人禽流感检测结果的解释分析课件

1、 中国人禽流感（H5N1）病例的 实验室诊断及结果分析 2007年流感培训 中国疾病预防控制中心 病毒病预防控制所 国家流感中心Country20032004200520062007TotalcasesdeathscasesdeathscasesdeathscasesdeathscasesdeathscasesdeathsAzerbaijan000000850085Cambodia000044221177China110085138322516Djibouti000000100010Egypt00000018102053815Indonesia000020135545282410382Iraq

2、000000320032Lao000000002222Nigeria000000001111Thailand0017125233002517Turkey00000012400124Total4446329843115795735320193全球人禽流感（H5N1）病例数Total number of cases includes number of deaths.WHO reports only laboratory-confirmed cases.16 August 2007呼吸道标本的采集血清标本的采集组织标本的采集消化道标本的采集第一部分 检测标本的采集人禽流感病例呼吸道标本的采集尽量采

3、集深部呼吸道标本肺穿刺液深部吸取液血清标本的采集采集急性期和恢复期血清；急性期血清应该在病人发病后立即采集，一般不要超过7天，如果没有采集到7天内血清，在能够采集的时候立即采集；恢复期血清在病人发病后2-3周采集，如果病人死亡，在死亡前应采集一份血清；病人恢复后应定期采集血清；采集血清时不能少于5ml；最好采集全血和血清标本。消化道标本的采集粪便标本特别是有腹泻症状的粪便标本PBS 或者细胞培养液 加入以下抗菌素和0.5%BSA -氨苄青霉素 (2 x 106 IU/litre)- 链霉素 (200 mg/litre) - 多粘菌素 B (2 x 106 IU/litre) - 庆大霉素 (2

4、50 mg/litre) - 制霉菌素 (0.5 x 106 IU/litre) - 盐酸氧氟沙星 (60 mg/litre), and - sulfamethoxazole (0.2 g/litre) 标本的保存和运输用于病毒分离的标本应保存于4oC,并且立即送往实验室进行接种，如果在48小时内不能进行接种，应该保存于-70oC；标本应避免反复的冻融（降低分离率）；血清标本可以在4oC保存7天，否则应保存于-20oC；标本的保存和运输应遵守国家生物安全的相关规定。任何成功的流感大流行的准备都依赖于对新出现毒株的正确的诊断。对于可能是A型流感病毒的每个样品最初的实验室检测应该快速并且能够排除其

5、它常见的呼吸道病毒感染。最理想检测方法是24小时内能出结果。第二部分：H5N1检测方法的建立符合以下标准之一的病例可以诊断为疑似或可能的H5N1病例H5N1 病毒分离阳性。 两种不同的引物针对不同的片段检测H5病毒的PCR结果为 阳性。微量中和实验恢复血清(发病后2-4周)比急性期血清(发病后7日内) H亚型抗体滴度升高4倍或以上， 且恢复期的血清的微量中和抗体滴度在1:80以上。单份恢复期血清(症状出现后的14天或以后)的微量抗体滴度在1:80或 以上,这以阳性结果采用不同的血清学方法可以验证,如马血球实验凝集抑制实验抗体滴度在1:160 以上或 H5特异的 westerblot 检测结果阳

6、性。确认人禽流感病例(notify WHO) 核酸检测抗体检测病毒分离抗原检测HIMNSRH细胞分离鸡胚分离PCRMDDNASBAIFAELISA胶体金诊断快速；特异度高；灵敏度低；诊断快速；特异度高；灵敏度高；得不到病毒；特异度高；无法进行早期诊断；金标准；病毒可用于其它研究；需要较严格的实验条件；H5N1病毒的检测方法 2005年以来中国人禽流感病例的实验室诊断结果（续） Methods Cases RT-PCRReal-time PCRMDDHIMNELISA Virus IsolationSichuan case1+NDNDND+case2+NDNDND+case3+NDNDND+Sh

7、anghaicase1+NDNDND+Guangdongcase1+NDNDND+case2+NDNDND+Hubeicase1+NDNDND+Zhejiangcase1+NDNDND+Liaoning case1-+-Xinjiangcase1-NDNDND+RT-PCR技术的建立Real-time PCR 灵敏度检测临床标本的Real-time PCR检测Luminex xMAP technologyMDD 对呼吸道病毒的诊断MDD对流感病毒亚型的鉴别1号为气管吸取物标本一临床病例的MDD检测结果NASBA H5N1 标本的检测结果VirusesH5 NASBA results N1NAS

8、BA resultsH1N1NegativeNegativeH3N2NegativeNegativeFlu BNegativeNegativeANHUI/1/2005/H5N1PositivePositiveGUANGDONG/1/2005/H5N1PositivePositiveXINJIANG/1/2006/H5N1PositivePositivesamplesH5 NASBA results N1 NASBA resultsH5 Real-time RT-PCR resultsH5N1(100TCID50)PositivePositivePositiveH5N1(10TCID50)Pos

9、itivePositivePositiveH5N1(1TCID50)PositivePositivePositiveH5N1(10-1TCID50)PositivePositivePositiveH5N1(10-2TCID50)NegativeNegativeNegativeH5N1(10-3TCID50)NegativeNegativeNegativeH5N1(10HA)PositivePositivePositiveH5N1(1HA)PositivePositivePositiveH5N1(10-1HA)PositivePositivePositiveH5N1(10-2HA)Negativ

10、ePositiveNegativeH5N1(10-3HA)NegativeNegativeNegative NASBA 与Real-time RT-PCR敏感性的比较血清学检测方法 HI( HRBC)ELISASRHMNHI assayRBCChickenTurkeyGuinea pigHuman (o)Horse Conc0.5%0.5%0.75%0.75%1%Plate shapeVVUUUIncubation temperature RTRTRTRT4oCIncubation time30 min30 min60min60 min60 min不同种属 红细胞HI实验的比较RBCSerum

11、 (USCDC)Serum (CNIC)ChickenTurkeyHorse16016016032051205120H5 HI titer using different red blood cellHRCCRCH5（ positive ，HS)16020H5（ positive ，USCDC)1280640H5（negative，Human)1010H5（ negative ，Human)1010H5（ negative ，Chicken)1010H5（ negative ，Chicken)10108day101017day 32016022day320160Antigen: A/chick

12、en/hunanxiangtan-he/21/2005(H5N1)(provided by HARBIN Veterinary Research Institute )Serum:HS: Chicken serum from HARBIN Veterinary Research Institute USCDC: goat serum from USCDCHI(HRBC) assay使用U型板4 代替室温孵育。每次病毒的滴度要回滴马血球要新鲜采集，一周内使用。SRH原理示意图SRH 实验的优化最适的抗原浓度是常规血凝板滴定 的1000 HAI。1% agarose。鸡血球更适合于SRH实验中的溶

13、血圈的产生。SRH技术的特点简便、可靠、重复性好、特异性强、敏感性高，不受血清中非特异性抑制素影响。血清用量少, 并不必进行稀释。可对大量血清标本进行测定。一般实验室均可开展，特别适用于我国基层地区。单扩溶血的特异性对H5N1患者血清的测定微量中和实验 (MN）的特点微量中和实验 是诊断人感染禽流感的最好的血清雪方法。 敏感性高。特异性强( strain selection) 。每次实验要设病毒和细胞对照，病毒要回滴A/CK/LNHS-JHL/23/05A/Anhui/1/2005A/CK/Hunanxiangtan-he/21/05Case 1Acute serum202020Case 1C

14、onvalescence serum2080160Case 2Acute serum202020Case 2Convalescence serum1604040Case 3Acute serum202020Case 3Convalescence serum208080Positive control1280640640MN assay of confirmed human cases 第三部分: 检测结果和分析antigenserumA/CK/HN/21/2005(H5N1)A/CK/LN/23/2005(H5N1)A/AH/1/2005(H5N1)A/AH/2/2005(H5N1)A/FJ/

15、1/2005(H5N1)A/GX/1/2005(H5N1)A/JX/1/2005(H5N1)NIBRGA/CK/HN/21/2005(H5N1)121.412112.8A/CK/LN/23/2005(H5N1)12.85.785.72.82A/AH/1/2005(H5N1)11.41112.8A/AH/2/2005(H5N1)12.81.414A/FJ/1/2005(H5N1)111.42A/GX/1/2005(H5N1)115.7A/JX/1/2005(H5N1)18NIBRG1The antigenic ratio of Human H5N1 isolates by HI assayAnt

16、igen Serum A/CK/HN/21/2005(H5N1)A/CK/LN/23/2005(H5N1)A/AH/1/2005(H5N1)A/AH/2/2005(H5N1)A/FJ/1/2005(H5N1)A/GX/1/2005(H5N1)A/JX/1/2005(H5N1)NIBRGA/CK/HN/21/2005(H5N1)121.41.41222A/CK/LN/23/2005(H5N1)12.8222.848A/AH/1/2005(H5N1)111.411.42.8A/AH/2/2005(H5N1)11.412.85.7A/FJ/1/2005(H5N1)1125.7A/GX/1/2005(

17、H5N1)115.7A/JX/1/2005(H5N1)14NIBRG1The antigenic ratio of Human H5N1 isolates by MN assayReference ferret serumA/Vietnam/1203/2004A/Indonesia/5/2005A/Turkey/15/2005A/Anhui/1/2005A/Anhui/2/2005A/Guangxi/1/2005A/Fujian/1/2005A/Sichuan/1/2006A/Jiangxi/1/2005VN/1203IND/5TK/15AH/1AH/232020104040206404016

18、080803201280802040320101280640201605320320208056403201080516016040160101280640806404025601280Antigenic analysis of human H5N1isolates by HI assay Antigenic analysis of human H5N1isolates by HI assay using chicken serumAntigen Reference chicken serumAH/1AH/2XJ/1VN/1194A/Anhui/1/20053201608080A/Anhui/

19、2/2005 640 64016080A/Xinjiang/1/2006401016020A/Vietnam/194/2004-PR8RG404020320South China North China VietNam Antigenic assay of human H5N1 isolates By MN assay using confirmed human case serumAntigen Confirmed casescase1*case2#A/Anhui/1/200516040A/Xinjiang/1/200640320case1 is a survived confirmed h

20、uman H5N1 case from Anhui province(southern china), the serum was collected 4 weeks after the illness onset.#Case 2 is a survived confirmed human H5N1 case from Liaoning province(northern china), the serum was collected 4 weeks after the illness onset.Human H5N1 confirmed cases in mainland of china

21、since Nov,2005death(15)survival(9)China CDCClade 1Clade2.1Clade2.3 Clade 2.2HA Phylogenetic tree generated by the neighbor-joining method using the Mega3.1 program and the boottrap value 1000, the result showing the relationships of HA genes of representative influenza A H5N1 virus from different re

22、gions. The red dots denote viruses isolated from humans in China; green dots denote viruses isolated from Viet Nam and were recommended as vaccine strains by the WHO. NAPB2PB1NPNSPAMA/JX/1/2005A/Ck/GXNN/6/2004Clade IA/VN/1194/2004Clade 2.1A/BHG/5/2005A/XJ/1/2006Clade II 2.2A/IND/5/2005Clade II 2.3A/

23、AH/1/2005A/AH/2/2005A/SC/1/2006A/SC/2/2006A/SC/3/2006A/HB/1/2006A/ZJ/1/2006Virus isolatesHA(226-228)a 2-3specific A/AH/1/05QSGA/AH/2/05RSG?A/GX/1/05QSGA/JX/1/05QSGA/FJ/1/05QSGA/SC/1/06QSGA/SC/2/06QSGA/HN/1/06QSGA/AH/1/06QSGA/ZJ/1/06QSGA/GD/1/06QSGA/SH/1/06QSGA/HB/1/06QSGA/SC/3/06QSGA/GD/2/06QSGA/XJ/

24、1/06QSGA/FJ/1/2007QSGA/AH/1/2007QSGReceptor Specificity (HA 226-228)Virus isolatesM2(25-42)Sensitive A/AH/1/05PLVVAASIIGILHLILWILA/AH/2/05PLVVAASIIGILHLILWILA/GX/1/05PLVVAASIIGILHLILWILA/JX/1/05PLVVAASIIGILHLILWILA/FJ/1/05PLVVAASIIGILHLILWILA/SC/1/06PLVVAASIIGILHLILWILA/SC/2/06PLVVAASIIGILHLILWILA/H

25、N/1/06PLVVAASIIGILHLILWILA/AH/1/06PLVVAASIIGILHLILWILA/ZJ/1/06PLVVAASIIGILHLILWILA/GD/1/06PLVVAASIIGILHLILWILA/SH/1/06PLVVAASIIGILHLILWILA/HB/1/06PLVVAASIIGILHLILWILA/SC/3/06PLVVAASIIGILHLILWILA/GD/2/06PLVVAASIIGILHLILWILA/XJ/1/06PLVVAASIIGILHLILWILA/FJ/1/2007PLVVAANIIGILHLILWILXA/AH/1/2007PLVVAASII

26、GILHLILWILDrug surveillance (M2 inhibitor drugs)Drug surveillance ( NA inhibitor drugs)Virus isolatesNA(274)Sensitive(H)Resistant(Y)A/AH/1/05LNAPNYHYEEA/AH/2/05LNAPNYHYEEA/GX/1/05LNAPNYHYEEA/JX/1/05LDAPNYHYEEA/FJ/1/05LDAPNYHYEEA/SC/1/2006LNAPNYHYEEA/SC/2/2006LNAPNYHYEEA/HN/1/2006LNAPNYHYEEA/AH/3/06L

27、DAPNYHYEEA/ZJ/1/06LNAPNYHYEEA/GD/1/06LDAPNYHYEEA/SH/1/06LDAPNYHYEEA/HB/1/06LNAPNYHYEEA/SC/3/2006LNAPNYHYEEA/GD/2/06LNAPNYHYEEA/XJ/1/06LDAPNYHYEEA/FJ/1/07LNAPNYHYEEA/AH/1/07LDAPNYHYEEVirus isolatesHA connecting peptideHPAIVA/AH/1/05LRERRRKRGA/AH/2/05LRERRRKRGA/GX/1/05LRERRRKRGA/JX/1/05LRERRRKRGA/FJ/1

28、/05LRERRRKRGA/SC/1/2006LRERRRKRGA/SC/2/2006LRERRRKRGA/HN/1/2006LRERRRKRGA/AH/3/06LRERRRKRGA/ZJ/1/06LRERRRKRGA/GD/1/06LRERRRKRGA/SH/1/06LRERRRKRGA/SC/3/06LRERRRKRGA/HB/1/06LRERRRKRGA/GD /2/06LRERRRKRGA/FJ/1/07LRERRRKRGA/AH/1/07LRERRRKRGA/XJ/1/06QGERRRKKRGHA connecting peptideWHO recommended candidate

29、 H5N1 vaccine viruses for potential use as pre-pandemic vaccines A/Hongkong/213/03 A/Vietnam/1194/04 A/Vietnam/1203/04 A/Indonesia/5/2005 A/BHG/QH/1A/2005 A/WPS/Mongolia/244/2005 A/Turkey/Turkey/1/2005A/AH/1/2005 printable version Availability of new recombinant H5N1 vaccine virus21 December 2006The

30、 WHO Global Influenza Programme is continuing to monitor the antigenic and genetic evolution of circulating H5N1 viruses and their human isolates. In August 2006, WHO reported1 that analysis of newly isolated viruses collected from both animals and humans indicated that the H5 haemagglutinin (HA) ge

31、nes had become genetically distinguishable from the H5N1 viruses that had previously been selected for vaccine development. Furthermore, there was also evidence of antigenic variation among the recent viruses. Since then, WHO Collaborating Centres (WHO CCs) and H5 Reference Laboratories have been de

32、veloping several new recombinant H5N1 vaccine viruses that are representative of this newly discovered genetic sub-group of clade 2 viruses.The WHO CC in the Centers for Disease Control and Prevention (CDC), Atlanta USA and the China Centers for Disease Control, Beijing China have together developed

33、 a new recombinant H5N1 virus from A/Anhui/1/2005 selected from clade 2, sub-clade 3. The recombinant vaccine virus is available for distribution, under a Material Transfer Agreement (MTA). The sequences of haemagglutinin (HA) and neuraminidase (NA) of the new H5N1 recombinant vaccine virus can be f

34、ound on the public web site of Los Alamos National Laboratory database together with all other WHO selected and developed influenza vaccine viruses for both seasonal and H5N1 influenza.Institutions, companies and others interested in pandemic vaccine development, who wish to receive this recombinant

35、 vaccine virus should contact either the WHO Global Influenza Programme at or WHO CC CDC at the address below:WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, 1600 Clifton Road, Mail stop G16, Atlanta, GA 30333, USA (fax: +1 404 639 23 34; e-mail: ).Review of the needs and the actual development of H5N1 vaccine viruse