巨细胞病毒早期抗原pp65和晚期抗原pp67-mRNA在移植术后活动性巨细胞病毒感染诊断中的价值

目的：通过对器官移植术后受者外周血中巨细胞病毒（CMV）早期抗原pp65和晚期抗原pp67-mRNA的监测,评价其对于诊断器官移植术后CMV感染、发病及预后的价值,为临床准确有效地分析理解检测结果提供依据。方法：收集28例器官移植受者EDTA抗凝外周血标本共120份,用免疫荧光技术（IFA）检测其中CMV早期抗原pp65;用核酸基础序列扩增法（NASBA）测定晚期CMVpp67-mRNA,绘制2组用于诊断CMV的受试者特征曲线（ROC曲线）,比较2组曲线下面积。结果1120份样本中,42份为CMVpp65阳性（≥1个阳性细胞／2×105白细胞）,23份为CMVpp67-mRNA阳性。28例器官移植受者中,9例CMVpp65和pp67-mRNA均为阳性（其中3例pp65和pp67-mRNA在同一时间为阳性）,8例pp65为阳性而pp67-mRNA为阴性,2例pp65为阴性而pp67-mRNA为阳性。临床诊断感染CMV的患者4例,在感染早期无临床症状病毒潜伏期间,受试者工作特征（ROC）曲线下面积（AUC）pp65为0．9542,pp67-mRNA为0．6611,即pp65检测的灵敏度和特异性高于pp67-mRNA;而在出现临床症状并治疗后期,pp65的AUC为0．8300,pp67-mRNA为0．9232,pp67-mRNA的灵敏度和特异性高于pp65。根据ROC曲线查得,以每2x105白细胞中有10个阳性细胞为诊断CMV活动性感染的最佳临界值。结论：AUC结果表明,pp65、pp67-mRNA均具有诊断意义。pp65检测更适于早期CMV活动性诊断,对于提示临床开始抗病毒治疗具有早期快速的意义;pp67-mRNA检测快速、结果准确,可作为结束抗病毒治疗的参考指标;两者联合检测用于监测CMV,对于辅助临床诊断有很好的价值。     

检测巨细胞病毒(CMV)DNA及其即刻早期抗原、pp65和pp67在肾移植受者术后巨细胞病毒感染早期诊断中的价值

目的探讨检测巨细胞病毒(CMV)DNA及其即刻早期抗原(IE)、巨细胞病毒pp65和pp67抗体对肾移植受者术后巨细胞病毒感染早期诊断的临床应用价值。方法按肾移植术受者术后3个月外周血是否出现CMV抗原,将71例患者分为CMV感染组(56例)和CMV未感染组(15例),肾移植术受者手术前和术后第1个月每周检查1次,第2、3个月每2周检查1次外周血巨细胞病毒pp65和巨细胞病毒pp67、即刻早期抗原(immediate early antigen,IE),巨细胞病毒DNA和IgM、IgG,共8次;以监测与分析评价肾移植术受者手术前后各项指标变化。结果肾移植术前71例肾移植受者PP65、PP67、IE、CMV DNA均为阴性;肾移植术后CMV感染组的pp65、pp67、IE、CMV DNA阳性率分别为67.8%(38/56)、66.1%(37/56)、64.2%(36/56)和48.2%(27/56),CMV未感染组4项指标值分别为0%、0%、13.3%(2/15)、和0%,两组差异均有统计学意义(P均0.01)。肾移植术后CMV感染组(56例)和CMV未感染组(15例)CMV IgG均为阳性,而IgM阳性率在CMV感染组仅为3.5%(2/56),在CMV未感染组为0%,IgM表达率在CMV感染组和未感染组无统计学差异(P0.05)。观察期内感染组与未感染组相比,术后CMV pp65,pp67,CMV DNA和IE指标出现阳性的例数及阳性出现的具体时间均有显著性差别(P均0.01),而IgM和IgG则均无显著性差别(P均0.05)。结论肾移植术后患者外周血CMV DNA,IE,pp65和pp67抗原检测阳性与其术后巨细胞病毒感染相关。检测CMV DNA、IE、pp65和pp67抗原可能更早更准确反映器官移植术后CMV活动性感染。而CMV IgG和IgM不能作为肾移植后患者CMV感染的诊断指标。     

A follow up study of cytomegalovirus infection in a group of Turkish renal transplant recipients using molecular assays

The clinical value of an in-house cytomegalovirus nested polymerase chain reaction (CMV-PCR) and a commercial molecular assay hybrid capture CMV DNA assay (HCA) was evaluated in monitoring a group of renal transplant patients for six months follow up. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of nested CMV DNA PCR assay and HCA at the beginning of the study were 70, 42.9, 46.7, 66.7, and 60, 78.6, 66.7, and 73.3% respectively. After six months, they were 80, 66.7, 80, 66.7 for CMV PCR and 73.3, 88.9, 91.7, 66.7% for HCA respectively. These results indicate that in monitoring and predicting CMV infections in renal transplant recipients, not only qualitative but also quantitative assays must be used together in order to decide the preemptive strategies.     

Identification of cytomegalovirus (CMV) infection by different laboratory methods in renal transplant recipients undergoing triple-drug immunosupressive treatment.

Early diagnosis of CMV infection is very important mainly in transplant recipients because CMV infection is a frequent complication after transplantation. In this work we compared different laboratory methods: ELISA (IgG, IgM), Western blot,shell vial, antigenemia assay (pp65), the immunofluorescent method with epithelial cells from urine (IF), DNA in leukocytes by PCR and DNA in leukocytes by hybridization (HCS) to estimate the most proper method for diagnosis of CMV in renal transplant recipients. This preliminary study showed that HCS, PCR and Western blot are sensitive methods for detecting CMV infection. Using HCS in quantitative variant we obtained a very good correlation between DNA load and clinical symptoms.     

肾移植术后巨细胞病毒pp65检测的临床意义

目的探讨肾移植受者术后,巨细胞病毒被膜磷蛋白pp65的检测在活动性巨细胞病毒感染中的意义。方法用间接免疫荧光法检测肾移植受者术后HCMV-pp65,同时用酶联免疫捕获法检测HCMV-IgM抗体,共采集91份血标本。结果 91份血标本中,HCMV-pp65阳性24份（26.4%）,HCMV-IgM抗体阳性4份（4.4%）,在24例HC-MV-pp65抗原血症阳性的患者中,有20例出现HCMV感染症状及HCMV病。结论 HCMV-pp65在活动性巨细胞病毒感染的检测中具有早期、准确的优点,可辅助临床对HCMV感染进行早期诊断与治疗。     

Quantification of Cytomegalovirus Glycoprotein Bn DNA in Hematopoietic Stem Cell Transplant Recipients by Real-time PCR

Based on sequence variation in the N-terminus of the UL55 gene, which encodes glycoprotein B (gB), human cytomegalovirus (CMV) can be classified into four gBn genotypes. We assessed the distribution of CMV gBn genotypes and the correlation between CMV gBn DNA (detected by real-time PCR) and CMV-positive pp65 cells (identified by immunohistochemical staining) in a cohort of hematopoietic stem cell transplant patients. The distribution of gB genotypes was as follows: gBn1, 60% of patients; gBn2, 13.3%; mixed gBn1 and gBn3 infection, 26.7%; and gBn4 and other mixed infections, 0%. CMV gBn1 was the most common genotype. The detected level of CMV gB DNA correlated well with the number of CMV-positive pp65 cells detected by immunostaining (r = 0.585).     

Correlation between CMV genotypes,multiple infections with herpesviruses (HHV-6, 7) and development of CMV disease in kidney recipients in Kuwait

The possible correlation between cytomegalovirus, human herpesvirus types 6, 7 and cytomegalovirus-related clinical symptoms was studied in kidney transplant patients in Kuwait. Cytomegalovirus infection was diagnosed using the pp65 antigenemia assay. DNA of cytomegalovirus was detected by nested polymerase chain reaction (nested-PCR). PCR was also used to amplify the genes coding for structural proteins of human herpesvirus-6 (240 bp) and human herpesvirus-7 (186 bp). Glycoprotein B genotypes of cytomegalovirus were determined by restriction fragment length polymorphism. The average number of cells positive for cytomegalovirus pp65 antigen showed a steady increase with the severity of the cytomegalovirus-related symptoms. Furthermore, cytomegalovirus pp65 antigen positivity was significantly more frequent among recipients of cadaver kidney (45.5%) than among those who received live related kidneys (22.6%). Cytomegalovirus gB genotype 1 was detected more frequently (P<0.036) in recipients with live related donor kidney (38%) than in patients of cadaver kidney (13%). The genome of human herpesvirus-6 was detected at the same rate in patients with or without cytomegalovirus-related symptoms. However, the genome of human herpesvirus-7 was detected significantly more frequently (P<0.0001) in asymptomatic patients (41.7%) than in recipients with symptomatic cytomegalovirus infection (17%). We conclude that cytomegalovirus gB genotypes are not associated with the outcome of a cytomegalovirus infection in kidney transplant patients, that human herpesvirus-6 does not play a role in cytomegalovirus pathogenesis and that the role of human herpesvirus-7 in cytomegalovirus-related morbidity in kidney recipients remains unclear.     

Multicenter evaluation of prototype real‐time PCR assays for Epstein‐Barr virus and cytomegalovirus DNA in whole blood samples from transplant recipients

Quantitative PCR is becoming widespread for diagnosing and monitoring post‐transplantation diseases associated with EBV and CMV. These assays need to be standardized to manage patients in different facilities. Five independent laboratories in Japan compared home‐brew assays and a prototype assay system to establish a standard quantitative procedure for measuring EBV and CMV. Reference standards and a total of 816 (642 EBV and 174 CMV) whole blood samples from post‐transplantation recipients were used for this multicenter evaluation. The prototype reference standard for EBV was compared to a panel of samples, with a theoretical expected value made using EBV‐positive cells containing two virus genome copies per cell. The mean ratio of the reference standard at each site to the standard of the prototype assay was ≤4.15 for EBV among three different sites and ≤3.0 for CMV between two laboratories. The mean of the theoretical expected number of the EBV genome: prototype reference was close to 1.0. The correlation coefficients between the viral copy numbers determined using the prototype assay and those using each home‐brew assay were high (EBV, 0.73–0.83, median = 0.78; CMV, 0.54–0.60, median = 0.57). The dynamics of the EBV and CMV loads in transplant recipients were similar between the assay types. There was an inter‐laboratory difference among the quantification results, indicating that a unified protocol and kit are favorable for standardizing the quantification of EBV and CMV. Such standardization will help to standardize the diagnosis and monitoring of diseases associated with EBV and CMV.     

T and B cell responses to cytomegalovirus antigens in healthy blood donors and bone marrow transplant recipients

Abstract We measured the production of interferon-gamma (IFN-γ) from single T cells and the T cell proliferative response to different cytomegalovirus (CMV) antigens in healthy blood donors and bone marrow transplant recipients. The antigens consisted of a CMV nuclear antigen (CMV na) containing the pp65-kDa matrix protein and the immediate early antigens but lacking CMV glycoproteins, and an antigen comprising native CMV glycoproteins (CMV gp). We also measured the IgG antibodies to CMV na and CMV gp. The T cells reacted to CMV na in CMV seropositive blood donors both with the production of IFN-γ and with proliferation, while bone marrow transplant recipients had a deficient T cell response. After stimulation with CMV gp, no T cell response could be observed in CMV seropositive subjects. IgG antibodies to CMV na coexisted in plasma with similar levels of antibodies to CMV gp.     

Prevalence of human herpesvirus-8 infection in HIV-positive patients with and without Kaposi's sarcoma in Hungary

Human herpesvirus-8 (HHV-8) infection of 130 Hungarian HIV-positive individuals with or without Kaposi's sarcoma was investigated from 158 serum and 122 peripheral blood samples using anti-latency-associated nuclear antigen (LANA) indirect immunofluorescence assay (IFA), recombinant orf65 and orfK8.1 antigen enzyme-linked immunosorbent assays (ELISAs), Western blot assays and orf26 specific nested polymerase chain reaction (PCR). The overall prevalence of HHV-8 infection was found to be 31.5% (41/130) among the Hungarian HIV-positive patients. This seroprevalence rate is 7-11-fold higher than that of healthy HIV-negative blood donors in Hungary. The highest prevalence of HHV-8 infection (36.1%, 35/97) was observed in homo- or bisexual patients. Similar to the serologic results, HHV-8 DNA was not always detectable in all serial samples previously shown to be positive for HHV-8 DNA.     

Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus,Epstein-Barr virus,and polyomavirus BK in whole blood from transplant candidates

Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR) method to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R² = 0.997) and reproducibility (CV range, 0.95–2.38%, 0.52–3.32%, and 0.31–2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.     

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA-peptide complexes

BACKGROUND: Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS: CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT: Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION: This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.     

sodC-based real-time PCR for detection of Neisseria meningitidis

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (C(t)) value of 35, with 100% average reaction efficiency and an average R(2) of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average C(t) values from 13.0 to 29.5. The mean sodC C(t) value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.     

Heat denaturation increases the sensitivity of the cytomegalovirus loop‐mediated isothermal amplification method

A new protocol for CMV LAMP with an additional heat denaturation step was developed. While the sensitivity of the original CMV LAMP method was 500 copies/tube, sensitivity was increased by up to 100 copies/tube by additional heat denaturation. CMV DNA was detected in 103 of 350 samples (29.4%) by the original CMV LAMP procedure and 148 of 350 samples (42.3%) by the new CMV LAMP protocol. When the pp65 antigenemia assay was used as the standard method, the sensitivity, specificity, PPV, and NPV of the new protocol were 92.9%, 77.7%, 62.2%, and 96.5%, respectively.     

Changes in cell-mediated immunity in kidney transplant recipients with active CMV infection

This study was aimed at determining (a) the extent of proliferation of peripheral blood mononuclear cells (PBMC) in response to stimulation by cytomegalovirus (CMV)-infected fibroblasts and (b) the levels of Th1 and Th2 cytokine production in kidney transplant recipients with and without active CMV infection. Thirty patients with, and 39 without active CMV infection, diagnosed by a CMV antigenemia assay (AA), were studied. PBMC of patients with active CMV infection showed significantly lower proliferation than those without ongoing CMV infection (P<0.0001). The levels of Th2-type cytokines (interleukin (IL-) 4 and IL-10) in AA-negative and AA-positive kidney transplant recipients were similar but the levels of the Th1-type cytokines interferon-gamma, tumor necrosis factor-alpha (P<0.05) and IL-2 were significantly lower in AA-positive kidney transplant recipients (P<0.0005).     

Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors

CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i.e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors.