病毒感染检查概要.ppt

病毒感染检查,approaches to the diagnosis of viral infection(1),There are two major approaches to the diagnosis of viral infection: virologic and serologic. The virologic approach includes (1) isolation of infectious virus in cell culture or in laboratory animals, (2) detection of viral antigen by immunologic methods such as immunofluorescence assay (IFA) or enzyme immunoassay (EIA), (3) identification of viral particles by electron microscopy (EM), and (4) detection of viral nucleic acid.,approaches to the diagnosis of viral infection(2),The serologic approach includes (1)demonstration of a fourfold or greater rise in antiviral antibody using acute and convalescent sera and a variety of methods that measure predominantly immunoglobulin (Ig) antibody and (2)demonstration of virus-specific IgM antibody in a single late acute or recovery phase serum.,标本处理,必须经滤过除菌，标本需迅速冷藏、运送。 Optimal Temperature For Storage And Transport Of Specimenfor viral culture and antigen detection is 4(refrigerator or wet ice temperature). Freezing at -20(ordinary freezer temperature) destroys or significantly reduces infectivity of virtually all viruses. If specimens must be stored for longer than 3 to 4 days, an ultra-low temperature freezer(-70)should be used, and specimen transported to the virology laboratory on ice.,细胞系选择与细胞病变特征,1.Several Different Types Of Cells： Because not all culturable viruses will grow in a single cell line, several different types of cells are required. 2.病毒分离培养后，根据细胞病变特征确定病毒的种， Cytopathic Effect (CPE) ：Many viruses will demonstrate a cytopathic effect (CPE) within a few days, whereas some may take as long as a week or two . Some viruses will do not cause typical CPE and are identified by the adsorption of red blood cells to the surface of virus-infected cells in the culture.,Confirmation Of a Specific Virus :,对已分离的病毒用已知参考血清作中和试验、补体结合试验、血凝抑制试验鉴定 Confirmation of a specific virus serotype or of a virus (e.g.HSV-2, influenza A H3N2, echovirus11)requires use of immunologic methods with antibody of known specificity. In recent years, fluorescence or peroxidase-conjugated monoclonal antibodies(MABs)have become available commercially to detect viral antigen in cell.,早期诊断技术,核酸杂交技术、 1、Solid phase 2、Liquid-phase 3、In Situ HybridizationIn PCR技术,Diagnosis of viral infections,The primary diagnostic technique for most viral infections is the isolation of the virus in cell culture. Serologic techniques may also be useful, especially if the virus was isolated from a nonsterile site. In some instances, serologic diagnoses is the only practical approach in a clinical laboratory. Direct detection of antigen in body fluids or tissues has also been effective for some viruses. The list of agents for which direct detection of antigen is useful will undoubtedly continue to expand.,The compare of the advantages and disadvantages of each of these approaches: Culture: Time:Days to weeks Advantages:Specificity and sensitivity maximum;isolate available for characterization Disadvantages:Cell culture facilities needed;time for diagnosis may be long,Direct detection: Time:Hours to 1 day Advantages:Speed of diagnosis;used for viruses difficult to culture Disadvantages:False-positives and negatives;hard to batch tests Serology: Time:Weeks Advantages:Assessment of immunity or response to virus isolated from nonsterile site; used for viruses difficult to culture Disadvantages:Potential cross-reaction;need for acute and convalescent specimens,检测项目的选择(1),选择合适检测项目检出病原是临床诊断病毒感染的关键之一，常依据感染的病毒生物学特性而不同。一般而言，病毒分离和血清学检查是病毒感染诊断的常规实验室方法，；而光学显微镜和电子显微镜直接检查病毒体仅被选择性使用。对于那些能在细胞培养中复制病毒的感染，采集合格标本后，选择恰当细胞系进行接种，根据病毒增殖指标识别，以血清学方法进行鉴定；而对于不能在细胞培养中快速复制的病毒，利用细胞培养和抗原检测组合，即低速离心接种有标本病毒的细胞培养瓶，经1620小时h孵育后，用单克隆抗体染色，可早期、快速诊断病毒感染（如巨细胞病毒感染）。,检测项目的选择(2),那些不能在细胞培养中增殖的病毒则使用核酸检测方法，快速提供检测结果，但它其不能无法证实明标本中病毒是具有感染性的。尽管过去认为血清学检查是实验室诊断病毒感染的主要手段，但更应注意是否能及时得到感染的信息。对那些可能新出现的病毒，只有采用分离方法可取得最好的诊断结果。,病毒感染实