微生物檢驗理論課 13臨床常見病毒檢驗2 學(xué)習(xí)資料

臨床常見病毒檢驗二南方醫(yī)院醫(yī)學(xué)檢驗系李海俠肝炎病毒

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體生物學(xué)性狀屬腸道病毒72型形態(tài)：呈球形結(jié)構(gòu)：直徑27～32nm,單股正鏈RNA，無包膜動物模型：黑猩猩對HAV易感細胞培養(yǎng)：原代獼猴肝細胞、猴胚腎細胞，一般不產(chǎn)生細胞病變抗原型別單一抵抗力：抵抗力強，對乙醚、酸、熱等較穩(wěn)定致病性與免疫性傳染源：病人傳染方式：糞口途徑致病機制：在肝細胞內(nèi)增殖，不直接造成肝細胞損傷，免疫病理作用導(dǎo)致肝細胞損傷免疫性：刺激機體產(chǎn)生抗HAVIgG和IgM

抗HAVIgG對再感染具有免疫力微生物學(xué)檢驗免疫電鏡檢測病毒顆粒免疫學(xué)方法檢測病毒抗原檢測抗HAVIgM是確診甲型肝炎的依據(jù)方法：ELISA、化學(xué)發(fā)光等PCR法：檢測病毒核酸檢測抗HAVIgG主要用于流行病學(xué)調(diào)查防治原則特異性預(yù)防：接種甲型肝炎病毒減毒活疫苗緊急預(yù)防：肌肉注射丙種球蛋白一般性預(yù)防：加強飲水和食品的衛(wèi)生監(jiān)督管理乙型肝炎病毒

hepatitisBvirus,HBVHBsAgPrevalence>8%-High

2-7%-Intermediate

<2%-Low全球約有20億人曾感染過乙肝其中3.5億人為慢性HBV感染者,幾乎一半在中國/mediacentre/factsheets/fs204/en慢性乙肝全球流行分布圖china亞洲是乙肝病毒肆虐的重災(zāi)區(qū)，全球2/3以上的慢性乙肝患者生活在亞洲。而根據(jù)1992年全國乙肝血清流行病學(xué)調(diào)查的結(jié)果，中國的乙肝病毒攜帶者近1.3億人，平均每10個人中有一個，感染率更高達57.6%。中國乙肝病情發(fā)展情況肝癌肝硬化慢性肝炎

HBV攜帶者40%20%80%1～4%Everyyear,1millionpeopledieofHBV-relatedlivercirrhosisorlivercancer=>HBVclaimsalifeevery30secondsHBV的結(jié)構(gòu)42nm17-25nmlengthvariableppolHBcpreS1preS2tubularparticlesphericalDaneparticleStructureofHBVHBsAg

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

不完全雙鏈環(huán)狀DNAfouropenreadingframesthatencodesevenpolypeptides.含4個ORF,編碼7個蛋白SHBsAg,Pre-s1,Pre-s2CHBcAg，HBeAgPpolymerase多聚酶XHBxAg基因抗原組成AntigenofHBVhepatitisBsurfaceantigenHBsAg表面抗原說明病毒在肝中復(fù)制（機體受感染標志）fourphenotypes：adr,adw,ayr,aywanti－HBs:neutralizationantibody中和抗體抗原組成AntigenofHBVhepatitisBcoreantigenHBcAg核心抗原notfoundinblood一般不能檢出anti－HBcnon－neutralizationantibody非中和抗體CoreIgMindicatesrecentinfection.抗－HBcIgM說明HBV復(fù)制CoreIgGindicatesexposuretoHBV抗原組成AntigenofHBVhepatitisBeantigene抗原HBeAgthebestcorrelatetothepresenceofinfectiousvirus.感染性病毒存在的最有效證據(jù)（復(fù)制及具傳染性的標志）anti-HBeindicateslowinfectivityinacarrier抗HBe說明病毒感染性較低（是預(yù)后良好的征象）HBV其他生物學(xué)性狀培養(yǎng):

◆黑猩猩是HBV最敏感的動物模型

◆細胞培養(yǎng)已獲成功抵抗力:強

◆能耐受低溫、紫外線、干燥和一般消毒劑100℃10分鐘、高壓蒸汽滅菌、0.5％過氧乙酸等能使HBV滅活致病性1.傳染源：主要為急、慢性乙肝患者或病毒攜帶者2.傳播途徑：水平傳播最主要途徑是注射或粘膜接觸含HBV的血液及分泌物性傳播：不少見密切接觸：有可能飲食傳播：可能性較小垂直傳播主要途徑：圍產(chǎn)期（新生兒皮膚、粘膜有破損而接觸母血）和產(chǎn)后密切接觸經(jīng)胎盤及生殖細胞傳播：有可能醫(yī)源性傳播輸血或注射器、血透機、內(nèi)窺鏡等消毒不嚴格垂直傳播是我國HBV感染的主要模式3.易感人群（1）感染者年齡高峰：低發(fā)病區(qū)：20～40歲高發(fā)病區(qū)：4～8歲（2）男女感染率相近，但發(fā)病者男多于女（3）感染時年齡越小，越易形成慢性肝炎、肝硬化或慢性HBV攜帶狀態(tài)（免疫耐受）。（4）感染后對相同HBsAg亞型的HBV再感染有持久免疫力，但對不同亞型的保護力不完全。微生物學(xué)檢驗免疫學(xué)檢測：HBV五項指標

HBsAg、抗HBsHBeAg、抗HBe

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

time-resolvedfluoroimmunoassayc）CLIAchemiluminescentimmunoassay血清標志物檢測方法AutomaticenzymeimmunoassayanalyzerElectroChemiLuminescenceAssy(ECLA)三種方法比較methodAdvantagesdisadvantagesELISAeconomy，shorttimeinBulkdetectionLowSensitivityLowspecificityqualitativeTRFIAHighSensitivityquantitativeSpecificitynothighSemi-automaticECLIAHighSensitivityhighspecificityquantitativeHighercost

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

QuantitativedetectionofsurfaceantibodyClinicalAgreementsofWHOEuropeanregionrecommends100mIU/mlasthethresholdneedtostrengthentheimmune.疫苗接種后HBsAb監(jiān)控具有重要意義>10mIU/ml

認為是陽性，>100mIU/ml才具備保護意義疫苗接種完成后1個月抗體濃度達到高峰乙肝表面抗原攜帶者乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb+----++++++++++++++急性肝炎早期，傳染性強乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb++-+--++++++++++++急性或慢性現(xiàn)癥感染，傳染性強。“大三陽”乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb++++-+-++++++++++乙肝趨向恢復(fù)，屬慢性攜帶，傳染性弱“小三陽”乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb+++++++--++++++++急性感染或是慢性乙肝表面抗原攜帶，傳染性弱“小二陽”乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb++++++++++---+++++急性乙肝恢復(fù)期或既往感染乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb+++++++++++-+-+++乙肝疫苗接種后乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb++++++++++++++-+---未感染過HBV乙肝兩對半常見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb+++++++++++++++-----乙肝兩對半罕見結(jié)果模式及其意義HBsAgHBsAbHBeAgHBeAbHBcAb+-+++++++++++急性感染趨于恢復(fù)，血清學(xué)轉(zhuǎn)換不同亞型HBV二次感染；血清學(xué)轉(zhuǎn)換HBsAgHBsAbHBeAgHBeAbHBcAb+++++++-+++++乙肝兩對半罕見結(jié)果模式及其意義急性感染趨向恢復(fù)HBsAgHBsAbHBeAgHBeAbHBcAb+++++++++--+-++乙肝兩對半罕見結(jié)果模式及其意義基因突變；鉤狀效應(yīng)；爆發(fā)性肝炎；抗原表達量低，檢測不出HBsAgHBsAbHBeAgHBeAbHBcAb++++++++++--+-+乙肝兩對半罕見結(jié)果模式及其意義微生物學(xué)檢驗免疫學(xué)檢測：HBV五項指標

HBsAg、抗HBsHBeAg、抗HBe

抗HBc分子生物學(xué)檢測：血清中HBVDNA1)Detectionmethod:

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

HBVDNA的檢測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

治療中表面抗原定量測定可監(jiān)測復(fù)發(fā)和應(yīng)答，HBV難于發(fā)現(xiàn)HBsAg轉(zhuǎn)換可作為持久免疫控制指標即使用高靈敏度試劑測不到HBVDNA并不表示無循環(huán)的HBV和/或HBV感染已清除時點評價病毒載量可作為抗病毒治療有效和持續(xù)應(yīng)答HBsAg定量檢測可確定宿主免疫系統(tǒng)是否已有效控制HBV感染Controlthesourceofinfection控制傳染源Cutdowntherouteoftransmission切斷傳播途徑

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

hepatitisCvirus,HCV

predominantcauseofnonAnonBhepatitis

HCV的結(jié)構(gòu)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.傳播途徑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(+)檢測方法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聯(lián)合檢測臨床意義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;Carre?o.WorldJGastroenterol2006DiagnosisofHCV:anti-HCVassayWHO2003.SupplementarytestingConfirmationofantibodypositiveresultswithHCVRNAtestImmunoblotassayRetestInduplicateAnti-HCVassayInitialscreeningtestNon-reactiveBothresultsImmunoblotassayorRNApositiveConfirmedresultHCVpositiveNon-reactiveHCVnegativeInitiallyreactiveRepeatedlyreactive

EitherresultreactiveHCVnegativeAreportWindowperiod:falsenegative

Itistheperiodbetweenwhenapartyisexposedtoaninfectiousorganismandwhenthatorganismbecomesdetectableviaaserummarker.Whatisthewindowperiod?ALT?Anti-HCV?HCVRNA+Symptoms?Contagious+Becauseof“windowperiod”,somedonatorswhocarryinfectiousdiseaseintheirbloodarenotdetected.Therefore,thereceivermaybeindangerofgettingsickafterbloodtransfusion.ThisproblemisespeciallycommoninthecaseofHepatitisC.2012亞太肝臟研究學(xué)會(APASL,Asian-PacificAssociationfortheStudyoftheLiver)丙型肝炎病毒感染共識和治療程序HCV感染和實驗室檢測抗-HCV抗體應(yīng)采用經(jīng)批準的第三代或第四代酶免疫法（EIA）或化學(xué)發(fā)光免疫法（CIA）測定采用經(jīng)批準的EIA或CIA法測定抗體陰性的標本可報告為抗-HCV抗體陰性。但是，在血液透析人群或HIV共感染者可能出現(xiàn)HCVRNA陽性而抗-HCV陰性在標本吸光度值/臨界質(zhì)控吸光度值（S/CO）比值足夠高可以預(yù)測真陽性的情況下，采用經(jīng)批準的單一EIA法測定有活性的標本可報告為抗-HCV抗體陽性對于未達到預(yù)測真陽性閾值的標本或活性接近臨界指控的標本，應(yīng)考慮進行敏感的HCVRNA檢測和（或）隨訪復(fù)查抗-HCV及HCVRNA核酸擴增檢測（NAT）HCVRNA檢測需要適當(dāng)?shù)奈廴究刂谱詈檬褂脤ｉT的樣本進行HCVRNA檢測而非來源于其他檢測目的的樣本HCVRNA定量應(yīng)以“IU/ml”為單位報告（也可選擇“拷貝/ml”）在治療期間監(jiān)測HCV病毒載量對應(yīng)答指導(dǎo)治療中決定治療方案和療程是重要的HCV基因型檢測對評估抗病毒治療療程和療效是重要的。所有檢測項目加入外部質(zhì)量保證方案是理想的所有檢測項目進行內(nèi)部質(zhì)量保證測試是必需的通過一次性使用采血針取樣且在室溫保持穩(wěn)定的干血斑（DBSs）檢測法在靜脈吸毒人員（IDUs）中可加強對HCV的公共衛(wèi)生監(jiān)測肝纖維化的非侵襲性檢測方法對于慢性丙型肝炎患者有助于區(qū)分無纖維化和進展期纖維化，可以預(yù)測臨床預(yù)后的差異。一種結(jié)合非侵襲性方法的逐步算法可以提高診斷的準確性，顯著減少肝組織活檢的需要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導(dǎo)致呼吸道病變或以呼吸道途徑感染而主要引起呼吸道以外組織器官病變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、風(fēng)疹病毒等特點：傳染源為病人及病毒攜帶者飛沫傳播，傳染性強潛伏期短，發(fā)病急感染可發(fā)生在呼吸道任何水平病后免疫力不牢固同一病毒的反復(fù)感染，不同病毒引起同一疾病表現(xiàn)流行性感冒病毒流感病毒(influenzavirus)為流感的病原體分三型甲(A)型—大流行乙(B)型—局部暴發(fā),致病性低丙(C)型—嬰幼兒（病情輕微,抗原穩(wěn)定）生物學(xué)性狀形態(tài)與結(jié)構(gòu)形態(tài)球形（80-120nm）絲狀、桿狀、長短不一結(jié)構(gòu)流感病毒為包膜病毒,由三部分組成核心及核蛋白組成RNA核蛋白（NP）