Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting

Epstein-Barr virus (EBV) is associated with a wide range of benign and malignant diseases, including infectious mononucleosis, lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. Measurement of EBV viral load in plasma is increasingly used for rapid assessment of disease status. We evaluated the performance characteristics of an EBV polymerase chain reaction assay that uses commercial reagents and instruments from Roche Diagnostics (Indianapolis, IN). DNA was extracted from plasma using a MagNaPure instrument, and viral load was measured by real-time polymerase chain reaction on a LightCycler. Analyte-specific reagents included primers and hybridization probes targeting the EBV LMP2 gene and a spiked control sequence. Accuracy and reproducibility were established using DNA from three cell lines. The assay was sensitive to approximately 750 copies of EBV DNA per milliliter of plasma and was linear across at least four orders of magnitude. The assay detected EBV DNA in three of five samples from nasopharyngeal carcinoma patients, seven of nine infectious mononucleosis samples, and 34/34 samples from immunosuppressed patients with clinically significant EBV-related disease, whereas EBV DNA was undetectable in plasma from 21 individuals without EBV-related disease. In conclusion, this LightCycler EBV assay is rapid, sensitive, and linear for quantifying EBV viral load. The assay appears to be useful for measuring clinically significant EBV levels in immunodeficient patients.     

Genetics of Epstein-Barr virus infection.

Epstein-Barr (EBV) virus is a member of the human herpesvirus family. EBV is the etiologic agent of acute infectious mononucleosis and is closely associated with the genesis of Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma. EBV is also implicated in a variety of other diseases, such as X-linked lymphoproliferative syndrome, T-cell non-Hodgkin's lymphoma, Hodgkin's disease, and NK-cell granular lymphoproliferative disorder. Recently, lymphoepithelial carcinoma of the stomach, gastric carcinoma, pyothorax-associated lymphoma, and smooth muscle tumors were also recognized as EBV-associated diseases. It is therefore important to review the genetics and immunological response of EBV infection. In this review we summarize the genetics of EBV, immunological responses and clinical findings of EBV-associated diseases, which would help us to understand the pathophysiology of EBV-associated disease and develop specific treatments.     

淋巴组织增生性疾病患者外周血EBV DNA定量检测临床意义的研究进展

目前发现爱泼斯坦巴尔病毒(Epstein-Barr virus,EBV)与多种淋巴组织增生性疾病的关系越来越密切,主要包括EBV相关淋巴瘤、EBV阳性淋巴组织增殖性疾病(EBV+LPD)以及传染性单核细胞增多症(infectious mononucleosis,IM)等,以往研究认为EBV+LPD及IM的外周血EBV ...     

Epstein-Barr viral load measurement as a marker of EBV-related disease.

Epstein-Barr virus (EBV) is associated with a wide variety of benign and neoplastic diseases. EBV viral load assays that may prove useful in rapid assessment of disease status are now available. The two most common approaches to viral load measurement are quantitative, competitive PCR, and real-time PCR. Laboratory studies have shown that these assays are sensitive and specific for measuring EBV DNA in blood samples. Clinical investigations suggest a role for viral load measurement in predicting and monitoring EBV-associated tumors, including nasopharyngeal carcinoma, post-transplant lymphoproliferative disorder, Hodgkin's disease, and AIDS-related lymphoma. These new laboratory tools show promise in improving clinical management of affected patients.     

Epstein-Barr virus and rheumatoid arthritis: are rheumatoid arthritis associated nuclear antigen and Epstein-Barr virus nuclear antigen different?

In order to investigate the relationship between antibodies to RANA and other Epstein-Barr virus induced antigens, we have tested 50 sera from subjects without rheumatoid arthritis and with various EBV serology patterns for aRANA, aEA, aVCA, aEBNA. Patients were either suffering from Burkitt's lymphoma, infectious mononucleosis, nasopharyngeal carcinoma, Hodgkin's disease or immunodeficiencies, or were healthy controls. RANA was detected by indirect immunofluorescence on G1 synchronized Raji cells. The correlation was very strong between aEBNA and aRANA (r = 0.86) without any correlation between aRANA and aVCA. These data not only strongly support the opinion that aRANA is frequently found in non-rheumatoid diseases but cast doubt on the distinction between RANA and EBNA.     

The many faces of Epstein-Barr virus

Epstein-Barr virus (EBV) persists for life in a person who has been infected. Investigators are uncertain what this means to the host, particularly an immunocompromised one. EBV is the most common cause of infectious mononucleosis, but the diagnosis must be based on clinical, hematologic, and serologic criteria because other agents are the cause in about 10% of cases. EBV is the first virus to be associated with a neoplasm--Burkitt's lymphoma. This childhood malignancy is relatively common in central Africa, and EBV is a cofactor in its development, although direct evidence for a causal relationship has not been found. In southern China, nasopharyngeal carcinoma is an important health problem in adults in areas where the infection rate of EBV in childhood is high. Rare cases of primary EBV infection that evolved into uncontrolled lymphoproliferative disease have also been reported. EBV's relationship to these diverse diseases, its varying effects on certain individuals and in certain geographic locations, and the testing of a vaccine against the virus are areas of ongoing study.     

Epstein-Barr virus infections: prospects for treatment

Epstein-Barr virus (EBV) causes infectious mononucleosis and oral hairy leucoplakia, and is associated with a number of malignancies. There are, however, no regulatory agency-approved treatments for EBV-related diseases. Several antiviral drugs inhibit replication of EBV in cell culture including acyclic nucleoside and nucleotide analogues and pyrophosphate analogues, all of which inhibit the EBV DNA polymerase. Despite their potency in vitro, these drugs have limited use in vivo for treatment of acute primary EBV infection as well as EBV-associated malignancies for several reasons. Here we discuss novel anti-EBV compounds, including maribavir, potentially useful for the treatment of acute EBV infections. A number of experimental approaches for treatment of EBV-related malignancies that are not susceptible to conventional antiviral drug treatment are also discussed.     

Synthetic peptide derived from the epstein-barr virus encoded early diffuse antigen (ea-d) reactive with human antibodies

Primary infection with Epstein-Barr virus (EBV) and reactivation of latent virus are associated with increased antibody titers against the diffuse early antigen (EA-D). In order to better define the antigenic epitopes recognized by antibodies from patients with infectious mononucleosis (IM) and with other disease states, a series of synthetic peptides were prepared based on the DNA sequence encoding the EA-D molecule. One synthetic peptide (K7b) was reactive with the majority of sera from patients with acute IM. Anti-K7b activity was most readily detected among IgM and IgA antibodies and to a lesser extent among IgG antibodies. In contrast, significant elevations of anti-K7b activity were observed in less than 5% of healthy adults. Serial analysis of samples from individuals prior to and after exposure to EBV demonstrated increased anti-K7b reactivity associated with the symptoms of acute IM. Elevated anti-peptide K7b titers also were found in sera of patients with nasopharyngeal carcinoma and with Sjogren's syndrome (an autoimmune disease involving the salivary glands). Four different synthetic peptides from other regions of the EA-D molecule were not reactive with antibodies from these patients nor from IM patients. These results suggest that peptide K7b defines an antigenic epitope recognized during primary EBV infection and during viral reactivation occurring in patients with autoimmune and neoplastic disease.     

A Rare Presentation of Epstein–Barr Virus Infection

BackgroundEpstein–Barr virus (EBV) is a herpesvirus spread by intimate contact. It is known to cause infectious mononucleosis. Complications, including hematologic pathology and splenic rupture, are uncommon. This report is a case of EBV-induced autoimmune hemolytic anemia and biliary stasis.Case ReportAn 18-year-old man presented to the emergency department with abdominal pain, nausea, vomiting, and jaundice. He did not have risk factors for liver injury or hepatitis. His vital signs were notable for a fever. On examination, he was obviously jaundiced, but not in distress. Laboratory evaluation showed hemolytic anemia and biliary stasis. Ultimately, his inpatient workup yielded positive EBV serology and a positive direct agglutinin test with cold agglutinins. He made a full recovery with supportive care.Why Should an Emergency Physician Be Aware of This?EBV is a widely disseminated herpesvirus. Infectious mononucleosis is a common presentation of acute infection, and treatment of EBV-related diseases are largely supportive. Complications, such as splenic rupture and hematologic pathology, are uncommon. Biliary stasis and autoimmune hemolytic anemia in the form of cold agglutinin disease secondary to EBV is rare, and typically resolves with supportive care and cold avoidance. More advanced treatment methods are available in the setting of severe hemolysis. Elevated transaminases, direct hyperbilirubinemia, or evidence of hemolytic anemia in the setting of a nonspecific viral syndrome should raise suspicion for EBV infection. Rapid recognition can lead to more prompt prevention and treatment of other EBV-related complications.     

Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens

We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.     

The role of Epstein-Barr virus in cancer

Epstein-Barr virus (EBV), discovered > 40 years ago from a Burkitt's lymphoma biopsy, was the first virus to be directly associated with human cancer. EBV has two distinct life cycles in the human host; a lytic form of infection that produces new infectious virions, and a latent form of infection that allows the virus to persist in a dormant state for the lifetime of the host. EBV has evolved a life cycle that mimics the natural differentiation pathway of antigen-activated B cells, giving the virus access to its site of latent infection, the resting memory B cell. By steering infected cells through the various stages of lymphocyte differentiation, EBV is able to enter a cell type suitable for long-term latent persistence and periodic reactivation. However, its presence in various stages of B-cell development, and its ability to infect certain epithelial cells, can have pathogenic consequences, and can contribute to the development of a diverse group of lymphomas and carcinomas. The presence of EBV in the tumour cells of EBV-associated cancers might provide a basis for specific therapy. This article focuses on the contributions that the virus may play in different types of human cancer, particularly Burkitt's lymphoma, Hodgkin's lymphoma, lymphomas and lymphoproliferative diseases in the immunocompromised, and nasopharyngeal and gastric carcinoma.     

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.     

Epstein-Barr virus quantitation by real-time PCR targeting multiple gene segments: a novel approach to screen for the virus in paraffin-embedded tissue and plasma

Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.     

Epstein-Barr virus in patients with immunodeficiency disorders.

The Epstein-Barr virus (EBV), one of eight known human herpesviruses, causes a wide spectrum of diseases under certain conditions. In particular, in the setting of immunodeficiency, which includes primary or secondary/acquired immunodeficiencies, they have been increasingly reported. The major clinical phenotype is the EBV genome-positive lymphoproliferative disorder, which ranges from benign lymphoproliferation to malignant lymphoma with cytogenetic alterations. Severe or fatal infectious mononucleosis may develop in some patients with immunodeficiencies such as X-linked lymphoproliferative disease.     

荧光定量PCR检测传染性单核细胞增多症不同血样本EBV的研究

[目的]寻找荧光定量PCR检测传染性单核细胞增多症（IM）EBV DNA拷贝量的最佳样本。[方法]采用荧光定量PCR分别检测了IM病人的外周血单个核细胞、全血基因组DNA及血浆的EBV DNA拷贝量,并比较各种方法的阳性率及拷贝量。[结果]三种不同来源的样品荧光定量PCR方法的阳性率分别为76％、69％、23％。全基因组及单个核细胞组阳性率比较经x^2检验,差异无显著性,且两组拷贝量比较差异亦无显著性。血浆组阳性率及拷贝量与前两种方法比较差异均有显著性。[结论]外周血单个核细胞是荧光定量PCR检测IM的EBV DNA量的最佳样本。     

HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection

Infectious mononucleosis (IM) is an immunopathological disease caused by EBV that occurs in young adults and is a risk factor for Hodgkin lymphoma (HL). An association between EBV-positive HL and genetic markers in the HLA class I locus has been identified, indicating that genetic differences in the HLA class I locus may alter disease phenotypes associated with EBV infection. To further determine whether HLA class I alleles may affect development of EBV-associated diseases, we analyzed 2 microsatellite markers and 2 SNPs located near the HLA class I locus in patients with acute IM and in asymptomatic EBV-seropositive and -seronegative individuals. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the 2 SNPs were also significantly more frequent in patients with IM than in EBV-seronegative individuals. IM patients possessing the associated microsatellite allele had fewer lymphocytes and increased neutrophils relative to IM patients lacking the allele. These patients also displayed higher EBV titers and milder IM symptoms. The results of this study indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection, suggesting that genetic variation in T cell responses can influence the nature of primary EBV infection and the level of viral persistence.     

Comparison of diagnostic criteria for Epstein-Barr virus infection in children

Serum samples from 159 children with clinically diagnosed infectious mononucleosis, were compared by different serodiagnostic tests for Epstein-Barr infection. These were: 1. detection of viral capsid antigen IgM; 2. detection of viral capsid antigen IgG in absence of nuclear antigen antibodies; 3. detection of IgG to early antigen; and 4. detection of heterophil antibodies. Heterophil antibodies detection is not suitable for diagnosing infectious mononucleosis among children below 6 y. The serological diagnosis of Epstein-Barr virus (EBV) infections must be done by detecting viral capsid antigen IgM, and/or viral capsid IgG in the absence of Epstein-Barr nuclear antigen antibodies. Early antigen IgG was shown on its own not to be a reliable diagnostic marker.     

基于ddPCR技术分析EB病毒载量特征及与qPCR技术的比较研究

目的解决临床工作中应用实时荧光定量PCR(qPCR)方法检测EB病毒(EBV)载量与临床诊断不符的问题,探讨微滴式数字PCR(ddPCR)和qPCR方法检测EBV载量的能力并为临床提供可能的解决方案。方法收集510例疑似EBV感染相关疾病患者血浆标本,采用ddPCR和qPCR两种方法测定同一血浆标本的EBV-DNA载量。结果 EBV感染人群中,EBV-DNA载量较其他地区低,载量中位数仅360copies/mL,其中初诊未治鼻咽癌患者中位病毒载量为4 590copies/mL,治疗后鼻咽癌患者中位病毒载量下降为430copies/mL,免疫力低下者中位病毒载量为130copies/mL,而淋巴瘤患者中位病毒载量为840copies/mL;qPCR检测EBV感染以400copies/mL为界值,高于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈中度相关(r=0.533,P<0.05),低于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈弱相关(r=0.299 5,P<0.05);以ddPCR为标准,qPCR检测EBV-DNA的灵敏度仅为0.317,以ddPCR检测结果为标准,构建qPCR的受试者工作特征曲线下面积为0.871,此时临界值(qPCR)为10copies/mL,灵敏度为0.824,特异度为0.780。结论采用ddPCR方法或优化qPCR的临界值去检测EBV-DNA载量更能为临床诊断EBV感染提供有利支持。