CMV感染细胞核抗早期抗原抗体(PENA)的测定及临床评价

目的对目前国内各种广泛应用于婴、幼儿的CMV实验室检测方法进行初步评价.方法本研究建立了应用间接荧光免疫法测定CMV细胞核抗早期抗原抗体(PENA)的新方法,同时进行PCR检测和ELISA检测IgM、IgA、IgG实验,对491例临床可疑为CMV感染的患儿进行检测,同时进行PCR方法及ELISA-IgM、IgA、IgG方法的检测,观察结果,并与尿中寻找CMV包涵体方法结果相比较.结果PENA(1/20)阳性率54.99%,PENA(1/100)阳性率21.70%,PCR方法的阳性率55.80%,ELISA-IgM阳性率8.76%,ELISA-IgA阳性率5.50%,ELISA-IgG阳性率68.43%.结论PENA方法可以特异的检测出病人血清中的抗病毒早期抗原抗体的存在,并具有敏感、特异、简便的特点,是一种实验室检测CMV的新方法,可作为临床检测CMV感染的补充方法,尤其适用于婴儿.     

应用荧光免疫法检测PENA-IgG及PCR检测HCMV感染的比较

目的 建立应用间接荧光免疫法测定CMV抗早期核抗原抗体（PENA）的方法。同时进行PCR检测实验，对两种应用于幼儿的CMV实验室检测方法进行初步评价。方法 应用二种实验对552例临床可疑为CMV感染的患儿进行实验观察。结果 PENA（1/20）阳性率49.46%，可疑阳性率6.16%,PENA(1/100)阳性率15.49%，可疑阳性率4.89%;PCR阳性率41.30%。结论 PENA方法可以特异的检测出病人血清中的抗病毒早期抗原抗体的存在，并具有感染，特异，简便的特点，可为初筛CMV感染的检测方法之一。尤其适用于婴儿。     

单克隆抗体与原位杂交检测早期培养细胞中巨细胞病毒的比较(文摘)

巨细胞病毒(CMV)感染人群(如AIDS病人,化疗患者和新生儿等)日益增多,迫切需要一种快速敏感检测诊断CMV的方法。目前,检验诊断CMV的方法有:用免疫荧光法检测CMV早期抗原和分离培养病毒,观察CPE的出现等。本文详细地比较了4种检测早期培养细胞CMV诊断方法。这4种方法是 (1)直接McAb荧光染色法(DFA);(2)间接McAb荧光染色法(IFA);(3)生物素标记DNA探针的原位杂交及 (4)辣根     

联合检测巨细胞病毒感染在原位肝移植术后诊断的应用

目的比较外周血巨细胞病毒（CMV）定量PCR检测,CMV-pp65抗原检测及组织病理学检查对原位肝移植术后CMV感染诊断的价值。方法回顾分析45例肝移植患者临床资料,统计三种检查结果,并结合文献进行讨论。结果三种方法检查均显示,有18例患者发生巨细胞病毒感染,18例均为无临床症状的巨细胞病毒感染。经更昔洛韦治疗,18例全部治愈。结论外周血CMV定量PCR,CMV-pp65抗原检测及组织病理学检查均能作为肝移植术后CMV感染早期诊断的有效手段,CMV定量PCR更敏感,CMV-pp65抗原检测更特异,组织病理学检查则具有创伤性,三者的共同应用能够对CMV感染患者做出早期诊断并且指导治疗。更昔洛韦能够有效治疗CMV感染。     

巨细胞病毒感染对细胞肾素基因表达的影响及其生物学意义

目的 探讨巨细胞病毒(cytomegalovirus,CMV)感染肾球旁细胞肾素基因表达的变化及其意义.方法 用病毒感染复数(MOI)为10、0.1和0的鼠CMV分别与鼠肾球旁细胞模型As4.1细胞共育5 d作为实验组;用紫外线灭活病毒的假感染(mock感染)对照组.RT-PCR检测感染细胞中CMV即刻早期基因1(IE1)的表达;免疫荧光观察肾素阳性细胞和肾素阳性颗粒在细胞的分布;双色免疫荧光染色观察肾素阳性颗粒是否出现在CMV阳性细胞;RT-PCR和Western blot检测肾素基因在感染细胞内的表达.结果 CMV感染As4.1细胞后出现典型的病毒空斑;病毒感染细胞CMV IE1 RT-PCR产物阳性;肾素阳性细胞集中在病毒空斑周围CMV新感染细胞区,肾素阳性荧光颗粒主要以块状和环状存在于病毒感染细胞质中;双色免疫荧光染色显示肾素阳性颗粒和CMV阳性颗粒出现在同一细胞;CMV感染细胞肾素基因的表达随病毒感染量增加而增加.结论 CMV感染并导致其宿主细胞肾素基因表达,可能涉及CMV加速心血管疾病发生发展的新机制.     

巨细胞病毒感染对细胞肾素基因表达的影响及其生物学意义

目的 探讨巨细胞病毒(cytomegalovirus,CMV)感染肾球旁细胞肾素基因表达的变化及其意义.方法 用病毒感染复数(MOI)为10、0.1和0的鼠CMV分别与鼠肾球旁细胞模型As4.1细胞共育5 d作为实验组;用紫外线灭活病毒的假感染(mock感染)对照组.RT-PCR检测感染细胞中CMV即刻早期基因1(IE1)的表达;免疫荧光观察肾素阳性细胞和肾素阳性颗粒在细胞的分布;双色免疫荧光染色观察肾素阳性颗粒是否出现在CMV阳性细胞;RT-PCR和Western blot检测肾素基因在感染细胞内的表达.结果 CMV感染As4.1细胞后出现典型的病毒空斑;病毒感染细胞CMV IE1 RT-PCR产物阳性;肾素阳性细胞集中在病毒空斑周围CMV新感染细胞区,肾素阳性荧光颗粒主要以块状和环状存在于病毒感染细胞质中;双色免疫荧光染色显示肾素阳性颗粒和CMV阳性颗粒出现在同一细胞;CMV感染细胞肾素基因的表达随病毒感染量增加而增加.结论 CMV感染并导致其宿主细胞肾素基因表达,可能涉及CMV加速心血管疾病发生发展的新机制.     

FQ-PCR检测尿巨细胞病毒在婴儿CMV感染中的应用价值

目的 探讨并分析荧光定量聚合酶链反应(FQ-PCR)法检测尿巨细胞病毒(CMV)在婴儿CMV感染的诊断与治疗动态监测中的应用价值.方法 以120例确诊或疑似CMV感染观察组患儿(观察组)和120例健康对照组儿童为研究对象,采用荧光定量聚合酶链反应(FQ-PCR)对尿CMV-DNA进行检测,对血细胞pp65抗原及血清CMV-IgM同时进行检测,并且对检测结果进行比较分析.另外,对30例确诊CMV感染患儿给予更昔洛韦治疗后尿CMV-DNA拷贝进行动监测.结果 FQ-PCR检测的灵敏性和特异性均高于pp65抗原检测,两种方法的灵敏性比较有统计学意义(x2=8.004,P＜0.05),特异性比较有统计学差异(x2=10.047,P＜0.05);FQ-PCR法的灵敏性、特异性均较CMV-IgM法高,两法的灵敏性比较有统计学意义(x2=14.468,P＜0.05),特异性比较有统计学意义(x2=37.604,P＜0.05).30例确诊患儿尿CMV-DNA病毒拷贝数治疗前后有统计学差异(x2=10.55,P＜0.05).结论 FQ-PCR法检测尿CMV在婴儿CMV感染的诊断与治疗动态监测中有良好的应用价值,值得临床推广.     

巨细胞病毒潜伏感染在母婴及乳汁传播中的影响

目的探讨巨细胞病毒(CMV)潜伏感染在母婴及乳汁中的传播。方法选取CMV-IgG阳性潜伏期感染母亲及出生婴儿,将婴儿分为新生儿组和1～6月龄组,分别对母亲乳汁、婴儿全血及尿进行CMV病毒载量PCR检测。结果 71对新生儿组中母亲乳汁阳性51人占71.7%,婴儿全血及尿荧光定量PCR检测阳性8人占11.3%;87对1～6月龄组中母亲乳汁阳性71人占81.6%,婴儿全血及尿荧光定量PCR检测阳性63人占72.4%。婴儿检测PCR结果与母亲乳汁PCR两组数据相符情况,新生儿组为0.366,1～6月龄组为0.793,表明1～6月龄组婴儿CMV感染与母亲乳汁的关系较新生儿组大。结论 CMV潜伏期感染母亲垂直传播风险低,乳汁是婴儿CMV感染的高风险因素。     

单克隆抗体与原位杂交检测早期培养细胞中巨细胞病毒的比较（文摘）

巨细胞病毒(CMV)感染人群(如AIDS病人，化疗患者和新生儿等)日益增多．追切需要一种快速敏感检测诊断CMV的方法。目前，检验诊断CMV的方法有：用免疫荧光法检测CMV早期抗原和分离培养病毒，观察CPE的出现等。本文详细地比较了4种检测早期培养细胞CMV诊断方法。这4种方法是(1)直接McAb荧光染色法(DFA)；(2)间接McAb荧光染色法(IFA)；(3)生物素标记DNA探针的原位杂交及(4)辣根过氧化物酶直接标记DNA(HRP—DNA)探针的原位杂交。     

癌-睾丸抗原基因在神经胶质瘤干细胞中的表达

背景：癌-睾丸抗原基因在胶质瘤干细胞表达还不甚清楚。 目的：检测癌-睾丸抗原基因在神经胶质瘤干细胞中的表达。 方法：通过神经球培养的方法而分离干细胞。以半定量PCR及实时荧光定量PCR检测癌-睾丸抗原基因在母细胞、干细胞与分化细胞中的表达。 结果与结论：以神经球培养方式从胶质瘤细胞系中可分离出肿瘤球,将肿瘤球培养在含血清的培养基中,它的形态呈贴壁分化,且与母细胞没有区别。半定量PCR及实时荧光定量PCR检测发现与母细胞和分化细胞相比,癌-睾丸抗原基因在干细胞中的表达最高。提示癌-睾丸抗原基因可能为肿瘤干细胞的表面抗原。     

Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treatment in renal transplant patients

Cytomegalovirus (CMV) infection is a significant problem in transplantation. In this study, a quantitative PCR test was compared with the CMVpp65 antigenemia assay not only in the diagnosis CMV infections but especially in the monitoring of viral loads during ganciclovir treatment of CMV disease in individual renal transplant patients. Altogether 342 blood specimens were obtained from 116 patients. Blood specimens were used for Cobas Amplicor Monitor plasma PCR and for the pp65 assay. Also shell vial culture was performed. The patients with a positive pp65 finding were monitored for CMV weekly during ganciclovir treatment and/or until the antigenemia subsided. CMV was detected in 31/116 (27%) patients, of whom 14 (12%) developed CMV disease and were treated with ganciclovir. CMV was found by shell vial culture in 13/14 cases, but by PCR and pp65 test in all 14 patients. CMV was detected in 156 (45%) samples; by PCR in 121/156 (range 344-103,000 copies/ml) and by pp65 test in 138/156 (range 1-1,000 positive cells/50,000 leukocytes) and by culture in 59/156 (38%) only. The peak viral loads were significantly (P<0.0001) higher in CMV disease than in untreated infections (19,650 vs. 379 copies/ml, and 100 vs. 5pp65 positive cells). In the monitoring of individual patients, the time-related CMV-DNAemia and pp65 antigenemia correlated well during the treatment of CMV disease. In conclusion, Cobas Amplicor Monitor plasma PCR and CMVpp65 antigen assays can be equally used in the diagnosis CMV infection and in the monitoring of viral load during antiviral treatment.     

Detection of cytomegalovirus (CMV) in granulocytes by polymerase chain reaction compared with the CMV antigen test.

To compare the sensitivity and suitability of detection of active cytomegalovirus (CMV) infection by using monoclonal antibodies against CMV antigen (antigen test to detect antigenemia) and the polymerase chain reaction (PCR; to detect viral DNA) in granulocytes, 19 heart and 2 lung transplant recipients were closely monitored by these tests for at least 3 months after transplantation. All patients were CMV seropositive or had a seropositive donor. In total, 201 samples were tested; 46 were positive by both tests, 9 samples showed only antigenemia, 54 samples were positive by PCR only, and 102 samples were negative by both tests. PCR was positive earlier after transplantation in eight patients, whereas antigenemia was positive earlier after transplantation in one patient. In another four patients, both tests were positive at the same time. PCR was, on average, positive for a longer period of time. Discordant results showing a positive antigen test and a negative PCR were partly due to sampling error; some were positive by PCR on retesting. Samples which were negative by the antigen test and positive by PCR were taken at the beginning or at the end of an active CMV infection. In two patients, no active CMV infection was detected by the antigen test, cultures of urine and saliva, or serology, although PCR was positive for a long period of time in the two patients.     

A new microsphere-based immunofluorescence assay using flow cytometry

The quantitative and qualitative capacities of flow cytometric analysis that have made it such a powerful tool in studies of cellular antigens have not previously been exploited when dealing with non-cellular antigens. A new immunofluorescence assay technique was developed, using an indirect staining procedure with monoclonal anti-kappa antibodies, to detect human free kappa light chains covalently bound to microspheres of a size suitable for flow cytometry. The strength of the fluorescent signal produced on the microspheres was related to the amount of antigen bound and the size of the beads. At the time of this work large microspheres (i.e., greater than 3 micron in diameter) suitable for this application were only available as suspensions of polysized beads. The fluorescent signal detected on labelled beads was optimized by selecting for analysis, on the basis of the forward angle laser scatter, only those beads of largest diameter. There are many potential applications for this technique - microspheres can be used for the presentation of virtually any antigen or antibody. The analytical benefits inherent in flow cytometry would be a significant advantage in the development of quantitative assays using this method.     

Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow cytometry.

A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to HIV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.     

巨细胞病毒抑制树突状细胞抗原递呈功能

目的： 探讨巨细胞病毒（CMV）对单核细胞来源的树突状细胞（DC）的感染效率及对受染细胞的功能影响。方法: 以50半数细胞培养感染量（TCID50）滴度的CMV与未成熟及成熟DC（imDC，mDC）共培养，逆转录－聚合酶链反应（RT-PCR）方法检测细胞内CMV即刻早期抗原（IEA）mRNA水平，间接免疫荧光技术检测受染细胞内早期抗原（EA）阳性率，流式细胞仪检测细胞胞内病毒晚期抗原pp65表达，BrdU ELISA法检测受染DCs（cmv-imDC,cmv-mDC）刺激异基因T细胞增殖能力。结果: 感染 12 h，cmv-mDC内IEA mRNA水平低于cmv-imDC，相对表达量分别为0.102±0.020和0.862±0.124（P<0.05）。24 h，imDC组EA阳性率高于mDC组，分别为(62.32±14.20)%和(10.78±3.04)%（P<0.01）。72 h，cmv-DC胞内低表达pp65抗原，imDC和mDC中阳性率分别为4.86%和0.82%。与未处理mDC相比，cmv-imDC经成熟诱导因子LPS作用后，其刺激异基因T细胞增殖能力较弱（均P<0.05）；而cmv-mDC，仅当DC/T细胞为 1∶1 时，刺激能力下降（P<0.05）。结论: CMV可有效感染imDC，并在细胞内复制活化；cmv-DC的抗原递呈能力下降。     

Improved monitoring of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation by an ultrasensitive plasma DNA PCR assay

Cytomegalovirus (CMV) DNA amplification assays in plasma have shown limited sensitivity compared to the detection of pp65 antigen in leukocytes. Our goal was to increase the sensitivity of a commercial CMV DNA PCR quantitative assay. After modification, the new assay was able to reproducibly detect 20 CMV DNA copies/ml of plasma. We compared this new ultrasensitive PCR assay with the standard PCR and the pp65 test for CMV detection and quantification in 22 consecutive allogeneic hematopoietic stem cell recipients. CMV infection or reactivation was detected in 84 of 319 (26%) samples by the ultrasensitive PCR assay compared to 38 of 319 (12%) samples by the pp65 assay (P < 0.01). All samples positive by the pp65 assay were positive by the ultrasensitive PCR, and CMV episodes were detected on average 4 days earlier and 7 days later than the first and the last pp65-positive test, respectively. In addition, during CMV episodes, the ultrasensitive assay identified positive samples that were inconsistently detected by the pp65 assay. The ultrasensitive assay was also much more sensitive than the standard PCR, with 26 versus 12% of CMV DNA-positive samples (P < 0.01). This assay improved the monitoring of CMV infection or reactivation in hematopoietic allogeneic stem cell recipients.     

Monitoring low cytomegalovirus viremia in transplanted patients by a real-time PCR on plasma

Until recently, human cytomegalovirus (hCMV) infection and anti-CMV treatment in transplanted patients have been monitored essentially by pp65 antigenemia, which is time-consuming and requires experienced operators. For the last two years, pp65 antigenemia levels have tended to be lower than previously in our laboratory, which could be due to better monitoring of CMV-related risk. Results obtained by real-time PCR with a LightCycler instrument or by pp65 antigen assay were compared on 145 serial samples from bone marrow or kidney transplant recipients under the usual conditions of our laboratory. CMV DNA was extracted from plasma and quantified by using primers and probes directed to HXFL4 gene. The plasma CMV DNA load was measured by using a standard curve constructed with a commercially available quantified CMV DNA suspension. Among the 145 samples, 139 showed a pp65 antigen which was negative or lower than 20 positively stained cells per 200,000 leukocytes. In the patients with positive pp65 antigenemia, the corresponding values of CMV DNA copy number/ml were significantly higher than those observed in patients without antigenemia (P < 0.001). CMV DNA was detected from 4 up to 52 days before pp65 antigen. Elsewhere, between two dates at which pp65 antigen was positive, intermediate PCR results could be positive while the pp65 antigen was negative. This real-time quantitative PCR assay is a rapid technique adapted to monitor plasma CMV DNA in transplant setting, even for low viremia.     

Qualitative and semiquantitative polymerase chain reaction testing for cytomegalovirus DNA in serum allows prediction of CMV related disease in liver transplant recipients

AIM: To identify cytomegalovirus (CMV) infection in liver transplant recipients by polymerase chain reaction (PCR) techniques and to separate the cases in which CMV related disease will occur, for whom treatment is indicated, from those in whom infection will remain innocuous. METHODS: The combination of qualitative and semiquantitative PCR of serum and urine was assessed to determine whether these assays can identify those at risk of CMV related disease and compared their performance with conventional approaches to diagnosis. RESULTS: Qualitative PCR of serum had superior specificity, sensitivity, and positive and negative predictive values compared with urine DEAFF (detection of early antigen fluorescent foci) and PCR of urine. All episodes of CMV related disease were associated with the presence of CMV DNA by PCR in serum or urine; CMV was detected before clinical onset in 70% and 60% of cases, respectively. The period over which CMV DNA could be detected was not correlated with CMV related disease. Both peak viral load and cumulative viral load estimated using a semiquantitative PCR method on serum samples positive by the qualitative method could be used to distinguish asymptomatic infection from CMV related disease with 100% specificity and sensitivity. In contrast semiquantitative PCR of urine was of little value. CONCLUSIONS: An approach based on PCR testing with a combination of qualitative and subsequently semiquantitative serum samples would improve the diagnosis of CMV infection and aid identification of those patients at risk of CMV related disease, allowing treatment to be targeted specifically.     

Kinase-deficient CMVpp65 triggers a CMVpp65 specific T-cell immune response in HLA-A*0201.Kb transgenic mice after DNA immunization

CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) activity that could affect vaccine safety. A mutated form of CMVpp65 substituting asparagine for lysine at the adenosine triphosphate (ATP)-binding site (CMVpp65mII) is kinase-deficient. Using DNA immunizations in a transgenic human leucocyte antigen (HLA)A*0201.Kb mouse model, the mutated CMVpp65 induced cytotoxic T lymphocytes (CTL) immunity similarly to native CMVpp65. Murine CTL lines generated from these immunizations killed human cells either after sensitization with CMVpp65-specific peptides or after infection with either CMV-Towne strain or rvac-pp65. It is proposed that CMVpp65mII be evaluated in candidate vaccines for CMV.     

Flow cytometric indirect immunofluorescence assay with high sensitivity and specificity for detection of antibodies to human immunodeficiency virus (HIV)

A flow cytometric immunofluorescence assay (FIFA) was developed to detect antibodies to human immunodeficiency virus (HIV) using live cell indirect immunofluorescence and analysis by flow cytometry. A panel of 107 sera, previously tested for anti-HIV antibody with the Abbott Enzyme-Linked Immunosorbent Assay test and Western blot (WB), was rescreened by FIFA. Antibodies were tested on HIV-infected and uninfected H9 cells in the FIFA. Although ELISA results indicated seven false positive results by comparison with the WB, 46 of 46 FIFA positive results tested WB positive and 61 of 61 FIFA negative results were WB negative. The results of FIFA showed 100% sensitivity and 100% specificity compared with WB. This rapid, quantitative, relatively easy assay makes FIFA an alternate confirmatory test for the presence of HIV antibodies.