Citation
Epidemiology and Transmission Dynamics of Arbovirus in Latin America

Material Information

Title:
Epidemiology and Transmission Dynamics of Arbovirus in Latin America
Creator:
Rojas Alvarez, Diana Patricia
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Epidemiology
Committee Chair:
COOK,ROBERT L
Committee Co-Chair:
LONGINI,IRA M
Committee Members:
PRINS,CINDY A
YANG,YANG
HALLORAN,M ELIZABETH

Subjects

Subjects / Keywords:
arbovirus
epidemiology

Notes

General Note:
Arthropod-borne viruses (arboviruses) are considered global public health priorities. Arbovirus are considered the cause of some of the most important emerging infectious diseases currently threatening the globe. Approximately three billion people worldwide live in areas infested with Aedes mosquitoes and are at risk for arbovirus infections like dengue virus, chikungunya virus and Zika virus. Dengue virus has been circulating since the 1980s in Latin America and its incidence has increased significantly worldwide and in this region. Aedes aegypti is the main vector for most of the arbovirus transmitted in Latin America. In the last three years two arbovirus had emerge in the Western Hemisphere causing important outbreaks. In 2013, Chikungunya virus emerged in the Caribbean and spread rapidly through the Caribbean, Central and South America with 1.8 million cases reported. Similarly in 2015 Zika Virus was isolated in Brazil and since then it spread in areas infested with Aedes in Latin America and the Caribbean and this time also with authoctonous transmission in the United States. The aim of this dissertation is to understand better the epidemiology and transmission dynamics of Arbovirus in Latin America. Three studies were done: 1) To design an epidemiological study to understand the full burden and the dynamics of DENV and other arbovirus in Yucatan, Mexico; 2) Understand the epidemiology, transmission dynamics and risk factors associated with dengue seroprevalence in Yucatan, Mexico; 3)Characterize the epidemiology and transmissibility of ZIKV in Colombia using surveillance data to define the epidemiological features of local ZIKV outbreaks, to estimate corresponding transmission parameters and potential risk factors associated with ZIKV transmission. Understanding the epidemiology of ZIKV with other arboviruses (DENV and CHIKV) is important for Aedes infested areas. It is needed to characterize symptoms, potential risk factors for severe disease, and predict long term dynamics. This study offers a unique perspective on the epidemiology of arbovirus in Latin America in unique timeframe where for the first time three arbovirus are co-circulating in this region.

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Source Institution:
UFRGP
Rights Management:
All applicable rights reserved by the source institution and holding location.
Embargo Date:
8/31/2018

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EPIDEMIOLOGYANDTRANSMISSIONDYNAMICSOFARBOVIRUSINLATINAMERICAByDIANAP.ROJASALVAREZADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOLOFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENTOFTHEREQUIREMENTSFORTHEDEGREEOFDOCTOROFPHILOSOPHYUNIVERSITYOFFLORIDA2017

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c2017DianaP.RojasAlvarez

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Idedicatethisworktomyfamily,speciallytomysupportinghusbandanddaugtherfortheirunconditionalsupport,loveandpatiencethroughalltheseyears

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ACKNOWLEDGMENTSFirstly,IwouldliketoexpressmysinceregratitudetomyadvisorProf.IraM.LonginiforthecontinuoussupportduringmyPh.Dstudyandrelatedresearch,forhispatience,motivation,andimmenseknowledge.Hisguidancehelpedmeinallthetimeofresearchandwritingofthisthesis.Besidesmyadvisor,Iwouldliketothanktherestofmydissertationcommittee:Dr.RobertCook,Dr.CindyPrins,Dr.YangYang,andProf.M.ElizabethHalloran,fortheirinsightfulcommentsandencouragement,butalsoforthehardquestionwhichintendedmetowidenmyresearchfromvariousperspectives.Withouttheirprecioussupportitwouldnotbepossibletoconductthisresearch.IwouldalsowanttothanktotheUniversidadAutonomadeYucatanandtheFamiliassindengueeldteam,speciallytoHectorGomezDantes,NormaPaviaRuz,PabloManriqueSaide,andGloriaBarreraforallthehardeldworkinYucatanthatmadepossiblethisdissertation.IthanktheFulbright-ColcienciasprogramforthefundingreceivedtopursuemyPhDtraining.AlsoIthankmyfellowlabmatesinforthediscussions,thesleeplessnightswewereworkingtogetherbeforedeadlines,andforallthefunwehavehadinthelastfouryears.Lastbutnottheleast,Iwouldliketothankmyfamily:myhusbandandtomydaughterforsupportingmeunconditionallythroughoutwritingthisthesisandmylifeingeneral. 4

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TABLEOFCONTENTS page ACKNOWLEDGMENTS ................................... 4 LISTOFTABLES ...................................... 8 LISTOFFIGURES ..................................... 9 ABSTRACT ......................................... 10 CHAPTER 1INTRODUCTION ................................... 12 1.1EpidemiologyofArbovirus ............................ 12 1.1.1EpidemiologyofDengueVirus ...................... 12 1.1.2EpidemiologyofZikaVirus ........................ 14 1.2TransmissionDynamicsofArbovirus ...................... 16 1.3FieldStudiestoUnderstandTransmissionDynamicsofArbovirus ....... 17 2METHODSFORTHEBASELINEFIELDSTUDIESINYUCATAN,MEXICO .... 19 2.1StudyComponents ................................ 19 2.2StudySitesandOrganization .......................... 19 2.3SeroprevalenceSurvey .............................. 21 2.4CohortStudy ................................... 23 2.4.1SelectionofRiskAreas .......................... 23 2.4.2RecruitmentandEnrollment ....................... 24 2.4.3Follow-upVisits ............................. 25 2.4.3.1Suspectedcasedenition ................... 25 2.4.3.2Identicationofdengueinfectionsandcases ......... 26 2.4.3.3Enhancedsurveillance ..................... 26 2.4.3.4Investigationofgeographicassociatedcases(clusters) .... 27 2.4.3.5Case-controlstudy ....................... 28 2.5DataCollectionandStorage ........................... 28 2.6StatisticalAnalysis ................................ 29 3DENGUESEROPREVALENCESURVEYINTHREEURBANSETTINGSINYUCATAN,MEXICO ........................................ 32 3.1Background ................................... 32 3.2Aims ....................................... 33 3.3Methods ..................................... 33 3.3.1Sampling ................................. 33 3.3.2StudyProcedures ............................. 34 3.3.3EthicalReview .............................. 35 3.3.4StatisticalAnalysis ............................ 35 5

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3.4Results ...................................... 36 3.4.1DescriptionofthePopulationofStudy ................. 36 3.4.2DengueSeroprevalence .......................... 37 3.5Discussion .................................... 38 4EPIDEMIOLOGYOFDENGUEANDOTHERARBOVIRUSINASCHOOL-BASEDCOHORTINYUCATAN,MEXICO:BASELINEANDFIRSTYEARFOLLOW-UP 44 4.1Background ................................... 44 4.2Aims ....................................... 45 4.3Methods ..................................... 46 4.3.1CaseDenitions ............................. 46 4.3.1.1Denguecasedenition ..................... 46 4.3.1.2Denguewithoutwarningsigns ................. 46 4.3.1.3DenguewithwarningSigns: .................. 46 4.3.1.4Severedengue: ......................... 46 4.3.1.5Laboratorycriteriaforconrmationfordengue ........ 46 4.3.1.6Denguepriorexposure ..................... 47 4.3.2Chikungunyacasedenition ....................... 47 4.3.2.1Acutechikungunyainfection .................. 47 4.3.2.2Laboratorycriteriaforconrmationforchikungunya ..... 48 4.3.3Zikacasedenition ............................ 48 4.3.3.1Zikavirusdisease ....................... 48 4.3.3.2LaboratorycriteriaforZikaconrmation ........... 48 4.3.4EthicsStatement ............................. 48 4.3.5StatisticalAnalysis ............................ 48 4.4Results ...................................... 51 4.4.1BaselineCharacteristicsoftheParticipants ............... 51 4.4.2DengueBaselineSeroprevalence ..................... 53 4.4.3SuspectedSymptomaticArboviralInfections .............. 55 4.4.4ConrmedArboviralSymptomaticInfections .............. 55 4.4.5TotalDengueInfections ......................... 57 4.4.6PrimaryDengueInfectionsintheNavePopulation ........... 59 4.4.7IncidenceRateRatioofArbovirusConrmedSymptomaticCasesandDengueInfections ............................ 60 4.4.8SurvivalModelforDengueInfections .................. 60 4.5Discussion .................................... 61 5THEEPIDEMIOLOGYANDTRANSMISSIBILITYOFZIKAVIRUSINGIRARDOTANDSANANDRESISLAND,COLOMBIA ...................... 66 5.1Background ................................... 66 5.2Aims ....................................... 67 5.3Methods ..................................... 67 5.3.1Settings .................................. 67 5.3.1.1SanAndres ........................... 67 6

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5.3.1.2Girardot ............................ 68 5.3.2CaseDenitionandLaboratoryAnalysis ................. 68 5.3.3DataCollection .............................. 70 5.3.4StatisticalAnalysis ............................ 70 5.4Results ...................................... 71 5.4.1SanAndres ................................ 71 5.4.2Girardot .................................. 73 5.4.3EstimationsoftheBasicReproductiveNumber ............. 74 5.5Discussion .................................... 75 6CONCLUSIONS .................................... 80 REFERENCES ........................................ 87 BIOGRAPHICALSKETCH ................................. 104 7

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LISTOFTABLES Table page 3-1DemographiccharacteristicsofthestudypopulationinYucatan,Mexico(N=1,667) 37 3-2DengueseroprevalencetoindirectIgGbyagegroupandcity,2014 ......... 40 4-1Demographiccharacteristicsofthecohortpopulationenrolledin2015Yucatan,Mexico(N=3,400) ....................................... 52 4-2Baselineexposuretodengueintheparticipantsofthecohortenrolledin2,015inYucatan,Mexico(n=2,732) .............................. 54 4-3ProbablesymptomaticcasesofarboviralinfectionsinthecohortofYucatan,Mexico(N=199) ........................................ 56 4-4Symptomsofthefebrilecasesstudiedduringtherstyearfollow-upinthecohortofYucatan,Mexico(N=199) ............................. 57 4-5Incidencerates(IR)per1,000person-yearsofArboviralconrmedsymptomaticinfections 58 4-6Incidencerates(IR)per1,000person-yearsofalldengueinfections(N=1,890) ... 59 4-7Seroconversioninthedengue-naveatbaseline(n=478) ............... 60 4-8Incidencerateratioforallarboviralinfectionsintherstyearoffollow-upofthecohortinYucatan,Mexico. .............................. 60 4-9Hazardratiosfortotaldengueinfectionsintherstyearoffollow-upofthecohortinYucatan,Mexico. .................................. 61 5-1CharacteristicsofreportedcasesofZikaVirusDisease ................ 71 5-2EstimatesofR0 .................................... 75 8

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LISTOFFIGURES Figure page 2-1OverviewofthebaselinedenguestudiesinYucatan ................. 20 2-2MapoftheYucatanandgeographicallocationsofthestudysites .......... 22 3-1LocationoftheselectedschoolsforthedengueseroprevalencesurveyinYucatan,Mexico. ........................................ 34 3-2LocationofthehouseholdsoftheparticipantsindengueseroprevalencesurveyinYucatan,Mexico. ................................... 36 3-3DengueseroprevalencetoIndirectIgGbyagegroupandcity,2014 ......... 39 4-1Flowchartofparticipantsinthedenguecohort,Yucatan,Mexico. ........... 51 4-2Age-specicbaselinedengueseroprevalenceinthecohort,Yucatan,Mexico. ..... 53 5-1MapofColombia ................................... 69 5-2DailyZVDincidenceforSanAndres,Colombia .................... 72 5-3Age-andgender-specicZVDattackratesforSanAndres,Colombia ........ 72 5-4DailyZVDincidenceforGirardot,Colombia ..................... 73 5-5Age-andgender-specicZVDattackratesforGirardot,Colombia .......... 74 5-6Estimatesofeectivereproductivenumber ...................... 76 9

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AbstractofDissertationPresentedtotheGraduateSchooloftheUniversityofFloridainPartialFulllmentoftheRequirementsfortheDegreeofDoctorofPhilosophyEPIDEMIOLOGYANDTRANSMISSIONDYNAMICSOFARBOVIRUSINLATINAMERICAByDianaP.RojasAlvarezAugust2017Chair:RobertCookCochair:IraM.LonginiMajor:EpidemiologyArthropod-borneviruses(arboviruses)areconsideredglobalpublichealthpriorities.Arbovirusareconsideredthecauseofsomeofthemostimportantemerginginfectiousdiseasescurrentlythreateningtheglobe.ApproximatelythreebillionpeopleworldwideliveinareasinfestedwithAedesmosquitoesandareatriskforarbovirusinfectionslikedenguevirus,chikungunyavirusandZikavirus.Denguevirushasbeencirculatingsincethe1980'sinLatinAmericaanditsincidencehasincreasedsignicantlyworldwideandinthisregion.AedesaegyptiisthemainvectorformostofthearbovirustransmittedinLatinAmerica.InthelastthreeyearstwoarbovirushademergeintheWesternHemispherecausingimportantoutbreaks.In2013,ChikungunyavirusemergedintheCaribbeanandspreadrapidlythroughtheCaribbean,CentralandSouthAmericawith1.8millioncasesreported.Similarlyin2015ZikaViruswasisolatedinBrazilandsincethenitspreadinareasinfestedwithAedesmosquitosinLatinAmericaandtheCaribbeanandalsowithauthoctonoustransmissionincontinentalUnitedStates.TheaimofthisdissertationistoprovidenewestimatesabouttheepidemiologyandtransmissiondynamicsofArbovirusinLatinAmerica.Intherstpaper,theestimatesofdengueseroprevalenceinthreesettingsinYucatan,Mexicoarepresentedtodescribethebaselineconditionsofthesepopulationsandpotentialriskfactorsassociatedwithdenguetransmission.Thesecondpaperhastherstresultsofthebaselinedengueseroprevalenceandrstyearfollow-upofacommunitybasedcohortinYucatan,Mexico;andthethirdpaper 10

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characterizetheepidemiologyandtransmissibilityofZikavirusinColombiausingsurveillancedatadeningtheepidemiologicalfeaturesoflocalZIKVoutbreaksinColombiaandestimatingthetransmissionparametersandpotentialriskfactorsassociatedwithZikatransmission.ThisstudyoersauniqueperspectiveontheepidemiologyofarbovirusinLatinAmericagiventhetimeframewhereforthersttimethreearbovirusareco-circulatinginthisregion.Duetotherobustdatasetsavailable,thisdissertationisoneoftherststudiesdescribingtheepidemiologyandtransmissiondynamicsofthethreevirusesintheWesternHemisphere. 11

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CHAPTER1INTRODUCTIONArthropod-borneviralinfections,orarboviralinfections,arecommoncausesofdisablingfeversyndromesworldwide,buttheircumulativeimpactonglobaldiseaseburdenhasnotbeenfullyassessed.Intheiracutestages,arboviralinfectionscauseabroadspectrumofdisease,rangingfromasymptomaticinfectiontoseveredisease.Theycanalsoprogresstomuchmorecomplexsecondaryconditions,orsequelae,suchasencephalitisorhemorrhagicdiathesis,whichresultinlong-termphysicalandcognitiveimpairmentorinearlydeath[ 1 ].Morethan100arbovirusesareknowntocausediseaseinhumans.Asignicantsubset,includingmembersoftheFlaviviridae,Bunyaviridae,andTogaviridaefamilies,aretransmittedwidelyindierentareasoftheworld.Arbovirusesarealsoconsideredtobeemergingpathogensbasedontheirgeographicspreadandtheirincreasingimpactonsusceptiblehumanpopulations[ 1 { 3 ].ThisdissertationwillprovidenewevidenceabouttheepidemiologyandtransmissibilityofarbovirustransmittedbyAedesmosquitoesfromtwocountriesinLatinAmerica. 1.1EpidemiologyofArbovirus 1.1.1EpidemiologyofDengueVirusDenguevirus(DENV)isthemostrapidlyspreadingarbovirusworldwide[ 4 ].ItistransmittedpredominantlybyAedesmosquitoes,mainlyAedesaegyptiandAedesalbopic-tus.Inthelast50years,itsincidencehasincreased30-foldgloballywithgrowinggeographicexpansiontonewcountriesandfromurbantoruralsettings[ 5 ].Currentlymorethan40%oftheworldpopulationisatriskofDENVinfection,andapproximately390millionDENVinfectionsareestimatedgloballyeachyear,ofwhich96millionhaveclinicalmanifestationsofthevirus[ 4 ].TheburdenofthediseaseishigherincountriesofSouth-EastAsiaandtheWesternPacicregions,however,adramaticincreaseofcaseshasbeenreportedintheLatinAmericaandtheCaribbeanduringthelastdecade[ 6 ].DENVhasfourcloselyrelatedserotypes(DENV1,DENV2,DENV3,andDENV4)[ 7 ].Theinfectionwithanyserotypeconfers 12

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life-longprotectiveimmunitydirectedagainsttheserotypeofinfectionbutdoesnotconferlong-termprotectionagainstinfectionbyotherserotypes[ 8 9 ].Eachofthefourserotypescanberesponsiblefordengueepidemicsandcanalsobeassociatedwithseveredenguediseasedependingonthesequenceandtimebetweeninfections,amongotherfactors[ 10 ].Theclinicalspectrumofdenguegoesfromasymptomaticinfectionstolife-threateningseveredisease;theproportionofasymptomaticinfectionsrangesfrom50%to90%[ 11 12 ].DENVinfectiondoesnothavespecictreatmentorclinicalpredictorstopreventseveredisease[ 7 ].SeveralfactorsareinvolvedintheincreasingincidenceofdengueintheAmericas.Themainfactorsaretheunsustainableandineectivevectorcontrolstrategiesthattargetthevectorinallstagesofdevelopmentanditstillremainsastheonlymeasurefordengueprevention[ 2 13 14 ].AlsothePopulationgrowth,unplannedurbanizationwithpoorsanitaryconditions,lackofthepublichealthinfrastructure,anddecreasedaccesstohealthcarehasalsocontributedtotheincreaseofdiseaseburden.Globalizationoftheeconomy,internationaltravel,andclimaticchangesmightalsoexplainthediseaseexpansion[ 6 ].Theestimatesofthetransmissionparametersfordenguefevervaryconsiderablybetweenstudies[ 15 { 19 ]andthereasonsofthevariabilityontheparametersarenotcompletelyunderstood.Itcouldbeexplainedbythehistoryofdenguetransmissioninthepopulation,thequalityoftheepidemiologicalsurveillance,andtheclimatologicalconditionsthatcanaectvectordensitiesandthemacrofactorsalreadymentioned.Methodsforestimatingthereproductivenumber(R)usedataontheintrinsicgrowthrateoftheepidemic[ 16 17 ];ontherelationbetweenthereproductivenumberandthenalepidemicsize[ 15 ];andage-stratiedseroprevalencesurveys[ 18 20 21 ].Theestimationsofthebasicreproductivenumber(R0)forDENVepidemicsrangefrom2to6[ 15 { 19 22 { 24 ].Givingthecomplexityofdenguetransmission,denguepreventionrequiresinnovativeinterventionse.g.vaccines,eectivevectorcontrolandgeneticallymodiedmosquitoestoprovidemoreeectiveandsustainablestrategies,especiallyingrowingandcomplexurbanenvironments[ 25 26 ]. 13

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CurrentlyveDENVvaccinecandidatesareinclinicalstagesofdevelopment[ 27 ].TwoofthemareinadvancedstagesandonlyonevaccinehascompletedtwoPhaseIIItrials(Dengvaxia)anditisbeingapprovedforintroductioninsomecountries[ 28 { 30 ].Thisvaccinehasanestimatedecacyof64.7%and56.5%intheLatinAmericaandSouthEastAsiatrials,respectively.Thevaccineecacyinthepooledanalysisofbothtrialswasfoundtobesignicantlyhigherinparticipantswithpre-existingdengueneutralizingantibodiescomparedtothosewhowereseronegative.Theserotype-specicvaccineecacywasheterogeneousinbothtrials,andthevaccineecacyagainsthospitalizationfordengueinSouthEastAsiawas67.2%andinLatinAmerica80.3%[ 28 29 31 ].InApril2016,theWorldHealthOrganization(WHO)StrategicAdvisoryGroupofExpertsonImmunization(SAGE)recommendedtothedengueendemiccountriestoconsidertheintroductionofDengvaxiaonlyintransmissionsettingswithhighendemicity,measuredbyseroprevalencesurveysofapproximately70%orgreaterintheagegrouptargetedforvaccination(9yearolds).Thevaccinewasnotrecommendedwhendengueseroprevalenceisbelow50%[ 32 ].BasedontheWHOrecommendationsandtheparticularitiesofthisvaccine,eldstudiesareneededtounderstandbetterthetransmissiondynamicsofdengueandthefullburdenofdisease.Alsodengueseroprevalencesurveysarearequirementtointroducethevaccineinendemiccountriessothisprojectwillbekeytoplanvaccineintroductionindierentdenguetransmissionsettingsandtoevaluateeectivenessofdierentinterventionslikevaccineeectiveness,vectorcontrolactivitiesorthecombinationofboth. 1.1.2EpidemiologyofZikaVirusZikavirus(ZIKV)isavivirusinthesamegenusasdenguevirusandyellowfevervirus.ZIKVwasrstisolatedintheZikaForestofUgandain1947andprimarilytransmittedbyAedesmosquitoes[ 33 ].AlthoughZIKVhascirculatedinAfricaandAsiasincethe1950s,littleisknownaboutitstransmissiondynamics[ 34 ].RecentoutbreaksinYapIslandintheFederatedStatesofMicronesia(2007),FrenchPolynesia(2013),andotherPacicislands,includingCookIslands,EasterIsland(Chile),andNewCaledonia(2014),indicatethatZIKVhasspread 14

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beyonditsformergeographicrange[ 35 { 38 ].InApril2015ZIKVwasisolatedintheNortheastofBrazil[ 39 ]andsincethenitspreadrapidlythroughLatinAmericaandtheCaribbean.ZIKVInfectiontypicallycausesaself-limiteddengue-likeillnesscharacterizedbyexanthema,low-gradefever,conjunctivitis,andarthralgia[ 40 ].Whileillnessisbelievedtobemildorasymptomaticinapproximately80%oftheinfections[ 41 ],anincreaseinratesofGuillain-Barresyndrome(GBS)hasbeenobservedduringZIKVoutbreaks[ 42 { 44 ].Furthermore,inOctober2015,theBrazilianMinistryofHealthreportedadramaticincreaseincasesofmicrocephalyinNortheastBrazilwhereZIKVhadbeencirculating[ 45 ].OnthebasisofthepossiblelinkbetweenZIKV,GBSandmicrocephaly,theWorldHealthOrganizationdeclaredapublichealthemergencyonFebruary1,2016[ 46 47 ].AsofAugust2016,around500,000Zikavirusdisease(ZVD)caseshavebeenestimatedinBrazil,andautochthonouscirculationhasbeenobservedin45countriesintheAmericasregionincludingUnitedStateswithlocaltransmissionintwocountiesintheStateofFlorida[ 48 ].ZIKVisspreadprimarilythroughAedesmosquitoes,especially.Ae.aegypti.ThecapacityofAe.albopictustotransmitZIKVisstillunknownbutisbelievedtobelowerthanAe.aegypti[ 49 50 ].Alsosexual[ 51 { 54 ],perinataltransmissionofZIKV[ 55 { 58 ]andthepotentialfortransmissionbytransfusionisbeingdescribed[ 43 ].MostoftheR0estimationsforZIKVhavebeenestimatedfromZIKVoutbreakinFrenchPolynesiain2013[ 59 ].TheZIKVbasicreproductivenumber(R0)wasestimatedaroundof2.33(95%CI[2.15-2.51])[ 59 { 61 ].TheR0fortheLatinAmericanoutbreakisbeingestimatedfromBrazil.TheR0estimatesforZIKVinRiodeJaneiro(R0=3.9,95%(95%CI:[3.1-5.3])[ 62 ].ThecountrieswithZIKVtransmissionfocusedtheirresponsesonvectorcontrolduringtheoutbreakandadvicetodelaypregnancy,followedbyanextendedrecommendationtoallaectedcountriesbyWHOinJune2016[ 63 ].CountriesintheAmericasreportingthehighestincidenceofZIKVdiseasearealsocountrieswithhistoricallyendemicDENVtransmissionandrecentCHIKVoutbreaks.Currently,othercountrieswithZIKVoutbreaksbesidesBrazilhave 15

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reportedcasesofmicrocephalyandotherbirthdefectsassociatedwithZIKVinfectionduringpregnancy(ZikaCongenitalSyndrome)[ 64 ].ThemagnitudeandpublichealthimpactofthisZIKVoutbreakisduetothespreadinareasinfestedbyAe.aegypti,thefullpopulationwassusceptibleandineectivevectorcontrolactivities.SomefactorscancontributetounderestimationofthediseaseburdenofZIKVinfectione.g.lackofadequateinfrastructureinlaboratoryperformZIKVdetection,ZIKVinmostofthecasesisverymildorasymptomaticinfections,andpatientsmaynotseekmedicalcare[ 65 ].AsthetransmissionintheAmericasissorecentveryfewisknownabouttheepidemiologyandtransmissionparameterstounderstandbetterthedynamicsofZIKVandhowitstransmissioncouldbeaectedbytheco-circulationwithotherarboviruses. 1.2TransmissionDynamicsofArbovirusTransmissionofanarbovirusinapopulationisafunctionoflocalenvironment,thenaturalhistoryofinfectionandthesusceptibilityofthepopulationtoinfection.Thesuitabilityofthelocalenvironmentforarbovirustransmissionandtheimpactofthenaturalhistoryofthediseaseareusuallycapturedbythebasicreproductivenumber(R0),denedasthenumberofsecondaryinfectionsexpectedfromasinglecaseinapopulationwithnopreexistingimmunity.R0andthetransmissibilityofvector-bornediseasesisassociatedwithstrongspatialheterogeneity,drivenbyvariabilityinvectorabundanceandcharacteristicsoftheexposedpopulations[ 3 ].Thecombinedimpactofthesefactorsandsusceptibilityarecapturedbythereproductivenumber(R),whichisrelatedtoR0bytheequationR=R0S,whereSisproportionofthepopulationsusceptibletoaparticulararbovirus.Thisvalue,combinedwiththeserialinterval(thetimeseparatingtwoconsecutiveinfectionsinachainoftransmission)isimportanttounderstandhowapathogenwillspreadinapopulation.ThesizeofanoutbreakafteranintroductionwilldependonR(R0inanaivepopulation),withsmall,self-limitingoutbreaksbecomingmorelikelyasRapproachesone,andincreasingepidemicswithlargerRs.Hence,ZIKVcansuccessfullyspreadtoanewregionifR>1,whichrequires,amongotherfactors, 16

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sucientdensityofthevectorpopulation[ 3 ].Theforceofinfection()isameasurethatisusedtoestimatetheintensityoftransmission(transmissionhazard)inagivensettingandevaluatestherateofacquisitionofinfectionamongsusceptibleindividuals[ 18 21 66 ].Theseparameterscouldbeestimatedfromsurveillancedatawhendatafromeldstudiesarenotavailable.ForDENVasisbeingcirculatingforlongtimeatleastnon-serotypesspecicserosurveysareneededtoestimateRandformonotypicinfections.ToestimateR0andforceofinfectionformultitypicinfectionslongitudinaleldstudiesanddatacollectionareneeded.EstimatethesetransmissionparametersforCHIKVandZIKVislesscomplexbecauseitisjustoneserotypeandasmostoftheviralinfectionswewillassumethatafteraninfectionthereislife-longimmunityagainstthevirus. 1.3FieldStudiestoUnderstandTransmissionDynamicsofArbovirusSeroprevalencesurveysareusefultoestimatethebaselinehazardofDENVinfectionandbasicreproductivenumberinaspecicpopulation,toestablishthemostrecentserotypescirculating,andtodeterminewhetherpeoplehavebeenexposedtooneormoreserotypesofdengue[ 67 { 69 ].ThisinformationallowstoidentifyhighriskpopulationsfordengueinfectionwheretargetedinterventionscouldbeimplementedandaccordingtotheWHO-SAGErecommendationswillbeneededtoplantheintroductionofthedenguevaccine.Additionally,testingforspecicantibodies(monotypicvs.multitypicimmunity)isconsideredessentialtounderstandthetransmissiondynamicsofdengueinanspecicpopulation[ 18 70 71 ]Prospectivecohortstudiesarekeytoestimatetheriskofinfectionbydenguevirus.Currently,thereareseveralongoinglongitudinalstudiesindengueendemicareasinSouthEastAsiaandLatinAmericathatarecollectinginformationconsideringalloftheclinicalspectrumofdengueinfections[ 72 { 79 ].MostofthesestudiesincludeenhancedsurveillancebecauseitimprovesthedetectionofdengueinfectionsandcasesintheacutephasethatwillallowthevirologicalconrmationoftheinfectionsusingRT-PCRtoidentifytheinfectingserotypeimprovingtheregularvirologicalsurveillancethatisdonebythelocalpublichealthlaboratories[ 76 80 ].Theincidenceofseverecasesamongdengueinfectionsarekeyto 17

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determinepossibleriskfactorsthatcouldbeassociatedwiththeseverityofdenguediseaseincludingviral,social,cultural,environmentalandhealthcare-relatedfactors[ 74 75 81 ].Tobetterunderstanddenguetransmissionandestimatetheburdenofdiseaseinendemicregions,severallong-termprospectiveepidemiologicalstudieshavebeenestablishedinSouthEastAsiaandLatinAmerica[ 19 72 80 82 { 85 ].Additionalresearchisneededtoaddresssomequestionsthatarecriticaltofuturevaccineevaluation:estimationofdiseaseburdenincountrieswheredengueisnotwellcharacterized,explorethedengueserotype-specictransmissionanddengueseverity,determineuniqueviralandhostfactorsthatcancontributetodierencesindenguerisk,andassessthepotentialimpactofavaccine,vectorcontrolinterventionorthecombinationofboth[ 86 ].TheaimofthisdissertationistobetterunderstandtheepidemiologyandthetransmissiondynamicsofarbovirusindierentLatinAmericansettings.ThersttwopapersaimedtoassessthebaselineepidemiologytoidentifythedenguetransmissiondynamicsinthreedierenturbansettingsinYucatan,Mexico.Wedesignaeldstudythatincludesaseroprevalencesurveyinthegeneralpopulationandaschool-based,prospectivecohortstudysetinurbanenvironmentswithdierentsocial,demographic,andeconomicproles,anddierentdenguetransmissionpatternsbasedonepidemiologicalsurveillancedata.Aninitialhouseholdsurveywillbeusedtoassessriskfactors,riskbehavior,andclinicalsymptoms,accompaniedbyserologicaltestingonallmembersofthefamily.ThethirdpaperisananalysisofZikaVirusoutbreakandestimationoftransmissionparametersintwosettingsinColombiausingepidemiologicalsurveillancedata. 18

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CHAPTER2METHODSFORTHEBASELINEFIELDSTUDIESINYUCATAN,MEXICO 2.1StudyComponentsUnderstandingthefullburdenofdengueinfectionsandotherarbovirusdynamics,includingasymptomaticinfections,andthedegreeofunderreportingondierenttransmissionsettingshaveimportantpublichealthimplicationsintransmissiondynamics.Thisstudywillalsocharacterizethediseasespectrumandtheepidemiologicalparametersassociatedwithdenguetransmissionthatmaybeinuencedbyinterventions.ThisstudyisdesignedtocollectprospectivebaselinedatainYucatan,Mexicotomeasure:1)age-specicseroprevalence,2)age-specicattackratesofdenguedeterminedbyserologicalconrmation,3)prevalenceofasymptomaticinfections,and4)proportionofunderreportedasymptomaticandclinicaldenguecases(includingseveredenguecases).Therststudyisabaselineseroprevalencesurveythatincludesarandomsampleofthepopulationinthethreedierentsettingstoestimatethepreviousexposureofthesepopulationstodenguevirus.Second,aschool-basedprospectivecohortstudybeginningenrollmentoncetheseroprevalencesurveyiscompleted.Theaimofthissecondstudyistocharacterizethelocalepidemiologyofdengueincludingage-specicattackrates,proportionofasymptomaticinfections,andtheestimatedunderreportinglevelsinthestudyarea.Theactivitiesplannedforthecohortincludeactivesurveillance,febriledetection,andanannualfollow-upvisittoallparticipants.Theseactivitieswilllastforthedurationofthestudy.(Fig. 2-1 ).Duringthedengueseasonotherstudieswillbeaddedtotheactivefollow-up.Clusterstudiesfordenguetransmissionandcase-controlstudiesofseveredenguewillbehelpfultobetterunderstandthediseasespectrumofdengueinYucatan,Mexico. 2.2StudySitesandOrganizationThestateofYucatanislocatedinthesoutheastpeninsulaborderingtheGulfofMexicoandtheCaribbeanSea.Meridaisthelargesturbancenterintheregionandcapitalcity 19

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Figure 2-1. Overview of the baseline dengue studies in Yucatan 20

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with814,000habitants(2013).Theweatheriswarmandhumid,andtherainyseasonfallsbetweenJuneandOctober;themeanannualtemperatureis25.9C(19.5to33.6)andannualprecipitationis1050(mm).Progresoisthemainseaportinthestate,32kms.,awayfromMerida,with54,000habitants(Fig. 2-2 ).Thistownhassimilarweatherconditionsandistheweekendandholidayresort(July-August)forthepeoplelivinginMerida,aswellasatouristresortatthenationalandinternationallevels.Ticulisatown82kms.,southfromMeridawith34,000habitantswhosemaineconomicactivityisdedicatedtotheproductionofshoes[ 87 ].ThecitiesofMerida,ProgresoandTiculwereselectedasthreedierentepidemiologicalsettingsfordenguetransmissionduetothedistinctintensityofdengueoutbreaksinthepastandtheco-circulationofmorethantwoserotypesinrecentyears.Incidencerateswerecalculatedforeachcityofsuspectedandconrmedcasesfrom1979to2013.Thesethreecitiesalsohavedierentdengueincidencerates,allowingustoexplorepossibledierencesindenguetransmissionscenarios[ 88 ].ThisstudyisbeingconductedbytheRegionalResearchCenter"HideyoNoguchi"oftheUniversidadAutonomadeYucatan,inMerida,thecapitalofthestateofYucataninMexicowiththeparticipationoftheMinistryofHealthofYucatanandtheStateReferenceLaboratory.OtherresearchinstitutionsinvolvedintheprojectaretheUniversityofFlorida,UniversityofWashington,andtheFredHutchinsonCancerResearchCenter,creatinganinterdisciplinaryresearchnetwork.ThisstudywasapprovedbytheInstitutionalReviewBoardatUniversidadAutonomadeYucatan.Writteninformedconsentandassentformswillbesignedduringenrollmentandbeforesamplesareobtained.Writteninformedconsentwillbeobtainedfromparticipantsolderthan18years,andfromparentsofparticipantsyoungerthan18yearsold.Theanalysisusingde-identieddatawasapprovedatFredHutchinsonCancerResearchCenter,Seattle,WA. 2.3SeroprevalenceSurveyInthispartofthestudy,weaimtocharacterizethebaselineage-specicprevalenceofantibody-mediatedimmunitytodenguethroughacross-sectionalserologicalsurveyinthethree 21

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Figure 2-2. Map of the Yucatan and geographical locations of the study sites 22

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selectedcities.Serumsampleswillbecollectedfromarandomsampleofchildrenandadultsaged2to65yearsoldwholiveinthestudyareas.Thesedatawillprovideinformationaboutthehistoryofdenguetransmissionineachcity.Thesamplingschemefortheseroprevalencesurveyincludestwodierentstrategies.Therstisaschool-basedsurveywitharandomselectionofschoolsinthethreesites,followedbyarandomselectionofchildren(5to19yearsold)inthedierenteducationallevels(primary,secondaryandhighschool).Theadultpopulationwillbeselectedatrandomfromthepopulationattendingthehealthcentersinthethreecities(onehealthcenterpercity).Inclusioncriteriaincludeadults(18yearsold)whowerenotsueringfromanacutefebriledisease,andwenttothehealthcarecenterforanannualphysicalexaminationorwereunderclinicalcontrol,andthereforerequiredabloodsamplefortheirconsultation.Individualswererandomlyrecruiteduntilthetargetsamplesizeisreached.Informedconsentwasrequestedandsignedbyadults(18yearsold)andfromparentsofminors(<18yearsold).Bloodsamplewastakenintheschooloncetheinformedconsentformsweresignedfromtheparents.Resultsweredeliveredpersonallytotheparentsofminorswhilethosefromtheadultpopulationweredeliveredbytelephone.Theage-groupspecicprevalenceofpre-existingantibody-mediatedimmunitytoanyofthedenguevirusesisdenedastheproportionofthesampledindividualsintheagegroupwhoseserologicspecimenispositivetoanyserotypeusingPanbioDengueIgGIndirectELISA(titer0.12).Condenceintervals(95%)fortheseprevalenceestimateswillbeestimated[ 67 { 69 ]. 2.4CohortStudy 2.4.1SelectionofRiskAreasThispartofthestudyinvolvesaschool-based,prospectivecohortinthelow-to-middle-incomedistrictsofMerida,ProgresoandTicul.TheprimaryaimofthisstudyistocharacterizethelocalepidemiologyandtransmissiondynamicsofdengueinYucatan.Toassessinitiallytheriskoftransmissionbycitythreeconsiderationswereadopted:1)Proportionofepidemiological 23

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weekswithreporteddenguecasescomparedtoepidemiologicalweekswithnoreportedcases;2)Durationofthedengueepidemicwavesasaproxyofpersistentdenguetransmission;3)Intensityoftransmissionmeasuredastheincidencerateduringthegivenperiod.Theschool-basedstudyisdenedbyarandomselectionofveextensivegeographicareaswithdierentdenguetransmissionrisks(high,mediumorlow).Thedataobtainedfromtheepidemiologicalsurveillancesystemidentiedthoseareaswheredenguehasbeenhistoricallyhigher,persistentandwherethetransmissionriskisdenedashigh,mediumandlow.Thesampleincludeslow-riskareasinMeridaandProgreso,medium-riskareasinMeridaandTicul,andahigh-riskareainMerida.Eachareaoftransmissionincludesseveralprimarypublicschools,fromwhichthecohortofchildrenfromrsttothirdgradewillberandomlyselected.Primarypublicschoolswillbeselectedrandomlyandinvitedtoparticipateinthisstudybasedontheircriteriaofrisk(high,mediumandlow). 2.4.2RecruitmentandEnrollmentChildrenwillbeenrolledatthebeginningoftheacademicyearfromrstthroughthirdgradeandareeligibletoremaininthestudyuntiltheygraduatefromsixthgrade.Duringeachsubsequentyear,children9-12yearsoldwillberecruitedfromtheirprimaryandmiddleschoolsintheareasofstudy.Theonlyexclusioncriteriaisintenttomoveoutsideofthestudyareaduringthemonthsfollowingenrollment.Thestudentsenteringelementaryschoolandthoseinsecondandthirdgradewillbeselectedandincludedinthecohortstudywithwrittenconsentgrantedbytheirparents.Eachgroupofchildrenwhoareenrolledandtheirrespectivefamilieswillbefollowed-upduringtheireducationtrackfortheperiodofthestudy.Thestrategyofcreatingandmaintainingacohortofindividualsandtheirfamiliesparticipatinginlong-termprojectrequiresongoingeort.Ithighlightstheimportanceofsharinginterimresults,advancesintheproject,andunderstandingthecommunity-levelprotectivemeasuresagainstdengueepidemics.Therefore,eachclusterwillhaveateamofcommunityspecialistsconsistingoflocalphysicians,nurses,laboratorytechnicians, 24

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microbiologists,andanthropologistswhowillgatherinformationandorganizeactivitiesdirectedtosensitizeandeducatecohortmembersandtheirfamilies.Theywillparticipateintheenrollmentofnewrstgradestudentsandtheirfamiliesduringtheentirestudyperiod. 2.4.3Follow-upVisitsEvaluationsofthestudypopulationincludingbaselinedemographicinformation,biologicalandbloodsampleswillbeobtainedonceeveryyear.Bloodsampleswillbetakenduringthesurveillanceperiodeachyear(January-July).ThestudyparticipantsandtheirfamilieswillbefollowedfromJanuary2015throughDecember2020at12-monthintervalsforserologicalevidenceofdengueinfectionafterthedengueseasoneveryyear.Participantswillbeconsideredlosttofollow-upafterafullyearhaspassedsincetheirpreviousblooddraw,despiterepeatedattemptstolocatetheparticipant,orifthereisaveriablereasonfordroppingthemfromthestudy(e.g.,directrequestfromtheparticipant,movementfromthestudyarea,ordeath).Thefollow-upperiodproposedallowsustomeasuretheinfectionratesindierentagegroupsaswellastheproportionofasymptomaticcasesorunder-reportingoffebrilecasesifanoutbreakshouldoccur.Theprospectivefollow-upoftheprimaryschoolcohortandtheirfamilieswillprovideinformationfortheestimationoftheagegrouphazardsofinfectionforthoserepresentedinthissample.Wewillinvestigatetheeectsofinfectionhistoryandheterotypicantibody-mediatedimmuneresponsesontheriskofsevereillnessonceinfected. 2.4.3.1SuspectedcasedenitionParticipantsfromthecohortareclassiedassuspecteddenguecasesiftheysatisfythefollowingcriteria:(1)areover24monthsinage;(2)haveanoraltemperature37.2Candonsetofsymptomswithinthelast72hours;and(3)presentatleastoneclinicalmanifestationlistedintheWorldHealthOrganization(WHO)dengueguidelines.Oncethesubjectislaboratoryconrmedasadenguecase,thefamilyhouseholdwillbestudiedclinicallyandserologicallytodeterminedenguetransmissioninthehousehold,includingthedetectionofasymptomaticcasesinallmembers2years. 25

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2.4.3.2IdenticationofdengueinfectionsandcasesTheidenticationofsymptomaticandasymptomaticdenguecasesrequiresestablishingdierentstrategiestoincreasedenguecasedetection.Sincenoteveryfebrileorclinicaldenguecaseseekshealthcare,wewanttoidentifymoredenguecasesthroughthreestrategies:enhancedsurveillance,geographicalrelatedcasesorclusters,andseveredengueorcasecontrolstudies. 2.4.3.3EnhancedsurveillanceThisstrategyaimstoidentifydenguefebrilecasesintheschool-basedcohortthroughabsenteesurveillanceatschoolanditssurroundingarea,andalsofromthetraditionalsurveillancesystem.Thisactivityincludesthedetectionoffebrileillnessinstudyparticipantsusingschool-basedsurveillanceintheselectedareasbyweeklyvisitstoinvestigateschoolabsenteeismandcontinuousactivesurveillanceforfebrilecasesthroughmobileapplicationandfamilyuseofadenguealertnumber(1-800).Cohortparticipantswillbefollowedcloselyforallfebrileillnesses,andchildrenandtheirrelativeswhoalsohavefeverwillbescreenedclinicallyandbylaboratorytesting,forsignsandsymptomsofdengue.Subjectswithacutefebriledengue-likeillnesswillbetestedandidentiedbytheeldgroupworkinginthecommunity;theabsenteesurveillancesystemthatwillbeimplementedineachschooland/orbythetraditionalsurveillancesystemwillprovidedataonsuspectedcasesoccurringintheselectedareas.Thesurveillancesystemwillbeusedfortheidenticationofsuspectedorprobabledenguecasesthatareclinicallyandserologicallystudiedtoconrmdengueinfection.Duringschoolvacation,childrenwillbevisitedorcontactedweeklyattheirhomes.Symptomaticdengueinfectionisclassiedassymptomaticnon-hospitalizedorsymptomatichospitalized,basedonadmissionintothehospitalasdecidedbythetreatingphysician.Hospitalizedsymptomaticcaseswillbefurtherdenedaseitherdengueorseveredengueusingthe2009guidelinesfromtheWorldHealthOrganization[ 5 ]. 26

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Acute-phaseserumsamplesaretestedwithNS1,anti-DENVIgMandIgGantibodiesbyIgMcaptureenzyme-linkedimmunosorbentassay(ELISA).FebrileepisodeswillbeclassiedasdengueinfectionsbasedonNS1andIgMpairedserology.Infectionsidentiedwithafour-foldincreasetitersIgMorIgGtiterwillbecountedasseroconversionsinallincidencecalculations.Inapparent(subclinical)dengueinfectionwillbedenedasapositiveIgMantibodyagainstanyDENVserotypeinthesera,obtainedduringthesurveillancemonthswithoutafebrileillnessorschool-absenceidentiedduringactivesurveillanceinthetimeperiod,oraseroconversionofthecohortparticipantintheyearlyfollow-upsamples.Clinicaldenitionsofserologicallyorvirologicallyconrmeddengueinfectionwillbebasedonevidenceofacutedengueinfection. 2.4.3.4Investigationofgeographicassociatedcases(clusters)Duringadengueoutbreakorhightransmissionseason,caseswillbedetectedthroughthestandardsurveillancesystemthatidentiesclustersof5to10conrmedandprobablecasesofdengueinaradiusof500metersduringtheprevious2weeks.Priorityselectionofcasestobestudiedwillbeperformedintheareasunderfollow-up,andoneclusterwillberandomlyselectedeveryweek.Oncedenguecasesandtheirfamiliesareidentied,wewillselectacontrolfamily(withoutdenguelikesymptoms)pairedbysexandagegroupoftheindexcaseinthesurroundinghouseholds.Wewillinterviewandtakethebloodsamplesfortheidenticationofasymptomaticandclinicalcasesinthehouseholdperimeternotcapturedbythesurveillancesystem.Thisapproachisjustiedbyevidencethatdemonstratesthatdenguecasesaregeographicallyclusteredduringtheinitialdaysofinfectionandaroundtheindexcases.Theoutbreakinvestigationsofclustersofdenguecaseswillhelpusdeterminewhetherthenumberofcasessurroundingaconrmedcasevariesaccordingtothetransmissionintensitydenedbytheemergenceofaclusterofgeographicallyassociatedcases,aswellaspotentialindividualandhouseholdlevelfactorsthatmayaecttransmissiondynamics. 27

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2.4.3.5Case-controlstudyOneofthemostimportantoutcomesofdengueisseveredengueandassociatedmortality.Tomeasureitsimpact,weproposeacase-controlstudyforseveredenguecasesthatwillbepairedtoacontrol(mild-denguecase)basedonagegroup,sex,hospital,andplaceofresidence.Theaimofthisstudyistomeasurethenumberofcasessurroundingseverecasestodistinguishapotentiallydierentormoreintensivetransmissionpattern.Patientsofallageswillbeeligibleforrecruitmentwhenadmittedtoareferencehospitalinandoutsidetheselectedareaswithaclinicaldiagnosisofseveredengueaccordingtothe2009WHOguidelines[ 5 ].Patientswillbeexamineddailyduringhospitalizationbyadedicatedteamofphysicianswithexperienceindenguediagnosisandtreatment.Signsandsymptomsofseveredengue(hemorrhage,capillarypermeability,shock)alongwithotherrelevantclinicalandlaboratoryinformationwillbeextractedfromtheclinicalrecordsandwillbeprospectivelyrecordedusingstandardizedcaserecordforms.Familymembersofbothcasesandcontrolswillberecruitedandbloodsampleswillbetakentomeasureandcomparetheirdengueimmunologicalorserological(IgMandIgG)prole.Ahouseholdcontrol(matchedbyageandsex)andtheirfamilymemberswillalsobeselectedwiththepurposeofcomparingthefamilyimmunitystatusorclinicalproleandtoidentifyifrecentandmoreintensiveintra-householdtransmissionofdengueactsasariskfactorfordengueseverity.Thisparticularissuewillbeimportanttoestimatetheindirecteectofvaccinationonceadenguevaccineisavailable. 2.5DataCollectionandStorageDatafromthisstudywillbecollectedandenteredintoanelectronicdatabasethatcanbeaccessedthroughaweb-basedsystem.Thedatawillbecapturedusingamobileplatformusingmobiledevicessuchastabletsandsmartphones.ThisinformationwillbesynchronizedwhenthereisaccesstotheInternetandwillbecontinuouslyupdated.Theapplicationwillallowustomakepredesignedandcustomizedconsultationsinrealtime.Thisapplicationincludes 28

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basicdemographicdata(age,sex,educationlevel,occupation,etc.),withaspecicsectionregardingclinicalhistoryofdengue(clinicalsignsandsymptoms,datesofoccurrence,accessordemandofhealthservices,hospitalization,etc.).Datafromthebehavioral,febrileandabsenteequestionnaireswillalsobeincludedintheapplication.Thebehavioralquestionnairewillexploremovementsbetweenhouseholds,vicinity,neighborhood,localityandoutsidethecities.Thefebrilequestionnaire(regularhousetohousevisits)includesdataregardingfebrileperiods,symptoms,dates,duration,severity,movementsoutsidethearea,demandofhealthservices,contactsandbloodsampleresults(serology)includingallfamilymembers.Theabsenteequestionnaire(activeschool-basesurveillance)willbeusedweeklyateachschooltoidentifychildrenwhowereabsentinthepreviousweekandreasonsfortheirabsencewillberecorded.Bloodsampleresultswillberecordedfromschoolchildrenandfamilymembers. 2.6StatisticalAnalysisTheage-stratiedspecicprevalenceofpre-existingantibody-mediatedimmunitytoanyofthedenguevirusesisdenedastheproportionofthesampledindividualsintheagegroupwhoseserologicspecimenispositivetoanyserotypeusingPanbioDengueIgGIndirectELISA[ 67 { 69 ].Weplantoestimatecondenceintervals(95%)fortheseprevalenceestimatesthatwillbebasedonthestandarddeviationfromtheBinomialdistribution,assumingindependent,randomlyselectedobservations.Itisalsopossibletoestimatethebaselinehazardofinfectionaswellasthebasicreproductivenumberusinginformationfromthisage-stratiedserologicalsurveys[ 18 20 71 ].Theprospectivecohortwillestimatebaselineseroprevalencefordenguevirusandagespecicdengueincidenceratesandincidencerateratios.Wewillinvestigatetheeectsofinfectionhistoryontheriskofsevereillnessonceinfected[ 9 80 89 ].Wewillalsoestimatethelevelofassociationbetweenriskfactorsandtheriskofsevereillnessgiveninfection(i.e.,pathogenicity).Riskfactorsforpathogenicitywillincludeage,gender,city,aswellastheindividualsinfectionandimmunologichistory[ 11 ]. 29

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Toanalyzetheinformationderivedfromtheclusterinvestigationswewillemploymethodspreviouslyimplementedbyourgrouptoestimatetheeectsofindividualandneighborhood-levelpredictorsofthetransmissionpotentialofaninfection[ 90 ].Fortheoutbreakdatacollectedfromthisstudy,wewillusestatisticalmethodsthattachain-binomialmodelforthetransmissionofinfection.Thebasicmodelwillestimatetwotransmission-relatedparameters,theprobabilityofwithin-householdtransmissionandtheprobabilityofinfectionduetoexposure(viaamosquitovector)toaninfectiousindividualwhoisnotamemberofthesamehousehold(community-to-person)[ 91 ].Thispartitioningofinfectionriskbythesourceofexposurewillhelpdescribeandquantifythespatialandtemporalheterogeneityoftheriskofdenguetransmission.Theprobabilityofwithin-householdtransmissionwillbequantiedbythehouseholdsecondaryattackrate(SAR),whichisdenedastheaverageprobabilitythataninfectedindividualwithdenguewilltransmitthevirustoanotherhouseholdmemberduringhis/herinfectiousperiod(intrinsic+extrinsicinfectiousperiods).Incontrast,theprobabilityofcommunity-to-persontransmissionwillbequantiedasthecommunity-to-personprobabilityofinfection(CPI).TheCPIwillbedenedasthecumulativeprobabilitythatoverthecourseoftheintensivefollow-upperiodforaclusterofdenguecases,asusceptibleindividualwillbecomeinfectedduetoexposure(viaamosquito)toacommunity-basedsource.Thismodelingapproachpermitstheestimationoftheeectsofriskfactorsonboththeinfectiousnessandsusceptibilitytoinfection.Sincedenguecasestendtoclusterintimeandspace,theSARisexpectedtoprovideasubstantialamountofinformationaboutthetransmissionofthisvirus.Specialconsiderationwillbetakentoestimatetherolesofinfectionandimmunologichistoriesonsusceptibilitytoinfection,aswellasoninfectiousness.Thematchedcase-controlsub-studywillbeanalyzedusingastandardconditionallogisticregressionapproach,withage,sex,andlocation(hospitalwherethepatientwasseen)includedasindependentpredictors.Allotherimportantriskfactorswillbedeterminedaprioriandincludedintheregressionmodel.Inclusionofinformationregardingimmuneprole,characteristics,andrecenttransmissionhistoryofcaseandcontrolhouseholdsasriskfactors 30

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forseveredenguemayrequiretheadaptationofexistingstatisticalmethods[ 92 ].Wewilldevelopandvalidatetheperformanceofthesemethodspriortoconductingsuchanalysis.Apreliminaryapproachfordealingwiththisissuewillbetoaggregatetheindividual-levelinformationaboutimmunityandtransmissionhistoryatthehousehold-level,therebylosingsomeinformationbutallowingustousestandardanalyticmethodsformatchedcase-controldata.Thehospital-based,matchedcase-controlsub-studywillprovideinformationaboutriskfactorsforseveredengue,includingacharacterizationoftheimmuneprolesofthemembersofthecasesandcontrolshouseholds,aswellasthehistoryoftransmissioninthesefamilies. 31

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CHAPTER3DENGUESEROPREVALENCESURVEYINTHREEURBANSETTINGSINYUCATAN,MEXICO 3.1BackgroundDenguehasbecomethemostrapidlyspreadingvector-borneviraldiseasetransmittedthroughouttheAmericas.Brazil,ColombiaandMexicoaccountingforthelargestburdenofdiseaseintheregion[ 6 93 ].Whilethecasesreportedareasmallproportionofthetotalburdenofdiseaseoccurringinvulnerablecommunities,thereisscantinformationregardingtheextentoftransmissionincitiesandsuburbanareasinendemiccountrieslikeMexico.Theestimationoftherealburdenofdenguediseaserequiresstrongepidemiologicalsurveillancemethodsandmodelingtechniques[ 94 { 96 ].Toidentifytherealburdenofdengue,itisimportanttoacknowledgemultiplechallenges:1)Onlyasmallfractionofsymptomaticcasesarediagnosedandreportedthroughthesurveillancesystem;2)Datadescribingtheproportionofasymptomaticandmildinfectionsislimited,andhospitalrecordsonlypartiallyreporttheincidenceofseveredenguecases[ 97 ];3)Dierentialdiagnosisbetweendengueandotherendemicinfections(leptospirosis[ 98 ],rickettiosis[ 99 ]andtherecentlyintroducedchikungunyavirus[ 100 ]andZikavirus[ 101 ]isoftendicultintheabsenceofadequateserologicalandvirologicaltests;4)Surveillancedatacanhavereportingbiasesinsettingswheredierentendemicpathogensarecommonlyco-circulating[ 102 103 ]Tollthegapsoftheepidemiologicalsurveillancesystems,dengueseroprevalencesurveysareneededtocharacterizethespectrumanddynamicsofdengueinfectionsinselectedpopulationgroupsinendemicareas.DenguevirushasbeencirculatinginMexicosincethelateseventies.Despiteanincreaseinthereportednumberofdengueandseveredenguecases[ 104 ],seroprevalencesurveysareverylimitedandwillbehelpfultounderstandthechangesintheepidemiologyofthediseaseoverthelastthreedecades.Thisisparticularlyimportantbecauselargerandmorefrequentdengueoutbreakswithanincreasingproportionofseverecaseshavebeenreportedtotheepidemiologicalsurveillancesystemthroughoutthecountry.Alsoco-circulationofthefourserotypesofdenguevirusismore 32

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frequentlydetected[ 105 ].ThestateofYucataninMexicoemergesasaregionwithalonghistoryofdenguetransmission.DenguewasrstreportedintheYucatanin1979,followedbylargedengueoutbreaksandannualco-circulationofatleasttwoserotypesduringthelasttwodecades[ 104 ].Cross-sectionaldengueserosurveysprovidethebaselineinformationofdengueanditiskeytoassesstheeectivenessofdierentinterventionsavailablelikeinnovativevectorcontrolinterventionsandvaccines,amongothers[ 27 106 { 110 ] 3.2AimsEstimatethebaselinedengueseroprevalenceinthreedierenttransmissionsettingsinYucatan,Mexico.Thisstudywillprovideinformationthatwillbekeytoassesstheeectivenessofdierentinterventionsavailablelikeinnovativevectorcontrolinterventionsandvaccinesamongothers. 3.3Methods 3.3.1SamplingThesurveysamplewasestimatedbasedon50%prevalencewith3%errorand95%condenceinterval(95%CI)thatgaveasamplesizeof1,307individuals.Weincreasedthesamplesizeto1,700inordertohavehalfofthesamplesfromthechildrenpopulation(<18yearsold)anddistributedaccordingtothepopulationsizeinthethreesettings,withahigherproportionofsamplesfromMerida,followedbyProgresoandTicul.Thesamplingschemeincludedtwodierentapproaches.Therstonewasaschool-basedsurveywitharandomselectionofschoolsinthethreecitiesfollowedbyarandomselectionofchildren(5to18yearsold)intheselectedelementary,middleandhighschoolsofMerida,TiculandProgreso:eightschoolswereselectedfromMerida(vewereelementaryschools),sixschoolsfromProgreso(fourwereelementaryschools)andvefromTicul(threewereelementaryschools)(Fig. 3-1 ).PrioragreementsandanexplanationofthestudyaimsweremadewiththelocalministriesofHealthandEducationofYucatan.Werequestedthelistsofstudentsbygradetoobtainagesoftheparticipantsandtheirinformation.Wedidastratiedrandomizationbygradeandschooltohaveaproportionalsampleineachagegroup.We 33

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excludedparticipantswhosesiblingswerepreviouslyrandomizedandhaveprovidedsignedinformedconsent.InthecityofMerida,weconnedthestudytoschoolsinthecentralmetropolitanarea.Bloodsampleswereobtainedintheschoolaftertheparentssignedtheinformedconsent. Figure3-1. LocationoftheselectedschoolsforthedengueseroprevalencesurveyinYucatan,Mexico.A)LocationoftheschoolsinMerida;B)LocationoftheschoolsinProgreso;andC)LocationoftheschoolsinTicul. Theadults(18yearsold)wererandomlyselectedandstratiedbyagefrompeopleattendingpublicprimaryhealthcarecentersinMerida,ProgresoandTicul.Weincludedindividualswhodidnothavesignsofacutefebrileillness,whowererequestingahealthcerticateorwereunderclinicalfollow-upforanon-communicabledisease.Bloodsampleswereobtainedaftertheadultssignedtheinformedconsent.Theresultsweredeliveredpersonallytotheparentsofthechildrenparticipantsintheschools.Fortheadultpopulationtheresultswerereportedbytelephoneorhomevisitsdirectlytotheparticipants. 3.3.2StudyProceduresParticipantswereaskedtoprovidea5mLvenousbloodsampleandtocompleteabriefquestionnairewithbasicdemographicinformation.Bloodsampleswerecollectedinanticoagulant-freetubesbytrainedandcertiedhealthpersonnel,centrifugedwithin1.3hoursofcollection,andtransportedtothestatelaboratory.Sampleswerestoredat-705Cuntiltheserologicaltestingwasdone.Serumsampleswereobtainedfromindividualsandtestedagainstdengueinfectioninstatepublichealthlaboratory(LESPRE)oftheMinistryofHealth. 34

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Priorexposuretodengueandage-specicserostatusweredeterminedusingPanbioIgGindirectELISA.Priorexposuretodengueandage-specicseroprevalencesweredeterminedusingthestandardcut-opointstodenepositive(12Panbiounits)fromnegativesamples(<9Panbiounits)andindeterminateforthoseinbetween. 3.3.3EthicalReviewThisstudywasapprovedbytheinstitutionalreviewboardsatFredHutchinsonCancerResearchCenterandtheGeneralHospitalAgustnO'HoranofYucatan.Writtenconsentwasobtainedfromalladultparticipants(18yearsold)afterprovidingthemwithadetailedexplanationofthestudyandprocedures.Parents/guardiansofallchildparticipants(<18yearsold)wereaskedtoprovidewrittenconsentontheirbehalf. 3.3.4StatisticalAnalysisDescriptiveanalysisofthedemographicvariablesandalogisticregressionmodelweredoneusingR(version3.2.2).Wegroupedagein8yearsold,9-14,15-19,20-49and50.Weestimatedtheagespecicdengueseroprevalenceandtalogisticregressionmodelofthevariablesindependentlyassociatedwithdengueseropositivityastheoutcomevariable.Theindependentvariablesconsideredforthemodelwereage,gender,placeofresidence,previoushistoryofdengueandpreviousdengueconrmation.Theage-stratiedspecicprevalenceofpre-existingantibody-mediatedimmunitytoanyofthedenguevirusesisdenedastheproportionofthesampledindividualsintheagegroupwhoseserologicspecimenwaspositivetoanyserotypeusingPanbioDengueIgGIndirectELISA[ 67 { 69 ].Weplantoestimatecondenceintervals(95%)fortheseprevalenceestimatesthatwillbebasedonthestandarddeviationfromtheBinomialdistribution,assumingindependent,randomlyselectedobservations[ 18 20 71 111 ].43 35

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3.4Results 3.4.1DescriptionofthePopulationofStudyAtotalof1,731serumsampleswereobtainedforthisstudy.ThesampleswereobtainedfromJanuarythroughJune2014inthecitiesofMerida,ProgresoandTicul.Mostofthesamples(43%)werecollectedinMerida(748),27%inProgreso(472)and30%inTicul(511).Mostofthesampleswereobtainedfromhealthcarecenters(56%),24%fromelementaryschoolsand20%frommiddleandhighschools.Afterprocessingthesamples3.6%(63/1,731)hadindeterminateresultsfortheIgGELISAandwereexcludedfromthisanalysis.Weanalyzed1667bloodsamplesfrompeoplefromthethreecities:Merida(700),Progreso(469)andTicul(498).Thesampleswerelocatedgeographicallywidelythroughalltheneighborhoodsintheselectedcities(Fig. 3-2 ).WhilesomeclusteringofcasesappearedinMeridaandProgreso,thismayresultfromoversamplinginoneortwoschools. Figure3-2. LocationofthehouseholdsoftheparticipantsindengueseroprevalencesurveyinYucatan,Mexico.A)LocationoftheparticipantsinMerida;B)LocationoftheparticipantsinProgreso;andC)LocationoftheparticipantsinTicul.Thereddotsshowdengueseropositiveindividualsandgreentheseronegatives. Themeanageintheoverallpopulationwas25.8years(SD:18.04).Themeanageoftheindividualsdidnothavesignicantdierencesbetweenthreecities(p=0.172).Thepopulationwasbalancedbyagegroupswiththeexceptionofthe9to14and15to19yearoldsgroupsinProgresothatcontributedwithdierentproportionscomparedtotheothercitiesinthisstudy(Table 3-1 ).Mostofthepopulationofthisstudy(94%)wasborninthe 36

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Yucatan.ThisproportionissignicantlyhigherinTicul(98%),followedbyProgreso(93.8%)andMerida(89.1%).TiculisaruraltownsoitispossiblethatthemobilityofitspopulationismorewithintheYucatanstatecomparedwiththeothercities.Afewproportionoftheparticipants(5%)mentionedhavingprevioushistoryofdengue.ThisproportionwasfoundhigherinMerida(8.6%),followedbyProgreso(3.4%)andTicul(2%).Wealsofoundasmallproportionofpeoplerecalledhavingpreviouslaboratoryconrmationofdengue.Just2.8%hadpreviouslaboratoryconrmationfordengueandmostoftheparticipantsconrmedwerefromMerida(3.9%).Anotherinterestingndingwasthattheproportionoflaboratoryconrmationincreasedwithage.ApossibleexplanationforthesetwolastvariablescouldbethattheaccessibilitytohealthcareinMeridathatisthecapitalofYucatanishighercomparedwiththeothercitieslikeProgresoandTicul. Table3-1. DemographiccharacteristicsofthestudypopulationinYucatan,Mexico(N=1,667) VariableMeridaProgresoTiculTotal Age(Years)8101(14.5%)71(15.1%)69(13.8%)241(14.5%)9-1490(12.8%)110(23.4%)75(15.0%)275(16.5%)15-19128(18.3.%)164(13.6%)93(18.7%)285(17.0%)20-49292(41.7%)175(37.5%)188(37.8%)655(39.3%)5089(12.7%)49(10.4%)73(14.7%)211(12.7%)GenderMale291(41.5%)157(33.1%)184(36.9%)632(37.9%)Female409(58.5%)312(66.9%)314(63.1%)1035(62.1%)BorninYucatanYes624(89.1%)440(93.8%)487(97.8%)1551(94%)No76(10.9%)29(6.2%)11(2.2%)116(7%)HistoryofpreviousdengueYes60(8.6%)16(3.4%)10(2.0%)86(5.2%)No640(91.4%)453(96.6%)488(98%)1581(94.8%)PreviousconrmationofdengueYes27(3.9%)12(2.6%)8(1.6%)47(2.8%)No673(96.1%)457(97.4%)490(98.4%)1620(97.2%) 3.4.2DengueSeroprevalenceTheoverallestimateddengueseroprevalenceinYucatanwas73.6%(95%CI71.4%-75.7%)showingthatmostofthepopulationhadalreadybeenexposedtodengueinfection. 37

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ThedengueseroprevalenceintheYucatanincreasedwithageinallthreestudysettings(Fig. 3-3 ).Thelowestoverallseroprevalencewas51.4%(95%CI45%-57.9%)inthegroup8yearoldsandthehighestforthepopulation50yearsold(83.4%(95%CI77%-88.2%)(Figure5A).TheseroprevalenceinMeridawas68.6%(95%CI65%-72%),inProgreso68.7%(95%CI64.2%-72.8%)andinTicul85.3%(95%CI81.9%-88.3%).TheestimateddengueseroprevalenceinMeridawentfrom43.6%(95%CI33.7%-53.8%)inthegroup8yearoldsto79.8%(95%CI69.9%-87.6%)inthepopulation50yearsold(Figure5B).InProgresotheseroprevalencewentfrom45.1%(95%CI33.2%-57.3%)intheyoungeragegroupto71.4%(95%CI56.7%-83.4%)inthepopulation50yearsold(Figure5C).Ticulhadthehighestdengueseroprevalenceinallagegroupscomparedwiththeothertwocities.InTicultheseroprevalencewentfrom69.6%(95%CI57.3%-80.1%)inthe8yearsoldto95.9%(95%CI88.5%-99.1%)inthepopulation50yearsold(Figure5D).Wettedasimplelogisticregressionmodelfordengueseropositivity.Weestimatedtheoddsratioforeachofthevariablescollectedinthesurvey(age,sex,city,borninYucatan,andpriordengueclinicalorlaboratorydiagnosis).Theanalysisshowedthattheoddsofbeingseropositivealsoincreasewithage.Thereferencegroupforagewasthe8yearsoldgroup.The9-14yearsgrouphas2.43(95%CI1.69-3.50)timestheoddsofbeingseropositivecomparedtothereferencegroup(Table 3-2 ).The50yearsgrouphas4.74(95%CI3.08-7.46)timestheoddsofbeingseropositivecomparedtothereferencegroup.Femaleshad1.27(95%CI1.02-1.59)timestheoddsofbeingseropositivecomparedtomales.TheparticipantsfromTiculhad2.67(95%CI1.99-3.60)timestheoddsofbeingseropositivecomparedtotheparticipantsfromMerida.ThevariablesbeingbornintheYucatan,havinghistoryofpreviousinfectionswithdengueandhavingapreviousconrmationofdenguewerenotassociatedwithseropositivity(Table 3-2 ). 3.5DiscussionThisisthemostrecentcross-sectionaldengueserosurveyconductedinthreecitiesinthestateofYucatan,Mexico.Ourstudyestimatedanoveralldengueseroprevalenceof 38

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Figure 3-3. Dengue seroprevalence to Indirect IgG by age group and city, 2014 39

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T able3-2.DengueseroprevalencetoindirectIgGbyagegroupandcity,2014 V ariables Total(N=1667)Seropositives(N=1227)Seronegatives(N=440)OddsRatio95%CI Age (Years) 8 241(14.5%)124(10.1%) 117(26.6%) RefRef 9-14 275(16.5%)198(16.1%) 77(17.5%) 2.431.69-3.50 15-19 285(17%)209(17%) 76(17.2%) 2.591.81-3.75 20-49 655(39.3%)520(42.4%) 135(30.7%) 2.632.65-4.99 50 211(12.7%)176(14.3%) 35(8.0%) 4.743.08-7.46 Gender Male 632(37.9%)445(36.3%) 184(36.9%) RefRef Female 1035(62.1%)781(63.7%) 254(58.0%) 1.271.02-1.59 BorninYucatan No 116(6.9%)77(6.3%) 39(8.9%) RefRef Yes 1551(93.1%)1150(93.7%) 401(91.1%) 1.450.96-2.16 Historyofpreviousdengue No 1581(94.9%)1163(94.8%) 418(95.2%) RefRef Yes 86(5.1%)64(5.2%) 21(4.8%) 1.090.67-1.86 Previousconfrmationofdengue No 1620(97.2%)1190(97.0%) 428(97.3%) RefRef Yes 47(2.8%)37(3.0%) 12(2.7%) 1.250.64-2.70 40

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73.6%,similartootherdengueserosurveysdoneinMexicoandtheYucatan[ 15 ].In1985,theprevalenceofanti-dengueantibodiesintheurbanpopulationofYucatanwas72.5%[ 112 ].Anotherstudyinacohortofschoolchildrenfrom8to14yearsold(1987-1988)includingurbanandrurallocalitiesofMeridareportedanoveralldengueseroprevalenceof56.3%inchildrenlivinginurbanareascomparedto63.7%inchildrenlivinginruralareas[ 113 ].Morerecentstudieshavefoundanoverallseroprevalenceof59.9%and81.5%in1996and2006respectively[ 114 ].Thecasesreportedtothesurveillancesystemindicatedthatbothwomen(51%)andmen(49%)weresimilarlyexposedtodengue[ 113 ],whichisadierentndingcomparedtoourstudywherewomenweremorelikelytobeseropositivecomparedtomen(OR:1.14).Theagespecicseroprevalencewasaslowas51.5%inchildren8yearsoldandincreaseswithageupto83.4%intheagegroupof50yearsold.TheseroprevalenceinMeridawas68.6%,Progreso68.7%andTicul85.3%.Ticulhadthehighestseroprevalenceinallagegroups.ThisserosurveyofthreedierenturbanareasinYucatanhighlightstheheterogeneouslevelofexposuretodenguevirusthesesettings.TheseroprevalenceestimatedinourstudywassimilartoestimatesfromVenezuelaandBrazilamongotherendemiccountriesfromLatinAmericabutlowercomparedwiththeestimatesfromNicaragua[ 76 103 115 116 ].Inourstudytheoveralldengueseroprevalenceinchildrenandadolescents9-14yearsoldwas72%.ThisisarelevantndinggivingtheWorldHealthOrganization(WHO)recommendationsforintroductionoftherstdenguevaccinethatisalsolicensedinMexico(CYD-TDV-Denvaxia)[ 117 ].Thisvaccinewaslicensedtargetingthepopulationfrom9to45yearsold.TheWHOrecommendsintroducingthedenguevaccineCYD-TDVonlyingeographicsettingswhereepidemiologicaldatasuggestahighburdenofdiseaseinthepopulationstobetargetedforvaccination.Thishighburdencanbeobjectivelymeasuredbydengueseroprevalencesurveys.Theseroprevalenceshouldbe>70%intheagegroupthatisgoingtobetargetedforvaccinationinordertomaximizepublichealthimpactandcost-eectiveness[ 117 118 ]Thisdengueserosurveyprovidesdatafromschoolchildrenandtheadultpopulationattendingpublichealthservicesprovided 41

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bytheMinistryofHealthandmayrepresentthelowerandmediumsocioeconomiclevels,nevertheless,thedistributionofthesampleinthethreecitiesdemonstratedthatexposuretodengueinfectioniswidespread.Datafromtheearly80'sshowthatexposuretospecicserotypesinthepasthasalsobeenintenseandthattheexposuretodengueserotypes1and2hasbeenpredominantinthelasttwodecades[ 15 112 ].TheregressionmodelresultsshowedthatthemainriskfactorassociatedwithdengueseropositivityinYucatanwereage,sexandcity.ThevariablesbeingbornintheYucatan,havinghistoryofpreviousinfectionswithdengueandhavingapreviousconrmationofdenguewerenotassociatedwithseropositivityinthisstudy.Populationbasedserosurveysareusefultoidentifythoseagegroupsmoreatriskandwhichoneswouldbethebesttargetforinterventions[ 80 108 119 120 ].SimilarstudiesshouldbepursuedelsewhereinMexicoandotherendemiccountriestobetterunderstandthetransmissiondynamicsofdengueandtohelpevaluatetheeectivenessofinterventionslikevectorcontrolanddenguevaccinesinthenearfuture.Dengueserosurveyshavealsobecomekeyformodelingpotentialimpactsofthedierentinterventionsalreadyavailableandindevelopment[ 110 121 { 126 ].PreviousdengueserosurveysweredoneinMexicointheearly1980'sbutthesedataareverylimitedtocertainpopulationgroupsorlocalitiesneverthelessallofthemdemonstratedhighdengueexposureindierenttimeperiods[ 127 { 130 ].Oneofthemainlimitationsofthisstudyisthelackofdengueserotypespecicseroprevalencethatiskeytobetterunderstandtheintensityoftransmission,andestablishwhetherthepopulationhasbeenexposedtooneormoreserotypesaswellasthelevelofexposureinthedierentagegroups.Anotherlimitationisthatitisnotatruerandomsampleofthepopulationgivingthesamplingstrategytoenrolladultsinthehealthcarecentersinsteadofthecommunityrandomsample.Alsoadegreeofrecallbiasispresentwheninterviewingpeopleaboutpastexperiences,suchashavinghaddengueDengueepidemicshaveincreasedsignicantlyworldwide[ 4 7 131 ]Theglobalestimatesofdenguedistributionanddiseaseburdenremainimpreciseinmostoftheendemicareas[ 132 133 ]Therealburdenofthe 42

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dengueisunknownsincemostofthedengueinfectionsareasymptomatic,misdiagnosedornotreported[ 134 ].Someofthemainissuesindenguesurveillanceincludethelackofstandardizedreportingprocedures,variablediagnosticlaboratorycapacityintraditionalsurveillancesystems,alongwiththeabsenceofreportingfromtheprivatehealthsector,whichinourstudypopulationrepresentanimportantproportionofallhealthcareproviders[ 135 ].Seroprevalencesurveysareinvaluableidentifyingtheburdenofbothsymptomaticandasymptomaticinfectionsandquantifyinginfectionprevalenceandincidenceindierentepidemiologicalsettings[ 119 122 129 136 137 ].AsestablishedbytheMexicanDengueExpertGroupitisessentialtodevelopanevidence-basedproactivestrategythatprovidesevidencetodecisionmakers[ 135 ].Thesedatashouldincluderesultsfromclinicaltrials,epidemiologicalstudiesandbetterburdenofdiseaseestimatesinordertocreateasustainableimmunizationprogramwithalltheresourcesrequiredfortheadoptionofthenewvaccineanditsfurtherevaluation[ 135 ].Thisparticularstudycontributestoprovideevidenceregardingtheserologicalstatusofpotentialtargetpopulationsinahighlyendemicregionofthecountry. 43

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CHAPTER4EPIDEMIOLOGYOFDENGUEANDOTHERARBOVIRUSINASCHOOL-BASEDCOHORTINYUCATAN,MEXICO:BASELINEANDFIRSTYEARFOLLOW-UP 4.1BackgroundDengueisthemostrapidlyspreadingmosquito-bornevirusworldwide.Inthelast50years,itsincidencehasincreased30-foldwithgrowinggeographicexpansiontonewcountriesandfromurbantoruralsettings[ 5 ]Currentlymorethan40%oftheworldpopulationisatriskofinfection,andapproximately390millioninfectionsareestimatedgloballyeachyear,ofwhich96millionhaveclinicalmanifestationsofthevirus[ 4 ].CurrentlythereisnospecictreatmentforDENVinfectionorclinicalpredictorstopreventseveredisease[ 7 ].DengueVirus(DENV)hasfourcloselyrelatedserotypes,eachofthefourserotypescanberesponsiblefordengueepidemics.Eachofthefourserotypescanalsobeassociatedwithseveredenguediseasedependingonthesequenceandtimebetweeninfections,amongotherfactors[ 8 { 10 ].Theclinicalspectrumofdenguerangesfromasymptomaticinfectionstolife-threateningseveredisease;approximately50%to90%ofdengueinfectionsareasymptomatic[ 11 12 ].Measuringtheburdenofdiseaseattributabletodengueinfectionisneededtounderstandthepotentialimpactofdenguecontrolinterventions.Thereforeitisveryimportanttostrengthenandincreasesurveillancetoimproveourknowledgeofhowmanycasesareactuallyoccurringinthecommunity.Anaccurateriskanalysisandallocationofresourcesfordenguecontroldependsontimelydiseasesurveillance[ 138 ].Surveillancesystemsbasedonthemonitoringandnoticationofsymptomaticcaseshavelowsensitivityandrarelydetectloworsporadictransmission[ 139 140 ].Theproportionofasymptomaticinfectionsvarywidelywithinthesamepopulations,geographicalareas,andoverdierentepidemiologicalperiods[ 141 ].Underreportingofdenguecasestonationalsurveillancesystemshindersaccuratelocal,regionalandglobalcalculationsofdiseaseburden[ 142 ].Itisneededtostrengthenpassivesurveillancesystemsbyincorporatingactivesurveillancemethods(e.g.,house-to-housevisits,schoolabsenteeismorself-identicationoffeverepisodes)and 44

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improvetherecognitionofinapparentinfectionsandunderreportingofunspeciedfebriledengueinfections.Vectorcontrolstrategiesstillremainastheonlymeasureofdengueprevention[ 13 14 ].Thesetraditionalapproachesthattargetthevectorinallstagesofdevelopmenthaveproventobeunsustainableandineectiveatpreventingdenguetransmission[ 2 ].Denguepreventionrequiresinnovatione.g.vaccines,eectivevectorcontrolandgeneticallymodiedmosquitoestoprovidemoreeectiveandsustainablestrategiesfordenguecontrol,especiallyingrowingandcomplexurbanenvironments[ 25 26 ].Currentlyvevaccinecandidatesarenowinclinicalstagesofdevelopment.TwovaccinesareinPhaseIItrials,andtwoareinPhaseItesting[ 27 ].OnlyonevaccinehascompletedtwoPhaseIIItrialsanditisbeinglicencedforintroductioninsomecountries[ 28 { 30 ].Thisvaccinehasanestimatedecacyof64.7%and56.5%intheLatinAmericaandSouthEastAsiatrials,respectively.Thevaccineecacyinthepooledanalysisofbothtrialswasfoundtobesignicantlyhigherinparticipantswithpre-existingdengueneutralizingantibodiescomparedtothosewhowereseronegative.Theserotype-specicvaccineecacywasheterogeneousinbothtrials,andthevaccineecacyagainsthospitalizationfordengueinSouthEastAsiawas67.2%andinLatinAmerica80.3%[ 28 29 31 ].Theavailabilityofalicensedvaccineposesdierentchallengestocurrentdenguecontrolprogramssinceitisnotexpectedtouniversallycoverallsusceptibleandat-riskpopulationsorevenprovidecompletecoverageintargetgroups.Vaccineintroductionwillthereforebeagradualprocess.Ineachofthesepopulationsthereareseveralquestionsthatneedtobeaddressedregardingtheclinicalspectrumandtransmissionriskswherethevaccinemayhaveapotentialbenet[ 143 ]. 4.2AimsTheaimofthisstudyistocharacterizethecurrentlocalepidemiologyofarboviralinfectionsestimatingage-specicattackrates,theproportionofasymptomaticinfectionsin 45

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thestudyarea,incidencerateratiosandassessthecovariatesassociatedwithinfectionsinaprospectivecohortstudyinthreetransmissionsettingsinYucatan,Mexico. 4.3MethodsThedescriptionofthestudysites,enrollmentactivitiesandthestudyproceduresweredescribedindetailinthesecondchapterofthisdissertation. 4.3.1CaseDenitions 4.3.1.1DenguecasedenitionDenguecaseswereclassiedaccordingtothe2009WHOCaseClassication[ 5 ]. 4.3.1.2DenguewithoutwarningsignsDenguewithoutwarningsignsisdenedbyacutefever(38.5)andtwoormoreofthefollowingsymptoms:headache,myalgia,arthralgia,retroorbitalpain,rash,hemorrhagicmanifestationsorleukopenia[ 5 ]. 4.3.1.3DenguewithwarningSigns:Denguefeverwithanyofthefollowingwarningsigns:abdominalpain,persistentvomiting,uidaccumulation,lethargy,mucosalbleeding,uidaccumulation,liverenlargement,orincreasinghematocritwithdecreasingplatelets[ 5 ]. 4.3.1.4Severedengue:Denguefeverwithanyofthefollowing:severebleeding,severeplasmaleakageleadingtoshockoruidaccumulationwithrespiratorydistress,ororganfailureorinvolvementasevidencedbyliverALTorAST1,000,impairedconsciousness,failureoftheheartorotherorgans[ 5 ]. 4.3.1.5LaboratorycriteriaforconrmationfordengueAcutedenguecaseAsymptomaticparticipantwhotestedpositivefordengueevidencedby:1)detectionofDENVRNAbyRT-PCR,2)viralisolationofDENV,3)seroconversionasdeterminedbyaDENV-specicimmunoglobulinM(IgM)captureenzyme-linkedimmunosorbentassay(ELISA)usingpairedacuteandconvalescentsera,and/or4)a4-foldriseintotalantibodytiterbetweenacuteandconvalescentseraasmeasuredbyInhibitionELISA[ 144 { 146 ]. 46

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InapparentdengueinfectionAparticipantwhosepairedannualserumsamplesdemonstratedseroconversionas4-foldincreaseinantibodytiterasdeterminedbyInhibitionELISAandwhodidnotexperienceasymptomaticdengueinfectionduringthetimeoffollow-up[ 144 { 147 ].PrimaryandsecondarydengueinfectionAdengueinfectionisclassiedasaprimaryDENVinfectionifseroconversionwasobservedandwasconsideredasecondaryDENVinfectionifa4-foldincreaseinantibodytiterwasobservedinpairedconsecutiveannualsamples,asdeterminedbyInhibitionELISA.Ifaparticipant'sserumcontainedanti-DENVantibodyatenrollmentortheparticipantexperiencedapreviousDENVinfectionduringthecohort,priortoadocumentedsubsequentDENVinfection,thiswasalsoconsideredasecondaryDENVinfection[ 148 ].FirstandsecondDENVinfectionsareidentiedbycountingthenumberoftheinfectionsdocumentedinaparticipantwhoenteredthecohortdengue-nave,usinga4)]TJ /F1 11.955 Tf 9.29 0 Td[(foldriseintiterbyInhibitionELISAinpairedannualsamplestoidentifyinapparentinfectionsordiagnosticassaysinacutesamplesforsymptomaticcases[ 145 147 148 ]. 4.3.1.6DenguepriorexposureDengue-nave.Aparticipantwhodidnothavedetectableanti-DENVantibodyatenrollmentasevidencedbyInhibitionELISAandhasnotexperienceaDENVinfectionduringthecohortstudy.Non-dengue-nave.Aparticipantwhohadanti-DENVantibodyatenrollmentasevidencedbyInhibitionELISA[ 145 147 148 ]. 4.3.2Chikungunyacasedenition 4.3.2.1AcutechikungunyainfectionAparticipantwithabruptonsetoffever38.5accompaniedbyjointpain,musclepain,headache,nausea,fatigueandrash.Thejointpainisoftenverydebilitating,butusuallylastsforafewdaysormaybeprolongedtoweeks[ 149 ]. 47

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4.3.2.2LaboratorycriteriaforconrmationforchikungunyaAsymptomaticparticipantwhotestedpositiveforchikungunyaevidencedby:1)detectionofCHIKVRNAbyRT-PCR,2)viralisolationofCHIKV,3)enzyme-linkedimmunosorbentassays(ELISA)conrmingthepresenceofIgMandIgGanti-chikungunyaantibodies[ 149 ]. 4.3.3Zikacasedenition 4.3.3.1ZikavirusdiseasePatientwithrashusuallypruriticandmaculopapularwithtwoormoreofthefollowingsignsorsymptoms:fever,usually38.0,conjunctivitis(non-purulent/hyperemic),arthralgiaormyalgia[ 150 151 ]. 4.3.3.2LaboratorycriteriaforZikaconrmationParticipantwhomeetsthecriteriaforasuspectedcaseANDhaslaboratoryconrmationofrecentZikavirusinfectionby1)RNAorZikavirusantigeninanyspecimen(serum,urine,saliva,tissueorwholeblood);2)PositiveZikaIgMantibodiesANDPlaquereductionneutralization(PRNT90)forZikavirus(titers20)andfourormoretimesgreaterthanthetitersforotheraviviruses;ANDexclusionofotheravivirus[ 152 ]. 4.3.4EthicsStatementThisstudywasconductedasacollaborationbetweentheMinistryofHealthofYucatanandtheCenterforInferenceandDynamicsInfectiousDiseases.ThisstudywasapprovedbytheInstitutionalReviewBoardsatFredHutchinsonCancerResearchCenterandtheGeneralHospitalAgustnO'HoranofYucatan.Writtenconsentwasobtainedfromalladultparticipants(18yearsold)afterprovidingthemwithadetailedexplanationofthestudyandprocedures.Parents/guardiansofallchildparticipants(18yearsold)wereaskedtoprovidewrittenconsentontheirbehalf. 4.3.5StatisticalAnalysisTheage-specicbaselineseroprevalencefordengueviruswasestimatedfortheentirecohort.Fortherstyearoffollow-up,thefollow-uptimewasestimatedasthetimebetweenenrollmentandtheendofthereportedstudyperiod(August2016),orwithdrawfromthe 48

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study.Forthosewhowerelostoffollow-up,person-yearswerecalculatedasthetimebetweenenrollmentandthelastcontactwithstudypersonnel,plusone-halfthetimebetweenthelastcontactandthedaterecordedaslosttofollow-up.Theanalysisofdengueinfectionswaslimitedtothoseparticipantswhocompletedtheyearandcontributedabloodsampleatthebeginningandtheendoftheyear.Inordertoprovideconservativeestimatesofdengueinfectionincidence,sincetheexacttimingofthedengueinfectioncouldnotalwaysbeascertained,personswhoexperiencedadengueinfectionintherstyearoffollow-upcontributedperson-timeforthatentireyear.Fortheanalysisofdengueinfections,theageoftheparticipantwasdenedastheagewhentheirannualsamplewascollected.Thecrudeincidenceper1,000person-yearswas1,000timesthenumberofdengueinfectionsdividedbythenumberofperson-yearsinthenewdataset(=1,000Xinfections/(sum(days)/365.25))[ 103 144 145 ].Theincidencerateratiowasalsoestimated[ 153 { 155 ].Theincidenceratewasestimatedforeachgroup(non-naveanddenguenave).Thentheincidenceratesindenguenon-naveandnavepeople,respectively,are: ^r0=m0 T0inthenon-nave(controlgroup), (4{1) ^r1=m1 T1inthenave(exposedgroup). (4{2) Then,theincidencerateratiois IRR=m1 T1 m0 T0=m1 T1T0 m0,(4{3)whichcantakeanyvaluein[0,1).TocalculateacondenceintervalforthetrueIRR,werstusealogtransformation: ln^IRR=lnm1 T1)]TJ /F7 11.955 Tf 11.96 0 Td[(lnm0 T0.(4{4)Sinceoutcomesinthetwogroupsareindependent, 49

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Varln^IRR=Varlnm1 T1+Varlnm0 T0.(4{5)Bythedeltamethod,wegettherst-orderappoximatevariances Varlnmi TiTi mi2Varlnmi Ti(4{6)Usinganexponentialmodelforthetimestoevents,i.e.,Poissonprocess,weget Varlnmi Ti=mi T2i.(4{7)Substitutingthisbackintoequation 4{6 ,weget Varlnmi Ti=Ti mi2mi T2i=1 mi.(4{8)fori=0,1.Therefore,theestimatedvarianceoflnIRRisapproximately 1 m0+1 m1(4{9)andanapproximate95%condenceintervalforthetruelnIRRis lnm1T0 T1m01.96r 1 m0+1 m1(4{10)Thecorresponding95%condencelimitsforIRRare m1T0 T1m0exp1.96r 1 m0+1 m1(4{11)Thehazardratios(HRs)and95%condenceintervals(95%CIs)fordengueinfectionswereestimatedusingCoxproportionalhazardsmodelsandadjustingforpotentialconfounders[ 155 156 ].Oneadvantageofthesurvivalmodelisthatitisvalidwithrightcensoring(lost-offollow-up).LetT0andT1bethetotalperson-timecontributedbeforeanalysistimetinthecontrolgroupandtheexposedgroup,respectively.Wedenethehazardfunctionattimetforsubjectiwithpcovariates(explanatoryvariables)xi=(xi1,.....,xip)as 50

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(tjXi)=0(t)exp(1Xi1+...........+pXip) 4.4Results 4.4.1BaselineCharacteristicsoftheParticipantsAtotalof767familiescorrespondingto3,400participantswereenrolledfromJanuarytoJune,2015fromthethreesitesinYucatan.ThemajorityofthefamiliesandparticipantswerefromMerida(2,021(59.4%)),followedbyTicul(738(21.7%))andProgreso(641(18.9%)).Amongtheparticipants1,869(54.97%)werefemales,1,463(43.03%)were14yearsoldoryounger,and2,970(87.35%)wereborninthestateofYucatan(Table. 4-1 ).Theseparticipantscontributedwith3430.87person-yearsfortherstyearoffollow-up.Themeanparticipationtimeis1.01years(368.31days)perparticipant(range0.04-1.94).Atotalof1,094(32.2%)participantsdidnotcompletetherstyearoffollow-up.Atotalof320(29.25%)oftheparticipantswerelosttofollow-up,210(19.20%)movedoutofthestateofYucatanand564(51.55%)askedforvoluntarywithdrawnfromthestudy(Fig. 4-1 ). Figure4-1. Flowchartofparticipantsinthedenguecohort,Yucatan,Mexico. 51

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T able4-1.Demographiccharacteristicsofthecohortpopulationenrolledin2015Yucatan,Mexico(N=3,400) V ariable Merida(N=2,021)Progreso(N=641)Ticul(N=738)Total Age (Years) < 8 593(29.34%)188(29.33%)198(26.83%)979(28.79%) 9-14 289(14.30%)86(13.42%)109(14.77%)484(14.24%) 15-19 85(4.21%)33(5.15%)40(5.42%)158(4.65%) 20-49 842(41.66%)277(43.21%)337(45.66%)1456(42.82%) > 50 212(10.49%)57(8.89%)54(7.32%)323(9.5%) Gender Male 917(45.37%)286(44.62%)328(44.44%)1531(45.03%) Female 1104(54.63%)355(55.38%)410(55.56%)1869(54.97%) Numberoffamilies 463(60.36%)153(19.95%)151(19.69%)767(100%) Averagenumberofpeopleperfamily(Range)4.36(2,11)4.18(2,11)4.88(2,16)4.43(2,16) BorninYucatan Yes 1775(87.83%)550(85.80%)645(87.40%)2970(87.35%) No 129(6.38%)52(8.11%)46(6.23%)227(6.68%) Noinformation 117(5.79%)39(6.08%)47(6.37%)203(5.97%) Withdrawn Yes 778(38.50%)123(19.19%)195(26.42%)1096(32.24%) No 1243(61.50%)518(80.81%)543(73.58%)2304(67.76%) 52

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4.4.2DengueBaselineSeroprevalenceThebaselineseroprevalencewasestimatedin2,732participantswhoauthorizedtotakeabloodsampleatenrollment.Theoverallbaselinedengueseroprevalenceinthecohortwas73.39%(95%CI71.7%-75.0%)showingthatmostofthepopulationhadalreadybeenexposedtodengueinfection(Fig. 4-2 ). Figure4-2. Age-specicbaselinedengueseroprevalenceinthecohort,Yucatan,Mexico. ThehighestdengueseroprevalencewasfoundinTicul(81.59%),followedbyMerida(73.99%)andProgreso(61.37%).Thedengueseroprevalenceincreaseswithageandfemalesinthecohorthadhigherseroprevalenceestimatescomparedwithmales(Table 4-2 ). 53

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T able4-2.Baselineexposuretodengueintheparticipantsofthecohortenrolledin2,015inYucatan,Mexico(n=2,732) Baseline exposureDengue-nave(n=708)Denguenon-nave(n=2005)Indeterminate(n=19)Total Cit y Merida 404(25.38%) 1178(73.99%) 10(0.63%) 1592 Progreso195(38.24%) 313(61.37%) 2(0.39%) 510 Ticul 109(17.30%) 514(81.59%) 7(0.69%) 630 Age(Years) 8 418(55.36%) 329(43.57%) 8(1.05%) 755 9-14 150(36.85%) 253(62.16%) 4(0.98%) 407 15-19 30(25.64) 86(73.50%) 1(0.85%) 117 20-49 96(7.67%) 1149(91.84%) 6(0.48%) 1251 50 14(6.93%) 188(93.07%) 0(0%) 202 Gender Male 352(30.39%) 972(68.39%) 14(1.21%) 1158 Female 356(22.62%) 1213(77.06%) 5(0.32%) 1574 54

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4.4.3SuspectedSymptomaticArboviralInfectionsSincetheenrollmentandbaselinebloodsampletotherstyearfollow-upofthecohort,199suspectedarbovirusinfectionscasesorundierentiatedfebrileillnesseswereidentiedinthe3,400population.Theseparticipantscontributedwith3430.87person-yearsfortherstyearoffollow-up.Themostcommonclinicaldiagnosiswasundierentiatedfever105(52.76%),followedby76suspecteddengue(38.19%),chikungunya17(8.54%)andonesuspectedcaseofZika.(Table 4-3 ).Thedengueincidencerateforsuspectedcasesof21.86per1,000person-years.Alltheconsultationswereintheoutpatientclinic.Themostcommonsymptomspresentedbythecohortpopulationwere:fever,headache,myalgia,arthralgia,rashandconjunctivitis.Notsignicantdierenceswerefoundamongallthearboviralclinicaldiagnosis(p=0.560)(Table 4-4 ).Noseveresymptomswereidentiedinthestudypopulation.Asthesymptomsareunspecicamongthearboviralinfections,noincidencerateswereestimatedforprobabledenguecases.Theonlyincidenceratethatcouldbeestimatedistheincidencerateofarbovirussuspectedfeveranditwas58.02per1,000personyears. 4.4.4ConrmedArboviralSymptomaticInfectionsThisanalysiswasdoneusingasdenominatorthe199participantswithsuspectedarbovirussymptomaticinfectionsorundierentiatedfebrileillnesseswereidentiedinthe3,400cohortpopulation.Amongthesymptomaticarboviralinfections12(6.03%)werelaboratory-conrmedasdengue-positive,30(15.08%)wereconrmedaschikungunya-positive,8(4.02%)wereconrmedasZika-positive,and149(74.87%)wereconsideredfeverofunknownorigen.Theseparticipantscontributedwith3430.87person-yearsfortherstyearoffollow-up.Theoverallincidencerateofarboviralconrmedsymptomaticinfectionswas14.57per1,000person-years(95%CI:10.82,19.21).Theincidencerateofconrmedsymptomaticdenguewas3.49casesper1,000person-years(95%CI:1.87,5.86).Intherstyearofthestudy,themajorityofsymptomaticcaseswerecausedbytheserotypeDENV1(52%);followedbyserotypeDENV2(36%)andDENV4(12%).DENV3wasnotisolatedduringthestudyperiod. 55

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Table 4-3.ProbablesymptomaticcasesofarboviralinfectionsinthecohortofYucatan,Mexico(N=199) DengueZik aChikungunyaIndeterminateNegativeTotal Probablesymptomatic cases Dengue 7(58.33%)1(12.5%)11(36.67%)1(100%)56(37.84%)76(38.19%) Zika 1(8.33%)6(75%)0 0 10(6.76%)17(8.54%) Chikungunya 1(8.33%)00 0 0 1(0.50%) Undierentiatedfever3(25%)1(12.5%)19(63.33%)0 82(55.41%)105(55.41%) 56

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Table4-4. Symptomsofthefebrilecasesstudiedduringtherstyearfollow-upinthecohortofYucatan,Mexico(N=199) SymptomsYesNo Fever189(94.97%)10(5.03%)Headache158(79.40%)41(20.60%)Conjunctivitis107(53.77%)92(46.23%)Myalgia140(70.35%)59(29.65%)Rash102(51.26%)97(48.74%)Abdominalpain13(6.53%)186(93.47%)Upperrespiratorysymptoms11(5.53%)188(94.47%) Nohospitalizationsordeathsduetodenguesymptomaticinfectionwerereportedduringtherstyearofthecohort.Thehighestincidenceofdenguewasobservedintheparticipantsinthegroupfrom15-19yearsofage,femalesandintheparticipantsfromMerida(Table 4-5 ).Theincidencerateofconrmedsymptomaticchikungunyawas8.74casesper1,000person-years(95%CI:5.81,12.30).Thehighestincidenceofchikungunyawasobservedintheparticipantsinthegroup50yearsoldormore,femalesandinTicul.(Table 4-5 ).TheincidencerateofconrmedsymptomaticZikawas2.33casesper1,000person-years(95%CI:0.989,4.529).ThehighestincidencerateforZikawasestimatedalsoinTicul(10.25per1,000person-years).Onecaseofco-infectionofdengueandchikungunyawasalsoidentiedThemajorityofcasesoccurredfromAugusttoDecember2015thatishistoricallythedengueseasoninYucatan,Mexico. 4.4.5TotalDengueInfectionsTheanalysisofdengueinfectionswaslimitedto1,890participantswhocompletedtherstyearfollow-upandcontributedwithabloodsampleatthebeginning(January2015)andattheendoftherstyearoffollow-up(June-October2016).Therewere1,037(69.15%)participantsfromMerida,379(20.05%)fromProgresoand474(25.08%)fromTicul.Intotal,the1,890participantscontributed2271.14person-yearsand89dengueinfectionswereconrmed,foranincidencerateof39.2infectionsper1,000person-years(95%CI31.66,48.01)(Table 4-6 ).Theoverallratioofsymptomaticcasesamongdengueinfectionswas7.41dengueinfectionsperdenguesymptomaticcase.Thehighestincidenceofdenguewasobservedinthe 57

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T able4-5.Incidencerates(IR)per1,000person-yearsofArboviralconfrmedsymptomaticinfections P erson-yearsatriskDengueIR(95%CI)ChikungunyaIR(95%CI)ZikaIR(95%CI) All participants3430.87 123.45(1.87,5.86)30 8.62(5.81,12.3)82.3(0.99,4.53) Agegroups(years) 8 1001.4 32.99(0.602,8.753)10 9.99(4.78-18.37)43.99(1.08,10.23) 9-14 502.5 11.99(0.03,11.07)4 7.96(2.14,20.38)23.98(0.45,14.37) 15-19 145.3 16.88(0.09,38.29)1 6.88(0.09,38.29)00) 20-49 1490.1 74.69(1.88-9.68)11 7.38(3.68,13.21)21.34(0.15,4.85) 50 291.5 00 4 13.72(3.69,35.13)00 Gender Male 1511.68 00 9 5.95(5.81,12.3)82.29(2.72,11.30) 3 1.985(0.39,5.79) Female 1919.19 126.25(3.23,10.92)21 10.942(6.78,16.73)52.61(0.84,6.08) City Merida 2164.26 94.16(1.89,7.89)18 8.32(4.93,13.14)20.92(0.13,3.34) Progreso 601.76 23.32(0.37,12.01)5 8.31(2.68,19.29)11.66(0.02,2.25) Ticul 682.85 11.46(0.02,8.15)7 10.25(4.11,21.12)57.32(2.36,17.09) 58

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participantsonthegroupfrom15-19yearsofagefollowedbythe8yearolds.ThehigherincidencerateswereobservedalsoMerida,females,navesandintheagegroupfrom20-49(Table 4-6 ).Thelowestratesofsymptomaticcaseswereseenintheoldestagegroup,50years-oldwithnonsymptomaticcasesandthe9to14years-oldgroupwithrates6.66casesper100infections. Table4-6. Incidencerates(IR)per1,000person-yearsofalldengueinfections(N=1,890) Person-yearsatriskDengueinfectionsIR(95%CI) Allparticipants22718933.9(31.7,48.0)Age(Years)8692.843550.5(35.7,69.5)9-14359.741747.3(28.5,74.1)15-1992.91996.9(47.2,177.8)20-49959.682323.9(15.6,35.4)50166.11530.1(11.0,66.7)GenderMale984.492727.4(18.4,39.4)Female1286.796248.2(37.3,61.4)CityMerida1248.145140.9(30.7,53.3)Progreso481.762756.1(37.7,80.4)Ticul540.381120.4(10.7,35.4)PriorexposuretodengueNave645.880123.9(98.9,153.4)Non-nave1625.295.5(2.7,10.2) 4.4.6PrimaryDengueInfectionsintheNavePopulationAmongthe708participantswhoenteredthecohortasdengue-nave,478dengue-naveparticipantscompletedtherstyearfollow-upandcontributedwith490.31person-yearsoftime.Overtherstyearoffollow-up80dengue-naveparticipantsexperiencedadengueprimaryinfection.Theoverallproportionofseroconversionwas16.74%.Theincidencerateofprimarydengueinfectionswas163.16infectionsper1,000person-years(95%CI:130.2,202.1).ThehighestproportionofseroconversioninnaveswasestimatedinMerida(18.47%),followedbyProgreso(17.48%andthelowestwasinTicul(10.47%)(Table 4-7 ). 59

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Table4-7. Seroconversioninthedengue-naveatbaseline(n=478) Merida(n=249)Progreso(n=143)Ticul(n=86)Total SeroconvesioninnavesYes46(18.47%)25(17.48%)9(10.46%)80(16.74%)No203(81.53%)118(82.52%)77(89.53%)398(83.26%) 4.4.7IncidenceRateRatioofArbovirusConrmedSymptomaticCasesandDengueInfectionsTheincidencerateratio(IRR)wasalsoestimatedandthe95%condenceintervalusinganexponentialmodelforthetimestoevent.TheIRRassumingasexposedgroupthepopulationthatwasnaveatbaselineandtheunexposedthenon-naves.TheIRRswereestimatedfordengueconrmedsymptomaticcases,chikungunyaconrmedsymptomaticcases,Zikaconrmedcasesandoveralldengueinfections.TheIRRfortotaldengueinfectionsandZikasymptomaticcasesweresignicantusingtheLogranktest(Table 4-8 ).Morezikaconrmedcasesweredetectedinnavesandmorechikungunyaconrmedcasesweredetectedinnon-naves. Table4-8. Incidencerateratioforallarboviralinfectionsintherstyearoffollow-upofthecohortinYucatan,Mexico. EventIRR(95%CI)Logrank Dengueconrmedcases1.4(0.47,4.14)p=0.539Denguetotalinfections22.2(11.13,44.18)p=0.001Chikungunyaconrmedcases0.5(0.21,1.24)p=0.114Zikaconrmedcases3.7(1.06,13.26)p=0.041Anyarboviralinfection0.9(0.49,1.61)p=0.625 4.4.8SurvivalModelforDengueInfectionsACoxproportionalhazardmodelwasttedfortotaldengueinfections.Thevariablesincludedinthemodelwere:Age,denguepriorexposure,gender,city,overcrowdinginthehousehold(morethan5peoplelivinginthesamehousehold),andoneormoreinfectionsinthesamehousehold.Intheunivariateanalysis,ageasacontinuousvariablewassignicantasaprotectivefactorfordengueinfections(HR=0.98,95%CI:0.96,0.99).Therewasnosignicantassociationbetweentotaldengueinfectionsandovercrowdinginthehouseholdbuttherestofthevariablesweresignicantlyassociatedwiththehavingadengueinfection. 60

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Thehighesthazardratioswereestimatedforbaselineexposuretodengue(HR=20.5,95%CI:10.30,40.91)andhavingmorepeopleinfectedwithdengueinthehousehold(HR=57.9,95%CI:37.21,90.08).Thosetwovariableswereincludedinthenalmodelaspotentialconfounders.Inthenalmodel,havingadengueinfectionduringtherstyearoffollow-upwassignicantlyassociatedwithfemalegender,livinginTiculorProgreso,oneormoreinfectionsinthehousehold,andbeingdenguenaveatbaseline.Agewasnotsignicantlyassociatedwiththeoutcome,itwasconfoundedbypriorimmunitytodenguethatincreaseswithage.(Table 4-9 ). Table4-9. Hazardratiosfortotaldengueinfectionsintherstyearoffollow-upofthecohortinYucatan,Mexico. VariableHazardratio(95%CI) Age8RefRef9-141.060.59,1.8915-191.770.79,3.9620-491.270.72,2.24501.170.44,3.09GenderMalesRefRefFemales1.651.04,2.62CityMeridaRefRefTicul7.173.12,16.49Progreso2.041.12,3.76BaselinestatusDenguenon-naveRefRefDenguenave15.357.19,33.08Oneormoreinfectionshousehold22.3114.01,35.55 4.5DiscussionOverthelastvedecades,denguehasemergedasamajorhealthprobleminthetropicalregionsworldwideincludingLatinAmericaandtheCaribbean[ 6 7 29 146 ]andmorerecentlychikungunyaandZikavirusthatemergedintheAmericascausingsignicantepidemicsinmostofthecountriesinfestedwithAedesaegypti[ 157 { 160 ].Mexicoisconsideredoneof 61

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theendemicdenguecountriesintheregionandsince2015thethreearbovirushavebeenco-circulatinginmanyareasofthecountry[ 15 104 134 ].Thiscohortenrolled767familiesand3,400participantsfromthreecitiesinthestateofYucatan,Mexico.ThemajorityofthefamilieswerefromMerida(59.4%)thecapitalcityofthestateofYucatan,followedbyTicul(21.7%)andProgreso(18.9%).Themedianageofthecohortwas24.8yearsandaround46%oftheparticipantsare14years-oldoryounger.Theratiofemale:maleis1.2similartotheratiointhestateofYucatan[ 87 ].MostoftheparticipantswereborninthestateofYucatan(87.35%),theaveragenumberofpeopleperfamilyis4.43witharangefrom2to16.ThelargestfamiliesareinTiculthatisalsothemostruralsettingofthestudy.MostofthedenguecohortsinLatinAmericaandSouthEastAsiaarepediatriccohorts[ 76 84 92 103 144 145 147 148 161 ].TheYucatancohortisuniquegiventhatithasparticipantsfromallagegroupsbuttherecruitmentstartsattheschoolsanditextendstothefamilies.Thiscohortgivestheopportunitytocollectprospectivedatainallagestounderstandbetterthefullburdenofdengueandotherarbovirusthathaveemergedintheregion.The3,400participantscontributedwith3,480person-yearsduringtherstyearoffollow-up.Theestimatedbaselineseroprevalenceinthecohortwas73.39%.ThecitywiththehigherseroprevalencewasalsoTicul(81.9%),followedbyMeridaandProgreso.Theseroprevalenceincreasedwithageasexpected.Theseseroprevalenceestimatesareverysimilartotheseroprevalencesestimatedinthedengueseroprevalencesuveyinthepreviouschapterofthisdissertation(73.6%).Atotalof199suspectedarboviralsymptomaticinfectionsweredetectedduringtherstyearoffollow-up.Themostcommonsymptomwasfever(95%),followedbyheadache(79%)andmyalgia(70.35%).Thesesymptomsaresimilaramongarboviralinfectionsthatiswhytheclinicaldiagnosismightnotbeaccurate.Itisneededtoconrmthesuspectedcasestoknowwhichvirusarecirculatinginendemicareas.Theincidencerateofarboviralsuspectedcaseswas58.2per1,000personyears.Thedengueincidencerateforsuspectedcasesof21.86 62

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per1,000person-years(95%CI17.32,27.25).ThisestimatefallsinthecondenceintervalofincidenceratesestimatedintheNicaraguandenguecohort[ 145 162 163 ].Amongthesymptomaticarboviruscasesjust6.60%wereconrmedasdengue.Theincidencerateofconrmedsymptomaticdengueinfectionswas3.49per1,000person-years.Thisproportionofconrmationandtheincidencewerelowerthantheseestimatesinotherdenguecohorts[ 76 80 84 92 103 144 145 147 148 161 ].Thiscanbeexplainedthatduringthisrstyearoffollow-upchikungunyaviruswasintroducedinYucatan,MexicoandthesearbovirusthataretransmittedbyAedesaegyptimightcompeteinthevectorcausinglowercirculationoftheotherarbovirusinthiscasedengue[ 164 { 166 ]ThemajorityofthesymptomaticcaseswerecausedbyDENV1,followedbyDENV2andDENV4.DENV3wasnotisolatedinthecohortsamples.TheseresultsareconsistentwiththeisolatesfromthenationalvirologylaboratoryinYucatan[ 88 ].AlldenguesymptomaticcaseswereclassiedasdenguewithoutwarningsignsaccordingtotheWHO2009casedenitionfordengue[ 5 ]TheincidenceratesofconrmedsymptomaticZikaandconrmedchikungunyawere2.33casesper1,000person-yearsand8.74casesper1,000person-yearsrespectively.Onecasewithco-infectionofdengueandchikungunyawasalsodetectedasinsomeotherendemiccountrieswithco-circulationofmultiplearbovirus[ 167 ].Fromtheoriginalcohortatotalof1,094(32.2%)participantsdidnotcompletetheactivitiesoftherstyearoffollow-upbut2,304arebeingfollowedfromtheoriginalcohort.Mostoftheparticipantswithdrewfromthestudyatthetimeoftakingtherstyearfollow-upbloodsampleortheywereconsideredlostoffollow-up.Themobilityonthispopulationisveryhighsoassessingpotentialmobilityisimportantforthefutureofthecohorttobeabletoassesstransmissionofarbovirusasplanforthispopulation.Theanalysisofthedengueinfectionswasdonein1,890participantswhohadabaselineandfollow-upbloodsample.Theincidenceratefordengueinfectionswas39.19infectionsper1,000person-years.Thisincidencerateislowercomparedwithothercohortsaroundthe 63

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worldbutitisshowingthatthedenguetransmissionwaslowduringthisrst-yearoffollow-up[ 76 80 84 92 103 144 145 147 148 161 ].Thehighestincidenceofdenguewasobservedintheparticipantsonthegroupfrom15-19yearsofagefollowedbythe8year-olds.Thegroupfrom15-19yearsofageisjust4.65%ofthecohortandjust25.64%ofthemwerenaveatbaseline.Moreparticipantsfromthisagegrouphavebeenenrolledinthelastmonthssoitisexpectedtohavebetterdenominatorsforthenextyearsoffollow-up.Among708participantswhowerenaveatbaseline,478(67.51%)completedtherstyearoffollow-up.Inthenaveswereconrmed80dengueinfectionsforanoverallseroconversionrateof16.74%.Theincidencerateonprimaryinfectionswas123.88per1,000person-years.Thisisverysimilartoestimatesfromotherdengueprospectivecohortsinendemicareas[ 144 145 ]Theincidencerateratioofconrmedoveralldengueinfectionsamongnavesandnon-naveatbaselinewas22.179(95%CI11.13,44.18).IntheYucatancohortthepopulationthatwasdenguenavehad22.17timestheincidenceofdengueinfectionsasthosethatwerenon-naveatbaseline.TheincidenceratioofZikaconrmedcaseswas3.7(95%CI1.1,13.3).TheotherIRRestimatedwerenotsignicant.Thehazardratiosestimatedfordengueinfectionsduringtherstyearoffollow-upweresignicantforfemalegender,livinginTiculorProgreso,oneormoreinfectionsconrmedinthehousehold,andbeingdenguenaveatbaseline.Inthenalmodelaftercontrollingforbaselinedenguestatusagewasnotsignicantlyassociatedwiththeoutcome.Toourknowledge,thisistherstdenguecohortstudythatusessurvivalanalysisasatooltounderstandbetterthetransmissiondynamicsofdengueandotherarbovirus.Thelimitationsofthisstudyincludeascertainmentofcasesthroughenhancedpassivesurveillance.Thus,somedenguecasesmaynothavebeendetectedduetoparticipantsnotseekinghealthcare.Anotherlimitationisthatserumsamplesforcohort-wideserologicaltestingareonlyavailableforparticipantsonceperyearsoitwillbeidealtohavemoreseroprevalencedatapointsintimebutlogisticallyisdiculttomanage.ThisstudyreliesonInhibitionELISA 64

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toassessinapparentorasymptomaticinfections.ThegoldstandardtoassessDENVinfectionistheplaquereductionneutralizationtest(PRNT)butitisverylaborintensiveandexpensivesotestingalltheparticipantswasnotfeasible.Insummary,thisstudyreportedbaselineseroprevalence,incidenceofdengueinfections,denguecasesandotherarboviralcasesinacommunity-basedcohortfromthreesettingsinYucatan,Mexicousinganalyticmethods.Thisistherstreportoftheresultsfromthisprospectivecohortthatistheonlywaytodeterminetheincidenceofarboviralinfectionstoestimatethetruerateofdisease,whichisusuallyunderestimatedbypassivesurveillance.Thesedatawillbeusedtoestimatediseaseburden.Theincidenceestimateswillbeusefulforpolicymakersandtoevaluateinterventionslikevectorcontrolstrategiesandvaccines.Futureanalysisofhouseholdtransmission,clusteranalysisandeectivenessofinterventionsareplanned. 65

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CHAPTER5THEEPIDEMIOLOGYANDTRANSMISSIBILITYOFZIKAVIRUSINGIRARDOTANDSANANDRESISLAND,COLOMBIA 5.1BackgroundFirstisolatedintheZikaForestofUgandain1947,Zikavirus(ZIKV)isanarbovirusprimarilytransmittedbyAedesaegyptimosquitoes[ 33 ].AlthoughZIKVhascirculatedinAfricaandAsiasincethe1950s,littleisknownaboutitstransmissiondynamics[ 34 ].RecentoutbreaksinYapIslandintheFederatedStatesofMicronesia(2007),FrenchPolynesia(2013),andotherPacicislands,includingCookIslands,EasterIsland,andNewCaledonia(2014),indicatethatZIKVhasspreadbeyonditsformergeographicrange[ 35 { 38 ].InApril2015ZIKVwasisolatedintheNortheastofBrazil[ 39 ].AsofMarch11,2016,around500,000Zikavirusdisease(ZVD)caseshavebeenestimatedinBrazil,andautochthonouscirculationhasbeenobservedin31countriesintheAmericasregion.FurtherspreadtocountrieswithinthegeographicalrangeofAe.aegyptimosquitoesisconsideredlikely[ 42 ].ZIKVisaavivirusinthesamegenusasdenguevirusandyellowfevervirus.Infectiontypicallycausesaself-limiteddengue-likeillnesscharacterizedbyexanthema,low-gradefever,conjunctivitis,andarthralgia[ 40 ].Whileillnessisbelievedtobemildorasymptomaticinapproximately80%oftheinfections[ 41 ],anincreaseinratesofGuillain-Barresyndrome(GBS)hasbeenobservedduringZIKVoutbreaks[ 42 { 44 ].Furthermore,inOctober2015,theBrazilianMinistryofHealthreportedadramaticincreaseincasesofmicrocephalyinNortheastBrazilwhereZIKVhadbeencirculating[ 45 ].OnthebasisofthepossiblelinkbetweenZIKV,GBSandmicrocephaly,theWorldHealthOrganizationdeclaredapublichealthemergencyonFebruary1,2016[ 46 47 ].ZikaviruswasrstdetectedinColombiainmid-September2015inamunicipalitycalledTurbacoontheCaribbeancoast.Turbacoislocatedapproximately20minutesfromCartagena,awellknowncommercialandtouristhub(Figure 5-1 ).InOctober2015,ZIKVspreadthroughthecentralregionofthecountry,appearinginareaswithendemicdengueandongoingcirculationofchikungunya(CHIKV)since2014.ThroughMarch2016,Colombiahasreported 66

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over50,000casesofZVD,makingitthesecond-mostaectedcountryinthisoutbreak,afterBrazil.Therewere2,090laboratory-conrmedcaseswiththerestbeingsuspectedcasesorconrmedbyclinicalndings[ 157 168 ].UptoMarch2016,280casesofneurologicalcomplicationsincludingGuillain-Barresyndrome(GBS)andthreedeathspossiblyassociatedwithZVDhavebeenreportedinColombia[ 169 ].AsofMarch2016,therehavebeenseveralsuspectedbutnoconrmedcasesofZIKV-associatedmicrocephalyinColombia[ 157 168 ].InthispaperwedescribelocalZIKVoutbreaksinColombiainGirardotandSanAndresIslandbetweenSeptember2015andJanuary2016forwhichdetailedepidemiologicaldataareavailable.Weconductaninvestigationtodenetheepidemiologicalfeaturesoftheseoutbreaksandtoestimatecorrespondingtransmissionparameters. 5.2AimsTodescribelocalZIKVoutbreaksinColombiainGirardotandSanAndresIslandbetweenSeptember2015andJanuary2016forwhichdetailedepidemiologicaldataareavailable.Weconductedaninvestigationtodenetheepidemiologicalfeaturesoftheseoutbreaksandtoestimatecorrespondingtransmissionparameters. 5.3Methods 5.3.1Settings 5.3.1.1SanAndresSanAndresisthelargestislandinaColombianarchipelagointheCaribbeansealocatedabout750kmnorthofmainlandColombiaand230kmeastofNicaragua(Figure 5-1 ).Theislandhasanareaof27km2,apopulationof54,513inhabitantsacross13,652households,andapopulationdensityof2,932habitantsperkm2in2010[ 170 171 ].Theaveragetemperatureis273C,and80%ofthetotalannualrainfallof1,700mmoccursduringtheheavyrainyseasonbetweenOctoberandDecember.Theweatherishumidsubtropicalwithoccasionaltropicalcyclonesandhurricanes.ThepopulationinSanAndreshastwomainethnicgroups:Afro-Colombians(17.5%)andRaizal(anethnicgroupofmixedAfro-CaribbeanandBritishdescent)(39.2%)[ 171 ].ThemostproductivebreedingsitesofAe.aegyptiinSanAndres 67

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areunprotectedwatercontainerslocatedinthehouseholds.SanAndreshasexperiencedlowdenguetransmissionsince1983.Since1995,thefrequencyofdengueoutbreaksincreasedtoeverytwotoveyearswithameanannualincidenceof436casesper100,000inhabitantsbetween1999and2010[ 170 ].In2014,CHIKVbegancirculatinginSanAndres,reachinganannualincidenceof3651casesper100,000inhabitants[ 172 ]. 5.3.1.2GirardotThecityofGirardotislocated134km(2hoursdrive)fromthecapitalcityofBogota(Figure 5-1 ).Girardothas102,225inhabitantsacrossapproximately23,000householdsbasedonthemostrecentcensusfromtheNationalStatisticsDepartment(NSD)[ 173 ],thoughthepopulationtriplesduringweekendsandholidays.Girardotis289metersabovesealevel.Theaveragetemperatureis33.3C,andtherelativehumidityis66%.Themeanannualprecipitationis1,220mmwitharainyseasonextendingfromMaythroughOctober[ 174 ].ThemostproductivebreedingsitesofAe.aegyptiinGirardotareunprotectedprivatewatercontainers,suchaswaterstoragetanksusedinthehouseholdsduringthedryandrainyseasons,whilepublicspacesprovidemorebreedingsitesduringtherainyseason[ 175 ].Girardothasexperiencedhyperendemictransmissionofdenguesince1990withsimultaneouscirculationofallfourserotypes;themeanannualincidencewas572.2per100,000inhabitantsbetween1999and2010[ 170 ].Inlate2014,CHIKVbegancirculatinginGirardotreachinganannualincidenceof394per100,000inhabitantsin2014and8,416per100,000inhabitantsin2015. 5.3.2CaseDenitionandLaboratoryAnalysisWeanalyzedsurveillancedatafromninelocalhealthcaresitesinSanAndresandtwenty-twolocalhealthcaresitesinGirardot.StandardizedcasedenitionsusedinbothareasweredenedbytheMinistryofHealth(MoH)andColombianNationalInstituteofHealth(C-NIH)atthebeginningoftheZIKVepidemic.AsuspectedZVDcaseisdenedasapersonwholivedortraveledinanareabelow2,000metersabovesealevelwhopresentswithmaculopapularexanthema,temperaturehigherthan37.2C,andoneormoreofthefollowing:non-purulentconjunctivitis,arthralgia,myalgia,headache,ormalaise.Alaboratoryconrmed 68

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Figure 5-1. Map of Colombia 69

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caseisasuspectedcasewithaZIKVpositivereverse-transcriptase-polymerase-chain-reaction(RT-PCR)resultasdeterminedbytheC-NIHvirologyreferencelaboratory.AclinicallyconrmedcaseisasuspectedcasethatlivedortraveledinanareawithlaboratoryconrmedZIKVcirculationpriortoonsetofsymptoms[ 176 ].Atthestartoftheoutbreak,allsuspectedcaseswerereportedbasedonthesuspectedcasedenitiontotheColombiannationalsurveillancesystem.OncelaboratoryconrmationfromC-NIHwasperformedforcasesinGirardotandSanAndres,thenewsuspectedZIKVcaseswerelaboratoryconrmediftheyfellintotheriskgroupsdenedbytheC-NIH:newborns,age<1year,age>65years,andcaseswithco-morbidities[ 176 ].AfterZIKVcirculationwasconrmedintheregions,allsuspectedcaseswerereclassiedasclinicallyconrmed.[ 177 ] 5.3.3DataCollectionThedatawascollectedusingtheC-NIHstandardreportformforZikasurveillance.Theformincludessocio-demographicvariables.Weanalyzedadeidentieddatasetwiththefollowingvariables:gender,age,pregnancystatus,dateofsymptomonset,datethecasevisitedthehealthcarefacility,datethecasewasreportedtothenationalsurveillancesystem,andcasetype(suspected,laboratoryconrmed,clinicallyconrmed)[ 178 179 ]. 5.3.4StatisticalAnalysisWecalculatedoverallandage/gender-specicattackratesusingpopulationcensusdatafromNSD[ 173 ].SurveillancedatawereanalyzedusingRversion3.2.0[ 180 ].Fordescriptiveresults,categoricalvariablesarepresentedasproportionsandcontinuousvariablesbythemedianandinterquartilerange(IQR)orrange.Theimpactofageandgenderonattackrateswastestedusinglog-linearmodelsforcasecountswithagecategory,gender,andaninteractionasindependentvariableswithpopulationsizeasanoset.ToestimatethebasicreproductivenumberR0ineachpopulation,weusedmaximumlikelihoodmethodstotachain-binomialmodeltodailyincidencedata[ 181 ].R0isthemedianeectivereproductivenumberduringthegrowthphaseoftheepidemic,after 70

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accountingforearlyunder-reporting.(SeeSupplementaryOnlineMaterialsforadditionaldetailsonthemodel.)[ 36 40 182 183 ] 5.4Results 5.4.1SanAndresInSanAndres,weidentied928reportedZVDcases(Table 5-1 ).Ofthesecases,52(5.6%)werelaboratoryconrmedbyRT-PCRonacutephasesamplescollectedwithinvedaysofsymptomonset,and876(94.4%)caseswereclinicallyconrmed. Table5-1. CharacteristicsofreportedcasesofZikaVirusDisease RegionSanAndresGirardot Totalnumberofcases9281936Laboratoryconrmedcases52(5.6%)32(1.7%)Clinicallyconrmedcases876(94.4%)1904(98.3%)Female589(63.5%)1138(58.8%)Medianage,years(IQR)31(15,47)34(24,46)Mediantime,days,tovisithealthcarefacilityfromsymptomonset(IQR)4(1,16)1(1,2) ThedatesofsymptomonsetamongcasesinSanAndresrangedfromSeptember6,2015,toJanuary30,2016(Figure 5-2 ).ThoughtheearliestcasereportedsymptomonsetonSeptember,6,2015,thelocalhealthcareauthoritiesdidnotreceivelaboratoryconrmationofZIKVuntilOctober22,2015.Thenumberofcasespeakedinepidemiologicalweek45(November8to14)andsubsidedinthelastweekofDecember.Themediantimebetweensymptomonsetandvisitingahealthcarefacilitywas4days(IQR1to16).79%ofcaseswerereportedtothenationalsurveillancesystemonthesamedaytheyvisitedthehealthcarefacility.ThemedianageofreportedZVDcasesinSanAndreswas31yearsold(IQR15to47;range12daysto82years).589(63.5%)ofthereportedcasesoccurredinfemales.Duringthistimeperiod70denguecasesand10CHIKVcaseswereconrmedinSanAndres.TheoverallattackrateforZVDreportedbylocalsurveillancewas12.13per1,000SanAndresresidents.Thegender-specicattackrateswere15.34per1,000femalesand8.91per1,000males;thedierencewassignicantadjustingforage(p<0.001).Casesoccurredamongallagegroups,buttheincidenceofZVDdetectedbylocalsurveillancewashighestamong 71

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Figure5-2. DailyZVDincidenceforSanAndres,Colombia persons20to49yearsold(Figure 5-3 );therewassignicantheterogeneityacrosstheagegroups(p<0.001).Attackrateswerehigherinfemalesacrossallagegroups10yearsoldandabove;therewasasignicantinteractionbetweenageandgender(p<0.001). Figure5-3. Age-andgender-specicZVDattackratesforSanAndres,Colombia ThirtytwopregnantwomenwithZVDwerereportedinSanAndresandarebeingfollowedaccordingtonationalguidelines.ByMarch2016,fourteenofthemhadgivenbirthwithnomicrocephalyreported.TherewereeightneurologicalsyndromesreportedinSanAndres,includingGBSandmeningoencephalitisattributedtoZIKVandamongthemonedeathwas 72

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reported.TheincidencerateofneurologicalsyndromesamongZVDcasesinSanAndresis8.6per1,000cases. 5.4.2GirardotInGirardot,weidentied1,936reportedZVDcases(Table 5-1 ).Ofthesecases,32(1.7%)werelaboratoryconrmedbyRT-PCRonacutephasesamplescollectedwithinvedaysofsymptomonsetand1,904(98.3%)wereclinicallyconrmed.Duringthistimeperiodwereconrmed67denguecasesand37CHIKVcasesinGirardot.ThedateofsymptomonsetamongcasesinGirardotrangedfromOctober19,2015,toJanuary22,2016(Figure 5-4 ).TherstsuspectedcasewasreportedonOctober23,2015,withlaboratoryconrmationobtainedonJanuary27,2016.Thenumberofcasespeakedinepidemiologicalweek48(November29toDecember5)andsubsidedinearlyJanuary.Themediantimebetweensymptomonsetandvisitingahealthcarefacilitywas1day(IQR1to2days).89%ofcaseswerereportedtothenationalsurveillancesystemonthesamedaytheyvisitedthehealthcarefacility.ThemedianageofconrmedZVDcaseswas34yearsold(IQR24to46;range15daysto92years).1138(58.8%)ofthecasesoccurredinfemales. Figure5-4. DailyZVDincidenceforGirardot,Colombia TheoverallattackrateforconrmedZVDdetectedbylocalsurveillancewas18.43per1,000Girardotresidents.Thegender-specicattackrateswere20.53per1,000femalesand16.07per1,000males;thedierencewassignicantadjustingforage(p<0.001).Cases 73

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occurredamongallagegroups,buttheincidenceofZVDdetectedbylocalsurveillancewashighestamongpersons20to49yearsold(Figure 5-5 );therewassignicantheterogeneityacrosstheagegroups(p<0.001).Attackrateswerehigherinfemalesinallagegroupsexceptinthose10to14and65to69yearsold;therewasnosignicantinteractionbetweenageandgender. Figure5-5. Age-andgender-specicZVDattackratesforGirardot,Colombia SixteenpregnantwomenwithZVDwerereportedinGirardotandarebeingfollowedaccordingtonationalguidelines.ByMarch2016,sevenofthemhadgivenbirthwithnocomplicationsormicrocephalyreported.NinecaseswithGBShavebeenreportedafteraninitialsuspectedZIKVinfection;laboratory-conrmationofZIKVispending.TherewerenodeathsattributedtoZIKV.TheincidencerateofneurologicalsyndromesamongZVDcasesinGirardotis4.6per1,000cases. 5.4.3EstimationsoftheBasicReproductiveNumberThebasicreproductivenumber(R0)wasestimatedusingdailyincidencedata.Themodelassumesameanserialinterval(timebetweensuccessivecasesinachainoftransmission)of22days,basedonameanincubationperiodinhumansof5days,anextrinsiclatentperiod(timefrominfectiontoinfectiousnesswithinthemosquito)of10days,andameaninfectiousperiodinmosquitoesof10days.Under-reportingisassumedtobehigh(only10%ofcasesreported)atthestartoftheoutbreakandfullreportingisassumedtobeachievedinfourweeksafter 74

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theoutbreakbeginstogrow.TheestimatedR0fortheZikaoutbreakinSanAndreswas1.41(95%CI1.15to1.74),andtheR0inGirardotwas4.61(95%CI4.11to5.16)(Table 5-2 andFigure 5-6 ).Oddsratiosforgenderandageeectswereobtainedfromthelikelihoodmodel,indicatingincreasedoddsoftransmissionamongfemalesandadultsaged20to49yearsoldinbothSanAndresandGirardot(Table 5-2 ).TheestimationprocedurewasalsoappliedtodailyincidencedatafromapublishedoutbreakinSalvador,Brazil,thatoccurredbetweenFebruary15,2015,andJune25,2015;14,835caseswerereportedwithanoverallattackrateof5.5casesper1,000Salvadorresidents[ 39 ].TheestimatedR0oftheZikaoutbreakinSalvador,Brazilwas1.42(95%CI1.35to1.49).SensitivityanalysesarereportedintheSupplementaryOnlineMaterials,includingvaryingtheincubationperiodinhumans,theinfectiousperiodinhumans,theinfectiousperiodinmosquitoes,thedurationofunder-reporting,andthelevelofunder-reportingatthestartoftheoutbreak. Table5-2. EstimatesofR0gender-specicoddsratiosfortransmission,andage-specicoddsratiosfortransmissionforZVDinSanAndresandGirardot,Colombia. SanAndresGirardot ParameterLevelEst.95%CIEst.95%CI R01.41(1.15,1.74)4.61(4.11,5.16)ORgenderMale----Female1.71(1.50,1.95)1.28(1.17,1.40)ORage20-49----0-190.86(0.74,0.99)0.37(0.33,0.42)500.74(0.63,0.88)0.46(0.41,0.52) 5.5DiscussionWereportsurveillancedataonZIKVoutbreaksintworegionsinColombiabetweenSeptember2015andJanuary2016.Therstregion,SanAndres,isasmall,densely-populatedislandthatisrelativelyisolatedfromcontinentalColombia.Thesecondregion,Girardot,isatypicalmoderatelysizedColombianmunicipality.BothregionshaveendemictransmissionofdengueandexperiencedrecentoutbreaksofCHIKV.WedescribekeyepidemiologicalfeaturesoftheZikaoutbreaksandestimatetheR0fromdailyincidencedata. 75

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Figure 5-6.Estimatesofeectivereproductivenumber.A)Estimatesofeective R (red)and model-ftteddailycasenumbers(green)fortheoutbreakofZVDinSanAndres, Colombia.Thereportingratioisassumedtoincreaselinearlyfrom10%onand beforeSeptember30,2015,to100%in4weeks.Dashedcurves(bothredand green)areconservative95%CIs.Histogramingreyshowstheepidemiccurve.The horizontalorangelineindicatesthereferencevalueof1.Thetwoverticalorange linesindicatethetimeintervalusedfortheestimationof R 0 .B)AsA)for Girardot,Colombia.ThereportingratioincreasesonOctober19,2015. 76

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TheoverallattackratesforZVDasdetectedbylocalsurveillancewere12.13casesper1,000residentsofSanAndresand18.43casesper1,000residentsofGirardot.TheseattackratesaresimilartothosereportedfromYapIsland(14.3per1,000)[ 35 ]buthigherthanthosereportedinSalvador,Brazil(5.5per1,000)[ 39 ].Inbothareas,signicantlyhigherattackratesareobservedamongwomen,especiallythoseofchild-bearingage.TheColombiangovernmentissuedanepidemiologicalalertonDecember2015toactivelysearchforpregnantwomenwithZika-likesymptomsinareaswithactivetransmission[ 184 185 ].Thiseortmaypartiallyexplainthendings,thoughdierencesingender-specicattackratespersistwhenonlycasesoccurringpriortoDecemberareconsidered[ 186 { 188 ].Casesoccurredinallagegroups,butthemostaectedagegroupwas20to49yearofage,similartopreviouslypublishedoutbreaksinYapIsland,FederatedStatesofMicronesia,andinSalvador,Brazil[ 35 39 ].AsthepopulationwasfullysusceptibletoZikatransmissionbeforetheoutbreaks,itisreasonablethatallagegroupswouldbeaected.Forty-eightpregnantwomenwithZVDwerereportedfromSanAndresandGirardot.Thesewomenarebeingfollowedaccordingtonationalguidelines[ 184 185 ]withnoconrmedcasesofmicrocephalyobservedyet.Seventeenneurologicalsyndromes,includingGBSandZIKV-associatedmeningoencephalitis,wereidentied,similartoreportsfromFrenchPolynesiaandBrazil[ 44 189 ].Laboratory-conrmationofthesecasesischallengingbecauseneurologicalsymptomsgenerallyappeartwoweeksafteracutesymptoms[ 190 ]atwhichtimeZIKVdiagnosisbyRT-PCRisnotpossibleandserologicaltestsareunreliablebecauseofcross-reactivitywithdengue[ 167 191 ].AsZIKVcanbedetectedinurinelongerthaninblood[ 192 ],usingurinesamplestoconrmZIKVinGBScasesmaybeanalternative[ 193 ].ThesechallengesunderscoretheneedforreliablediagnosticteststhatcandetectZIKVaftertheviremicperiod.Thebasicreproductivenumber(R0)wasestimatedineachareausingdailyincidencedata.OurestimatedR0fortheZikaoutbreakinSanAndreswas1.41(95%CI1.15to1.74),andtheR0forGirardotwas4.61(95%CI4.11to5.16).Applyingthesamemethodswith 77

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previouslypublisheddata,weestimatedthattheR0forZikavirusinSalvador,Brazil,was1.42(95%CI1.35to1.49)[ 39 ].WeconsidertheestimatefromSanAndrestobethemostreliablebecauseitisasmall,denselypopulatedisland,whileGirardothasahigherriskofimportationbecausethepopulationuctuatesduringweekendsandholidays.TherelativemagnitudesofR0areconsistentwiththehigherdenguetransmissionobservedinGirardotversusSanAndres.EstimatesofR0inZikaarenotwidelyavailable,thoughreportssuggestanR0of4.3to5.8inYapIslandandR0of1.8to2.0inFrenchPolynesia[ 194 ].ArecentmanuscriptconsideringtheFrenchPolynesianoutbreakreportedarangefrom1.9to3.1[ 59 ].Relativelyfewcaseswerelaboratoryconrmed.Themajorityofcaseswereclinicallyconrmed,andthesymptomscouldbecausedbyotheretiologiessuchasdengue.Thisreportonlyincludessymptomaticcaseswhoattendedahealthcarefacilityandwerecapturedbythesurveillancesystems.ZIKVusuallycausesrelativelymildillnesslastingseveraldays,andaround80%ofinfectionsarecurrentlybelievedtobeasymptomatic,sowearelikelymissingmanymildorasymptomaticcases[ 41 ].Wealsodonothaveareliableestimateofunder-reportingatthesesites.Earlyunder-reportingappearedtobeespeciallyapparentintheGirardotoutbreak,andthesharpincreaseincasesobservedmaybeduetoincreasedpublicawarenessofthedisease.ThisphenomenoncanresultinanoverestimateofR0.Well-designedstudiescanprovidevaluableinsight.PhylogeneticanalysesofcirculatingZIKVstrainswillbecriticalforunderstandingwhethermutationsintheviralgenomeareassociatedwithanincreasedseverityofdisease,asmanifestedbymicrocephalyandGBSinthisoutbreak.Householdstudiescanallowformoreaccurateestimationoftransmissiondynamicsandenhanceunderstandingofasymptomaticinfection.StudiesarerequiredtounderstandtheinteractionsbetweenZIKV,dengue,CHIKV,andotherco-circulatingarbovirusesandtheirimpactondisease.ItisalsonecessarytoincreasesurveillanceofneurologicalsyndromesassociatedwithZVD,likeGBSandencephalitis.TheevidenceforacausalrelationshipbetweenZIKVandmicrocephalyisstrengthening[ 195 { 197 ].RecentevidencefromtheFrenchPolynesiaoutbreaksuggestsanestimatednumber 78

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ofmicrocephalycasesassociatedwithZIKVisaroundoneper100womeninfectedinthersttrimester[ 198 ].CurrentlytheColombianGovernmentisfollowingacohortofpregnantwomenthatreportedZika-likesymptomsanytimeduringtheirpregnancy.ThosewhoaredetectedduringtheacutephasearebeingdiagnosedwithZIKVRT-PCR.Allwomenwillbefolloweduntiltheendofpregnancy,andthefetuswillbeevaluatedduringpregnancyandpost-natallyfortwelvemonths.Theprospectivecollectionofdatathroughthisandothersimilarnationalcohortswillbeessentialforassessingcausality,determiningriskfactors,andestimatingratesofbirthdefects.TheresultsofthisandotherreportsconcludesthattransmissionofZIKVmaybewidespread.Vectorcontrolhashadlimitedsuccessincontrollingotherarboviruses,suchasdengue.Asafeandecaciousvaccine,especiallyforwomenofchild-bearingage,maybeneededtoreducethediseaseburden. 79

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CHAPTER6CONCLUSIONSArboviralinfectionsareconsideredpublichealthproblemsworldwidebuttheircumulativeimpactonglobaldiseaseburdenhasnotbeenfullyassessed.Aproximately40%oftheworldpopulationisatriskofarboviralinfectionsbecausetheyliveinareasinfestedbyAedesmosquitoes,mainlyAedesaegyptiandAedesalbopictusthevectorsfordengue,chikungunyaandZikavirus.Intheiracutestages,arboviralinfectionscauseabroadspectrumofdisease,rangingfromasymptomaticinfectiontoseveredisease.Thiswidespectrumofthediseasemakesdiculttoassessthefullburdenandtransmissionparametersoftheseviraldiseasesusingepidemiologicalsurveillancedata.TobetterunderstandtheepidemiologyandtransmissionofarbovirusinLatinAmerica,aeldstudywasdesignedtocollectthebaselinedengueseroprevalenceandaprospectiveschool-basedcohorttoassessincidenceandpotentialriskfactorsassociatedwithtransmissionofthesevirusesinthepopulationofYucatan,Mexico.ThestateofYucatanisconsideredadengue-endemicstatewithheterogeneouslocaltransmissionamongthedierentcities.InitiallythebaselineeldstudiesinYucatanweredesigntounderstandbetterthetransmissiondynamicsofdenguebutchikungunyaandZikavirusemergedinMexicoandinLatinAmericain2015-2016providingtheopportunitytoanalyzetheepidemiologyandtransmissiondynamicsofthesevirustoo.TherstcasesofZikaviruswereconrmedinLatinAmericain2015-2016andColombiawasthesecondcountryintheregionmoreaectedbyZikavirusafterBrazil.ThelastchapterofthisdissertationincludestheanalysisofepidemiologicalsurveillancedatafromtwoZikaoutbreaksinColombiaandtheestimationsoftransmissionparametersusefultoestimatetheimpactoftheoutbreakandplanningfurtherinterventions. 80

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Epidemiologicaleldstudiesarekeytoassessthebaselinedenguetransmissioninendemicpopulations.Thepopulationbasedserosurveysareextremelyusefultoidentifytheagegroupsmoreatriskandwhichoneswouldbethebesttargetforinterventions.ThebaselineseroprevalencesurveywasconductedinarandomsampleofthepopulationfromthreeurbansettingsinYucatan,Mexico.thesesettingswereclassiedbasedonepidemiologicaldataashigh,mediumorlowtransmission.MeridadecapitalofYucatanwasclassiedinitiallyashighrisk,TiculwasclassiedasmediumriskasProgresoaslowrisk.InthesurveytheoverallestimateddengueseroprevalenceinYucatanwas73.6%(95%CI71.4%-75.7%)showingthatmostofthepopulationhadalreadybeenexposedtodengueinfection.WealsoobservedthatdengueseroprevalenceintheYucatanincreasedwithageinallthreestudysettings.Thelowestoverallseroprevalencewas51.4%inthegroup8yearoldsandthehighestforthepopulation50yearsold(83.4%)asexpectedgiventhattheyoungeragegroupshavebeenexposedlesstodenguecomparedwiththeoldestagegroups.ThebaselineseroprevalenceinMeridawas68.6%,inProgreso68.7%andinTicul85.3%.TheseseroprevalenceswereexpectedgiventhatthestateofYucatanisawell-knowndengueendemicareainMexico.Thesendingswereveryinterestinggiventhattheinitialriskclassicationusingtheepidemiologicalsurveillancedatawasnotcompletelyaccurate.Ticulendedupbeingthehighestriskpopulation,andMeridaandProgresoweremediumrisk.ItisimportanttotakeintoaccountthesizeofthecityandpossibleheterogeneitiesindenguetransmissioninlargercitieslikeMeridacomparedwithsmallestcitieslikeTicul.Inordertoknowthebaselineexposureconditionstodengueandotheravivirusinendemicareas,eldstudiesarerequiredgiventhecomplexityofthetransmission,thebroadclinicalspectrumofthedisease(largeproportionofasymptomaticinfections),andtheunderreportingtothesurveillancesystems.ThemodelresultsshowedthatthemainriskfactorassociatedwithdengueseropositivityinYucatanwereage,sexandcity.ThevariablesbeingbornintheYucatan,havinghistoryofpreviousinfectionswithdengueandhavingapreviousconrmationofdenguewerenotassociatedwithseropositivityinthisstudy. 81

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Prospectivecohortstudiesareimportanttoestimatetheriskofinfectionbydenguevirus.Currently,thereareseveralongoingcohortstudiesindengueendemicareasinSouthEastAsiaandLatinAmerica.TheYucatancohortstudyisthesecondstudyfromthebaselinedenguestudies.Duringtherstyearoffollow-updengueandotherarbovirusincidencerateswereestimated.Thiscohortisuniquegiventhatithasparticipantsfromallagegroups.Theenrollmentwasinitiallyatelementaryschoolsandextendedtothefamilies.ThiscohortgivestheopportunitytocollectprospectiveincidencedatatounderstandbetterthelocaltransmissiondeterminantsandfullburdenofdengueandotherarbovirustransmittedbyAedesmosquitosthathaveemergedrecentlyintheAmericas.Thiscohortenrolled767familiesand3,400participantsfromthreecitiesinthestateofYucatan,Mexico.ThemajorityofthefamilieswerefromMerida(59.4%)thecapitalcityofthestateofYucatan,followedbyTicul(21.7%)andProgreso(18.9%).Themeanageofthecohort18.40yearsandaround46%oftheparticipantsare14years-oldoryounger.MostoftheparticipantswereborninthestateofYucatan(87.35%),theaveragenumberofpeopleperfamilyis4.43witharangefrom2to16.ThelargestfamiliesareinTiculthatisalsothemostruralsettingofthestudy.Theestimatedbaselineseroprevalenceinthecohortwas73.39%.ThecitywiththehigherseroprevalencewasalsoTicul(81.9%),followedbyMeridaandProgreso.Thedengueseroprevalenceinthecohortincreasedwithageasobservedintheinitialdengueseroprevalencesurveyoftherandomsampleofthepopulation(73.6%).ThematchontheresultsofthetwodengueseroprevalenceisreassuringthatthethreesettingsareendemicareasandthatTiculhasthehighestriskfordenguetransmission.Themostcommonsymptomofthesymptomaticcaseswasfever(95%),followedbyheadache(79%)andmyalgia(70.35%).Thesesymptomsaresimilaramongarboviralinfectionsthatiswhytheclinicaldiagnosisandcasereportingtotheepidemiologicalsurveillancesystemarenotaccurateasexpected.Itisneededtoenhanceepidemiological 82

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andlaboratorysurveillancetobeabletodetectnotjustthevirusesthatarecirculatingbutalsogiveinformationthatcouldbeusefulforpolicymakers.Thecliniciansalsoneedtoknowtheindividualdiagnosistobeabletopreventparticularcomplicationsthatareassociatedwitheacharbovirusandsuspectco-infections.Amongthesymptomaticarbovirussuspectedcasesjust6.60%wereconrmedasdengue.Theincidencerateofconrmedsymptomaticdengueinfectionswas3.49per1,000person-years.Thisproportionofconrmationandtheincidencewerelowerthantheseestimatesinotherdenguecohorts.OnepossibleexplanationisthatthereweremorearboviruscirculatinginYucatan,Mexicoduringthisrst-yearoffollow-upsothesevirusesmightcompiteanddecreasedenguetransmission.Currentlywearealsoplanningtoreviewandupdatetheprotocolsforconrmationofacutecasesgiventhecurrentconditionsofco-circulatingvirusesintheareathatcouldbeleadingtobiasesinthediagnosis.Theincidenceratefordengueinfectionswaslowercomparedwithothercohortsaroundtheworld.Itcouldbeusefultosupportthefactthatthisrstyearoffollow-upwasalowtransmissionseasonfordengue.Thehighestincidenceofdenguewasobservedintheparticipantsonthegroupfrom15-19yearsofagefollowedbythe8yearolds.Thegroupfrom15-19yearsofagerepresents4.65%ofthecohortandjust25.64%ofthemwerenaveatbaseline.Duringthesecondsemesterof2016wereenrolledparticipantsformiddleandhighschoolstohavearepresentativenumberofparticipantsofthisparticularagegroupthatisalsotheagegroupthatcouldbepotentiallytargetedfordenguevaccination.Amongtheparticipantswhowerenaveatbaseline,theoverallseroconversionrateof16.74%.Theseroconversionrateisthebestestimateofforceofinfectiongiventhatthosewereprimaryinfections.IntheYucatancohortthepopulationthatwasdenguenaveatbaselinehad22.17timestheincidenceofdengueinfectionsasthosethatwerenon-naveatbaseline.Alsothepopulationthatwasdenguenaveatbaselinehad3.743timestheincidenceofZikasymptomaticinfectionsasthosethatwerenon-naveatbaseline.Theseareveryinteresting 83

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resultsgiventhatbeingnon-naveforavivirusinfectionscouldbepotentiallybeprotectiveforsymptomaticZikabutthishypothesiswouldneedmoredetailedimmunologicalresearch.Theotherincidencerateratiosestimatedwerenotsignicant.Thehazardratiosestimatedfordengueinfectionsduringtherstyearoffollow-upweresignicantforfemalegender,livinginTiculorProgreso,oneormoreinfectionsconrmedinthehousehold,andbeingdenguenaveatbaseline.Thisistherstdenguecohortstudythatusessurvivalanalysisasatooltounderstandbetterthetransmissiondynamicsofdengueandotherarbovirus.Themainlimitationofthisrstyearfollow-upresultsisthat32.2%oftheparticipantsduringtherstyearoffollow-upwerelostoffollow-uporwithdrewfromthestudy.Therewerenotdiferenciallostoffollow-upandwithdrawsamongnavesandnon-naves.Mostoftheparticipantswithdrewfromthestudyatthetimeoftakingtheyearlyfollow-upbloodsampleorbecausetheymovedoutofthestateofYucatan.Currentlynewstrategiestoincreaseadherencetothecohorthavebeenimplemented.TheincidenceratesofconrmedsymptomaticZikaandconrmedchikungunyawere2.33casesper1,000person-yearsand8.74casesper1,000person-yearsrespectively.ThestateofYucatanhadachikungunyaoutbreakin2016sothatexplainswhythisratesishigherthanthedenguerates.ZikaemergedalsoinYucatanbutitwasintroducedattheendofthetraditionaldenguetransmissionseason(July-December).Fewnumberofcaseswerereportedandthiscouldbeconsistentwiththeincidencerateestimatedinthecohortstudy.ThisinformationisusefultodesignstudiestoevaluateeectivenessofpublichealthinterventionslikevectorcontrolorZikavaccinetrials.IncontrastwithMexico,ColombiawasthesecondmostaectedcountrybyZikavirusduring2015-2016outbreakintheAmericas.Colombiahasagoodsurveillancesystemcomparedwithothercountriesintheregion.WeanalyzeepidemiologicalsurveillancedatafromtwolocalZikavirusoutbreaksbetweenSeptember2015andJanuary2016.AtthebeginningoftheZikavirusoutbreakmostofthetransmissionparameterswereunknownasitwasa 84

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virusthatrecentlyemergedtothewesternhemisphere.InthelastpaperwedescribedkeyepidemiologicalfeaturesoftheZikaoutbreaksandestimatetheR0fromdailyincidencedatafromtwosettings:1)SanAndresthatisasmall,densely-populatedislandthatisrelativelyisolatedfromcontinentalColombia;2)Girardot,thatisatypicalmoderatelysizedColombianmunicipality.BothregionshaveendemictransmissionofdengueandexperiencedrecentoutbreaksofCHIKV.TheoverallattackratesforZikavirusdiseaseanddetectedwere12.13casesper1,000residentsofSanAndresand18.43casesper1,000residentsofGirardot.Inbothareas,signicantlyhigherattackratesareobservedamongwomen,especiallythoseofchild-bearingage.ThesehigherattackratescouldbeexplainedbytheeortoftheColombiangovernmenttocaptureallthepregnantwomenpossiblyinfectedwithZikavirustounderstandbetterthespectrumofcongenitalZikasyndrome.InbothsettingsasthepopulationwasfullysusceptibletoZikatransmissionbeforetheoutbreaks,allagegroupswereaected.Forty-eightpregnantwomenwithZikavirusdiseasewerereportedfromSanAndresandGirardot.Thesewomenwerefollowedduringpregnancyaccordingtonationalguidelinesandnoconrmedcasesofmicrocephalywereobserved.Seventeenneurologicalsyndromes,includingGBSandZika-associatedmeningoencephalitis,wereidentied.Thelaboratory-conrmationofZikainfectionsischallengingandinZikainfectionswithneurologicalsymptomscouldbemorechallengingbecausethesesymptomsgenerallyappeartwoweeksafteracutesymptoms,atwhichtimeZikavirusdiagnosisbyRT-PCRisnotpossibleandserologicaltestsareunreliablebecauseofthehighcross-reactivitywithdengue.AsZIKVcanbedetectedinurinelongerthaninblood,usingurinesamplestoconrmZikainGBScasesshouldbeanalternative.ThesechallengesunderscoretheneedforreliablediagnosticteststhatcandetectZikaaftertheviremicperiod.Thebasicreproductivenumber(R0)forZikawasestimatedineachareausingdailyincidencedata.OurestimatedR0fortheZikaoutbreakinSanAndreswas1.41(95%CI1.15to1.74),andtheR0forGirardotwas4.61(95%CI4.11to5.16). 85

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WeconsidertheestimatefromSanAndrestobethemostreliablebecauseitisasmall,denselypopulatedisland,whileGirardothasahigherriskofimportationbecausethepopulationuctuatesduringweekendsandholidays.TherelativemagnitudesofR0areconsistentwiththehigherdenguetransmissionobservedinGirardotversusSanAndres.EstimatesofR0inZikaintheAmericasarenotwidelyavailable,sotheseparametershavebeenusedtomodeltransmissiondynamicsofZikaintheAmericasandtoestimateprobableareaswhereZikavaccinetrialsshouldberuninordertodetectcasesduringthetrial.WiththeprospectivecohortinYucatanweexpecttocollectprospectivedataaboutZikatransmissionandvalidateourestimationswiththeColombiandata.Insummary,itisauniquemomentforarbovirusinLatinAmericagiventhatthreevirusesthataretransmittedbythesamevectorareco-circulatinginmostoftheareasatrisk.TheYucatancohortwillprovideinformationtobetterunderstanthetransmissiondynamicsoftheseviruses.VectorcontrolhadlimitedsuccessincontrollingthesearbovirusestransmittedbyAedesmosquitoes,suchasdengue,Zikaandchikungunyasonewapproachestocontrolmosquitodensitiesareneeded.Alsosafeandecaciousvaccinestopreventarboviralinfectionsandsymptomaticdiseasewillbekeytoreducetransmissionandburdenofdisease.InthecaseofZikavirus,thevaccineshouldtargetespeciallywomenofchild-bearingagetopreventZikacongenitalsyndrome. 86

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BIOGRAPHICALSKETCHDianaP.RojasAlvarezisaMedicalDoctorfromColombia,specialistintropicalinfectiousdiseases.HerresearchinterestfocusesonepidemiologyandtransmissiondynamicsoftropicalinfectiousdiseasesinLatinAmerica.DianawasthedirectorofthenationalepidemiologicalsurveillancesystemintheColombianNationalInstituteofHealthforseveralyears.SheworkedalsoasaresearcherintheUniversidadIndustrialdeSantander(Bucaramanga-Colombia)doingobservationalstudiestomeasureimpactofdengueinfectionintheColombianpopulationandphase3trialsofadenguevaccine.In2013,shegotawardedaFulbright-ColcienciasdoctoralscholarshipthatallowedhertostartherPhDtraininginEpidemiologyatUniversityofFlorida.Dianaisbeeninvolvedinmultipleprojectssinceshestartedherprogrammainlyindengue,chikungunyaandZikavirustransmission.ShereceivedherPh.D.fromtheUniversityofFloridainthesummer2017 104