异基因造血干细胞移植中巨细胞病毒的检测及临床意义
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摘要
背景与目的：异基因造血干细胞移植(allo-HSCT)成为近年来治疗恶性血液病、再生障碍性贫血、自身免疫性疾病、某些遗传性疾病及实体肿瘤的一种重要的有效手段。人类巨细胞病毒(human cytomegalovirus，hCMV)感染是造血干细胞移植患者早期的主要感染并发症及死亡原因，既往因CMV感染导致的间质性肺炎死亡率高达80％以上。CMV属于人类疱疹病毒乙组DNA病毒，在人群中感染非常普遍(80％～100％)，正常人感染CMV后，由于机体免疫功能正常，CMV在感染细胞内复制水平低下，以一种潜伏整合状态存在，通常无临床症状。当机体免疫功能尤其是细胞免疫功能受到抑制，潜伏的CMV可被重新激活，从而持续高水平的复制，引起CMV活动性感染，产生有严重临床症状的CMV感染性疾病。移植物抗宿主病(GVHD)是异基因造血干细胞移植的主要障碍，CMV感染可以诱导GVHD出现：CMV感染还可影响造血干细胞的植入，使造血重建延迟；引起间质性肺炎；是继发曲霉菌感染的高危因素，易并发细菌、真菌、原虫等混合感染，从而造成移植失败。近年来，随着大量新型免疫抑制剂的出现及多种免疫抑制剂联合应用，非亲缘关系造血干细胞移植、单倍体造血干细胞移植、非清髓造血干细胞移植广泛开展，造血干细胞移植得到快速发展。同时巨细胞病毒感染率也不断增高，已引起医学界高度重视，CMV感染引起严重疾病再次成为医学领域的研究热点。虽然目前更昔洛韦的使用使CMV疾病的治疗有较大转机，但取得抗CMV显著疗效的切入点还在于早期诊断和及时治疗。在CMV病发生之前，及时应用抗病毒治疗，可以预防和减少CMV病的发生，
郑州大学2004届硕士研究生毕业论文
异基因造血干细胞移植中巨细胞病毒的检测及临床意义
降低移植受者的死亡率。巨细胞病毒活动性感染早期所致疾病临床表现多为非特
异性的，早期诊断主要依靠实验室检测。尽管目前诊断CMV手段日臻完善，但
诊断方法多种多样，哪种方法更能简便快捷、灵敏可靠地早期诊断，从而进行早
期干预性治疗争议很大。本研究分别应用ELISA、流式细胞仪、荧光定量PCR
检测巨细胞病毒IgG、lgM抗体、pp65抗原、DNA负荷定量，旨在探讨HcMv
的早期有效诊断方法。
材料与方法:本研究分别采用酶联免疫吸附法(ELISA)检测血清巨细胞病
毒抗体IgG、IgM;流式细胞仪检测巨细胞病毒抗原pp65;荧光聚合酶链反应
(FQ一PCR)核酸扩增技术检测血浆巨细胞病毒DNA负荷量。选择造血干细胞
移植术受者(移植组)预处理前常规检测lgG、IgM抗体，排除IgM抗体阳性移
植患者。于回输当天、回输后每10天左右;正常造血干细胞移植健康供者(正
常组)，于粒细胞集落刺激因子(G一CSF)动员之前;长期化疗的白血病患者(化
疗组)于化疗后白细胞升至2.0xl夕/L时，同时检测巨细胞病毒血清IgG、IgM
抗体、粒细胞pp65抗原、血浆DNA负荷量。ELISA采用间接法，待测抗原包
被微孔、洗板、加抗原一酶结合复合物、孵育后洗板，加底物液后置室温暗处显
色，用酶联免疫检测仪(波长45Onln)测定OD值。流式细胞仪检测采用单色间
标法，取标本分离单个核细胞、固定、破膜、分别加入pp“抗体和小鼠IgGI
抗原(对照)，再加FITC标记的兔抗鼠IgGI抗体(二抗)，用流式细胞仪检测
阳性细胞数比例。荧光定量PCR检测采用外标法，用DNA提取液提取血浆DNA
后和阴、阳性对照一起加入荧光引物、Taq酶等反应液，置于荧光PCR扩增仪
测定DNA含量。统计学分析:所有资料采用SPSS 10.0软件处理，a=0.05为显
著性检验水准。
结果:
(l)巨细胞病毒IgG抗体阳性检出率在正常组、化疗组、移植组之间分别
为82.24%，81 .48%和86.91%，三者之间的差别无统计学意义(尸>0.05)。
(2)在化疗组中IgM抗体、pp65抗原、DNA定量三者阳性检出率差别无
统计学意义(P>0.05)o
(3)正常组中巨细胞病毒lgM抗体和DNA荧光定量、pp65抗原流式细胞
仪检测结果基本相同。
郑州大学2004届硕士研究生毕业论文
异基因造血干细胞移植中巨细胞病毒的检测及临床意义
(4)移植组中巨细胞病毒IgM抗体阳性检测率明显低于DNA荧光定量和
pp65抗原流式细胞仪检出率，其差别有统计学意义(p<0.05)。两种检测方法
有关系，但关系不密切。
(5)无论在移植组还是化疗组中巨细胞病毒DNA荧光定量检测与pp65抗
原流式细胞仪阳性检出率的差别都无统计学意义(尸>0.05)，两种检测方法有明
显的相关性。
(6)lgM抗体、I〕NA荧光定量和pp65抗原流式细胞仪检测阳性率各自在
化疗组和移植组之间的差异具有显著性(P<0.05)。
(7)移植组治疗中应用免疫抑制剂三种及三种以上与三种以下在DNA荧
光定量和pp65抗原流式细胞仪检测阳性率的差异有统计学意义(尸<0.05)。
结论:
(1) ELIsA法检测血清cMV IgG和IgM抗体相结合可以用于造血干细胞
移植术供者的选择。IgM抗体不适用于移植术后早期诊断CMV激活感染，但可
用于化疗后CMV活动性感染的诊断，还可以用于筛选阳性受者及时采取措施。
(2)流式细胞仪检测CMV PP65抗原快捷、客观、易于定量，适用于造血
干细胞移植术后造血恢复以后CMV感染的诊断。
(3)荧光定量PCR检测血浆CMV DNA病毒方法简单快速、
Background and Objective: Allogeneic hematopoietic stem cell transplantation, allo-HSCT has been an effective way to treat malignant hematologic disease, aplastic anemia, autoimmune diseases and some solid tumors. Human cytomegalo-virus (hCMV) infection is the early main infection and death-cause to the recipients of allo-HSCT. The prevenient mortality of interstitial pneumonia (I?) caused by CMV infection was above 80 precent. CMV belongs to DNA virus of human herpersviruses, and CMV is very common among people. When infecting normal people, because of the normal immune function of the host, CMV reproduces slow, stays in a latent and concordant state and usually causes no clinical syndrome. When the host immune function especially the cellar immunofunction is suppressed, the latent CMV can be reactivated, and CMV reproduce in a high level constantly, the causes CMV active infection and CMV infectious disease with serious clinical syndromes. At the same time the infectious rate of CMV has been increasing con
stantly and drawing much attention to by the medical field, then the serious disease caused by CMV infection became a focal point of medical research again. Graft-verse-host disease, GVHD is a main obstacle to the allo-HSCT, and CMV can induce GVHD; affluence the survival of HSCT, delaying the hematopoiesis rebuilding; causing IP. It is a high-dangerous factors of secondary aspergillosis infection, also can be subject to bacteria, fungi and
protozoon infections ,thus lead to to the failure of HSCT. As the occurrence and combined application of many new immune inhibitors, unrelated-HSCT, haploid-HSCT, non-myeloablative-HSCT developed extensively, and HSCT has been developed quickly in recent years. Though the treatments of CMV using ganciclovirus have turned for better, the key of antivirus therapy depends on early-stage diagnose and timely treatment. Using antivirus treatment in time before the occurrence of CMV diseases can prevent and decrease the occurrence of the disease and lower the mortality of HSCT recipients . The early clinical signs and symptoms of the disease by CMV active infection are mostly non-specific, and its early diagnosis mainly depends on laboratorial examination. At present methods for CMV diagnosis are various. Inspite of the more intact diagnostic methods, there is much controversy on which method is simpler, more sensitive and reliable to diagnose CMV early, and then early intervening treatment is in great dilemma. In this paper, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), fluorescence quantitative PCR (FQ-PCR/real-time PCR) to examine IgG antibodies and IgM antibodies of CMV, pp65 antigen, quantity of DNA for exploring an effective way to diagnose early CMV infection.
Methods and Material: In our research, we used ELISA to detect IgG antibodies, IgM antibodies of CMV; used FCM to detect pp65 antigen; used FQ-PCR to examine HCMV DNA load of blood plasma. Before conditioning we examined CMV IgG, IgM and excluded IgM positive HSCT recipients from our research. We simultaneously examined CMV IgG, IgM, pp65 antigen and DNA load of recipients (the transplantation group) at the day of transfusion of hematopoietic stem cell, and every 10 days after the transfusion of hematopoietic stem cells, of normal HSCT donors (the normal group) before G-CSF mobilization; of leukemic patients (the chemotherapy group)with long term chemotherapy when their WBC to 2.0x109/L after chemo- therapy. We used indirect ELISA: the antigen to examine was in lamellan aperture, washing plate, added Ag-enzyme complex, washing plate again after incubation added substratum, put in darkness to colorate in room temperature,
then examined OD valves by enzyme immunoassay (X=450nm); we used indirect simple color method by FCM selecting single-nuclear cells, then fixing, effecting cell membrance, adding pp65 antibodies and mouse IgG1 antigen (the control) then adding rat-anti-mouse IgGl (antibody), then detecting the proportion of positive cells. The FQ-PCR took the outer-marking method: abstrac
郑州大学2004届硕士研究生毕业论文
异基因造血干细胞移植中巨细胞病毒的检测及临床意义
降低移植受者的死亡率。巨细胞病毒活动性感染早期所致疾病临床表现多为非特
异性的，早期诊断主要依靠实验室检测。尽管目前诊断CMV手段日臻完善，但
诊断方法多种多样，哪种方法更能简便快捷、灵敏可靠地早期诊断，从而进行早
期干预性治疗争议很大。本研究分别应用ELISA、流式细胞仪、荧光定量PCR
检测巨细胞病毒IgG、lgM抗体、pp65抗原、DNA负荷定量，旨在探讨HcMv
的早期有效诊断方法。
材料与方法:本研究分别采用酶联免疫吸附法(ELISA)检测血清巨细胞病
毒抗体IgG、IgM;流式细胞仪检测巨细胞病毒抗原pp65;荧光聚合酶链反应
(FQ一PCR)核酸扩增技术检测血浆巨细胞病毒DNA负荷量。选择造血干细胞
移植术受者(移植组)预处理前常规检测lgG、IgM抗体，排除IgM抗体阳性移
植患者。于回输当天、回输后每10天左右;正常造血干细胞移植健康供者(正
常组)，于粒细胞集落刺激因子(G一CSF)动员之前;长期化疗的白血病患者(化
疗组)于化疗后白细胞升至2.0xl夕/L时，同时检测巨细胞病毒血清IgG、IgM
抗体、粒细胞pp65抗原、血浆DNA负荷量。ELISA采用间接法，待测抗原包
被微孔、洗板、加抗原一酶结合复合物、孵育后洗板，加底物液后置室温暗处显
色，用酶联免疫检测仪(波长45Onln)测定OD值。流式细胞仪检测采用单色间
标法，取标本分离单个核细胞、固定、破膜、分别加入pp“抗体和小鼠IgGI
抗原(对照)，再加FITC标记的兔抗鼠IgGI抗体(二抗)，用流式细胞仪检测
阳性细胞数比例。荧光定量PCR检测采用外标法，用DNA提取液提取血浆DNA
后和阴、阳性对照一起加入荧光引物、Taq酶等反应液，置于荧光PCR扩增仪
测定DNA含量。统计学分析:所有资料采用SPSS 10.0软件处理，a=0.05为显
著性检验水准。
结果:
(l)巨细胞病毒IgG抗体阳性检出率在正常组、化疗组、移植组之间分别
为82.24%，81 .48%和86.91%，三者之间的差别无统计学意义(尸>0.05)。
(2)在化疗组中IgM抗体、pp65抗原、DNA定量三者阳性检出率差别无
统计学意义(P>0.05)o
(3)正常组中巨细胞病毒lgM抗体和DNA荧光定量、pp65抗原流式细胞
仪检测结果基本相同。
郑州大学2004届硕士研究生毕业论文
异基因造血干细胞移植中巨细胞病毒的检测及临床意义
(4)移植组中巨细胞病毒IgM抗体阳性检测率明显低于DNA荧光定量和
pp65抗原流式细胞仪检出率，其差别有统计学意义(p<0.05)。两种检测方法
有关系，但关系不密切。
(5)无论在移植组还是化疗组中巨细胞病毒DNA荧光定量检测与pp65抗
原流式细胞仪阳性检出率的差别都无统计学意义(尸>0.05)，两种检测方法有明
显的相关性。
(6)lgM抗体、I〕NA荧光定量和pp65抗原流式细胞仪检测阳性率各自在
化疗组和移植组之间的差异具有显著性(P<0.05)。
(7)移植组治疗中应用免疫抑制剂三种及三种以上与三种以下在DNA荧
光定量和pp65抗原流式细胞仪检测阳性率的差异有统计学意义(尸<0.05)。
结论:
(1) ELIsA法检测血清cMV IgG和IgM抗体相结合可以用于造血干细胞
移植术供者的选择。IgM抗体不适用于移植术后早期诊断CMV激活感染，但可
用于化疗后CMV活动性感染的诊断，还可以用于筛选阳性受者及时采取措施。
(2)流式细胞仪检测CMV PP65抗原快捷、客观、易于定量，适用于造血
干细胞移植术后造血恢复以后CMV感染的诊断。
(3)荧光定量PCR检测血浆CMV DNA病毒方法简单快速、
Background and Objective: Allogeneic hematopoietic stem cell transplantation, allo-HSCT has been an effective way to treat malignant hematologic disease, aplastic anemia, autoimmune diseases and some solid tumors. Human cytomegalo-virus (hCMV) infection is the early main infection and death-cause to the recipients of allo-HSCT. The prevenient mortality of interstitial pneumonia (I?) caused by CMV infection was above 80 precent. CMV belongs to DNA virus of human herpersviruses, and CMV is very common among people. When infecting normal people, because of the normal immune function of the host, CMV reproduces slow, stays in a latent and concordant state and usually causes no clinical syndrome. When the host immune function especially the cellar immunofunction is suppressed, the latent CMV can be reactivated, and CMV reproduce in a high level constantly, the causes CMV active infection and CMV infectious disease with serious clinical syndromes. At the same time the infectious rate of CMV has been increasing con
stantly and drawing much attention to by the medical field, then the serious disease caused by CMV infection became a focal point of medical research again. Graft-verse-host disease, GVHD is a main obstacle to the allo-HSCT, and CMV can induce GVHD; affluence the survival of HSCT, delaying the hematopoiesis rebuilding; causing IP. It is a high-dangerous factors of secondary aspergillosis infection, also can be subject to bacteria, fungi and
protozoon infections ,thus lead to to the failure of HSCT. As the occurrence and combined application of many new immune inhibitors, unrelated-HSCT, haploid-HSCT, non-myeloablative-HSCT developed extensively, and HSCT has been developed quickly in recent years. Though the treatments of CMV using ganciclovirus have turned for better, the key of antivirus therapy depends on early-stage diagnose and timely treatment. Using antivirus treatment in time before the occurrence of CMV diseases can prevent and decrease the occurrence of the disease and lower the mortality of HSCT recipients . The early clinical signs and symptoms of the disease by CMV active infection are mostly non-specific, and its early diagnosis mainly depends on laboratorial examination. At present methods for CMV diagnosis are various. Inspite of the more intact diagnostic methods, there is much controversy on which method is simpler, more sensitive and reliable to diagnose CMV early, and then early intervening treatment is in great dilemma. In this paper, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), fluorescence quantitative PCR (FQ-PCR/real-time PCR) to examine IgG antibodies and IgM antibodies of CMV, pp65 antigen, quantity of DNA for exploring an effective way to diagnose early CMV infection.
Methods and Material: In our research, we used ELISA to detect IgG antibodies, IgM antibodies of CMV; used FCM to detect pp65 antigen; used FQ-PCR to examine HCMV DNA load of blood plasma. Before conditioning we examined CMV IgG, IgM and excluded IgM positive HSCT recipients from our research. We simultaneously examined CMV IgG, IgM, pp65 antigen and DNA load of recipients (the transplantation group) at the day of transfusion of hematopoietic stem cell, and every 10 days after the transfusion of hematopoietic stem cells, of normal HSCT donors (the normal group) before G-CSF mobilization; of leukemic patients (the chemotherapy group)with long term chemotherapy when their WBC to 2.0x109/L after chemo- therapy. We used indirect ELISA: the antigen to examine was in lamellan aperture, washing plate, added Ag-enzyme complex, washing plate again after incubation added substratum, put in darkness to colorate in room temperature,
then examined OD valves by enzyme immunoassay (X=450nm); we used indirect simple color method by FCM selecting single-nuclear cells, then fixing, effecting cell membrance, adding pp65 antibodies and mouse IgG1 antigen (the control) then adding rat-anti-mouse IgGl (antibody), then detecting the proportion of positive cells. The FQ-PCR took the outer-marking method: abstrac
引文
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and US11 Differ in Their Ability To Attack Major Histocompatibility Class Ⅰ Heavy Chains in Dendritic Cells. J Virol, May 2002; 76(10): 5043-5050.
23. Raftery MJ, Schwab M, Eibert SM, et al. Targeting the function of mature dendritic cells by human cytomegalovirus: a multilayered viral defense strategy. Immunity, Dec 2001; 15(6): 997-1009.
24. Kline JN, Hunninghake GM, He B, et al. Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines. Exp Lung Res, Jan 1998; 24(1): 3-14.
25. Crapnell K, Zanjani ED, Chaudhuri A, et al. In vitro infection of megakaryoeytes and their precursors by human cytomegalovirus. Blood, Jan 2000; 95:487-493.
26. Lagneaux L, Delforge A, Snoeck R, et al. Decreased production of cytokines after cytomegalovirus infection of marrow-derived stromal cells. Exp Hematol, Jan 1994; 22(1): 26-30.
27, Mori T, Ando K, Tanaka K, et al. Fas-Mediated Apoptosis of the Hematopoietic Progenitor Cells in Mice Infected With Murine Cytomegatovirus. Blood, May 1997; 89:3565-3573.
28. Sindre, H Rollag, H Olafsen MK, et al. Human eytomegalovirus induces apoptosis in the hematopoietic cell line MO7e. APMIS, Mar 2000; 108(3): 223-230.
29. Sarniento JM, Dockrell DH, Schwad TR, et al. Mycophenolate mofetil increases cytomegalovirus invasive organ disease in transplant patients. Clin Transplant 2000; 14: 136-138.
30. Mansy F, Brancart F, Liesnard C, et al. A PCR based DNA hybridisation capture system for the detection of human cytomegalovirus. A comparative study with other identification methods [J]. J Virol Methods, Jul 1999; 80(2): 113-122.
31. Ruellan-Eugene G, Barjot P, Campet M, et al. Evalution of virological procedures to detect fetal human cytomegalovirus infection: Avidity of IgG antibodies virus detection in amniotie fluid and maternal serum [J]. J Med Virol, 1996; 50: 9-15.
32. Sia IG, Patel R. New strategies for prevention and therapy of cytomegalovirus infection and disease in solid-organ transplant recipients. Clin Microbiol Rev 2000; 13(1): 83-122.
33. Eing BR, Baumeister H G., Kuehn J E, et al. Long-Term Persistence of Immunoglobulin A (IgA) and IgM Antibodies against Human Cytomegalovirus in Solid-Organ Transplant Recipients. Clin Diagn Lab Immunol, Jul 1999; 6(4): 621-623.
34. Bendiksen S, Van Ghelue M, Rekvig OP, et al. A longitudinal study of human cytomegalovirus serology and viruria fails to detect active viral infection in 20 systemic lupus ergthematosus.patients. Lupus, 2000; 9:120-126.
35. Weber B, Berger A, Rabenau H, et al. Human cytomegalovirus infection: diagnostic potential of recombinant antigens for cytomegalovirus antibody detection[J]. J Viorl Methods, 2001; 96(2): 157-170.
36. Pande H, Campo K, Tanamachi B, et al. Human cytomegalovirus strain Towne pp65 gene: nucleotide sequence and expression in Escherichia coli. Virology, May 1991; 182(1): 220-228.
37. Grefte JMM, van der Gun BT, Schmolke S, et al. The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocytes during an active cytomegalovirus infection. J Gen Virol, Nov 1992; 73:2923-2932.
38. Gyulai Z, Endresz V, Burian K, et al. Cytotoxic T lymphocyte(CTL) responses to human cytomegalovirus pp65, IE_1-Exon 4, gB, pp150 and pp28 in healthy individual: reevalution of prevalence of IEl—specific CTLs. J Infect Dis, 2001; 181(5): 1537-1546.
39. Wills MR, Carmichael AJ, Mynard K, et al. The human eytotoxic T lymphocyte (CTL) responses to CMV is dominated by structural protein pp65:frequency, specificy and T-cell recpptor usage of pp65-specific CTL. J Virol, 1996; 70: 7569-7579.
40. Boppana SB, Britt WJ, et al. Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells. Virology, Aug 1996; 222(1): 293-296.
41. Wills MR, Carmichael AJ, Mynard K, et al. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol, Nov 1996; 70(11): 7569-7579.
42. Beninga J, Kropff B, Mach M ,et al. Comparative analysis of fourteen individual
human cytomegalovirus proteins for helper T cell response. J Gen Virol, Jan 1995; 76(pt.1): 153-160.
43. Weekes MP, Wills MR, Mynard K, et al. The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual PeptideSpecific CTL Clones That Have Undergone Extensive Expansion In Vivo. J Virol, Mar 1999; 73(3): 2099-2108.
44. Hensel G, Meyer H, Gartner S, et al. Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32). J Gen Virol, Jul 1995; 76: 1591-1601.
45. van der Bij W, van Dijk RB, van Son WJ, et al. Antigen test for early diagnosis of active cytomegalovirus infection in heart transplant recipients. J Heart Transplant, Mar 1988; 7(2): 106-109.
46. ST George K, Boyd MJ, Lipson SM, et al. A multisite trail comparing two eytomegalovirus (CMV) pp65 antigenemia test Kits, biotest CMV bdte and Bartel/argene CMV antigenemia. J Clin Microbiol, 2000; 38:1430-1433.
47. Morello SC, Cranmer LD, Spector DH. In vivo replication, Latency, and Immunogenicity of murine cytomegalovirus mutants with detection in ate M83 and M84 genes, the putative homologs of human cytomegalovirus pp65(UL83). J Virology, 1999; 73: 7678-7693.
48. Borzi R M, Piacentini A, Monaco MC G, et al. A fluorescent in situ hybridization method in flow cytometry to detect HⅣ-1 specific RNA [J]. J Immunol Methos, 1996; 193: 167-176.
49. Kas-Deelen AM, Harmsen MC, De-Maar EF, et al. A sensitive methods for quantifying cytomegalic endothelial cells in peripheral blood from cytomegalovirus-infected patients [J]. Clin Diagn Lab Immunol, 1998; 5: 622-626.
50. Fowler KB, Dahle AJ, Boppana SB, et al. Newborn hearing screening:will children with hearing loss caused by congenital cytomegalovirus infection be missed? J Pediatrics, 1999; 135: 60-64.
51. Woodard P, Tong X, Richardson S, et al. Etiology and outcome of graft failure in pediatric hematopoietic stem cell transplant recipients. J Pediatr Hematol Oncol, 2003
Dec;25(12):955-999
52. Gault E, Michel Y, Dehee A, et al. Quantification of human cytomegalovirus DNA by real-time PCR. J Clin Microbiol, Feb 2001; 39(2): 772-775.
53. Nakamura Y, Sakarna SA, Ohta Y, et al. Detectation of the human cytomegalovirus gene in placental chronic villitis by polymerase chain reaction[J]. Human Pathology, 1994; 25(8): 815-818.
54. Sia IG, Wilson JA, Espy M J, et al. Evaluation of the COBAS AMPLICOR CMV MONITOR Test for Detection of Viral DNA in Specimens Taken from Patients after Liver Transplantation. J Clin Microbiol, Feb 2000; 38:600-606.
55. Rowley A H, Wolinsky S W, Sambol S P, et al. Rapid detection of cytomegalovirus DNA or RNA in blood of renal transplant patients by in vitro enzymatic amplification. Transplantation, 1991; 51 (5): 1028-1033.
56. Hamprecht K, Witzel S, Maschmann J, et al. Rapid detection and quantification of cell free cytomegalovirus by a high-speed centrifugation-based microculture assay: comparison to longitudinally analyzed viral DNA load and pp67 late transcript during lactation. J Clin Virol, Dec 2003; 28(3): 303-316.
57. Funato T, Satoh J, Sasaki T. Quantitative PCR system [J]. Rinsho Byori 1998; 46(5): 399-405.
58. Najioullah F, Thouvenot D, Lina B. Developmennt of a real-time PCR procedure including an internal control for the measurement of HCMV viral load. J Virol Methods, Mar 2001; 92(1): 55-64.
59. Livak KJ, Flood SJA, Marmaro J, et al. Oligonudetide with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Applic, 1995; 4(6): 357-362.
60. Higuehi R, Dollinger G, Walsh PS, et al. Stimustaneous amplification and detection of specific DNA sequence. Biotechnology, 1992; 10(4): 413-417.
61. Keams AM, Guiver M, James V, et al. Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus [J]. J Viorl Methods, Jun 2001(1-2); 95: 121-131.
62. Roberts TC, Brennan DC, Buller RS, et al. Quantitative polymerase chain reaction
to predication occurrence of symptomatic cytomegalovirus infection and assess response to ganciclovir therapy in renal transplant recipients. Infect Dis, Sep 1998; 178(3): 626-635.
63. Li H, Dummer JS, Estes WR, et al. Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J Clin Microbiol, Jan 2003; 41(1): 187-191.
64. Guiver M, Fox AJ, Mutton K, et al. Evaluation of CMV viral load using taqman CMV quantitative PCR and comparison with CMV antigenemia heart and lung transplant recipients [J], Transplantation, 2001; 71 (11): 1609-1615.
65. Flexman J, Kay I, Fonte R, et al. Differences between the quantitative antigenemia assay and the cobas amplicor monitor quantitative PCR assay for detecting CMV viraemia in bone marrow and solid organ transplant patients. J Med Virol, 2001 Jul; 64(3): 275-282.
66. Boeckh M, Huang M, Ferrenberg J, et al. Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol, Mar 2004; 42(3): 1142-1148.
67. Gouarin S, Vabret A, Gault E,et al.Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay. J Clin Virol, Mar 2004; 29(3): 194-201.
68. Cortez KJ, Fischer SH, Fahle GA, et al. Clinical trial of quantitative real-time polymerase chain reaction for detection of cytomegalovirus in peripheral blood of allogeneic hematopoietie stem-cell transplant recipients. J Infect Dis, 2003 Oct 1; 188(7): 967-972.
69. Barrett-Muir W, Breuer J, Millar C, et al. CMV viral load measurement in whole blood and plasma-which is best following renal transplantation? [J]. Transplantation, Jul 2000; 70(1): 116-119.
70．谢万灼，林茂芳．骨髓移植后巨细胞病毒感染的防治研究．国外医学输血及血液学杂志，2001；24：69-71．
71. Tanaka Y, Kanda Y, Kami M, et al. Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after
hematopoietic stem cell transplantation. Bone Marrow Transplant, 2002; 30:315-319.
72. Evans PC, Gray JJ, Wreghitt TG, et al. Comparison of three PCR techniques for detecting cytomegalovirus (CMV) DNA in serum, detection of early antigen fluorescent foci and culture for the diagnosis of CMV infection. J Med Microbiol, Nov 1999; 48(11): 1029-1035.
73. Butt N M, Clark R E. High frequency of positive surveillance for cytomegalovirus by PCR in allograft recipients at low risk of CMV. Bone Marrow Transplant, 2001; 27: 615-619.
74. Sia IG, Wilson JA, Groettum CM, et al. Cytomegalovirus (CMV) DNA load predicts relapsing CMV infection after solid organ transplantation. J Infect Dis, Feb 2000; 181(2): 717-720.
75. Solano C, Munoz I, Gutierrez A. Qualitative plasma PCR assay (AMPLICOR CMV test) versus pp65 antigenemia assay for monitoring cytomegalovirus viremia and guiding preemptive ganciclovir therapy in allogeneic stem cell transplantation. J Clin Microbiol, 2003; 39: 3938-3941.
76. Pellegrin I, Garrigue I, Ekouevi D, et al. New molecular assays to predict occurrence of cytomegalovirus disease in renal transplant recipients. J Infect Dis, Jul 2000; 182(1): 36-42.
77. Hanfler J, Kreuzer KA, Laurisch K, et al. Quantitation of cytomegalovirus (hCMV) DNA and beta-actin DNA by duplex real-time fluorescence PCR in solid organ (liver) transplant recipients. Med Microbiol Immunol, Nov 2003; 192 (4): 197-204.
78. Li Haijiang, Dummer JS, Estes WR. Measurement of cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J Clinical Microbiology, 2003; 41: 187-191.
79. Kerns AM, Guiver M, James V, et al. Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus. J Virological Methods, 2001; 95: 121-131.
80. Weinberg A, Schissel D, Giller R, et al. Molecular methods for cytomegalovirus surveillance in bone marrow transplant recipients. J Clin Microbiology 2002; 40:
4203-4206.
81. Nakamura R, Cortez K, Solomon S, et al. High-dose acyclovir and preemptive gancielovir to prevent cytomegalovirus disease in myeloablative and nonmyeloablative transplantation allogeneic stem cell transplantation. Bone Marrow Transplant, 2002; 30: 235-242.
2. Machado CM, Dulley FL, Vilas Boas LS, et al. CMV pneumonia in allogeneic BMT recipients undergoing early tréatment or pre-emptive ganciclovir therapy. Bone Marrow Transplant, 2000; 26: 413-417.
3. Trenschel R, Ross S, Husing J, et al. Reduced risk of persisting cytomegalovirus pp65 antigenemia and cytomegalovirus interstitial pneumonia following allogeneic PBSCT. Bone Marrow Transplant, 2000; 25: 665-672.
4. Taplitz RA, Jordan MC. Pneumonia caused by herpersviruscs in recipient of hematopoietic cell transplants. Seminars in Respiratory Infections, Jun 2002; 17 (2): 121-129.
5. Zaia JA. Prevention of cytomegalovirus disease in hematopoietie stem cell transplantation. Immunoeompromised Hosts, 2002; 35: 999-1002.
6. Einsele H, Hebart H, Kauffmann-Sehneider C, et al. Risk factor for treatment failure in patients receiving PCR-based preemptive therapy for CMV infection. Bone Marrow Transplant, 2000; 25: 757-763.
7. Emery VC. Investigation of CMV disease in immunoeompromised patients. J Clin Pathol, 2001; 54: 84-88.
8. Keever-Taylor CA, Bredeson C, Loberiza FR, et al. Analysis of risk factors for the development of GVHD after T cell-depleted allogeneic BMT: effect of HLA disparity, ABO incompatibility, and method of T-cell depletion. Biol Blood Marrow Transplant, Jan 2001; 7(11): 620-630.
9. Takemoto Y, Takatsaka H, Wada H, et al. Evaluation of CMV/herpes virus-6, positivity in bronehoalveolar lavage fluid as early detection of acute GVHD following BMT: evidence of a significant relationship. Bone Marrow Transplant, 2000; 26 (1): 77-81.
10. Miehelson S. Interaction of human eytomegalovirus with monocytes/maerophages: a love-hate relationship. Pathol Biol (Paris), 1997; 45: 146-158.
11. Miller DM, Cebulla CM, Rahill BM, et al. Cytomegalovirus and transcriptional down-regulation of majior histocompatiblity complex class Ⅱ expression. Semin Immunol, 2001; 13:11-18.
12. Marr KA, Carter RA, Boeekh M, Ivsive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors. Blood, 2002; 100: 4358-4366.
13. Revello MG, Gerna G. Diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant. Clin Microbiol Rev, 2002; 15(4): 680-715.
14. Zaia JA, Prevention and management of CMV-related problem after hematopoietic stem cell transplantation. Bone Marrow Transplant, 2002; 29: 633-638.
15. Koffron AJ, Hummel M, Patterson BK, et al. Cellular Localization of Latent Murine Cytomegalovirus. J Virol, Jan 1998; 72(1): 95-103.
16. Zhuravskaya T, Maciejewshi JP, et al. Spread of Human Cytomegalovirus (HCMV) After Infection of Human Hematopoietic Progenitor Cells: Model of HCMV Latency. Blood, Sep 1997; 90(6): 2482-2491.
17. Soderberg-Naucler C, Fish KN, Nelson JA. Reactivation of latent human eytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell, Oct 1997; 91(1): 119-126.
18. Lee CH, Lee GC, Chan YJ, et al. Factors affecting human cytomegalovirus gene expression in human monocyte cell lines. Mol Cells, Feb 1999; 9(1): 37-44.
19. Fish KN, Britt W, Nelson JA. A novel mechanism for persistence of human cytomegalovirus in macrophages. J Virol, Mar 1996; 70(3): 1855-1862.
20. Grigoleit U, Riegler S, Einsele H. Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-dedved immature dendritic cells. Br J Haematol, Oct 2002; 119(1): 189-198.
21. Moutaftsi M, Mehl AM, Borysiewicz LK. Human cytomegalovirus inhibits maturation and impairs function of monocyte-derived dendritic cells. Blood, Apr 2002; 99(8): 2913-2921.
22. Rehm A, Engeisberg A, Tortoella D. Human Cytomegalovirus Gene Products US2
and US11 Differ in Their Ability To Attack Major Histocompatibility Class Ⅰ Heavy Chains in Dendritic Cells. J Virol, May 2002; 76(10): 5043-5050.
23. Raftery MJ, Schwab M, Eibert SM, et al. Targeting the function of mature dendritic cells by human cytomegalovirus: a multilayered viral defense strategy. Immunity, Dec 2001; 15(6): 997-1009.
24. Kline JN, Hunninghake GM, He B, et al. Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines. Exp Lung Res, Jan 1998; 24(1): 3-14.
25. Crapnell K, Zanjani ED, Chaudhuri A, et al. In vitro infection of megakaryoeytes and their precursors by human cytomegalovirus. Blood, Jan 2000; 95:487-493.
26. Lagneaux L, Delforge A, Snoeck R, et al. Decreased production of cytokines after cytomegalovirus infection of marrow-derived stromal cells. Exp Hematol, Jan 1994; 22(1): 26-30.
27, Mori T, Ando K, Tanaka K, et al. Fas-Mediated Apoptosis of the Hematopoietic Progenitor Cells in Mice Infected With Murine Cytomegatovirus. Blood, May 1997; 89:3565-3573.
28. Sindre, H Rollag, H Olafsen MK, et al. Human eytomegalovirus induces apoptosis in the hematopoietic cell line MO7e. APMIS, Mar 2000; 108(3): 223-230.
29. Sarniento JM, Dockrell DH, Schwad TR, et al. Mycophenolate mofetil increases cytomegalovirus invasive organ disease in transplant patients. Clin Transplant 2000; 14: 136-138.
30. Mansy F, Brancart F, Liesnard C, et al. A PCR based DNA hybridisation capture system for the detection of human cytomegalovirus. A comparative study with other identification methods [J]. J Virol Methods, Jul 1999; 80(2): 113-122.
31. Ruellan-Eugene G, Barjot P, Campet M, et al. Evalution of virological procedures to detect fetal human cytomegalovirus infection: Avidity of IgG antibodies virus detection in amniotie fluid and maternal serum [J]. J Med Virol, 1996; 50: 9-15.
32. Sia IG, Patel R. New strategies for prevention and therapy of cytomegalovirus infection and disease in solid-organ transplant recipients. Clin Microbiol Rev 2000; 13(1): 83-122.
33. Eing BR, Baumeister H G., Kuehn J E, et al. Long-Term Persistence of Immunoglobulin A (IgA) and IgM Antibodies against Human Cytomegalovirus in Solid-Organ Transplant Recipients. Clin Diagn Lab Immunol, Jul 1999; 6(4): 621-623.
34. Bendiksen S, Van Ghelue M, Rekvig OP, et al. A longitudinal study of human cytomegalovirus serology and viruria fails to detect active viral infection in 20 systemic lupus ergthematosus.patients. Lupus, 2000; 9:120-126.
35. Weber B, Berger A, Rabenau H, et al. Human cytomegalovirus infection: diagnostic potential of recombinant antigens for cytomegalovirus antibody detection[J]. J Viorl Methods, 2001; 96(2): 157-170.
36. Pande H, Campo K, Tanamachi B, et al. Human cytomegalovirus strain Towne pp65 gene: nucleotide sequence and expression in Escherichia coli. Virology, May 1991; 182(1): 220-228.
37. Grefte JMM, van der Gun BT, Schmolke S, et al. The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocytes during an active cytomegalovirus infection. J Gen Virol, Nov 1992; 73:2923-2932.
38. Gyulai Z, Endresz V, Burian K, et al. Cytotoxic T lymphocyte(CTL) responses to human cytomegalovirus pp65, IE_1-Exon 4, gB, pp150 and pp28 in healthy individual: reevalution of prevalence of IEl—specific CTLs. J Infect Dis, 2001; 181(5): 1537-1546.
39. Wills MR, Carmichael AJ, Mynard K, et al. The human eytotoxic T lymphocyte (CTL) responses to CMV is dominated by structural protein pp65:frequency, specificy and T-cell recpptor usage of pp65-specific CTL. J Virol, 1996; 70: 7569-7579.
40. Boppana SB, Britt WJ, et al. Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells. Virology, Aug 1996; 222(1): 293-296.
41. Wills MR, Carmichael AJ, Mynard K, et al. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol, Nov 1996; 70(11): 7569-7579.
42. Beninga J, Kropff B, Mach M ,et al. Comparative analysis of fourteen individual
human cytomegalovirus proteins for helper T cell response. J Gen Virol, Jan 1995; 76(pt.1): 153-160.
43. Weekes MP, Wills MR, Mynard K, et al. The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual PeptideSpecific CTL Clones That Have Undergone Extensive Expansion In Vivo. J Virol, Mar 1999; 73(3): 2099-2108.
44. Hensel G, Meyer H, Gartner S, et al. Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32). J Gen Virol, Jul 1995; 76: 1591-1601.
45. van der Bij W, van Dijk RB, van Son WJ, et al. Antigen test for early diagnosis of active cytomegalovirus infection in heart transplant recipients. J Heart Transplant, Mar 1988; 7(2): 106-109.
46. ST George K, Boyd MJ, Lipson SM, et al. A multisite trail comparing two eytomegalovirus (CMV) pp65 antigenemia test Kits, biotest CMV bdte and Bartel/argene CMV antigenemia. J Clin Microbiol, 2000; 38:1430-1433.
47. Morello SC, Cranmer LD, Spector DH. In vivo replication, Latency, and Immunogenicity of murine cytomegalovirus mutants with detection in ate M83 and M84 genes, the putative homologs of human cytomegalovirus pp65(UL83). J Virology, 1999; 73: 7678-7693.
48. Borzi R M, Piacentini A, Monaco MC G, et al. A fluorescent in situ hybridization method in flow cytometry to detect HⅣ-1 specific RNA [J]. J Immunol Methos, 1996; 193: 167-176.
49. Kas-Deelen AM, Harmsen MC, De-Maar EF, et al. A sensitive methods for quantifying cytomegalic endothelial cells in peripheral blood from cytomegalovirus-infected patients [J]. Clin Diagn Lab Immunol, 1998; 5: 622-626.
50. Fowler KB, Dahle AJ, Boppana SB, et al. Newborn hearing screening:will children with hearing loss caused by congenital cytomegalovirus infection be missed? J Pediatrics, 1999; 135: 60-64.
51. Woodard P, Tong X, Richardson S, et al. Etiology and outcome of graft failure in pediatric hematopoietic stem cell transplant recipients. J Pediatr Hematol Oncol, 2003
Dec;25(12):955-999
52. Gault E, Michel Y, Dehee A, et al. Quantification of human cytomegalovirus DNA by real-time PCR. J Clin Microbiol, Feb 2001; 39(2): 772-775.
53. Nakamura Y, Sakarna SA, Ohta Y, et al. Detectation of the human cytomegalovirus gene in placental chronic villitis by polymerase chain reaction[J]. Human Pathology, 1994; 25(8): 815-818.
54. Sia IG, Wilson JA, Espy M J, et al. Evaluation of the COBAS AMPLICOR CMV MONITOR Test for Detection of Viral DNA in Specimens Taken from Patients after Liver Transplantation. J Clin Microbiol, Feb 2000; 38:600-606.
55. Rowley A H, Wolinsky S W, Sambol S P, et al. Rapid detection of cytomegalovirus DNA or RNA in blood of renal transplant patients by in vitro enzymatic amplification. Transplantation, 1991; 51 (5): 1028-1033.
56. Hamprecht K, Witzel S, Maschmann J, et al. Rapid detection and quantification of cell free cytomegalovirus by a high-speed centrifugation-based microculture assay: comparison to longitudinally analyzed viral DNA load and pp67 late transcript during lactation. J Clin Virol, Dec 2003; 28(3): 303-316.
57. Funato T, Satoh J, Sasaki T. Quantitative PCR system [J]. Rinsho Byori 1998; 46(5): 399-405.
58. Najioullah F, Thouvenot D, Lina B. Developmennt of a real-time PCR procedure including an internal control for the measurement of HCMV viral load. J Virol Methods, Mar 2001; 92(1): 55-64.
59. Livak KJ, Flood SJA, Marmaro J, et al. Oligonudetide with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Applic, 1995; 4(6): 357-362.
60. Higuehi R, Dollinger G, Walsh PS, et al. Stimustaneous amplification and detection of specific DNA sequence. Biotechnology, 1992; 10(4): 413-417.
61. Keams AM, Guiver M, James V, et al. Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus [J]. J Viorl Methods, Jun 2001(1-2); 95: 121-131.
62. Roberts TC, Brennan DC, Buller RS, et al. Quantitative polymerase chain reaction
to predication occurrence of symptomatic cytomegalovirus infection and assess response to ganciclovir therapy in renal transplant recipients. Infect Dis, Sep 1998; 178(3): 626-635.
63. Li H, Dummer JS, Estes WR, et al. Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J Clin Microbiol, Jan 2003; 41(1): 187-191.
64. Guiver M, Fox AJ, Mutton K, et al. Evaluation of CMV viral load using taqman CMV quantitative PCR and comparison with CMV antigenemia heart and lung transplant recipients [J], Transplantation, 2001; 71 (11): 1609-1615.
65. Flexman J, Kay I, Fonte R, et al. Differences between the quantitative antigenemia assay and the cobas amplicor monitor quantitative PCR assay for detecting CMV viraemia in bone marrow and solid organ transplant patients. J Med Virol, 2001 Jul; 64(3): 275-282.
66. Boeckh M, Huang M, Ferrenberg J, et al. Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol, Mar 2004; 42(3): 1142-1148.
67. Gouarin S, Vabret A, Gault E,et al.Quantitative analysis of HCMV DNA load in whole blood of renal transplant patients using real-time PCR assay. J Clin Virol, Mar 2004; 29(3): 194-201.
68. Cortez KJ, Fischer SH, Fahle GA, et al. Clinical trial of quantitative real-time polymerase chain reaction for detection of cytomegalovirus in peripheral blood of allogeneic hematopoietie stem-cell transplant recipients. J Infect Dis, 2003 Oct 1; 188(7): 967-972.
69. Barrett-Muir W, Breuer J, Millar C, et al. CMV viral load measurement in whole blood and plasma-which is best following renal transplantation? [J]. Transplantation, Jul 2000; 70(1): 116-119.
70．谢万灼，林茂芳．骨髓移植后巨细胞病毒感染的防治研究．国外医学输血及血液学杂志，2001；24：69-71．
71. Tanaka Y, Kanda Y, Kami M, et al. Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after
hematopoietic stem cell transplantation. Bone Marrow Transplant, 2002; 30:315-319.
72. Evans PC, Gray JJ, Wreghitt TG, et al. Comparison of three PCR techniques for detecting cytomegalovirus (CMV) DNA in serum, detection of early antigen fluorescent foci and culture for the diagnosis of CMV infection. J Med Microbiol, Nov 1999; 48(11): 1029-1035.
73. Butt N M, Clark R E. High frequency of positive surveillance for cytomegalovirus by PCR in allograft recipients at low risk of CMV. Bone Marrow Transplant, 2001; 27: 615-619.
74. Sia IG, Wilson JA, Groettum CM, et al. Cytomegalovirus (CMV) DNA load predicts relapsing CMV infection after solid organ transplantation. J Infect Dis, Feb 2000; 181(2): 717-720.
75. Solano C, Munoz I, Gutierrez A. Qualitative plasma PCR assay (AMPLICOR CMV test) versus pp65 antigenemia assay for monitoring cytomegalovirus viremia and guiding preemptive ganciclovir therapy in allogeneic stem cell transplantation. J Clin Microbiol, 2003; 39: 3938-3941.
76. Pellegrin I, Garrigue I, Ekouevi D, et al. New molecular assays to predict occurrence of cytomegalovirus disease in renal transplant recipients. J Infect Dis, Jul 2000; 182(1): 36-42.
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