2001年徐州地区暴发性病毒性脑膜炎病原的研究

目的 确定引起2001年江苏省徐州地区无菌性脑膜炎流行的病原体。方法 组织培养法从患者脑脊液分离病毒，标准血清中和试验鉴定分离毒株；中和试验检测双份血清中和抗体效价；逆转录聚合酶链反应（RT-PCR）检测肠道病毒特异性基因片段。结果 22份脑脊液中分离出4株柯萨奇B5型、2株柯萨奇B3型、1株艾可7型肠道病毒，分离阳性率31．8％。RT-PCR检测脑脊液21份，肠道病毒阳性11份。阳性率52．4％。19例双份血清中11例中和效价呈4倍以上增长或转阳，阳性率57．9％。结论 此次江苏省徐州地区无菌性脑膜炎流行的病原体是以柯萨奇B5为主要血清型的肠道病毒。     

一起新生儿柯萨奇病毒B组引起的暴发流行

对1993年9月至10月沈阳某妇婴医院发生的一起新生儿集体感染,用间接免疫荧光法检测了36例患儿血清中的柯萨奇B组病毒的特异性IgM抗体,28例阳性,阳性率为77.8％。病毒分离结果为:12例患儿咽拭子中1例阳性;9例患儿便标本中4例阳性;4例患儿骨髓标本中3例阳性。同时,3例死亡患儿的9份解剖标本(包括心肌、心血、心包积液、脑组织和肠组织)全部病毒分离阳性。其中2例死亡患儿的心肌分离株经中和试验鉴定为柯萨奇病毒B组3型。同时对部分(9例)患儿母亲和全部(20例)有关医护人员进行的柯萨奇B组病毒IgM抗体检测结果为,2例患儿母亲阳性。对1993年10月30日至1994年5月31日期间1616例临产孕妇的柯萨奇B组病毒IgM抗体检测中,65例阳性,阳性率为4.02％。其中1993年10月30日至11月17日期间的146例被检孕妇中,12例阳性,阳性率为8.22％。表明,1993年9～10月期间沈阳市有柯萨奇病毒流行。     

云南省病毒性脑炎和不明原因发热患者的相关病毒IgM抗体检测及分析

目的 调查云南省病毒性脑炎和不明原因发热病例的发病病因.方法 在云南省部分地区采集病毒性脑炎和不明原因发热患者血清和脑脊液标本,用ELISA法检测患者标本中包括虫媒病毒、肠道病毒和呼吸道病毒中15种病毒的IgM抗体.结果 在云南省共采集病毒性脑炎患者血清和脑脊液标本526份,不明原因发热患者血清标本221份.经ELISA法检测,病毒性脑炎患者标本中乙型脑炎病毒、柯萨奇病毒、埃可病毒、版纳病毒、单纯疱疹病毒、腮腺炎病毒、登革热病毒和罗斯河病毒的IgM抗体阳性率依次为50.95％(阳性数=268)、21.48％ (113)、19.58％(103)、7.41％ (39)、3.04％(16)、1.71％(9)、1.00％(5)和0.38％(2);不明原因发热患者血清中乙型脑炎病毒、登革热病毒、单纯疱疹病毒、版纳病毒、腮腺炎病毒、柯萨奇病毒、埃可病毒、罗斯河病毒和巴马森林病毒的IgM抗体阳性率依次为21.27％ (47)、20.36％ (45)、13.12％(29)、12.67％ (28)、11.76％ (26)、4.52％(10)、1.81％(4)、1.81％(4)和0.45％(1).乙型脑炎病毒、登革热病毒、版纳病毒、罗斯河病毒和巴马森林病毒感染以7-10月为主,柯萨奇病毒、埃可病毒、单纯疱疹病毒和腮腺炎病毒感染以7-8月居多.结论 乙型脑炎病毒是云南省夏秋季病毒性脑炎流行的主要病原体,柯萨奇、埃可和单纯疱疹病毒是散发性病毒性脑炎的重要病原;登革、版纳、单纯疱疹和腮腺炎病毒是不明原因发热的重要病毒病原并可能存在罗斯河和巴马森林等蚊媒病毒的流行.     

应用MAC-ELISA方法检测呼吸道合胞病毒IgM抗体

建立酶联免疫吸附试验(MAC-ELISA)检测呼吸道合胞(RS)病毒特异性IgM抗体方法,并与中和试验(NT)进行对比。 取35例患毛细支气管炎婴幼儿的急性期及恢复期双份血清共70份标本进行研究。MAC-ELISA测得急性期血清中RS病毒特异性IgM抗体的阳性率为27/35(77.14％),而恢复期血清中和抗体呈≥4倍升高者为23/35(65.71％)。凡NT阳性病例,MAC-ELISA也呈阳性;凡MAC-ELISA阴性病例,NT也呈阴性。另4例NT阴性而MAC-ELISA呈阳性。两者阳性符合率为88.57％。 本研究也观察了IgM抗体与年龄的关系。在6个月龄以前,MAC-ELISA的阳性率远远高于NT。 实验说明采用MAC-ELISA检测RS病毒特异性IgM是可靠的快速诊断方法。     

脑炎与人微小病毒B19感染-附1例PCR检测结果分析

我室用多聚酶链反应技术及ELISA法对1例临床拟诊为脑炎志儿的血清及脑脊液分别作了人微小病毒B19-DNA及该病毒特异性IgM、IgG抗体检测。其结果血清及脑脊液B19特异性IgM抗体均为强阳性；血清B19特异性IgG抗体阴性．脑脊液B19特异性IgG抗体强阳性；血清中检出B19-DNA。在国内首次提示人微小病毒B19感染与脑炎的发生有关。     

6种虫媒病毒蛋白芯片检测方法的建立

目的 建立针对6种虫媒病毒的蛋白芯片检测方法,用以检测流行性乙型脑炎病毒、蜱传脑炎病毒、登革病毒(1～4型)、西尼罗病毒、西部马脑炎病毒和东部马脑炎病毒的特异性抗体.方法 将病毒特异性抗原作为捕获抗原点样制备蛋白芯片,利用双抗夹心ELISA原理检测血清中的病毒特异性抗体.首先利用免疫兔血清进行特异性诊断抗原的筛选,并对抗体芯片检测条件进行优化,然后采用56份临床疑似的阳性血清标本及阴性对照标本对该方法进行验证,并与常规ELISA方法进行比对.结果 共筛选出11个特异性较好的重组诊断抗原.抗原点样浓度在0.125 ～0.900mg/ml时可获得良好的检测效果,血清检测范围为1:100～1:1000.对26份临床疑似的蜱传脑炎病毒血清标本,22份登革病毒血清标本及8份流行性乙型脑炎病毒临床血清标本的检测结果为:共检测出蜱传脑炎病毒IgG阳性血清标本20份,阳性检出率为76.9％,IgM阳性血清标本17份,阳性检出率65.3％,与ELISA检测符合率分别为96.1％和84.6％.乙型脑炎病毒IgG阳性血清4份,阳性检出率50.0％,IgM阳性血清5份,阳性检测率62.0％,与ELISA检测符合率分别为87.5％和100％.登革病毒IgG阳性血清标本13份,阳性检出率63.6％,IgM阳性血清标本14份,阳性检测率68.1％,与ELISA检测符合率分别为86.3％和90.1％,结果经一致性Kappa检验后,与ELISA检测结果一致性良好.阴性对照血清结果显示检测特异性为100％.结论 本研究建立的虫媒病毒抗体芯片检测方法具有较高的特异性和可靠性,可用于6种虫媒病毒抗体的临床检测.     

TORCH-IgM捕获法ELISA试剂盒的研制和应用

目的 为了发展TORCH近期感染的IgM酶免疫诊断技术。方法 以抗人IgM McAb包板，加待检血清、TORCH抗原，接着加酶标记的抗TORCH—McAb，最后加3，3’，5，5’-四甲基联苯胺(TNB)显色。用这种自制的捕获法ELISA(C—ELISA)试剂盒和HOPE公司的间接法ELISA(I—ELISA)试剂盒平行检测1196份孕妇血清TORCH-IgM。结果C—ELISA试剂盒检出阳性血清67份，并经其他试验证实为阳性。其中4份用I—ELISA试剂盒检测为阴性。由乳胶凝集试验诊断为类风湿因子(RF)阳性的2份血清，经I—ELISA试剂盒检测为阳性，但经C—ELISA试剂盒检测为阴性。结论 在检测孕妇TORCH—IgM方面，C—ELISA比I-ELISA有更强的敏感性和更高的特异性。C—ELISA试剂盒是诊断TORCH近期感染的良好工具。     

一起成人群体发热患者的病毒分离与鉴定

目的 从一起成人群体发热患者咽拭子标本中分离、鉴定病毒并研究其与疾病的关系.方法 采集患者咽拭子标本,接种细胞分离病毒,以中和试验和分子生物学技术鉴定分离病毒;用分离的病毒制备抗原片,采用间接免疫荧光法检测患者血清中特异性IgM、IgG抗体.结果 从10份发热患者咽拭子标本中分离到2株病毒,经核苷酸序列分析和中和试验证实为腺病毒11型;10例发热患者血清中,6例抗腺病毒11型特异性IgM阳性,3例特异性IgG阳性,1例特异性IgM、IgG均阴性.结论 从患者咽拭子标本中分离到的腺病毒B组11型可能为引起此次群体发热的病原体.     

血清中丙型肝炎NS3抗原ELISA检测方法的建立和初步应用

目的 评价血清中丙型肝炎病毒(HCV)游离NS3抗原的酶联免疫吸附(ELISA)检测方法的特异性和灵敏度,初步探讨该方法在临床应用中的意义.方法 对77例正常人血清标本,173例抗-HCV阳性标本和3708例抗-HCV阴性的其他类型肝炎血清标本检测HCV游离NS3抗原;对部分HCV NS3抗原阳性标本进行验证,包括HCV RNA测定、中和试验和免疫斑点试验;对11例患者的25份系列血清标本进行了HCV游离NS3抗原、HCV RNA和HCV抗体的联合检测,并结合临床资料综合分析.结果 3708例抗-HCV阴性的其他类型肝炎血清标本中有48例为HCV NS3抗原阳性,其中3030例单纯乙型肝炎和445例其他类型肝炎血清标本中分别有44例和4例为HCV NS3抗原阳性;173例HCV抗体阳性标本中有42例为HCV NS3抗原阳性;77例正常人血清标本的HCV NS3抗原检测结果均为阴性;15例HCV NS3抗原阳性标本中有9例为HCV RNA阳性;23例HCV NS3抗原阳性标本的中和率和免疫斑点试验的阳性率分别为87.0%和69.6%;25份系列血清标本的检测结果显示其HCV NS3抗原的吸光度值与时间呈负相关,并有2例HCV NS3抗原阳性标本随着血清中HCV NS3抗原的吸光度值下降,其HCV抗体转阳.结论 血清中HCV游离NS3抗原的ELISA检测方法有较好的特异性和敏感度,在发展中国家应用此方法进行HCV感染的早期诊断有一定的临床意义和推广价值.     

我国部分地区病毒性脑炎标本的实验室检测

目的 初步了解我国病毒性脑炎的病原种类及其分布特征.方法用ELISA方法对2004年至2006年从我国6个省份收集的771例临床诊断为病毒性脑炎患者的急性期血清和脑脊液标本检测乙脑病毒IgM抗体,然后对乙脑IgM抗体阴性的所有血清标本检测其他7种常见病毒IgM抗体.此外,用PCR方法对54例脑脊液标本检测肠道病毒、版纳病毒和辽宁病毒的基因.结果经血清学检测,771例患者中的567例(73.5%)检测出病毒特异性IgM抗体,构成顺序为乙脑病毒(47.0%)、腮腺炎病毒(10.6%)、肠道病毒(8.8%)、单纯疱疹病毒(5.7%)、麻疹病毒(0.4%)、水痘-带状疱疹病毒(0.4%)、EB病毒(0.4%)、巨细胞病毒(0.3%);经分子生物学检测,在54例脑脊液中检测到8例(14.8%)肠道病毒基因阳性标本,未检测到版纳病毒与辽宁病毒基因阳性标本.结论乙脑病毒是我国病毒性脑炎的首要病原,腮腺炎病毒次之,肠道病毒和单纯疱疹病毒也是重要的病原.     

Diagnosis of spirochetal meningitis by enzyme-linked immunosorbent assay and indirect immunofluorescence assay in serum and cerebrospinal fluid.

The antibody response against a spirochetal strain isolated from Swedish Ixodes ricinus ticks was determined by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay of cerebrospinal fluid (CSF) and serum specimens from 45 patients with chronic meningitis. Samples of CSF, serum, or both from patients with various infections of the central nervous system, multiple sclerosis, syphilis, or infectious mononucleosis and from healthy individuals served as control samples. Probable spirochetal etiology could be demonstrated for 41 of 45 (91%) patients with clinical symptoms of chronic meningitis. Approximately 25% of the patients had significantly elevated titers of antibody to the spirochete in CSF but not in serum. The highest diagnostic sensitivity, 91%, was demonstrated by measurement of CSF antibodies and calculation of a spirochetal CSF titer index, which is the ratio of (ELISA titer in CSF/ELISA titer in serum) to (albumin in CSF/albumin in serum) and which also considers the degree of blood-CSF barrier damage. The highest specificity, 98%, was obtained by calculation of a CSF titer index. Patients with short duration of disease were especially prone to be antibody negative in serum but positive in CSF. Significant rise in serum antibody titers was seldom demonstrated in patients treated with antibiotics. It is concluded that measurement of CSF antibodies, especially by ELISA, is a highly sensitive and specific method for the immunological diagnosis of spirochetal meningitis.     

Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.

BACKGROUND: Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. OBJECTIVE: To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. STUDY DESIGN: After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. RESULTS: Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80). CONCLUSIONS: The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.     

Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the “gold standard” were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.     

Rapid diagnosis of gram-negative bacterial meningitis by the Limulus endotoxin assay.

The Limulus amoebocyte lysate endotoxin assay was evaluated as a method for rapid diagnosis of acute bacterial meningitis in a series of 305 patients. The results of Limulus assays on cerebrospinal fluid (CSF) samples from these patients were compared with the results for each patient of routine bacterial cultures and Gram stains. Positive Limulus tests were obtained on initial CSF specimens from 84% of patients with culture-proven bacterial meningitis, including all patients with meningitis due to gram-negative organisms. Initial Gram-stained smears revealed the presence of organisms in 68% of the patients. One patient with pneumococcal meningitis had a weakly positive Limulus assay, whereas patients with meningitis due to other gram-positive organisms, those with aseptic meningitis, or patients without meningitis had negative CSF Limulus tests. The Limulus assay also demonstrated the persistence of endotoxin in the CSF of certain patients during antibiotic therapy, especially patients with Haemophilus influenzae meningitis. The Limulus test proved to be a rapid, reliable indicator of the presence of gram-negative organisms in the CSF of patients suspected of acute bacterial meningitis.     

Correlation between culture of Mycobacterium tuberculosis and detection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis

A retrospective study was done to correlate culture of Mycobacterium tuberculosis and detection of mycobacterial antigen in cerebrospinal fluid (CSF) by an inhibition enzyme-linked immunosorbent assay (ELISA). M. tuberculosis was cultured from CSF of 14 out of 70 patients with a clinical diagnosis of tuberculous meningitis (TBM). Mycobacterial antigens were demonstrated in CSF specimens by inhibition ELISA in all 14 culture-positive patients with antigen concentrations of 14.5-295 ng/ml (mean 158.8 ng/ml). Thus there was positive correlation between the detection of mycobacterial antigen and isolation of M. tuberculosis. Based on this observation, 56 CSF specimens from culture-negative patients with clinically diagnosed TBM were examined for mycobacterial antigen and the data were compared with those from culture positive patients. ELISA gave positive results in 38 specimens, with antigen levels of 12.5-280 ng/ml (mean 152.6 ng/ml). In 70 CSF specimens from patients with non-tuberculous neurological disease (control group), ELISA results were negative. Thus, detection of mycobacterial antigen in CSF specimens by inhibition ELISA had a specificity of 100% and a sensitivity of 67.8% for the diagnosis of TBM and is of potential value in the laboratory diagnosis of TBM.     

ELISA for the detection of herpes simplex virus antigens in the cerebrospinal fluid of patients with encephalitis

An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of herpes simplex virus antigens in cerebrospinal fluid (CSF) has been developed. A Triton X-100 extract of herpes simplex virus type 1 (HSV-1) infected HEp-2 cells was used to coat wells of polyvinyl chloride plates. Rabbit anti-HSV-1 globulin served as the reference antibody and the CSF specimens were tested at a final dilution of 1:4. Positive results were obtained in CSF specimens from 11/18 (61%) neonates with HSV infection, 15/23 (65%) older individuals with HSV culture positive brain biopsies, and in 4/29 (14%) patients with culture negative brain biopsies. The assay was negative with CSF from 14 infants without HSV infections, from 30 patients with bacterial meningitis and 10 with cryptococcal meningitis. The test was positive in 10/21 patients within 10 days of onset, 11/14 within 11-20 days, and in 5/6 more than 20 days after onset of the herpetic infection. The overall sensitivity of the assay was 63% and the specificity was 95%.     

PCR-Enzyme Immunoassay for Detection of Streptococcus pneumoniae DNA in Cerebrospinal Fluid Samples from Patients With Culture-Negative Meningitis

A PCR-based assay was developed to amplify a conserved region of the pneumococcal autolysin gene. The amplified product was labelled with digoxigenin-labelled dUTP and was detected with a biotin-labelled probe in an enzyme immunoassay (EIA). The assay was initially tested with suspensions of various serotypes of Streptococcus pneumoniae and other gram-positive and gram-negative bacteria and was then applied to cerebrospinal fluid (CSF) specimens from patients with meningitis and those with other neurological disorders. The assay detected all the serotypes of S. pneumoniae tested, whereas all the other bacterial strains tested were negative. Seven of the 8 CSF specimens positive for pneumococcus by culture or latex agglutination (LA) were positive by PCR-EIA, whereas all 10 specimens positive for other organisms were negative. Among 11 patients with clinically diagnosed meningitis but with negative culture and LA results, 5 were positive by PCR-EIA. The assay was negative for all but one patient without meningitis; it was positive with the CSF from a child with immunodeficiency and pneumococcal abscesses on the scalp. PCR-EIA is a useful tool for the diagnosis of meningitis, especially when culture and LA are negative because of prior antibiotic treatment.     

单克隆抗体ELISA法快速诊断疱疹性脑炎

本文用单纯疱疹病毒单克隆抗体（ＨＳＶ－ＭｃＡｂ）ＥＬＩＳＡ法检测了１２８例病毒性脑炎患儿脑脊液中ＨＳＶ特异性抗原（ＨＳＶ—Ａｇ），２０例其他神经系统疾患也作了测定，３２例脑炎患儿同时取脑脊液和血清作ＨＳＶ—ＩｇＧ抗体水平测定比较，病毒性脑炎患儿的ＨＳＶ－Ａｇ阳性检出率为１９．５％，与血清／脑脊液的ＨＳＶ－ＩｇＧ测定比较，敏感性８４．６％，特异性９４．７％。用ＥＬＩＳＡ法检测脑脊液中ＨＳＶ－Ａｇ是一种简便、快速、敏感、特异的方法，对疱疹性脑炎具有早期诊断价值。     

Evaluation of the roche AMPLICOR enterovirus PCR assay in the diagnosis of enteroviral central nervous system infections.

BACKGROUND: Enteroviruses cause a substantial number of cases of aseptic meningitis annually in the USA. While culture has been useful in the detection of patients with viral meningitis it is time-consuming and lacks sensitivity. Detection of viral nucleic acid in patient specimens has been demonstrated to improve enteroviral detection. OBJECTIVES: A research use only commercial amplification assay, the Roche AMPLICOR EV test, was compared to culture for the diagnosis of enteroviral meningoencephalitis. STUDY DESIGN: Four-hundred and sixty-five consecutive CSF samples sent prospectively for suspicion of enteroviral infection were evaluated by PCR and shell-vial culture. Clinical information and CSF analysis were used to resolve PCR positive, culture negative samples. Sensitivity and specificity were calculated using resolved data. RESULTS: There were 138 samples which met the definition of a true positive. Of these culture detected 77 (sensitivity 55.8%) and PCR detected 136 (sensitivity 98.6%). PCR missed two culture positive samples. Upon repeat testing, these CSF samples were found to contain inhibitors. CONCLUSIONS: The Roche AMPLICOR EV-PCR test was statistically more sensitive than culture (P<0.001) in the detection of enteroviruses in CSF in patients suspected of having enteroviral meningitis. This assay also has the advantage of a rapid turnaround time of 5-6 h compared to 3-5 days for culture.     

Diagnosis of tuberculous meningitis by enzyme-linked immunosorbent assay to detect mycobacterial antigen and antibody in cerebrospinal fluid

Enzymed-linked immunosorbent assay (ELISA) was used to detect mycobacterial antigen and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of 50 patients with tuberculous meningitis (TBM) and 50 patients with non-tuberculous neurological diseases (control group). The assay gave no false negative results in 10 culture-positive patients with TBM. Detection of mycobacterial antigen in CSF is more sensitive and specific for the diagnosis of TBM than detection of antibody. ELISA should be considered as one of the alternative methods in the laboratory diagnosis of TBM, particularly in culturenegative patients with TBM.