微生物检验理论课 13临床常见病毒检验2 学习资料

临床常见病毒检验二南方医院医学检验系李海侠肝炎病毒

HEPATITISVIRUSESHAV甲型肝炎病毒HBV乙型肝炎病毒HCV丙型肝炎病毒HDV丁型肝炎病毒HEV戊型肝炎病毒HFV已型肝炎病毒HGV庚型肝炎病毒甲型肝炎病毒

HepatitisAvirus，HAV是引起甲型肝炎的病原体生物学性状属肠道病毒72型形态：呈球形结构：直径27～32nm,单股正链RNA，无包膜动物模型：黑猩猩对HAV易感细胞培养：原代猕猴肝细胞、猴胚肾细胞，一般不产生细胞病变抗原型别单一抵抗力：抵抗力强，对乙醚、酸、热等较稳定致病性与免疫性传染源：病人传染方式：粪口途径致病机制：在肝细胞内增殖，不直接造成肝细胞损伤，免疫病理作用导致肝细胞损伤免疫性：刺激机体产生抗HAVIgG和IgM

抗HAVIgG对再感染具有免疫力微生物学检验免疫电镜检测病毒颗粒免疫学方法检测病毒抗原检测抗HAVIgM是确诊甲型肝炎的依据方法：ELISA、化学发光等PCR法：检测病毒核酸检测抗HAVIgG主要用于流行病学调查防治原则特异性预防：接种甲型肝炎病毒减毒活疫苗紧急预防：肌肉注射丙种球蛋白一般性预防：加强饮水和食品的卫生监督管理乙型肝炎病毒

hepatitisBvirus,HBVHBsAgPrevalence>8%-High

2-7%-Intermediate

<2%-Low全球约有20亿人曾感染过乙肝其中3.5亿人为慢性HBV感染者,几乎一半在中国/mediacentre/factsheets/fs204/en慢性乙肝全球流行分布图china亚洲是乙肝病毒肆虐的重灾区，全球2/3以上的慢性乙肝患者生活在亚洲。而根据1992年全国乙肝血清流行病学调查的结果，中国的乙肝病毒携带者近1.3亿人，平均每10个人中有一个，感染率更高达57.6%。中国乙肝病情发展情况肝癌肝硬化慢性肝炎

HBV携带者40%20%80%1～4%Everyyear,1millionpeopledieofHBV-relatedlivercirrhosisorlivercancer=>HBVclaimsalifeevery30secondsHBV的结构42nm17-25nmlengthvariableppolHBcpreS1preS2tubularparticlesphericalDaneparticleStructureofHBVHBsAg

StructureofHBVc:Daneparticleb：tubularparticlea：sphericalparticleelectronmicroscopyGenomeofHBVacircular,double-strandedDNAcontainingsingle-strandbreaks

不完全双链环状DNAfouropenreadingframesthatencodesevenpolypeptides.含4个ORF,编码7个蛋白SHBsAg,Pre-s1,Pre-s2CHBcAg，HBeAgPpolymerase多聚酶XHBxAg基因抗原组成AntigenofHBVhepatitisBsurfaceantigenHBsAg表面抗原说明病毒在肝中复制（机体受感染标志）fourphenotypes：adr,adw,ayr,aywanti－HBs:neutralizationantibody中和抗体抗原组成AntigenofHBVhepatitisBcoreantigenHBcAg核心抗原notfoundinblood一般不能检出anti－HBcnon－neutralizationantibody非中和抗体CoreIgMindicatesrecentinfection.抗－HBcIgM说明HBV复制CoreIgGindicatesexposuretoHBV抗原组成AntigenofHBVhepatitisBeantigene抗原HBeAgthebestcorrelatetothepresenceofinfectiousvirus.感染性病毒存在的最有效证据（复制及具传染性的标志）anti-HBeindicateslowinfectivityinacarrier抗HBe说明病毒感染性较低（是预后良好的征象）HBV其他生物学性状培养:

◆黑猩猩是HBV最敏感的动物模型

◆细胞培养已获成功抵抗力:强

◆能耐受低温、紫外线、干燥和一般消毒剂100℃10分钟、高压蒸汽灭菌、0.5％过氧乙酸等能使HBV灭活致病性1.传染源：主要为急、慢性乙肝患者或病毒携带者2.传播途径：水平传播最主要途径是注射或粘膜接触含HBV的血液及分泌物性传播：不少见密切接触：有可能饮食传播：可能性较小垂直传播主要途径：围产期（新生儿皮肤、粘膜有破损而接触母血）和产后密切接触经胎盘及生殖细胞传播：有可能医源性传播输血或注射器、血透机、内窥镜等消毒不严格垂直传播是我国HBV感染的主要模式3.易感人群（1）感染者年龄高峰：低发病区：20～40岁高发病区：4～8岁（2）男女感染率相近，但发病者男多于女（3）感染时年龄越小，越易形成慢性肝炎、肝硬化或慢性HBV携带状态（免疫耐受）。（4）感染后对相同HBsAg亚型的HBV再感染有持久免疫力，但对不同亚型的保护力不完全。微生物学检验免疫学检测：HBV五项指标

HBsAg、抗HBsHBeAg、抗HBe

抗HBc分子生物学检测：血清中HBVDNASymptomsHBeAganti-HBeTotalanti-HBcIgManti-HBcanti-HBsHBsAg0481216202428323652100WeeksafterExposureTitreAcuteHepatitisBVirusInfectionwithRecoveryTypicalSerologicCourseAcute(6months)Chronic(Years)IgManti-HBcTotalanti-HBcHBsAgHBeAganti-HBe0481216202428323652YearsWeeksafterExposureTitreProgressiontoChronicHepatitisBVirusInfectionTypicalSerologicCoursea）ELISAenzymelinkedimmunosorbentassayb）TRFIA

time-resolvedfluoroimmunoassayc）CLIAchemiluminescentimmunoassay血清标志物检测方法AutomaticenzymeimmunoassayanalyzerElectroChemiLuminescenceAssy(ECLA)三种方法比较methodAdvantagesdisadvantagesELISAeconomy，shorttimeinBulkdetectionLowSensitivityLowspecificityqualitativeTRFIAHighSensitivityquantitativeSpecificitynothighSemi-automaticECLIAHighSensitivityhighspecificityquantitativeHighercost

1.HBsAb<10mIU/ml，noresistance,inoculationimmediately2.HBsAb10-100mIU/ml,weakimmunity，inoculationshouldbestrengthenedduring3-6month.

QuantitativedetectionofsurfaceantibodyClinicalAgreementsofWHOEuropeanregionrecommends100mIU/mlasthethresholdneedtostrengthentheimmune.疫苗接种后HBsAb监控具有重要意义>10mIU/ml

认为是阳性，>100mIU/ml才具备保护意义疫苗接种完成后1个月抗体浓度达到高峰乙肝表面抗原携带者乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb+----++++++++++++++急性肝炎早期，传染性强乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb++-+--++++++++++++急性或慢性现症感染，传染性强。“大三阳”乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb++++-+-++++++++++乙肝趋向恢复，属慢性携带，传染性弱“小三阳”乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb+++++++--++++++++急性感染或是慢性乙肝表面抗原携带，传染性弱“小二阳”乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb++++++++++---+++++急性乙肝恢复期或既往感染乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb+++++++++++-+-+++乙肝疫苗接种后乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb++++++++++++++-+---未感染过HBV乙肝两对半常见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb+++++++++++++++-----乙肝两对半罕见结果模式及其意义HBsAgHBsAbHBeAgHBeAbHBcAb+-+++++++++++急性感染趋于恢复，血清学转换不同亚型HBV二次感染；血清学转换HBsAgHBsAbHBeAgHBeAbHBcAb+++++++-+++++乙肝两对半罕见结果模式及其意义急性感染趋向恢复HBsAgHBsAbHBeAgHBeAbHBcAb+++++++++--+-++乙肝两对半罕见结果模式及其意义基因突变；钩状效应；爆发性肝炎；抗原表达量低，检测不出HBsAgHBsAbHBeAgHBeAbHBcAb++++++++++--+-+乙肝两对半罕见结果模式及其意义微生物学检验免疫学检测：HBV五项指标

HBsAg、抗HBsHBeAg、抗HBe

抗HBc分子生物学检测：血清中HBVDNA1)Detectionmethod:

PCR(Qualitativeandquantitative)2)Referencevalue:negative3)Clinicalsignificance：Positive:areliablediagnosticindicatorforacuteHBVinfection.Quantitativetestcanbeusedasaindicatorfortreatingeffectandthereplicatingconditionofthevirus.

HBVDNA的检测resultsevaluationofHBVDNAandseromarkers1.HBVDNAandHBsAg:Generally:HBsAg（+）,HBVDNA（+）

But:HBsAg（-）,HBVDNA（+）Reason:thesensitivityofELISAislow,itcouldnotdetectverylowconcentrationsofHBsAg.2)mutationofHBsAg,ELISAcouldnotdetectit.Reason:HBVDNAhasnotcompletelyeliminatedfromtheliver.Only(-)ofHBVDNAindicatevirusesareclearedcompletely.HBVDNAandHBsAb:

Generally:

HBsAb(+)(recovery),HBVDNA（-）

But:(alittle)HBsAb(+)(recovery),HBVDNA（+）3.HBVDNAandHBeAg、HBeAb、HBcAb:

HBeAg(+),HBVDNA(+)

HBeAg(-)HBeAb(+)HBcAb(+)，HBVDNA(+)Reason:HBeAg(-)HBeAb(+)HBcAb(+)onlyindicateviralreplicationisreducing,butnotclearedcompletely.

HBsAgandHBVDNAarecomplementarymarkersformonitoringCHBpatients

治疗中表面抗原定量测定可监测复发和应答，HBV难于发现HBsAg转换可作为持久免疫控制指标即使用高灵敏度试剂测不到HBVDNA并不表示无循环的HBV和/或HBV感染已清除时点评价病毒载量可作为抗病毒治疗有效和持续应答HBsAg定量检测可确定宿主免疫系统是否已有效控制HBV感染Controlthesourceofinfection控制传染源Cutdowntherouteoftransmission切断传播途径

Passiveimmunization-HepatitisBimmuneglobulin（HBIg）抗-HBs人血清球蛋白ActiveimmunizationsHBsAgvaccine防治原则丙型肝炎病毒

hepatitisCvirus,HCV

predominantcauseofnonAnonBhepatitis

HCV的结构Nucleicacid—singleplus-strandRNA,containing9genezonescapsid—composedofproteinCcapsule—containingE1、E2/NS1geneencodingproteincoreLipidmembraneE2E1Single-strandedRNAgenomeModeofHCVgenestructuresingleplus-strandRNAC:coregeneE:envelopegeneNS:non-structuralproteinsgeneThehepatitisCvirusproteinssingleplus-strandRNAThehighlightedproteinsareusedintheRocheAnti-HCVIIassay.Acombinationoftheseproteinsisusedtoincreasethesensitivityoftheassay.First-andsecond-generationassaysusedfewerpeptidesandwerefoundtobelesssensitive.ThehepatitisCvirusproteinssingleplus-strandRNAonethird(1/3)nearto5‘terminalisthecodingregionofstructuralprotein,anditisdividedintothreeparts(core,E1andE2/NS1),mainlycodeviralnucleocapsidproteinandenvelopeprotein;ThehepatitisCvirusproteinssingleplus-strandRNATwo-thirds(2/3)nearto3’terminalisthecodingregionofnon-structuralprotein,anditincludesNS2\NS3\NS4\NS5,mainlycodeNS2,NS3(helicaseandserineprotease),NS4andNS5(RNAdependedRNApolymerase)protein.Over50subtypeshavebeenidentified,classifiedinto6genotypesHCVRNAreplicationispronetoerrorsresultinginmutationsthatproducenewstrains.Asaresult,thevirusexistsasaquasispeciesinthebloodThisisonereasonwhyithasbeendifficulttoproduceavaccineforhepatitisC.

ClassificationofhepatitisCsubtypesiscomplexHoofnagle.Hepatology2002;Lemonetal.Field’sVirology2007.传播途径Transmittingthroughbloodandbloodproducts90%patientswithHCVareinfectedthroughthisway.Sexualcontact.Ithasbecomeanimportantnewwaysofinfection

IncidenceofhepatitisCinSTDpatientsishighto10%-30%.PercutaneousexposureIntravenousabuse2)Contaminatedtubes、syrings、needle3)Piercing,tattoos4)Transplantations5)dialysis

MothertochildFactorsaffectingtransmissionofHCVInjected

druguseMajorroutesoftransmissionIncountrieswithdonorscreeningprograms:IntravenousdruguseIncountrieswithoutdonorscreeningprograms:ContaminatedbloodtransfusionororgantransplantContaminatedmedicalequipmentLowerriskoftransmissionBirthtoaninfectedmotherSexualintercoursewithaninfectedpartnerNeedle-stickinjuryTattooingLavanchy.LiverInt2009;Lemonetal.Field’sVirology2007;WHO2003.WhathappenswheninfectionwithHCVoccurs?BloodPersistenceofvirus

ChronichepatitisCAcutehepatitisCInitialinfectionClearanceofvirus

Resolutionofinfection70–85%ofHCVinfectionsprogresstochronicHCVinfectionLong-termlivercomplicationsinpatientswithchronichepatitisCNormalliverChronichepatitisCirrhosisEnd-stageliverdiseaseChronicinflammationandincreasingliverdamageovertimeCancer(HCC)WHO2003.Aimoftherapy:Preventprogressiontocirrhosis,end-stageliverdisease,HCCanddeathX

4.RIBA（Recombinantimmunoblottingassay)--HCVAbIfHCVAbdetectedbyELISAandchemiluminescentimmunoassayis(+),RIBAmustbedoneinordertoconfirmHCVAb(+)检测方法ELISA–HCV-Ab,HCV-Ag2.chemiluminescentimmunoassay–HCVAb,HCVAgSensitivityandspecificityarehigherthanELISA,shortenWindowperiod3.RT-PCR—VirusRNA1、HCV-AbNormalreferencevalue：Negative（-）

HCV–Ab(+)：acuteorchronicinfectedorpastinfection,combinedwiththeclinicalsymptomstodiagnose

clinicalvalue2、HCV-Ag

Normalreferencevalue：Negative（-）

HCV-Ag(+):acute

3、ViralRNANormalreferencevalue：negative

HCVRNA(+)：indicatethatpatientshavebeeninfectedbyHCVwhichareinactivereplicatingandhavehighlyinfectious.HCVRNA和HCVAb联合检测临床意义HCVRNA(+),HCV–Ab(+)thepatientsisinthepresentinfection.HCVRNA(-),HCV-Ab(+)thepatientsisinthepreviousinfection.EvaluationforHCVRNAandSerologicandmolecularmarkersareimportantinthediagnosisofhepatitisCAdiagnosisofacuteinfectionismadeifHCVantibodiesorRNAaredetected.ChronichepatitisisdefinedasthepersistenceofHCVRNAformorethan6monthsHoofnagle.Hepatology2002;Lemonetal.Field’sVirology2007.Earlyacutephase–risingALTanddetectableviralRNA

ALT+Anti-HCV−HCVRNA+Symptoms−Contagious+EvaluationforHCVRNAandHCVAb

EvaluationforHCVRNAandHCVAb

Acutephaseofinfectionwithdetectable

antibodies–patientcontagious

ALT+Anti-HCV+HCVRNA+Symptoms+Contagious+EvaluationforHCVRNAandHCVAb

RecoveryfromHCVinfection–antibodiesdetectablewithnormalALTlevelsandundetectableHCVRNAALT−Anti-HCV+HCVRNA−Symptoms−Contagious−EvaluationforHCVRNAandChronicHCVinfection–nochangeindiseasecoursefor>6monthsALT+Anti-HCV+HCVRNA+Symptoms−Contagious+Clinicalvalue

OccultinfectionALT+(butcanbenormal)Anti-HCV+(butcanbenegative)HCVRNA−(unlesssensitiveassaysareused)Symptoms−Contagious+(potentiallycontagious)Michalaketal.FutureVirology2007;Carreño.WorldJGastroenterol2006DiagnosisofHCV:anti-HCVassayWHO2003.SupplementarytestingConfirmationofantibodypositiveresultswithHCVRNAtestImmunoblotassayRetestInduplicateAnti-HCVassayInitialscreeningtestNon-reactiveBothresultsImmunoblotassayorRNApositiveConfirmedresultHCVpositiveNon-reactiveHCVnegativeInitiallyreactiveRepeatedlyreactive

EitherresultreactiveHCVnegativeAreportWindowperiod:falsenegative

Itistheperiodbetweenwhenapartyisexposedtoaninfectiousorganismandwhenthatorganismbecomesdetectableviaaserummarker.Whatisthewindowperiod?ALT−Anti-HCV−HCVRNA+Symptoms−Contagious+Becauseof“windowperiod”,somedonatorswhocarryinfectiousdiseaseintheirbloodarenotdetected.Therefore,thereceivermaybeindangerofgettingsickafterbloodtransfusion.ThisproblemisespeciallycommoninthecaseofHepatitisC.2012亚太肝脏研究学会(APASL,Asian-PacificAssociationfortheStudyoftheLiver)丙型肝炎病毒感染共识和治疗程序HCV感染和实验室检测抗-HCV抗体应采用经批准的第三代或第四代酶免疫法（EIA）或化学发光免疫法（CIA）测定采用经批准的EIA或CIA法测定抗体阴性的标本可报告为抗-HCV抗体阴性。但是，在血液透析人群或HIV共感染者可能出现HCVRNA阳性而抗-HCV阴性在标本吸光度值/临界质控吸光度值（S/CO）比值足够高可以预测真阳性的情况下，采用经批准的单一EIA法测定有活性的标本可报告为抗-HCV抗体阳性对于未达到预测真阳性阈值的标本或活性接近临界指控的标本，应考虑进行敏感的HCVRNA检测和（或）随访复查抗-HCV及HCVRNA核酸扩增检测（NAT）HCVRNA检测需要适当的污染控制最好使用专门的样本进行HCVRNA检测而非来源于其他检测目的的样本HCVRNA定量应以“IU/ml”为单位报告（也可选择“拷贝/ml”）在治疗期间监测HCV病毒载量对应答指导治疗中决定治疗方案和疗程是重要的HCV基因型检测对评估抗病毒治疗疗程和疗效是重要的。所有检测项目加入外部质量保证方案是理想的所有检测项目进行内部质量保证测试是必需的通过一次性使用采血针取样且在室温保持稳定的干血斑（DBSs）检测法在静脉吸毒人员（IDUs）中可加强对HCV的公共卫生监测肝纤维化的非侵袭性检测方法对于慢性丙型肝炎患者有助于区分无纤维化和进展期纤维化，可以预测临床预后的差异。一种结合非侵袭性方法的逐步算法可以提高诊断的准确性，显著减少肝组织活检的需要Clinicalvalue

WhoshouldbetestedforHCVinfection?CurrentguidelinessuggestscreeningthefollowingindividualsGhanyetal.Hepatology2009.HCV

screenIntravenous

drugusersPeoplefromhigh

prevalencepopulationsPeoplewithaHCV-infected

sexualpartnerAnywhoreceivedabloodor

organtransplantbefore1992Exposedworkers(e.g.,from

aneedle-stickinjury)Childrenof

HCV-infectedmothers呼吸道病毒指能侵犯呼吸道并导致呼吸道病变或以呼吸道途径感染而主要引起呼吸道以外组织器官病变的病毒前者如流感病毒、鼻病毒、呼吸道合胞病毒等后者如麻疹病毒、腮腺炎病毒、风疹病毒等特点：传染源为病人及病毒携带者飞沫传播，传染性强潜伏期短，发病急感染可发生在呼吸道任何水平病后免疫力不牢固同一病毒的反复感染，不同病毒引起同一疾病表现流行性感冒病毒流感病毒(influenzavirus)为流感的病原体分三型甲(A)型—大流行乙(B)型—局部暴发,致病性低丙(C)型—婴幼儿（病情轻微,抗原稳定）生物学性状形态与结构形态球形（80-120nm）丝状、杆状、长短不一结构流感病毒为包膜病毒,由三部分组成核心及核蛋白组成RNA核蛋白（NP）