnosis of viral infections. However, CAEBV patients do not necessarily have high titers of Epstein-Barr virus (EBV)-specific antibodies. Hosts that are immunocompromised after hematopoietic stem cell transplantations sometimes suffer from systemic EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV-positive lymphoma. Patients with EBV-associated diseases are often diagnosed by analyses of bone marrow. Cytomegalovirus (CMV) can cause serious pneumonia or retinitis in immunocompromised hosts. In order to noninvasively understand the clinical status of patients with EBV-associated diseases, we conducted real-time polymerase chain reaction (PCR) methods in their peripheral blood in order to quantify EBV and CMV DNA levels, which reflect viral activity. Here, we describe a 30-year-old Japanese female patient with CAEBV. The patient had repeated fever, fatigue, and liver dysfunction. The histopathological results of liver biopsies were positive for EBV-encoded RNA-1. Acute hepatitis was associated with the EBV infection. The whole-blood EBV DNA levels were high and above 1.0 × 10⁷ copies/mL. After immunosuppressive and antiviral therapies, EBV DNA levels lowered. However, she had to receive bone marrow transplantation because of her EBV-HLH. As the number of lymphocytes increased in the post-transplantation period, EBV DNA levels gradually increased again. The simultaneous detection of CMV DNA was more sensitive than the CMV antigenemia test that is often used to diagnose CMV infections. Unfortunately, the patient died due to a fungal infection. Observing EBV DNA levels closely with real-time quantitative PCR methods is helpful for evaluating the changes in the clinical course.

Quantification of whole-blood EBV DNA in immunocompetent patients with lymphoma.

e18524 Background: Epstein-Barr virus (EBV) is an oncovirus associated with several lymphomas. Quantification of EBV viral loads has been reported as a potential biomarker for EBV+ lymphoma. The goal of our study was to detect whole blood EBV DNA viral load at lymphoma diagnosis and correlate it with expression of EBV-encoded RNA (EBER) in tumor tissue. Methods: Between January to December 2009, 59 immunocompetent patients with a diagnosis of lymphoma were included. Five cc of whole blood were taken at diagnosis to determine EBV viral loads using real-time quantitative PCR. EBV in the tumor cells was evaluated by detecting EBER using a chromogenic in situ hybridization (ISH) technique. Results: From 59 patients, 46 (78%) had diffuse large B-cell lymphoma (DLBCL), 3 (5%) Hodgkin, 3 (5%) NK/T-cell extranodal (NKTCL), 4 (7%) adult T-cell leukemia/lymphoma (ATLL), 1 (2%) peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), 1 (2%) subcutaneous T-cell panniculitis-like lymphoma (SQPL) and 1 (2%) Hydroa-vacciniforme-like lymphoma (HVLL). Twelve cases (20%) showed elevated levels of EBV DNA ranging between 0.25-46,400 copies/ul. Six (50%) were DLBCL, 2 (17%) were NKTCL, 2 (17%) were ATLL, 1 (8%) Hodgkin and 1 (8%) HVLL. Ten cases (83%) expressed EBER in tumor tissue. Eight (80%) were EBER+ within tumor cells and 2 (20%) were positive within the microenvironment. Five (out of 6, 83%) DLBCL cases were positive for EBER. These cases were later defined as EBV+ DLBCL of the elderly (WHO). Two ATLL cases were EBER+ in the microenvironment. Two (out of 3, 66%) NKTL patients had detectable levels of EBV DNA as well as our case of HVLL; these 3 cases were positive for EBER. Conclusions: These results suggest that whole blood EBV DNA quantification correlates with the expression of EBER, detected by ISH, within the tumor tissue and might be of value as a biomarker in patients with EBV+ lymphomas, especially EBV+ DLBCL of the elderly. ATLL expresses EBER within the tumor microenvironment.

Anatomical mapping of human herpesvirus reservoirs of infection

Following primary infection, all eight human herpesviruses persist lifelong in the human host. However, a mapping of all anatomic sites of human herpesvirus persistence is lacking. Fresh tissue specimens representing approximately 40 major anatomic sites from eight autopsies were screened using a recently developed real-time PCR method for detection of all eight human herpesviruses. Patients with evidence of active herpesvirus infection (herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), herpesvirus 6 (HHV-6), herpesvirus 7 (HHV-7), and herpesvirus 8 (HHV-8)) at the time of death were excluded to avoid detection of widely disseminated infection. Despite this precaution, widespread HSV-1 positivity (with blood positivity) was detected in one case—an elderly male who died of cardiac arrest. In a middle-aged male with HIV-AIDS, HSV-1 was found in neural and pharyngeal tissues, skin, cartilage, bone, and urinary bladder, whereas in two other cases, HSV-1 was restricted to neural tissues. HSV-2 was detected in a single site, the anus, in the male with HIV-AIDS. VZV was detected only twice, once in the adrenal gland and once in the small intestine. CMV was detected in three cases, most commonly in nasal mucosa, trachea, thyroid, intestine, and liver. EBV was detected in all eight cases, especially in nasal mucosa, tonsil, spleen, lymph node, tongue, and intestine, but in only two of six whole-blood specimens. HHV-6, like EBV, was detected in all eight cases, most commonly in salivary glands, thyroid, stomach, intestines, liver, and pancreas. HHV-7, like EBV and HHV-6, was detected in all eight cases, most commonly in salivary glands, tonsil, lymph nodes, and bone marrow. HHV-8 was detected in only two sites (both lymph nodes) from two cases. Herpesviruses were detected in three of six whole-blood specimens, including HSV-1, EBV, HHV-6, and HHV-7. These results represent the most comprehensive mapping of herpesvirus tissue distribution in humans reported to date.