Longitudinal analysis of human cytomegalovirus glycoprotein B (gB)-specific and neutralizing antibodies in AIDS patients either with or without cytomegalovirus end-organ disease

Serum neutralizing and glycoprotein B (gB)-specific antibody levels were monitored prospectively in AIDS patients who either did or did not develop human cytomegalovirus (HCMV) end-organ disease, to delineate further the role of antibodies in protecting against HCMV disease. Antibody levels declined substantially (at least 4-fold) only in patients who developed HCMV disease; this decline in turn occurred concurrently with antigenemia. Nevertheless, AIDS patients who remained free of HCMV disease and did not become antigenemic during the follow-up period maintained stable levels of serum antibodies, with only minor fluctuations. The impact of HAART on the levels of functional anti-HCMV antibodies was investigated in a number of AIDS patients. Serum levels and kinetics of gB and neutralizing antibodies did not differ significantly between patients who responded biologically and virologically to therapy and those who failed to respond. In addition, CD4 + cell counts and HIV viral RNA levels did not correlate with anti-HCMV antibody titers.     

实时定量PCR检测技术应用于孕妇人巨细胞病毒感染

目的：用荧光定量聚合酶链反应（FQ-PCR）准确地定量检测孕妇血清中人巨细胞病毒（HCMV）的数量及其在与临床上的意义。方法：用FQ-PCR和ELISA方法同时检测300例孕妇及59例新生儿血清标本。结果：胎儿宫内感染的发生与孕妇血清中HCMVDNA数量有关,发生宫内胎儿感染的孕妇,其HCMVDNA拷贝数达10^6copies/ml以上,结论：孕妇血清中HCMVDNA含量升高是胎儿发生HCMV感染的重要因素之一,FQ-PCR可以准确定量检测HCMV的真实感染和制情况。对于临床诊断与治疗有一定的指导意义。     

Human cytomegalovirus glycoprotein B genotypes in blood of AIDS patients: Lack of association with either the viral DNA load in leukocytes or presence of retinitis

It has been suggested that human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes could be used as a marker for viral virulence in patients with AIDS. The present study was designed to evaluate a possible association between specific gB genotypes, the presence of HCMV retinitis, and the HCMV viral load. Fifty-four blood samples were obtained from 54 HIV- and HCMV-infected patients. Twenty-seven of these patients were asymptomatic for HCMV, whereas the other 27 patients had been diagnosed recently with HCMV retinitis. HCMV gB genotyping was carried out by using restriction enzyme analysis of PCR-amplified PMNL extracts. Determination of the HCMV viral load in the same specimens was carried out using a quantitative-PCR. HCMV gB genotype 2 was found more frequently than other genotypes in PCR-amplified polymorphonuclear leukocytes (PMNL) of patients with AIDS (P < 0.05) but not more frequently in samples from patients with HCMV retinitis. No significant association was found between any HCMV gB genotypes and the viral load in blood. In conclusion, the actual HCMV gB genotyping system using PMNL provides no additional benefit over the viral load in blood for identification of HIV-infected subjects at risk of HCMV disease. J. Med. Virol. 59:98–103, 1999. © 1999 Wiley-Liss, Inc.     

Development of two potential diagnostic monoclonal antibodies against human cytomegalovirus glycoprotein B

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.     

Lack of association between the kinetics of human cytomegalovirus (HCMV) glycoprotein B (gB)-specific and neutralizing serum antibodies and development or recovery from HCMV active infection in patients undergoing allogeneic stem cell transplant

The kinetics of the gB-specific and neutralizing antibody responses to human cytomegalovirus (HCMV) were analyzed in 26 allogeneic stem-cell transplant recipients who either did (n = 20) or did not (n = 6) develop asymptomatic HCMV active infection during the study period. Antibody response profiles varied widely among individuals in both groups, irrespective of whether HCMV active infection did or did not occur. Development of HCMV active infection was not preceded by a decline in functional serum antibody levels. Neither the absence nor the presence of HCMV active infection correlated with either high or low serum levels of gB-specific and neutralizing antibodies, respectively. In most patients, episodes of HCMV replication were not followed by a marked increment in functional serum antibody titers. Therefore, resolution of an ongoing HCMV active infection was not associated with a vigorous antibody response to viral replication. In addition, this study supports previous data indicating that passive transfer of human immunoglobulins may result in an increment in gB-specific and neutralizing serum antibody levels, the magnitude of which varies among recipients; however, both patients with and without measurable increments in serum antibody levels developed HCMV active infections with comparable frequency.     

荧光定量分型PCR、多重PCR和测序检测HBV基因型的方法学比较

目的 比较荧光定量分型PCR、直接测序和多重PCR三种HBV基因分型法,对本实验室建立的荧光定量分型PCR方法进行评价.方法 用多重PCR、直接测序和荧光定量分型PCR法检测113份HBV DNA阳性的临床样本.结果 荧光定量分型PCR和直接测序法检出率100%,多重PCR法检出率94.69%,6份样本未能分型.荧光定量分型PCR和多重PCR的Kappa系数0.915,荧光定量分型PCR和直接测序法的Kappa系数0.742,一致性均较好.对B/C基因型混合感染样本,荧光定量分型PCR法检出28例(24.78%),多重PCR法检出19例(16.81%),直接测序法检出13例(11.50%).结论 荧光定量分型PCR分型结果准确可靠,对基因型混合感染样本的检出率明显高于多重PCR及直接测序法,是一种高效、简便、快速、准确的HBV基因分型法,适用于大规模流行病学调查.     

Cytomegalovirus glycoprotein B genotypes and central nervous system disease in AIDS patients

To investigate any association between cytomegalovirus glycoprotein B (CMV gB) subtypes and central nervous system (CNS) disease in AIDS patients, proportions of different gB genotypes detected in AIDS patients with CNS disease were compared with the gB genotypes detected in AIDS patients with no neurological disorder. The patients were matched by CD4+ cell counts. CMV was detected by PCR in cerebrospinal fluid (CSF) samples obtained from AIDS patients with CNS disease and from urine and saliva samples obtained from AIDS patients without CNS disease. CMV strains obtained were digested by restriction enzymes HinffI and RsaI to classify the genotypes. The CMV gB genotype was determined in 26 CSF samples. Of these, 11/26 (42.3%) typed as gB group 1, seven (26.9%) as gB2, four (15.4%) as gB3, and four (15.4%) as gB4. The CMV gB genotype frequency distribution in the 42 AIDS patients without CNS disease showed that 18/42 (42.8%) were classified as gB group 1, 10 (23.8%) as gB2, seven (16.6%) as gB3, and seven (16.6%) as gB4. In the present study, no association was found between CMV gB genotypes and CMV-related central nervous system disease.     

Detection of human cytomegalovirus DNA in perilymph of patients with sensorineural hearing loss using real-time PCR

Although cytomegalovirus (CMV) has been detected in the inner ear fluid of patients who succumbed to the complications of symptomatic congenital CMV infection, it has not been detected in the inner ear fluid of living patients. In this study, real-time polymerase chain reaction (PCR) was used to measure CMV DNA in clinical samples (including perilymph) collected from five patients with deafness. In case 1, diagnosed as a symptomatic congenital CMV infection, 3 copies/microl of CMV DNA were detected in perilymph, although no viral DNA was detected in peripheral blood mononuclear cells (PBMCs) or urine samples. In case 4, a suspected asymptomatic congenital CMV infection, 36 copies/microg of CMV DNA were detected in PBMCs, but neither perilymph nor urine contained viral DNA. Likewise, in case 5, a case of deafness of unknown origin, 48 copies/microg of CMV DNA were detected in the PBMCs, but none in the perilymph or urine. CMV DNA was not detected in the samples obtained from the remaining two cases with deafness of unknown etiology. To our knowledge, this is the first report to detect CMV DNA in an inner ear sample obtained from a living human subject.     

Human cytomegalovirus glycoprotein B genotypes in renal transplant recipients

Based on sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) strains can be classified into four gB genotypes. In a previous study of bone marrow transplant recipients, infection with the gB type 1 correlated with a more favorable clinical outcome than infection with the gB types 2, 3, or 4. The gB type was determined in 60 renal transplant and in 47 bone marrow transplant recipients using PCR and restriction analysis. All HCMV variants in patient specimens could be assigned to one of the four previously described gB types. Two or more specimens obtained from 39 patients were analysed; in 31 of these patients the gB type was the same in all samples. The gB type did not correlate with the clinical outcome or the level of viremia in renal transplant recipients. © 1996 Wiley-Liss, Inc.     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的 建立检测HBV病毒DNA的荧光定量PCR法（FQ-PCR）,并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBV DNA314bp特异保守序列的1对引物及1条带2个荧光基团的寡核苷酸探针。用PE-5700型定量PCR仪完成PCR反应及产物的荧光定量检测。同是PCR产物经琼脂糖凝胶电泳,EB染色,UVP（凝胶成像仪）检出有314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术,用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct,原始拷贝数在10^5/ml以上者为阳性,用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性,阳性率29.2%;用定性PCR观察到193例有阳性特异带,阳性检出率为27.65%。没有发现用定性PCR检测为阳性而用FQ-PCR检测为阴性者。结论 FQ-PCR检测HBVDNA较普通定性PCR技术具有操作简便,灵敏度更高、减少发生污染可导致假阳性结果的可能性及自动化程度高等优点,值得在临床检验中推广应用。     

人巨细胞病毒临床分离株糖蛋白B基因型分析

目的 分析巨细胞病毒临床分离株糖蛋白B基因型。方法 以巢式聚合酶链反应(nested-PCR)检测器官移植受者外周血细胞和输卵管妊娠患者生殖道组织中的巨细胞病毒gBn和gBcls基因并测序分型。结果 分离到44例HCMV gBn、gBcls阳性基因，32例有HCMV gBn基因分型结果，其中l型8例(25％)，2型2例(6％)，3型22例(69％)，未见4型，分布以gBn3型为主。HCMV gBcls基因分型结果为26例，其中l型14例(54％)，2型5例(20％)，3型7例(26％)，未见4型，gBcls基因型则散在分布，以1型为多见。21例同时得到gBn、gBcls基因分型结果，其中8例(36％)表现出基因型别不一致。表现为gBclsl-gBn3型。结论 HCMV临床分离株，其糖蛋白基因分别属于gB基因型l-3型，并存在较多的gBcls与gSn基因型别的不对应现象，提示HCMV临床株有较高频率株内基因重组发生，是否由此导致病毒抗原性发生改变，值得进一步研究.     

实时荧光定量PCR方法检测非霍奇金淋巴瘤患者B淋巴细胞刺激因子表达水平

目的:建立实时荧光定量PCR方法(RFQ-PCR)检测非霍奇金淋巴瘤(NHL)患者B淋巴细胞刺激因子(BAFF)表达水平。方法:47例NHL患者及20例健康对照,采用实时荧光定量PCR方法检测其外周血单个核细胞中的B淋巴细胞刺激因子表达水平。结果:RFQ-PCR检测BAFF含量的示灵敏度为10 pg/ml,低浓度样本批内和批间变异系数(CV)分别为8.56%和11.32%,高浓度样本批内和批间CV分别为0.76%和4.58%。NHL患者和健康对照者BAFF mRNA表达量分别为(0.48±0.023,0.25±0.023),两者具有显著性差异(P=0.0001)。结论:RFQ-PCR检测BAFF mRNA含量的方法,具有较好的检测灵敏度和重复性。NHL患者BAFF mRNA高表达,提示BAFF可能在NHL发生发展中发挥重要作用。     

Improved prenatal diagnosis of congenital human cytomegalovirus infection by a modified nested polymerase chain reaction

Two major variables may cause false-negative results in prenatal diagnosis of congenital human cytomegalovirus (HCMV) infection: sensitivity of the technique(s) used; and time elapsed between maternal infection and antenatal testing. Previous results indicated that rapid HCMV isolation from amniotic fluid samples and viral DNA detection in amniotic fluid by nested polymerase chain reaction (nPCR) had comparable levels of sensitivity (69.2% and 76.9%, respectively). The nPCR protocol was reviewed following two additional false-negative antenatal diagnosis in a twin pregnancy during which two procedures were performed at 18 and 23 weeks of gestation, respectively. In the new assay, multiple (instead of single) and 100 (instead of 20) μl amniotic fluid aliquots were individually amplified and tested by nPCR. By using this approach, low DNA levels (1–10 genome equivalents) were detected in 1–5/8 replicates of amniotic fluid samples taken from both twins during both procedures. In addition, viral DNA was detected in 5/6 replicates from two amniotic fluid samples still available from two previous false-negative cases. However, nPCR on multiple amniotic fluid replicates did not anticipate positive prenatal results in a retrospective case, which required two procedures for correct diagnosis and, when prospectively employed, did not avoid one additional false-negative prenatal diagnosis 8 weeks after maternal infection. Thus, delayed intrauterine transmission of the infection may be a potential cause of false-negative results. However, the combination of a very sensitive technique with appropriate timing of prenatal testing can substantially increase the reliability of prenatal diagnosis results. J. Med. Virol. 56:99–103, 1998. © 1998 Wiley-Liss, Inc.     

聚合酶链反应-反向杂交快速检测巨细胞病毒糖蛋白gB、gH基因型变异

目的 建立一种简便、快速、敏感和特异的检测巨细胞病毒（HCMV）糖蛋白gB、gH基因型变异的方法。方法 以HCMV糖蛋白基因gH、gBn、gBclv为靶序列，设计17条具有型特异性的寡核苷酸探针，聚合酶链反应(PCR)扩增目的片段，反向杂交检测23例中国和6例德国移植患者HCMVgH、gBn、gBclv基因型变异，其结果与直接测序进行比较。结果 23例中国患者HCMV糖蛋白为gH1和2型，gB1、2和3型，未见gB4型；而6例德国患者的HCMV糖蛋白涵盖gH1和2型，gB1、2、3及4型。中、德国两患者均可同时感染2种不同基因型的病毒株。反向杂交技术对检测患者同时感染不同基因型病毒株优于直接测序。结论 PCR－反向杂交技术具有简便、快速、敏感和特异的特点，适用于临床实验室检测HCMV糖蛋白基因型变异。     

Antibody response to human cytomegalovirus (HCMV) glycoprotein B (gB) in AIDS patients with HCMV end-organ disease

Human cytomegalovirus (HCMV)-specific antibody responses in HIV-1 infected individuals either with or without HCMV end-organ disease were examined to determine the whether development of HCMV disease was associated with a particular deficit in the antibody response. Anti-whole HCMV, anti-glycoprotein B (gB), and neutralizing antibody levels were higher in HIV-1 infected individuals than in healthy immunocompetent subjects, particularly in patients with AIDS either with or without HCMV-associated disease. Irrespective of location and spread of HCMV disease, patients who had received anti-HCMV therapy prior to sampling exhibited significantly higher anti-gB and neutralizing antibody titers than those who remained untreated. Likewise, patients with HCMV disease who were antigenemic or viremic had significantly lower anti-gB and neutralizing antibody titers than those who tested negative in either assay. Patients with untreated HCMV disease had significantly lower antibody titers than AIDS patients without disease. Analysis of the IgG subclass antibody responses to gB revealed no significant differences among HIV-1 infected individuals. These results suggest that levels of detectable anti-gB and HCMV neutralizing antibodies are inversely related to systemic viral load. Thus, antibodies with such specificities may be relevant in preventing the establishment of HCMV-associated disease or in modulating its progression. J. Med. Virol. 55:272–280, 1998. © 1998 Wiley-Liss, Inc.     

Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis

Hepatitis B virus (HBV) genotype and HBV DNA levels have been implicated in clinical evaluation and prognosis of patients with chronic HBV infection. The aim of the present study was to develop a rapid and sensitive method for simultaneous HBV DNA quantitation and differentiation between HBV genotypes B and C in a single-step reaction by real-time PCR and melting curve analysis using SYBR Green I fluorescent dye. The genotypes obtained by this method were compared with those examined by PCR-RFLP and direct sequencing on 52 serum samples of patients with chronic HBV infection. Using the results obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of HBV genotyping by PCR-RFLP and melting curve analysis was 90.38 and 92.31%, respectively. The geometric mean of HBV DNA levels was 3.42×10⁶, 2.10×10⁶, 1.19×10⁵ and 3.10×10⁴ copies/μl in asymptomatic carriers, patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma, respectively. It is concluded that this method has the advantages of rapidity, reproducibility and accuracy, which would be feasible and attractive for large-scale analysis, particularly in regions where HBV genotypes B and C are prevalent.     

Detection of human cytomegalovirus DNA in various blood components after liver transplantation

The quantification of human cytomegalovirus (HCMV DNA) by real-time PCR is currentlya primary option for laboratory diagnosis of HCMV infection. However, the optimalsample material remains controversial due to the use of different PCR assays. Toexplore the best blood component for HCMV DNA surveillance after livertransplantation, whole blood (WB), serum (SE), and plasma (PL) specimens werecollected simultaneously from targeted patients and examined for HCMV DNA using onecommercially available assay. The HCMV DNA-positive rate with WB (16.67%) was higherthan that with either SE or PL (8.33%, both P<0.01). Quantitative DNA levels in WBwere of greater magnitude than those in SE (WB-SE mean log-transformed difference,0.99; 95%CI=0.74-1.25; P<0.0001) and PL (WB-PL mean log-transformed difference,1.37; 95%CI=1.07-1.66; P<0.0001). Dynamic monitoring revealed that HCMV DNA in WBwas positive sooner and had higher values for a longer period of time during therapy.With earlier positive detection, higher sensitivity, and yield of greater viralloads, WB compared favorably to SE or PL and hence is recommended as the superiormaterial for HCMV DNA surveillance after liver transplantation. In addition, infantrecipients require more intensive monitoring and prophylactic care because of theirhigher susceptibility to primary HCMV infection.     

人巨细胞病毒对药疹病情发生发展的影响

目的 探究人巨细胞病毒对药疹病情发生发展的影响.方法 选取2012年9月-2014年6月在我院就诊的50例药疹患者作为观察组,同期选取50例体检健康者作为对照组,采用Taqman实时荧光定量PCR检测外周血单一核细胞中巨细胞病毒(CMV) DNA阳性率及载量,用酶联免疫吸附试验(ELISA)检测血清中CMV IgM阳性率,对结果进行对比分析.结果 观察组中出现32例CMV DNA阳性患者,阳性率为64％.对照组中出现13例CMV DNA阳性患者,阳性率为26％,两者相比.观察组阳性率显著高于对照组,其差异具有统计学意义(P＜0.05);观察组中CMV DNA载量为28188.824±19525.632拷贝,对照组中CMV DNA载量为3018.9532±1 761.958拷贝,观察组显与对照组比较差异显著,具有统计学意义(P＜0.05);观察组中出现11例CMV IgM阳性患者,阳性率为22％,对照组中出现5例阳性患者,阳性率为10％,两者相比.观察组CMV IgM阳性率显著高于对照组,差异具有统计学意义(P＜0.05).结论 药疹患者中存在CMV感染,其在药疹的发生和发展过程中可能起到一定的促进作用,是药疹发病的因素之一.     

实时定量PCR在强直性肌营养不良1型重复突变检测中的应用

目的 鉴定一个强直性肌营养不良(dystrophy myotonica,DM)家系的致病突变,探讨实时定量PCR能否用于检测导致DM1的CTG重复延展突变.方法 采集家系成员外周血,提取基因组DNA.针对DM1位点DMPK基因内的CTG重复区和DM2位点CNBP基因内的CCTG重复区进行普通PCR、实时荧光定量PCR、微卫星标记连锁分析.结果 CTG重复区普通PCR产物电泳显示患者特有大于2 kb的弥散带.定量PCR显示,患者CTG重复区相对拷贝数约为0.5,CCTG重复区相对拷贝数约为1.在DM1位点的微卫星标记,患者均有共享等位基因;而在DM2位点的大部分标记,患者没有共享等位基因.结论 此DM家系的致病突变是DM1位点的CTG重复延展突变;应用实时定量PCR可以确定高度重复延展片段扩增失败,从而推断重复延展突变的存在,达到快速检测DM1的目的.