荧光定量聚合酶链反应检测解脲支原体感染的临床意义

目的　采用荧光定量聚合酶链反应 (FQ -PCR)技术 ,准确定量检测待检标本中解脲支原体 (UU)的感染状况。方法　采用FQ -PCR技术 ,检测男女生殖道标本 76 5份 ,并同时用常规PCR技术 ,对比检测了 85例。结果　FQ -PCR检测76 5份标本 ,UU -DNA阳性 2 79份 ,阳性率 36 .5 % ,其DNA平均拷贝数为 2 .4× 10 4。 38例阳性患者治疗两周内复查 ,转阴率仅为 4 4 .7% ,治疗 3w后复查 ,转阴率达 76 % (P <0 .0 0 5 )。常规PCR结果重复检测时 ,2 5例阳性中 2例变为阴性 ,而 6 0例阴性中 4例变为阳性。FQ -PCR结果重复时完全吻合。结论　FQ -PCR检测UU ,具有快速、特异性高、定量准确等优点 ,并可为临床评价疗效提供依据。     

EB病毒与乳腺癌的相关性

目的：探讨EB病毒( Epstein-Barr virus, EBV)与乳腺癌发生、发展的相关性。方法随机选取不同发展阶段的乳腺病变组织246例,采用聚合酶链反应( polymerase chain reaction, PCR)、原位杂交( in situ hybridization, ISH)、激光捕获显微切割( laser capture microdissection, LCM)、免疫组化染色EnVision法检测EBV DNA、RNA、蛋白质水平,分析EBV与乳腺癌发生、发展的关系。结果免疫组化标记246例乳腺病变均未检测到EBV潜伏膜蛋白LMP1的表达。 PCR法在乳腺癌(12/23)、原位癌(0/10)以及乳腺良性病变(0/15)中检测到EBV DNA,但用地高辛标记的EBV DNA探针对48例乳腺良、恶性病变组织(包括PCR扩增阳性的12例乳腺癌标本)进行ISH检测,在癌细胞、乳腺上皮细胞和间质淋巴细胞内均未检测到阳性杂交信号。采用LCM PCR检测12例PCR扩增阳性和12例阴性乳腺标本的乳腺上皮细胞和间质淋巴细胞,在12例PCR阳性标本的间质细胞中扩增出EBV DNA,癌组织中未检出EBV DNA。用EBV RNA探针和ISH方法在75例乳腺良恶性病变组织中均未检测到EBER表达。结论在该文选择的标本中,乳腺癌发生、发展与EBV感染无关。     

宫颈癌与人乳头状瘤病毒16/18型的关系探讨

目的　应用荧光定量聚合酶链反应 (FQ -PCR)检测宫颈癌患者人乳头状瘤病毒 (HPV) 16 / 18型感染率 ,探讨HPV 16 / 18型与宫颈癌的关系。方法　应用荧光探针标记引物的荧光定量聚合酶链反应对 88例宫颈癌患者的宫颈分泌物进行了HPV 16 / 18型检测。结果　 88例宫颈癌患者宫颈分泌物FQ -PCR阳性率为 78% ,阳性样品定量对数平均值 (ml-1)为 5 .33× 10 6,定量测值范围 (ml-1)为 1.2 0× 10 3 ～ 2 .4 1× 10 7。对照组 85例全部阴性。结论人乳头状瘤病毒 (HPV) 16 / 18型感染与宫颈癌的发生发展关系密切。     

不同亚型T细胞淋巴瘤与EB病毒的关系

目的 :探讨不同组织学类型T细胞淋巴瘤 (TCL)与EBV感染的关系。方法 :对 83例 (6种类型 )TCL进行研究 ,包括低度恶性 30例 (其中小多形 2 1例、小淋巴细胞性 9例 ) ,高度恶性 5 3例 (其中大 /中多形 31例、淋巴母细胞性 9例、间变性大细胞性 7例、透明细胞性 6例 )。采用PCR检测EBV特征性的DNA序列 (EBV DNA)和ISH法检测EBV编码的RNA(EBER 1/2 )。结果 :每种类型TCL均有EBV的阳性表达 ,低度恶性组与高度恶性组之间EBER 1/ 2表达率差别有显著性 (P <0 0 5 )。结论 :各种类型TCL的发生发展均与EBV感染有关 ,但高度恶性组EBER 1/ 2的表达比低度恶性组更显著。     

乙肝病毒前S1抗原与传统二对半及HBV-DNA-PCR的相关性探讨

目的　检测前S1抗原与传统二对半中HBsAg、HBeAg及HBV -DNA -PCR的相关性 ,进一步推论前S1抗原在乙型肝炎的诊断与治疗中的临床作用 .方法　对本院 2 0 0 3年 10月至 2 0 0 3年 12月的门诊及住院病人所有乙肝六项 (二对半和前S1抗原 )检测结果进行分析 ,对其中所有HBsAg阳性者 1170例按二对半结果模式进行分类 ,计算前S1抗原在各类模式中的阳性率 ,以及前S1抗原与HBsAg、HBeAg的相关性 ,另外留取 30份HBV -DNA -PCR阳性标本 ,检测其前S1抗原及HBeAg结果 ,研究此三项指标的相关性 .结果　前S1抗原在不同模式中的阳性表达率各不相同 ,与HBsAg阳性相关率为 6 3% ,与HBeAg阳性相关率为 88.6 % ,HBV -DNA -PCR阳性者的前S1抗原阳性率为 90 %,HBeAg阳性率为 73% .结论　前S1抗原与两对半结果有很好的相关性 ,与HBV -DNA -PCR高度相关 ,优于HBeAg ,能准确客观地反应乙肝病毒感染与复制 ,是乙型肝炎诊断和治疗的一种敏感有效的指标     

肺癌组织中EB病毒感染的检测

目的探讨原发性肺癌中EB病毒（Epstein—Barr virus，EBV）的存在情况及EBV与原发性肺癌的关系。方法唐山市人民医院和开滦医院病理科储存的2001--2006年肺癌手术切除石蜡包埋肺癌组织108份，癌旁组织22份，以EBV阳性鼻咽癌组织为阳性对照，用原位杂交法（ISH）检测肺癌患者石蜡包埋组织标本中EBV编码的小RNA（EBERl），并采用图像分析法进行形态学定量。结果癌组织及癌旁组织EBERl的阳性率分别为33．3％（36／108）和4．5％（1／22），二者间差异有统计学意义（P〈0．01）。鳞癌、腺癌、小细胞癌及大细胞癌中EBV感染率分别是35．9％（14／39）、31．6％（12／38）、31．0％（9／29）和1／2。EBV感染与患者年龄、性别和组织学类型无关，但与肺癌的部位、癌组织分化程度有关，右肺明显高于左肺，中低分化癌明显高于高中分化癌。结论唐山地区原发性肺癌组织中EBV感染率为33．3％，EBV感染可能是肺癌的潜在病因之一，在癌组织分化的不同阶段有不同的作用。     

粤东地区不同人群丙型肝炎病毒感染情况分析

目的 :了解粤东地区不同人群丙型肝炎病毒 (HCV)感染情况 ,探讨HCV感染途径。方法 :运用酶联免疫吸附试验 (ELISA)及荧光定量聚合酶链式反应 (FQ -PCR)检测不同人群血清中抗 -HCV及HCV -RNA。结果 :抗 -HCV阳性率在一般人群中为 0 89% (5 8/ 6 4 6 8) ;医护人员 2 93% (5 / 171) ;乙型肝炎病人11 5 % (47/ 4 10 ) ;普通孕妇 0 93% (17/ 1836 ) ,其中 12例HCV -RNA阳性 ,所生 12名婴儿 2名阳性 ,母婴传播率 16 7% ;血液透析病人 5 1 4 % (5 4 / 10 5 ) ,其中有输血史的 5 8% (5 1/ 88) ,无输血史的 17 7% (3/ 17) ,两者有极显著性差异 (χ2 =7.72 ,P <0 .0 1)。结论 :血途径是HCV感染的主要传播途径。母婴传播及医务人员的被感染值得重视。     

荧光定量PCR技术检测乳腺癌CK19 mRNA表达过量的临床应用

目的 :探讨荧光定量PCR(FQ -PCR)检测细胞角蛋白 19(CK - 19)在乳腺癌诊断中的应用。方法 :采用FQ -PCR法 ,并以磷酸甘油醛脱氢酶 (GAPDH)为内对照测定 30例健康女性体检者、30例良性乳腺疾病患者和 76例乳腺癌患者的外周血中CK19mRNA的表达量。结果 :正常对照组和良性乳腺疾病组CK19和CK19/GAPDH无显著性差异 ,乳腺癌组均显著高于前 2组 ,GAPDH在 3组间均无显著性差异。若以CK19基因表达量和CK19/GAPDH高于正常对照组 x± 2s为阳性 ,76例乳腺癌患者的阳性数为 4 1,阳性率 5 3 9% ,良性乳腺疾病组阳性数为 0。 76例乳腺癌中 2 7例患者从手术前到第 3次检测 ,CK19/GAPDH均显下降趋势 ,但第 1次和第 2次与手术前比较 ,无显著性差异 ,而第 3次与手术前比较 ,CK19/GAPDH均显著低于手术前 ,存在显著性差异。结论 :FQ -PCR技术是高度灵敏、高度特异的快速定量检测CK19基因的方法 ,可有效监测乳腺癌的诊断、治疗和预后。     

荧光定量PCR检测不明原因流产组织及血清中巨细胞病毒和弓形体感染

目的　探讨用荧光定量PCR检测不明原因流产组织及血清中巨细胞病毒 (CMV)和弓形体 (TOX)感染。方法　检测了 6 6名妊娠 70天内流产患者的绒毛和蜕膜组织中CMV、TOX ,探讨了与血清中IgM抗体和DNA拷贝数的关系。结果　不明原因流产者查出血清CMV感染者 6例 (9 1% ) ,其中血清、蜕膜和绒毛均感染者有 3例占 (4 5 % ) ;1例血清、蜕膜感染但未采到绒毛标本 ;2例 (3 0 % )母血清中查到CMV ,流产组织中未查到。查出血清、蜕膜和绒毛TOX均感染者 1例。FQ -PCR检测出CMV阳性者 5例 ,TOX 1例 ,而IgM无 1例阳性。当流产妇女外周血CMV -DAN≥ 10 3 时 ,有 6 7%在流产组织中查到CMV -DAN。结论　荧光定量PCR在血清、脱膜和绒毛组织检测中显现的高敏感和高特异性 ,应推荐作为妊娠初期筛查的首选方法。     

EB病毒感染对鼻咽癌细胞生长和凋亡的影响

目的:探讨EB病毒(EBV)感染对鼻咽癌细胞系生长和细胞凋亡的影响。方法:用EBV直接感染人鼻咽癌细胞系CNE1;用免疫组化(LSAB)法检测EBV-LMP1和bcl-2蛋白的表达;用MTT法测定鼻咽癌细胞系的生长能力;用流式细胞术和TUNEL法检测癌细胞凋亡。结果:感染EBV的鼻咽癌细胞系(E-CNE1)的EBV-LMP1表达阳性,生长能力较未感染EBV的CNE1明显增强(P＜0.01),2种鼻咽癌细胞系均无凋亡发生,而均仅有2%~3%的细胞表达bcl-2蛋白。结论:EBV感染和LMP1表达可促进鼻咽癌细胞生长,但对鼻咽癌细胞的bcl-2表达和细胞凋亡无影响。     

Assessing for primary oropharyngeal or nasopharyngeal squamous cell carcinoma from fine needle aspiration of cervical lymph node metastases

In fine needle aspirates of cervical lymph nodes with metastatic squamous cell carcinoma (SCC), the site of origin may not be clinically evident. The distinction between oropharyngeal and nasopharyngeal primary SCC has important management consequences. In the current study, we evaluated metastatic SCC for HPV types 16, 18, 31, 33, 51 (by in situ hybridization[ISH]), p16 and ProExC (surrogate HPV markers), and Epstein Barr Virus reported in nasopharyngeal SCC. Forty patients diagnosed between 2004 and 2008, with adequate cell block material were identified. ISH for high risk HPV and EBV (EBER), and immunohistochemistry for p16 and ProExC were performed. Primary site was designated in 31 cases with 26 head and neck including 11 oropharyngeal and 2 nasopharyngeal, and 5 other sites. High risk HPV was detected in 9 cases (22.5%), p16 in 16 (40%), ProExC in 35 (87.5%), and EBER in 2 (5%). All cases with high risk HPV ISH also showed overexpression of p16. The sensitivity for HPV infection by both surrogate markers was 100%; specificity for p16 and ProExC was 78.7 and 16.1%, respectively. Seven (63.6%) oropharyngeal SCC were positive for HPV ISH and negative for EBV; one nasopharyngeal SCC (50%) was EBER positive and HPV negative. HPV and EBER detection can serve as indicators for oropharyngeal and nasopharyngeal primary SCC, respectively, however our data show that only a subset (63.6%) of oropharyngeal SCC are high risk HPV‐related. Additionally, despite their high sensitivity for HPV infection, surrogate markers, especially ProExC, lack specificity. Diagn. Cytopathol. 2010;38:795‐800. © 2009 Wiley‐Liss, Inc.     

Prevalence and relevance of EBV latency in nasopharyngeal carcinoma in Israel

BACKGROUND: Nasopharyngeal carcinoma (NPC) is frequently associated with Epstein-Barr virus (EBV). The incidence of NPC in Western countries is lower than in the Far East, and EBV latency in NPC is less prevalent. Israel, as a part of the Mediterranean area, is one of the countries with an intermediate risk for NPC. METHODS: Immunohistochemistry (IHC) for latent membrane protein 1 (LMP-1) and in situ hybridisation (ISH) for EBV encoded RNA (EBER) were used to evaluate the prevalence and possible prognostic value of EBV latency among Israeli patients with NPC. Forty five patients with different NPC histologies were studied. RESULTS: LMP-1 IHC was positive in six samples only, all with undifferentiated histology. EBER ISH was positive in 40 of the 45 samples. According to histological type, three of five patients with squamous cell carcinoma were EBV positive and 37 of 40 non-keratinising and undifferentiated carcinoma cases were positive. Although EBV was more prevalent in patients with non-squamous carcinoma, the difference was not significant, probably because of the small number of patients with keratinising carcinoma. With regard to the clinical categories and survival, no significant difference could be detected between patients who were positive or negative for EBER ISH. No association was found between EBV latency and patient sex, age, origin, stage, or survival. CONCLUSIONS: NPC in Israel is highly associated with EBV latency as detected by EBER ISH. LMP-1 IHC is considerably less sensitive in detecting EBV latency in NPC among the same patient group.     

鼻咽癌颈淋巴结转移EB病毒基因的检测

目的　观察鼻咽癌(NPC)颈淋巴结转移EB病毒基因情况。方法　采用细针穿刺结合聚合酶链反应(PCR)技术对50例颈部肿块穿刺液进行EBVDNA检测。结果　30例鼻咽癌颈淋巴结转移者EBVDNA阳性数27例,阳性率为900%。7例恶性淋巴瘤者EBVDNA阳性数1例,阳性率143%。8例非NPC颈部转移癌者EBVDNA阳性数1例,阳性率为125%。5例鼻咽部炎症伴颈淋巴结增生者均未检测到EBVDNA。结论　细针穿刺结合PCR方法快速灵敏,所取标本量少,特异性高,尤其对于临床难以确认的隐匿性鼻咽癌及原发灶不明的颈部转移癌,具有一定的临床诊断价值,值得推广应用。     

Detection of Epstein–Barr virus small RNAs in routine paraffin sections using non-isotopic RNA/RNA in situ hybridization

The Epstein–Barr virus (EBV) is associated with an increasing range of reactive and neoplastic lesions. There is a need for a sensitive and specific method for detecting latent EBV in routine histological sections. We report the use of a highly sensitive paraffin section RNA/RNA in situ hybridization (ISH) technique using digoxigenin-labelled antisense riboprobes for demonstrating EBV encoded-small RNAs (EBERs), EBV gene products that are transcribed in abundance during latent EBV infection. We applied EBER-ISH to 846 paraffin embedded specimens, including cases of reactive lymphoid hyperplasia ( n = 28), infectious mononucleosis (16), Burkitt's lymphoma (44), immunodeficiency-associated lymphomas in transplant recipients (9) and AIDS patients (128), Hodgkin's disease (130), CD30 antigen positive lymphomas (106), peripheral T-cell lymphomas (104), sporadic B-cell non-Hodgkin's lymphomas (162), undifferentiated nasopharyngeal carcinoma (86), salivary gland lymphoepithelioma (11), and oral hairy leukoplakia (5). Strong, reproducible EBER staining was seen in EBV latently infected cells in archival surgical biopsy and autopsy specimens. EBER-ISH is specific, has a sensitivity comparable to that of the polymerase chain reaction, and is now the method of choice for the in situ detection of latent EBV infection.     

Absence of latent Epstein-Barr virus in thymic epithelial tumors as demonstrated by Epstein-Barr-encoded RNA(EBER) in situ hybridization

BACKGROUND: Several studies have established that Epstein-Barr virus (EBV) is associated with lympho-proliferative disorders such as Burkitt's lymphoma and Hodgkin's disease. EBV is also present in undifferentiated nasopharyngeal carcinomas and in tumors of similar morphology (lymphoepithelioma-like carcinomas) arising in a variety of organs, predominantly in stomach, salivary gland and thymus. As reports of EBV-positive thymic epithelial tumors (TET) have been divergent and as different methods have been used to detect EBV, the aim of this study was to investigate the possible role of EBV in TET of Danish patients. MATERIAL AND METHODS: Archival material of 157 cases of TET (105 thymomas and 52 thymic carcinomas, including 4 lymphoepithelioma-like thymic carcinomas (LELTC)) was analyzed for EBV by applying a sensitive and specific method for detecting latently EBV-infected cells (in situ hybridization for EBV-encoded RNA (EBER)). RESULTS: All investigated cases were EBER negative. CONCLUSIONS: EBV does not seem to be implicated in the pathogenesis of TET. However, a review of the literature showed that 28% of LELTC were EBER ISH positive. As they occurred in young people (mean 18 years), at an age when the patients were susceptible to infection by EBV, it is suggested that EBV merely acts as an innocent bystander.     

Association of Epstein-Barr virus with undifferentiated gastric carcinomas with intense lymphoid infiltration. Lymphoepithelioma-like carcinoma.

Some undifferentiated gastric carcinomas with intense lymphoid infiltration have a striking resemblance to nasopharyngeal lymphoepithelioma. The authors identified eight such cases (seven patients from Japan and one from the United States) of undifferentiated gastric carcinoma (lymphoepithelioma-like carcinoma [LELC]) and examined them for Epstein-Barr (EBV) viral sequences using polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques. EBV was detected in seven of the eight cases by PCR, including a lymph-node metastasis. ISH that was performed in six of these cases showed EBV genomes to be uniformly present in the carcinoma cells and not present in the reactive lymphoid infiltrate or normal gastric mucosa. PCR of a polymorphic EBV locus (lymphocyte-determined membrane antigen) showed that a single genotype was present in each gastric LELC, consistent with a clonal process. These findings suggest that some undifferentiated gastric carcinomas are EBV-related and that focal EBV infection occurs before transformation.     

Epstein-Barr virus genomes in Hodgkin's disease and non-Hodgkin's lymphomas

Seventeen of 40 cases of Hodgkin's disease (HD) and eight of 46 non-Hodgkin's lymphomas (NHL) were associated with Epstein-Barr virus (EBV) infection, judged by the EBER-1 in situ hybridization (ISH) method. Approximately 40% incidence in HD was comparable to previous reports. Young children and elderly HD patients were more prone to be found EBV positive. Fourteen of 17 HD and two of 8 NHL cases with positive EBER-1 ISH were also positive on LMP-1 immunostaining. EBV might have a role in lymph-omagenesis in these cases. The fact that 7 of 8 EBV-related NHL were peripheral T cell lymphoma indicates the necessity of a larger-scale survey on this subject. As the present study revealed four cases with positive LMP-1 immunostaining but negative EBER-1 ISH (1 HD, 3 NHL), LMP-1 alone should not be regarded as a tool to prove EBV infection.     

Epstein-Barr virus-associated gastric adenocarcinoma.

The Epstein-Barr virus (EBV) has been detected in certain types of lymphoma and some epithelial neoplasms including nasopharyngeal lymphoepithelioma, and rare lymphoepithelioma-like carcinomas occurring in a variety of organs including, most recently, the stomach. The authors investigated the possibility that EBV may be present not only in the rare gastric cancers that resemble nasopharyngeal lymphoepithelioma, but also in typical gastric adenocarcinoma. EBV sequences were detected in 22 of 138 (16%) cases of typical gastric adenocarcinoma by polymerase chain reaction and in situ hybridization (ISH) techniques. The EBV genomes were specifically present within the gastric carcinoma cells in an even distribution. The EBV genomes were also present in adjacent dysplastic epithelium but were absent in surrounding lymphocytes, other normal stromal cells, intestinal metaplasia, and normal gastric mucosa. The EBV genomes in the infected gastric carcinoma cells are expressed as EBV RNA was detected by ISH. EBV was most often detected in gastric tumors from men (21%) compared with women (3%). Thus some cases of gastric adenocarcinoma are EBV-associated.