Effectiveness of rotavirus vaccine against hospitalized rotavirus diarrhea: a case-control study

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ContentslistsavailableatScienceDirect

Vaccine

jou rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Effectiveness

of

rotavirus

vaccine

against

hospitalized

rotavirus

diarrhea:

A

case–control

study

Maria

Y.T.

Ichihara

a,∗

_,

_Laura

_C.

_Rodrigues

b

_,

_Carlos

_A.S.

_Teles

_Santos

c

_,

Maria

da

Gloria

L.C.

Teixeira

a

_,

_Sandra

_R.

_De

_Jesus

d

_,

_Sheila

_M.

_Alvim

_De

_Matos

a

_,

Jose

P. Gagliardi

Leite

e

_,

_Mauricio

_L.

_Barreto

a

a_Institute_of_Collective_Health,_Federal_University_of_Bahia,_Rua_Basílio_da_Gama,_s/n,_Campus_{Universitário}_do_Canela,_CEP_40110-040,_Salvador,_Bahia,

Brazil

b_Department_of_Infectious_Disease_{Epidemiology,}_London_School_of_Hygiene_and_Tropical_Medicine,_Keppel_Street,_WC1H_7HT_London,_United_Kingdom

c_Department_of_Exact_Sciences,_University_of_the_State_of_Feira_de_Santana,_Avenida_{Transnordestina,}_Novo_Horizonte,_CEP_44036-900,_Feira_de_Santana,

Bahia,Brazil

d_Institute_{Multidisciplinar}_of_Health,_Campus_Anísio_Teixeira,_Federal_University_of_Bahia,_Vitoria_da_Conquista,_Bahia,_Brazil

e_Avenida_Brasil,_4365-Pav._Hélio_&_Peggy_Pereira,_CEP_21040-360,_Rio_de_Janeiro,_Manguinhos,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20September2013

Receivedinrevisedform

13December2013

Accepted2January2014

Availableonline5February2014

Keywords:

Rotavirusdiarrhea

Vaccineeffectiveness

Acutediarrheahospitalization

Childdiarrhea

Vaccine Hospitalization

a

b

s

t

r

a

c

t

Rotavirusisoneoftheleadingcauseofhospitalizationandoutpatientsvisitsamongchildrenunder ﬁveyears.Thisstudyevaluatedoverallandgenotype-speciﬁcvaccineeffectivenessoforalmonovalent rotavirusvaccine(G1P[8]strain)inpreventinghospitaladmissionofBrazilianchildrenwithrotavirus acutediarrhea.

Ahospitalbasedcase–controlstudywasconductedinﬁveRegionsofBrazilusingtheNationalRotavirus AcuteDiarrheaSurveillanceSystemfromJuly2008toAugust2011.Atotalof215cases(aged4–24 months)admittedwithconﬁrmedrotavirusdiarrheawererecruitedand1961controlshospitalized withoutdiarrheawerefrequencymatchedbysexandagegrouptocases.

Two-doseadjustedvaccineeffectiveness(adjustedbyyearofbirthandthefrequencymatching vari-ables)was76%(95%CI:58–86)lastingfortwoyears.Effectivenesscontrolledbytheavailablepotential confounderswas72%(95%CI:44–85),suggestingnoappreciableconfoundingbythosefactorsforwhich adjustmentwasmade.InahalfofthecasestherotavirusgenotypewasG2P[4]andin15%G1P[8]. Genotype-speciﬁcVE(twodoses)was89%(95%CI:78–95),forG1P[8]and76%(95%CI:64–84)forG2P[4]. ForallG1,itwas74%(95%CI:35–90),forallG2,76%(95%CI:63–84),andforallnonG1/G2genotypes, 63%(95%CI:−27–99).Effectivenessforonedosewas62%(95%CI:39–97).

Effectiveness of two-dose monovalent rotavirus vaccine in preventing hospital admission with rotavirusdiarrheawashigh,lastedfortwoyearsanditwassimilaragainstbothG1P[8]andG2P[4].Based ontheﬁndingsofthestudywerecommendthecontinueduseofrotavirusintheBrazilianNational Immu-nizationProgramandthemonitoringoftheearlyemergenceofunusualandnovelrotavirusgenotypes.

∗ Correspondingauthorat:RuaBasíliodaGama,s/n,4◦_._Andar,_Campus

Univer-sitáriodoCanela,Canela–CEP40110-040,Salvador,Bahia,Brazil.

Tel.:+557132837445;fax:+557133360695;mobile:+557188453456.

E-mailaddresses:my.ichihara@gmail.com(M.Y.T.Ichihara),

laura.rodrigues@lshtm.ac.uk(L.C.Rodrigues),carlosateles@yahoo.com.br

(C.A.S.TelesSantos),magloria@ufba.br(M.d.G.L.C.Teixeira),sandrego@hotmail.com

(S.R.DeJesus),sheilaalvim@hotmail.com(S.M.AlvimDeMatos),

jpgleite@gmail.com(J.P.GagliardiLeite),mauricio@ufba.br(M.L.Barreto).

1. Introduction

Acutediarrhea(AD)isafrequentcauseofchildhospitalization andoutpatientvisitsinchildrenunder5years[1].InBrazil,before introductionoftherotavirusvaccinein2006,about120.000 hos-pitalizationsayearoccurredduetoADinchildrenunderﬁveyears (DATASUS/MinistryofHealthofBrazil,2006).

Rotavirusistheleadingcauseofsevereacutediarrheain chil-drenindevelopedandindevelopingcountriesandisthemajor causeofdeathinpoorcountries[2,3].Sevengroupsofrotavirus havebeenidentiﬁed(AtoG)andgroupA(RV-A)isresponsiblefor morethan90%ofhumanrotavirusinfections[4].RV-Ahasgreat geneticdiversityduealmost60serotypes(GandP)andthemost

http://dx.doi.org/10.1016/j.vaccine.2014.01.007

Open access under CC BY-NC-SA license.

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commonstrainsare:G1P[8],G2P[4],G3P[8],G4P[8]andG9P[8][5]. InBrazil,between12%and42%ofchildrenunder5yearswith diar-rheahadpositivestoolsamplesforRV-Abeforetheintroduction oftheRV-Avaccine.Thisincreasedfrom22%to38%inchildren hospitalizedfor AD[6,7]. Morethan 51 genotypecombinations werereportedandthemostcommongenotypesdescribedwere G1P[8],G9P[8]andG2P[4][8].

Vaccinationisthebettermeasuretopreventrotavirus[1,2,9] anditsadoptionhasbeenrecommendedbyWorldHealth Organi-zation[10].AnattenuatedmonovalenthumanRV-A(G1P[8]strain; Rotarix®)andapentavalentbovine-humanreassortant(G1,G2,G3, G4andP[8]strains;RotaTeq®)arelicensedworldwide.Rotarix® wasintroducedintheBrazilianNationalImmunizationProgram (BNIP)in2006inatwo-dosescheduleat2and4monthsofageand co-administered withtetravalent, pneumococcaland poliovirus vaccines.

RV-AvaccineefﬁcacyagainstsevereRV-AADvariedbetween morethan90%EuropeandAsia,85%inLatinAmerica,72%inSouth Africato49%inMalawi[11–14].Threecase–controlstudies car-riedoutinahighincomecountry(Belgium)[15]and inlowto middle-incomecountries(ElSalvadorandBolivia)[16,17]founda two-dosevaccineeffectivenessof90%;76%and77%respectively and a one-doseeffectiveness of91%; 51%and 56% respectively againsthospitalizationbyRV-AAD.InBrazil,twosmallcase con-trolsstudiesshowedarangeof40–85%effectivenessinpreventing hospitalization caused by G2P[4] [18,19]. Thereason for varia-tion in vaccineprotection is not clearand has beenattributed toantigendiversity,malnutritionand higherincidenceofother entericpathogens[20].Thereisstrongsuggestionofcross protec-tionamonggenotypes[11–14].

TheintroductionofRV-Avaccinationwasfollowedbya reduc-tioninchildhospitalizationdue toallcausesofADinBrazil,El SalvadorandMexicorangingfrom17to51%[21–23]anda reduc-tioninmortalityfromADinchildrenunder5yearsinBrazilof22% andinMexicoof41%[24].

Thisstudywill evaluatetheoveralleffectiveness of theoral monovalentvaccine,usedinroutinehealthservices,inpreventing BrazilianchildhospitalizationwithRV-AAD.Itwillalsoevaluate overallandgenotype-speciﬁcVEbytimesinceseconddose vacci-nation(uptotwoyears),andgenotype-speciﬁcVE.

2. Methods 2.1. Studydesign

This was a hospital based case–control study, frequency-matchedbysexandagegroup.Hospitalsweregeneralhospitals whichreceivedchildrenwithalargerangeofdiseasescomingfrom asimilargeographicalcatchmentarea.Seventeenofthehospitals enrolledintheRV-AADNationalSurveillanceSystemwereinvited toparticipateinthestudy,basedonhavinghadalargenumberof RV-Apositivesamplesin2007,adequateleveloforganizationof theunitanddataaccessibility.Afterconsultationandagreement onlogisticalarrangementswiththeFederalHealthSurveillance (SVS/MS),theepidemiologicalsurveillanceofthehospitalsandof thestates,theCentralPublicHealthandNationalReference Labo-ratories,10hospitalslocatedinﬁvemacro-regionsofBrazil(6state capitalcitiesand4municipalities)wereselected.

3. Participants 3.1. Eligiblechildren

Childrenwereeligibletoparticipateinthestudyiftheywere admitted in the study hospitals, were aged 4 to 24 months (andthereforeoldenoughtohavereceivedtheirseconddoseof rotavirusvaccine)and didnothave diarrheauptothreeweeks beforeadmissionor duringhospitalization. Alleligible children

werelistedandscreenedtoexcludechildrenwhohadanyhealth conditionpresumedtoreducevaccineeffectiveness (immunodefi-ciency,gastrointestinaldisease(e.g.diverticulitis),malformations orneoplasmconditionsrelatedtovaccineeffectiveness,general signsandsymptoms,infectiousandparasiticdiseases),thosewho hadreceivedtheseconddoseofvaccineinthe15daysbefore hospi-talization,orwhosevaccinationdidnotfollowtheBNIPschedule. Allthatfulfilledthespecificcriteriaforeithereffective’scaseor controlwereincluded.Thisaimedtoselectcontrolsfromthe pop-ulationthatproducedthecases,ascaseshospitalizedbyADorby otherdiseaseswerelikelytocomefromthesamepopulationgiven theuniversalhealthcaresysteminBrazil.

3.2. Potentialcasesandcontrols

Inclusioncriteriaforpotentialcaseswere:admissionwithAD (deﬁnedasthreeormoreliquidstoolsin24h,upto14daysbefore admission),stoolsamplewascollecteduntil48hafteradmission andpositiveforRV-Aandstayinhospitalforatleast24h.Children wereincludedinthestudyintheﬁrsthospitalizationonlyandhad noassociatedisease.

Inclusioncriteriaforcontrolswere:admissionfromthesame hospitals of the cases with respiratory, genitourinary, muscu-loskeletal,nervoussystems,skinandsubcutaneoustissue,earand mastoidprocesses,eyeandadnexadiseases,andexternalcauses. Controlswerenotincludediftheyhadaprevioushistoryof RV-Adiarrheaorhadavaccine-preventabledisease(aschildrenwho didnotreceiveonevaccinearemorelikelytonotreceiveother vaccines).

Allpotentialcontrolsfulﬁllingthecriteriaaboveundergonea furtherselectionforfrequencymatching,sothatthealleffective controlshadthesamedistributionofthemainconfounding vari-ables(sexandagegrouponadmission:4–6months;7–11months and12–24months)asthecases.Thisapproachaimedtoselectfrom thepoolofpotentialcontrols,aneffectivecontrolgroupwiththe samedistributionofconfoundersastheeffectivecases;inthe sit-uationinwhichmorecontrolsthanneededwereavailableinthe frequencymatchedgroupstheywereselectedatrandom.Random selectionoffrequencymatchedeffectivecontrolsfromthepoolof potentialcontrolswasdoneusingthe“sample”commandofthe Stataversion11.0

3.3. Effectivecasesandcontrols

Cases:Allpotentialcasesfulﬁllingthecriteriaaboveandhad stoolspositiveforrotavirusconﬁrmedbythereferencelaboratory wereincluded.

Controls:Allpotentialcontrolsfulﬁllingthecriteriaaboveand randomselectedforfrequencymatchingwereincluded.

Onestoolsamplewascollectedupto48hafteradmissionas partof theRV-A AD SurveillanceSystem.Samples werestored andtransportedtotheLACENsofeachStatewherethehospital waslocated,accordingtotheguidelinesof theGeneral Coordi-nationofPublicHealthLaboratories/MinistryofHealthofBrazil (CGLAB/SVS/MS).RV-AinvestigationwasdonebyEnzymeImmune Assay(EIA),usingcommercialkits,followingthemanufacture’ rec-ommendation(Dako®orOxoide®).

3.4. Laboratoryinvestigationofpotentialcases

AllpositivesamplesforRV-Aand25%ofnegativesampleswere senttoareferencelaboratory.AccordingtotheLACEN localiza-tion,thiswaseithertheNationalReferenceLaboratory(Evandro ChagasInstitute[Belém,PA],oraRegionalReferenceLaboratory (AdolfoLutzInstitute[SãoPaulo,SP],andOswaldoCruzInstitute [RiodeJaneiro,RJ]).ResultswereconﬁrmedbyEIAand polyac-rylamidegelelectrophoresis(PAGE)accordingtoLeiteetal.[25].

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Fecalsuspensionsand nucleicacidsextractionwerecarried out accordingtoLeiteet al.[25] andBoom etal.[26],respectively. TheRV-GenotypingwasconductedusingRT-PCRasdescribedby Dasetal.[27](“G”genotype)andGentschetal.[28](“P” geno-types).RV-Agenotypesweree-mailedtoCGLAB/SVS/MSandsent totheInstituteofCollectiveHealth, FederalUniversityofBahia (ISC/UFBa).

4. Datacollection

Informationfromcasesandcontrolswascollectedby interview-erswhovisitedallhospitalsdaily,fromJuly2008toAugust2011. Medicalrecordswerereviewedandthechild’scareranswereda standardquestionnaireonidentification,clinicalhistoryand evo-lution,socio-economicstatus,sanitation,feedingandnutritional statusofthechild,andmaternalreproductiveaspects.The vac-cinationstatusofthechildwasassessedthroughthevaccination card,askedforduringhospitalization.Also,datawereobtainedby homevisits,telephoneorthefamilyhealthteamoftheareaof resi-denceofthechild.Vaccinationstatuswasclassifiedaccordingtothe presenceandnumberofdosesandtimebetweenlastdoseand hos-pitalization.Weightatadmissionwastakenfromhospitalrecords anditsdeficitevaluatedaccordingtotheweight-agestandardsof theNationalCentreforHealthStatistics(NCHS)forboysandgirls [29].Mother’sskincolorwasselfreported.

Questionnairesforallpotentialcasesandcontrolsweresentto ISC/UFBaandreviewersconﬁrmedtheclassiﬁcationofcasesand controlsbyassessingtheinclusionandexclusioncriteria.

Tocomplementdataonmaternalreproductiveperiodandchild birthweconsultedlivebirthsroutinedata(SINASC)from7cities. Thissystemcovers80–90%of birthsinBrazil.Thechildageon admissionandonadministration ofﬁrst and seconddoses and breastfeedingdurationwerecalculatedindaysatthedateof admis-sion. Casesand controls were classiﬁedinto three age-groups, accordingtoage onadmission:4–6months, 7–11months and 12–24months.

5. Samplesize

Theminimumsamplesizerequired(usingEPI-INFO6.0)was88 casesand88controls(forvaccinecoverageof70%,VEof65%,95% conﬁdenceintervaland90%power.Theachievedsamplesizeof215 casesand1961controlsenabledestimationofgenotype-speciﬁc vaccineeffectiveness.

6. Statisticalanalysis

Vaccineeffectivenesswasobtainedbymultivariable uncondi-tionallogistic regression, which is appropriate when frequency matching is used. Theodds ratio wasadjusted for: a) sex and agebothusedforfrequency-matching,b)yearofbirth,tocontrol coverageofvaccinebyyearandc)robustvarianceestimationof Jackknife,withclustersbeinghospitals.Potentialconfounderswere includedintheﬁnallogisticmodelwhenthep-valueof associa-tionwas<0.20(bivariateanalysis).Weusedthebackwardmethod toanalyzethepresenceofconfounding.Thebestadjustmentwas givenby theAkaikeinformation criterion (AIC) [30].Given the absenceof confoundingby measuredvariables apparentin the analysisbynumberofdoses,thesubsequentanalysisbytimesince seconddosevaccination,genotype-speciﬁcwasconducted with-outcontrollingforconfoundersotherthanage,sex,yearofbirth, androbustvarianceestimationofJackknife.Thefrequencyof miss-ingvaluesforanyconfoundingvariablewasverylow(lessthan 1%),andtheywereattributedtothecategoryofreference (consid-erednotexposed)tokeepallcasesintheanalysis.Werepeatedthe

analysisstratiﬁedbyyearofadmissiontocontrolforincreasing vac-cinecoveragewithtime.Asensitivityanalysis(fortwodosesonly) wasdone,inwhichcasesandcontrolswithmissingvaccination cardsweretreatedasvaccinatedorunvaccinated,soassumingnon differentialmissingness.TheVEwascalculatedbythefollowing formula:VE=(1−oddsratioofvaccination)×100.Statistical anal-ysiswasperformedwithStataversion12.1(Copyright1985–2011 StataCorp).

Ethics:ThisstudywasapprovedbytheCommitteeofISC/UFBa (Protocol017-08/CEP/ISC-2008).Carersof participatingchildren signedawritteninformedconsentform.

7. Results

7.1. Studypopulation

Atotalof4955eligiblechildrenagedbetween4and24months wererecruitedintothestudyfromJuly2008toAugust2011.Of these,697childrendidnotfulfillthecriteriaofinclusionrelatedto informationonvaccination:268didnothaveavaccinecard;299 hadreceivedvaccinationinadifferentschedulefromthat recom-mendedbytheBNIP;and130hadreceivedtheseconddosefewer than15days beforeadmission. (Fig.1showsthebreakdownof exclusionsforeffectivecasesandcontrols).Inaddition,298 eligi-blechildrenwithADdidnotfulfillthecriteriaofinclusionrelatedto thestoolsamplecollection:in202astoolsamplewasnotcollected; in33thesampleswerelost,andin63thesamplewascollectedtoo longafteradmission.Samplesof965potentialcasesweretested forRV-Awiththefollowingresults:722werenegative(ofwhich 142hadanothervirusidentifiedand28werepositiveonthefirst testbutnegativeinthereferencelaboratory)and215werepositive forRV-AconfirmedbyEIAand/orPAGEandRT-PCR.Ofalleligible childrenforcontrols,191haddevelopeddiarrheaduring hospital-izationandwerenotselectedtothestudyand843werenotneeded giventhefrequencymatch.Atotalof215effectivecasesand1961 effectivecontrolswererecruited.

Characteristics of thestudy populationare presented inthe Supplementarytables(1a,1b,1c).Themeanageofthecasesand controlswas14months.Comparedtocontrols,caseshadlower socio-economicstatusandsanitarylevel,theirmothershadfewer yearsofschoolingandtheirfamilieslivedinsmallerhouseswith manyfamily membersand more thanonechild under5 years. Smokingandalcoholconsumptionduringpregnancyanddelayed startofprenatalcareweresigniﬁcantlyhigheramongcases.Also, oneormorevisitstohealthservicesorhospitalizationsdueto diar-rheabeforethecurrentadmissionweremorefrequentincasesthan controls.Therewasahigherproportionofcontrolswhowerenever exclusivelybreastfed(12.1%)comparedwithcases(7.4%).

Theuseofvaccinebetweencasesandcontrolswassigniﬁcantly different:31.2% (67)cases werenot vaccinated compared with 10.3%(201)ofcontrols,whereas53.5%(115)ofthecasesand75.5% (1481)ofthecontrolshadreceivedtwodosesofvaccine.

OfthechildrenuptotwoyearsadmittedtohospitalwithAD, 22.3%wereRV-Apositiveand156(73%)weregenotyped.The dis-tributionof RV-AGand Pgenotypes ispresented inFig.2 and SupplementaryTable2GandPgenotypeswereidentiﬁedin135 (63.3%)ofallRV-Apositivesamples(n=215),andonly“G”or“P” typesin 21samples.Therewasapredominanceof theG2P[4] genotype(51.3%,n=80)followedbyG1P[8](15.4%,n=24).Ofall observedgenotypes,G2wasfoundin57%(n=89)andG1in23% (n=36).The othergenotypescharacterizedwere: mixedgroups (n=14),G9(n=6),G3(n=3),andunusualstrainssuchasG12(n=2) andGroupC(n=1).Mixedinfectionsandunusualgenotypeswere identiﬁedin10.9%oftheRV-Apositivesamples.

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Fig.1.Studypopulation.

Fig.2.GenotypescirculatinginBrazilfrom2008to2011.

8. Vaccineeffectiveness

Thetwo-doseadjustedVE(adjustedforyearofbirthandthe frequencymatchingvariables)was76%(95%CI:58–86)(Table1).

Effectiveness controlled by the available potential confounders was very similar (72%, 95%CI: 44–85), suggesting no apprecia-ble confounding by those factors for which adjustment was made.

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Table1

EffectivenessoforalmonovalentrotavirusvaccineinpreventinghospitaladmissioninBrazilianchildrenwithrotavirusacutediarrheabynumberofdosesreceived.

Vaccinationby Case Control ORa _VEa _ORadjb _VEb

Numberofdoses n n (95%CI) (95%CI) (95%CI) (95%CI)

Unvaccinated 67 201 1 – 1 –

Fullyvaccinated(twodoses) 115 1481 0.24(0.14–0.42) 76(58–86) 0.28(0.15–0.56) 72(44–85)

Partiallyvaccinated(onedose) 33 279 0.38(0.23–0.61) 62(39–77) 0.40(0.25–0.63) 60(37–75)

AICc _1318.253 _1194.454

a_Odds_ratio_adjusted_by_year_of_birth_and_variables_(sex_and_age_group)_used_for_frequency_matching,_and_robust_variance_estimation_of_Jaccknife,_where_the_clusters_were

thehospitals.Thevaccineeffectiveness(VE)wascalculatedby(1−OR)×100%.

b _Odds_ratio_adjusted_by_year_of_birth_and_variables_(sex_and_age_group)_of_frequency_matching_and_confounders_(mother’s_schooling,_mother’s_absence_from_home,_smoking

duringpregnancy,typeandregularityofwatersupply,numberofADhospitalizationsbeforecurrentadmissionandexclusivebreastfeeding)androbustvarianceestimation

ofJaccknife,wheretheclusterswerethehospitals.c_Akaike_information_criteria_for_measuring_the_goodness_of_ﬁt_of_the_statistic_model.

Weexcludedasimilarproportionofcases(5.7%)andcontrols (5.3%)becausetheydidnothavecards.Sensitivityanalysisshowed thatiftheywereincludedasvaccinated,VE(twodoses)wouldbe 66%(95%CI:42–80)andifincludedasunvaccinatedVEwouldbe 74%(95%CI:53–86).

TheVE(adjustedforyearofbirthandthefrequencymatching variables)foronedosewas62%(95%CI:39–97)andonedoseVE adjustedforotherpotentialconfounderswas60%(95%CI:37–75). Table2showsthatVEwassimilarinthosewithtimesince sec-onddosevaccinationuntilhospitalization stratifiedbyoneyear (71%;95%CI:54–82)andtwoyears(78%;95%CI:52–90).TheVE forG1P[8]andG2P[4]bytimesinceseconddosevaccinationwas marginallyhigherforG1P[8](90%;95%CI:-0.92–-100foroneyear and89%;95%CI:0.01–-99fortwoyears)thanG2P[4](77%;95%CI: 57–88foroneyearand75%;95%CI:56–86fortwoyears)significant. Table3presentsgenotype-specificVEbynumberofdoses.VE (twodoses)was89%(95%CI:78–95)forG1P[8];76%(95%CI:64–84) forG2P[4];74%(95%CI:35–90)forallG1;76%(95%CI:63–84)for allG2and63%(95%IC:−27–99)forallthenonG1/G2genotypes.

EstimatedVEremainedverysimilarwhenanalysiswas strati-ﬁedbyyearofadmissionsuggestingthatVEdidnotchangewith increasingvaccinecoverage(datanotpresented).

9. Discussion

Two-doseVEwas76%(95%CI:44–85),inspiteofthegreat diver-sityofrotavirusgenotypescirculatinginBrazilandapredominance ofG2P[4]genotype(51.3%).Wefounda10.9%mixedandunusual genotypesas expected in developing countries[31,32]. The VE

lastedfortwoyearsafterseconddosevaccinationanditwashigher forG1P[8]thanG2P[4].

VariationofRV-Avaccineefficacyandeffectivenesshavebeen reportedintheliterature: efficacywashigherinEurope(96.4% againstRV-AsevereAD)[11]thaninalowincomecountry(Malawi, 49.2%againstalldiarrheaand57.5%againsthospitalizeddiarrhea) [13]andincountrieswithhighmortality(63%)[33].Inthemiddle incomecountriesofLatinAmerica[12],efficacywas84.8%against severeAD;inSouthAfricaitwas72.2%againstalldiarrhea[13].This studyshowedsimilareffectivenesstothatfoundinElSalvador[16] andBolivia[17](73%and76%forseverediarrhea)andinasmaller studyinBrazil[18](75.8%againsthospitalizeddiarrhea),butlower thaninBelgium(90%)[15].

Two-doseVEremainedhighfortwoyears.Thisissimilartoother countrieswithlowmortality;butdifferentfromsomecountries withhighmortalitywhereVEdecreasesinthesecondyearafter vaccination[5].ArecentstudyinNicaraguaalsofoundno wan-ing for thepentavalent vaccinein children aged12 monthsor morewithverysevereAD[34].Otherreasonsfortheﬁndingthat effectivenessdidnotdecreaseinthesecondyearinourstudyare: weexploredVEfromtimesinceseconddosevaccinewhilemost countriesestimatedVEbytimesincebirth;andweestimatedVE againstseverecasesonly.Besides,declinesobservedinother stud-iescouldberelatedtothesmallnumberstoestimateeffectiveness inthesecondyearoflife[35].Thereisnoagreementastothe rea-sonsforthevariationinVEandindurationofVEintheliterature. ThefactthateffectivenessinBrazilwassimilartoothermiddle incomecountriesintermsofoverallprotectionagainst hospital-izedADandsimilartoEuropeancountriesinrelationtowaning mighthelptoadvanceinthisexploration.

Table2

EffectivenessoforalmonovalentrotavirusvaccineinpreventinghospitaladmissioninBrazilianchildrenwithrotavirusacutediarrhea(overallandgenotype-speciﬁc)

stratiﬁedbytimefromreceivingtheseconddoseofvaccineuntilhospitaladmission.

Timesincethe Case Control ORa _VEa

Seconddose n n (95%CI) (95%CI)

Overall Unvaccinated 67 201 1 – <1year 71 938 0.26(0.16–0.42) 74(58–84) 1to2years 44 543 0.22(0.10–0.46) 78(54–90) AICb_(1114.384) G1P[8] Unvaccinated 9 201 1 – <1year 4 938 0.10(0.005–1.92) 90(-0.92–100) 1to2years 3 543 0.11(0.01–99) 89(0.01–99) AICb_(168.4906) G2P[4] Unvaccinated 25 201 1 – <1year 22 938 0.23(0.12–0.43) 77(57–88) 1to2years 19 543 0.25(0.14–0.44) 75(56–86) AICb_(533.5553)

a_Odds_Ratio_adjusted_by_year_of_birth_and_variables_(sex_and_age_group)_used_for_frequency_matching,_and_robust_variance_estimation_of_Jaccknife,_where_the_clusters_were

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Table3

Genotype-speciﬁcvaccineeffectivenessoforalmonovalentrotavirusvaccinebynumberofdosesreceived.

Vaccinationby Case Control ORa _VEa

Rotavirusgenotype n n (95%CI) (95%CI)

G1P[8]

Unvaccinated 9 201 1 –

Fullyvaccinated(twodoses) 7 1481 0.11(0.05–0.22) 89(78–95)

Partiallyvaccinated(onedose) 8 279 0.69(0.30–1.57) 31(−57–70)

AICb_(236.815)

G2P[4]

Partiallyvaccinated(onedose) 14 279 0.43(0.22–0.85) 57(15–78)

AICb_(641.170)

G1

Partiallyvaccinated(onedose) 8 279 0.62(0.22–1.79) 38(−79–78)

AICb_(350.510)

G2

Partiallyvaccinated(onedose) 17 279 0.44(0.26–0.74) 56(26–74)

AICb_(743.862)

NonG1/G2

Fullyvaccinated(twodoses) 6 1481 0.37(0.11–1.27) 63(−27–99)

Partiallyvaccinated(onedose) 2 279 0.47(0.72–3.09) 53(−2.09–28)

AICb_(137.232)

a_Odds_ratio_adjusted_by_year_of_birth_and_variables_(sex_and_age_group)_used_for_frequency_matching,_and_robust_variance_estimation_of_Jaccknife,_where_the_clusters_were

b_Akaike_information_criteria_for_measuring_the_goodness_of_ﬁt_of_the_statistic_model.

Asingledoseofferedsomeprotection,consistentwiththe lit-erature(althoughtheVEwashigherthaninElSalvador[16]and Bolivia[17]andlowerthaninBelgium(91%))[15].

Thegoodeffectivenessidentiﬁedisconsistentwiththe reduc-tionintherateofchildhospitalizationandmortalitybyADinBrazil followingtheintroductionofvaccineinBrazil[21].

Genotype-speciﬁcVE washighforG1P[8] (89%)and slightly lower for G2P[4] (76%) indicating a degree of cross protec-tion.Animalmodelsshown thatimmunitytogroupArotavirus (RVA) present homotypic and heterotypic components. Repeat RVAinfectionsacquirednaturallyorbyvaccination,increase pro-tectiveimmunitytoincludemultipleserotypes,as indicatedby developmentofcross-neutralizingantibodiesandcross-reactive epitope-blockingantibodiesspeciﬁcforVP7andVP4antigens.In thehumanvaccineclinicaltrials (monovalent,Rotarix®; penta-valent,RotaTeq®)aswellasinthefollow-upstudies,bothvaccines presented homotypic as well as heterotypic protection against differentRVAgenotypes,includingG2P[4]andG9P[8]genotypes [12,19,36,37].

GenotypespeciﬁcVEalsoremainedhighinthesecondyear,in contrastwiththeﬁndingsformiddleincomecountries.VEwas74% forallG1types,76%forallG2typesandlowerforthenonG1/G2 type(63%),althoughnumbersweresmall.TheresultofVEagainst G2P[4]issimilartothetwosmallstudiescarriedoutinBrazil(75.4% to77%toG2P[4])butunlikethem,effectivenessagainstbothG1P[8] andG2P[4]didnotfallinthesecondyear[18,19].

Thereisadiscussionastowhethervaccineuseleadstoserotype replacement[19].ThehigheffectivenessagainstbothG1P[8]and G2P[4]suggeststhatthepredominanceofG2P[4]ismostlikelya cyclicalpatternofrotavirusstrainsoccurrenceinBrazilas previ-ouslyreported[38,39].

Thisstudyavoidedthepossibilityofartiﬁciallyreducing effec-tivenessbyusingcontrolswithoutdiarrhearatherthancontrols withdiarrheaand(potentialfalse)norotavirusinstool.UsingEIA, PAGEandRT-PCRweconﬁrmedthatallcasesweretruecasesof RV-A.

Thedatacollectionstrategyallowedustoobtainindividualdata, tocontrolforpossibleconfoundingandverifyinteractionsin over-allVE.Aftercontrollingforsevenvariables,noconfoundingwas identiﬁed.

Wewereunabletoinvestigateeitherifeffectivenessdeclines aftertwoyearsofseconddosevaccineorwhetherthereisan inter-actionwithoralpoliovirusvaccineasthetwovaccinesaregiven atthesametime.Weassumednondifferentialmissingnessinthe sensitivityanalysis.Althoughthiswasacasecontrolstudyrecall biasisnotrelevantbecausewedidnotrelyonrecallof vaccina-tion;weusedarecord(vaccinecard)forestablishmentofthemain exposure.

Only73%ofgenotypesoftheRV-Apositivesamplewere iden-tiﬁed.Thiscouldhidethecirculationofothergenotypes,although, wewereabletoestimategenotype-speciﬁcVEforthemost com-moncirculatingstrains.

Inconclusion,weshowedconsistenteffectivenessoftwo-dose oral monovalent vaccine in preventing hospital admissions of BrazilianchildrenwithRV-AAD,closertoEuropeanthanAfricaVE. ProtectionlastedfortwoyearsanditwassimilaragainstG1P[8]and G2P[4]andslightlyloweragainstnonG1/G2.Theﬁrstdosealready conferredsomeprotection.

Theﬁndingsofthestudysupportsthecontinueduseofrotavirus intheBrazilianNationalImmunizationProgramandthe monitor-ingforearlydetectionofemergenceofunusualandnovelrotavirus genotypes.

Sincethis vaccine(whichrequires onlytwodosesandis co-administeredwithothervaccines)providesadequateprotection, thebeneﬁtsofachangetoamultivalentvaccinerequiringthree dosesmightarequestionable:thismaynotincreaseprotectionand leadtoincompletevaccinationschemes.

It might be useful to conduct cost-effectiveness stud-ies to inform national immunization policy. In addition, other effectiveness studies should investigate what is behind the observed variation in monovalent rotavirus vaccine VE.

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Finally,itisimportanttoidentifyearlyemergenceofunusual andnovelrotavirusgenotypessothatthevaccineeffectivenesscan beveriﬁed.

Conﬂictofintereststatement

Allauthorsconfirmthattherearenoknownconflictsofinterest associatedwiththispublicationandtherehasbeennosignificant financialsupportforthisworkthatcouldinfluenceitsoutcome. Contributors

MYTIdesignedthestudy,managedthefieldwork,analyzedand interpretedthedataandwrotethepaper.LCRcontributedtothe analysisandtheinterpretationofthedataandwroteup.CASTS contributedtoanalysisandinterpretationofthedata;MdaGLCT contributedtointerpretationofthedata;SRdidtheinitial analy-sisofthedata;SMAMcontributedtopreparethedatatoanalysis; JPGLcontributedwiththedesignofthestudyandinterpretation ofthedata;MLBcontributedwiththedesignofthestudy, analy-sisandinterpretationofthedata.Alltheauthorscontributedto editthepaper.Themanuscript hasbeenread and approvedby allnamedauthorsandthattherearenootherpersonswho sat-isfiedthecriteriaforauthorshipbut arenotlisted.Theorderof authorslistedinthemanuscripthasbeenapprovedbyallofus. Allauthorshavegivendueconsiderationtotheprotectionof intel-lectualpropertyassociatedwiththisworkandthatthereareno impedimentstopublication,includingthetimingofpublication, withrespecttointellectualproperty.Insodoingtheauthors con-firmthattheyhavefollowedtheregulationsoftheirinstitutions concerningintellectualproperty.

Ethicalapproval

This study was approved by the Committee of Institute of Collective Health, Federal University of Bahia (Protocol 017-08/CEP/ISC-2008), by four local ethics committees. Consent to participatewasobtainedfromallthehospitals.Carersof partici-patingchildrensignedwrittenaninformedconsentform. Funding

ThisworkwassupportedbyHealthSurveillanceofMinistryof HealthofBrazilwhocollaboratedinrecruitmentofsitesbutno roleinstudydesign,incollection,analysis,interpretationofdata, inthewritingofthereportorinthedecisionofsubmitthearticle forpublication.

Acknowledgments

WerecognizethecontributionoftheROTAVACGroupwhich includesalltheprofessionalsenrolledintherotavirusAD Surveil-lanceSystem whoparticipated inthe conduction ofthe study: Alessandra Araújo Siqueira, GreiceMadeleine, Rejane Maria de SouzaAlves,VivianeMartins,MarliCosta,ErnaniRenoir,Eduardo doCarmoHage(HealthSurveillanceofMinistryofHealth,Brasilia, Brazil); Alexandre Madi Fialho, Rosane Santos Maria de Assis (RegionalReferenceLaboratory,FIOCRUZ,RiodeJaneiro,Brazil); RitaCássiaCompagnoliCarmona(RegionalReferenceLaboratory, AdolfoLutz,SaoPaulo,Brazil);JoanaD’ArcPereiraMascarenhas, LuanadaSilvaSoares(NationalReferenceLaboratory/Evandro Cha-gas,Belém,Brazil);AcáciaPerolina ResendeSetton,Adelaideda SilvaNascimento,AnaGabrieladeAndradeCarreira,ÂngelaMaria RodriguesFerreira,FabíulaMariadeAlmeidadeHolandaTormenta, JaneteXavierdosSantos,TeoníliaLoulaDourado,MaraEspíndola

CardosoAraújo,MarcoAureliodeOliveiraGoes,MariaElisaPaula deOliveira,MaríliaReicheltBarbosa,MariaCristinaToledoCoelho, SandraCristinaDeboni(ADSurveillanceSystemoftheStatesand Municipalities,Brazil);IvanaR.S.Varella,EleniceBrandãoCunha, EmersonHenklainFerruzz,MaríciadeMacedoMoryKuroki,Maria deFátimaRezendeDóriaPinto,MariaRoseildaB.BarretodaSilva, NiedjaMaria de OliveiraCosta MedeirosNetto,Sandra Mendes deMagalhãesdaSilva(TechniciansofCore Surveillanceof hos-pitals,Brazil);AnaAcáciaPereira,AuxiliadoraNovais,LinaMaria Matos,LeonardoBertollo,MariadoRosárioMachadoLisboaLuna, Raimunda Carvalho,Renilda BetoneMoura,BrenoBrito, Sandra MariaAraújoMenezesCavalcante(PublicHealthCentral Labora-toriesoftheStates).

AppendixA. Supplementarydata

Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.vaccine. 2014.01.007.

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