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蕪ぷ・童； 警志恵無意無慧 範疇攣樟碍輔

1亨ttitre：糾○（D【N・1）．51罰（D【N・礼1200（D【鵬hl加の別刊；加dt‡tre：5120（D【N－1い02側（D【N－2い02側（D【N一礼10i側（D【NJI

deYelQPedgeneralisedskin rashand▲attendedthe Accident and Emergency Departmentof・Ⅵn Chaj HospiIal・IrtOctobeちhewassubsequentlYPi∈kedup

一一asone▼Pfthedenguecas甲basedoTISerOlogYreSu］t5

during．theadivecase牒ndingexerci甲わMaWan■

MoleculartestingperformedLOJlthedonat如 produdYaSPO坤ivefor・申喝u号了▲Yirus明把1－坤e

wo亘an recal一号dfo

foundtQbepositiveforcorrespqndingJgNIantibodies

よndトa如haern褐ghJtination－inhibitiontjtreofli2560・

Thisinddentwasthefirst．do⊂Um寧nted⊂iscs6fsuch

trans画S！ionln theliteIature，and sin⊂e October

仙唱ぬ咽M由JⅦ14トb3■血鋤・●w叩止血中和

macro町ti⊂anaemia一∴and panqtopenia∴She■・Wa5

diagnosedtohavevitanin812deficiencyanaemia， Whi⊂hwasrtreatedbYYitaminB12replacementand

rec占ivedabloodtFanSftJSionon24August2002．On day2post・tranSfusion，She developed、low瑠rade

feveちbLJtnOSkinrash，

OrretrO－Orbitalpain．ThepatiehtYVaStriatedwith antibioticsasbraurinarγtract・inねdionibasモdon thernicrobiologICalfindin名声・Thefever＿Subsided3

daγ亨（aterandthepatientrecovereduneventful如The

bloodprodudshe，re⊂ejvedwasdona！edby．a17－year－ Oldasymptomaticpatientliving恒Mり崎n，during

hisv行emicphぉeon17J叫200乙0ム寧4July2叩ちh寧

166

■Chu訂喝dal●

2002，（heHongKongRedCrossBloodltansfusion SerYice（BTS）hasintensifiedit5 donor deferra（

SySlemst｛〉COunterthispossibility・SpecificaltYlitnow

asksaboutsymptomsotdenguefeverinthe8lood DonorRegistrationForm（Supp（ement）byremjnding aJIprospectivedonorstoinformtheBT5staffofaIl instancesforflu′feverlheada⊂he，eyePa］n′mtJSCJe／

ioint patn′VOmiting，and skin rash experienced2 weeksbeforeorafterblooddonation．

1n our stud）ldengue fe）er was far more COrnmOn than DHF・and dengue sho⊂k syndrome， Whichwererareevents，Ourpatient50nJymanifested mildbleedingwithgood⊂lini⊂a］out⊂OmeSand no

fataJities・ThecJinica）presentationsofdenguefeveT，

SUCh as feverlmYalgia，headache′and arthraJgia′ Were ⊂OmParable to findings reportedin other

Studies・10h120urpatients（35％）presentedwithfewer

gastroenteritissymptomscomparedtothoseofothers （50－98％）．11・12 Lyrnphadenopathy was documented in▲OnIy16％ofour patients，Whichis rnuchJower

than the fjgure ofSO％reported elsewhere・13This difference maY be a⊂⊂OUnted for by］ess－than－

adequatephysiQlexamination・Gumbleedingand epjstaxiswerereportedin12％andlO％ofourpatients

respectivel≠Whichwas atso muchIowerthan that

reported previousIYn・12such diffeTenCeS COuld be duetothepopulationsstudied；Patientsre⊂ruitedin

endemkcountriesweremainlYen⊂OUnteredduring Outbreak5．inwhi⊂hbothdenguefeverandDHFwere COmmOn．Previousstudie5Showeddenguediseas

SeYeritycorre［atedwithhighviremiatitre5，SeCOndary

infection．and DEN－2seroけPeinfedion・－叩 Our findingsshowedthatthehaemorrhagktenden⊂ies anddurationofhospitaIisationtverenotrelatedto SPeCiEicserotypes・＾Jthough some ofour patients

did receive pIate暮et transfusjons′the eff桓虻y Of

SuCh treatmentin speeding recoYerγ remains

COntrOVerSiaI・＾ccordingtoThaiexperts′PlateJetsare

atmostimmediate）ydestroYedbYimmunelysisafter admini5tration．1‘

Our studY had severa（limitation5・Fi（5t／the

targetpatientswerelimitedtothosewithlaboratory－ COn（irmed dengue admitted to public hospitals・ During1998 to 2005，DH received notification

Of 203 dengue case5．incIuding 77 who were admjtted topr．vatehospitalsorconsultedgeneraJ PraCtitionersonly・ThediseaseburdenmightaIsobe Underestjmated′be⊂aUSeSOmePatientsmighthave re⊂OVered．withoutseekingrnedicalattention，Whi（e Othersmightnothaveunde｛gOneSerO［oglCaltesting・ Second，Statisti⊂aJana］YSIS⊂OUldnotbecarriedout

to compare cJinicaIandlaboratory parametersin PatientswithdenguefeveTandDH仁asthereweretoo

fewofthetatter・Third，］aboratoryresultsbefore2002

WerenOtaVaiJabIein thePubIicHealthしaboratorY ）nforTnationSYStem・Fourth′nOta＝clinicafsYmPtOmS

and5jgnslistedinTable2couJd be retrievedfrom the medicalrecords′a5SOme maYnOt havebeen SPedficatlYaSkedfororlookedfor．

fn conc暮usion．dengue fever should be COnSideredin the differentialdiagnosis of febrile Patientswithorwithout’atraVe＝1istory．Healthcare PrOViders should therefore have an understanding Oftheinfe⊂tion，thespectrumofitsc］inicalfeatures， andmethodsofdiagnosi5andappropriatetreatment・ UntiltheAede5mOSquitocanbeeffectivelycontroJ［ed Or a COSトeffective va⊂Ci，neis deve（oped，dengue

feverwillremainapublichealthconcern，eSPeCiallY in South－East＾sia．ControIat sourceis one of the

keYStO⊂Ombatingdenguefever、andrequJreSaCtive ParticipationfromaflsectorsofthecommunitY

Acknowledgemehts

The rnembers of D甲唱Ue ReviewWbrkingGroup gratefu］ly acknow］edge the CentralCommittee Onlnfe⊂tious Disease′Hospital＾uthoritY for the

PrOVisionofpatientmedicaIrecordsandSurYeiflance and EpidemiologY Bran⊂h′ Centre for Protection for the epidemiolog．calandlaboratory lnPUt・Withouttheirsupport′OUrreViewwouldno（ havebeenachieved．

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おuth－Eas一心iaRegion．WHqRegiona卜0相ceforSouth

亡astA5iaweb5ite：htや仙父arO・Who・in昨N侶edionlO／

5ection332＿1100．htm．Acce5Sed2ユAug2007・

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Singapore，Minis吋O一日ei帆Singapore：h呼仙moh・

g仇・5g仙ohc叩漬b血i⊂5W㌍klγbu＝e如5・aSPX・∧ccessd23

Aug2（氾7・

8．しimkittikuIKⅥng～akmongkonS．Jit帥血aph叩A．etal・ Clinioldi侮rencesamong PCR－PrOVen血nguese和t押e inkdions．ぬutheast A5ianJ甘甲州dれblk Hea仙

l・Oengリddenguehaemo汀ha由⊂毎柁－・Wkly【pi血mio＝l∝

2000；75：193－も．

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diagnosis．打eatme叫1椚Ⅵndon and co柑Ot・2nd ed・

Geneva：肌伽IdHea叫Organizatbn；1997：ト11・

3・＾uyeungTWQueTしLamK5．NIHL，SzetoML・The（irst Patientw仙l∝allya⊂qUireddenguek帽rinHongKong・ HongKongMdJ2003β：127－9・

4・Nimman＝吋a S．CtiniQIsp血m and managem即tOf denguehaemo一打－a8ic組立田山e鮎tAsian＝加甲州d PublicHea肋1987；柑：392－7．

5・NimmanityaS，Car（b91■DenguehaemorrhagicfeveLln： 他theraIl印．LedinghamJGG．ⅥねrreIIn～ed如鵬・Oxぬd

176 トb唱侮喝M由」＼鳳†4No3■血2∝氾●v刷〟．hk画一αg

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8h訂U′鮎Ianta～MaIaysia du血唱Ik y問rS1998－2003・

Southeast∧5ian）170PMedPubJicHealth2005；36：1179－

86．

13．Ryan FLWt50n M【′Kain KC．川舵SSa鮎rinternationat

tr訓el．NEngりルkd2002；347：50i－16．

14・VaughnDWGreenS，馳l甲nar叫iS′da】．Dengueviremia 【it叫anti叫re叩n軍門愉n′andviM梵碑⊂αdate Wi血dis飽父S餅訂吋JlnkdDi52000；18上2－9．

15・価nS．∧ungM♪れShwe‘TN．dal．Riskねcb帽indengue Sh∝ksyndrome．AmJlt甲山dHγg1997；56：56ト72．

托・∧l飲

Plateldね∩5血－Sbnsin Dengue Ha酬¶0汀hagic h佗r and

DengueSh∝k5yndrome・Dengue8u＝din2003；27：138■3．

ZOO5；3も：t432－8．

9・血er喝e M卯仙y Ovi廿ap tndex－ 2000－2006‥HKSAR bd andtEnvirdhh即ta1日ygi印e Depa沌ment wd汚ite： h牧P伽ww．khd・gOV・h兢afefoo仇kn糾Lkve爪ndex・hlml・

∧cces5d23∧喝2007・

10．払Iayanar∝，jS′Vaughn▼D叫Nimmann吋aSJdal・Early

⊂伽i00landlah洞tαγindicab帽Ofacutede爪印ei11ness・J

lnfectDis・1997；176：ユ1ユー21．

1l．SinghN8伽mbR，∧gan伯ISK，etal．≠e之0030u由一eakof De咽Ue如rjnDelhi．lnd軋知utheasl人sianJnopルId PublicHea仙2005；ユ6：11744．

1j・H＝SSin N．Jaafarj．Naing NN．MatHA．Muhamad人目． MamalMN・Areviewofde叩uekⅦr涙idenceinKota

ミう

ト輌K叩M由JⅦ14No3■血相2∝冶■柵〟．叫．αg 177

168

別紙様式第2 別紙（3）－3

医薬品 研究報告 調査報告書 報告日 一報入手日 新医薬品等の区分 摩生労働省処醐

識別番号・報告回数 2008年8月11白 該当なし

一般的名称 フィブリノゲン加第XIII因子 ClearanCeOfdenguevirusinthe

ベリブラストPコンビセット 販売名（企業名）

（CSLベーリング株式会社） 研究報告の公表状況 中国

2008Feb22．

問題点（血嬉分画製剤でのデングウイルスの不括化・除去） 使用上の注意記載状況・ デングウイルスは年間に世界で5千万から1億人が感染し、感染者の数十万人がより重篤で生命を脅かすデング出血熱やデングショック症 その他参考事項等 候群に進展する。

デングウイルスはフラピウイルス科に属し、直径50nmのエンベロープを有するRNAウイルスである。一般に血液などの高蛋白な体液で長

期間生存するので、輸血により感染する可能性がある。針刺し事故や骨髄移植、分娩での血液に関連するデングウイルス感染が報告されて

研 しlる。

究

報 出

⊂】 の 低温エタノール分画、陰イオン交換クロマトグラフィー、パスツリゼーション、S／Ⅷ処理とウイルスろ過を含むアルブミンやグロブリンの

概 各製造工程前に、高力価の培養デングウイルスセロタイプ2を正常人血紫にスパイクし、各製造工程でのデングウイルスのクリアランスを

要 TCID50アッセイ、RT－PCRで測定した。

デングウイルスの不括化・除去に対して、各製造工程前は全てで有効であった。

また、アルブミンの全製造工程（低温エタノール分画、パスツリゼーション）で少なくとも10．1210g減少すること、グロブリンの全製造

工程（低温エタノール分画、ウイルスろ過、S／D処理、クロマトグラフィー）では少なくとも14．24log減少することが証明された。

現在実施されている血躾分画製剤の製造方法は、デングウイルス伝播に関して安全である。

報告企業の意見 今後の対応

本剤の製造工程（低温エタノール分画、パスツリゼーション、イオン

交換樹脂等）でデングウイルスが不括化・除去できると考えられる。

170

AB了】仇㈲封沌

T札AⅣSFUS川Ⅳ COMPL川ATI（川S

Clearanceofdenguevirusintheplasma－derived

therapeutic proteins

n一仇加盈e，Pα〟J麒5．Cわα托，Cわ吉幻r5zeわ，5以fⅥ肋ノ0た，招αル乙rCわ〟，5揖〟町5，上．CJ勒

／りJ‥人■．川州／J．ゞ．・＼′′川iJJ川わ〃パ．仙JJい／〃仙■′‖i‥・l／い＝′‖汎用一丁州Il叫〔

enguevirusinfects50to100mi11ionpeople WOrldwide ayear；Ofthoseinfected，SeVeral

hundredthousanddevelopthemoresevere andlife－threateningdiseases，denguehemor－ D BACKGROUND：Viratsafetyisofparamountimpor－ tanceforhumanplasma－derivedtherapeuticproteins． Recentrepo丘SOfb100dTaSSOCiatedtransm桓Sionand

COntinuousreg10naJoutbreaksofdenguefeverhave PrOmPtedavaJjdatjonofclearanceofdenguevirusin themanufactureprocessesoflheplasma－derived PrOducts． STUDYDESJGNANDMETHODS：Ahightiterofcul－

tureddenguevirusserotype2wasspikedintoprocess SamPlesbeforeindividuaJstepsofalbuminandimmu－ nogtobuIinmanufactureprocesses．includingcotd ethanoIprecJPitation，Cation－eXChangechromatography．

PaSteurjzation，S｛）lvent／detergenttreatment，andvirus

firtratjon．Clearanceofdengueviruswasquantifiedwjth TCID5。aSSaySinthecultureofVeroE6ceIIsand，When

appropriate，reaトtimepolymerasechainreaction（RT－

PCR）assays・

RESULJS：TheindividualprocessstepswereaJleffec・

tiveintheinactivationand／orremovalofdenguevjrus，

andthedataobtainedclearlydemonst（atethattherisk OfdenguevirustransmissionwasreducedcumulativeLy byatleastlO．12andatleast14．24loginthealbumin

andimmunoglobuIinmanufactureprocesses． respectively． CONCLUS（ON：Thededicated viralinactivation andJor

removalapproachescurrentTyimplementedinthe manufactureofplasma－derivedproductsprovideagood SafetymarginwithTegardtothetransmissionofdengue Vims．

rhagicfeveranddengueshocksymdrome・DengueviruS belongs tothe family Flaviviridae，Whichin generalis known to survive overlong periodsinfluidswithhigh proteincontents，forexample，blood．Therefore，dengue viruSeS maybe transmittedviatransfusionofblood or bloodcomponents．Albeitrare，ithasindeedbeendocu－ mented that blood－aSSOCiated transmission of dengue virus occursvia routesincludingneedle－Stickiniuries，1 marrowtransplantation，2intrapar［umandverticaltrans－ mission，2andmucocutaneoustransmission，3Thiscanbe

aseriouspublichealthproblemwithoutpropercontrol meaSureS．

DengueviruSisalipid－enVelopedRNAviruS，Witha diameter ofapproximately50nm．4ReportedlY．dengue virus has been e圧ectivelyinactivated byphotosensitiz－ ers5・6andissensitivetohightemperaturesandacidicpH．7 Thisstudyaimstodemonstrateforthe丘rsttimethatthe

ABBREVIATION：BVDV＝bovineviraldiarrheavirus．

FromAdvantekSerumLaboratoriesLimitedandtheDepart－

mentofMicrobiologyPrinceofWhlesHospital，TheChinese UniversityofHongKong，Shatin．N．T，HongKong．

AddTeSSrePrlntTt！queStStO：Bing－LouWong，PhD，Advantek

Seruml月boratoriesLimited，3／RHKIB，2Biotechnology

Avenue，12Miles，niPoRoad，Shatin，N．T．HongKong；e－mail：

blwong＠advantekbiologlCS．COm．

Theauthorsa∫etha∫1k魚11forpartialfundingsupportfrom

theInnovationandTbchnologyFund（S／P583／03）．theGovern－

mentoftheHongKongSpecialAdministrativeRegion・Some

resultsofthepfeSentStudyareusedintheU・S・PatentaPplica－

tionnumberll／294，432andChinesepatentapplication

numberZOO610150052．2．

ReceivedforpubllCationMarch27，2007；revisionreceived

November12，2007，andacceptedNovember19，2007・

doi：10．1111／j．1537－2995．200乱m647・Ⅹ

MSFUSION2008；48：1342－1347．

1342 TRANSFUS10N Volume48，July2008

EuMtNAJ10NOFt）ENGUEVIRUS［NPLASMAFRACT10NAT10N

riskofdengueviruStranSmissioninplasmaderivativesis elimina【edbyspeci魚cvirusremovalandinactivationpro－ Cedures．LogreductionofdengueviruSisinvestigatedat individualstepsofthemanuhctureprocessesofplasma－ derivedalbumin andimmunoglobulins，Whichinclude COldethanolpreclpitation，Cation－eXChangechromatog－ raphy．pasteurization，SOlvent／detergent（S／D）treatment． andvirusfi1tration．The evaluation ofthe manufacture

processesprovidesameasureofconfidenceforeliminat－ ingdengueviruS・

Samplewithasequencedetectionsystem（ABI7900HTI AppliedBiosystems），andthecyclingconditionswere500C fbr2minutes・950CforlOminutesfollowedby40cyclesof 95bCfor15seconds，and600Cforlminute，aSWe11asa

dissociation stage of950C for15minutes，600C for 15minutes，and950Cfor15minutes．

FractionIVprecipitation

Thesupematantll＋ⅠⅠIwaspreparedfromfrozenhuman

plasmathroughtwoconsecutivestepsofcoldethanolpre－ Cipitationwith8percentethanolatpH7・1followedby

19percentethanolatpH5・85・8Duplicates of20mLof

SupematantII＋ⅠⅠIwerespikedwith7・00logpermLeach

OfdengueviruSataratio（VOl／vol）ofl：10．EthanOl（95％）

WaSaddeddropbydropintothesupematantII＋ⅠⅠItoa

丘nalethanoIconcentration of40percent，Which was

hrthermixedaト5to5・50Cforlhour，beforebeingcen」 trifugedat2300×gtOSeparatethefraction（F）Ⅳ＆omthe

SuPernatantⅣThesupematantII十III，theFIV and，the

SupernatantⅣweretitratedforquantityofvirusesby

TCID50aSSayOrRTIPCRassay．

MATERJALSAND METHODS

Rawmaterial＄

Normalhumanplasmawasobtainedfromtheplasma fractionator Shenzhen Weiwu Guangming Biological ProductsCo．（Shenzhen，China）．All．chemicalsusedinthis

Studywereofeitherpharmaceuticalgradeoranalytical grade・ViruSfilters（Planova35N，10cm2）wereagiftfrom AsahiKasei（Tbkyo，Japan）．

Vjruscultureandquantification

DengueviruSSerOtype2（SO47］00froInEnvironmental

HealthInstitute．Singapore）waspropagatedinC6／36cells （CDCGuangdong，China）inminimalessentialmedium

withlpercentfetalbovineserum（Gibco．GrandIsland．

NY）・DengueviruSWaSquantifiedwithTCID50aSSaySin

thecultureofVeroE6cells（ATCC，ManaSSaS，1乳）．VeroE6

CenS（2・5×105cells／mL）wereseededin96－We11platesina

VOlumeoflOOpLperwe11．A托erldayofincubatiozt，50llL

Of medium was added to each we11．Each diLution of

Samplewasaddedat50pLperwe11，andfurtherincuba－ tionwascarriedoutat36±20Cwith5percentCOz．Plates WereaSSeSSedforTCID5Oendpointascytopathice鮎cts

developedonthefifthdaylTheTCID50endpointwascal－ CulatedaccordingtotheSpearman－Karbermethod，and the Poisson distribution was used when no virus was

detectedinsamples．Quantitativereal－timepolymeraSe Chainreaction（RT－PCR）wasusedtodetermineviruStiter

inthe chromatographyandcold ethanolprecipitation StepS・RNAofdengueviruSWaS eXtraCtedinduplicate fromsampleswithaviralRNAminikit（QLAamp，Qiagen， Hilden．Germany）accordingtotheproceduTeprOvidedby themanufacturer・DengueviruSCDNAwasreversetran－ SCribedwithrandomhexamerswithreversetranscriptase （SupercriptIII，Invitrogen，C訂1sbad，CA）．Quantitative

RTPCRutiuzingThqMantechnology仏ppliedBiosystems， FosterCity，CA）wasperformedonsamplesandproper COntrOIswithspecific primers（GTCん如コuAGAAGm－ GjuCCTCCA and CTCⅧrGATGÅTGTAGCTGTCTCCG） andSYBRGreenfluorescentprobeswithconditionsopti－ mi21edtodetect4・67copleSOfviralRNAfordengueviruS・ Duplicate PCR procedures were performedfor each

Pa＄teurization

The puri丘ed albumin solution was diafiltrated with

8volumesofwaterandthenconcentratedtoaconcentra－

tionof22percentwitha30－kDacutoffcassette（Millipore，

Bedford，MA）．Sodiumcaprylatewasaddedtotheconcen－

trated albumin solution to a final concentration of

32mmolperL，beforeadjustmentofpHto6．8tomake

20percentalbumin bulk＿1加0－hundred miuiliters of

albumin bulk and duplicates ofthe steri1e一fi1tered

20percentalbuminina50一喝Lbottlewasheatedto590C

inawaterbath．fb1lowedbyspikingwithdengueviruS （6・67，7．50，Or7・67log／mL）ataratio（vol／vol）ofl：20and

l：25，reSpeCtivelytGentlemixingwithamechanicalstirrer

（Stainlesssteel）wasappliedtothebulkpasteurization・

SamplesweretakenoutforviruStitrationduringthetime COurSeOfalO－hourtreatmentat59to600C．

F‖precipitation

TheFII＋IIIsepaLatedfromthesupernatantII＋ⅠⅠIabove

WaS redissoIved．and NaAc－HAc bu鮎一（0．8mol／L－

4mol／L，pH3・9）wasaddeddropwisetoadjustpHto5・1・ Denguevirus（7・170r7．67log／mL）was畠pikedataratio （VOl／vol）ofl‥10into duplicates of 20mL of the pH－aqjustedFIl十III・Ethan01（95％）wasaddeddropby dropintotheFII＋ⅠIItoa丘nalethanoIconcentrationof

15percent，Whichwasfurthermixed．at－5to5．5OCfor

l・5hour・beforebeingcentrifugedat2300×gtOSeparate

theFIII丘omthesupematantIII・TheFII＋III，thesuper－ natantIII，andtheFIIIweretitratedforquantityofviruSeS byTCID50aS＄ay．

VoJurne48，July2008「隅ANSFUS10N1343

XIE ETAL．

Virus用tration

Aquantityof196mLofpartial1ypuri丘edimmunoglobulin WaSSPikedwith7．67logpermLdenguevirusataratio （VOl／vol）ofl：49．fouowedbyfiltrationwithaO．22－Pmfi1ter

（Steritop，Millipore）toremoveviralaggregates・Thefi1－ teredimmunoglobulinwassubjecttovirusBltrationwith the35Nfuterinanormal－flowmanner，underconstant

pressureof80kPa．Samplesweretitratedforquantityof viruSeSbyTCIDsoassayorRT－PCRassay

RESULJS

FIV precipitation

Afterthe40percentedlanOlprecipitationofthesuperna－ tantII＋III．nodengueviruswasdetectedwiththeTCID50

assayinboththesupernatantⅣandtheFrVrrablel）． DespiteitsdirectcytotoxicitytothevirusdetectorVeroE6 Cells，Whendiluted500－fold．40percentethanoldidnot

a仔ectthedeterminationofvirustiter・Resultsofquantita－ tive RT-PCR clearly showed that genetic materials of denguevirusWere COnCentratedin the FⅣ（Tもblel）， Whichis discarded during thealbumin manufacture． Becausechemicalinactivationbyhighconcentrationsof ethan01ismechanistical1ydifferentfromthephysicalparL titionlnge庁ectsbetweenfractions，thisFⅣpreclpitation StepprOvidesanextrasafetymarginintheeffectivecleaト anceofdengueviruses．

S／D treatment

Duplicates of27mL of theimmunoglobulin puri丘ed throughviruSfi1trationwereheated to280Cinawater bath，fouowedbyspikingwith7・160r7・83logpermL 〔kngueviruSataratio（VOl／vol）ofl二9・rItitonXLlOOand

tri－n－butylphosphatewereaddeddropbydropintothe immunoglobulin to a 再nalconcentration ofland O・3percent，reSpeCtivelyGentlemJjnngWaSaChievedwith amechanicalstirrer（Stainlesssteel）forthetimecourseof 16－hourtreatmentat28to300C，duringwhichsamples WCreremOVedforviruStitrationbyTCID50aSSay

Pasteurization

The kinetics ofinactivation of dengue viruSin the 20percentalbumirldufingthelO－hourpasteurizationat 59to600C afe ShowninFig・1・Thepasteurizationwas

CarriedoutatO．50Cbelowwhatisnormallyusedinthe manufacture，repreSentlngaWOrSt－CaSeSCenario．Dengue Cation－eXChangechromatography

A chromatography column oflO－mm diameter was packed to a bed height ofllcmwitheithernewCM SepharoseFastFlowresin（PharmaciaBiotech，Uppsala，

Sweden）orthe used resin that had previously been recycled476tjmeswiththeimmunoglobulinpuri丘cation process．Thecolumnwasequilibratedwith20mmolperL NaAcbu仔er，pH4．0．AdjustedtoapHof4・Owith1MHCl andanionicstrengthofl・4mSpercmwithpuri丘edwater． duplicatesof75mLoftheS／D－treatedimmunoglobulin SOlutionwerespikedwith7．670r7・83dengueviruSata ratio（vol／vol）ofll：20．Thevirus，Spikedimmunoglobulin SOlutionwasappliedtothecolumnatalinearflowrateof 40cm／hratambienttemperature．Afterwashingofthe CO】umnwithlOcolumnvolumesoflOmmolperLglyclne， pH7．0．immunoglobulinswereelutedwithlOOmmolper LglyclnetOgetherwith150mmolperLsodiumchloride， pH9．0．Thecolumnload．thenow－throughfraction．and the eluate fraction containing immunoglobulins were titratedforquantityofviruSeSbyTCID50aSSayOrRTTPCR aSSay・

（冨」）s⊃」S¢コぎ0凸

窟一裾

‾－・∵∴∵．

10

Pasteurization（hr）

Fig・1・InactivationofdenguevimsiJlalbtlminbypasteuri組一

也onovertime．（ロ）もu址pasteurimtiom；（◆）terminalpa5tel甘－

ization・Stockdenguevirus飴（6．670r7．50log／mL）were

SPikeda（aratio（vol／vol）ofl：20andl：25，reSpeCtively，inthe

t）ulkpastell血tionandterminalpasteurization・

● ； ● 、 】 壷．；．壷‥、こ壷≠－＝－。一重モ．．、…腰弓；…・‥．．こ●．．．…．：l．。…．義男。ミ，．‥、．＝．幸．．。音．．． 1344 TRANSFUS10N VoIume48，Juty2008

EuM］NAT10N OFDENGUEVIRUS fN PLASMAFRACT10NAT10N

7hours．NoinfectiousviruSWaSdetectablebytheTCID50 assayintheimmunoglobulinfiltrate，thesampleobtained WhentheviruSfi1terwasreverselyflushedwithpurified Water，OrtheviruS－SPikedimmunoglobulincontroIstand－ 1ngalongthewholeviruSfi1trationprocess・Tbditrerenti－ atephysicalseparationfromthechemicalinactivationby thelow pH，the samples werefurther quantified for dengueviruSwiththe quantitative RトPCR assay．The RT，PCRdatashowthatdengueviruSWaSmuChmorecon－ Centratedintheback－nuShfractionthanintheimmuno－ globulin丘Itrate・Viralreductionbythevirusfi1trationwas Calculatedandshowninlもbles3and4．Theseresultsindi－ CatethatdengueviruSiseffectivelyremovedbythe35N viruSaltration．

viruSWaSquicklyinactivatedbytheheattreatment，and infectiousvirus became undetectablewithin5minutes （Fig・l）・Tbtalviralreductionfbrboththebulkpasteuri－ Zation andthe terminalpasteurizationis shownin 職ble2．

F＝precipitation

Afterthe15percentethanolprecipitation，denguevirus WaSdetectablewiththeTCID50aSSayinboththesupema－ tantIIIandtheFIII．withamajorityofinfectiousvirusin theFIII．ViralreductionfromtheFII＋ⅠⅠItothesuperna－ tantIIIwascalculatedandisshowninThble3．

Virus filtration

TheimmunoglobulinspikedwithdengueviruSWaSPrO－ CeSSedat24to250Cthroughthe35Nvirusfilterwithin

S／Dtreatment

ThepresenceofS／Dwascytotoxictothevirusdetector Ⅵ汀OE6cells；Whendiluted1000－fold，S／Pdidnota仔ect

the determinationofviruS titer．Thekinetics ofinactiva－

tionofdengueviruSintheimmunoglobulinduringthe 16－hourS／Dtreatmentat28to300CisshowninFig．2．

DengueviruswasquicklyinactivatedbytheS／D treat－

ment，andinfectiousvirus became undetectablewithin lminute．TbtalviralreductionfortheS／Dtreatmentwas

Shownin恥ble3．

TÅBLE2．Clearanceofdengueviru＄lnthe albuminpro¢eSS■

Process step Reductionofvirus（10g）

≧5．18†／≧5．35†

≧4．61

≧4．94／≧5．44

≧10．12‡／≧10．79‡

FIV precipitation Bulk pasteurization lbrmina（pasteurization Cumulative

★ Dataareshownfromduplicateexperiments，eXCePtbulkpas－ teurization．whichwasconductedonce．

†ThedatafromtheTCID50aSSay．butnottheRT－PCRassay， areinchJded．

‡“8ulkpasteurization”isno＝ncludedinthe”cumulative，” becauseitissimilarmechanistjca11ytolermina暮 PaSteUrization．”

（ロ0」）s≡S¢⊃P－0凸

3 2 1 0

16 1

SIDTreatment（hr）

0

XIE ETAL．

Cation－eXChangechrornatography

Afterthe chromatographystep oftheimmunoglobulin process，nOinfectiousviruSWaSdetectablebytheTCID50 assayinthecolumnload，theflow－throughfraction，the eluatefraction，Orthevirus－SpikedloadcontroIstanding along the whole chromatography process．Tbtalviral reductionfromthecolumnloadtotheeluatefractionwas

atleast7－04andatleast6・93logforthenewresinandthe 476－CyCledusedresin，reSpeCtively（Thble5）．BecauseS／D WaSpreSentinthe startingmaterial，theeliminationof

denguevirus could be a result ofinactivation by the Chemicals▲Tbdifferentiatephysicalremovalfromchemi－ Calinactivation．thesampLeswerefurtherquanti魚edfor denguevirus with the quantitative RT－PCR assay・The RT－PCRdatashowthatamajorityofdengueviruSWaS Observedintheflow－throughfraction・Tbtalviralremoval bythechromatographyprocesswascalculatedtobe3．66 and4・18logfbrthenewresinandthe476－CyCledused resin，reSpeCtively（rIhbles3and5）．

D［SCUSSTON

Viralsafetyis of paramountimportance for human Plasma－derivcdtherapeutic proteins such asalbumin， αl－prOteinaseinhibitor，Clottingfactors．andimmunoglo－ bulins．Recent documentation of blood－aSSOCiated

transmissionlJ3and continuous reg10naloutbreaks of denguefeverhavepromptedavalidationofclearanCeOf dengueviruSinthemanufactureprocessesoftheplasma， derived products．Itwas theintention ofthis study to investlgateCleaEanCeOfdenguevirusinindividualstepsof manufacture processes ofplasma－derivedalbuminand immunoglobulins．The results showninThbles2and3 Clearlydemonstrateforthe鮎sttimethatspecificvirus removalandinactivationprocedures reducethe riskof denguevirustranSmissionbymorethanlOlogcumulaL tivelyinplasma－derivedalbuminandimmunoglobulins・

Inthisstudy．coldethanolprecipitationisverye鮎c－ tiveininactivatlngdengueviruSinthealbuminprocess． butmi1dly efFectivein removlng dengueviruSin the immunoglobulinprocess、Thisdi鮎renceine鮎ctiveness isprobablyduetothefac［thathigherconcentrationsof ethanoIwereusedinthealbuminprocess・Itisfairlyrea－ SOnabletospeculatethatothertherapeuticproteinspre－

1346 TRANSFUSION Volume48，Juty2008

paredfrom plasmabysimilarlyhighconcentrations of ethanol，forinstance，α1－prOteinaseinhibitoraJldtrans－ ferrinpuriRedfromtheCohnFrV；wouldhaveagood Safbtymarginwithregardtotransmissionofdenguevirus・

Pasteurizationinactivateddenguevirusveryquickly ande鮎ctivelyinthealbuminprocess．Thepresenceofa highconcentrationofalbuminorthealbuminstabilizing agentsodiumcaprylatedidnotseemtoprotectdengue virusfromthe heatinactivation．Caprylate has been Showntobeaneffectivevirus，inactivatingagentatmilli－ molar concentrations under acidic conditions；9‾】Z

however，Caprylateappearsunlikelytocontributemuchto theviralinactivationcapacityofthepasteurizationstepas inthealbuminformulationitisusedunderneutralpH， Which do not favor the formation of the active

COmpOnent－thenonionizedformofcaprylate．Asshown

byalbumin’slonghistoryofviralsafetyinclinicalapplト Cations，thededicatedviralinactivationstepinalbumin manufactureprocesseshasbeenveryrobustintheinac－ tivationofmanydifferentvirusesincludingWestNileviruS and bovineviraldiarrheaviruS伴ⅣDV），bothfrom the

SameFlaviviridaefamilyasdenguevirus・13r14 Virus filtration was very effective in separating

denguevirusfromthe、immunoglobulinRltrate（Table4）．

ThedatasuggestthatchemicalinactivationbythelowpH COnditioncanprobablycontributetotheviralclearance CapaCityofthis process step．In aseparate studywith BVDVwhichisofsimilarsizebutnotsensitivetolowpH treatment，amqjorityofthespikedBVDVwastrappedin thePlanova35N丘Iter，Whichwasrecoveredintheback－

nushsample（unpublishedobservation）． Likepasteurization，S／Dtreatmentveryquicklyand

e鮎ctivdyinactivateddenguevirusintheimmunoglobu－ 1inprocess・Thisdedicatedviralinactivationstepinthe immunoglobulin manufacture processes has been very robustintheinactivationofmarrydifferentviruSeSinclud－ ingWestNilevirusandBVDV13・14

Thecation－eXChangeChromatographywasorlginal1y intended to remove S／D from theimmunoglobulin process；however，itwasalsoobservedinthisstudyto e任ectivelyremovedengueviruSbya臨nityadsorption・In addition，thispuri丘cationstepwasmildlyeffectiveinthe

physicalremovalof BVDV（unpublished observation）． Althoughthe chromatographic process may not be a

ELIMINÅT10NOFDENGUEVIFtUSJNPLASMAFRACT嘉ONAT10N

robustviralremovalsteplngeneral，itisindeede仔ectivein

theclearanceofden許IeViruS．

InsummarylthisstudyhasshoⅥlthate鮎ctiveclear－

anceofdenguevirusisachievedinthemanufacturepro－ CeSSeS Ofalbumin andimmunoglobulins，prOviding additionalevidencesupportingtheviralsafetyofplasmaT derivedproducts．

REFERENCES

l・deWazieresB．G辻H，Ⅵ1ittonDA．DupondlL．Nosocomial

transmissionofdenguefromaneedlestickinjury．Ⅰ．ancet

1998；351：498．

2・Rigau－PerezJG，VomdamAV．ClarkGG・Thedengueand

denguehemorrhagicfeverepidemicinPuertoRico，1994－

1995・AmJTfopMedHyg2001；64：67－74．

3・ChenLH．WilsonME・TraLnSmissionofdengueviruS

Withoutamosquitovector：nOSOCOmialmucocutaneOuS

transmissionandotherroutesoftranSmission．ChnInfect Dis2004；39：e56－60．

4・m血nRl，ZhangW．RossmarmMG，PlentnevSV，CorverJ，

LenchesE，lonesCT．StruCtureOfdengueviruS：implica－

tionsforflaviviruSOrganization，maturation，andfusion．

Ceu2002；108：717－25．

5・LinYL，LeiHY．WenYYILuhTY，ChouCK．LiuHS．Light－

independentinactivationofdengue－2viruSbycarboxy－

fullereneC3isomer．Ⅵrology2000；275：258－62．

6・HuangQ．FuWL，ChenB，HuangJF．ZhangX，ⅩueQ．hac－

tivationofdengueviruSbymethyleneblue／narrowband－

widthlightsystem．JPhotochemPhotobioIB2004；77：39－

43．

7・Man血gJS，CollinsIKE鮎ctsofcellcultureand

laboratoryconditionsontype2derLgueviruSinfectivity・

JClinMicrobiol1979；10：235－9．

8・CohnEJ，StrongLE，HuglesWLMulbrdDl，AshworthlN．

MelinM・TbylorHL・Preparationandpropertiesofserum

andplasmaproteins・ⅣAsystemfortheseparationinto

fractionsoftheproteinandlipoproteincomponentsof biologicaltissuesarLdnuids．1AmChemSoc1946；68：459－

75．

9・LundbladTL，SengRL・Inactivatironoflipid－enVeloped

viruSeSinproteinsbycaprylate・VoxSang1991；60：75－81・

10・KorneyevaM・Hottal．LebingW，RosendlalRS．Fran寧SL．

PettewaySRlr・Envelopedvirusinactivationbycaprylate：

arobustalternativetosoIvent－detergenttreatmentin Plasmaderivedintermediates・Biologicals2002；30：

153－62．

11・LebingW・RemingtonKM・SchreinerC′PaulHI・Properties

Ofanewintravenousimmunoglobulin（IGrV－C，10％）pro－

ducedbyvirusinactivationwithcaprylateandcolumn Chromatography・VbxSang2003；84：193－201．

12・JohnstonA．UrenE，lohnstOneD，WuJ．LowpH，Caprylate

incubationasasecondvifaiinactivationstepinthemanu－ factureofalbumin．Parametricandvalidationstudies．Bio＿

lo由Cals2003；31：213－21．

13・KreilTR，8ertingA，XistnerO，KindermamJ．WestNile

viruSandthesaLetyofplasmaderivatives：Veri丘cationof

highsafetymargins，andthevalidityofpredictionsbased OnmOdelvirusdata．TtanSfusion2003；43：1023－8．

14・RemingtonKM．TtejoSR，BuczynskiG，LiH，OsherofFWP，

BromllP，RenfrowH，ReymOldsR．PifatDY．Inactivationof

鴨stN辻evirus，VaCCiniaviruSandviralsurrogatesforreト

evantandemergentviralpathogensinplasma－derived products．Ⅵ）ⅩSang2004；87：10－8．E）

Volume48，July20081TIANSFUSTON1347

別紙様式第2－1

番号10 医薬品

医薬部外品 研究報告 調査報告書 化粧品

識別・雛回数 離日 新医区分 厚生労働省処理欄

①乾燥抗HBs人免疫グロブリン 公表国

一般的名称 ②ポリエチレングリコール処理抗HBs人免疫グロブリン 研究報告の プエルトリコ

販売名 公表状況 1348－1354 （企業名）

＜背景＞

輸血を介したデングウイルス感染が1例報告されている。しかし、流行地域でのデング熱の発生率が高いこと、無症候感染の割合が高い 使用上の注意記載状況・

こと、およびウイルス血症の中央値が5日であることから、輸血に関連したデング感染は、報告されているよりもさらに広範匪＝こ拡がっ その他参考事項等

ている可能性がある。 代表として静住用へブスプリンーIHの記載を示 ＜研究デザインおよび方法＞

研 す。 ■2005年9月20日から12月4日までにプエルトリコにある米国赤十字へ提供された全ての血液ドネーションのデングウイルス（DENA）紺A 2．重要な基本的注意 究 の陽性率を、tranSCriptionmediatedamplification（TMA）法と呼ばれる特異タイプの核酸増幅検査を使って測定した。TMAの測定結 （1）本剤の原材料となる血液については、KBs抗

報 果が2回くり返して陽性であったものを、TMA法陽性のドネーションと定めた。TMA脇性のドネーションについて、IgM抗体のELISA、RT－PCR 原、抗HCV抗体、抗HIV－1抗体、抗HIV－2抗体陰性 およびウイルス培養による検査を行った。 で 、かつALT（GPT）値でスク1」－ニングを実施し 告 ている。更に、プールした試験血某については、

KIV－1、HBV及びHCVについて核酸増幅検査（NAT）を

の

、 るが、当該NATの検出限界以下のウイルスが混入

と、5つのドネーションだけがTMA陽性であった。

要 I している可能性が常に存在する。本剤は、以上の

検査に適合した高力価の抗HB5抗体を含有する血 紫を原料として、Cobnの低温エタノール分画で得

た画分からポリエチレングリコール4000処理、

DEAEセファデックス処理等により抗HBs人免疫グ

れるであろう。輸血後のデング感染の患者において、輸血感染を考慮すべきである。 ロブリンを濃縮・精製した製剤であり、ウイルス

‾報告企業の意見 今後の対応 不括化・除去を目的として、製造工程において

60℃、10時間の液状加熱処理及びろ過膜処理（ナ

ある。 影響を与えないと考える ノフィルトレーション）を施しているが、投与に

血典分画翠剤からのデングウイルス伝播の事例は報告されていない。万一、原料血紫にデングウイルスが混入し ので、特段の措置はとらな 際しては、次の点に十分注意すること。 ても、脚Dをモデルウイルスとしたウイルスバリデーション試験成績から、本剤の製造工程において十分に不括 い。

化・・除去されると考えている。

′＼・・′7「rlI、．

178

TRAⅣSFUSI州1≠椚几川椚Ⅰ州S

Denguevirustinblooddonations，PuertoRico，2005

肋m助肋わαmme4蜘肱エgれ托e托，カ唱eエ．肋和之－力感血，疋卸乃m“わeた，Gre卯叩艮）∫励1

Arry且β和㍑枕身iePg短nerl，α托dSl措肌エS汀αmgr

engueviruS（DENⅥisamosquito－bome瓜a－ viviruStranSmittedbythebiteofaninfected Aedksspp．mosquito．Inkctionbyeachofthe antigenical1ydistinctserotypesmENVll，－2， D

BACKGROUND：Asingfeinstanceoft（anSfusion－

transmitteddengueinfectjonhasbeenreported．The highincidenceofdengueinendemiccountries．thehjgh PrOPOrtionofasympIomaticinfection，andthemedian 5－dayviremia．however，SUggeStthattransfusion－

assocjateddenguetTanSmissionmaybemorewide－ SPreadthandocumented． STUDYDESIGNANDME丁目ODS：Theprevalenceof

denguevirus（DENV）RNAwasdeternlinedina［IbIood

donationstotheAmerican Red Crossin Puerto Rico

fromSeptember20toDecember4，20O5，uSingaspe－

Cifictypeofnuc［eicacidamplificationtestca＝ed

transcription－mediatedamp（ification

POSitivedonationsweredefinedasthosehavingtwo repeatedlyreactiveTMAresults・TMA－POSitivedona－

tionsweretestedbyenzyme－tinkedimmunosorbent assayforimmunogJobulinM（lgM）antibodies．by

reversetranscriptionl⊃Olymerasechainreaction（RT

PCR），andbyvira］culture・

RESULJS：’Twelve（0．O7％）of16；521btooddonations

testedwereTMA－POSitive．Fourwerepositiveby

RTPCR（DENVserotypes2and3）・Ⅵruswascultured

from30f4RT，PCR・やOSjtivedonations．Oneofthe12

TMA－POSjtivedonationswas暮gM－POSitive・Only5dona－

tionsremainedTMA・POSitivewhendiI血edl：16，aSis

doneforroutinemlnlPOOIscreenlngforother transfusion－tranSmissibleviralinfections（hepatitisC，

humaninlmunOdeficiency，WestNilevirus占spvNVsl）．

CONCLUS］ON：Near（ylinlOOOblooddonationscon－

tainedDENVRNA，andviru＄COu（dbecu（turedfrom

TMA－POSitivedonations，SuggeStingatran去fusiontrans－

missionrisksimilartothatwhichexistedintheUn托ed

StatesforWNVbeforeuniversaldonationscreenIng， Simi（artoWNV，JgMantjbodyscreenIng’islikelytobe

in畠fFective，andsomepotentia（Iyinfectiousdonations

Wi”bemiss寧dbyminipooIscreenlng・Transfusiontrans一

画SS！onsho山1dbeconsideredinpatientswithdengue

a代erbtoodtransfusion．

ー3，and－4）conferslifelongserotype－SpeCifici皿munity． Subsequentinfectionwithan0therserotypeispossible becauseimmunityto heterologous serotypesis short－ uved・Most（53％－87％）dengueinfections are asymP－

tomatic ormi1dly symptOmatic．l‾3Dengueinhctionis Characterizedbyamedian5－dayvmmia．andincli血cauy apparentinfections，SymPtOmOnSetOCCurSldayaf［er

OnSetOfviremia・4・5TheclinicalspectrumOfdengueinftc－ donrangesfrom denguefeverto deng叫ehemorrhagic

fever，dengue shock syndrome，and death．PrimarY

denguein飴ctionsof［enpresentwithfbaturescLfclassic

dengueftverincludingacuteorlSetOf輌r；arthralgia， myalgia，retrOOrbitalpain，headache，and rash．Subse－

quentinfectionwithaseconddenかesirbtypeincr8aSeS theriskofdevelopingdenguehemorrhagicfever，Whichis Characterizedbyfever，thrombocytoperda（plateletcount

≦100×109／L），hemorTha由Cmanifbstations，andevidence

ABBREVIAT10NS：ARC＝AmericanRedCross；DENV＝dengue

ViruS；IC＝intemalcontrol；m＝initial吋reactive；S／CO

ratio＝Signal－tO－CutOffratio；TMA＝tranSCription－mediated

ampl通cation；W＝Ⅵ屯stNile血s．

FromtheDengueBranCh，DivisionofVector－Bomehdectious

Disease，CentersforDiseaseG⊃ntrOlandPrevendon，SanJuan，

PuertoRico；AmericanRedCross．Gaithersburg，Maryland；Gen－

Probe，hc・，SanDiego．Califomia；andtheDivisionofVector－

、BomeInfectiou云Disease，CentersforDiseaseControland

Prevendon，FortCouins，Colorado．

舶加即御伽旭叩醸敵地misbMobammed，phD，

DengueBranch，DivisionofⅥ∋CtOr－Bomeh鮎ctiousDiseases，

NationalCenterforZoonotic，Vtctor－Bome，andEntericDis－

eases，CentersforDiseaseControlandPrevention，1324CkⅢe

Cafiada，Sanluan，PROO920；e－maiLhmohammed＠cdc・gOⅥ

ConAictofinterest：JMLandASBamemployeesofGen－

Probe，Inc・andareownersofequitystockopdonsin

Gen－Probe．

ReceivedforpublicationDecember13．2007；revision

receivedMarch31，2008，andaccepted．March31，2008・

doi：10．1111／j・1537－2995．2008．01771．x

WS叩SION2008；48：1348－1354．

料 デ

1348 TRANSFUS］ON Volume48，July2008

179

DENGUEIN BLOOD DONÅTlONS

dona也onsare CO11ectedannuallybytheAmerican Red Cross仏RC）collectioncentersandblooddonationdrive sitesinPuertoRico．Thesedonationsareusedinthecon－

tinentalUmitedStates，PuertoRico，andelsewhereinthe

Caribbean．Tb assess the potentialfor transfusion－ associateddengueinLectioninPuertoRico．wetestedall blooddonadonstotheARCfordengueviralnucleicacid uslngareCendydevelopeddengue－SPeCi負cNATduringan u－Weekperiodofseasona止yh9ighteneddengueac

h2005．

MJn’ERIALSAND METHODS

Weanalyzed demographic data collectedfrom blood donorsandplasmaspe？imensfromanblooddonationsto ARCbloodcollectionc？nterSandblooddrivesinPuerto RicofromSeptember20toDecember4，2005．Thisstudy periodcommenced2weeksafterthepeakofseason叫 heighteneddengueactivityinPuertoRico（Fig・1）．Plasma SpeCimenscontaimingethylenediaminetetraacetateasan anticoagulant（BDVacutainerPPTplasmチPreparatipn tubes，BD．FraJiklinu止es，N7）from血1blooddonations

duringthis＄tudyperiodwereretainedinarepositoryat theARCfacilityinGaithersburg，Maryland．

Allspecimenswerefirstscreenedforthepresenceof DENVRNAusing a DENVIspecific N瓜■develop虐d by Gen－Probe，Inc．（SaLnDiego，WthatusestranSCription－ mediatedampli丘cation（TMA）．Specimensweretestedby TMAatGcn－ProbebytrainedARCsta鼠Allinitiglyreac－ tive（m）specimens were retestedand TMA－pOSitive

ofincreasedvascularpermeabilityandplasmaleakage■6・7 Withtime吋SuppOrtivecare，denguehemorrha由Cfever case－fatalityratescanbereducedtolessthanLLperCent・8・9

TheprlnCipaldenguevectorisAedesaegypti・Itis fbundthroughoutthetropicsandsubtropicsandinlimited areas ofsome statesinthe southeasternUnited States． Aedesalbopictusisalsoacompetentvectorfordengueand hasbeenimplicatedpreviouslyindengueoutbreaks・10 AlthoughithasnotbeehdetectedinPuertoRico・Aalbop－ ictLLSeXistsinsomepartsoftheAmericasincludingmore than20statesintheeasternhalfoftheUnitedStates．11 Antochthonous dengue transmission doe＄SPOradical吋 occurinsouthemTbxasalongtheUmitedStates－Mexico border，withthemostrecentoutbreakoccumnginthe conti糾OuSbo王dertoⅥmSOfBrownsville／rbxas，andMata－ moro占，恥Ⅰ血adipas（Mexico）・12・13Thissuggeststheende－

micityOf denguein SouthTbxas andthe risk of reemergenceofdengueinstatesthatborderMexicoaswell asinsoutheastemstateswithcompetentvector（S）and

Subtropicalclimates・Research．however・hasfoundthat diEerencesinhousing（e．g．，uSeOfaircondidoAmgand screens）andlifbstylemayp㌍Ventthisfromhappe血ngl12・13

Although飴wreportsdocu皿entDENVtransmission throughreceiptofinfectedblood，14tissues，150rOrganS，16 tranSfusion－aSSOCiatedden卯etranSmissionmaybemore

COmmOnthanPreviouslyrecognized・Thehighproportion ofasymPtOma也cinfections，themedian5－dayperiodof detectable viremia，andthe highincidence，eSpeCiany

ddringoutbreaks，SuggeStthatasubstantialnumberof donorscqddbeviremicatthetimeofdonation．haddi－

tion．nosocomialtranSmissionofDENV vianeedle－StickinjurY17●21hr血erindi－ 450 CateSthe．tranSmissibilityofDENVby infected tblood．Viremicindividuals mayunkn0winglydonatebloodbefore SymPtOm OnSet Orif they remain asymptOmatic．WtstNileviruS（WNⅥ，a

relatedmosquito－borne且aviviruS，may

PrOvide a use餌・mOdelfor assesslng tranSfusion－aSSOCiatedDENVtranSmis－

sion．廿anSfusiontranSmissionofW

iswe11documented．andal1blooddona－

tionsintheUmitedStatesarescreened usingW7Wspeci8＝CnuCleicacidampli一

丘cadontests（NÅ恥）．22B

Dengue was first identified in ∴

i（year－rOundwithoccasionalishndwide Outbreaks．AmeanOf5446（range，2416－

10，048）suspectedcaseswerereported annu姐yduringthenonoutbreakyears 仕om1990to2004，Wheteas6039cases

Were rePOrtedin2005（incidences of 151versus159perlOO，000populadon／

year）．Approximately 77，000 blood

〇 ．〇

5 0

乍V ▲3

∽○∽■0－OJOq∈コN

0 0

■－J nV

▲フー 2

14 71013 代19 22 25 28 31封 3740 43 46 49 52

Weekofonsotof写ymPtOmS

Rg．l．Numberof＄u叩eCted■岬）mdco鱒md†（¶denguec鮎朗吋Weekof synptomonset，PuertoRico，2005・■S11SPeCted＝rePOrtedc鮎eOfden糾ewitha dinicalstLSPiciomofdenpe．†Co血ed＝1aboratory－COnfimed巾y＄erOlogyor

drologカC耶eOfden卵色

Volume48，Ju（y2008 TRANSFUS］ON1349

180

MOHAMMED ETAL．

SpeCimenswerethosethatwererepeatedlyreactive；al1 OtherswereconsideredtobeTMA－negative・Bothinitial

and repeatTMAscreeningwereperfbmedusingindi－ vidualspecimens．AllIRspecimensweresenttotheCDC’s Dengue BranCh1月boratoryin SanJuan，Puerto Rico，

fbr supplemental testing by reverse tranSCription－ polymeraSeChainreaction（虹PCR）．24TbstingofdonaT tionswas unlinked to donorpersonalidentiBers；thus，

Subsequent contactwithdonors orrecipientswas not POSSible．Deidentifieddata丘■Omblooddonationrecords

WereuSedinthestatisticalanalYSis（describedbelow）．The datawerestoredonaslnglepassword－PrOteCtedterminal attheCDC，andnoattemptwasmadetotracethedonors．

The study protocoIwas approved bytheInstitudonal ReviewBoardoftheARC．

WereteStedusingareal－timeRT－PCRassayforthedetec－ tionofNS5genesequence（恥qMan，AppliedBiosystems， FosterCity，CA）．24ThisRT－PCRtestis multiplexedand

detectsthefourdengueserotypesinonereaction．Itcan alsobeusedtoquandtativelymeasureviralRNAinblood SpeCimenswithasensidvityofapproximatelylxlO3to 5×103viralRNAcopleSPermLThesamplevolumeis

20pLderivedfromalOO－uLRNAextractobtained丘oma

O．24－mLserumSPeCi皿en．山1TMA－pOSitiveandIRspeci－

menswerealsotestedforthepresenceofimmunoglobu－ 1inM（1gM）andimmunoglobulinG（IgG）antibodyusing IgMMAC－enZyme－linkedimmunOSOrbentassay（モLISA）

andIgG EuSA，reSpeCtively131Y32ⅥruS WaSisolated on C6／36cellsandbymosquitoisola・don・33・34

Stati＄ticalanaIysis

AtrendanalysisuslngSimplelinearregressionwasper－ formedtodeterm血eiftherewasachangeinthenumber Ofblooddonationscollectedduringthestudyperiod．The prevalenceofDENVRNAwasdeterminedbydividingthe number ofTMA－pOSitive donations bythe number of blQOddonadonscollectedduringthestudyperiod．InLbr－ mation about donor characteristics（Seelもblel）was

ObtainedfromtheARC，＄electronic donor database and

incl。deddate。fc。u。。ti。n，g。nd。r，date。frth，Zipc。de Ofresidence，ZlpCOdeofdonationsite，donationstatus （むst－timedonororrepeatdonor），phlebotomyprocedl，lre （WllOle blood，plateletpheresis，Orleukapheresis），and donationtype（allogeneic，directed，OrautOlogous）．Both donorresidenceanddonationsitewererecodedintothe

threereglOnSOfPuertolruCOSetbytheUhitedStatesPostal Service（SanJuanMetropolitanArea，WeSt．andeast）and

thentreatedasbinarYVariablesintheanalyses（i．e．．San JuanmetropolitanareaVerSuSOther）．Agewasstratifiedby itsmedianandconsideredasabinarycategoricalvariable inanalyses，

DifferencesinTMApositivitybydonorcharacteristics WereaSSeSSedbytheFisher’sexacttestandexactlo由Stic regression．Potential、covariatesidentiBedforinclusionin the丘nalmultivariablehodelincludedcovariateswithap ValuelessthanO．200nbivariateanalysis．Agewasadded tothemodelapnonglVenitsassociationwithdengue infection．ZAl1comparisonsweremadewiththeuseofa two－tailedtest，andaTypeIerrorrateofO．05wasusedto assesssigni丘caLnCe．

RESULTS

Atotalof16，521blooddonadonswereco11ectedduringthe ll－Weekstudyperiod（mean，1502donationsperYeek ［range・28ト1864】withoutasi卯i丘canttrend中donation

frequency）・TⅣelvedonadons（0．73perlOOOdonations）

Were TMA－pOSitive，dthtwo orlessidenti丘ed per Week．Eleven of these －12 donations were whole

TMA

Tbstingwasperfohnedusingaprototypedengue・TMA assayonafu11yautomatedsystemforNÅrbloodscreen 伊rocleixTigrjssystem．ChironCorp．，EmerYvi11e，CA）・The assayusesth（∋SameChemistryasotherhumanimmun0－

deficiencyviruS－1／hepatitisCviruSandWassays伊ro－ cleixandUltrio，reSPeCtively，ChironCorp．）25－27andtargets SequenCeSthatareconservedacross allfourserotypes． Thustheassayusediscapableofdetectingal1fourdengue SerOtyPeS．TMAisanisothermalRNAtranscriptionampli一

丘cationsystemusingbacteriophageT7RNApolymeraSe andMoloneymtuineleukemiaviruSreVerSetranSCriptase （MMlVRT）toproduceRNAampliconsviaDNAinterme－ diates．Viral1ysis andmagnedc－basedtargetcaptureof viralRNAarefouowedbyamPl迫cationanddetectionwith

theuseofchemi1uminescentprobes．26Thisteclmiqueis abletodetect3．4WestNileviralcopleSpermLata50 PerCentdetecdonrate・28Theanalyticalsensitivityofthe DENVTMAassayusedinthisstudyisverysimilaT，with50 PerCentdetectionat3・5vird「RNAcopleSPer如Landa SamPlevolumeofO．50mL．29Assayresultswerereported inrelativelightunits，Whichwereusedtoderivesignal－tO－ CutOff（S／CO）ratios．Cutoffvaluesforthe DengueTMA assayinternalcontrol（IC）andanalytesignalswerecalcu－ latedusingthesameformulaeusedfortheProcleix Assay．30Asamplewasconsideredreactiveifthea皿alyte

S／COratiowasatleastl．0，nOnreaCtiveiftheanalyteS／CO ratiowaslessthan1．0andtheICsignalwasabovetheIC CutOff，andinvalidiftheanaiyteS／COratiovaluewasless

than1．0andtheICsignalwasbelowtheICcuto氏

Supp］ementa］testing

AllTM－pOSitivespecimenswereretestedatal：16dilu－ tioninplasmascreenednegativeforal1infecdousdisease markersincluding dengue RNA at Gen－Probe to de－ terminethe e伍cacy of testing blood donations by

mlnlPOOledmethods．TheTMA－pOSidveandIRspecimens

13501月ANSFUS10N Vo山me48，」uly2008

181

DENGUEINBLOODDON〝nONS

blood・CO11ections，Whiletheotherwasaplateletpheresis fromanO－donor．Donoranddona仕onchamcteristicswere

Simi1aramOngtheTMA－POSitiveand－negative＞donations （p＞0．05forallvariables．Fisher’sexacttest）．hamuldvari－ ′ablemodeladjustedfbrage．donorswithresidenceinthe San如anmetrOpOlitan arear扉ere approximately three times morelikelythan donors residingoutside ofthe metropolitanareatObeTMA－pOSidve（a句ustedoddsratio， 3．0；95％con丘denceinterval，0．9－10．1）．

The丘代blooddonationsthathadthehighestS／CO ratiosoninidalTMAtestingweretheo山yspecimenstobe Thn－reaCtiveatadilutionofl：16恥ble2）．Fourofthese

fivespecimenswereposidvebyRT－PCRandhadquanti－ fiablevirdloadsrangingfrom2×103to8×107viral

COpleSpermL・ThreewereidentifiedasDENV2andthe Otheras DENV二3．DENVwas culturedfromthreeofthe

fburspecimens．twobymosquitoinoculadonandonein CeuCulture・DENV・2andDEN¢3werethepredominant

SerOtypeSincirculationinPuertoRicoin2005． Serologictestingofthe12TMA－pOSitiveblooddona－

donsrevealedthatonlylwasIgM－pOSitiveand9were IgG－pOSi伽ebyELISACrhble2）・ThelackofIgGan仕body dtersinSpecimensl，4．and8indicates no previous

dengueinfectionsinthesepatients・ThepresenceofIgG andbodiesintheabsenceofIgMand－ bodiescouldre瓜ectevidenceofprevi－ OuSin飴CdonsinSpecimens2，3，5，7，9， 10，11，and12，andIgGdtersequalor greaterthanl：163，840inSpecimens3，7，

9．andlOindicate arecentor current

SeCOndaryinfectioninthosepatients．32 ThepresenceofIgGinthesoledonor withIgMantibodies（Speci皿en6）could

Sim血中 be re8．ective of recent or

Currentinftction．

0血er血an血e12TMA－pOSidve

SPeCimens．there wereanadditional threemspecimenswithS／COra孟son initialtestingofl．00，1．03．andll・58and

OnrePeatteStingofO・92，0・40，andO・07，

respectively：Al1werenega仕veonPCR，

IgM MAC－EuSA，and virus recovery．

neywere，however，一positive pnIgG

ELISA．htheWNVTMAassay，anS／CO rati00fgreaterthanOrequalto17has a positive predictive value fbr con一

点rmation of95 percent 仏RC data．

unpub止＄hed）；itislikelythatthisrela－

tionshipisthe same for DENVTMA．

TABLEl．Character‡＄tjcsofal】andTMA－POSltjveblooddonors雷n Pue代OR‡co，＄¢ tember20toDecember5，2005’

All donors TMA－POSitjv8 Characten＄tic （n＝…16，521） doncrs（n＝12）

Ago（years） Ma18

Donation status Rlちt欄m8donor Repeat donor

Region of residence SanJuanMetropolitanArea East W9扇

PhlebotomyprDCedure Wh010blood Plat81占中ho帽5鹿 Pl坤叫咋卸叩吟鳳RBCpheresis Doubl8RBCpherosi＄ Louk畠Pheresiさ

Donaゼm吋PO A1logoneiG Direded A山0logous

Regionofdonaぬn＄it8 SanJuanMetropoIibnAr由 Ea＄t Wost

37．0（1＆85）

10．654（64．5）

5，056（30．6） 11．485（69．4）

6，631（40．1）

5，182（31．4） 4，706（28．5）

15，838（95．9） 627（3．8）

48（0．3）

7（0．0）

1（8．0）

16，400（99．3） 67（0．4）

54（0．3）

8，9糾（54．4）

3，870（23．4）

3，667（22．2）

囲刑㈹剛刑期

57 831 111000 200

●Dataarereporteda＄rnedian（range）ornumber（％）．

TABLE2．fl毎un＄Of＄uPPJementarytestingofTMAIRspecirTm（，）＝12） TMAtestG即トProbe（SJCOrab）● Supph）mentaryt融ngCDCdenguebranch

Numberviml Mosquito Spedmen tnitialtest Secondtest l：16 PCR† RNAImL lgM‡ lgG Cell culture inoculation

D2 7．14×108 D3 8．12×107 D2 7．74×105 D2 2．OxlO3 Nega伽8 Und8t郎鹿d Nega厄ve Undetected Negative Und8teCted Negaゼv8 Undotected Nega伽e Undetected Negative Undetected Negative Undetected Ne9ath，e Undetected

1 31．96

‘2 30．31

3 29．22

4 29．17

5 23．89

6 21．22

7 17，78

8 17．41

9 17．24

10 5．97

11 4．08

12 1．53

9

．g．28．86朋．59．28．10〟．〇5調．15鹿

2

737812g2劇21153133151300

． 2728．27と8〇．〇．〇．〇．〇．〇．〇．

0．229． Nega厄Ve Nega伽e D2 0．幻7 1：10，248 Nega一行e ［〉3 0．409 1：163β㈹ D2 Negative O．22g Nega削ら N09血e Nega也ve O．469 1：2，560 Negative Nega伽e 8．870 1：160 Nega伽e Nega伽e O．4O9 1：655，360 Negative Negative O．198 Negative Negative Nega伽e l．封0 1：163，840 Neg血e Neg血8 0．440 1：65年，360 Negative Negative O．368 1：10，240 Negat毎e Nega血相 0．270 1：2，560 Nega伽o Negative

嵐ざ

■ S／CO≧1con＄id8redtoboread山嵐 †D2＝DEN≠2．D3＝DENV－3． ‡＞2．00ロeonsid餅edpo＄柑V8．

肋Iumeヰ8，July2008 TRÅNSFUStON1351

182

MOHAMMED ETAL．

Nineofthe12repeat－reaCdvesampleshadS／COvaluesin OneOrbothtestsof17－Orgreater・

DISCUSS（ON

Thisstudy；andasimi1aronerecendyconductedusing donationsinfIonduras，Brazil，andAnstralia，29arethefirst todocumentthepresenceofdengueviralnucleicacidin blooddonations・hPuertoRico，nearlylinlOOOdona－ thnswaspositiveforthepresenceofdengueviraln11Cleic acidbyTMAFurthermore，1iveviruSWaSreCOVeredfrom threeofthe12TMA－pOSitivedonations．indicatingthatat leastthese3were capableoftranSmittinginfectionto recipients・Theprevalenceofdengueviralnucleicacidin

blooddonationsinthisstudywassimilartothatestimated forWNVintheareasexperienclngOutbreaksintheconti－ nentalUnitedStatesin200235beforeuniversalscreerung using minipooINÅr wasimplementedinJuly2003．22 AssuminganarmualprevalencerateofO・73per1000（as bundinthisstudy）andthateachdonationismadeintoa meanOfl．45tranSfusablecomponents，36theremaybeas manyaS56potentialrYviremicdonadonsand81compo－ nents generated h’Omthe approximately77，DOO blood donationsco11ectedaLnnuauybytheARCinPuertoRico・ Dengueincidenceishigh吋SeaSOnalandv虹iesconsider－ ably丘■OmyeartOyear，37r38however．sotheprevalenceof potential1yviremicdonorscouldbecorlSiderablyhigher Orlowerthanthis丘gureatanyglVentime．FurthermOre， theth工ee m SPeCimenslackingreproducible results repe？tTMAtestingmayhavebeentrue－pOSitivespeci－

mensbutwithlowerviralloads．Ifthecase．thiswould

underestimatethetruepreValenceofTMApositivity． The unlinkedstudydesign didnotpermitcontact

Withthe recipients ofthe TMA－pOSitive donations to assesswhethertransmissionoccurred．Nevertheless，viruS

WaSCulturedh－Omthreedonationsandtheviralloadsof

thefburRTIPCRl）OSidve donationsindicatethattheir tranSfusionwouldhaveresultedininoculaordersofmag－ nitudegreaterthantheamountofviruSSeCretedinthe SalivaofAedesmosquitoes．documentedtobeaslowas lO2viralparticlespersecredon．39TheRIしPCRassayusea inthisstudyhadlowersensitivitythantheTMAassay；and itwasnotpossibletoassesstheviral1oadoftheRT－PCR－

negativespecimens． OurresultsindicatethefeasibihtyOfNÅTasascreen－

1ngStrategyforDENV；ashasbeensuccessfuJyusedfor WNVOfconcem．wefbundthatsimulatedmimipooINAr （dilutionl：16）wouldnothavedetectedthemajority（70f 12，Or58％）oftheTMA－POSitivespeci皿enS；howevef．the

experiencewithWsuggeststhatnotallofthesedona－ tionsmaybeinfectious．Approximatety30percentofWNV NÅトpositivedonationshaNeviral，loadsbelowthelimits

OfdetectionbyminipooINArandcanonlybedetectedby SCreerungOfindividualdonations．23・40AlthQughⅥWVha5

beentranSmitted打omtranSfusionsdetectableonlyby

1352 TRANSFUS10N VoIume48，July2008

183

indjvidualunitscreenmgandwithanestimatedlevelof ViremiaaslowasO・06plaque－fbrming皿its（PFUs）permL

（1PFUisapproximateけ400viralcopies），41mostdona－ tionsonlydetectablebyindividualunitscreqninghadIgM andlgGantibodiesandwerehkelynotinfec也ousgiventhe fhctthatnear吋al1WNVtrans鮎siont＝anSmissionshave OCCurred丘■Omantibody－negadvedonadons．22・25・42unLbト

tunately；thissamemarkerofinfectivityisnotappucable todenguet）eCauSeOfthehighprevalenceofpreexisting， CrOSS－reaCtivedengueantibodiesinthepopulationand thecomplexandvariableserologicresponseaftersecond－ arydengueinftction．32・43

Theglobalincidenceofdenguehasrisenmorethan 30－foldinthepast50years・hareaswhereden計1eis endemic，however，tranSfusiontranSmissionoftheagent israrelyirⅣeStigatedformanyreasons，indudingthefact

thatthismodeoftransmissionisd臆culttoproveagainst abackgroundofendemicdengue．Insuchcases，thedis－

tinctionbetweenarecipientinLectionviamosquito－bome transmission as opposed to transfusion tranSmission maybetoocomplextodistinguish・Furthermore，many dengue－endemic countrieslackhemovignance systems withsu瓜cientresourcestoinvestigatecasesofrecipient infectionthat are potentiaⅡyrelated to trdnsfusion of blood components・Finany，SOphisticatedlaboratory testingmaynotbereadilywailableinm査nydengue－ endemiccotntriesandsuchtestingisrequiredtodis血－

guish denguefrom other arboviralinLbctions as well as distinguishing cu汀ent dengueinfection＆om pnor infections．

Incontrast，WhenWNVenteredtheUhitedStates，it

WaSagalnStabackgroundofana伽epopuladon．ThisperL

mittedthelaboratorylinkage of multiple transfusion recipientswithWNVin血ctiontoasingleinfecteddonor

withinseveralclusters ofW cases．hfectiousviruS

and／orviralRNAcouldalsoberecovered仕omretrieved COCOmPOnent plasma units；inthese cases，WNVwas

readilyidendjiedintheabsenceofcompetingarboviral infections・ThetranSmissibnityofWNVviabloodtranSfu－ Sionhasbeenestal）lished，andourfindingsdocumenting thepresence ofDENVRNAinthePuerto Ricanblood SuPply，at alevelcomparible tothatwhich triggered 戸CreemingoftheUSbloodsupplyforWin2003，highー

Iighttheriskstotrqnsfusionsafetyposedbyemerging diseases such asthe vector－borne naviviruSeS．Further

evaluationisrequiredtoassesstheriskofdenguetranS－ missionbyTMA－pOSitive donationsandthe cost and bene丘tofroutinedenguescre阜ninginendemicreglOnS・

ACKNOWLEDGMENTS

Theauthorsacknowledgethefollowhgindividualsfortheircon一

雨bu血nto也isrese8∫Ch：M∫5Di鮎Mag由yBorge＄a皿dMf AntoniodeV岳ra（ARC－PuertoRico）fbrprovidingdataspecificto

thePRJre如nofARC；DrMark寧eattyO｝ediatricDengueⅥlCCine

註ア

DENGU∈INBLOODDONÅT10NS

hitiadve）forassistingwithprotocoldevelopment；MrGilberto

SantiagoandMrMarkVerduinのengueBranch，CDC）fbrlabo一

円血Ⅳ飢甲p“七山MkhaeⅢ山肌SSOn（DengueBran叫FDqfbr

Stadsticalsupport；andDrEd■Notari（ARC），DrMathewmlelmert （0伍ce ofBlood．Orga皿，and otherTi＄Sue Safety（proposed），

CDC），MrJoshuaSmith，Dr払teMcElroylDrD．FermhArgiie1lo，

andDrWel山IgtOnStm（DengueBranch，CDC）fortheirscientific

mputinprepaLingthemanuscript・

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RothmanAL，VaughnAI・，Emis臥Epidemi0logyof

inapparentand＄ymptOmaticacutedengueviruSinFbction：

apro叩eC伽esmdyofp血むγSFhooIc出1血enin伽n－

phaengphet．Thai1and・Am1Epidemio12002；I56：40－5l・

2．Rodrisuez－FigueroaL，Rigau－Pere211G，SuarezELReiterP．

昆5k払ctorsぬrdenguein飴dondu血ganoutb∫eakh

lねnes，PuertoRicoin1991．Am1TtopMedtlyg1995；52：

496－502．

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1988；38：172－80．

4．Ⅵlughn叫GfeenS．Kalayanar005S．hmisBL

Niman血中S，Suntay＊omS，EndyTP，Raeng古址dl

B．RothmanALErmisEA．NisalakA．Dengueviremiatiter，

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Wi血曲easeseved呼．Jb鮎∝Dis2000；18l：2－9．

5．mshitmH，HalsteadSB．NatdalhistorYOfdenguevims

（DENV）－1andDEⅣ¢4infections：reaha＆sisofclassic

Studies．J恥ctDis2007；195：1007－13．

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1980；58：1・2l．

7・Haemo汀ba癖CD・nⅣer：鵬a伊10由．虻eatmenもpreVendon

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Org血鮎n；1997．

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9・ChareonsookO，FbyH．恥eratkulA．SilamgN．Changing epidemi0logyofdenguehemorrhagicfeverinThai1and． Epidemiol地眈1999；122：161－6．

10・EE）erPVIPangLKitsutanjP．Ⅵ｝mdamV．NahtaM，Ayers

T，ElmJ，TbhT．ReittrP．RigzLu－PerezIG，HayesJM，Mills

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11・MooreC・Aede＄albopictu＄intheUnitedStates：Current

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MH．bpeヱJL，Fo血erM，叫山oA札Bu正OnR，Bn∬止訂d

JM，Ana阿一山pezL，ぬ山dゴAんMor山部PK放出也乱

MumOZIL，WatermanSH，TheDengueSerosurveyWorking

Grolユp・Epidemicdengueanddenguehemorrhagicfeverat

theTbxas－MexicoBorder：reSultsofahousehold－based

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13・ReiterP．hthropS，BunningM．BiggerstaffB，SingerD，

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MendezJ，RamirezB，RobinsonJ，RavdingsJ，Ⅵ）mdamV，

WhtermanS，GublerD．ClarkG．HayesE．Tbxasliftstyle

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9：86－9．

14・T5angC・Localden即eftvercasesin2002・HongKong－

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取ansplant2005；20：447－8．

17．ChenLN．WilsonME．Nosocomialmucutaneoustransmi－

Sionandotherroute台OftranSmission・EmergInfectDis

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18．DeWhzier6sB，G辻H，Ⅵ止托OnD，Dupondl．Nosocomial

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20・Ⅵ独伊erD，deⅦ血KHu坤D，Hu食止F，ⅥねidmamM，

BreisingerS．EppingerS．KemWⅥBauerTM．Nosocomial

acquisitionofden伊1e．EmergI唾ctDis2004；10：1872・3．

21・Hkschl．DeschamPe亭C，u山山ierM．MetropolitantranS－

missionofden訃1ebyaccidentalinocdationatahospital・

AnnhtemMed1990；Ⅰ41：629．

22．PealerLN，MarfinAんPetersentR，1且mdottiRS，PagePL．

▲S仕amerSLStobierskiMGiSignsKNewmanB．KapoorH，

GoodmanJL，ChambedandME；WtstNileV辻u＄取ansmi－

sionInvestigatibnTbam，TtanSmissiotlOfWestNnevirus throughblood En由JMed2003；349：1236－45．

23．StramerSL．FhngCれFosterG九WhgnerAd，BrodskYJP，

DoddRY・WestNileviruSamOngblooddonorsinthe

UnitedStates，2003and2004．NEnglJMed2005；353：i51－9・

24．ChienU，LiaoTL．ShuPY．HllangJH，GublerDl，ChangGl・

Developmentofreal－timereversetranscriptasePCRassays todetectandserotypedengueviruSeS．JClinMicrobiol 2006；44：1295－304．

25・Bu＄血MP．Tbblerul，SaldanhaJ，Ca9iotiS，ShyamalaV，

LinnenTM，Ganardal．PhelpsB，SmithRl，DrebotM，

紀einmanSH．AnalyticalandclinicalsensitivityofWest

NileviruSRNAscreenln8and盲upplementalassaysaⅥ山一

／ ablein2003．取anSfusion2005；45：492－9．

26．GiachettiC，LinnenTM，Kb敗DP，DockterJ．G山otte－1払ylor

K払止叱Ho－Shg一山yM．McCo血改MKMimⅢu、，

Volume48，July2008 TRANSFUS10N1353

184

MOHAMMED ETAL．

McDonoughSH．High吋SenSitivemdtiplexassayfDr

detectionofhumaLnimmunOdeficiencyviruStyPeland

hepatidsCviruSRNA．JClinMicrobioljoo2；40：2408－19．

27．McCormickMK，DockterJ．LinnenJM，KolkD．WuY，

Giachetti（：・baluationofanewmQlecularassayfordetec－

donofhulnanimmunOde丘ciencyviruStyPelRNA．hepa－

titisCviruSRNA．anbhepatidsBviruSDNん1ClinVkoI

ZOO6；36：166－76．

28．LinnenlM，DerasML，ClineJ．WuW．Bro山腹AS，CoryRE，

mightJL，CassMM．CollinsCS，GiachettiC．PerformanCe

evaluationofthePROCLEⅨWtstNileviruSaSSayOnSemi一

automatedandautomatedsystems．JMedV加12007；79：

1422－30．

29．LinnenJM，VinelliE，SabinoEC，TbblerLH，Ⅰ坤and’c，Lee

TH．Kon（DP，BroulikAS．Col追nsCS．LanciottiRS，Busch

MP．DengueviremiainblooddonQrSf［OmHonduras，

BrazilandAustralia．廿ansfusionDOI：10．1111／j．1537－

2995．2008．（）1772．x

30．LinnenTM，DerasML，ClineJ，帆W，BroulikAS．CoryRE，

KnightTL．（：assMM．CollinsCS．GiachettiC．PerfbrmanCe

evaluationofthePROCI丘ⅨWtstNileviruSaSSayOnSemi－

automatedandautomatedsystems．JMedV辻012007；79：

1422－30．

31．BurkeDS．NisAlakんUsseryMAAndbodycapttueimmu－

noassaydetectionofJapaneSeenCePhalidsviruSinmun0－ globulinMandGantibodiesincerd）TOSpinalnuid．JChn

Microbiol198Z；16：1034－42．

32．MiagostovichMP，NogueiraRM．dosSanto＄FB．Schatz－

mayrHG．AraujoES，VomdamV・EvaluationofaLnIgG

enzyme－1inkedinlmunOSOrbentassayfordenguedia伊0－

Sis．JClinMicrobiol1999；14：183－9．

33・RosenL．GllblerD・Theuseofmosquitoestodetectand

propagatedengueviruses．AmJnopMedHyg1974；23：

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65．

35・Biggersta茸BJ．PetersenI且Estimatedriskoftransmission

OftheWestNileviruSthroughbloodtransfusionintheUS， 2002．Ttansh5ion2003；43：1007－17，

36・Marshal1DA．KleinmanSH．WongJB，Al通uchonJP，Grima

DT，粗11inNA，WeinsteinMC．Cost－effectivenessofnucleic

acidtestscreenlngOfvolunteerblooddonationsforhepa一

tidsB・hepatitisCandhumanimmunodeficiencyviruSin

theUrdtedStates・＼わⅩSang2004；86：28－40．

37・Rigau－Pere乙JG・DengueactivityinPuertoRicoduringan

interepidemicperiod（1995－1997）・Am1TfopMedHyg

2001；64：75－83，

38・Rigau－PerezJG・Thereappearanceofdengue－3andasub－

Sequentdengue－4anddengue－1epidemicinPuertoRico

in1998・Am1取opMedHyg2002；67：355－62．

39■ KraiselburdE，GublerDl，KesslerMl・Quantityofdengue

virusrequiredtoinfectrhesusmonkeys・nanSRSocTh）p

MedHyg1985；79：248－51．

40・BuschMP．CagliotiS，Rober【sonEF．McA11leyTD．Tbbler

LH．XamelH．LinnenJM．ShyamalaV．TbmasuloP．Klein－

manSH・ScreeningthebloodsupplyforWestNileviruS

RNAbynucleicacidamPlificationtesting・NEngllMed

2005；353：460－7．

41・MontgomerySP．BrownJA．XuehnertM，SmithTL，Cr山1N．

hnciottiRS．MacedodeOuveiraA，BooT．Ma血AA；2003

WestNileViruS取ansfusion－AsscciatedTIanSmission

，と hlVeStigationTban取ansfusion－aSSOCiated任ansmissionof

WestNileviruS，UnitedStates2003thro11gh2005．取ansfu－

SionZOO6；46：2038－46．

42・PetersenLR，EpsteinJS．ProblemsoIved？WestNilevirus

andtransfusionsafety．NEn由JMed2005；353：516－7．

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denguehemorrha如虎ver．In：HommaA．CunhaTF， editors・ProceedingsoftheintemationalsympOSiumon

yellowfeveranddengue．RiodeJaneiro，Braヱil：Riode

ねnei∫0；1988．p．29ト322．口

ふ ′

1354 TRANSFUSION Volume48．July2008

185

叫パ隼噂担ゆ㌣噂叫声‘u

186

医薬品

医薬部外品 研究報告 調査報告書

化粧品

別紙3イ

報告日

年 月 日

第一報入手日

2008年6月13日 新医薬品等の区分

該当なし 識別番号・報告回数

総合機構処理欄

一般的名称 r 】 nid

】

研究報告の公表状況 Culture． オーストリア

販売名（企業名） Ehrlich，H．］．etal，

N帥Engl．J．Med．，358－2573－2584

（2008）

本稿では，抗H5Nl型鳥インフルエンザワクチンの安全性及び有効性を検討する無作為化用量漸増第l・2相試験の試験成績につい 使用上の注意記載状況・

て記載した。BaxterBioscience社が同ワクチンを開発し，試験を実施した。当該ワクチンの主な特徴は，サルの腎臓細胞の培養株（ベ その他参考事項等 ロ細胞）で作成された自然発生するウイルス株〟Vietnam／1203／2004を利用していることである。ミョウバンアジュバントによる作 BYL－2008－0338

研 用も検討し，ウイルス全体をワクチンとして使用した。有効性エンドポイントとして，ワクチンの（i）へマグルチニン阻害を生じさ

究 せる能力，（ii）中和抗体を誘発する能力，（iii）注射21日後にセロコンバージョンを生じさせる能力を検討した。各被験者に対し，

報

告 の

概

要 有効性に関する限り，免疫応答はアジュバントなしの処方を投与した被験者において最も高い割合で認められたが，いずれの処方で

も第21日目から第42 日目で中和抗体の抗体価は同程度に増加した。さらに，ウイルス株A／lndonesia／05／2005及びA／Hong Kong／156／1997に対する交差中和が認められた。再鼠アジュバントなしの処方が最も高い免疫原性を示した。本試験では重要な用量

反応性の関連が示されなかったことから，今後の開発にあたってアジュバントなしの7．5ugの処方が選択された。

報告企業の意見 今後の対応

抗H5Nl型鳥インフルエンザワクチンが利用可能となれば、パンデミックの 現時点で新たな安全対策上の措置を講じる必要はないと考え

発生及び拡大を防ぐために有効であろう。血焚由来製剤工程におけるウイ

ルス除去は、インフルエンザウイルス除去に対しても有効と考えられる。

弊社製品の製造工程に使用されている血渠分画成分は，製造工程中のウイ ルスバリデーションにおいて，インフルエンザウイルスと同様の土ンベロ

ープRNAウイルスであるH川（レトロウイルス）の不括化・除去能が確認さ れでいる。各成分の製造工程における不活化・除去能は以下のとおり。

●アルブミン・カッター及びコージネイトFSの製造工程培地に使用され七いるヒト血清アル

プミン：17．8log以上

・プラスマネートカッター，コージネイトFS及びコージネイトFSバイオセットの製造工程

培地に使用されている加熱ヒト血梁タンパク：15log以上

●コージネイトFSの製造工程に使用されているトランスフェリン：9．1log以上

●べタフエロン皮下注，ゼヴァリンイットリウム（90Y）静注用セット及びゼヴァリンインジウ

ム（川‖静注用セットの製造工程に使用されているヒト血滑アルブミン：9．98log以上

I I l

ー ノ

■、■

188

BYL－2008－0338

7’わ己 Nf二W ENGLANr＝OURNAL∂＿「入†E「＝Cl三JE

ORIG王NAL AR．■11CLE

AClinicalTrialofaWhole－Virus H5Nl

Vaccine Derived丘om Ce11Cul［ure

．・い・・－∴∵ ∴∴、∴：∴ ∴・、、■」‥・∴－ ‥‥・・、．ミー ー∴・．

， Paui良丁ヨr門口yah，M・B■，B－S‥Ci－r≡5t血1joukhadar，M．D・、EmanueieMontorno‡LPh．D．，

Da‡eFニisher，F叔．A▼⊂．P・．GregBerezしk，吼S．，SandorF再sれPh．D．，

Akx抑draLわW一己ase！雪盲，Ph・Dt，Nin3Vart盲an．Ph・DリRomanBobrovsky，Ph．D，，

Bor言s一品vaG．Pav室ova，Ph．D．，EvaMar毒aP6貼bauer，M．D．｝○モ育iedKistner，Ph、D．，

andP．Noe‡Barreモモ，Ph．D．－わrthe8axterH5NIPandem盲clnポリenza

VaccineC圭in妄caiStudyTearn

ABSTRACT

B入⊂XGlモOUIヾD

Widespreadinfさctionsofavian specieswithavianinfluen2，aH5Nlvir11Sandits limitedspreadtohumanssuggestthattheviruShas【hepoten亡ial亡OCauSeahuman influenzapandemic．AnurgentneedexistsfbranH5NIvacciflethatise晩ctive agalnStdivergen亡StrainsofH5NIvirus・

ME丁目ODS

Inarandomized，dose・ざSCalation，Phaseland2studyirNOlvingsiXSubgro11pS，We inves［1gatedthesafityofanH5NIwhole－VirusvaccineproducedonVbrocelicul－ turesanddetermineditsabili［ytOinducean［ibodiescapableoFneutralizingvarious H5NIs亡rains・lz］［WOVisi亡S21daysapart，之75volunteersbetweentheagesof18and 45yearsreceivedtwodosesofvaccinetha［eaehcontained3・75FLg，7．5〃ノg，15jLg， Or30FLgOfhernagglutininantlgenwithalumadjuvantor7・5FLgOr15FLgOfhemag－ glutininantlgenwithou亡a4jwant・SeroIoglCanalysiswasper丘）rmedatbaselineand Ondays21alld42．

RESULTS

Thevaccineinducedaneu［ralizinglmmunereSPOflSenO亡Onlyagainstthecladel （AIVietnamI1203！2004）viruSStraint）utaIsoagainsttheclade2and3strains．The useo（a4iuvantsdidnotimprovetheantibodyresponse・Maximumresponses【0【he VaCCirleStrainwereobtainedwithformulatiorlSCOntaining7・5FLgand15fLgOf hemagglutininantigenwithouta嘩uvant．Mildpaina亡theiftiectionsite（in9【027％ OfLsubiects）andheadache（in6to31％ofsu旬ects）werethemostcommonadverse eventsider】tified氏〉ra11vaccinefbrmulations．

CONCLUS10NS

Atwo－dosevaccinereglmenOfeither7．5FLgOr15iLgOf－hernagglutininantigen Withou［a句uvantindueedneutralizingantibodiesagainstdiverseH5NIviruSStrains inabigbpercentageoFsu句ects，SuggeStlng亡batthisrnavbeause知IH5Nlvaccine． （ClinicalTrials，gOVnumber，NCTOO349141．）

FromtheDepartmentoFGloba‡Research and Development】Baxter BjoS⊂－帥Ce （H．j．【．，G．8．，S．FリんL．－臥．N．V．．R．8．．B．G．P‥

E．ト九R．0．K．，P・N．B－），andtheDepartment O†⊂tini亡alpharma⊂Ology，Medi⊂∂1Uni－

Ve（Sityofvienna，ViennaGeneralHospi－ ta＝M．M・，CJ・）・・】・▼bothinVienna；⊂hangl

Genera＝」ospitat（H・M．L・0．）andtheNa－ tionatUniversityofSingaporeandNation－ a＝JniversityHospital（P・A・T・．P・F・）→－－al】 inSingapore；andtheUniversttyofsie＝a， Siena，舶‡y（E・M－）．Address reprint re－

queststoDr－Mt｝llerattheDepartmento（ ⊂linica】Pharmacology．Medi⊂at Univeト SityofVienna．ViennaGenerat Hosp■ta霊 （AfH），W紬ringerGt・rte118－20．1O90Vi－

enaa，Austria，Or at markus，mue＝即＠ meduniwien＿a⊂．at，

Drs・Ehr＝chandM白＝即COntributedequaト tytothisarti⊂te・

N【ngtjMedZOO3；35g：2573一旦4・

⊂opy廟t◎2α鳩肌】S8√た〟iC拝5M‘d血f∫ock吋・

257∋ N ENGLJM亡Dう58；2ヰ WWW・Nり軋ORG jUNElコ．2008

Downloadedfrornwww．nqm．orgatBAYER－YAKUHINCO．，OnAugust17．2008． Copydght◎2008MassachusettsMedicalSociety．A］川ghtsreserved．

189

T■たどNEWl三NGLÅがDJOUliNALげ川El）1Cl三ヾE

agesof18and45yearsinarandomized，partially blinded（betweengroups）clinicaltrialatthree Sites：OneinAustriaandtwoinSingapore．The Studywasdesignedbyitssponsor，Baxter．Data WereeOlleetedbytheinvestlgatOrSandwereheld and analyzed by Baxter．The manuserlptWaS Writtenbyasubgroupofindustryandacademic authors；allauthorscontribu【edtothecontent， had fh11access to the data，andvouch fbrthe COmpletenessandaccuracyofthedataandda【a analysis．

Theapproprlatelocalreviewboardsandethics COmmitteesapprovedtheprotocolfbrthestudy， WhichwasconductedineompliancewithGood ClinicalPracticeguidelinesandtheprovisionsoF theDeclarationofHelsinki．Thestudyinvestiga－ torswereunawareofassignmentstostudygroups． （Fordetailsofthestudydesign，SeetheSupple－ mentaryAppendix，aVailablewiththefu11textof thisarticleatwww．nqim．org．）

Theo切eetivewastoidentifytheimmunoge－ nicityandsa丘q，Ofvariousdosesorinactivated H5NIwhole－virusvaccinein氏）rmulations with andwithouta4iuvant．TheprlmarylmmunOge－ nieiryoutcomewasthenumberof’subjectswith hemagglutinationrinhibidonandneutralizinganti－ bodiestothevaccinestrain（A／Vietnam11203I2004）

21daysafterthefirs［andseconddosesofvac－ Cine．TheprlmarySafさtvou亡COmeWaSanySyS－ temic reactionafterthefirstand second doses．

V▲C⊂lNE

Tbe monovalent avianinnuenza H5NIwho王e一

viruSVaCCine（BaxtezうWaS琶rOducedwiththewi1d－

typestrainAIVietnamI1203I2004，Whichwasob－ tained丘omtheCentersfbrDiseaseControland

Prevemionand叩aSinactivatedwithformalinand ultravioletlight．Thevaccinewasmanufactured inVerocellcultureinanenhancedRSし3hcility 匝Srequiredfbrwild－typeH5NIvirus），aSde－ SCribedpreviously．9

R▲NDOMほATtON▲NDFOLLOW・UP

Su句ectswereeligibletopartlCIPateiftheywere Clinica11yhealthy，understoodthestudyproce－ dures，prOVidedwritteninfbrmedconsent，and agreedtokeepadai＆recordofsymptoms・Wom－ enwererequiredtohaveanegativepregzlanCy testatscreenlngandbeforeeachvaccination・

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HE EMERGENCE OF A NEW HU」ⅥANIN－ fluenzapandemiccausedbyanavianviruS

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ableearlyenoughattheonsetoFapandemicto haveam毎o