慢性活动性EB病毒感染的研究进展

机体感染EB病毒(EBV)后,可以表现为持续性或反复发作性发热,肝、脾、淋巴结大,以及肝丙氨酸氨基转移酶水平异常增高等传染性单核细胞增多症(IM)样症状.伴随EBV特异性抗体谱表达与外周血EBV-DNA拷贝数显著增高的EBV感染,称为慢性活动性EB病毒感染(CAEBV).CAEBV为一种临床少见的致命性疾病,预后凶险,导致的患者病死率极高.目前,CAEBV的发病机制尚未完全阐明,亦无标准治疗方案.笔者拟就CAEBV的发病机制、临床表现、诊断及治疗研究的最新进展进行综述,旨在提高临床医师对CAEBV的认识.     

Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus,comparison of TaqMan probes,and molecular beacons

Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients’ samples. The TaqMan and molecular beacon assays were linear between 10 to 10⁷ viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.     

Hemophagocytic syndrome due to Epstein-Barr virus and cytomegalovirus coinfection in a patient on adalimumab

IntroductionHemophagocytic syndrome (HPS) is a rare but potentially fatal complication of viral infections. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) often infect patients receiving TNF-alpha inhibitors (TNF-α inhibitors). While EBV and CMV are well established infections for the development of infectious mononucleosis, coinfection with EBV and CMV is common among immunosuppressed patients and can result in a fatal course. In addition, such viral infections can cause HPS. To the best of our knowledge, we present here the first report of HPS induced by EBV and CMV coinfection during anti-TNFα inhibitor use.Case reportA 23-year-old man hospitalized with fever, elevated liver enzymes, lymphadenopathy, and hepatosplenomegaly was diagnosed with HPS associated with EBV and CMV coinfection while using adalimumab. No clinical improvement was observed after discontinuation of adalimumab. HPS complicated by EBV and CMV coinfection was finally diagnosed, and immediate administration of ganciclovir and prednisone was considered to have prevented a lethal clinical outcome.ConclusionFor cases showing unexplained fever, elevated liver enzymes, and lymphadenopathy while using anti-TNFα inhibitors, screening for EBV and CMV coinfection should be encouraged. In addition, HPS should be considered in patients with EBV and/or CMV infection receiving anti-TNFα inhibitors to facilitate early definitive therapy.     

儿童Epstein—Barr病毒感染临床分析

目的探讨慢性活动性Epstein—Barr病毒（EpsteinBarrvirus,EBV）感染、急性EBV感染及正常儿童EBV—DNA及适应性体液免疫的差异。方法慢性活动性EBV感染患儿8例（慢性组）,急性EBV感染患儿13例（急性组）,正常儿童12例（对照组）外周血单个核细胞采用实时荧光定量PCR法检测,3组EBV—DNA水平,采用ELISA、抗体稀释试验评价EBV适应性体液免疫,分析其与EBV感染不同转归的关系。结果慢性组EBV—DNA载量、病毒壳蛋白抗原-IgA、-IgG及早期抗原-IgA水平明显高于急性组和对照组（P〈0．01）,病毒壳蛋白抗原-IgM水平及早期抗原-IgG滴度改变速率低于急性组（P〈O．01）;慢性组与急性组EBV核抗原-IgG抗体水平均低于对照组（P〈0．01）。结论慢性活动性EBV感染存在不同EBV核抗原-LP拷贝数及不同亲和力的抗体谱,可能与慢性活动性病程相关,对早期识别诊治有重要意义。     

慢性活动性EB病毒感染临床分析

目的通过总结、分析慢性活动性EB病毒感染（CAEBV）患者的临床资料,以提高对CAEBV的诊断、治疗的认识。方法回顾性分析2008年10月-2013年1月诊治的9例CAEBV患者的临床症状特点,以及实验室检食、病原学相关检查、影像学和病理学检佥结果等,并评估其对药物治疗的反应和随访、预后表现。结果CAEBV临床症状缺乏特异性,主要表现为发热、肝脾和淋巴结肿大,其他尚有乏力、恶心、皮疹、黄疸等。辅助检查的异常发现包括：贫血、白细胞降低、中性粒细胞降低、血小板减少、乳酸脱氧酶及羟丁酸脱氢酶升高等肝功能异常及胸部影像学的异常表现等。病原学相关检查结果：6例患者抗EB病毒衣壳抗原IgA抗体升高,8例EB病毒早期抗原IgG为阳性,实时定量聚合酶链反应俭测外周血EB病毒DNA载量（中位数）为3．07×10^5copies／mL。9例患者中6例死亡,其中1例死于颅内出血,1例死于多脏器功能衰竭,1例死于噬血细胞综合征,1例死于肺部感染4例患者进展为淋巴瘤,在抗肿瘤治疗过程中1例死于肝功能衰竭,1例死于严重感染。结论CAEBV临床表现多样且缺乏特异性,常伴有各系统严重并发症,部分患者可进展为淋巴细胞增殖性疾病,预后差,病死率高,应引起临床重视。     

儿童慢性活动性EB病毒感染的相关血液疾病特征

本研究通过4个病例分析儿童慢性活动性EB病毒（CAEBV）感染相关的血液学病征。总结了临床特点，应用显微镜观察骨髓涂片的细胞形态，流式细胞仪分析淋巴细胞亚群，免疫组织化学检测肝穿刺组织，酶联免疫吸附试验（ELISA）检测血浆EBV抗体，实时定量PCR检测血浆EBV—DNA，原位杂交检测外周血单个核细胞（PBMNCs）EBV编码小RNA-1（EBER-1）。结果显示：4例患儿有持续或反复的发热、肝脾肿大、肝功能异常、贫血、血小板减少症、全身炎症反应；骨髓象表现增生减低、成熟障碍、病态造血和噬血细胞增多；4例患者均有CD8^＋T淋巴细胞增多，其中1例进展为T细胞淋巴瘤；2例EBV—VCA-IgG滴度≥1：5120，1例血浆EBV-DNA拷贝数为3．26×10^3／ml，1例PBMNC的EBER1阳性率为1．7％；最后，4例患者均被诊断为CAEBV。结论：免疫相关性血细胞减少、巨噬细胞活化综合征和淋巴细胞增殖性疾病等是本研究中4例CAEBV患儿的血液学病征。     

Chronic active Epstein-Barr virus infection treated with PEG-aspargase: A case report

BACKGROUNDChronic active Epstein-Barr virus infection (EBV) is a systemic EBV-positive lymphoproliferative disease, which may lead to fatal illness. There is currently no standard treatment regimen for chronic active EBV (CAEBV), and hematopoietic stem cell transplantation is the only effective treatment. We here report a CAEBV patient treated with PEG-aspargase, who achieved negative EBV-DNA.CASE SUMMARYA 33-year-old female Chinese patient who had fever for approximately 3 mo was admitted to our hospital in December 2017. EBV-DNA was positive with a high copy number. She was diagnosed with chronic active EB virus infection. PEG-aspargase was administered at a dose of 1500 U/m² at a 14-d interval, resulting in eradication of EBV for more than 6 mo. The effect of PEG-aspargase in this patient was excellent.CONCLUSIONA chemotherapy regimen containing PEG-aspargase for CAEBV may be further considered.     

Assessment of serologic markers for epstein-barr virus

Antibody responses to early antigen (EA) and viral capsid antigen (VCA) were analyzed in 48 proven cases of Epstein-Barr Virus infection and in 48 age- and sex-matched healthy controls to establish optimal cutoff values for diagnosing EBV infection. Predictive values were determined for individual EA and VCA antibody titers and for EA to VCA antibody ratios and the optimal dilution cutoff values for positivity of EA (1:20), VCA (1:640), and EA to VCA (0:031) were selected. When evaluated on a subset of 10 VCA IgM positive cases and 35 negative controls, the three selected cutoff values identified as infections nine of 10, four of 10, and 10 of 10 of the cases and one of 35, none of 35, and one of 35 of the controls, respectively. When evaluated individually on 22 cases of suspected EBV infection who were heterophile antibody-negative and presented with symptoms compatible with EBV infection, an equal number of VCA IgM-positive and negative cases were identified as EBV infections. Overall, the cutoffs EA, VCA, and ratio identified 19 of 22 (86.4%), 14 of 22 (63.6%), and 18 of 22 (81.8%), respectively, and all cases could be identified using combinations of these values. Although these serologic values may be used with some accuracy, until more definitive markers are described a combination of heterophile responses, lymphocyte analysis, clinical symptoms, and serologic cutoff values should be used to assess the role of EBV in patient evaluation.     

Evaluation of the Epstein-Barr virus R-gene quantification kit in whole blood with different extraction methods and PCR platforms

Reliable real-time quantitative PCR assays to measure Epstein-Barr virus (EBV) DNA load (EBV) are useful for monitoring EBV-associated diseases. We evaluated a new commercial kit, EBV R-gene Quantification kit (Argene, Varilhes, France) to quantify EBV DNA load in whole blood. Assay performance was assessed with two PCR platforms (LightCycler 2.0 and SmartCycler 2.0) and three commercial DNA extraction methods. The assay was compared with our in-house real-time EBV PCR using samples from the Quality Control for Molecular Diagnostics 2006 EBV proficiency program and using 167 whole-blood specimens from individuals with infectious mononucleosis, from transplanted or HIV-infected patients, and from EBV-seropositive healthy carriers. The EBV R-gene assay was sensitive to 500 copies of EBV DNA per milliliter of whole blood with the two PCR platforms and the three extraction methods and was linear across 4 orders of magnitude. Intra- and interassay coefficients of variations were less than 20%. Nine of 10 samples tested with the EBV R-gene were in agreement with the expected qualitative results of the Quality Control for Molecular Diagnostics 2006 EBV proficiency program, and 7 of 10 samples were within +/-0.5 log units of the expected quantitative values, with discrepant results mostly observed for low viral load (ie, <1000 copies/ml). In the clinical specimens, the correlation between the R-gene assay and the in-house PCR was high (r=0.92). In conclusion, the EBV R-gene assay accurately assesses the EBV DNA load in whole blood of patients with various forms of EBV infections.     

Chronic active Epstein–Barr virus infection with intrapulmonary shunting: A case report

Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is a high-mortality form of EBV infection. However, chronic hypoxemia is rare in these patients. We herein reported a case of severe hypoxemia due to intrapulmonary shunting in CAEBV. A 17-year-old girl presented with fever, dyspnea, cyanosis, and hepatosplenomegaly. Laboratory tests showed mild liver dysfunction and high copy numbers of EBV-DNA in the peripheral blood. A left supratrochlear lymph node biopsy showed infiltration of highly proliferative T lymphocytes with positive EBV encoded small RNA by in situ hybridization. Technetium-99m-labeled macroaggregated albumin and contrast-enhanced echocardiography confirmed the existence of intrapulmonary shunting, which was probably related to hepatopulmonary syndrome. The final diagnosis was CAEBV with intrapulmonary shunting. The patient was treated with cyclosporine A, etoposide, and dexamethasone. Finally, the patient died of respiratory failure. Intrapulmonary shunting is a rare complication of CAEBV. Early recognition and exploring the cause of hypoxemia should be highlighted in patients with CAEBV.     

Real-time Epstein-Barr virus PCR for the diagnosis of primary EBV infections and EBV reactivation.

BACKGROUND: The serological diagnosis of primary Epstein-Barr virus (EBV) infections is often difficult, whereas the relevance of elevated immunoglobulin G (IgG) antibodies against early antigen (EA) for the diagnosis of EBV reactivation has increasingly become a matter of dispute. Recently, EBV PCR has been added as a diagnostic tool. Positive EBV PCR has been demonstrated in the serum of patients with primary EBV infections and EBV reactivation. OBJECTIVES: To compare classical serological diagnosis of primary EBV infection and EBV reactivation with real-time EBV PCR. STUDY DESIGN: Sera from 45 patients were selected with detectable immunoglobulin M (IgM) antibodies against EBV viral capsid antigen (VCA), and 62 sera were selected with a reactivation profile. A real-time EBV PCR was performed with DNA extracted from these sera. RESULTS: Based on serological data, the diagnosis of primary EBV infection was established for 24 of the 45 IgM VCA-positive patients. By performing PCR, seven extra cases of primary infection were diagnosed for which no heterophilic antibodies could be detected. In five cases of primary infection, no EBV DNA could be detected by PCR. Only in two of the 62 sera with a reactivation seroprofile could EBV DNA be detected. CONCLUSIONS: Based on these data, we suggest that for the diagnosis of primary infections, EBV PCR could lead to an increase of >16% in the number of positive diagnoses by confirming a positive IgM VCA in the absence of heterophilic antibodies. Furthermore, EBV PCR is positive in only 3% of sera with elevated antibodies against EA, raising doubt as to the utility of EA titers for diagnosing EBV reactivation.     

Advanced therapeutic and prophylactic strategies for Epstein-Barr virus infection in immunocompromised patients

Epstein-Barr virus (EBV) is an ubiquitous human herpesvirus. Primary infection is generally subclinical but in certain circumstances, such as in patients with either hereditary or secondary immunodeficiency, EBV infection may cause overt disease that is often lethal. Strategies for the prophylaxis and treatment of these potentially life-threatening complications of EBV infection have advanced dramatically. They include immunological-based approaches targeted at EBV-infected cells, as well as improvement in the treatment of the underlying and predisposing disease. This review will discuss EBV biology and immune events that occur in both immunocompetent and immunocompromised individuals and introduce the novel prophylactic and therapeutic strategies for EBV-associated life-threatening diseases.     

Predictive value of quantitative PCR-based viral burden analysis for eight human herpesviruses in pediatric solid organ transplant patients

Human herpesviruses can cause significant morbidity and mortality in pediatric solid organ transplant recipients. It was hypothesized that viral burden quantification by polymerase chain reaction using an internal calibration standard could aid in distinguishing between viral disease and latency. Here we report the results of a 2-year prospective study of 27 pediatric solid organ (liver, kidney, or heart) transplant recipients in which multiple samples were analyzed for levels of all eight human herpesviruses by internal calibration standard-polymerase chain reaction. Herpes simplex viruses 1 and 2, varicella-zoster virus, and Kaposi’s sarcoma-associated herpesvirus were not detected in any of these samples. Human herpesvirus types 6 and 7 were detected in half of the patients, but were present at low levels, similar to those found in reference populations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were detected in 89% and 56% of the patients, respectively. Viral burden analysis suggested distinct patient populations for CMV, with a natural cutoff of 10,000 viral targets/ml blood strongly associated with disease. In some cases, a dramatic increase in CMV levels preceded clinical evidence of disease by several weeks. EBV viral burden was relatively high in the only patient presenting with an EBV syndrome. However, two other patients without evidence of EBV disease had single samples with high EBV burden. Rapid reduction in both EBV and CMV burden occurred with antiviral treatment. These data suggest that viral burden analysis using internal calibration standard-polymerase chain reaction for CMV, and possibly other herpesviruses, is an effective method for monitoring pediatric transplant patients for significant herpesvirus infection and response to therapy.     

Identical IgM antibodies recognizing a glycine-alanine epitope are induced during acute infection with Epstein-Barr virus and cytomegalovirus.

We studied antibody production in serial serum samples from patients with acute Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections. Sera were analyzed both by enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide (P62) derived from the glycine-alanine repeating region of the Epstein-Barr nuclear antigen (EBNA-1) and by immunoblotting. In prior studies, we have shown that patients with acute EBV infection make IgM antibodies that react with this peptide, that recognize a viral-specific protein (EBNA-1), and that bind with a number of proteins present in uninfected cells; however, antibody binding to these autoantigens was inhibited by the peptide. IgG antibodies reactive with the peptide did not appear until months after the disease and were specific for the EBNA-1 protein. We now find that patients with acute CMV infection but not those with acute infections from a variety of other nonherpes organisms also produce IgM antibodies that recognize the EBV-derived peptide P62. These antibodies also appear to recognize the same cellular proteins as the EBV-induced IgM antibodies. The IgM antibodies appeared in all acutely infected CMV patients studied and occurred both in patients with previous EBV infections and in one patient studied who had not previously been exposed to EBV. It appears that infection with EBV or CMV can induce the synthesis of a very similar or identical set of IgM antibodies.     

Evaluation of antibodies to the Epstein-Barr virus immediate early gene product ZEBRA by a new enzyme-linked immunosorbent assay.

For the serodiagnosis of Epstein-Barr virus (EBV) infections, we have developed a new enzyme-linked immunosorbent assay (ELISA) for antibodies to the ZEBRA product of EBV immediate early gene BZLF1. ZEBRA protein fused with glutathione-S-transferase (GST) was expressed in Escherichia coli and purified by affinity chromatography with glutathione-Sepharose 4B. An ELISA sandwich capture system was constructed with the GST-ZEBRA immobilized on plastic microtiter plates which had been coated with a mouse monoclonal antibody to GST. ZEBRA-IgG antibodies in patients' sera with chronic active EBV infection (CAEBV) and infectious mononucleosis (IM) had, respectively, very high and high titers. Anti-ZEBRA antibodies were also detected at low titers in sera of some healthy controls. ZEBRA-IgM antibodies were detected in sera of patients with IM and CAEBV but not in sera of healthy controls. In sera of patients with CAEBV, the titers of IgG antibodies to ZEBRA correlated with the antibody titers to early antigens obtained with an immunofluorescence assay, but not to EBV nuclear antigens. This ELISA is a useful diagnostic and prognostic test for EBV infection.     

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.     

Cytomegalovirus infection

Interest has focused on cytomegalovirus (CMV) infections during recent years for two reasons: First, the number of immunocompromised patients with CMV infections has risen continuously and, secondly, recent advances in basic research have clarified some of the mechanisms of persistent and recurrent CMV infection. Three different clinical pictures can arise with CMV infection. In healthy individuals most of the CMV infections are not clinically apparent. In immunocompromised patients CMV causes a wide spectrum of diseases and is one of the predominant causes of death in AIDS patients and bone marrow transplant recipients. The most important problem for public health associated with CMV are connatal and perinatal CMV infections. Progress has been made with treatment of CMV infection in immunocompromised patients with inhibitors of viral replication.     

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.