8 人检测结果的解释分析

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Country20032004200520062007TotalcasesdeathscasesdeathscasesdeathscasesdeathscasesdeathscasesdeathsAzerbaijan000000850085Cambodia000044221177China110085138322516Djibouti000000100010Egypt00000018102053815Indonesia

000020135545282410382Iraq000000320032Lao000000002222Nigeria000000001111Thailand0017125233002517Turkey00000012400124Total4446329843115795735320193全球人禽流感〔H5N1〕病例数Totalnumberofcasesincludesnumberofdeaths.

WHOreportsonlylaboratory-confirmedcases.16August2007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局部检测标本的采集人禽流感病例呼吸道标本的采集

鼻拭子鼻咽拭子鼻咽吸取物鼻洗液咽洗液气管吸取物气管肺灌洗液

人禽流感病例呼吸道标本的采集尽量采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。组织标本的采集肺活检组织尸检组织，尸检组织标本应尽可能采集各种器官组织，在采集过程中应尽可能防止交叉污染，每种尸检组织标本可分为两局部，一局部立即用于病毒的别离检测，如果不能马上进行，应该保存于-70oC；另外一局部保存于福尔马林中，用于病理的研究。消化道标本的采集粪便标本特别是有腹泻病症的粪便标本PBS或者细胞培养液参加以下抗菌素和0.5%BSA-氨苄青霉素(2x106IU/litre)

-链霉素(200mg/litre)

-多粘菌素B(2x106IU/litre)

-庆大霉素(250mg/litre)

-制霉菌素(0.5x106IU/litre)

-盐酸氧氟沙星(60mg/litre),and

-sulfamethoxazole(0.2g/litre)

标本的保存和运输用于病毒别离的标本应保存于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；标本应防止反复的冻融〔降低别离率〕；血清标本可以在4oC保存7天，否那么应保存于-20oC；标本的保存和运输应遵守国家生物平安的相关规定。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。第二局部：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病毒别离阳性。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(病症出现后的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检测结果阳性。确认人禽流感病例(notifyWHO)

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

MethodsCasesRT-PCRReal-timePCRMDDHIMNELISAVirusIsolation

Hunancase1---+++-case2++++++-case3+++NDNDND+

Anhuicase1+++NDNDND+case2+++NDND++case3+++NDNDND+case4++++++-case5+++NDNDND+Guangxicase1+++NDNDND+Jiangxicase1+++NDNDND+Fujiancase1+++NDNDND+case2++++++-case3++NDNDNDND+case4++++++-2005年以来中国人禽流感病例的实验室诊断结果〔续〕Methods

CasesRT-PCRReal-timePCRMDDHIMNELISAVirusIsolationSichuancase1+++NDNDND+case2+++NDNDND+case3+++NDNDND+Shanghaicase1+++NDNDND+Guangdongcase1+++NDNDND+case2+++NDNDND+Hubeicase1+++NDNDND+Zhejiangcase1+++NDNDND+Liaoningcase1---+++-Xinjiangcase1---NDNDND+RT-PCR技术的建立临床标本的RT-PCR检测H5N1Real-timePCR灵敏度检测临床标本的Real-timePCR检测LuminexxMAPtechnologyMDD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NASBAH5N1标本的检测结果VirusesH5NASBAresultsN1NASBAresultsH1N1NegativeNegativeH3N2NegativeNegativeFluBNegativeNegativeANHUI/1/2005/H5N1PositivePositiveGUANGDONG/1/2005/H5N1PositivePositiveXINJIANG/1/2006/H5N1PositivePositivesamplesH5NASBAresultsN1NASBAresultsH5Real-timeRT-PCRresultsH5N1(100TCID50)PositivePositivePositiveH5N1(10TCID50)PositivePositivePositiveH5N1(1TCID50)PositivePositivePositiveH5N1(10-1TCID50)PositivePositivePositiveH5N1(10-2TCID50)NegativeNegativeNegativeH5N1(10-3TCID50)NegativeNegativeNegativeH5N1(10HA)PositivePositivePositiveH5N1(1HA)PositivePositivePositiveH5N1(10-1HA)PositivePositivePositiveH5N1(10-2HA)NegativePositiveNegativeH5N1(10-3HA)NegativeNegativeNegativeNASBA与Real-timeRT-PCR敏感性的比较NASBA技术的特点NASBA技术,与Real-PCR方法灵敏度相当。NASBA技术操作简便，而且是一种高通量的方法。与PCR的产物DNA分子不同，RNA分子不易对实验仪器和环境造成污染。

血清学检测方法

HI(HRBC)ELISASRHMNHIassayRBCChickenTurkeyGuineapigHuman(o)HorseConc0.5%0.5%0.75%0.75%1%PlateshapeVVUUUIncubationtemperatureRTRTRTRT4oCIncubationtime30min30min60min60min60min不同种属红细胞HI实验的比较RBCSerum(USCDC)Serum(CNIC)ChickenTurkeyHorse16016016032051205120H5HItiterusingdifferentredbloodcellHRCCRCH5（positive，HS)16020H5（positive，USCDC)1280640H5（negative，Human)

10

10H5（negative，Human)

10

10H5（negative，Chicken)

10

10H5（negative，Chicken)

10

108day

10

1017day32016022day320160Antigen:A/chicken/hunanxiangtan-he/21/2005(H5N1)(providedbyHARBINVeterinaryResearchInstitute)Serum:HS:ChickenserumfromHARBINVeterinaryResearchInstituteUSCDC:goatserumfromUSCDCHI(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1000HAI。1%agarose。鸡血球更适合于SRH实验中的溶血圈的产生。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(strainselection)。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/21/05Case1Acuteserum<20<20<20Case1Convalescenceserum<2080160Case2Acuteserum<20<20<20Case2Convalescenceserum1604040Case3Acuteserum<20<20<20Case3Convalescenceserum<208080Positivecontrol1280640640MNassayofconfirmedhumancases第三局部: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/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)18NIBRG

1TheantigenicratioofHumanH5N1isolatesbyHIassay

AntigenSerumA/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)1＞21.41.4122＞2A/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(H5N1)115.7A/JX/1/2005(H5N1)1＞4NIBRG1TheantigenicratioofHumanH5N1isolatesbyMNassayReferenceferretserumA/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/232020104040206404016080803201280802040320101280640201605320320208056403201080516016040160101280640806404025601280AntigenicanalysisofhumanH5N1isolatesbyHIassay

AntigenicanalysisofhumanH5N1isolatesbyHIassayusingchickenserumAntigenReferencechickenserumAH/1AH/2XJ/1VN/1194A/Anhui/1/20053201608080A/Anhui/2/200564064016080A/Xinjiang/1/2006401016020A/Vietnam/194/2004-PR8RG404020320SouthChinaNorthChinaVietNamAntigenicassayofhumanH5N1isolatesByMNassayusingconfirmedhumancaseserumAntigenConfirmedcasescase1*case2#A/Anhui/1/200516040A/Xinjiang/1/200640320case1isasurvivedconfirmedhumanH5N1casefromAnhuiprovince(southernchina),theserumwascollected4weeksaftertheillnessonset.#Case2isasurvivedconfirmedhumanH5N1casefromLiaoningprovince(northernchina),theserumwascollected4weeksaftertheillnessonset.HumanH5N1confirmedcasesinmainlandofchinasinceNov,2005death(15)survival(9)ChinaCDCClade1Clade2.1Clade2.3Clade2.2HAPhylogenetictreegeneratedbytheneighbor-joiningmethodusingtheMega3.1programandtheboottrapvalue1000,theresultshowingtherelationshipsofHAgenesofrepresentativeinfluenzaAH5N1virusfromdifferentregions.ThereddotsdenotevirusesisolatedfromhumansinChina;greendotsdenotevirusesisolatedfromVietNamandwererecommendedasvaccinestrainsbytheWHO.NAPB2PB1NPNSPAMA/JX/1/2005A/Ck/GXNN/6/2004CladeIA/VN/1194/2004Clade2.1A/BHG/5/2005A/XJ/1/2006CladeII2.2A/IND/5/2005CladeII2.3A/AH/1/2005A/AH/2/2005A/SC/1/2006A/SC/2/2006A/SC/3/2006A/HB/1/2006A/ZJ/1/2006VirusisolatesHA(226-228)a2-3specificA/AH/1/05QSG√A/AH/2/05RSG?A/GX/1/05QSG√A/JX/1/05QSG√A/FJ/1/05QSG√A/SC/1/06QSG√A/SC/2/06QSG√A/HN/1/06QSG√A/AH/1/06QSG√A/ZJ/1/06QSG√A/GD/1/06QSG√A/SH/1/06QSG√A/HB/1/06QSG√A/SC/3/06QSG√A/GD/2/06QSG√A/XJ/1/06QSG√A/FJ/1/2007QSG√A/AH/1/2007QSG√ReceptorSpecificity(HA226-228)VirusisolatesM2(25-42)SensitiveA/AH/1/05PLVVAASIIGILHLILWIL√A/AH/2/05PLVVAASIIGILHLILWIL√A/GX/1/05PLVVAASIIGILHLILWIL√A/JX/1/05PLVVAASIIGILHLILWIL√A/FJ/1/05PLVVAASIIGILHLILWIL√A/SC/1/06PLVVAASIIGILHLILWIL√A/SC/2/06PLVVAASIIGILHLILWIL√A/HN/1/06PLVVAASIIGILHLILWIL√A/AH/1/06PLVVAASIIGILHLILWIL√A/ZJ/1/06PLVVAASIIGILHLILWIL√A/GD/1/06PLVVAASIIGILHLILWIL√A/SH/1/06PLVVAASIIGILHLILWIL√A/HB/1/06PLVVAASIIGILHLILWIL√A/SC/3/06PLVVAASIIGILHLILWIL√A/GD/2/06PLVVAASIIGILHLILWIL√A/XJ/1/06PLVVAASIIGILHLILWIL√A/FJ/1/2007PLVVAANIIGILHLILWILXA/AH/1/2007PLVVAASIIGILHLILWIL√Drugsurveillance(M2inhibitordrugs)Drugsurveillance(NAinhibitordrugs)VirusisolatesNA(274)Sensitive(H)Resistant(Y)A/AH/1/05LNAPNYHYEE√A/AH/2/05LNAPNYHYEE√A/GX/1/05LNAPNYHYEE√A/JX/1/05LDAPNYHYEE√A/FJ/1/05LDAPNYHYEE√A/SC/1/2006LNAPNYHYEE√A/SC/2/2006LNAPNYHYEE√A/HN/1/2006LNAPNYHYEE√A/AH/3/06LDAPNYHYEE√A/ZJ/1/06LNAPNYHYEE√A/GD/1/06LDAPNYHYEE√A/SH/1/06LDAPNYHYEE√A/HB/1/06LNAPNYHYEE√A/SC/3/2006LNAPNYHYEE√A/GD/2/06LNAPNYHYEE√A/XJ/1/06LDAPNYHYEE√A/FJ/1/07LNAPNYHYEE√A/AH/1/07LDAPNYHYEE√VirusisolatesHAconnectingpeptideHPAIVA/AH/1/05LRERRRKRG√A/AH/2/05LRERRRKRG√A/GX/1/05LRERRRKRG√A/JX/1/05LRERRRKRG√A/FJ/1/05LRERRRKRG√A/SC/1/2006LRERRRKRG√A/SC/2/2006LRERRRKRG√A/HN/1/2006LRERRRKRG√A/AH/3/06LRERRRKRG√A/ZJ/1/06LRERRRKRG√A/GD/1/06LRERRRKRG√A/SH/1/06LRERRRKRG√A/SC/3/06LRERRRKRG√A/HB/1/06LRERRRKRG√A/GD/2/06LRERRRKRG√A/FJ/1/07LRERRRKRG√A/AH/1/07LRERRRKRG√A/XJ/1/06QGERRRKKRG√HAconnectingpeptideWHOrecommendedcandidateH5N1vaccinevirusesforpotentialuseaspre-pandemicvaccines

A/Hongkong/213/03A/Vietnam/1194/04A/Vietnam/1203/04A/Indonesia/5/2005A/BHG/QH/1A/2005A/WPS/Mongolia/244/2005A/Turkey/Turkey/1/2005A/AH/1/2005

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AvailabilityofnewrecombinantH5N1vaccinevirus21December2006TheWHOGlobalInfluenzaProgrammeiscontinuingtomonitortheantigenicandgeneticevolutionofcirculatingH5N1virusesandtheirhumanisolates.InAugust2006,WHOreported1thatanalysisofnewlyisolatedvirusescollectedfrombothanimalsandhumansindicatedthattheH5haemagglutinin(HA)geneshadbecomegeneticallydistinguishablefromtheH5N1virusesthathadpreviouslybeenselectedforvaccinedevelopment.Furthermore,therewasalsoevidenceofantigenicvariationamongtherecentviruses.Sincethen,WHOCollaboratingCentres(WHOCCs)andH5ReferenceLaboratorieshavebeendevelopingseveralnewrecombinantH5N1vaccinevirusesthatarerepresentativeofthisnewlydiscoveredgeneticsub-groupofclade2viruses.TheWHOCCintheCentersforDiseaseControlandPrevention(CDC),AtlantaUSAandtheChinaCentersforDiseaseControl,BeijingChinahavetogetherdevelopedanewrecombinantH5N1virusfromA/Anhui/1/2005selectedfromclade2,sub-clade3.Therecombinantvaccinevirusisavailablefordistribution,underaMaterialTransferAgreement(MTA).Thesequencesofhaemagglutinin(HA)andneuraminidase(NA)ofthenewH5N1recombinantvaccineviruscanbefoundonthepublicwebsiteofLosAlamosNationalLaboratorydatabasetogetherwithallotherWHOselectedanddevelopedinfluenzavaccinevirusesforbothseasonalandH5N1influenza.Institutions,companiesandothersinterestedinpandemicvaccinedevelopment,whowishtoreceivethisrecombinantvaccinevirusshouldcontacteithertheWHOGlobalInfluenzaProgrammeatwhoinfluenza@orWHOCCCDCattheaddressbelow:WHOCollaboratingCenterforSurveillance,EpidemiologyandControlofInfluenza,CentersforDiseaseControlandPrevention,1600CliftonRoad,MailstopG16,Atlanta,