8 人檢測結(jié)果的解釋分析

中國人禽流感〔H5N1〕病例的實驗室診斷及結(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呼吸道標本的采集血清標本的采集組織標本的采集消化道標本的采集第一局部檢測標本的采集人禽流感病例呼吸道標本的采集

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本的采集盡量采集深部呼吸道標本肺穿刺液深部吸取液血清標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本。組織標本的采集肺活檢組織尸檢組織，尸檢組織標本應(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。消化道標本的采集糞便標本特別是有腹瀉病癥的糞便標本PBS或者細胞培養(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)

標本的保存和運輸用于病毒別離的標本應(yīng)保存于4oC,并且立即送往實驗室進行接種，如果在48小時內(nèi)不能進行接種，應(yīng)該保存于-70oC；標本應(yīng)防止反復(fù)的凍融〔降低別離率〕；血清標本可以在4oC保存7天，否那么應(yīng)保存于-20oC；標本的保存和運輸應(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驗室檢測應(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準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。微量中和實驗恢復(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)方法可以驗證,如馬血球?qū)嶒災(zāi)种茖嶒灴贵w滴度在1:160以上或H5特異的westerblot檢測結(jié)果陽性。確認人禽流感病例(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；無法進行早期診斷；金標準；病毒可用于其它研究；需要較嚴格的實驗條件；H5N1病毒的檢測方法2005年以來中國人禽流感病例的實驗室診斷結(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驗室診斷結(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)的建立臨床標本的RT-PCR檢測H5N1Real-timePCR靈敏度檢測臨床標本的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號為氣管吸取物標本一臨床病例的MDD檢測結(jié)果NASBAH5N1標本的檢測結(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NASBA技術(shù),與Real-PCR方法靈敏度相當。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分子不易對實驗儀器和環(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不同種屬紅細胞HI實驗的比較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實驗的優(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實驗中的溶血圈的產(chǎn)生。SRH技術(shù)的特點簡便、可靠、重復(fù)性好、特異性強、敏感性高，不受血清中非特異性抑制素影響。血清用量少,并不必進行稀釋?？蓪Υ罅垦鍢吮具M行測定。一般實驗室均可開展，特別適用于我國基層地區(qū)。單擴溶血的特異性對H5N1

患者血清的測定微量中和實驗(MN〕的特點微量中和實驗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(strainselection)。每次實驗要設(shè)病毒和細胞對照，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

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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,