Characterization,of,the,infectious,reservoir,of,malaria,with,an,agent5based,model,calibrated,to,age5

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1 Characterization,of,the,infectious,reservoir,of,malaria,with,an,agent5based,model,calibrated,to,age5 stratified,parasite,densities,and,infectiousness, JalineGerardin 1 *,AndréLinOuédraogo 1,2,KevinA.McCarthy 1,BocarKouyaté 2,3,PhilipA.Eckhoff 1, EdwardA.Wenger 1 1 InstituteforDiseaseModeling,IntellectualVentures, nd AveNE,BellevueWA98005USA 2 DepartmentofBiomedicalSciences,CentreNationaldeRechercheetdeFormationsurlePaludisme, Ouagadougou,BurkinaFaso 3 MinistèredelaSanté,Ouagadougou,BurkinaFaso *Towhomcorrespondenceshouldbeaddressed. jgerardin@intven.com 1

2 Abstract, BackgroundEliminationofmalariacanonlybeachievedthroughremovalofallvectorsorcomplete depletionoftheinfectiousreservoirinhumans.mechanisticmodelscanbebuilttosynthesizediverse observationsfromthefieldcollectedunderavarietyofconditionsandsubsequentlyusedtoquerythe infectiousreservoiringreatdetail. MethodsTheEMODmodelofmalariatransmissionwascalibratedtoprevalence,incidence,asexual parasitedensity,gametocytedensity,infectionduration,andinfectiousnessdatafrom9studysites.the infectiousreservoirwascharacterizedbydiagnosticdetectionlimitandagegroupoverarangeof transmissionintensitieswithandwithoutcasemanagementandvectorcontrol.massscreen_and_treat drugcampaignsweretestedforlikelihoodofachievingelimination. ResultsThecompositionoftheinfectiousreservoirbydiagnosticthresholdissimilaroverarangeof transmissionintensities,andhigherintensitysettingsarebiasedtowardinfectionsinchildren.recent ramp_upsincasemanagementanduseofinsecticide_treatedbednetsreducetheinfectiousreservoir andshiftthecompositiontowardsubmicroscopicinfections.masscampaignswithantimalarialdrugs arehighlyeffectiveatinterruptingtransmissionifdeployedshortlyafteritncampaigns. ConclusionsLowdensityinfectionscompriseasubstantialportionoftheinfectiousreservoir.Proper timingofvectorcontrol,seasonalvariationintransmissionintensity,andmassdrugcampaignsallows lingeringpopulationimmunitytohelpdrivearegiontowardelimination. Keywords, mechanisticmodel,infectiousreservoir,diagnostics,infectivity,drugcampaigns 2

3 Background, Malariaisaglobaldiseaseresponsibleforhundredsofthousandsofdeathseachyear[1].Inthe lastdecade,manyregionshavemadeconsiderableprogressinmalariacontrolandarenowworking towardlocaleliminationofmalaria[2,3].technicalstrategiesforeliminationdifferfromthosefor control,anditiscriticaltounderstandthefactorsthatleadtosuccessfuloutcomesandmakethebest useofavailableresources. Eliminationofmalariarequiresinterruptingtransmission.Completedepletionoftheinfectious reservoirofmalariaparasitesrequiresclearingallmalariainfectionsinthehumanpopulation.inregions wheremalariaisendemic,accumulatedexposuretoinfectionleadsindividualstodevelopimmunityto clinicalsymptoms.properidentificationofparasitecarriersisthereforeconfoundedbyapopulationof asymptomaticpeople. Currentrapiddiagnostictests(RDT)canquicklyandcheaplyidentifyindividualswithparasite densitiesabove50_200parasitesperµl[4].however,inendemicareas,asignificantportionofthe populationharborsinfectionsthatareundetectablebyrdt,andmicroscopyormolecularmethodsare requiredtoidentifyinfectedindividuals.althoughcapableofdetectinginfectionsaslowas1_5 parasitesperµl,eventhemostsensitivemolecularteststodaycannotdetectallinfections[5]. Asexualparasitedensity,gametocytedensity,andhumaninfectiousnessareknowntobe linked[6,7].higherasexualdensitiesleadtohighergametocytedensities,whichincreasethechanceof infectingmosquitoes.however,therelationshipbetweengametocytedensityandinfectiousnessto mosquitoesiscomplex,asindividualswithhighgametocytedensitymayinfectfewornomosquitoesin xenodiagnosticstudies,andindividualswithnoobservablegametocyteshavebeenobservedtobe infectious[8,9].humansexual_stageandtransmission_blockingimmunityhavebeenproposedtoplay 3

4 rolesinmoderatingtherelationshipbetweengametocytedensityandinfectiousness[10],butthe magnitudeanddurationofsexualstageimmunityeffectsremainunclear. Whilemostmalariainfectionsinareasofevenmoderatetransmissionareasymptomatic[11], therelativecontributiontotheinfectiousreservoirofsymptomaticandasymptomatic,patentand subpatent,andadultandchildpopulationsremainsanareaofactiveinvestigation[12,13].subpatent individuals,eveniftheyoutnumberpatentindividuals,arelessinfectioustomosquitoes.inan eliminationsetting,itiscriticaltounderstandhowsuccessmayormaynotdependonappropriate targetingofsubpatentinfections,assensitivediagnosticscanbeveryresource_intensive. Mathematicalmodelingofmalariatransmissionandimmunitycanbegintoanswersomeof thesequestions[12,13].asinglemechanisticmodelcantietogetherapparentlycontradictoryfielddata thatdescribemalariatransmissioninavarietyofsettingsanddemographicgroups.withadetailed modelofwithin_hostinfection,includingparasiteandgametocytelifecyclesanddensitiesaswellas acquiredimmunity,therelativecontributiontotheinfectiousreservoirduetoeachsubsetofthe populationcanbecomparedandimplicationsforcontrolandeliminationcanbequantitativelyassessed. Arecentlongitudinalstudyinahigh_transmissionareaofBurkinaFasousedmolecularmethods toquantifyasexualandgametocytedensitiesandmembranefeedingteststomeasureinfectiousnessto mosquitoes,providinginvaluabledataformodelcalibration[14].usingthisdatasetandanagent_based stochasticmodelofmalariatransmission,wefirstcalibrateadaptiveimmunityparameterstobest capturetheage_dependentdistributionofasexualparasiteandgametocytedensities.next,weusethe membranefeedingdatatocalibratemodelparametersgoverninghumaninfectiousnesstomosquitoes. Thecalibratedmodelisthenusedtocharacterizethecompositionoftheinfectiousreservoirby detectionthresholdandagegroupthroughseasonalvariationsandatarangeoftransmission intensities.wedescribehowtheinfectiousreservoirchangeswhencasemanagementandvector controlaredeployed.finally,wediscusstheimplicationsforeliminationcampaignsbytestinghowmass 4

5 drugcampaignsmaysuccessfullyachieveeliminationbytargetingalloronlyaportionoftheinfectious reservoir. Methods, Malaria&transmission&model& AllsimulationswereconductedwithEMODDTKv1.6,anagent_basedmechanisticmodelof malariatransmission.duringthebloodstageofmalariainfection,asexualparasitedensitytriggers innateandadaptiveimmunitywithinthehost.innateimmunitystimulatescytokineproduction,leading tofeverinadensity_dependentmannerandlimitingthemaximumparasitedensity.adaptiveimmunity ismodeledusingthreetypesofantigens:p.&falciparumerythrocytemembraneprotein1(pfemp1) variants,merozoitesurfaceproteins(msp),andminorepitopevariants.fullerdiscussionofimmune systemmodelingwithintheemodmodelisdescribedin[15_17].asexualparasiteandgametocyte densitiesaretrackedwithineachhost.gametocytesdifferentiatefrominfectedredbloodcellsand matureinfivestagesovertendays,andafractionofgametocytesarelostateachstageofmaturation fromimplicitimmuneeffects.maturestagegametocytesaretakenupinamosquitobloodmealwith density_dependentprobability,andhumanandmosquitoimmunefactorsimplicitlylimitgametocyte survivalwithinthemosquito.infectiousnessofanindividualhumaniscalculatedasaxenodiagnostic experimentmeasuringthefractionofmosquitoesinfectedafterfeedingontheindividual.theemod modelisavailablefordownloadathttp://idmod.org/software. Calibration&of&asexual&parasite&and&gametocyte&densities&by&age&and&season& 5

6 Calibrationofparasitedensitiesbuiltonpreviousworkin[18].Datafromninestudysiteswere used,includingfoursiteswithage_stratifiedprevalencedata NamawalainTanzaniaandMatsari,Rafin Marke,andSugunguminNigeria andtwositeswithage_stratifiedclinicalincidencedata Dielmoand NdiopinSenegal usedinpreviouswork[19_22].recentlyavailabledatafromtwosites,dapelogoand LayeinBurkinaFaso,providedage_andseason_stratifiedasexualparasiteandgametocytedensitiesas measuredbymolecularmethods[14].malariatherapydataonpeakparasitemiaandgametocytemiaas wellasdurationofparasitemiaandgametocytemiaforsingleinfectionsinnaïveadultswerealso includedinthecalibration[23]. Parametersundercalibrationincludedthemaximumnumberofsimultaneousinfections, numberofpfemp1variantsavailabletotheparasitepopulation,switchingratebetweenpfemp1 variants,numberofmspvariants,numberofminorepitopevariants,killingstrengthsofanti_mspand anti_minorepitopeimmuneresponses,productionrateofgametocytes,andsurvivalrateof gametocytesastheyprogressthroughstagesofmaturation. Prevalenceandincidencesitesweresimulatedandlikelihoodsofcalibrationparameterswere calculatedasdescribedin[18]allsitesotherthanmalariatherapyweresimulatedwithforced entomologicalinoculationrate(eir);novectorsaresimulated,andinsteadthepopulationexperiencesa pre_setnumberofinfectiousbitespermonth.monthlyeirfordapelogoandlayefollowedburkinafaso seasonalityandwerebasedonavailableentomologicaldata,withanannualeirof300fordapelogoand 30forLaye[24].Malariatherapysimulationsincludedasingleinfectiousbiteonday0withnoother infectiousbites. Inthelikelihoodfunctionusedtocalibrateasexualparasiteandgametocytedensities,datafrom eachagegroup(under5,5_15,over15),season(startofwetseason,peakofwetseason,dryseason), studysite(dapelogo,laye),andparasitestage(asexual,gametocyte)wereconsideredseparately (Figure,1).AsexualparasiteandgametocytedensitieswerecollectedinsimulationsonJuly1(startof 6

7 wetseason),september1(peakofwetseason),andmarch1(dryseason),binnedforeachagegroup, andcomparedwithfielddatawithadirichlet_multinomialdistribution. Foreachparameterset!,theparasite/gametocytedensitydistributionsineachagegroup,site, andseasonarebinnedinto6densitybins.thelikelihoodofparameterset!&giventhedatad!(consisting! of!! totalmeasurements,!!!countsineachbin,!!!!!,! =!! )isapproximatedasfollows.theprior distributionforthemultinomialprobabilityvector!!associatedwiththe6binsisinitiallyassumedtobe asymmetricdirichletdistributionwithconcentrationparameter1.thesimulateddistributionofcounts!! isusedtoinformtheposteriordistributionof!:!#! 1!!!#! 1 +!!!! Thelikelihoodof!isthenapproximatedusingtheposteriorpredictivedistributioncomputedby marginalizingover!: L!!) =!!!! =!!!!!!!!! =!#$!!!!!,!!#(! 1 +!!! )!! =!!#$%&'!!!!, 1 +!!! )! Thejointlikelihoodistheproductoflikelihoodsforeachagegroup,season,studysite,and parasitestage,whichisalsomultipliedwiththelikelihoodsfromprevalence,incidence,and malariatherapycomparisonstodeterminetheoveralllikelihoodofeachparameterset.likelihoodsfor prevalenceandincidenceareapproximatedusingamethodologysimilartothatdescribedabove (detailedin[18]),usingbeta_binomialandgamma_poissondistributions,respectively. Forcalibrationtomalariatherapydata,patientswhoreceivedantimalarialtreatmentwere removedfromthecomparisondata.allpatientswereassumedtohaveonlyasingleinfection. Distributionsofpeakparasitemias,peakgametocytemias,parasitemiaduration,andgametocytemia durationswerecomparedtosimulationusingadirichlet_multinomiallikelihoodasdescribedabove. Parametersamplingwasdonebyincrementalmixtureimportancesampling(IMIS),asdescribed in[25].finalbest_fitparametervaluesareshownintable,s1,in,additional,file,1andcomparisonwith 7

8 studysitedatashowninfigure,s1,in,additional,file,2.malariatherapydatawasweightedat10% relativetodatafromendemicareasinthelikelihoodfunction,asfullweightingofmalariatherapydata resultedinpoorerfittingtoprevalencedata,particularlyattherafinmarkestudysite(figure,s2,in, Additional,file,2).Infectionsfrommalariatherapystudiesappeartohaverelativelyshortdurations comparedtowhatwouldbeexpectedbasedondatacollectedfromendemicsites.themalariatherapy datasetusedincalibration,whichdidnotincludeanypatientswhoreceivedcurativetreatmentwith drugs,maybebiasedtowardmilderinfections.straindifferencesbetweenparasitesusedfor malariatherapyandparasitesinendemicregionsmayalsoaccountforthediscrepancies.differencesin infectiondurationbetweenstrainshasbeenpreviouslynoted,withellimonshowinglonginfections, McLendonshowingshortinfections,andSanteeCoopershowingbothlongandshortinfections[17]. Calibration&of&infectiousness&by&age&and&season& Humaninfectiousnesstomosquitoeswascalibratedtomembranefeedingdatastratifiedby age,season,andstudysite[14].simulationsusedtheimmunity,infection,andgametocyte developmentparametersintable,s1,in,additional,file,1.apopulationof1000individualsofallages wassubjectedto50yearsofforcedeirtoinitializeage_appropriateimmunity,witheirvaluesas describedaboveforthedapelogoandlayesites.calibrationalsousedforcedeirandnovectors. Twoparameters,thesurvivalrateofgametocytesinthemosquitoandthemaximumprobability amosquitobecomesinfected,werecalibratedusingimisasdescribedabove.foreachsampled parameterset,thedistributionoffractionmosquitoesinfectedwascomparedtofielddatawitha Dirichlet_multinomialasdescribedabove.Infectiousnessbyagegroup(under5,5_15,over15)and season(startofwetseason,peakofwetseason,dryseason)wereconsideredseparately,and 8

9 likelihoodsweremultiplied.calibratedparametervaluesareshownintable,s2,in,additional,file,1and simulatedinfectiousnesscomparedwithfielddatainfigure,2. Characterization&of&the&infectious&reservoir&in&full&vector&transmission&model& Simulationsoftheinfectiousreservoirwereconductedwithafullvectortransmissionmodelina BurkinaFasoclimatewithvectorabundancecalibratedtoachieveannualEIRbetween200and300. VectorspeciesincludedA.&gambiaeandasmallamountofA.&funestus,andlarvalhabitatsweremodeled asin[26]withenoughconstantlyavailablehabitattoensurevectorsurvivalthroughthedryseason. Vectorfeedingbehaviorwas95%anthropophagicand95%endophagic;othervectorlifecycle parametersweremodeledasin[26].populationdemographicsfollowedtheburkinafasoagestructure [27],andallsimulationswereperformedonawell_mixedpopulationof1000individuals.Larvalhabitat wasscaledtosimulatesettingswithlowertransmissionintensity(table,s3,in,additional,file,1). Simulationofeachtransmissionintensitywasrepeatedfor1000stochasticrealizations,andallfigures showmeanmeasurementvalues.populationsweresimulatedfor50yearsateachtransmission intensityintheabsenceofinterventionstoinitializeage_appropriateimmunity.importationofcases occurredatarateof1/year.allsimulationsincluded1yearofequilibrationpriorto3yearsof measurement. Individualswereassignedtodetectiongroupsoneachdayofthedetectionperiod.Each individualwastestedforasexualparasitepositivityat3diagnosticsensitivitylevels:100,10,and5 asexualparasitesperµlandassignedtotheleastsensitivediagnostictowhichtheytestedpositive. IndividualstestedpositiveifarandomdrawfromaPoissondistributioncenteredatthetruenumberof asexualparasitesin1/(diagnosticsensitivity)µlofbloodwasnonzero.individualstestingnegativebyall diagnostics,butnonethelessinfectedwithasexualparasites,wereassignedtotheundetectablegroup. 9

10 Hostsizeisknowntopredictmosquitobitingbehavior,andanage_dependentbitingriskwas includedtoapproximatesurfaceareadependence[28_30].compositionoftheinfectiousreservoiris calculatedbynormalizingthescaledhumaninfectiousness.uncertaintyinparametervaluesand stochasticvariationsbetweensimulationrunsdonotqualitativelychangethecompositionofthe infectiousreservoir(figure,s3,in,additional,file,2). Modeling&case&management&and&campaigns&with&insecticide>treated&bednets&(ITNs)& Severalsimulationscenariosaremodeled:baseline nointervention scenarioswithnocase managementoritns;casemanagementscenarioswheresymptomaticindividualscouldaccess treatmentwithantimalarialdrugs,butnoitns;itncampaignscenarioswhereitnsweredistributedto thepopulationinanage_dependentmanner,butnocasemanagementwasavailable;andscenariosthat includedbothitndistributionandcasemanagement. Casemanagementwasavailabletosimulatedindividualswithclinicalorseveremalaria.A clinicalcaseoccurredwhenanindividual sbodytemperatureincreasedbyatleast1.5 C,andsevere casesoccurredwhenfever,anemia,orparasitedensityexceededthresholdsdescribedin[18].ninety_ fivepercentofthepopulationhadanyaccesstocasemanagement.ofthepopulationwithaccessto casemanagement,individualswithclinicalmalariahad60%probabilityofseekingcareforagiven episode.individualswithseveremalariahad95%probabilityofseekingcare.allindividualsseekingcare receivedtreatmentwithartemether_lumefantrine(al)within3daysforclinicalcasesor2daysfor severecases,andalltreatedindividualscompletedthefullcourseoftreatmentwithfulladherence. Age_dependentdosing,pharmacokinetics,andpharmacodynamicsofALweremodeledasdescribed in[31].simulationswithcasemanagementallowedcasemanagementduringboththeequilibrationand measurementperiods. 10

11 ITNsweredistributedatbirthwith90%coverageandwerealsomassdistributedonday200of thefirstmeasurementyearwith80%coverageforchildrenunder10and50%forindividualsover10 yearsofage.itnshadaninitialblockingrateof80%andkillingrateof60%,withefficacydecaying exponentiallywithhalf_livesof2yearsand4yearsrespectively.whenitnswerepresent,the contributionofitn_protectedindividualstotheinfectiousreservoirwasreducedbytheitnblocking rateafterscalingbysurfaceareaasdiscussedabove. AnnualEIRwasmeasuredbysummingthedailyinfectiousbitesperpersonoverthesecond measurementyear.forthesameamountoflarvalhabitat,casemanagementanditnsreducedthe apparenteirrelativetosimulationswithoutinterventions.whencasemanagementand/oritnswere present,theplottedeiristheapparenteirexperiencedbythesimulatedpopulation. Simulation&of&mass&drug&campaigns& Massdrugcampaignswereconductedwithdihydroartemisinin_piperaquine(DP),withimproved pediatricdosingasdescribedin[32]andpharmacokineticsandpharmacodynamicsasin[31].all individualsreceivingdpadheredtothefulltreatmentcourse.masscampaignswereconductedduring thesecondandthirdmeasurementyearsin3roundsperyearseparatedby6weeks,withthefirstround occurringonday60ofthesecondmeasurementyear.allcoveredindividualsreceiveddponthesame day.coveragewasindependentbetweenrounds.importationofcasesoccurredatarateof1/year. MasscampaignsweretestedatapparentEIRsbetween7and40forsettingswithnoother interventionsandsettingswithcasemanagementanditnuseasdescribedabove.drugcampaign coveragewastestedat0%,20%,40%,60%,80%,and100%.interpolationswerecalculatedwiththe SciPyv0.14.1interpolatefunctioninPython2.7.Massscreen_and_treat(MSAT)campaignsused 11

12 diagnosticswithsensitivity100,20,or2asexualparasitesperµl.simulationofeacheir,coverage,and MSATdiagnosticsensitivitywasrepeatedfor100stochasticrealizations. Eliminationwasdeterminedtobeachievediftrueasexualparasiteprevalencewas0forthelast 150daysofsimulationyear3.Probabilityofeliminationwasthefractionofthe100stochastic realizationsresultinginelimination. Results, Composition&of&the&infectious&reservoir&in&the&absence&of&interventions& Adaptiveimmunityandhumaninfectiousnessparameterswithinanagent_basedmodelwere calibratedtoage_andseason_stratifieddatafromtwohigh_transmissionareasinburkinafaso(figures,1, and,2)(seemethods).typicalinfectiontrajectoriesinthecalibratedmodelshowshorterdurationsof infectionandlowerparasitedensitiesinadults,especiallyinhigh_transmissionsettings(figure,1band Figure,S4,in,Additional,file,2).Gametocyteclearancelaggedbehindasexualparasiteclearance. Figure,3Ashowstheinfectiousreservoirover3yearsinamoderate_transmissionsettingwith annualeirof10andtheseasonalityofburkinafaso.totalasexualparasiteprevalencepeakstowardthe endoftherainyseasonandislargelyduetoalargeincreaseinprevalenceofrdt_positiveinfections. PrevalenceofRDT_negative,microscopypositiveinfectionsaswellasinfectionspositivebyPCRonlyand undetectableinfectiousremainedrelativelymoreconstantthroughtheyear,peakingtowardthe beginningofthedryseasonastherdt_positiveinfectionsbegintheclear. Totalinfectiousnesswasquantifiedbymeasuringtheprobabilityofeachindividualinfectinga mosquitoandscalingbyagetoapproximatethedecreasedbitingriskofchildrenduetosmallersurface area(seemethods).totalinfectiousnessexhibitsmoreseasonalvariationthanasexualparasite 12

13 prevalencesincetransmissiontomosquitoesisdominatedbyrdt_positiveindividuals,andhigher parasitedensityleadstogreaterinfectiousness. Undertheseconditionsofmoderatetransmissionandhighseasonality,thecompositionofthe infectiousreservoirshowsconsiderablevariationthroughtheyear.rdt_positiveindividualscomprise over60%oftheinfectiousreservoirduringthewetseasonbutonly30%duringthedryseason. Submicroscopicinfectionsaccountfor20_40%oftheinfectiousreservoiratanytimeofyear,a substantialportion. Whilechildrenhavehigherparasitedensitiesandarehighlyinfectious(Figure,1,and,2),their smallersurfaceareareducestheircontributiontothetotalinfectiousnessofthepopulation.children contributethemosttotheinfectiousreservoiratthebeginningofthedryseasonwhenadultimmune systemshavealreadyreducedparasiteloadinadults,leavingchildrentoslowlycleartheirlonger infectionsandcontinuetransmission(figure,1b). Tocomparetheinfectiousreservoiracrossarangeoftransmissionintensities,weaveragedthe seasonalvariationinasexualparasiteprevalenceandtotalinfectiousnessoverayearofsimulation (Figure,3B).TotalasexualparasiteprevalenceincreasessteeplyasEIRincreasesfrom<0.1to1 infectiousbiteperpersonperyear.aboveeir=1,asexualparasiteprevalenceincreasesmoregradually andapproaches100%foreir>100.totalpopulationinfectiousnessalsoincreasessteeplyaseir increasestoward1butisnearlyconstantforeirsbetween1and200,consistentwithobservationsina studyareawheretheproportionofinfectedmosquitoesremainedroughlyconstantwhileslide prevalenceinhumansdeclinednearlyfour_fold[33]. RDT_detectableinfectionscomprise20_30%ofallinfectionsovertheentiresampledrangeof transmissionintensities,withhighereirsettingsshowingrelativelymorerdt_detectableinfections (Figure,S5,in,Additional,file,2).Theseobservationsareconsistentwiththeproposedhypothesisthat individualsinhigh_transmissionsettingsareconstantlysubjectedtoburstsofhighparasitedensityfrom 13

14 newinfections,whileindividualsinlow_transmissionsettingsexperiencealongperiodoflow_density infectionasuntreatedsingleinfectionsareclearedbyimmuneactivity[12]. RDT_detectableinfectionsrepresentasimilarproportionoftheinfectiousreservoirregardlessof transmissionintensity,asdomicroscopy_detectableandpcr_detectableinfections.populationsin regionswithverylowtransmissionintensity(eir<1)haveslightlymoreoftheinfectiousreservoir contributedfromrdt_positiveindividuals,whilerdt_negativeindividualsformanincreasinglylarge portionofthereservoiraseirincreasesabove1. Childrencontributemoretotheinfectiousreservoiratthehighesttransmissionintensities(EIR> 100).Athightransmissionintensity,adultimmunityretainsmemoryofalargerepertoireofantigenic variants,andthereforeparasitedensityisunlikelytoremainabovetherdtdetectionlimitforlong.in thissituation,childrencomprisealargerportionoftherdt_positivepopulation.atlowtransmission(eir <1),childrenunder5barelycontributetotheinfectiousreservoir,andchildrenbetween5and15 contributeonly20%.adultsarethemajordriversoftransmissioninallbutthehighesttransmission settings. Composition&of&the&infectious&reservoir&after&recent&ramp>up&in&case&management&and&ITN&use& Regionsconsideringeliminationarelikelytohavealreadyimplementedvectorcontroland strengthenedtheirhealthsystems.weconsideredtheeffectofgoodcasemanagementandaggressive campaignswithinsecticide_treatednets(itns)ontheinfectiousreservoir.bothcasemanagementand ITNcampaignsdecreasedtheobservedEIR(Figure,S6,in,Additional,file,2). Undercasemanagementwithartemether_lumefantrine(AL),where95%ofthepopulationhas accesstocare,60%ofclinicalcasesseektreatment,and95%ofseverecasesseektreatment,thetotal infectiousreservoirisreduced(figure,4a)comparedtosimulationswithoutcasemanagement(figure, 14

15 3B).Atlowtransmission(EIR<1),casemanagementisparticularlyeffectiveatdepletingtheinfectious reservoir,asinfectionsaremorelikelytobecomeclinicalcasesandtreatmentofeachcasemaycleara significantportionoftheinfectiousreservoir. WesimulatedanITNcampaignwhereITNusewasskewedtowardchildren(seeMethods),as hasbeenrecommendedforcontrolsituations[34].deploymentofitnssubstantiallyreducesthe infectiousreservoiratalltransmissionlevels(figure,4b).childrenunder5compriseanevensmaller portionoftheinfectiousreservoirthanthecasewithnoitns,evenathightransmission. Relativetonointerventions,highcasemanagementandITNusagebiastheinfectiousreservoir towardrdt_negativeandsubmicroscopicinfections,especiallyforlow_transmissionsettings.atthe sameobservedannualeirof1,settingswhereeirhasrecentlybeenreducedbycasemanagementor ITNsshowalargerfractionoftheinfectiousreservoirstemmingfromsubpatentinfectionsrelativeto settingswhereeirhashistoricallybeenat1(figure,4c).settingswhereeirhasrecentlybeenreduced to1havepopulationswhoseimmunesystemsareadaptedtoeir>1,soquicklyreducingeirleadsto infectionswithlowerparasitedensity.ifthereducedeirismaintainedformanyyears,population immunitywillrebound[35],andthecompositionoftheinfectiousreservoirwillleanmoretowardrdt_ positiveindividuals. Depletion&of&the&infectious&reservoir&after&mass&drug&campaigns& Wetestedtheprobabilityofeliminationfollowingmassscreen_and_treat(MSAT)campaigns withdihydroartemisinin_piperaquine(dp)atvariouslevelsofdiagnosticsensitivity,coverage,and transmissionintensity(figure,5a).threeroundsofmsatwithindependentcoveragewereapplied duringthedryseasonfortwoyears.animprovedpediatricformulationofdpwasadministeredtoavoid under_dosinginyoungchildren[32,36].themsatcampaignoutcomeswerecomparedtomassdrug 15

16 administration(mda),whereallindividualsaretreated,aswellasmsatcampaignswherecase managementanditnshaverecentlyreducedeir. Highcoverage,highdiagnosticsensitivity,andlowtransmissionallincreasethelikelihoodof eliminationfollowinganmsatcampaign.highercoveragecannotcompletelycompensateforlower diagnosticsensitivity,andeliminationisnotpossibleforsettingswitheir>1whenanmsatis conductedwithcurrentrdts,whichhavesensitivityaround100parasitesperµl.afieldtrialofan MSATcampaigninaBurkinaFasositewithasexualparasiteprevalencebetween30and50%showed littlesuccesswithlong_termprevalencereduction[37]. CasemanagementandITNuseincreasethelikelihoodofeliminationbyMSAT.Underthesame transmissionintensity,lingeringpopulationimmunityhelpspusharegiontowardeliminationaftera drugcampaignifeirhasrecentlybeenreduced(figure,5b).thebonusfromlingeringimmunity increaseswithbettercoverage,underscoringthecriticalimportanceoftreatingasmanypeopleas possibleinadrugcampaign.simulationresultsalsosuggestthatlingeringimmunitymaybemost beneficialforeliminationeffortsateirbetween1and10,whereeirislowenoughthateliminationis possiblebuthighenoughthatadultimmunityisstrong. Discussion, Tointerruptthechainofmalariatransmission,itiscriticaltoidentifywhoistransmitting.Here wecalibrateamechanisticmodelofwithin_hostparasiteandimmunedynamicstofielddataon gametocytedensitiesandtransmissionprobabilities.themodelisthenusedtopredicttheinfectious reservoirofmalariabyagegroupanddiagnosticthresholdoverawiderangeoftransmissionintensities. Whilehigh_qualityfielddataisessentialforunderstandingmalariatransmission,modelsarealso invaluableforshowingthatdatafromdiversegeographic,demographic,andinterventionconditionscan 16

17 arisefromthesameunderlyingmechanisms.inthisworkwehavealsofoundthatinourmodel framework,malariatherapydataoninfectiondurationisincompatiblewithprevalencemeasurements fromendemicareas,highlightingalikelyareaformodelimprovement. Whileadaptiveimmunitytowardasexualparasitesisfairlywellunderstood,itremainsunclear howsexualstageimmunityaffectsgametocyteproduction,survivalwithinthehost,andabilityto continuetheparasitelifecyclewithinthevector[10,24,38_40].ourmodelofhostimmunitydidnot includeanyimmunitytowardgametocytes,andthetrendoflowergametocytedensitywithageisdue entirelytolowerasexualparasitedensities.comparisonofcalibratedsimulationsofgametocytedensity toobserveddistributionsofgametocytedensitiesdoesnotsuggestthatsexualstageimmunityplaysa strongrole,atleastinthehigh_transmissionsettingswherethedatawerecollected. Transmission_blockingimmunity,whereindividualswithhighgametocytecountsfailtoinfecta largeportionoffeedingmosquitoes,hasalsobeenproposed.ourcalibrationsuggeststhat transmission_blockingimmunitymayexist,asourmodelsystematicallyfailedtoreplicatedatawhere individualswithgametocytedensities>1000/µlweresometimesobservedtoinfect<5%ofmosquitoes. Ourmodelalsodoesnotaccountforcaseswhereindividualswithveryloworundetectableparasite densityhavebeenobservedtoinfectmosquitoes[41].wethusanticipatethatwemaybe overestimatingthecontributiontotheinfectiousreservoirofpeoplewithhighdensityinfections, implyingthatrdt_negativeinfectionsareevenmorecriticaltotargetthanouranalysissuggests. Measurementuncertaintyofmolecularmethodscouldalsoplayaroleinoverestimationofgametocyte densities[42].inaddition,directskinfeedingisknowntoinfectmosquitoesatahigherratethan membranefeeds[43].futuredatacollectionontransmissionbydirectskinfeedingorconcentrationof maturegametocytesintheskinwillbeinvaluableforimprovingmodelsofmalariatransmissionand understandingthenatureoftheinfectiousreservoir. 17

18 Oursimulationsshowthatchildrencomprisealargeportionoftheinfectiousreservoironlyat veryhightransmissionintensity,andadults(agedover15yearsinoursimulations)arethemaindrivers oftransmissioninlowtomoderatetransmissionsettings.theseresultsareforannualaverages,butthe infectiousreservoiralsovariesseasonally,andrelativecontributionfromchildrenincreasesattheendof thewetseasonwhenadultinfectionshavelargelycleared.theagestructureoftheinfectiousreservoir willalsochangeifmobileadultsarereimportinginfectionsfromanothersettingwheretransmissionis lessseasonal. BasedonwarningsthatpediatricdosingofDPisinsufficient,oursimulationsincreaseddosage ofdpovercurrentrecommendedlevels.wefindanimprovementindrugcampaignoutcomes comparedtopreviouswork,whichfollowedcurrentguidelinesfordosing,particularlyinincreasingthe probabilityofeliminationateir>10atmoderatelyhighcoveragelevels[31].whilecorrectdosingis importantforreducingthechanceofrecrudescenceinindividualpatients,under_dosingisparticularly criticaltoavoidinaneliminationscenario,asinterruptingtransmissioncannotoccurwhenasubgroup ofthepopulationcancontinuetoharborandtransmitinfectionorwhenasubgroupdoesnotreceive thebenefitsofprophylaxis. ComparedtosettingswherenointerventionshaveperturbedtheEIRforalongperiod,settings whereeirhasrecentlybeenreducedshowashiftintheinfectiousreservoirtowardyoungerindividuals andtowardlowerdensityinfections.thismaysuggestthatmsatcampaignsshouldachievelower successratesinsettingswithrecentlyreducedeirbecausediagnosticswillmissagreaterfractionofthe infectiousreservoir.however,oursimulationsshowthatmsatcampaignsareactuallymoresuccessful insettingswithrecentlyreducedeirbecausestrongpopulationimmunitymorethancompensatesfor ongoingtransmissionfromlow_densityinfections. OursimulationsofMSATcampaignspredictthatpropertimingofdrugcampaignsrelativetoITN deploymentmaybecriticaltoharnessthepoweroflingeringimmunityinordertodrivetheregion 18

19 towardelimination.however,thereisalackofdatatoproperlycalibratetherateofimmunitydecay, andourpredictionsoflikelyeliminationmaybeoptimistic.whilegatheringsuchdatamayprove extremelychallenging,weanticipatethatthoroughunderstandingofimmunitydecayiscriticalfor meaningfulmodelingofeliminationscenarios. Conclusions, Compositionoftheinfectiousreservoirvariesseasonally,withhigherdensityinfectionsforming alargerportionduringthehigh_transmissionseason.rdt_negativeinfectionsmakeupasubstantial portionoftheinfectiousreservoiroverawiderangeoftransmissionintensities.theincreased infectiousnessofchildrenduetohighergametocytedensitiesisbalancedbydecreasedpropensitytobe bittenduetosmallersurfacearea.adultscomprisethelargestfractionoftheinfectiousreservoiratlow tomoderatetransmissionintensities,whilechildrenformthelargestportiononlyinveryhigh transmissionsettings. InterventionssuchascasemanagementandITNusetilttheinfectiousreservoirtoward submicroscopicinfections.masscampaignswithantimalarialdrugsaremoresuccessfulwhentheyreach alargerportionoftheinfectiousreservoirthroughmoresensitivediagnosticsorhighercoverage. Propertimingofdrugcampaignswithseasonalvariationintransmissionintensityaswellasrecent deploymentofitnsallowslingeringpopulationimmunitytohelpdrivearegiontowardelimination. Competing,interests, Theauthorsdeclarethattheyhavenocompetinginterests. 19

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26 , Figure,legends, Figure1_Calibrationofasexualparasiteandgametocytedensities.(A)Thedistributionofasexual parasiteandgametocytedensitiesbyseasonandagegroupinreferencedata(opencircles)andpost_ calibrationsimulation(filledcircles)isshownfortwostudysites.circleareaisproportionaltonumberof individualsandnormalizedforeachseasonofeachagegroup.(b)exampleinfectiontrajectoriesof asexualparasites(blue)andgametocytes(green)aftercalibrationforanage6child(solidlines)andage 28adult(dottedlines)withimmunehistoriescorrespondingtohigh_transmission(top)andlow_ transmission(bottom)settings.eachindividualwaschallengedwithasingleinfectiousbiteonday0, withnosubsequentbiting.nodrugswereadministered. Figure2_Calibrationofhumaninfectiousnesstomosquitoes.Therelationshipbetweengametocyte densityandinfectiousnessbyseasonandagegroupinreferencedata(opencircles)andpost_calibration simulation(filledcircles)isshownfortwostudysites.circleareaisproportionaltonumberofindividuals andnormalizedforeachseasonofeachagegroup. Figure3_Compositionoftheinfectiousreservoirintheabsenceofinterventions.(A)Thecompositionof theinfectiousreservoirbydiagnosticthresholdandageinasettingwithmoderatetransmission.a regionofeir=10wassimulatedover3yearswithseasonalrainfallandtemperaturebasedonburkina Fasoclimate.Theentirepopulationwastestedforasexualparasiteprevalencedailyfor3yearswith diagnosticsat3levelsofsensitivity:100parasites/μl(bottom3stripes,pink),10parasites/μl(3stripes secondfrombottom,yellow),and5parasites/μl(3stripesthirdfrombottom,blue).infections undetectableat5parasites/μlareshowningray(top3stripes).infectiousnesswascalculatedby 26

27 simulatingamembranefeedingtestandsubsequentlyscalingbyagetoapproximatesurfacearea effects.thefractionoftheinfectiousreservoiristhenormalizationoftotalinfectiousness.theaverage of100stochasticrealizationsisshown.(b)annualaveragecompositionoftheinfectiousreservoirunder arangeoftransmissionintensities.allmeasurementswereaveragedoverthesecondyearof simulation.1000stochasticrealizationswereaveragedforeacheirlevel. Figure4_Compositionoftheinfectiousreservoirbydiagnosticthresholdandagewithcase managementanddeploymentofinsecticide_treatednets(itns)underarangeoftransmission intensities.(a)theinfectiousreservoirunderarangeoftransmissionintensitieswhencase managementisavailable.alltreatedindividualsreceivedafullcourseofartemether_lumefantrine(al) withage_dependentdosing.seemethodsfordetailsoftreatmentavailabilityandconditionsforseeking care.(b)theinfectiousreservoirunderarangeoftransmissionintensitiesafterdeploymentofitns.see MethodsfordetailsofITNdistributionandeffectiveness.Scalinghumaninfectiousnessforagebiting riskincludedeffectsofnetusage.(c)comparisonoftheinfectiousreservoiratapparenteirof1under conditionsofnointervention,casemanagementwithal,anditnuse. Figure5_Depletionoftheinfectiousreservoiraftermassdrugcampaignswithdihydroartemisinin_ piperaquine(dp).(a)probabilityofeliminationafter2consecutiveyearsofmassdrugcampaignsat3 roundsperyear.seemethodsfordetailsoftimingofcampaignrounds.casemanagementanditns weresimulatedasinfigure4.probabilityofeliminationwasthefractionof100stochasticrealizations resultingincompleteeliminationofallparasitesbytheendofyear3.coveragewasindependentforall roundsandallinterventions.forsimulationswithcasemanagementanditns,eiristheeirthatwould havebeenexperiencedduringthesecondyearofsimulationhadthedrugcampaignsnotbeen administered.crossesindicatetheeir,coverage,anddiagnosticsensitivitysimulatedinpanelb.(b)the 27

28 infectiousreservoirbefore,during,andaftermsatcampaignsinareaswithapparenteir=1,diagnostic sensitivity20/μl,and80%coverage.left:asexualparasiteprevalenceandhumaninfectiousnessinan endemicregionwitheir1andnointerventions.right:asexualparasiteprevalenceandhuman infectiousnessinaregionwherecasemanagementanditncampaignshavereducedeirto1. Additional,files, Additionalfile1 SupplementaryTables TableS1.Parametervaluesaftercalibrationtoprevalence,incidence,anddensitydata.TableS2. Parametervaluesaftercalibrationtoinfectiousnessdata.TableS3.ApparentEIRstunedbyscaling availablelarvalhabitat. Additionalfile2 SupplementaryFigures FigureS1.Comparisonofincidence,prevalence,peakdensity,andinfectiondurationbetweenreference dataandsimulationwithcalibratedimmunityandgametocytedevelopmentparameters.figures2. Comparisonbetweensimulationandreferencedataaftercalibrationofimmuneandgametocyte parameterswithfullweightingofmalariatherapydata.figures3.sensitivityofthecompositionofthe infectiousreservoirtostochasticvariationanduncertaintyinparametervaluesforhigh_andmoderate_ transmissionsettingsbyagegroupandasexualparasitedetectionlimit.figures4.trajectoriesof infectionforchildrenandadultsinhigh_andlow_transmissionsettingswithcalibratedimmuneand gametocyteparameters.figures5.unscaledtotalinfectiousnessbydiagnosticthresholdandagegroup. FigureS6.MalariatransmissionundercasemanagementandITNuse. 28

29 A DAPELOGO start wet peak wet dry LAYE start wet peak wet dry asexual density per μl <5x10 5 <5x10 3 <5x10 1 none gametocyte density per μl <5x10 5 <5x10 3 <5x10 1 none < < < < age group (years) simulation reference data < < age group (years) B number of parasites per μl high transmission region (EIR 220) low transmission region (EIR 2) asexual parasites gametocytes age 6 age days since infectious bite

30 season age % mosquitoes infected DAPELOGO start wet peak wet dry gametocyte density per μl gametocyte density per μl simulation reference data LAYE start wet peak wet dry < 5 years 5-15 years 15+ years

31 A daily EIR fraction with asexual parasites total infectiousness, scaled by age biting risk fraction of infectious reservoir simulation time (years) undetected PCR microscopy RDT 15+ yrs 5-15 yrs <5 yrs B fraction with asexual parasites 4 total infectiousness, scaled by age biting risk fraction of infectious reservoir annual EIR annual EIR annual EIR

32 A fraction with asexual parasites Composition of the infectious reservoir with case management by artemether-lumefantrine 4 2 total infectiousness, scaled by age biting risk annual EIR annual EIR annual EIR fraction of infectious reservoir undetected PCR microscopy RDT 15+ yrs 5-15 yrs <5 yrs B fraction with asexual parasites Composition of the infectious reservoir after campaign with insecticide-treated nets 4 2 total infectiousness, scaled by age biting risk annual EIR annual EIR annual EIR fraction of infectious reservoir C fraction of infectious reservoir Composition of the infectious reservoir at EIR = 1 no intervention with case management with ITNs

33 A Diagnostic sensitivity: 100/μL 20/μL 2/μL % drug campaign only drug campaign + case management and ITNs coverage coverage 50% 0% 100% 50% MSAT MDA 0% annual EIR annual EIR annual EIR annual EIR probability of elimination B fraction with asexual parasites 5 total infectiousness, scaled by age biting risk no interventions MSAT campaign simulation time (years) with case management and ITNs ITN campaign MSAT campaign simulation time (years) undetected PCR microscopy RDT 15+ yrs 5-15 yrs <5 yrs

34 Table&S1.&Parameter&values&after&calibration&to&prevalence,&incidence,&and&density&data& Parameter&name& Parameter&Value& Max_Individual_Infections& 3& Antigen_Switch_Rate& 2.96eH9& Falciparum_PfEMP1_Variants& 1112& Falciparum_MSP_Variants& 7& MSP1_Merozoite_Kill_Fraction& 3& Falciparum_Nonspecific_Types& 90& Nonspecific_Antigenicity_Factor& 2& Base_Gametocyte_Production_Rate& 44& Gametocyte_Stage_Survival_Rate& 2& & Table&S2.&Parameter&values&after&calibration&to&infectiousness&data& Parameter&name& Base_Gametocyte_Mosquito_Survival_Rate& Acquire_Modifier&(vector&species&parameter)& & Parameter&Value& 0088& & Table&S3.&Apparent&EIRs&tuned&by&scaling&available&larval&habitat& Larval&habitat& multipler& EIR&under&no& interventions& EIR&under&case& management& EIR&with&ITNs& EIR&with&case& management&and&itns& & 221& 201& 64& 37& & 145& 128& 24& 9.7& & 103& 88& 8.7& 3.4& & 58& 44& 1.5& 0.57& 0.1& 27& 16& 2& 72& 8& 16& 9.0& 0.11& 095& 6& 9.3& 4.2& 4& 051& 4& 5.5& 2.1& 2& 015& 15& 1.7& 0.17& 02& 0031& 10& 0.38& 08& 001& 0005& 08& 5& 003& 0005& 0001& &

35 A annual clinical incidence DIELMO simulation reference data age B prevalence NAMAWALA age prevalence MATSARI age annual clinical incidence NDIOP age prevalence RAFIN MARKE SUGUNGUM age prevalence age C fraction of patients simulation reference data <10 2 <10 4 peak parasitemia / μl <10 6 MALARIATHERAPY <20 <50 <100 <150 <200 <300 <1000 duration in days of asexual parasitemia fraction of patients <10 2 <10 4 peak gametocytemia / μl < <20 <50 <100 <150 <200 <300 <1000 duration in days of gametocytemia Figure S1. Comparison of incidence, prevalence, peak density, and infection duration between reference data and simulation with calibrated immunity and gametocyte development parameters. Simulation data corresponds to the parameter set with highest combined likelihood across all study sites.