病毒感染檢查概要

1、病毒感染檢查,,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

2、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 microscop

3、y (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 conv

4、alescent 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.,標(biāo)本處理,必須經(jīng)濾過除菌，標(biāo)本需迅速冷藏、運送。

5、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 red

6、uces 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.,細(xì)胞系選

7、擇與細(xì)胞病變特征,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.病毒分離培養(yǎng)后，根據(jù)細(xì)胞病變特征確定病毒的種，Cytopathic Effect (CPE) ：Many vi

8、ruses 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 t

9、he surface of virus-infected cells in the culture.,Confirmation Of a Specific Virus :,對已分離的病毒用已知參考血清作中和試驗、補體結(jié)合試驗、血凝抑制試驗鑒定Confirmation of a specific virus serotype or of a virus (e.g.HSV-2, influenza A H3N2, echovirus11)

10、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 cel

11、l.,早期診斷技術(shù),核酸雜交技術(shù)、1、Solid phase2、Liquid-phase3、In Situ HybridizationInPCR技術(shù),Diagnosis of viral infections,The primary diagnostic technique for most viral infections is the isolation of the virus in cell culture. Serol

12、ogic 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

13、 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

14、 of each of these approaches:Culture: Time:Days to weeks Advantages:Specificity and sensitivity maximum;isolate available for characterization Disadvantages:Cell culture facilities needed;tim

15、e 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

16、testsSerology: Time:Weeks Advantages:Assessment of immunity or response to virus isolated from nonsterile site; used for viruses difficult to culture Disadvantages:Potential cross-reaction;nee

17、d for acute and convalescent specimens,檢測項目的選擇(1),選擇合適檢測項目檢出病原是臨床診斷病毒感染的關(guān)鍵之一，常依據(jù)感染的病毒生物學(xué)特性而不同。一般而言，病毒分離和血清學(xué)檢查是病毒感染診斷的常規(guī)實驗室方法，；而光學(xué)顯微鏡和電子顯微鏡直接檢查病毒體僅被選擇性使用。對于那些能在細(xì)胞培養(yǎng)中復(fù)制病毒的感染，采集合格標(biāo)本后，選擇恰當(dāng)細(xì)胞系進(jìn)行接種，根據(jù)病毒增殖指標(biāo)識別，以血清學(xué)方法進(jìn)行鑒定；而對于不能在

18、細(xì)胞培養(yǎng)中快速復(fù)制的病毒，利用細(xì)胞培養(yǎng)和抗原檢測組合，即低速離心接種有標(biāo)本病毒的細(xì)胞培養(yǎng)瓶，經(jīng)16~～20小時h孵育后，用單克隆抗體染色，可早期、快速診斷病毒感染（如巨細(xì)胞病毒感染）。,檢測項目的選擇(2),那些不能在細(xì)胞培養(yǎng)中增殖的病毒則使用核酸檢測方法，快速提供檢測結(jié)果，但它其不能無法證實明標(biāo)本中病毒是具有感染性的。盡管過去認(rèn)為血清學(xué)檢查是實驗室診斷病毒感染的主要手段，但更應(yīng)注意是否能及時得到感染的信息。對那些可能新出現(xiàn)的病毒，只有

19、采用分離方法可取得最好的診斷結(jié)果。,病毒感染實驗診斷方法諸多，且各具不同的敏感度和特異度，臨床診斷選擇實驗項目應(yīng)以循證檢驗為依據(jù)。有關(guān)的醫(yī)學(xué)專業(yè)組織可充分利用已有的循證診斷試驗的文獻(xiàn)，制定科學(xué)的、嚴(yán)格的、用于臨床疾病診斷的指南。指南根據(jù)現(xiàn)有的最佳證據(jù)或?qū)＜业囊恢乱庖?，提供診斷建議，將實驗室診斷試驗項目分別按證據(jù)的等級水平進(jìn)行標(biāo)記。臨床實驗室人員、特別是臨床醫(yī)師可依據(jù)指南對同一組織器官、不同病毒感染或不同組織器官、同一病毒感染選擇最佳檢測