荧光定量聚合酶链反应检测技术在小儿巨细胞病毒感染诊断和治疗中的应用

目的 建立小儿人巨细胞病毒（HCMV）感染的分子诊断方法。方法 采用荧光定量聚合酶链反应（FQ－PCR）法测定45例疑诊HCMV感染的患儿，并与常规PCR及酶联免疫吸附试验（ELISA）比较。对确诊为HCMV肝炎的25例分别在5个时点（治疗前、出院时、3个月、6个月及9个月）监测其外周血白细胞中HCMV DNA拷贝（copies)数。结果 常规PCR、ELISA和FQ－PCR的阳性率分别为60．00％，33．33％和66．67％，灵敏度分别为84．38％，46．88％和93．75％。HCMV肝炎治疗组和对照组的HCMV DNA量在5个时点的差异有非常显著意义（P＜0．001）。以10^3 copies/ml的FQ－PCR值为临界值（≥10^3有症状，＜10^3无症状）可预测活动性HCMV感染的出现。结论 FQ－PCR可作为小儿HCMV感染的有效诊断方法之一，动态检测HCMV DNA含量有助于指导治疗、估计疾病的发展和预后。     

先天性巨细胞病毒感染新生儿gB基因分型研究

目的 探讨先天性巨细胞病毒（HCMV）感染的新生儿gB基因分型与临床表现的关系。方法采集67例经PCR方法确诊的有症状的先天性HCMV感染新生儿尿液标本。用巢式PCR方法扩增尿液标本中gB基因片段,用限制性片段长度多态性分析检测gB基因分型。结果 67例病例中最常见的基因型为gB1 （50.7％）,其次为gB3 （23.9％）,gB2 （17.9％）和gB1/gB3混合感染（7.5％）,gB4基因型未检测到。在有肝脏损害的患儿中,gB1基因型最为常见（27/37,73.0％）,高于无肝脏损害的其他有症状感染患儿（ 13/30,43.3％;P＜0.05）。结论 在有症状的先天性HCMV感染新生儿中最常见的gB基因型为gB1,其次是gB3、gB2及gB4。     

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。     

聚合酶链反应检测新生儿尿巨细胞病毒DNA

采用聚合酶链反应技术（PCR），对45例疑为巨细胞病毒（HCMV）感染的新生儿，测定其尿中的HCMV－DNA，同时与尿培养法进行比较．结果前者阳性为44．44％，后者为17．78％（P＜0．01）。从而证实PCR法具敏感、特异、简便、可靠等优点，达到对HCMV感染的早期诊断和及早干预的目的。     

HIV/HCV合并感染者淋巴细胞活化及第二受体表达的研究

目的了解中国不同疾病进展阶段人类免疫缺陷病毒和丙型肝炎病毒（HIV／HCV）合并感染者T淋巴细胞与自然杀伤细胞（natural killer cells，NK）数量变化及T淋巴细胞活化、受体表达情况，并探讨HCV感染对HIV感染免疫指标及疾病进展的影响。方法应用流式细胞术分析228例不同疾病进展阶段的HIV／HCV合并感染者及101例单纯HIV感染者外周血T淋巴细胞、NK细胞数量及T淋巴细胞活化受体（HLA-DR、CD38）、第二受体（CCR5、CXCR4）表达情况。结果（1）HIV／HCV合并感染组中，CD4^＋T淋巴细胞、NK细胞数量随疾病进展持续下降，其中艾滋病组（AIDS）明显低于无症状HIV感染组（HIV）（P〈0．05），HIV组明显低于长期不进展组（LTNP）（P〈0．01），LTNP组与健康对照组差异无统计学意义。LTNP组、HIV组及AIDS组CD4^＋、CD8^＋T细胞表面活化受体HLA-DR、CD38的表达依次升高，其中各组间CD8／CD38的升高差异均有统计学意义（P〈0．05），AIDS组CD4／HLA-DR、CD8／HLA-DR的升高明显高于LTNP组和HIV组（P〈0．01）。LTNP组、HIV组及AIDS组CD4^＋、CD3^＋T细胞表面CCR5的表达亦依次升高，各组间差异均有统计学意义（P〈0．05）；CD3^＋T细胞表面CXCR4的表达依次升高，AIDS组明显高于HIV组和LTNP组（P〈0．01）。（2）HIV／HCV合并感染组与单纯HIV感染组相比，AIDS组NK细胞明显下降（P〈0．05），CD4^＋T细胞下降，但无统计学意义，CD4／HLA-DR、CD8／HLA-DR、CD4／CXCR4、CD3／CXCR4明显升高（P〈0．01）；HIV组NK细胞明显下降（P〈0．01），CD4／CXCR4明显升高（P〈0．05）；LTNP组各项指标与单纯HIV感染组相比差异无统计学意义。（3）HIV／HCV合并感染组的HIV病毒载量随疾病进展不断升高，与单纯HIV感染组相比差异无统计学意义；HCV病毒载量在疾病不同阶段差异无统计学意义（P〉0．05）。结论随疾病进展，HIV／HCV合并感染者的免疫功能逐渐下降，HIV病毒载量逐渐升高。与单纯HIV感染相比，合并HCV感染可通过破坏机体天然免疫功能、促进免疫系统活化和受体表达，加速HIV感染的疾病进展。     

双色荧光定量PCR快速分型、定量高危型人乳头状瘤病毒

目的针对高危型人乳头状瘤病毒（humanpapillomavims，HPV）亚型的多样性以及感染病毒载量的高低，旨在建立高危型HPV的定量与分型的快速检测方法，为HPV筛查与治疗提供依据。方法利用2种荧光染料分别标记HPV-16、HPV-33／52／58／67和HPV-18／45、HPV-31探针，同时以B球蛋白基因作为内对照，对120份疑为HPV感染的宫颈脱落细胞样本进行I{I＇V分型与定量，定量范围为5×10^1-5×10^7拷贝/ml，以HC-2杂交捕获法作为“金标准”，评价该方法的特异性与灵敏度。结果荧光定量PCR阳性检出率为52．5％（631120），其中HPV-16、HPV-18／45、HPV-31和m．V-33，52，58，67阳性率分别为36．51％（23／63）、11．11％（7／63）、12．70％（8／63）、58．73％（37／63）；HPV感染的平均病毒载量为1．08×10^7拷贝/ml。荧光定量PCR的特异性为100％，灵敏度为81．82％。结论双色荧光定量PCR能快速分型、定量高危型HPV，可用于HPV感染的筛查与宫颈病变程度预测以及疗效观察。     

HMA-SSCP方法研究人类巨细胞病毒UL144基因在临床低传代分离株中的多态性

目的 探讨人类巨细胞病毒（HCMV）UL144序列在临床患儿低传代分离株中的多态性及与临床疾病的关系。方法 对65株HCMV临床低传代分离株及7例同年龄组HCMV，DNA定量PCR方法检测阳性无症状感染儿尿液进行HCMV-ULl44 PCR扩增及HMA-SSCP分析，并对其中32份阳性标本进行测序。结果 65株分离株中有55株UL144全序列引物PCR扩增阳性，7份QPCR检测HCMV—DNA阳性无症状感染儿尿液中5份UL144全序列引物PCR扩增阳性。60份UL144扩增阳性标本HMA-SSCP（异源双链泳动及单链构象多态分析）呈现3种典型带形，巨结肠患儿分离株序列、小头畸形患儿的序列分布以1型为主，巨结肠患儿分离株序列没有2型，黄疽患儿以3型为主。结论 HCMV-UL144广泛存在于临床低传代分离株中，用HMA-SSCP检测HCMV-UL144基因在临床低传代分离株中的多态性是一种可行的方法。HCMV不同疾病类型的HCMV-UL144序列不同，提示UL144基因可能对HCMV致病性起一定作用。     

中国北方地区隐源性肝炎及乙型肝炎表面抗原阴性肝癌患者中隐匿性HBV感染流行状况分析

目的调查中国北方地区隐源性肝炎及乙肝表面抗原（HBsAg）阴性肝癌患者中隐匿性乙型肝炎病毒（HBV）感染的流行状况。方法收集393个受试者的血清,其中包括隐源性慢性肝炎患者215名、HBsAg阴性肝癌患者178名。使用巢式PCR的方法检测血清中的HBV-DNA,同时使用实时定量PCR的方法检测HBV—DNA的载量。结果在隐源性慢性肝炎患者、HBsAg阴性肝癌患者中隐匿性HBV感染流行率分别为23．7％（51／215）和68．5％（122／178）。在IgGanti—HBc阳性者中,隐匿性HBV感染率较高。所有隐匿性感染者的病毒载量均较低（〈10^5拷贝／ml）。结论在中国北方隐源性肝炎及肝癌患者中,隐匿性HBV感染率较高。因此,对隐匿性HBV感染应给及足够的重视,避免因输血及器官移植造成HBV的传播。     

先天性HCMV感染胎鼠大脑皮层ET-1 mRNA的研究

目的 对先天性人巨细胞病毒（HCMV）感染的胎鼠大脑皮层内皮素－1（ET－1）mRNA进行测定,以探讨先天性HCVMV感染致脑损害的机制。方法 在建立先天性HCMV中枢神经系统（CNS）感染胎鼠模型的基础上,用逆转录－聚合酶链式反应（RT－PCR）测定受不同病毒剂量感染的胎鼠大脑皮层ET－1 mRNA,并用地高辛标记的ET－1寡核苷酸探针对大脑皮层细胞印片进行原位杂交以检测相应mRNA转录量及胞内定位。结果 在大脑皮层组织的上清液中HCMV分离阳性;病理学研究证实受染胎鼠大脑皮层表现为侵袭性脑膜脑炎性改变,并在神经细胞内发现特异性核内嗜碱性包涵体。RT－PCR和原位杂交研究发现,受染胎鼠大脑皮层内ET－1 mRNA转录量增加,以1．0ml和0．5ml组为显著,而0.25ml组与正常对照组比较无明显差别。结论 HCM可经胎盘垂直传播至胎鼠脑组织。先天性HCMV感染可刺激受染胎鼠CNS ET－1 mRNA的转录,且与母鼠所接种的病毒量存在一定的量效关系。这些结果提示,ET－1在先天性HCMV感染脑损害过程中,早期可导致组织缺血性改变,而晚期则与受损大脑皮层的功能恢复有关。这对了解先天性HCMV感染致NS损伤的机理将提供有价值的参考依据,同时也为临床防治和优生优育提供一种有价值的手段。     

人巨细胞病毒感染检测方法进展

人巨细胞病毒（HCMV）主要感染免疫力低下人群、妊娠期孕妇以及新生儿。我国新生儿先天感染率为1%,其中10%新生儿发生巨细胞包涵体病（cytomegalovirus inclusiondisease,CID）,表现为小头畸形、智力低下、性格行为改变和黄疸等[2-3];90%的先天性感染者虽然在新生儿期无临床表现,但5%~17%日后表现为感觉神经性耳聋、学习记忆能力缺陷及其他神经行为异常。     

Diagnostic and prognostic value of human cytomegalovirus load and IgM antibody in blood of congenitally infected newborns.

BACKGROUND: Diagnosis of congenital human cytomegalovirus (HCMV) infection relies on virus isolation from urine collected in the first 3 weeks of life. However, very little is known about the presence, levels and duration of HCMV pp65 antigenemia, viremia and DNAemia in congenitally infected newborns. OBJECTIVES: To investigate the diagnostic and prognostic value of HCMV load determination in blood of newborns/infants with congenital HCMV infection. STUDY DESIGN: HCMV pp65 antigenemia, viremia and DNAemia were investigated in 116 sequential peripheral blood leukocytes (PBL) samples from 41 newborns/infants with congenital HCMV infection and in 34 PBL samples from 34 uninfected newborn. Virus-specific IgM were determined in parallel on 145 sequential serum samples. RESULTS: Compared to virus isolation from urine, sensitivities of DNAemia, antigenemia, viremia, and IgM determination were 100, 42.5, 28.2, and 70.7%, respectively. Specificity was 100% for all assays. Antigenemia, viremia and DNAemia levels were significantly higher and persisted longer in newborns with symptomatic infection compared to subclinically infected babies, whereas no difference was observed for virus-specific IgM antibody between the two groups. CONCLUSIONS: (i) determination of viral DNA in blood at birth appears to be a sensitive and specific marker for diagnosis of congenital HCMV infection; (ii) significantly higher levels of HCMV load were detected in infants with symptomatic HCMV infection; and (iii) virus clearance from blood occurs spontaneously both in symptomatic and subclinically infected infants. However, the process takes longer in infants presenting with symptoms at birth.     

儿童巨细胞病毒感染与甘露聚糖结合凝集素表达相关性研究

目的探讨甘露聚糖结合凝集素（MBL）基因多态性及血浆蛋白水平与儿童巨细胞病毒（HCMV）感染的相关性。方法收集2007年3～11月在浙江大学医学院附属儿童医院诊断为HCMV感染的患儿作为HCMV感染组,选择同期行健康体检的儿童作为对照组。对两组行MBL基因检测和分型,并对HCMV感染组进行随访,分别测定其急性期和恢复期血浆MBL蛋白水平。分别比较两组基因变异频率和血浆MBL蛋白水平。结果HCMV感染组纳入104例,对照组纳入105例;HCMV感染组有50例患儿进行随访,HCMV感染组MBL基因启动子区-550位点的L型变异频率明显高于对照组（56.7%vs34.3%,P=0.001）,野生单体基因型HYPA的频率明显低于对照组（47.6%vs62.8%,P=0.002）,完整基因型中高水平表达基因型（YA/YA）的频率低于对照组（41.3%vs60.0%,P=0.007）,而低水平表达基因型（YA/XA,YA/YB,XA/XA）的频率高于对照组（52.9%vs29.5%,P=0.001）。HCMV感染组急性期和恢复期血浆MBL蛋白水平均低于对照组（P=0.019和0.000）。HCMV感染组急性期血浆MBL蛋白水平高于恢复期（P=0.000）。结论MBL基因多态性导致的血浆MBL蛋白水平低下与儿童HCMV感染相关,提示MBL可能对儿童HCMV感染具有保护作用。     

The relationship between polymorphisms of HCMV UL144 ORF and clinical manifestations in 73 strains with congenital and/or perinatal HCMV infection

Summary. Human cytomegalovirus (HCMV) displays genetic variability and can cause a wide range of diseases in neonates. To explore the relationship between polymorphisms and clinical manifestations, the UL144 genes from 73 clinical strains were sequenced. All of the strains, which came from 70 infants with suspected congenital and/or perinatal HCMV infection, were non-passage strains. Among them, 23 strains were from surgery specimens, and the others were from urine samples. Clinically, 12 infants displayed asymptomatic infection and 58 patients displayed symptomatic infection. The results showed that 36 patients (49.3%) were infected with strains belonging to UL144 group G1 (G1a 33/36, G1b 3/36), 19 patients (28.8%) were infected with strains belonging to group G2, and 15 patients (21.9%) were infected with strains belonging to group G3. This result indicated that UL144 group G1 was the predominant genotype in congenital and/or perinatal HCMV infection in northern China. Compared with the distribution pattern of strains in UL144 genotypes of data from Chicago, Iowa and Texas, and Japan by chi-square test, the difference was statistically significant. This suggested that the distribution pattern of strains in UL144 genotype was related to geographic location. However, no linkage was observed between the UL144 genotypes and the severity and/or outcome of HCMV disease.     

先天性巨结肠患者人类巨细胞病毒UL144基因多态性的研究

目的研究人类巨细胞病毒（human cytomegalovirus,HCMV）UL144基因在先天性巨结肠（Hirschsprung＇s disease,HD）临床株中的多态性,探讨HCMV UL144基因多态性与致病性之间的关系.方法随机选取53个先天性巨结肠患儿痉挛段结肠手术标本及经荧光定量PCR方法检测HCMV DNA为阳性的4个HD患儿的尿标本,对照组为无症状或仅有皮肤轻度黄疸的6个尿标本.应用巢式聚合酶链反应的方法,扩增HCMV UL144基因开放阅读框架（ORF）,扩增阳性的临床株进行双向DNA测序,最后通过DNAclub、Bioedit、DNAstar、GeneDoc等软件进行分析.结果23份HD痉挛段肠组织（46%）及4份尿标本HCMV UL144基因扩增阳性,并且完成测序.种系进化树分析结果显示25个HD患儿的DNA序列分为3个基因型,G1A型64.0%,G2型24%,G3型12%.与对照组比较,经χ^2检验,χ^2=10.93,P为0.012;其中HD临床株G1A和G3型基因经Fisher检验,P为0.015,差异具有统计学意义.全结肠型、长段型及普通型HD分散分布于UL144各个基因型中.结论HD与HCMV感染有关,HCMV可能是HD的病因之一;在HD患儿中,HCMV感染以UL144基因G1A型为主;HD的临床分型与HCMV UL144基因分型无关.     

Comparison of viral load and duration of virus shedding in symptomatic and asymptomatic neonatal rotavirus infections

A single rotavirus strain causing asymptomatic infections as well as severe gastrointestinal disease has been described in the neonatal nurseries of the Christian Medical College, Vellore. In this study, quantitative real‐time RT‐PCR was used to determine the association of viral load with the presence of gastrointestinal symptoms in neonates. Viral load was estimated in terms of the crossing point [C(t) value] at which the amplicon could be detected in the real‐time PCR assay. The study was carried out on 103 neonates, including 33 asymptomatic neonates and 70 neonates with different gastrointestinal symptoms. The duration of virus shedding was also compared between five symptomatic and four asymptomatic neonates using real‐time RT‐PCR. There was no significant difference in viral load between symptomatic and asymptomatic neonates (P = 0.087). Among neonates with different gastrointestinal symptoms, those presenting with feed intolerance and abdominal distension had a significantly higher viral load than those with other gastrointestinal symptoms (P = 0.02). For the study on virus shedding, nine neonates were followed up for a median duration of 53 days, with a median of 31 samples tested per child. Extended shedding of low copies of rotavirus was found, with no significant differences in pattern of shedding between symptomatic and asymptomatic neonates. The lack of correlation between viral load and gastrointestinal disease demonstrates yet another difference between neonatal rotavirus infection and infection in older children where higher viral load correlates with severe disease. J. Med. Virol. 82:1803–1807, 2010. © 2010 Wiley‐Liss, Inc.     

Primary human cytomegalovirus infections: Kinetics of ELISA-IgG and neutralizing antibody in pauci/asymptomatic pregnant women vs symptomatic non-pregnant subjects

BackgroundHuman cytomegalovirus infections are mostly asymptomatic in infants and young children, while they are often associated with overt clinical symptoms in adults.ObjectivesTo verify whether the antibody response to HCMV is more potent in symptomatic non-pregnant adults as compared to asymptomatic/paucisymptomatic pregnant women.Study designOverall, 36 consecutive pregnant women with primary HCMV infection were compared with 10 consecutive symptomatic non-pregnant subjects with primary HCMV infection and overt clinical symptoms. Levels of IgG antibody responses to HCMV-infected cell lysate and the pentamer gH/gL/pUL128L, gH/gL and gB HCMV glycoprotein complexes as well as neutralizing antibodies preventing infection of epithelial cells (ARPE-19) and human embryonic lung fibroblast (HELF) cells were compared at intervals of 1–30, 31–60, 61–90, 91–180 and 181–360 days after onset of infection. In parallel, viral load was quantified by real-time PCR.ResultsIn symptomatic non-pregnant subjects, the IgG responses to HCMV lysate as well as to gH/gL and ARPE-19 neutralizing antibodies were significantly higher from 31 to 60 through 180 days after infection onset. In the same patients, the IgG antibody responses to the pentamer and HELF-neutralizing antibody were significantly higher starting 90 days post-infection.ConclusionsThe presence of overt clinical symptoms is associated with a significantly higher antibody response (concomitantly with a higher viral load) in non-pregnant subjects with symptomatic primary HCMV infection as compared to pregnant women with paucisymptomatic/ asymptomatic primary infection (and lower viral load).     

Usefulness of blood and urine samples collected on filter paper in detecting cytomegalovirus by the polymerase chain reaction technique

A rapid test for the diagnosis of congenital CMV infection is still needed. This study evaluated the usefulness of dried blood and urine samples collected on filter paper for detecting cytomegalovirus (CMV) by the polymerase chain reaction (PCR) assay compared with the use of liquid urine. Samples were obtained from 332 infants aged 1-7 days. Liquid urine samples were collected into bags, cultured in human fibroblasts, and processed using a multiplex PCR technique. Dried urine samples were obtained by placing a piece of filter paper in contact with the infant's genitals. The heels of neonates were punctured and capillary blood was blotted onto filter paper and dried. Dried blood and urine specimens were analyzed by multiplex PCR and nested-PCR assays. A diagnosis of congenital CMV infection was established by isolating the virus, and by detecting viral DNA in the liquid urine. Of the 332 liquid urine samples collected from 332 neonates, seven (2.1%) were positive for CMV and 325 were negative, by both cell culture and PCR assay. In dried samples, CMV DNA was detectable only with a nested PCR assay. Compared with known CMV infection status, 5/7 (71.4%) neonates were positive for congenital CMV infection using dried blood samples. All 325 uninfected neonates were negative. In the dried urine samples, 4/4 CMV-infected infants gave positive tests, and all 262 uninfected infants were negative. Although further improvements in sample collection and/or processing are still needed, PCR testing on dried urine or blood collected on filter paper is a promising approach in the diagnosis of neonatal CMV infection.     

四种腺病毒核酸提取方法的比较

目的通过蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种腺病毒核酸提取方法的比较。为进一步研究腺病毒的分子生物学特性选择合适的方法提供参考。方法以腺病毒感染的A549细胞为样品．分别以蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸．核酸经紫外分光光度计检测A260／A280的比值后,用荧光定量PCR方法检测核酸中腺病毒拷贝数浓度。并记录四种腺病毒核酸提取方法所需的操作时间。结果蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸的A260／A280的比值依次为1．85532、1．7377、1．81474和1．43934,核酸中腺病毒拷贝数浓度依次为4．9×10^5copies／mL、3．94×10^3copies／mL、2．66×10^6copies／mL和6．15×10^6copies／mL,提取核酸所需的时间分别为1．5、15、0．5和0．5h。结论四种腺病毒核酸提取方法中,蛋白酶K法简单快捷、成本低廉,适合一般实验室使用;苯酚法适合用于提取细胞培养上清中病毒的核酸;病毒DNA／RNA试剂盒的优点是操作时间短,得到的病毒核酸纯度较高;腺病毒荧光PCR检测试剂盒对病毒的核酸损耗少．操作步骤少．适用于临床标本的腺病毒核酸检测。