8 人檢測結(jié)果的解釋分析

中國人禽流感〔H5N1〕病例的實(shí)驗(yàn)室診斷及結(jié)果分析

2007年流感培訓(xùn)中國疾病預(yù)防控制中心病毒病預(yù)防控制所國家流感中心Country20032004200520062007TotalcasesdeathscasesdeathscasesdeathscasesdeathscasesdeathscasesdeathsAzerbaijan000000850085Cambodia000044221177China110085138322516Djibouti000000100010Egypt00000018102053815Indonesia

000020135545282410382Iraq000000320032Lao000000002222Nigeria000000001111Thailand0017125233002517Turkey00000012400124Total4446329843115795735320193全球人禽流感〔H5N1〕病例數(shù)Totalnumberofcasesincludesnumberofdeaths.

WHOreportsonlylaboratory-confirmedcases.16August2007呼吸道標(biāo)本的采集血清標(biāo)本的采集組織標(biāo)本的采集消化道標(biāo)本的采集第一局部檢測標(biāo)本的采集人禽流感病例呼吸道標(biāo)本的采集

鼻拭子鼻咽拭子鼻咽吸取物鼻洗液咽洗液氣管吸取物氣管肺灌洗液

人禽流感病例呼吸道標(biāo)本的采集盡量采集深部呼吸道標(biāo)本肺穿刺液深部吸取液血清標(biāo)本的采集采集急性期和恢復(fù)期血清；急性期血清應(yīng)該在病人發(fā)病后立即采集，一般不要超過7天，如果沒有采集到7天內(nèi)血清，在能夠采集的時候立即采集；恢復(fù)期血清在病人發(fā)病后2-3周采集，如果病人死亡，在死亡前應(yīng)采集一份血清；病人恢復(fù)后應(yīng)定期采集血清；采集血清時不能少于5ml；最好采集全血和血清標(biāo)本。組織標(biāo)本的采集肺活檢組織尸檢組織，尸檢組織標(biāo)本應(yīng)盡可能采集各種器官組織，在采集過程中應(yīng)盡可能防止交叉污染，每種尸檢組織標(biāo)本可分為兩局部，一局部立即用于病毒的別離檢測，如果不能馬上進(jìn)行，應(yīng)該保存于-70oC；另外一局部保存于福爾馬林中，用于病理的研究。消化道標(biāo)本的采集糞便標(biāo)本特別是有腹瀉病癥的糞便標(biāo)本PBS或者細(xì)胞培養(yǎng)液參加以下抗菌素和0.5%BSA-氨芐青霉素(2x106IU/litre)

-鏈霉素(200mg/litre)

-多粘菌素B(2x106IU/litre)

-慶大霉素(250mg/litre)

-制霉菌素(0.5x106IU/litre)

-鹽酸氧氟沙星(60mg/litre),and

-sulfamethoxazole(0.2g/litre)

標(biāo)本的保存和運(yùn)輸用于病毒別離的標(biāo)本應(yīng)保存于4oC,并且立即送往實(shí)驗(yàn)室進(jìn)行接種，如果在48小時內(nèi)不能進(jìn)行接種，應(yīng)該保存于-70oC；標(biāo)本應(yīng)防止反復(fù)的凍融〔降低別離率〕；血清標(biāo)本可以在4oC保存7天，否那么應(yīng)保存于-20oC；標(biāo)本的保存和運(yùn)輸應(yīng)遵守國家生物平安的相關(guān)規(guī)定。任何成功的流感大流行的準(zhǔn)備都依賴于對新出現(xiàn)毒株的正確的診斷。對于可能是A型流感病毒的每個樣品最初的實(shí)驗(yàn)室檢測應(yīng)該快速并且能夠排除其它常見的呼吸道病毒感染。最理想檢測方法是24小時內(nèi)能出結(jié)果。第二局部：H5N1檢測方法的建立符合以下標(biāo)準(zhǔn)之一的病例可以診斷為疑似或可能的H5N1病例H5N1病毒別離陽性。兩種不同的引物針對不同的片段檢測H5病毒的PCR結(jié)果為陽性。微量中和實(shí)驗(yàn)恢復(fù)血清(發(fā)病后2-4周)比急性期血清(發(fā)病后7日內(nèi))H亞型抗體滴度升高4倍或以上，且恢復(fù)期的血清的微量中和抗體滴度在1:80以上。單份恢復(fù)期血清(病癥出現(xiàn)后的14天或以后)的微量抗體滴度在1:80或以上,這以陽性結(jié)果采用不同的血清學(xué)方法可以驗(yàn)證,如馬血球?qū)嶒?yàn)?zāi)种茖?shí)驗(yàn)抗體滴度在1:160以上或H5特異的westerblot檢測結(jié)果陽性。確認(rèn)人禽流感病例(notifyWHO)

核酸檢測抗體檢測病毒分離抗原檢測HIMNSRH細(xì)胞分離雞胚分離PCRMDDNASBAIFAELISA膠體金診斷快速；特異度高；靈敏度低；診斷快速；特異度高；靈敏度高；得不到病毒；特異度高；無法進(jìn)行早期診斷；金標(biāo)準(zhǔn)；病毒可用于其它研究；需要較嚴(yán)格的實(shí)驗(yàn)條件；H5N1病毒的檢測方法2005年以來中國人禽流感病例的實(shí)驗(yàn)室診斷結(jié)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年以來中國人禽流感病例的實(shí)驗(yàn)室診斷結(jié)果〔續(xù)〕Methods

CasesRT-PCRReal-timePCRMDDHIMNELISAVirusIsolationSichuancase1+++NDNDND+case2+++NDNDND+case3+++NDNDND+Shanghaicase1+++NDNDND+Guangdongcase1+++NDNDND+case2+++NDNDND+Hubeicase1+++NDNDND+Zhejiangcase1+++NDNDND+Liaoningcase1---+++-Xinjiangcase1---NDNDND+RT-PCR技術(shù)的建立臨床標(biāo)本的RT-PCR檢測H5N1Real-timePCR靈敏度檢測臨床標(biāo)本的Real-timePCR檢測LuminexxMAPtechnologyMDD對呼吸道病毒的診斷MDD對流感病毒亞型的鑒別1號為氣管吸取物標(biāo)本一臨床病例的MDD檢測結(jié)果NASBAH5N1標(biāo)本的檢測結(jié)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與Real-timeRT-PCR敏感性的比較NASBA技術(shù)的特點(diǎn)NASBA技術(shù),與Real-PCR方法靈敏度相當(dāng)。NASBA技術(shù)操作簡便，而且是一種高通量的方法。與PCR的產(chǎn)物DNA分子不同，RNA分子不易對實(shí)驗(yàn)儀器和環(huán)境造成污染。

血清學(xué)檢測方法

HI(HRBC)ELISASRHMNHIassayRBCChickenTurkeyGuineapigHuman(o)HorseConc0.5%0.5%0.75%0.75%1%PlateshapeVVUUUIncubationtemperatureRTRTRTRT4oCIncubationtime30min30min60min60min60min不同種屬紅細(xì)胞HI實(shí)驗(yàn)的比較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℃代替室溫孵育。每次病毒的滴度要回滴馬血球要新鮮采集，一周內(nèi)使用。SRH原理示意圖SRH實(shí)驗(yàn)的優(yōu)化最適的抗原濃度是常規(guī)血凝板滴定的1000HAI。1%agarose。雞血球更適合于SRH實(shí)驗(yàn)中的溶血圈的產(chǎn)生。SRH技術(shù)的特點(diǎn)簡便、可靠、重復(fù)性好、特異性強(qiáng)、敏感性高，不受血清中非特異性抑制素影響。血清用量少,并不必進(jìn)行稀釋?？蓪Υ罅垦鍢?biāo)本進(jìn)行測定。一般實(shí)驗(yàn)室均可開展，特別適用于我國基層地區(qū)。單擴(kuò)溶血的特異性對H5N1

患者血清的測定微量中和實(shí)驗(yàn)(MN〕的特點(diǎn)微量中和實(shí)驗(yàn)是診斷人感染禽流感的最好的血清雪方法。

敏感性高。特異性強(qiáng)(strainselection)。每次實(shí)驗(yàn)要設(shè)病毒和細(xì)胞對照，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:檢測結(jié)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

printableversion

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,