HCMVpp65抗原、HCMV mRNA和IgM抗体检测在诊断HCMV活动性感染中的比较

目的比较人巨细胞病毒（HCMV）pp65抗原、HCMV mRNA和血清HCMV—IgM抗体检测3种方法在诊断HCMV活动性感染中的实用意义。方法采集医院TORCH检查HCMV—IgM阳性的病人外周血（60份）。将标本分2份2ml和3ml,别用于HCMV mRNA和pp65检测。将三者结果进行比较。结果pp65抗原检测的结果与IgM抗体检测的阳性符合率为81．67％。与HCMV mRNA检测相比pp65抗原检测法的符合率、特异度和敏感度分别为81．67％,81．81％和81．63％。而且高pp65抗原血症与患者的临床症状密切相关。结论pp65抗原血症反映该病毒活动状况,可监测HCMV活动性感染,联合HCMV—IgM的检测可以提高临床的诊断率并可用于指导临床用药及监测药物疗效。     

血硒、GPX与外周血CD94在反复自然流产患者水平变化的临床意义

目的：检测原因不明反复自然流产（RSA）患者血硒、谷胱甘肽过氧化物酶（GPX）及外周血中CD94水平变化,探讨该类变化对反复自然流产发病中的作用。方法：采集RSA患者、正常妊娠妇女和非妊娠妇女（各35例）外周血检测硒、GPX与CD94含量。结果：RSA患者血硒、GPX水平均比正常妊娠妇女和非妊娠妇女呈现显著低下（P〈0．01）,CD94水平均亦比正常妊娠妇女和非妊娠妇女低（P〈0．05）。结论：血硒、GPX与CD94在外周血中的低表达可能与反复自然流产的发病和病理生理过程有关。     

CD4^＋CD25^＋FOXP3^＋调节性T细胞在与原因不明反复自然流产的相关性研究

目的探讨外周血中CD4＋CD25＋FOXP3＋调节性T细胞（CD4^＋CD25^＋FOXP3^＋Tr）在原因不明反复自然流产（URSA）发病中的作用。方法应用三色荧光标记流式细胞术检测21例原因不明反复自然流产妇女（URSA组）、20例正常妊娠妇女（正常对照组）外周血中CD4^＋CD25^＋FOXP3^＋Tr及T细胞亚群水平的表达情况。结果URSA组妇女外周血中CD4^＋CD25^＋FOXP3^＋Tr表达率为（1.36±0.61）%,明显低于正常对照组（5.59±1.02）%,两组比较差异显著（P（0.05）,但两组相比较外周血中T细胞亚群CD3^＋、CD4^＋、CD8^＋、CD4^＋/CD8^＋水平无显著差异（P（0.05）。结论原因不明反复自然流产的发生可能与患者外周血中CD4^＋CD25^＋FOXP3^＋Tr数量减少有关。     

106例反复自然流产患者封闭抗体的检测及临床应用

目的评价反复自然流产（RSA）患者封闭抗体（BA）的水平,并应用淋巴细胞免疫治疗的方法观察其治疗效果。方法用酶联免疫法（EL ISA）测定106例RSA患者BA抗体水平,选取其中92例BA抗体水平阴性者进行淋巴细胞免疫治疗,疗程结束后复查BA抗体水平。结果106例反复自然流产患者中,BA抗体阳性率13.21%（14/106）;其中92例BA抗体阴性者免疫治疗一个疗程后,BA抗体阳性率为55.43%（51/92）,2个疗程后BA抗体阳性率为75%（69/92）,3个疗程后BA抗体阳性率为87%（80/92）,三者与未治疗的106例RSA比较差异均有显著性（P〈0.01）;免疫治疗后BA抗体阳性的RSA再次妊娠的成功率为82.05%（64/78）。结论反复自然流产患者中BA抗体阳性水平较低,用淋巴细胞免疫治疗RSA患者能提高BA抗体阳性率及再次妊娠的成功率。     

2602例入巨细胞病毒感染跟踪调查研究的分析

目的探讨HCMV发生孕期活动性感染、引起宫内传播及对胎儿、婴儿影响的相关因素.方法ELISA孕前筛查HCMV-IgM、IgG抗体并分4组.用ELISA和PCR两种技术跟踪检测孕妇早、中、晚孕期外周血、羊水及新生儿脐带血感染情况、对新生儿行跟踪调查.结果孕前育龄妇女活动性感染率为10.2%;孕前4组感染类型发生在孕期活动性感染率高达24.1%,以原发、复发组发生率最高占孕期活动性感染的75%;孕期活动性感染自然流产率显著高于非活动性感染组;孕期活动性感染发生官内感染的危险度为34.6%;孕期活动性感染新生儿先天性感染检出率为8.9%;发生在孕早期活动性感染妊娠结局最为严重.结论孕前感染类型与孕后发生活动性感染危险度密切相关,且不同类型对妊娠结局影响不同;孕期活动性感染自然流产率高,能够引起官内垂直传播;胎盘及母体的特异性IgG抗体对胎儿的保护作用不可忽视;重点预防早孕期CMV感染.     

PCR技术和ELISA法诊断HCMV宫内感染的敏感性与可靠性比较

用聚合酶链反应(PCR)技术.对78例妊娠早期孕妇进行外周血、尿中人巨细胞病毒(HCMV)DNA测定;用酶联免疫吸附法对78例妊娠早期孕妇进行外周血中人巨细胞病毒IgG、IgM测定.结果:PCR技术中外周血阳性率10%、尿中阳性率11.4%;ELISA反应中血IgG阳性率1.1%、IgM阳性率1.1%.统计结果表明在同一孕妇中HCMV阳性检出率PCR技术明显高于ELISA法,在PCR技术中,尿的阳性率又高于血的阳性率,这进一步证实了PCR技术是诊断宫内感染较理想的手段.     

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.     

广州市天河区2006—2008年孕产妇梅毒感染状况调查

目的了解天河区孕产妇梅毒感染状况,为梅毒防治决策提供依据。方法对2006—2008年在本院产科门诊产检的孕妇10754例,采用TRUST作梅毒抗体初筛试验,采用TPPA作梅毒抗体确认试验。结果广州市天河区孕妇梅毒感染率逐年上升,其中2006年3012例孕妇梅毒感染率为1．39％,2007年3727例孕妇梅毒感染率为2．20％,2008年4015例孕妇梅毒感染率为2.81％。不同年龄组孕产妇梅毒阳性率差异无统计学意义（χ^2=0．0415,P〉0．05）;不同职业的孕产妇梅毒阳性率差异有统计学意义（χ^2=20．453,P〈0．05）;不同文化程度的孕产妇梅毒阳性率差异有统计学意义（χ^2=47．52,P〈0．05）。结论对孕妇进行梅毒血清学检测及规范治疗是杜绝先天梅毒、切断母婴垂直传播的有效方法和措施。     

秦巴山区孕妇HCMV感染率高的主要原因初步探析

目的探讨秦巴山区孕妇人巨细胞病毒(HCMV)感染率高的主要原因,初步研究缺锌与HCMV感染的相关性.方法ELISA和PCR相结合检测秦巴山区161名孕妇外周血特异性抗CMVI IgM抗体,CMV DNA,检测出活动性感染为病例组,无活动性感染为对照组.原子吸收光谱法测定其血清锌含量.双抗体夹心ELISA法检测孕妇外周血肿瘤坏死因子α(TNFα)水平.结果1.秦巴山区孕妇HCMV活动感染率19.25%,明显高于国内外报道.2.病例组的血清锌含量明显低于对照组,有显著性差异(P<0.05).3.病例组的TNFα含量与对照组相比有显著差异(P<0.05).4.对与孕期HCMV活动感染有关的因素,进行多元逐步回归分析,得出y=0.375X1(血清锌)-0.343X2(TNFα)-0.340X3(胎次)+0.209X4(年龄),血清锌含量与HCMV感染相关性最强(β=0.375,P<0.01).5.血清锌含量与TNFα含量有显著负相关(r=-0.571).结论秦巴山区孕妇HCMV感染率高于国内外报道;锌与该地区孕期HCMV活动性感染有明显相关性,缺锌可能是该地区HCMV感染率高的重要原因.     

2602例人巨细胞病毒感染跟踪调查研究的分析

目的：探讨HCMV发生孕期活动性感染、引起宫内传播及对胎儿、婴儿影响的相关因素。方法：ELISA孕前筛查HCMV－IgM、IgG抗体并分4组。用ELISA和PCR两种技术跟踪检测孕妇早、中、晚孕期外周血、羊水及新生儿脐带血感染情况、对新生儿行跟踪调查。结果：孕前育龄妇女活动性感染率为10．2％；孕前4组感染类型发生在孕期活动性感染率高达24．1％，以原发、复发组发生率最高占孕期活动性感染的75％；孕期活动性感染自然流产率显著高于非活动性感染组；孕期活动性感染发生官内感染的危险度为34．6％；孕期活动性感染新生儿先天性感染检出率为8．9％；发生在孕早期活动性感染妊娠结局最为严重。结论：孕前感染类型与孕后发生活动性感染危险度密切相关，且不同类型对妊娠结局影响不同；孕期活动性感染自然流产率高，能够引起官内垂直传播；胎盘及母体的特异性IgG抗体对胎儿的保护作用不可忽视；重点预防早孕期CMV感染。     

Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay.

BACKGROUND: Preemptive antiviral treatment of Human Cytomegalovirus (HCMV) disease is a major goal in the management of organ transplant patients. It requires sensitive diagnostic methods. Automated real-time PCR systems have been recently proposed to monitor HCMV infection in such patients. OBJECTIVE: Objectives of this study was to compare a real-time quantitative PCR on whole blood with the HCMV pp65 antigenemia assay in renal transplant recipients, and also to evaluate two different DNA extraction methods. STUDY DESIGN: A total of 248 specimens from 21 patients were tested by quantitative pp65 antigenemia and quantitative real-time PCR. DNA was extracted from whole blood samples using two different methods: a conventional column manual assay and an automated system. RESULTS: Quantification of HCMV DNA using the two extraction methods showed highly similar results (Spearman rank test, r=0.863). We found a significant correlation between DNA quantification by real-time PCR in whole blood and pp65 antigenemia test (Spearman rank test, r=0.767). This correlation was not modified when the HCMV DNA results were normalized by quantification of the albumin cellular gene. In eight patients, HCMV infection was detected earlier with quantitative PCR than with the antigenemia test (mean delay of 11.25 days). HCMV DNA load equivalent of 50 pp65 positive cells/200,000 polymorphonuclear leukocytes (PMNLs) is log4.095 copies per ml of blood. CONCLUSIONS: Real-time PCR in whole blood is a sensitive method for estimating the HCMV genome load in renal transplant patients, and is more rapid and practicable than using PMNLs for pp65 antigenemia tests.     

Low correlation of human cytomegalovirus DNA amplification by polymerase chain reaction with cytomegalovirus disease in organ transplant recipients

Seventy-five organ transplant recipients underwent prolonged virological and serological follow-up for early detection of human Cytomegalovirus (HCMV) infection after transplantation. HCMV DNA detection by nested polymerase chain reaction (PCR) and HCMV early structural antigen (pp65) detection were carried out in 576 peripheral blood leucocyte (PBL) samples. Furthermore, 563 blood specimens were investigated by a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins G, M, and A against HCMV structural antigens. In eight of nine symptomatic organ transplant recipients, HCMV DNA was detected in one or more consecutive blood samples. HCMV DNA PCR was also positive in one or more samples from eight patients who never developed HCMV-related symptoms. HCMV pp65 antigen was detected almost exclusively in PBL samples from organ transplant recipients suffering from HCMV disease. However, antigenaemia was not detected in four PCR positive patients presenting clinical signs attributable to HCMV infection. Two of the initially HCMV DNA positive samples were not confirmed by retesting and hybridisation. The results of the present study demonstrate that despite the high specificity of nested PCR, HCMV DNA may be detected in the absence of clinical symptoms attributable to HCMV infection. In asymptomatic reactivation, limited replication of viral DNA may be responsible for positive results of PCR without any clinical relevance. In this context, pp65-antigen detection from PBL seems to have a better prognostic value, but is not always detected when clinical symptoms are present. © 1994 Wiley-Liss, Inc.     

Detection of late pp67-mRNA by NASBA in peripheral blood for the diagnosis of human cytomegalovirus disease in AIDS patients.

OBJECTIVE: To evaluate the value of late-pp67-mRNA nucleic acid sequence-based amplification (NASBA) in comparison to DNA-PCR, blood culture and pp65-antigenemia assay for the detection of human cytomegalovirus (HCMV) disease in HIV-infected patients. METHODS: The results of pp67-mRNA NASBA, DNA-PCR, culture and pp65-antigenemia assay were compared in 402 whole blood specimens of 98 HIV-infected patients with a low CD4 lymphocyte count who had not yet received highly active antiretroviral therapy (HAART). Thirty-seven samples were obtained from 30 patients with a diagnosis of HCMV disease and 365 samples from 68 patients without HCMV disease. RESULTS: The highest agreement of test results was observed between pp67-mRNA NASBA and quantitative pp65-antigenemia, with a threshold of nine antigen-positive cells/10(5) leukocytes (kappa-value 0.70, 95% CI=0.58-0.82). The sensitivity of pp67-mRNA NASBA for the diagnosis of HCMV disease (59.3%) was identical to that of the quantitative pp65-antigenemia assay, higher than that of the blood culture (48.2%) but lower than that of the DNA-PCR (77.8%). Pp67-mRNA NASBA (92.3%), quantitative pp65-antigenemia assay (92.3%) and blood culture (93.9%) were highly specific for the diagnosis of HCMV disease and as a result, had a higher positive predictive value (76.2, 76.2 and 76.5%, respectively) than the qualitative DNA-PCR (58.3%) and the qualitative pp65-antigenemia assay (47.6%). CONCLUSION: pp67-mRNA NASBA, an easy and rapid to perform assay, well-standardised by virtue of co-amplified internal system control RNA, provides a high specificity and positive predictive value for the diagnosis of HCMV disease in HIV-infected patients, comparable to that of the quantitative pp65-antigenemia assay and blood culture.     

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.     

Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification

This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.     

Value of different assays for detection of human cytomegalovirus (HCMV) in predicting the development of HCMV disease in human immunodeficiency virus-infected patients

In the present prospective study, five blood tests for detection of human cytomegalovirus (HCMV), nucleic acid sequence-based amplification (NASBA) for detection of early (immediate-early antigen) and late (pp67) mRNA, PCR for detection of HCMV DNA (DNA PCR), culture, and pp65 antigenemia assay, and culture and DNA PCR of urine and throat swab specimens were compared for their abilities to predict the development of disease caused by HCMV (HCMV disease). Of 101 human immunodeficiency virus (HIV)-infected patients with 相似文献   

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.     

Development and application of a novel multiplex polymerase chain reaction for semi-quantitation of human Cytomegalovirus in clinical specimens

Human Cytomegalovirus (HCMV) is an important cause of morbidity and occasional mortality in immunocompromised patients. The aims of the study were to develop and apply a multiplex PCR for semi-quantitation of HCMV in clinical specimens, compare its efficiency with pp65 antigenemia assay and real-time PCR. A multiplex PCR combining the primers targeting three regions of the HCMV genome, viz. the morphological transforming region II (mtr II), UL-83 and glycoprotein O (gO) genes for the detection of the genome of HCMV was standardized with HCMV AD169 strain. This was evaluated against pp65 antigenemia assay by applying it on 70 peripheral blood specimens obtained from 70 post-renal transplant recipients. The multiplex PCR and a real-time PCR were prospectively applied to 31 clinical specimens from 29 immunocompromised patients. The multiplex PCR was specific for HCMV. The level of antigenemia and the copy number of the viral DNA as estimated by real-time PCR in the samples positive for all the three targets was significantly higher than in those that were positive for only one or two of the targets. The multiplex PCR provides a simple and effective means of quantifying HCMV in clinical specimens with efficiency equivalent to the pp65 antigenemia assay and real-time PCR.     

Monitoring of renal allograft recipients by quantisation of human cytomegalovirus genomes in peripheral blood leukocytes

The ratio of human cytomegalovirus (HCMV) genomes per cellular genomes in serial peripheral blood leukocyte (PEL) extracts of renal allograft recipients was quantitated by competitive nested polymerase chain reaction (PCR). Patients were also monitored for the development of acute HCMV infection by detection of HCMV pp65 antigenemia, HCMV IgM antibodies, and viruria. Compared to qualitative nested HCMV PCR, the frequency of positive PCR results in renal allograft recipients without further evidence of acute HCMV infection was significantly reduced by quantitative HCMV PCR. HCMV DMA levels ≥1,000 copies HCMV/10⁶ copies β-globin were found to be highly indicative for the development of a clinically symptomatic HCMV infection following renal allograft transplantation. In patients treated with ganciclovir, quantitation of HCMV target sequences allowed the assessment of the efficacy of antiviral therapy. © 1994 Wiley-Liss, Inc.