大连地区孕妇及新生儿巨细胞病毒感染的调查

本文采用间接酶联免疫吸附法(ELISA)检测1119例孕妇血清1人巨细胞病毒(HCMV)特异性抗体IgM，并用聚合酶涟反应(PCR)检测41例新生儿尿液中HCMV—DNA。结果：孕妇HCMV活动感染率10．10％(113／1119)，晚期孕妇感染率略高于早中期(7．43％和10．50％)，其感染与孕妇的文化程度和职业有关。母血HCMV—IgM阳性的21例新生儿尿液HCMVDNA阳性6例，(28．57％)，41例新生儿尿液检测结果可见表．母血HCMV—IgM阳性与新生儿尿液HCMVDNA阳性密切相关。     

识别HCMV早期抗原单克隆抗体的鉴定及应用

目的：建立人巨细胞病毒（HCMV）感染的实验室早期快速诊断方法。方法：从6株HCMV单克隆抗体（mAb）中，选择1株识别HCMV早期抗原（EA）的mAb进行了进一步鉴定，并用于临床标本中EA的快速诊断。用间接免疫荧光试验筛选出识别EA的mAb888，并用Western blot检测EA的相对分子质量（Mr），mAb888在免疫组化染色（IHC）方法中用于检测临床标本中的EA。结果：HCMV感染细胞4h时，用mAbSB8染色，见到核内荧光。该EA的Mr为72000。在73份HCMV阳性标本中测出EA59份（81％），而用常规细胞培养方法观测到细胞病变效应（CPE）的标本58份（79％），2种方法测定均阳性的44份（60％），仅EA测定阳性的15份（21％），仅检测到CPE的14份（19％），考虑到所有种类的样本，2种方法在检测HCMV方面无统计学意义。结论：mAbSB8可用于HCMV感染的实验室早期快速诊断。     

6种虫媒病毒蛋白芯片检测方法的建立

目的 建立针对6种虫媒病毒的蛋白芯片检测方法,用以检测流行性乙型脑炎病毒、蜱传脑炎病毒、登革病毒(1～4型)、西尼罗病毒、西部马脑炎病毒和东部马脑炎病毒的特异性抗体.方法 将病毒特异性抗原作为捕获抗原点样制备蛋白芯片,利用双抗夹心ELISA原理检测血清中的病毒特异性抗体.首先利用免疫兔血清进行特异性诊断抗原的筛选,并对抗体芯片检测条件进行优化,然后采用56份临床疑似的阳性血清标本及阴性对照标本对该方法进行验证,并与常规ELISA方法进行比对.结果 共筛选出11个特异性较好的重组诊断抗原.抗原点样浓度在0.125 ～0.900mg/ml时可获得良好的检测效果,血清检测范围为1:100～1:1000.对26份临床疑似的蜱传脑炎病毒血清标本,22份登革病毒血清标本及8份流行性乙型脑炎病毒临床血清标本的检测结果为:共检测出蜱传脑炎病毒IgG阳性血清标本20份,阳性检出率为76.9％,IgM阳性血清标本17份,阳性检出率65.3％,与ELISA检测符合率分别为96.1％和84.6％.乙型脑炎病毒IgG阳性血清4份,阳性检出率50.0％,IgM阳性血清5份,阳性检测率62.0％,与ELISA检测符合率分别为87.5％和100％.登革病毒IgG阳性血清标本13份,阳性检出率63.6％,IgM阳性血清标本14份,阳性检测率68.1％,与ELISA检测符合率分别为86.3％和90.1％,结果经一致性Kappa检验后,与ELISA检测结果一致性良好.阴性对照血清结果显示检测特异性为100％.结论 本研究建立的虫媒病毒抗体芯片检测方法具有较高的特异性和可靠性,可用于6种虫媒病毒抗体的临床检测.     

建立固相Capture-ELISA技术诊断糖尿病人HCMV活动性感染

目的建立检测HCMV抗原特异性IgM的抗体捕捉酶联免疫吸附试验（IgM antibody capture enzyme-linked immunosorbent assay,Capture-ELISA）,并分析糖尿病患者HCMV活动性感染状态。方法利用抗人IgM（μ链特异性）抗体包被固相载体,作为＂捕捉抗体＂吸附待检血清中的IgM,经过洗涤除去血清中IgG及其它成分,再加入特异性抗原与＂捕捉＂到的相应IgM抗体结合,然后加入酶标抗体和底物显色进行测定。同时,应用建立的酶联免疫捕获法诊断糖尿病患者HCMV近期感染状态,并与常用的间接酶联免疫吸附试验（In-ELISA）及RT-PCR进行比较。结果 Capture-ELISA的特异性及敏感性均高于间接法,且不受RF因子的影响。结论 Capture-ELISA操作简单、快速,且具有较高的特异性和灵敏度,是检测IgM抗体理想的方法之一。     

早期妊娠胚胎先天性人巨细胞病毒感染检测及危险因素分析

本文利用免疫组织化学方法(PAP)和ELISA法对400名早期妊娠胚胎绒毛组织进行人巨细胞病毒(HCMV)先天性感染和孕妇活动性HCMV抗体检测。结果表明早孕绒毛中有67例感染，阳性率为16．8％；孕妇血中HCMV—IgM( )22例，HCMV—IgA( )27例，孕妇活动性HCMV感染率为10、0%。并对早期妊娠胚胎感染HCMV危险因素进行分析，孕妇活动性HCMV感染传播给胚胎的危险度为32.5％．而且胚胎先天性感染与妊娠妇女的妊娠天数和性生活频度有着密切的相关关系。证明孕早期胚胎HCMV感染既可由母亲活动性HCMV感染垂直传播，也可来源父亲的传染途径。     

HLA高分辨等位基因与骨髓移植受者HCMV抗原血症研究

目的 分析HLA高分辨等位基因与骨髓移植术后HCMVpp65抗原血症的相关性.方法 选取2009年2月至2010年10月在我院行骨髓移植术患者48例;采用免疫组化方法检测患者HCMVpp65,采用直接测序分型方法（PCR-SBT）检测患者HLA-A＊1101、HLA-A＊0201、HLA-A＊2402、HLA-B＊4001、HLA-DRB1＊0901五个高分辨等位基因.结果 ①48例骨髓移植术后患者HCMV感染率100％;②HLA-A＊1101、HLA-A＊0201、HLA-A＊2402、HLA-B＊ 4001等位基因阳性率在pp65抗原血症12例低感染组和36例高感染组中比较没有统计学意义（P＞0.05）,其阳性率HLA-A＊1101为33.3％ （8/24）和20.8％ （15/72）、HLA-A＊0201为4.2％ （1/24）和13.9％ （10/72）、HLA-A＊ 2402为12.5％ （3/24）和19.4％ （14/72）、HLA-B＊ 4001 16.7％ （4/24）和12.5％ （9/72）;③HLA-DRB1＊0901等位基因阳性率在pp65抗原血症12例低感染组和36例高感染组中比较有统计学意义（P =0.048）,其阳性率为4.2％ （1/24）和19.4％ （14/72）;④HLA-DRB1＊0901组患者pp65抗原血症高于HLA-A＊2402组（P =0.007）和HLA-A＊1101组患者（P=0.028）,HLA-A＊0201组患者pp65抗原血症高于HLA-A＊2402组患者（P=0.02）,其他高分辨等位基因组之间pp65抗原血症差异没有统计学意义（P＞0.05）.结论 HLA-DRB1＊0901等位基因可能与骨髓移植术后患者发生高HCMVpp65抗原血症有关;HLA-A＊2402等位基因可能与骨髓移植术后患者发生低HCMVpp65抗原血症有关.     

温州市区育龄妇女孕前巨细胞病毒感染现状调查

目的了解温州地区育龄妇女孕前人巨细胞病毒（HCMV）感染的状况。方法收集2008年10月至2010年6日参加温州市龙湾区免费孕前优生筛查的妇女血标本2869份,采用酶联免疫吸附试验（ELISA）检测血清HCMV IgG/IgM抗体;HCMV IgM抗体阳性标本,采用实时荧光定量聚合酶链反应（FQ-PCR）检测血HCMV DNA载量;HCMV IgG/IgM抗体双阳性标本,采用尿素变性结合ELISA技术检测IgG抗体亲和力指数（AI）。结果 2869份孕前妇女血清中HC-MV IgG抗体阳性检出率为97.77%（2805/2869）,HCMV IgM抗体阳性检出率为0.77%（22/2 869）,IgG/IgM抗体均阳性检出率占0.17%（5/2 869）;22份HCMV IgM阳性标本中,血HCMV DNA阳性检出率为68.18%（15/22）;5份HCMVIgG/IgM双阳性标本中,检出低亲和力IgG抗体1份,中等亲和力IgG抗体2份,高亲和力IgG抗体2份。结论温州市区育龄妇女孕前HCMV IgG抗体阳性率高;对HCMV IgM抗体阳性孕前妇女应进行多指标检测以判断HCMV感染的状态,为减少出生缺陷、做好优生优育服务提供依据。     

HCMV pp65和gB联合核酸疫苗表达载体的构建及其体内生物学活性研究

人巨细胞病毒(HCMV)主要被膜磷蛋白pp65(phosphoprotein 65)及糖蛋白gB(glycoprotein B)在病毒感染及诱发机体免疫反应中起重要作用，近年来有不少学者对HCMV pp65和gB这两种抗原进行动物和临床试验研究，虽取得了一定效果，但仍存在免疫原性弱、免疫范围局限等缺点。本研究利用分子生物学方法将HCMV两种保护性免疫原性基因pp65和gB构建在一起，制备HCMV核酸疫苗，并研究其在小鼠体内的免疫活性。     

活动性巨细胞病毒感染与反复自然流产的关系探讨

目的探讨活动性人巨细胞病毒（human cytomegalovirus,HCMV）感染与反复自然流产（recurrent spontaneous abortion,RSA）的关系.方法采集反复自然流产孕妇和正常产前体检孕妇外周血,分离外周血单个核细胞（PBMCs）和血浆,分别用免疫荧光法和实时定量PCR检测HCMV pp65抗原和HCMV DNA,并比较2种方法的一致性.结果 65例RSA患者HCMV pp65抗原有20例阳性,阳性率30.8%,50例正常体检孕妇 HCMV pp65抗原有4例阳性,阳性率8.0%,2组孕妇HCMV活动性感染率有显著性差异（χ^2=8.87,P＜0.01）.孕妇HCMV pp65抗原阳性率升高,孕妇流产几率增加（χ^2=7.53,P＜0.01）. 免疫荧光法和实时定量PCR有较好的一致性（92.3%）.结论反复自然流产孕妇 HCMV活动性感染率显著高于正常孕妇,HCMV pp65抗原检测也许可作为RSA早期诊断指标之一.     

婴幼儿巨细胞病毒抗原血症检测法的研究

Van der bij(1988 J Med viol 25:79)报导的HCMV抗原血症试验是一项直接诊断活动性HCMV感染的新方法,其敏感性与特异性均在90％以上。 作者用改良的抗原血症试验对临床上诊断为HCMV肝炎患儿进行了检测,并同时与血清学检测、尿病毒分离和原位杂交作比较。取69例患儿微量静脉血(40μl),其中30例患儿同时采取微量手指血(40～60μl)收集白细胞制片,均用HCMV早期抗原单克隆抗体以间接酶免疫组化法检测抗原血症来诊断活动性HCMV感染。结果显示血清IgM、尿病毒分离和抗原血症阳性率分别为81.2％、40.7％和66.7％。三者结果经比较分析表明:抗原血症在诊断活动性HCMV     

Detection of human cytomegalovirus (HCMV) pp67-mRNA and pp65 antigenemia in relation to development of clinical HCMV disease in renal transplant recipients

Objective   To evaluate the performance of the recently introduced method based on detection of human cytomegalovirus (HCMV) pp67 mRNA in blood by the nucleic acid sequence-based amplification (NucliSens), in comparison to semiquantitative detection of pp65 HCMV antigen in white blood cells, in relation to development of clinical HCMV disease.
Methods   Thirty patients, recipients of renal transplants, were monitored prospectively for the presence of pp67 mRNA, the presence and level of pp65 antigenemia, IgG and IgM antibodies, and the development of clinical HCMV disease. A total of 148 samples were examined during the observation period.
Results   Twenty-five samples were positive for pp67-mRNA and 45 samples contained at least one pp65 positive cell, with 68% agreement between the two assays. Both assays predicted correctly the development of clinical disease in five patients, giving a sensitivity of 100%. However, the specificity of the pp67-mRNA test was 72%, and of the pp65 antigenemia test from 20 to 64%, depending on the level of antigenemia chosen for cut-off. pp67-RNA appeared somewhat earlier than pp65 antigenemia, and responded earlier to treatment. Sero-conversion and appearance of IgM antibodies were of very little clinical value.
Conclusion   Both the pp67-mRNA and the pp65 antigenemia assay predicted correctly the development of clinical HCMV disease in renal transplant recipients. However, the specificity of both tests with respect to development of HCMV disease, especially the pp65 antigen test was moderate. Significantly positive tests not necessarily prove the development of clinical disease. Testing for pp67-mRNA may improve the diagnosis and management of HCMV disease in renal transplant patients.     

Human cytomegalovirus infection: Recent developments in diagnosis and epidemiology

Summary Cytomegalic inclusion disease (CID) is caused by a horizontally or vertically transmitted human herpes virus infection and may persist for life without obvious clinical symptoms. A serious course of horizontal primary and recurrent infections, however, is often observed in immunocompromised persons such as recipients of organ transplants and patients receiving fresh blood transfusions. Vertical infection may cause fetopathies. The human cytomegalovirus (HCMV) is thought to inherit an oncogenic potential as lately discussed for AIDS and M. Kaposi. Laboratory diagnosis of HCMV infection is performed by light microscopy (inclusion bodies), electron microscopy, virus isolation in cell culture, demonstration of viral DNA and antigen in clinical specimens, by histochemical methods (e.g. immunoperoxidase technique) and by DNA and peptide analysis for identification of different isolates and viral finger prints. Evaluation of cell-mediated immunity in HCMV infection is performed quantitatively (assessment of T_helper/T_suppressor ratios) or qualitatively (specific lymphocyte stimulation by the antigen). In most cases laboratory diagnosis is achieved by serological methods, i.e. demonstration and quantitation of HCMV-specific antibodies. In this context, a number of liquid- and solid-phase immunoassays have been developed, of which immunofluorescence and ELISA are most commonly used, besides complement fixation and passive haemaglutination. These procedures on the one hand allow the use of different antigen preparations as early and late viral proteins, and on the other hand permit a specific determination of different Ig classes and subclasses. A variety of assays has been established especially for determination of virus-specific IgM antibodies, which are predominantly found in active infection. These, however, at least in part may show non-specific results caused by interference of rheumatoid factor or IgG competition. Such problems have now been dealt with and are avoided by IgG precipitation or IgM immunosorption (-capture technique). These recent methods allow an exact epidemiological identification of risk groups for CMV infection. Results from our laboratory revealed 13% HCMV-IgM positive patients among pregnant women, 16% IgM positive patients among renal transplant recipients, 4% igM positive cases in patients after cardiosurgery and 1.7% IgM positives among prostitutes. The prevalence of HCMV infection as indicated by specific IgG antibodies was 56%, 90%, 83%, and 90%, respectively. No IgM antibodies were found in haemophiliacs and healthy blood donours, which showed a prevalence of HCMV infection in 69% and 47% of tested serum samples.Abbreviations ACIFT anti-complement immunofluorescence test - AIDS acquired immunodeficiency syndrome - CFT complement-fixation test - CID cytomegalic inclusion disease - A₄₉₂ difference of absorption values (=492 nm) (virus — neg. control antigen) - DNA desoxyribonucleic acid - EBNA EBV-nuclear antigen - EBV Epstein-Barr virus - ELISA enzyme-linked immunosorbent assay - EM electron microscopy - EV enveloped virus - HBc, HBe, HBs ag hepatitis B virus c/e/s antigen - HBV hepatitis B virus - HCMV Human cytomegalovirus - HSV herpes simplex virus - (I)EA (immediate) early antigen - IFT immunofluorescence test - IgA, IgG, IgM immunoglobulin A, G, M - LA late antigen - NC nucleocapsid - n.d. not done - neg. negative - NT neutralization test - PHA passive haemagglutination - RIA radioimmunoassay - STR significant titer rise - VZV varicella-zoster virus Dedicated to Prof. Dres. mult. Wilhelm Doerr on his 70th birthday     

Diagnostic value of HCMV pp65 antigen detection by FCA for symptomatic and asymptomatic infection: compared to quantification of HCMV DNA and detection of IgM antibody in infants

Human cytomegalovirus (HCMV) can cause symptomatic or asymptomatic infection in infants. One hundred and twenty-six infants were assessed clinically for disease in infantile period. Eighty of them were classified as symptomatic infection on the basis of physical, instrumental, and laboratory findings, 5 were demonstrated by following up to have later developed HCMV disease, and the other 41 infants were classified as asymptomatic infection. HCMV DNA was positive in all urine samples of the symptomatic infants detected by quantitative polymerase chain reaction. HCMV-IgM antibody detected by chemiluminescent immunoassay (CLIA) was positive in 62 of the 85 symptomatic infants, but was negative in all of the samples of asymptomatic infants. HCMV pp65 antigen detected by flow cytometry assay (FCA) was positive in 77 of the 85 symptomatic infants and in none of the asymptomatic infants. The coincidence to symptom of HCMV pp65 antigen detection was higher than those of HCMV DNA and HCMV-IgM antibody detection. The sensitivity, specificity, positive prognostic value and the negative prognostic value of HCMV pp65 antigen detection for diagnosis of HCMV infection was 90.6, 100, 100 and 83.7%, respectively. We concluded that detection of pp65 antigen by FCA is more sensitive for diagnosis of HCMV infection than detection of HCMV-IgM antibody and is better than HCMV DNA quantification for distinguishing the symptomatic and asymptomatic HCMV infection in infants.     

Direct quantification of human cytomegalovirus immediate-early and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10(4) copies per 100 microl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.     

Quantitation of human cytomegalovirus in recipients of solid organ transplants by real-time quantitative PCR and pp65 antigenemia

Human cytomegalovirus (HCMV) infections and anti-HCMV treatment are usually monitored by measuring pp65 antigenemia. This method is time-consuming, labour-intensive and requires skilled operators. We have compared results obtained using real-time Light Cycler quantitative PCR (QPCR) and the pp65 antigen assay on serial samples collected from recipients of solid organ transplants. We collected 198 blood samples from 14 solid organ transplant recipients and assayed them for pp65 antigen and with Light Cycler PCR. HCMV DNA was extracted from leukocytes and measured using primers and probe located in the UL83 region. The quantity of HCMV DNA was calculated using a standard curve prepared from a plasmid containing the target sequence. There was a good correlation between the number of pp65-positive cells and the DNA copy number (r = 0.57, P < 0.0001). A clinical threshold of 50 positive polymorphonuclear leukocytes/200,000 cells was equivalent to two log10 genome copies per capillary by Light Cycler PCR. HCMV DNA was detected before pp65 antigen in three patients at a mean time of 10 days, whereas the two tests were positive simultaneously for eight patients. Both the pp65 antigen data and DNA copy number decreased over time during antiviral treatment, although the QPCR was positive 28.2 days after the pp65 antigen assay had become negative. The real-time Light Cycler quantitative PCR assay is a rapid and labour-saving technique. This molecular method could be useful for monitoring infections and antiviral treatment in recipients of solid organ transplants.     

核转录因子κB在中药金叶败毒制剂抗人巨细胞病毒感染中的作用

目的研究核转录因子NF-κB在金叶败毒制剂（简称中药）抗人巨细胞病毒（HCMV）感染中的作用。方法分别应用原位杂交和半定量RT-PCR方法检测中药和更昔洛韦（GCV）干预HCMV感染的细胞NF-κBp65mRNA及HCMV晚期mRNA的表达水平,观察两种药物对HCMV感染致人胚胎成纤维细胞（HEL）病变（CPE）的影响。结果两种药物对HCMV晚期mRNA的表达和HCMV致细胞病变CPE均有抑制作用,但中药能显著抑制NF-κBp65mRNA的水平,而GCV对此影响不明显。结论中药可能通过抑制核转录因子NF-κB而拮抗HCMV感染,NF-κB信号通路激活可能为HCMV致病机制之一。     

Monitoring for cytomegalovirus infection in organ transplant recipients: Analysis of pp65 antigen,DNA and late mRNA in peripheral blood leukocytes

The use of sensitive and specific methods for rapid and reliable diagnosis is required due to the considerable impact of human cytomegalovirus (HCMV) in organ transplant recipients. For this purpose the demonstration of the presence of viral antigens in peripheral blood leukocytes (PMNLs) and of viral nucleic acids in the same cells or in sera would seem to be of valid support. The present study was designed to test pp65 antigen, HCMV DNA and HCMV late mRNA in order to provide clinical information for the management of the infection. Fifty solid organ recipients were monitored for six months after transplant. The data obtained from the various tests were analysed from the first evidence of HCMV infection revealed by positive antigenaemia and/or DNA-polymerase chain reaction (PCR). In 3 asymptomatic and in 7 symptomatic patients, PCR became positive 1–2 weeks before antigenaemia but PCR did not discriminate the clinical evolution of HCMV infection. The antigenaemia test well correlated to the development of viral infection being positive in all symptomatics and in 31, 2% of asymptomatics. The antigenic load >100/2 × 10⁵ positive cells was always associated with clinical signs of illness. The detection of late mRNA was more indicative of the virus replicative status in the follow-up of patients treated with ganciclovir. In some cases there was evidence, prior to the other two tests, the block of viral replication due to the antiviral therapy and in others the onset of HCMV infection relapse. J. Med. Virol. 53:189–195, 1997. © 1997 Wiley-Liss, Inc.