5th FMR Abstract Book - malaria.jhsph.edu FMR Abstr… · THE JOHNS HOPKINS MALARIA RESEARCH INSTITUTE 5TH ANNUAL FUTURE OF MALARIA RESEARCH SYMPOSIUM, NOVEMBER 18TH, 2019 3 López-Uribe1,

THEJOHNSHOPKINSMALARIARESEARCHINSTITUTE5TH

ANNUALFUTUREOFMALARIARESEARCHSYMPOSIUM,NOVEMBER18TH,2019

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5thAnnual

FutureofMalariaResearchSymposium

TheJohnsHopkinsUniversityMontgomeryCountyCampus

Rockville,Maryland,USAMonday,November18th,2019

ABSTRACTS Oralpresentations..........................................p.1 Posterpresentations......................................p.5 Indexbyauthor..............................................p.34

ORALPRESENTATIONS

OP-01Impactofexpandingseasonalmalariachemopreventionacrossnewgeographicalareasandtochildrenunder10years–amodelingapproachKamaldeenOkuneye1,JalineGerardin1,2

1InstituteforGlobalHealth,2DepartmentofPreventiveMedicine,NorthwesternUniversitySeasonalmalariachemoprevention(SMC)withsulfadoxine-pyrimethamineplusamodiaquine(SP-AQ)isrecommendedasanadditionalinterventionagainstPlasmodiumfalciparummalariaforchildrenundertheageof5yearsintheSahelsub-regionofAfrica,whereover60%ofclinicalmalariacasesoccurduringthe3–4-monthrainyseasonandabout90%ofthemortalityandmorbidityoccursamongchildrenundertheageof5years.TheaimofSMCistopreventillnessbymaintainingtherapeuticantimalarialdrugconcentrationsinthebloodthroughoutthemalariaseason.RandomizedcontroltrialshaveshownSMCtobeeffective,safe,relativelycheapandtypicallyreducesclinicalmalariaepisodesby

approximately75%.ThisstudyaimstoquantifythepotentialburdenreductionachievablebyexpandingSMCacrossnewgeographicalareasandexpandingtheSMCagerangetochildrenunder10years,usingderivedpharmacologicalpropertiesofSP-AQ.WedevelopedacomputationalmodelframeworkinEMOD,anagent-basedstochasticmodel,thatintegratesthepopulationdynamicsofmalariatransmissionwithpharmacokineticandpharmacodynamicsmodelstocreatesimulatedclinicaltrialsofantimalarialdrugs.ThemodeloutputsarefittedtoclinicaltrialoutcomesofAQ,SP-AQandartesunate-AQinregionswherethesedrugsareusedascurativetreatmentofmalaria,toobtainthebestfitpharmacodynamicsparametersofSPandAQthatquantifythetherapeuticeffectiveness,i.e.,thecurativeandprophylacticproperties,ofSP-AQ.ThefittedmodelcanbeusedtodescribetheimpactofimperfectadherenceonthesuccessofSMC,theroleanovelsingle-doseSMCdrugcouldplayinincreasingSMCimpact,aswellasimproveimplementationofSMCbyprovidingstandardstrategiesandindividualapproachestoexistingandnewSMCregions.

OP-02

ClearanceofPlasmodiumfalciparum-infectedredbloodcellsbyNKcellsandmonocytes PadmapriyaSekar,GunjanArora,EricO.Long LaboratoryofImmunogenetics,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Maryland AntibodiesnaturallyacquiredduringrepeatedexposuretotheparasitePlasmodiumfalciparumareessentialforcontrolofblood-stagemalaria.NKcellsareinnateimmunecellswithanimportantroleincontainingviralinfectionsandtumorgrowth,mainlybydirectcytotoxicityandinflammatorycytokineproduction.Recently,wereportedthatprimaryhumanNKcellslysePlasmodiumfalciparum-infectedredbloodcells(iRBCs)viaantibody-dependentcellularcytotoxicity(ADCC)inpresenceofIgGisolatedfromadultslivinginamalaria-endemicregion.ThisNK-mediatedlysisresultsininhibitionofparasitegrowthinvitro.Liveimagingshowedreleaseofparasitophorousvacuoles(PV)fromiRBCsafterincubationwithNKcells.ManyofthesePVsgavepropidiumiodide-positivesignals,suggestinglossofviabilityofP.falciparuminsidethePVsafterNK-mediatediRBClysis.WeareinterestedtoknowwhetherandhowPVsareeliminatedfromcirculationandpostulatethatmonocytesplayaroleinclearanceofPVs,therebypreventinginflammatoryresponsesbytheinnateimmunesystem.PreliminaryliveimagingexperimentssuggestthatmonocytesphagocytosePVsthathavebeenreleasedfromiRBCsafterincubationwithNKcells.Wearetestingplasmasamplesofindividualsfrommalaria-endemicregionsforantibodiesreactivetoP.falciparumantigensatthesurfaceof



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PVs,andthecontributionofFcreceptorsonmonocytestophagocytosisofPVs.

OP-03Metabolomicsidentifiesglutathioneproductionascriticaltothemechanismofartemisininresistanceinmalariaparasites.GhizalSiddiqui,AmandaDePaoli,DarrenJ.CreekDrugDelivery,DispositionandDynamics,MonashInstituteofPharmaceuticalSciences,MonashUniversity,ParkvilleCampus,Parkville,Victoria,AustraliaArtemisininanditsderivatives(ARTs)underpinthemosteffectivetreatmentsforuncomplicatedPlasmodiumfalciparummalaria.However,resistancetoARTsisbecomingincreasinglyprevalentandthecellularmechanismofresistancerequiresfurtherelucidation.UntargetedmetabolomicsandpeptidomicsanalysisofPfKelch13-mutantP.falciparumrevealedadown-regulationofhaemoglobindigestion,andanincreasedabundanceofglutathione(GSH)anditsprecursorgamma-glutamylcysteineinART-resistantparasites.WeproposethatthesedifferencesenhancetheabilityofART-resistantparasitestoameliorateART-inducedfreeradicaldamage,asglutathioneconjugationhasbeenshowntofunctionasadetoxificationmechanismformanydrugsthatactviathegenerationofreactiveintermediates.ThisstudyinvestigatedtheroleofglutathioneinPfKelch13-mediatedARTresistanceinP.falciparumparasitesusingmetabolomics,andtestedwhetherinhibitionofthisdetoxificationsystemcanbeastrategyformodulationofresistancetoART.ART-resistantisolateswereincubatedwithbuthioninesulphoximine(agamma-glutamylcysteinesynthetaseinhibitor)inordertoinhibitglutathioneproduction,resultinginincreasedARTring-stageactivityintheART-resistantparasites(40%survivaldecreasedto18%,p<0.05)toalevelofactivitycomparabletosensitiveisolates.Onthecontrary,pre-incubationofART-sensitiveparasiteswiththeglutathioneprecursor,N-acetylcysteine,wassufficienttoreduceARTring-stageactivitytoalevelcomparabletotheresistantparasites.AtargetedquantitativeLCMSmethodforglutathionewasthenestablishedtoobtainaquantitativerelationshipbetweenintracellularglutathioneconcentrationandsusceptibilitytoART.Inconclusion,wehaveidentifiedalteredglutathionemetabolismasamajorcontributingfactortothemechanismofPfKelch13-mediatedARTresistance.Furthermore,theinvitroARTresistancephenotypecanbereversedbyinhibitingthisdetoxificationsystem.

OP-04

CharacterizingriskfactorsoftheresidualtransmissionvectorAnophelessquamosusinaneliminationsettinginChomaDistrict,Zambia

MaryE.Gebhardt1,KellyM.Searle2,TamakiKobayashi3,TimothyM.Shields3,HarryHamapumbu4,LimontySimubali4,TwigMudenda4,PhilipE.Thuma4,JenniferC.Stevenson1,4,WilliamJ.Moss3,DouglasE.Norris11DepartmentofMolecularMicrobiologyandImmunology,JohnsHopkinsMalariaResearchInstitute,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2DivisionofEpidemiologyandCommunityHealth,UniversityofMinnesota,Minneapolis,Minnesota,3DepartmentofEpidemiology,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,4MachaResearchTrust,Macha,Zambia Macha,inChomaDistrictinsouthernZambia,isworkingtowardanationalgoaltoeliminatemalariaby2021.MalariaprevalenceinMachadecreasedfrom9%in2008to1%in2013afterinsecticide-treatedbednet(ITN)campaigns,howeverresidualmalariatransmissionstilloccurswithyearlyinfectionprevalencefrom1-3%underactivecasesurveillance.Asvectorcontrolstrategiesaresuccessfullyimplemented,identificationofthevectorsofresidualtransmissionremainschallenging.WhileITNsreducethethreatfrompredominatelyendophagicandendophilicvectors,otheropportunisticforagingspeciesmaycontributeassecondaryvectorsinresidualtransmissionsettings.In2013,theZambiangovernmentimplementedareactivetest-and-treatstrategyinChomaDistricttopursuetheirgoalofelimination.WhenanindexcasewasidentifiedpositiveforP.falciparumbyrapiddiagnostictest(RDT)atalocalhealthfacility,ahealthcareworkervisitedtheirhouseholdandeveryhouseholdwithin140meters.Inthisstudy,theradiuswasextendedto250metersandallindividualswithintheradiuswerescreenedforP.falciparumusingaRDTandtreatedwithACTifpositive.Thisprocedurewasrepeated30and90daysaftertheinitialvisit.Additionally,entomologicalsampleswerealsocollectedbyplacingCDClighttrapsindoorsnexttoapersonsleepingunderabednetandoutdoorsnearanimalpensintheindexandoneneighboringhousehold.EachanophelinewasmorphologicallyandmolecularlyidentifiedtospecieslevelbyPCR,analyzedforrecentbloodmealcontentviaPCR,andevaluatedforsporozoiteprevalenceusingaP.falciparumELISA.Aknownsecondaryvectorinthisarea,Anophelessquamosus,wascapturedthroughoutallthreeyearsofthisstudy.Ananalysiswasperformedtoidentifyriskfactorsforthepresenceofthismosquitowiththeintentiontorecommendpotentialinterventionsthatwilltargetthisresidualmalariavector.

OP-05

AglanceofthebloodstagetranscriptomeofaSoutheastAsianPlasmodiumovaleisolate

AwtumBrashear1,2,WanlapaRoobsoong3,FaizaA.Siddiqui2,WangNguitragool4,JetsumonSattabongkot3,MargaritaM.



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López-Uribe1,JunMiao2&LiwangCui1,2

1DepartmentofEntomology,PennsylvaniaStateUniversity,DepartmentofEntomology,UniversityPark,Pennsylvania,2DepartmentofInternalMedicine,UniversityofSouthFlorida,Tampa,Florida,3MahidolVivaxResearchUnit,FacultyofTropicalMedicine,MahidolUniversity,Thailand,4DepartmentofMolecularTropicalMedicineandGenetics,FacultyofTropicalMedicine,MahidolUniversity,Thailand

Plasmodiumovaleaccountsforadisproportionatenumberoftravel-relatedmalariacases.Itisalsounderstudiedinpartduetoarelianceonclinicalsamples.WecollectedaP.ovalecurtisiparasiteisolatefromaclinicalcaseinwesternThailandandperformedRNA-seqanalysisonthebloodstagetranscriptomes,takingadvantageofbothdenovoassemblyandalignment-basedtechniques.Wedetectedtranscriptsfor6628outof7280annotatedgenes.Forthoselackingevidenceofexpression,thevastmajoritybelongedtothePIRandSTP1genefamilies.Weidentifiednewsplicingpatternsforover2500genes,andmappedatleastoneuntranslatedregionforoverhalfofallannotatedgenes.Ouranalysisalsodetectedanotablepresenceofanti-sensetranscriptsforover10%ofP.ovalecurtisigenes.Thistranscriptomicanalysisprovidesnewinsightsintotheblood-stagebiologyofthisneglectedparasite.

OP-06

AsymptomaticPlasmodiumfalciparumandPlasmodiumvivaxInfectionsunderdifferentTransmissionIntensities:WhocontributestoTransmission?CristianKoepfli1,2,WangNguitragool3,AnneCristineGomesdeAlmeida4,5,AndreaKuehn6,AndreeaWaltmann2,ElineKattenberg1,7,MariaOme-Kaius2,7,PatriciaRarau7,ThomasObadia8,9,JamesKazura10,WueltonMonteiro4,5,AndrewW.Darcy11,LyndesWini12,QuiqueBassat6,13,14,15,16,IngridFelger17,JetsumonSattabongkot18,ColinsOduma19,LeanneJ.Robinson7,MarcusLacerda4,IvoMueller2,9

1EckInstituteforGlobalHealth,UniversityofNotreDame,2PopulationHealth&ImmunityDivision,Walter&ElizaHallInstitute,2DepartmentofMedicalBiology,UniversityofMelbourne,Parkville,Australia,3DepartmentofMolecularTropicalMedicineandGenetics,FacultyofTropicalMedicine,MahidolUniversity,Bangkok,Thailand,4FundaçãodeMedicinaTropicalDr.HeitorVieiraDourado,5UniversidadedoEstadodoAmazonas,Manaus,Brazil,6ISGlobal,HospitalClínic,UniversitatdeBarcelona,Spain,7PapuaNewGuineaInstituteofMedicalResearch,Madang,PapuaNewGuinea,8HubdeBioinformatiqueetBiostatistique,DépartementBiologieComputationnelle,InstitutPasteur,9UnitéMalaria:parasitesetHôtes,DépartementParasitesetInsectesVecteurs,InstitutPasteur,Paris,France,10CenterforGlobalHealth&Diseases,CaseWesternReserveUniversity,Cleveland,Ohio,11NationalHealthTrainingandResearch

Institute,12VectorBorneDiseasesProgram,MinistryofHealth,Honiara,SolomonIslands,13CentrodeInvestigaçãoemSaúdedeManhiça,Maputo,Mozambique,14ICREA,Barcelona,Spain,15PediatricInfectiousDiseasesUnit,PediatricsDepartment,HospitalSantJoandeDéu,UniversityofBarcelona,16ConsorciodeInvestigaciónBiomédicaenReddeEpidemiologíaySaludPública,Madrid,Spain,17SwissTropicalandPublicHealthInstitute,Basel,Switzerland,18MahidolVivaxResearchUnit,FacultyofTropicalMedicine,MahidolUniversity,Thailand,19KenyaMedicalResearchInstitute,Kisumu,KenyaUnderstandingepidemiologicalvariablesaffectinggametocytecarriage,andthushuman-to-vectortransmissionpotentialanddensityisessentialtodesigninterventionsthatmosteffectivelyreducemalariahuman-to-mosquitotransmission.PlasmodiumfalciparumandPlasmodiumvivaxparasitesandgametocyteswerequantifiedbyqPCRandRT-qPCRassaysin5cross-sectionalsurveysinvolving16,493individualsinBrazil,Thailand,PapuaNewGuinea,andSolomonIslands.Theproportionofinfectionswithdetectablegametocytesrangedfrom44-94%forP.falciparumandfrom23-72%forP.vivax.Blood-stageparasitedensitywasthemostimportantpredictoroftheprobabilitytodetectgametocytes.Inmoderatetransmissionsettings(prevalencebyqPCR>5%),parasitedensitydecreasedwithageand53-84%ofthecumulativegametocytedensitywasfoundinchildren<6years.Inlowtransmissionsettings(prevalence<5%),noagetrendsingametocytecarriagewereevident.Persurvey,37-100%ofallindividualspositiveforgametocytesbyRT-qPCRwerepositivebylightmicroscopyforasexualstagesorgametocytes(overall:P.falciparum178/348,P.vivax235/398).66-70%ofthecumulativegametocytedensitywasfoundinlightmicroscopypositivesamples.Interventionstoreducehuman-to-mosquitomalariatransmissioninmoderate-highendemicitysettingswillhavethegreatestimpactwhenchildrenaretargeted.Incontrast,allagegroupsneedtobetargetedinlowendemicitysettingstoachieveelimination.Detectionofinfectionsbylightmicroscopyisavaluabletooltoidentifyasymptomaticbloodstageinfectionsthatlikelycontributemosttoongoingtransmission.

OP-07

TheassociationbetweenPlasmodiumfalciparuminfectioninthefirstsixmonthsoflifeandsubsequentinfectionamongchildrenunder24monthsinMalawi,2016-2018LianaR.Andronescu1,AndreaG.Buchwald2,AndyBauleni3,PatriciaMawindo3,DonP.Mathanga3,MiriamK.Laufer1

1CenterforVaccineDevelopmentandGlobalHealth,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,2UniversityofColoradoSchoolofPublicHealth,UniversityofColorado,Denver,Colorado,3MalariaAlertCenter,UniversityofMalawiCollegeofMedicine,Blantyre,Malawi



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Inhightransmissionsettings,infantsundersixmonthscompriseupto10%ofmalaria-relatedhospitalizationsinchildrenunderfiveyearsold.Despitetheprevalenceofmalariainfectionanddiseaseinthisagegroup,theepidemiologyisnotwellunderstoodandincidenceisrarelyassessed.Inthefirstsixmonthsoflife,infantsmayeitherhaveincreasedimmunitytoinfectionduetopresenceofmaternalantibodiesorincreasedsusceptibilityduetoexposureinutero.Theimpactofearlymalariainfectiononsubsequentdiseaseisunclear.WeaimedtodetermineifP.falciparuminfectioninthefirstsixmonthsoflifealterstheriskofsubsequentclinicalmalariaupto24monthsofage.Toaddressthisquestion,infantsinMalawiwerefollowedattwositesfrombirthuntil24monthsofage.Studyparticipantswerescheduledforclinicvisitsatthree-monthintervalsandaskedtovisittheclinicbetweenappointmentsintheeventofillness.RDTswereperformedatallscheduledappointmentsandifmalariasignsandsymptomswerepresentatsickvisits.PositiveRDTswereconfirmedbymicroscopy.Preliminarydatafromonestudysiteincluded94participantswithameanfollow-uptimeof21.48(SD:6.54)monthsandameanof2.37(SD:4.30)infections.Inthefirstsixmonthsoffollow-up,27infectionswerereportedforanincidencerateof5.1P.falciparuminfectionsper100person-monthsofobservation.Inthefollowing18months,therewere207infectionsforanincidencerateof14.6infectionsper100person-months.APoissonmodel,adjustedforseasonofbirth,indicatesthatforeveryinfectionoccurringbeforesixmonths,anadditional1.73(95%CI0.97,3.07)infectionsmaybeobservedbetweensixand24months,suggestingearlyinfectionisassociatedwithincreasedriskofsubsequentinfection.TheseresultsmightbeduetoincreasedexposuretoP.falciparumoranimpactonimmunityamonginfantswhoareinfectedinthefirstsixmonthsoflife.Analysisofourfinalanalysiswillincludedatafrombothhealthcentersandcovariatesforbednetuseandvillagetoassessexposure.

OP-08

AnophelesinnateimmunefactorsCTL4andCTLMA2:Solutionstructure,glycanspecificityandphenoloxidaseinhibitoryactivityRitikaBishnoi1,AliciaContet2,ChristopherJ.Day3,Chun-FengDavidHou1,LaurenA.Profitt4,DeepakSingla5,GregoryL.Sousa6,MichaelP.Jennings3,MichaelPovelones6,AnnM.Valentine4,RichardH.G.Baxter1

1Dept.ofMedicalGenetics&MolecularBiochemistry,LewisKatzSchoolofMedicineatTempleUniversity,Philadelphia,Pennsylvania,2Dept.ofChemistry,YaleUniversity,NewHaven,Connecticut,3InstituteofGlycomics,GriffithUniversity,Queensland,Australia,4Dept.ofChemistry,TempleUniversity,Philadelphia,Pennsylvania,5LaboratoryofHost-ParasiteInteractionStudies,NationalInstituteofMalariaResearch,

Dwarka,India,6SchoolofVeterinaryMedicine,UniversityofPennsylvania,Philadelphia,PennsylvaniaMalaria,theworld’smostdevastatingparasiticdisease,istransmittedbetweenhumansbymosquitoesoftheAnophelesgenus.An.gambiaeistheprincipalmalariavectorinSub-SaharanAfrica.TheC-typelectinsCTL4andCTLMA2cooperativelyinfluencePlasmodiuminfectioninthemalariavectorAnopheles.HerewereportthepurificationandbiochemicalcharacterizationofCTL4andCTLMA2fromAn.gambiaeandAn.albimanus.CTL4andCTLMA2areknowntoformadisulfide-bridgedheterodimerviaanN-terminaltri-cysteineCXCPCmotif.WedemonstrateinvitrothatCTL4andCTLMA2intermoleculardisulfideformationispromiscuouswithinthismotif.Furthermore,CTL4andCTLMA2formhigheroligomericstatesatphysiologicalpH.Bothlectinsbindspecificsugars,includingglycosamino-glycanmotifswithβ1-3/β1-4linkagesbetweenglucose,galactoseandtheirrespectivehexosamines.Small-anglex-rayscatteringdatasupportsacompactheterodimerbetweentheCTLdomains.RecombinantCTL4/CTLMA2isfunctionalinvivo,reversingtheenhancementofphenoloxidaseactivityindsCTL4-treatedmosquitoes.WeproposethesemolecularfeaturesunderlineacommonfunctionforCTL4/CTLMA2inmosquitoes,withspeciesandstrain-specificvariationindegreesofactivityinresponsetoPlasmodiuminfection.

OP-9MeasurementofPlasmodiumfalciparum-andP.vivax-specificantibodyprofilesonproteinmicroarraysfromdriedbloodspotsChristineF.Markwalter,1MyatHtutNyunt,2ZayYarHan,2AartiJain,3OmidTaghavian,3PhilipL.Felgner,3ChristopherV.Plowe,1KayThweHan,2MyaingM.Nyunt1

1DukeGlobalHealthInstitute,2DepartmentofMedicalResearch,Myanmar,3UniversityofCaliforniaIrvineSensitiveandfield-deployabledetectiontoolsareneededformalariasurveillanceandeliminationinlow-prevalencesettings.Anidealsuchtoolwouldnotonlymeasureparasiteprevalence,butalsoestimaterecentaggregatemalariaexposure,providingamorerobustcharacterizationofmalariatransmissioninapopulation.Serologicalbiomarkersarepromisingtargetsformalariasurveillancebecausetheycanindicatecumulativerecentexposureandareeasilyintegratedintoexistingpoint-of-careplatforms.SerologicalresponsestoP.falciparumandP.vivaxantigens,identifiedandmeasuredusingproteinmicroarrays,mayserveasusefultoolsforidentifyingantibodybiomarkersofexposure.Typically,serumsamplesareprobedonproteinmicroarraystomeasureantibodyresponsestomalariaantigens.Thecomplexityofsampleprocessingandcoldchainrequirementlimittheutilityofthismethodinremotehard-to-reach



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placeswheremalariaisprevalent.Driedbloodspotsarepotentiallyusefulalternativestoserum;theyareeasytocollect,durable,easytotransport,andpresentlittlebiohazardrisk.Inthisstudy,proteinmicroarrayswereusedtomeasureantibodyprofilesagainst250P.falciparumand250P.vivaxantigensorfragmentsinmatchedserumandDBSfrom100individualsfromIngapuTownship,Myanmarandfoundgoodcorrelationbetweenserologicalresponsesderivedfromthetwosamplematrices.WeappliedthesameDBSelutionmethodologyandproteinmicroarraystomeasureantibodyresponsesofindividualswithlow-densitymalariainfectionsandmatcheduninfectedindividualsfromInjangyangandAnnTownshipsinMyanmar.Thus,usingdriedbloodspotscanextendproteinmicroarraysero-surveillancetoremoteareaswherecoldchainsrequiredtocollectandtransportfrozensamplesdonotexist.

POSTERPRESENTATIONS

PP-01TheEffectsofpHandTemperatureParametersofWateronAbundanceofAnophelesMosquitoLarvaeinDifferentBreedingSitesofKapiriMposhiDistrictofZambia.

MosesMusonda,AlfredMatafwaliSichilima

CopperbeltUniversity,Ndola,Zambia MalariaisavectorbornediseaseamajorcauseofmorbidityandmortalityinZambia.ItistransmittedbyAnophelesmosquitoes.ThereispaucityofevidenceontheeffectsofphysicochemicalparametersonlarvaeinKapirimposhidistrictofZambia.ThestudyaimedatassessingtheeffectsofphysicochemicalparametersonAnopheleslarvaeabundanceindifferentbreedingsites.Thedistrictwasdividedintofourzonesandsurveyedfordams,rivers,swamps,marshlandsandtemporalwaterponds.TemperatureandpHmeasurementofwaterweretakenonaweeklybasisusingamultiparametermeter(explorerGLXPasco).450Anopheleslarvaewerecollectedand73.6%(n=331)emergedadults,siblingspecieswereidentifiedusingquantitativePolymeraseChainReaction(qPCR).TheabundanceofAn.gambiaes.swassignificantlythehighest(70.9%n=223),An.arabiensisPaton(29.1%=92),An.funestus(0%)and5.2%non-amplified.31.8%ofAn.gambiaes.swasfoundintemporalwaterpondswithanaveragepHandtemperatureof6.37and26.90C,respectively.Temporalwaterpondshad24.6%An.arabiensisPatonwithtemperatureandpHof26.90Cand6.37.ThedamsandrivershadnoAnophelessiblings.pHhasapositivecoefficientcorrelation(r=0.114p=0.05)whiletemperaturehasnegativecorrelation(r=-0.373,p=0.05).Therefore,pHandtemperatureaffectstheabundanceoftheAnophelesmosquitolarvaeinbreedingsitesofKapiriMposhidistrict.

PP-02MalariainNigeria,WestAfrica:AnOverview OlubunmiSharonObayemi1,JamesFlorence2,AdekunleOdunayoAdejuwon3,4

1DepartmentofPublicHealthandCommunityHealthPromotion,LibertyUniversity,2DepartmentofHealthProfessions,LibertyUniversity,Lynchburg,Virginia,3SchoolofHealthInformationManagement,UniversityCollegeHospital,Ibadan,Nigeria,4CommunityHealthOfficersTraining,UniversityCollegeHospital,Ibadan,Nigeria MalariacausedbyspeciesoftheparasitePlasmodiumisholo-endemicinNigeria,WestAfrica.Morethan90%oftheNigerianpopulationisatrisktothisdeadlyinfectionwithover100millioncasesyearlyinthelastdecade.Reportedcasesofdeathhasbeenestimatedtobeabout300,000peryearintropicalNigeria.ThepoorstandardoflivingintheruralNigeriancommunityisoneofthecausesofprotractedinfectionleadingtomalariadisease.SpeciesofPlasmodiumresponsibleformalariainNigeriaincludevivax,malariae,falciparumandovale.AccordingtotheWHOWorldMalariaReport,2017,Nigeriacontributedto27%ofglobalmalariaburdenin2016andaccountsfor26%ofglobalestimatedmalariadeaths.TheNigeriaGovernmenthasrecentlyreachedanagreementwiththeUnitedStatesPublicHealthontheeradicationofmalariainNigeria.TheproposedFiscalYear(FY)UnitedStatesPresident’sMalariaInitiative(PMI)budgetforNigeriawas65milliondollars.ControlstrategiesontheeradicationofmalariainNigeriainclude:Insecticide–treatednets,indoorresidualspraying,seasonalmalariachemo-prevention,casemanagement,pharmaceuticalmanagement.Surveillance,monitoringandevaluationreportshavealsobeenhelpfulinthecontrolofthisendemicdiseaseinNigeria,WestAfrica.

PP-03TheimportanceofcopynumbervariationinmetabolicinsecticideresistanceinAnophelesgambiaeLizzieTchongwe1,EricLucas2,MartinDonnelly2

1MalawiLiverpoolWellcomeTrust,Malawi,2LiverpoolSchoolofTropicalMedicine,Liverpool,UnitedKingdom,Insecticideresistanceisathreattothesuccessinmalariacontrolduetointensifieduseofthefewavailableinsecticides.GeneticvariationsintheAnophelesgambiaegenomeareimportantinincreasedresistanceasevidencedintargetsitemutations.However,geneticvariationssuchascopynumbervariations(CNVs)havebeenpoorlystudiedandtheirrolepoorlyunderstoodinmetabolicresistance.Theaimofthisstudywastoidentifymetabolicresistancegenesthatexhibitcopynumbervariationandtheirsignificancein



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metabolicresistance.PyrethroidphenotypedsamplesfromMalawi(59)andKenya(354)werescreenedforthepresenceofaspecificCyp6aa1duplicationknowntoexistinEastAfrica(Cyp6aa1-Dup1)withPCR.CNVdiscoverywasdoneusingtheAnophelesgambiae1000genomesphase3datafromUgandaandTanzania,focusingonknownmetabolicresistancegenes;Cyp6aa1,GstesandCyp9k1.AhighfrequencyofCyp6aa1-Dup1wasfoundinbothdeadandalivephenotypes.Atotalof177(43%)individualshadtheduplication,137(33%)didnothaveaduplicationand99(24%)weredropouts.TheCyp6aa1-Dup1duplicationwassignificantlyassociatedwiththeresistancephenotypes.TheCNVdiscoveryrevealedhighCNVfrequenciesinthecandidategenesscreened.Cyp6aa1-Dup1,sofaronlyknowntoexistasaduplication,wasfoundtoexistasatriplicationintheTanzaniadata,andanewduplicationwasfoundonGste2in2individualsfromUganda.Theseresultshaveshownanimportanceofgeneduplicationinpyrethroidresistance,aswellashighfrequencyofCNVsinthethreemetabolicresistancegenes.MoreworkshouldbedonetofunctionallyvalidateCyp6aa1inAnophelesgambiaeandunderstanditsroleininsecticideresistanceandasadiagnosticmarkerinmetabolicresistance.

PP-04Transcriptome-wideinvivomRNAtargetidentificationofRNA-bindingproteinsessentialforP.falciparumsexualdevelopmentAndrewJ.Stasic,HeatherJ.Painter

U.S.Food&DrugAdministration,CenterforBiologics&EvaluationResearch,DivisionofBacterial,Parasitic&AllergenicProducts,SilverSpring,MarylandEffortstoeradicatetheglobalmalariahealthburdenrelyonthedisruptionofPlasmodiumfalciparumtransmission.Understandinggeneregulatorymechanismsthatenablematurationofthetransmissiblesexualstagesisessentialtothiseffort.Itiswellestablishedthatbothpost-transcriptionalregulationandtranslationalrepressionplayamajorroleinmaturationandfertilizationduringsexualdevelopment.However,duetolackofeffectivemethodologies,invivoidentificationoftheexacttargetsofRNAbindingproteins(RNA-BPs)essentialforsexualdevelopmentremainedelusive.Here,weaimtoestablishtheuseofphotoactivatedribonucleoside-enhancedcrosslinkingandimmunoprecipitation(PAR-CLIP)forthetranscriptome-wideidentificationofmRNA-proteininteractions.ThismethodexploitstheuseofatransgenicP.falciparumexpressingayeastfusionofuracilphosposribosyltransferaseandcytosinedeaminase(yFCU)whichcanincorporatethiol-modifieduracilintomRNA(PMIDs:29985403&28416533),enablingtranscriptome-wideinvivoidentificationofthespecifictargetsofanyRNA-BPviaphotoactivatedcovalentcrosslinkingtothiolatedtranscripts.TovalidatePAR-CLIPinP.falciparum,wewillinvestigateRNA-BPsthathave

previouslydemonstratedtoplayaroleinsexualdevelopment,includingPUF2,DOZIandCITH.ThesegeneswillbeGFP-taggedinP.falciparumstrainsexpressingyFCUandthiol-labeledmRNAswillbephoto-crosslinkedtotheRNA-BP.TheresultingRNA-proteincomplexeswillbeimmunoprecipitatedandthecrosslinkedmRNAsequencedfortargetidentification(PMID27871973).TheexactRNA-BPbindingsiteswillbeidentifiedbytakingadvantageofaninducednucleotidemismatchsignaturewhichisaresultofcrosslinking.Overall,thisapproachwillprovideamethodforfullycharacterizingessentialRNA-BPsandbroadenourunderstandingofpost-transcriptionalregulationduringparasitetransmissionfortheidentificationofpotentialinterventionstrategies.

PP-05HealthProfessionalsPerceptionAndSatisfactionOnQualityOfLaboratoryMalariaDiagnosticService;TheCaseAwiZone,NorthEthiopiaAgajieLikieBogale1,JemalHaidarAli2,AsterTsegaye3,FatumaHassen31EthiopianPublicHealthInstitute,AddisAbaba,2SchoolofPublicHealth,AddisAbabaUniversity,AddisAbaba,3DepartmentofMedicalLaboratorySciences,SchoolofAlliedHealthSciences,CollegeofHealthSciences,AddisAbabaUniversity,EthiopiaInappropriateperceptionandinadequatesatisfactionofhealthworkersposesignificantchallengesonmalariadiagnosticservicesinthefightagainstmalaria.Suchinformationhoweverisagapinmostruralhealthfacilities.ThisstudyassessedHealthProfessionalsperceptionandtheirsatisfactiontowardsmalariaqualitydiagnosticserviceinNorthernEthiopia.Across-sectionalfacilitybasedstudywasconductedin2013among136participants(110cliniciansand26laboratoryprofessionals)engagedinmalariamanagement.Levelofperceptionandsatisfactionwasmeasuredusingavalidatedstructuredquestionnaire.ThestructureddatawereenteredusingEpi-Infoversion3.5.3andanalyzedbySPSSversion20.Thefindingindicatedthatabout61%(67/110)ofthecliniciansand50%(13/26)oflaboratoryprofessionalsweresatisfiedwiththequalityofwork.Thoseclinicianswhorequestlaboratorymalariadiagnosisbasedonsoundclinicaljudgmentweremoresatisfied(AOR=3.12,95%CI=1.06-9.13)thantheircounterpartswhilethosewhotrustlaboratorymalariadiagnosticresultasjustreliablewere68.0%(AOR=0.32,95%CI=0.13-0.83)lesslikelytobesatisfiedthanthereferentgroups.Laboratoryprofessionalswithnolimitingfactorsforlaboratorydiagnosiswere30.6times(AOR=30.6,95%CI=1.83-511.8)moresatisfiedontheservicecomparedwiththosewhohadconstraintsintheirhealthfacility.Thelevelofsatisfactionofhealthprofessionalsinthecurrentstudywasnotencouragingandislowerthansomeprevious



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studiesconductedinthecountry.Thus,targetingtheidentifiedlimitingfactorsarecrucialstepstoconsiderinthefightagainstmalaria.

PP-06Mitochondrialacetyl-coAbiosynthesisisessentialduringtheredbloodstagesofhumanmalariaparasitePlasmodiumfalciparum.SethuC.Nair,JustinMunro,ManuelLlinas,SeanT.PriggeJohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,MarylandAcetyl-coAisacentralmoleculeincarbonmetabolism.Malariaparasiteshavethreemajorpathwaystomakeacetyl-coAthatarelocateddifferentsubcellularcompartments:theapicoplast,mitochondrionandcytosol.Boththeapicoplastandmitochondrialpathwaysusethepyruvatederivedfromglycolysisandenzymaticallygenerateacetyl-coAusinganorganelle-specificpyruvatedehydrogenaseenzyme.Thecytosolicpathwayreliesonmillimolarlevelsofexogenousacetateanditsconversiontoacetyl-coAusingacytosolicacetyl-coAsynthetaseenzyme.Theapicoplastpathwayisnotessentialduringthebloodstages,leavingthemitochondrialsourceofacetyl-coAapotentiallyimportantandvitalsource.Inthepresentstudy,wegeneratedaseriesofdeletionmutantsofimportantenzymesinthemitochondrialacetyl-coAbiosyntheticpathwayandshowedthatmitochondrialacetyl-coAbiosynthesisisessentialforthesurvivaloftheparasite.Unexpectedly,wefoundthattwomitochondrialenzymesarecapableofmakingacetyl-coA(PyruvateDehydrogenaseandalpha-KetoglutarateDehydrogenase);deletionofbothenzymesisrequiredtokillbloodstageparasitesunlessexogenousacetateisprovided.Bothenzymesrelyonlipoicacidasacofactorandwewereabletosimultaneouslyblocktheactivityofbothenzymes,andtheproductionofacetyl-coAbygeneticallytargetinglipoicacidmetabolism.Westudiedtheproductionofacetyl-coAinwildtypeandgene-knockoutparasitesusing13C-labeledglucose,acetateorglutamineandshowedhowthesedifferentcarbonsourcesaremetabolizedindifferentmetabolicscenarios.Theseresultsprovidenovelinsightsintoparasitemetabolismandidentifyanessentialproductofthemitochondrion.

PP-07TheecdysonereceptorregulatesvitellogenesisinAnophelesarabiensisfemalemosquitoesElodieEkoka1,2,LuisaNardini1,2,AyeshaAswat1,2,YaelDahan-Moss1,2,andLizetteLKoekemoer1,2

1CentreforEmerging,Zoonotic&ParasiticDiseases,NationalInstituteforCommunicableDiseases,Johannesburg,2Wits

ResearchInstituteforMalaria,SAMRCCollaboratingCentreforMultidisciplinaryResearchonMalaria,SchoolofPathology,FacultyofHealthSciences,UniversityoftheWitwatersrand,Johannesburg,SouthAfrica

Anophelesarabiensis,oneofthemainmalariavectorsinSouthAfrica,isnotefficientlycontrolledbytheinsecticide-basedstrategiesusedlocallybecauseofitsincreasingresistance,andexophilic/exophagicbehaviour.Interestingly,severalreportssuggestthattransmission-blockingvaccinestargetingmosquitogenes,orgeneticallymodifiedmosquitoescouldofferanavenueforAn.arabiensiscontrol.Thesetechniquestargetgenesthatareessentialforthemosquitovectorialcapacity.Inthiscontext,onecandidateofinterestistheEcdysoneReceptor(EcR),asitwasshowntoregulateimmunity,bloodfeeding,vitellogenesis,fecundity,andPlasmodiumdevelopmentinmosquitoes.Currently,nofunctionalcharacterizationofEcRinAn.arabiensishasbeenreported.ThisstudyhypothesizedthatinAn.arabiensis,EcRregulatesvectordensitybycontrollingtheexpressionofkeyeffectorsofvitellogenesis.Totestthishypothesis,real-time-PCRandinsilicoanalyseswereusedtoisolateEcRfromAn.arabiensis.Then,EcRexpressionatdifferenttimepointsduringvitellogenesis(12hourspostbloodmeal[hPBM],24hPBM,36hPBM,and48hPBM)wasquantifiedwithqPCR.Finally,weusedRNAinterferencetoinvestigatehowsilencingEcRaffectstheexpressionoffourYolkProteinsPrecursors(YPPs):Vitellogenin(Vg),Lipophorin(Lp),VitellogeninCarboxypeptidase(VCP),andCathepsinb-likeprotease(VCB).PhylogeneticanalysesrevealedthatAn.arabiensisEcRorthologueis,asexpected,closertothatofAn.gambiae,assupportedbyabootstrapvalueof100%.Duringvitellogenesis,EcRexpressionincreasedfrom12to24hPBMandthendeclinedfrom36to48hPBM,similartofindingsinAedesaegypti,thevectorofdengueandzikaviruses.Finally,silencingEcRaffectedtheexpressionofVg,VCP,andVCB,whiletheexpressionofLpremainedunchanged.ThiswasthefirststudytocharacterizetheexpressionandfunctionofEcRinvitellogenesisinaSouthAfricanmosquitovector.

PP-08Diagnosisofasymptomaticmalariainfections:PCRmethodasgoldstandardoverultrasensitiverapiddiagnostictests RomanaAkter UniversityofDhaka MalariaisaleadingcauseofmorbidityandmortalityinmanydevelopingcountriesincludingBangladesh.RecenttrendsoflownumberofthecasesinChittagongHillTracts(HyperendemicareaofBangladesh)underscoretheneedtogiveattentiontotheeliminationofmalariafromBangladesh.



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WhiledealingwiththemalariaeliminationprogramthathasalreadybeenadoptedbyNMEP,Bangladesh,itisimportanttopayattentiontotheasymptomaticmalariainfections.Theobjectiveofthisstudywastoidentifytheindividualswithasymptomaticmalariaanditsrelationtoage,sex,andseasonalvariations.Across-sectionalstudywasconductedfromSeptember,2018toMay,2019inKuhalongandRajbilaUnionofBandarbantodiagnosetheasymptomatic-malariaindividualsusingNestedPCRandtoevaluatethesensitivityofUltrasensitiveRDTforfieldapplicationaswell.ThesensitivityandspecificityoftheUs-RDTwascomparedwiththatofthegoldstandardPCR.Among300samples,9sampleswereconfirmedtobepositivebyPCR.BothPlasmodiumfalciparumandPlasmodiumvivaxwerethedominantamongthePlasmodiumspecies.Intermsofsexandage,thefemales(66.67%)andtheadolescent(44.44%),respectively,wereharboringmoreinfectionscomparedtootherstudygroupsandalsotheratewashigherinSeptember(44.44%).TheUltrasensitiveRDThadthesensitivityof33.33%(95%CI;9.04%-69.08%)andthespecificityof90.3%(95%CI;86.24%-93.40%).PCRhasalwaysbeenthegold-standardforthediagnosisofasymptomaticmalaria.Moreover,thestudyfindingssuggestthattheUS-RDT,thoughconvenientanduser-friendly,stillcannotbeusedasatoolforthedetectionofasymptomaticcases.Nevertheless,forfieldlevelandmasssurveillanceofasymptomaticcases,thereisnootherbetteralternativethanUS-RDTandhence,itimposesthatthesensitivityandspecificityofthistechniquemustbeimprovedtomeetthediagnosticrequirement.

PP-09ABObloodgroupandmortalityinchildrenwithseveremalarialanemiaduetoPlasmodiumfalciparum Jean-BertinKabuya1,LucK.Kamavu2,MikeChaponda1,

JamesS.Lupiya1,ManuelaHauser3,WilliamJ.Moss4,5,PhilipThuma4,6,MatthewM.Ippolito4,7fortheSouthernandCentralAfricaInternationalCentersofExcellenceforMalariaResearch

1TropicalDiseasesResearchCentre,Ndola,Zambia,2SaintPaul’sGeneralHospital,Nchelenge,Zambia,3UniversityChildren’sHospital,Zurich,Switzerland,4MalariaResearchInstitute,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,5DepartmentofEpidemiology,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,UnitedStates,6MachaResearchTrust,Macha,Zambia,7DepartmentofMedicine,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MarylandPreviousstudiesofABObloodgroupandclinicaloutcomesinpatientswithPlasmodiumfalciparuminfectionsuggestaprotectiveeffectofbloodgrouptypeO.ToassessthepossiblerelationshipbetweenABObloodgroupandsurvivalinchildrenwithseveremalarialanemiaduetoP.falciparum

infection,wecombineddatafromtwoobservationalstudiesofseveremalariaconductedintwodistrict-levelhospitalsinZambia.Studyparticipantswerechildren≤12yearsadmittedtothehospitalwithadiagnosisofmalariawithsevereanemia,definedashemoglobinconcentration≤5g/dl.Forcomparison,weincludedchildrenwithsevereanemiaduetoacauseotherthanmalaria.WeexaminedassociationsbetweenABObloodgroupandmortalityinunadjustedandadjustedlogisticregressionmodels.Weidentified384childrenwithseveremalarialanemiaand45childrenwithsevereanemiaduetoanothercause.Othercausesincludedsicklecelldisease,malnutrition,renaldisease,andinfectionsotherthanmalaria.Amongchildrenwithseveremalarialanemia,bloodgrouptypeOwasthemostprevalent(43%)followedbytypeA(28%),typeB(23%)andtypeAB(5%).Themedianagewas23months(interquartilerange[IQR]:12-36)and52%weregirls.Themedianhemoglobinconcentrationwas3.8g/dl(IQR:3.1-4.3g/dl).Mostofthechildren(86%)receivedbloodtransfusionandthecasefatalityratiowas13%.Childrenwithothersevereanemiaweresimilaracrossallcharacteristics.Adjustedforage,sex,hemoglobinconcentration,andbloodtransfusiontherewasnostatisticallysignificantassociationbetweenABObloodgroupandmortalityinchildrenwithseveremalarialanemia,orinchildrenwithsevereanemiaduetoothercauses.Higherhemoglobinconcentrationandreceiptofabloodtransfusionwereassociatedwithimprovedsurvival(OR1.6,95%CI:1.1-2.3,p=0.02perg/dlincreaseinhemoglobin;OR3.3,95%CI:1.5-7.4,p<0.01forbloodtransfusion).AlthoughpreviousstudiesdemonstratedarelationshipbetweenABObloodgroupandmalaria-relatedoutcomes,wedidnotfindanassociationbetweenbloodtypeandmortalityinZambianchildrenwithseveremalarialanemia,orsevereanemiaduetoanothercause.FurtherstudiesthatassesstheassociationbetweenABObloodgroupandmalariamaylendinsightintothepathophysiologyofmalariacausedbyP.falciparum.

PP-10Developmentofmulti-epitopedrivensubunitvaccineinsecretoryandmembraneproteinofPlasmodiumfalciparumtoconveyprotectionagainstplacentalmalariainfection RajanKumarPandey,VijayKumarPrajapati DepartmentofBiochemistry,SchoolofLifeSciences,CentralUniversityofRajasthan,Bandarsindri,Kishangarh,Ajmer,Rajasthan,IndiaPlacentalmalariaistheseverehealthconcernforalongtimewhichtargetsthepregnantwomanregardlessofpreviousacquiredimmunity.ThisseverepathologicalconditionismediatedbytheVAR2CSA,aPlasmodiumantigenthatinteractswiththeplacentalchondroitinsulfate-A(CSA).AspertheWHOreports,themalarialinfectioncauseshugemortalityallaroundtheworldandisincomparablewithany



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otherinfectiousdiseases.Theabsenceofeffectivetreatmentoptionsandincreasingdrugresistancetotheavailabletherapeuticslikeartemisininandotherderivativesdemandanefficientalternativetoovercomethisdeathburden.WeperformedtheliteraturesurveyandsortedthePlasmodiumfalciparumsecretoryandmembraneproteinsincludingVAR2CSAtodesignmulti-epitopesubunitvaccineusinganadjuvant,B-cell-andT-cellepitopesincludingcytotoxicT-lymphocytes(CTL)andhelperT-lymphocytes(HTL)epitopes.EveryhelperHTLepitopewasIFN-γpositiveandIL-4non-inducer.Thephysicochemicalproperties,allergenicity,andantigenicityofdesignedvaccinewereanalyzedforthesafetyconcern.Homologymodelingandrefinementwereperformedtoobtainthefunctionaltertiarystructureofvaccineproteinfollowedbyitsmoleculardockingwiththetoll-likereceptor-4(TLR-4)immunereceptor.Moleculardynamicssimulationwasperformedtochecktheinteractionandstabilityofthereceptor-ligandcomplex.Multiepitopesubunitvaccineof704aminoacidresidueswasdesignedhavingitsentirecomponentasmentionedaboveincludingCTL,HTL,B-cellepitopes,adjuvantandlinkers.Immunoinformaticsevaluationrevealedtheimmunogenicandstablenatureofdesignedsubunitvaccinewhile,itsexperimentalvalidationmayconfirmitsabilitytoenhancethematernalimmunityagainstplacentalmalariainfection.Thisway,wedesignedthemulti-epitopesubunitvaccinetoservethematernalhealthlivingintheglobalendemiczone.

PP-11AchievementsofLiver-HumanizedMiceintheEliminationofMalariaLanderFoquetYecuris,Portland,OregonAlthoughP.falciparumcanbeculturedinvitro,severalaspectsofmalariainfectionscanonlybeaddressedbyinvivoresearch.Thecomplexmechanismsofmalariatransmissionthroughmosquitobites,themultipletissuebarrierscrossedbytheparasite,andthedifferentcelltypesthatareinfectedduringitslifecyclemakeitimpossibletostudyallaspectsinoneinvitrosystem.Thedevelopmentofliver-humanizedmousemodelshasmadeitpossibletostudyP.falciparumliver-stageinfectioninvivo.Humanizationofthemousebloodstreamisachievablebyfrequentinjectionsofhumanredbloodcells(hRBCs)andiscurrentlytheonlysystemwithwhichtostudyhumanmalariablood-stageinfectionsinasmallanimalmodel.Thetransferofhumanantibodiesgeneratedbyvaccinetrialsintoliver-humanizedmicehasenabledresearcherstodeterminetheirefficacyinpreventinginfectionofeithertheliver,ortransitiontoblood-stage.Infectionoftheliver-humanizedmousemodelwithP.vivaxhasallowedresearcherstostudythedevelopmentof

hypnozoitesandtestnoveldrugstokillthehibernatingparasites.Thecurrentliver-humanizedmousemodelslackanadaptiveimmunesystem.Thisisknocked-outtopreventhostvsgraftdisease.Asafurtherenhancement,wearedevelopingaliver-humanizedmousemodel,co-engraftedwithahumanimmunesystem.Thiswillallowourcollaboratorstostudypathogen-hostinteractionsbetweennotonlyhumanhepatocytes,butalsotheactivationofhumanimmunecells.

PP-12DualSiteandMechanismofActionofArtemisininAntimalarials WenchuanMa1,VictoriaBalta2,KatyN.Olafson1,OgnjenŠ.Miljanić3,DavidJ.SullivanJr.2,PeterG.Vekilov1,3,JeffreyD.Rimer1,31DepartmentofChemicalandBiomolecularEngineering,UniversityofHouston,Houston,Texas,2MalariaResearchInstitute,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,3DepartmentofChemistry,UniversityofHouston,Houston,TexasThedelayedclearanceofPlasmodiumfalciparumparasitesfollowingartemisinincombinationtherapyischaracterizedbyaringstage-resistancetoartemisininsmeasuredbyelevateddrugpulseconcentrationstoinhibitringstages.Thetrophozoitestagesremainsensitivetolownanomolardrug.ThephenotypeiscloselyassociatedwiththeKelch13genotypicchangesatnumerousaminoacids.Hereweinvestigatedforparasiticidalactivityandmechanismofactiononhemecrystallizationtheheme-artemisininadductmetabolite,previouslythoughttobeinactiveafterhemeorironcleavageoftheendoperoxidebridge.Heme-artemisininadductwassynthesizedinreducingconditions,columnpurifiedandvalidatedbymassspectrometrywithsharppeaksatm/z838and898,withtheabsenceofm/z282offreeartemisinin.Observationsofbeta-hematincrystalgrowthinhibitioninthepresenceofARTandH-ARTbytime-resolvedinsituatomicforcemicroscopy(AFM)revealedthattheadditionofnon-activatedARThadnoeffectonthesurfacefeaturesandthekineticsoflayernucleationandgrowth.Incontrast,hemeartemisininadductdemonstratednearirreversiblehemecrystalgrowthinhibitionbyabsorbingatgrowthsitestopreventsubsequentaddition.Biologicvalidationofexogenousheme-artemisininadducteffectonparasitesshowed50%inhibitioninthe10to70nmolarrangevaryingbyartemisininorartesunateasthehemeadductinbothresistantKelch13mutantandsensitiveparasites.Aftera6hourexogenous700nMH-ARTpulseoftrophozoites,followedbyextensivelySDS/bicarbonate/waterwashestopurifyhemozoin,H-ARTisdetectedwithhemozoinhemebyamassspectrometryatm/zs838.Thepreviouslythoughtinactiveheme-artemisininadductkillsparasiteswhenexogenousinculturemedium.Artemisininsworkinthe



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parasitecytosolatringstagesandinthecytosolanddigestivevacuoleatthetrophozoitestageviasuicideactivationbyironorhemetogenerateradicalswhichdamagebystanderproteins.Inthedigestivevacuolethereisanadditionalquinoline-likemechanismofhemecrystalinhibitionbytheheme-artemisininadduct.

PP-13DormancyandrecoveryofthearrestinPlasmodiumfalciparumisolatesagainstArtemisinin-basedCombinationTherapyRosaDelCarmenMiluskaVargasRodriguez1,AndrésJimenezGalisteoJr2,MariadeJesusCostadoNascimento3,SilviaMariaFátimaDiSanti3,41NationalUniversityofthePeruvianAmazon,Iquitos,Peru,2InstitutodeMedicinaTropicaldeSãoPaulo,SãoPaulo,Brazil,3NúcleodeEstudosemMalária,SuperintendênciadeControledeEndemias,SãoPaulo,Brazil,4FaculdadedeMedicina,UniversidadedeSãoPaulo,SãoPaulo,BrazilDormancyisaphenomenonthatPlasmodiumfalciparumusetotoleratetheactionofArtemisinin-basedCombinedTherapy(ACT).ThisstudyproposedtoevaluatedormancyofclinicalandreferenceisolatesofP.falciparumagainstfirst-lineregimensformalaria:artemether-lumefantrineandartesunatemefloquinebydormancyandflowcytometryassays.P.falciparumisolatesweretestedagainstdihydroartemisinin(DHA:4-1,000nM),artesunate(AS:0.1-100nM),lumefantrine(LMF:3.1-200nM),andmefloquine(MFQ:0.2-1,000nM)byex-vivoandinvitroassays.Therecrudescenceinvitrooftheseisolateswasaccompaniedinthedormancyassay.TheviabilityofthereferenceisolatewasassessedbyflowcytometryusingRhodamine123andDAPI.Inthedormancyassay,schizontswereobservedintheP.falciparumreferenceisolateNF54andintheclinicalisolateS-01/15afterpressurewith62.5nM,250nMand1,000nMDHA.Therecoveryperiodrangedfrom4to40days.ForLMF,dormancyonlywasobservedinNF54,inthedays7and12afterexposureto66.6nMand200nMofthedrug,respectively.SchizontswereseenintheP.falciparumclinicalisolateFS-08/15afterpressurewith100nMofAS,rightafterincubationperiodoftheex-vivoassay,withnodormancyoftrophozoitesinthedormancyassay.Inflowcytometryassay,viableyoungtrophozoitesofP.falciparumlabeledwithDAPIandRhodamine123wereobservedatthemaximumconcentrationsofDHA(1,000nM)andLMF(200nM).OurresultssuggestthatthedormancyisnotpresentedinallclinicalisolatesofP.falciparum.Flowcytometrywasabletoconfirmtheparasiteviabilityaccurately.

PP-14FLIMandImagingAnalysisofPlasmodiumfalciparumExposedtoArtemisinin

SeanV.Connelly1*,ZoeC.Levine1*,JavierManzella-Lapeira2,JosephBrzostowski2,LudmilaKrymskaya2,RifatS.Rahman1,JulianaM.Sá1,ThomasE.Wellems11LaboratoryofMalariaandVectorResearch,NationalInstituteofAllergyandInfectiousDiseases,2LaboratoryofImmunogenetics,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,Maryland,*equalcontributorsPlasmodiumfalciparumrecrudescenceafter3–7daysartemisininmonotherapyiswellknown.Recrudescenceisalsoobservedinvitrowhenearlyringstageparasitesareexposedtothedrug.Parasitesafterrecrudescencearejustassensitivetoartemisininasbeforetheinitialdrugtreatment,indicatingnochangeofdrugresponsephenotype.Microscopicobservationofadelayintheintraerythrocyticcyclewitharresteddevelopmentofthering-stageparasitessuggestedcelldormancymayprovideameanstoescapeartemisinintoxicity.Here,weapplyFluorescenceActivatedCellSorting(FACS),FluorescenceLifetimeImaging(FLIM),andsuper-resolutionAiryScanmicroscopytocomparering-stageparasitesexposedtodihydroartemisinin(DHA)ortocontrolmediumwithdimethylsulfoxide(DMSO,DHAvehicle).CellpopulationswereisolatedbyFACSineachsamplethroughstainingwithaDNAdye,SYBRGreen,andamitochondrialpotentialmarker,MitoTrackerDeepRedFM.Theputativedormantparasitefractionwasisolatedbasedonstainingofmitochondrialpotentialandwasverifiedbyre-culturingexperiments.FLIMofintrinsicreducednicotinamideadeninedinucleotide(NADH)wasusedtoquantifythemetabolicstateandAiryScanmicroscopywasusedtovisualizeparasitestructure.DHA-treatedparasitesshowedacollapseofthenucleusandmitochondriaaswellasalowermeanlifetime,whereasparasitesincontrolmediumshowedthesetwoorganellesapartfromoneother.Afterstandardizingthismethod,otherantimalarialswillbetestedtoseeifthesechangesareuniquelyassociatedwithDHAexposure.Furthercharacterizationofthisphenomenonmayprovideinsightsonpreventionofclinicalrecrudescence.

PP-15MalianchildrenwithseveremalariaexhibitdistinctPfEMP1antibodyprofilesthatdifferbybloodtypeAlbertE.Zhou1,PaulHan1,AndreaA.Berry1,DrissaCoulibaly2,EmilyM.Stucke1,AmedOuattara1,BirajShrestha1,MatthewB.Laurens1,RieNakajima3,AartiJain3,OmidTaghavian3,JoshuaM.Obiero3,LiLiang3,AlgisJasinskas3,AmadouNiangaly2,BouremaKouriba2,AbdoulayeK.Kone2,OgobaraK.Doumbo2†,J.AlexandraRowe4,ShannonTakala-Harrison1,KirstenE.Lyke1,ChristopherV.Plowe5,PhilipL.Felgner3,MahamadouA.Thera2,MarkA.Travassos1



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1MalariaResearchProgram,CenterforVaccineDevelopmentandGlobalHealth,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,2MalariaResearchandTrainingCenter,UniversityofSciences,TechniquesandTechnologies,Bamako,Mali,3VaccineResearch&DevelopmentCenter,DepartmentofPhysiology&Biophysics,SchoolofMedicine,UniversityofCalifornia,Irvine,Irvine,California,4CentreforImmunity,InfectionandEvolution,InstituteofImmunologyandInfectionResearch,SchoolofBiologicalSciences,UniversityofEdinburgh,Edinburgh,UnitedKingdom,5DukeGlobalHealthInstitute,DukeUniversity,Durham,NorthCarolina,†DeceasedSeveremalariacausedbyPlasmodiumfalciparumprimarilyaffectschildreninsub-SaharanAfrica.Severemalariapathogenesisispoorlyunderstoodbutlikelyinvolvesdeeptissuesequestrationandrosettingofinfectederythrocytes.ABObloodtypeandP.falciparumerythrocytemembraneprotein-1(PfEMP1)variantsurfaceantigenexpressionappeartoplaycriticalrolesinrosetting.Dependingonbloodtype,parasitesinfectingsubjectsmayexpresscertainsubsetsofPfEMP1s,suchasDBLα1domains,associatedwithseverediseasepathogenesis.Foreachbloodtype,wepredictedthatMalianchildrenwithseveremalariawoulddemonstrateauniquePfEMP1serologicalprofilethatreflectsthelackofantibodyresponsestoPfEMP1spotentiallyinvolvedinpathogenesis.WealsopredictedthatseveremalariacaseswouldhavelessrecognitionofandloweredseroreactivitytoasubsetofPfEMP1proteinfragmentsthancontrolswithuncomplicatedmalaria.Weprobedacustomproteinmicroarraypopulatedwithreferencestrainandfield-derivedPfEMP1sassociatedwithseveremalariapathogenesiswithserafroma2000-2003Malianseveremalariacase-controlstudy.OurfindingssuggestthatserafromchildrenwithseveremalariadifferedinthenumberofPfEMP1srecognizeddependingonthebloodtype:serafrombloodtypeAseveremalariacases(n=27)recognized91.6%ofPfEMP1fragments,serafrombloodtypeABcases(n=7)recognized21.8%ofPfEMP1proteinfragments,andserafrombloodtypeOcases(n=13)recognized81.6%ofPfEMP1proteinfragments.Inaddition,serafrombloodtypesA,AB,andOseveremalariacasesreactedlessintenselyto67,12,and34PfEMP1sfragments,respectively,thanuncomplicatedmalariasera.AnalysesinvestigatingthedifferentialserologicalresponsestoDBLα1-PfEMP1sarecurrentlyongoing.Thesefindingsmayinformourunderstandingofseveremalariapathogenesisandthedesignofvaccinestoprotectindividualsfromseveremalaria.

PP-16LacticacidsupplementedmediastimulatesgametocytogenesisinPlasmodiumfalciparumcultureRachelWest1,AbhaiTripathi1,DavidSullivan1,2

1JohnsHopkinsBloombergSchoolofPublicHealth,2JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MarylandMalariainfectionbyPlasmodiumfalciparumcontinuestoafflictmillionsofpeopleworldwide,withtransmissionmaintainedbythedefinitivehostmosquito.Transmissionisdependentuponmosquitoingestionofthegametocytestageoftheparasite.Thesesexuallycommittedstagesdevelopfromtheasexualstages,yetthefactorsbehindthistransitionarepoorlyunderstood.Invitrostudieshaverevealedthatextracellularfactors,suchasLysoPC,presentindifferentmediaandcellularenvironmentsinfluencegametocytogenesis.Parasitesreadilyproducemetabolitessuchaslacticacidinmillimolarconcentrationsduringmalariainfection.Wehypothesizegametocytogenesisisinducedthroughmolecularfactorsfoundinparasite-orreticulocyte-richenvironments.Wehavedemonstratedthatlacticacidsupplementedmediasignificantlyinfluencesgametocyto-genesisinanNF54strainP.falciparuminfection.Parasitesexposedtodifferentexposuretimesoflacticacidweremonitoredthroughoutgametocytedevelopmentinvitro.Wefoundthatcontinuouslacticacidsupplementationincreasedparasitemiaandgametocytemiaafter72hand168h,respectively.Usingreverse-transcriptaseqPCR,wealsofoundthatgametocytespecificgeneexpressionwasincreasedafter240hoursinlacticacidsupplementedcultures.Inamosquitoinfection,wefoundthatgametocytescontinuouslyexposedtolacticacidweremoreinfectioustoAnophelesstephensimosquitoes,increasingprevalenceofinfectionbutnotoocystdensity.Further,wefoundthatgametocytesproducelacticacidthroughoutdevelopment,andproduceitathigherlevelsthancontrolswhensupplementedwithlacticacid.Wethenadaptedagametocyte-specificquantitativePfs16promoterluciferaseassaytoexaminetheeffectsofmediaenvironmentongametocytedevelopment.Lacticacidsupplementedmediasignificantlyincreasedgametocyteequivalentnumbersafter72hoursofincubation,countedbyluciferaseassay.Wewillcontinuetotestvariousacidsproducedbyparasitesorreticulocytesinadoseandtimedependenttodeterminetheireffectsongametocytogenesis.

PP-17BacterialSuppressionofMalariaTransmissionbyMosquitoesWeiHuang1,JannethRodrigues2,AlfonsoMendoza-Losana2,MarceloJacobs-Lorena1.

1JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2GSKOpenLabFoundation,TresCantos,SpainTheintolerableburdenofmalariademandstheurgentdevelopmentofnovelapproachestofightthisdeadlydisease.Previously,wehaveshownthatengineeredmosquitosymbioticbacteriacanrendermosquitoesresistant



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totheparasite.However,translationofthesefindingstothefieldfacesmajorregulatorybarriers.Herewedescribetwonon-modifiedsymbioticbacteria–Delftiasp.andPseudomonassp.ThesebacteriawereoriginallyisolatedfrommosquitoesthathadlosttheabilitytosustainthedevelopmentofPlasmodiumfalciparumparasites.WhilePseudomonaseasilycolonizesthemosquitoandistransmittedvertically,itisapoorinhibitorofPlasmodiumdevelopment.Delftiaontheotherhand,isapotentinhibitorofPlasmodiumdevelopmentinmosquitoes.FurtherstudyshowedDelftiasecretssomemoleculesbelow3KDwhich100%inhibitookinetesandoocystofPlasmodiumfalciparuminmosquitomidgut.Thisbacteriumdoesnotimposeafitnessload:itdoesnotaffectmosquitosurvival,bloodfeedingbehavior,fertilityorfecundity.Delftiaanditssecretedmoleculesshowpromiseforuseinthecontrolofmalaria.

PP-18RelatingLarvalandAdultMosquitoSalivaryGlandBiologytoDevelopNewStrategiestoBlockDiseaseTransmissionMichelleChiu1,2,MichaelWells1,2,BrianTrigg1,2,DeborahAndrew1,2

1JohnsHopkinsSchoolofMedicine,2JohnsHopkinsMalariaResearchInstituteMosquitoesareadirethreattohumanhealth,accountingforhundredsofmillionsofinfectionsandover1milliondeathsannually.Thepathogensinvolvedmusttravelthroughtheadultfemalemosquitosalivaryglands(SGs)tobeinjectedintothenexthumanhostduringafuturebloodmeal.ThisfeaturemakestheSGsanidealtargetfortransmissionblockinginterventions.TheadultSGsforminthepupafromapopulationofcellslocatedinthelarvalsalivaryductbud,whiletheembryonic/larvalSGsaredestroyed.LittleisknownaboutcellarchitectureandsecretioninalarvalmosquitoSG,howtheadultSGacquiresitselaborateshape,andwhatgeneticregulationisatworkinlarvalandadultmosquitoSGs.WedevelopedarobustprotocolforfixingandstaininglarvalSGtissue(usingdyesandantibodies)andappliedthismethodtoover1000SGs,imagingmanyofthesesamplesusingconfocalmicroscopy.AcomparisonoflarvalandadultSGsmorphologyandgeneexpressionhasalsobeenconducted.AswiththeadultmosquitoSGs,thelarvalSGshaveunusualandinterestingmorphologiesthatarelikelylinkedtotheirfunction.Wehavelearnedaboutthelocalizationofkeycellularcomponentsandhavegatheredevidencesupportingthepresenceofextracellularvesiclesinlarvalsaliva.WehavefurtherdeterminedtherelatednessingeneexpressionprofilesbetweenlarvalandadultmosquitoSGs,aswellaslarvalmosquitoandfruitflySGs.Thisworkhelpstodescribetheformationandregulationofanorganwithaheavyimpactonhumanhealth:mosquitosalivaryglands.ThisinitialcharacterizationofthelarvalSGandthe

adultprecursorslaysthefoundationforexploringnewstrategiesforpreventingthespreadofmosquito-bornediseases.

PP-19Challenges,opportunitiesandupdates:Afocusonthe2019EasternBurmaRetrospectiveMortalitySurveyBreaghCheng1,SawNayHtoo2,NawPuePueMhote2,andColleenM.Davison1

1Queen’sUniversity,2BurmaMedicalAssociationImprovingdatacollectioninlow-incomesettingsisneededtosupporthealthequityanddiseasepreventionefforts.AlmostnoliteratureexistsintheSouthAsiancontextdescribingchallengesencounteredduringhouseholdsurveyscapturingdiseaseoutcomesanddeterminants.Wediscuss:(1)challengesandopportunitiesassociatedwithcollectionanduseofhealth,healthcareandmalaria-specificdatafromtheEasternBurmaRetrospectiveMortalitySurvey(EBRMS)inMyanmarand2)shareprogresstodateonthe2019EBRMS.Toexploretheseissues,aqualitativereviewofthenotesonfeedbackquestionnairesfromEBRMSfieldstaffattheBurmaMedicalAssociation(BMA)andHealthInformationSystemWorkingGroup(HISWG)wasundertaken.Opportunitiesandchallengesprecedingandduringthe2019datacollectionarehighlighted.Challengesincludedinaccessibilityofvillagesitesduetosecurityproblemsfromnearbyarmedconflict;uncertaintyinhandlingresponsestoquestionsonsensitivetopics;andtheimplicationsofalengthyquestionnaireonparticipationanddataquality.Despitetheselimitations,EBRMSreportsessentialinformation,includingthoserelatedtoemergentinfectiousdiseaseandmalariaamongdisplacedpopulationsinethnicstates.Thisinformationisnotavailablefromgovernmentserviceprovidersorotherhealthorganizations.The2013EBRMSfoundmalariawastheprimaryreportedcauseofdeathacrossallagegroupsand63.6%ofrespondentsreportedusingabednetasapreventativemeasureagainstmalaria.The2019EBRMShasimproveditscomparabilitytointernationaldemographicsurveysbyincludingnewindicatorsmeasuringincome,healthbeliefs,andpotentialemerginghealthissuesthatcanfurtherdescribediseasepatterns.Populationsinremoteandconflict-affectedregionsofeasternBurmacontinuetofacecriticalhealthandhealthequitychallenges.Continuedeffortsareessentialtosystematicallymeasurethenatureandimpactofthesechallenges.Byanticipatingsurvey-relateddifficulties,researchersinLMICsaremorelikelytobeabletoimplementstrategiestominimisetheireffects.

PP-20MutantPlasmodiumwithdelayedgrowthduringtheliverstagedevelopmentinducesbetterimmunitythanitswild



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typecounterpartinaChemoprophylaxisVaccinationregimenTejramSahu,GodfreeMlambo,EllaGehrke,JuliaD.Romano,YevelFlores-Garcia,FidelZavala,IsabelleCoppens

JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,MarylandChemoprophylaxisVaccination(CVac)isarelativelynewandpromisingvaccinationapproachagainstmalaria.UsingwholeorganismsporozoitescombinedwithChloroquinetreatment,CVacprovidesbettersterilizingimmunity,withtwo-loglesssporozoitesthantheRadiationAttenuatedSporozoite(RAS)vaccine.WehaveengineeredaPlasmodiummutantnamedPbATG8-OE(PlasmodiumbergheioverexpressingATG8)thatexhibitsaseveregrowthdelayinhepatocytes,thusexposingliverstageantigenstotheimmunesystemlongerthanWTparasites.WehypothesizethatPbATG8-OEparasiteswouldbemoresensitivetoachemoprophylaxistreatment,henceanidealantigenicconstituentofaCVacregimen.ComparingthevaccinepotentialofP.bergheiWTandPbATG8-OEusingaCVacregimen,weshowthatPbATG8-OEprovidessuperiormemoryresponse(100%)thanWTparasites(60%)andconfersbetterlong-termprotection(40%vs.20%forWT).PbATG8-OE-CVacgeneratesacomparableantibody-responseagainstsporozoitesandliverformsasWT-CVac.Interestingly,theprotectionelicitedbyPbATG8-OE-CVacisCD8-Tcell-dependentandIFN-gisplayngaminorrole.WearecurrentlyinvestigatingtheprotectivemechanisminmiceinfectedwithPbATG8-OE-CVac,whichwouldleadtothediscoveryofnewantigensandmemoryeffectorsandmayhelpdevelopbettermalariavaccine.

PP-21SubclinicalPlasmodiumfalciparuminfectionamongchildrenandadultsresidinginahighmalariatransmissioncommunityTamakiKobayashi1,MatthewM.Ippolito2,3,JaySikalima4,JamesS.Lupiya4,MikeChaponda4,WilliamJ.Moss1,3forSouthernandCentralAfricaInternationalCentersofExcellenceforMalariaResearch1DepartmentofEpidemiology,JohnsHopkinsBloombergSchoolofPublicHealth,2DivisionofClinicalPharmacology,DepartmentofMedicine,JohnsHopkinsUniversitySchoolofMedicine,3DepartmentofMolecularMicrobiologyandImmunology,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,4TropicalDiseasesResearchCentre,Ndola,Copperbelt,ZambiaChronic,asymptomatic,andsubpatentmalariaareoverlappingsubclinicalsyndromesclassifiedaccordingtotheextentordurationofparasitemiaandthepresenceor

absenceofparasite-attributablepathology.Subclinicalmalariaisincompletelycharacterizedintermsofitsnaturalhistory,clinicalconsequences,andepidemiologicalcontribution.Weinvestigateddemographicandclinicalfeaturesofsubclinicalmalariatoassesspotentialriskfactorsandelucidateitsrelevancetomalariacontrol.Longitudinalandcross-sectionalsurveyswereconductedbetween2013and2017inadultsandchildrenresidinginahighmalariatransmissionareaofnorthernZambia(n=2,249).Plasmodiumfalciparumparasitemiawasmeasuredbypolymerasechainreaction(PCR),microscopy,andrapiddiagnostictest(RDT).Demographic,clinicaldata(temperature,hemoglobin,self-reportedsymptoms),bednetuse,indoorresidualspraying,andrecentantimalarialtreatmentwererecorded.Parasiteprevalencewas31%bymicroscopy,53%byRDT,and55%byPCR.ParticipantswerestratifiedaccordingtoPCR,microscopy,andRDTresults:patent(+/+/+),subpatent(+/-/-),recent(-/-/+),andno(-/-/-)parasitemia.AmongPCR-positiveindividuals,66%(n=816)hadsubclinicalmalariadefinedastemperature<38°Candnoself-reportedfever,chills,headache,vomiting,diarrhea,orcoughwithintheprior48hours.Participantswithpatent(n=592)orrecent(n=183)parasitemiatendedtobeyounger(medianage=10y,IQR5-20)thanthosewithsubpatent(n=253)ornoparasitemia(n=762)(25y,IQR10-41).Participantswithpatentorrecentparasitemiaweremorelikelytohaveanemiacomparedtonoparasitemia(OR=1.8,95%CI1.5,2.2).Thosewithnoparasitemiaweremorelikelytoreportsleepingunderabednetcomparedtothosewithpatent,subpatentorrecentparasitemia(OR=2.5,95%CI1.7,2.5).Thisstudyidentifiedahighprevalenceofsubclinicalmalariainaholoendemicarea.Characterizationofthisdiversegroupwillinformpublichealthandclinicalapproachestomalariacontrolinsimilartransmissionsettings.

PP-22SevereMalariaSurveillanceinaRuralDistrictHospitalinNorthernZambiaBenjaminKussin-Shoptaw1,Jean-BertinKabuya2,JamesS.Lupiya2,LucK.Kamavu3,JaySikalima2,MikeChaponda2,ProscoviaBanda3,WilliamJ.Moss1,andMatthewM.Ippolito1,4fortheSouthernandCentralAfricaInternationalCentersofExcellenceforMalariaResearch1JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2TropicalDiseasesResearchCentre,Ndola,Zambia,3St.Paul’sGeneralHospital,Nchelenge,Zambia,4JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MarylandSeveremalariasurveillanceisroutinelyperformedinZambiabyhospital-basedHealthManagementInformationSystem(HMIS)personnel.HMISsurveillancereliesoninformationaggregatedfromwardregisters.SupplementationofHMISdatawithhospital-andpatient-leveldatafromadditional



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sourcesmayprovidegreaterresolutionofseveremalariaclinicalepidemiology.Toassesshospital-basedmethodsofseveremalariasurveillance,weconductedasinglecenterstudythatevaluatedfourdatacollectionapproachesovertheperiodJune2017toMarch2019.AggregatedatawerecollectedfromHMISrecordsandcentralpharmacyartesunateinventories.Individual-leveldatawerecollectedfromartesunateadministrationrecordsandlaboratorybloodtransfusionlogbooks.MeansandstandarddeviationsofcasespermonthwerecalculatedandcomparedusingStudent’sttestandone-wayanalysisofvariance.TheHMISmonthlycaseestimate(79±40)wassystematicallygreaterthanestimatesbasedonartesunateinventory(56±38),artesunatetreatmentcourses(54±32),andpediatricbloodtransfusions(45±18)(P=0.02).Agewassimilarbetweenartesunate-treatedpatientsandtransfusedpatients(median24months,interquartilerange14-43)andsimilaracrossyearsofsurveillance.Partialorcompletebloodproductstockoutswererecordedfor95daysoverthe667-daysurveillanceperiod(14%).Accurateaccountingofseveremalariacaseburdencanhelpfocusresourcesinatimelyandeffectivemanner.Shiftsovertimeincasevolume,agedistribution,andallocationofbloodtransfusionmayrevealunderlyingchangesinlocalmalariaepidemiology.Alternativedatasourcescansupplementexistingregister-basedHMISsurveillance.

PP-23Shelf-lifestudyofHumanErythrocytesCryopreservedfortheLaboratoryCultureofPlasmodiumfalciparumAbhaiTripathi1,AnnaliseArmstrong2,MadisonScialanca2,EdrasMendez-Hernandez2,SamuelStambaugh2,NicoleClemente2,andLawrenceMylin2

1JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2DepartmentofBiologicalSciences,MessiahCollege,Grantham,Pennsylvania

MalariaiscausedbymultiplespeciesoftheparasitePlasmodiumanddisproportionatelyaffectspeoplelivinginthedevelopingworldwhooftendonothaveaccessorfundstoallowforeffectivecontroloforprotectionfromtheparasite.ThisstudyisintendedtosupportongoingresearchattheMachaResearchTrust(MRT)[alsoknownastheMalariaInstituteatMacha(MIAM)],whichislocatedinMacha,aruralareaintheSouthernProvinceofZambiawherethevirulentspecies,Plasmodiafalciparumisprevalent.OurgoalistosupportthecapacityofthelaboratoryatMRTtoculture(propagateandpreserve)locally-isolatedorlaboratorystrainsofPlasmodium.LaboratorycultivationofPlasmodiumfalciparumrequiresfreshhumanblood.However,itisdifficulttoassurethesteadysupplyoffresh,uninfectedhumanbloodneededtosustaincultureexperimentsatMRTbecausebloodfromlocalresidentscannotbeused,andbecausemanyvisitingscientistsand

physiciansroutinelytakeprophylacticanti-malarialdrugswhichcanmaketheirerythrocytesunabletosupportasexualpropagationof,orgametocytegenerationbyPlasmodiuminculture.WeareinvestigatingmethodsthatshouldallowforthecryopreservationoferythrocytesobtainedfromuninfectedindividualsintheU.S.withsubsequentshipmenttoZambia.Wehavecryopreservedpreparationsoffresh,leukocyte-depletederythrocytesuspensionsusingminimalaqueousvolumesofsolutionscontainingthemacromolecularstarch-basedcyro-protectanthydroxyethylstarch(HES)withorwithoutpolyvinylalcohol(PVA).Suchcompoundsactasicerecrystallizationinhibitors(IRIs),preventingformationoficecrystalsduringthethawingprocess.ThispresentationwilldescribeongoingeffortstodetermineifcryopreservedRBCswilleffectivelysupportasexualpropagationorgametocyteformationforthePlasmodiumfalciparumlaboratorystrainNF54followinglong-termstorageat-80°C.Further,theeffectofasingleshort-termtransfertoliquidnitrogenduringthestorageperiod(tosimulatetransport)willbeassessed.

PP-24SmallmoleculescreenofepigeneticinhibitorsonPlasmodiumfalciparumbloodstagereplicationandgametocytematurationLeenVanheer,BjörnF.C.KafsackDepartmentofMicrobiology&Immunology,WeillCornellMedicine,NewYork,NewYorkTheregulationofgeneexpressionbyhistonemodificationsiscriticalforparasitesurvivalinbothasexualandsexualbloodstagesofP.falciparum,andchangessubstantiallyasparasitesdifferentiatefromonetotheother.Thismakesinhibitorstargetingthesepathwaysintriguingintermsoftheirpotentialasmulti-stageactiveanti-malarials.Therefore,wescreenedtwocherry-pickedlibrariesofsmallmoleculeepigeneticinhibitorsfromSelleckchem(142compounds)andCaymanChemicals(139compounds)fortheireffectonasexualbloodstagereplicationandgametocytedevelopment.Thesecompoundsincludeinhibitorsofepigeneticwriters,readers,anderasersknowninmammaliansystems,includinghistonedeacetylases(HDAC),histonedemethylases(HDM),histonemethyltransferases(HMT)andhistoneacetyltransferases(HAT).Weidentified74epigeneticinhibitorswithEC90sbelow10µMforinhibitionofgametocytedevelopment,ofwhich27compoundsretainedtheir>90%inhibitoryeffectat1µM.Asexualstagegrowthwasblocked>90%by73compoundsat10µM,ofwhich30compoundsmaintainedtheirinhibitoryeffectat1µM.Dose-responsecurvesweredeterminedforcompoundswithpotentactivityat1µM,withseveralthathadEC50concentrationsinthelownano-molarrangeagainstbothasexualandsexualbloodstages.TheseincludeinhibitorspreviouslyidentifiedtobeactiveagainstP.



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falciparumbloodstagesaswellasseveralthathadnotpreviouslybeentested.Additionally,weidentifiedmultiplecompoundsthatdisplayedhigheractivityagainstgametocytematurationcomparedtoasexualreplication.TheemergenceofdrugresistanceinP.falciparum,themostvirulenthumanmalariaparasite,necessitatesthedevelopmentofnovelanti-malarialdrugs.Thesefindingsprovidesupportforthedevelopmentofnewclassesofanti-malarialsthattargettheparasite’sepigeneticregulationmachinery.

PP-25MeasuringGeneExpressiontoEvaluatetheEffectofArtemether-LumefantrineonParasitemiaandGametocytemiainZambianChildrenwithUncomplicatedPlasmodiumfalciparumMalariaDeborahM.Stiffler1,MatthewM.Ippolito2,3,MwicheP.Siame4,PhilipE.Thuma5,V.AnnStewart11UniformedServicesUniversityoftheHealthSciences,Bethesda,Maryland,2JohnsHopkinsSchoolofMedicine,Baltimore,Maryland,3JohnsHopkinsBloombergSchoolofPublicHealth,4MinistryofHealth,Lusaka,Zambia,MachaResearchTrust,Macha,ZambiaArtemisinincombinationtherapy(ACT)iscurrentlythetreatmentofchoiceforuncomplicatedP.falciparummalaria.AlthoughACTsareknowntobehighlyeffectiveagainstasexualparasites,theinvivoeffectofACTsonvariousstagesofgametocytesisincompletelycharacterized.Samplesfrom75participantswereselectedfromatherapeuticefficacystudyof100childrenfromNchelenge,ZambiawithuncomplicatedP.falciparummalariatreatedwithartemether-lumefantrine(AL)andfollowedforfiveweeks.Driedbloodspots(DBS)werecollectedfromeachparticipantevery6hoursforthefirst48hours,at72hours,andweekly;inthisstudy,RNAwasextractedfromsamplescollecteduptothesecondweekoffollow-up.ThisRNAwasusedforquantitativereal-timePCR(qPCR)assaysthatquantifythreestagespecifictranscripts:Pfsbp1,Pfs16,andPfs25.Pfsbp1isexpressedbyring-stageasexualparasites,whilePfs16andPfs25aregametocyte-specificmarkers.Pfs16ismosthighlyexpressedbyearlygametocytesbutisexpressedatalowerlevelthroughouttheremainderofgametocytedevelopmentinbothmaleandfemalegametocytes,andPfs25ishighlyexpressedbystageVfemalegametocytes.WewereabletosuccessfullyextractRNAandamplifytranscriptspresentinquantitiesaslowas2.5copy/μlforPfsbp1,and1.25copies/μlforPfs16andPfs25.Inthiscohort,itwasfoundthattreatmentwithALwasassociatedwithan80-foldreductionofPfsbp1copynumberswithinthefirst36hours.Despitethisreductionincopynumber,75%oftheparticipantswerepositiveforPfsbp1byqPCRafter2weeks.17%ofsampleswerepositiveforPfs16and/orPfs25after2weeks,butPfs16andPfs25copynumbersweredifferentiallyaffectedby

treatment,consistentwithstage-dependentvariationsindrugsusceptibilitytoAL.Additionalstudiesareneededtoevaluatetheepidemiologicalimportanceofsuchlowlyexpressedtranscripts,todetermineifthesustainedprevalenceoftranscriptsduringthefollow-upperiodisduetopersistentparasitemia,reinfection,orrecrudescence,andtodeterminetheimpactofdrugtreatmentbeyondtwoweeks.

PP-26PerceptionsontheefficacyandbenefitsoftheIRSprogrammesbybeneficiarieshasabearingonsprayingcoverageNziraLukwa1,TonderaiChiwade1,TakaMduluza2,ChamunogwaNyoni3,MosesZimba41NationalInstituteofHealthResearch,Harare,Zimbabwe,2DepartmentofBiochemistry,UniversityofZimbabwe,Harare,Zimbabwe,3DepartmentofSocialWork,BinduraUniversityofScienceEducation,Bindura,Zimbabwe,4DepartmentBiologicalSciences,UniversityofZimbabwe,Harare,Zimbabwe

TwodeltamethrinformulationsweresprayedininsidehousesinPfungwedistrict,Zimbabwe,formalariacontrolpurposes.Adeltamethrinproductcontaining5%wettablepowder(WP)asactiveingredient,wassprayedin1899/2157(88%)oftheroomsandanotherdeltamethrinproductcontaining2.5%activeingredient(SuspensionConcentrate)(SC)wassprayedin1730/1892(91.4%)oftherooms.Roomcoveragewasnotsignificantlydifferentbetweenthe2insecticides(P=0.097).Roomcoveragewasnotassociatedwiththelevelofperceptiononwhetherproblemswerebeingencounteredbyhouseholderswhensprayingwasbeingconducted(noproblemsreportedby89.6%respondentsinSCvillagesand91.4%inWPvillages),butbythelevelofknowledgepertainingtothesprayingexercisemasteredduringtheinitialphase(95.8%respondentsknewinSCvillagesthan91.4%inWPvillages).Householdersopenedtheirdoorsforsprayingwhentheyobservedotherpublichealthpestsdyingfromtheirneighbor’shouse(72.9%and70.3%saidsoindeltamethrinSCandWPvillagesrespectively).Percentlockedroomsrangedfrom0.7-9.3%and3.4-14.4%indeltamethrinSCanddeltamethrinWPsprayedvillagesrespectively,althoughtheresultswerenotsignificantlydifferent(P=0.22).Theresidualeffectofbothformulationswas6monthsbothonmudwallsandthatchedroofs.Inconclusion,perceptionsofhouseholdersonthelevelofproducteffectivenessinfluencessprayingcoverageandeffortsshouldbemadetoincreaseprogramuptake.

PP-28

MolecularcharacterizationofrecombinantPlasmodiumfalciparumPHISTbproteinsaspotentialtargetsof



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naturallyacquiredimmunityagainstmalariatransmissioninhumans TonyI.Isebe1,2,JoelL.Bargul2,JamesTuju1andMartinRono1

2JomoKenyattaUniversityofAgricultureandTechnology,1KEMRI-WellcomeTrustResearchProgramme,CentreforGeographicMedicineResearch Plasmodiumfalciparumisknowntocausethedeadliestformofmalariainsub-SaharanAfrica.Uponinfection,remodelingofinfectedhostredbloodcells(iRBCs)bytheparasite-exportedsurfaceproteinsoccurstoprovideafavorablenicheforparasitedevelopmentandmaturation.SomeofthesemoleculesincludeproductsofthemulticopyfamilyofPlasmodiumHelicalInterspersedSub-telomeric(PHIST)proteins,whoseroleinmalariapathogenesislargelyremainsunknownatpresent.However,inarecenttranscriptomeanalysisofclinicalisolates,somemembersofthePHISTbgenefamilywereassociatedwithparasiteadaptationstomalariatransmissionintensityandgametocytedevelopment.ItwasfurtherestablishedthatPHISTbproteinscouldserveastargetsofnaturallyacquiredimmunitytomalariainindividualsfromthreedifferentgeographicalsitesinKenyaandTheGambiawithvaryingmalariatransmissionintensities.Tocharacterizethepossibilityoftheseproteinsaspotentialvaccinetargets,recombinantPHISTbproteinswereexpressedinbacteriafollowedbyELISA-basedevaluationofantibodyresponsesusingimmuneserafrommalaria-exposedindividuals.Ourfindingsshowthatchildrenandadultsfrommalaria-endemicregionrecognizedPHISTbproteins(i.e.PF3D7_0532400,PF3D7_1401600,andPF3D7_1102500),providingaclinicalevidencefortheroleofPHISTbantigensinimmuneresponseagainstP.falciparuminfection.AntibodyresponsesagainstthethreerecombinantPHISTbantigenswerehowevervariable.AnassociationstudyofantibodyresponsestothedifferentPHISTbantigenswithagerevealednocorrelationbetweentheageandantibodyresponsestoPF3D7_1102500andPF3D7_1401600(p<0.507andp<0.15,respectively,CI=95%),butasignificantcorrelationtoPF3D7_0532400(p<0.009).Furthermore,therewasstrongcorrelationofantibodyresponsestoboththeschizontextractandPF3D7_0532400(p<0.0001),equivalenttothoseagainstPF3D7_1102500andPF3D7_1401600(p<0.0001).Collectively,thesefindingsempiricallyprovideevidenceofrecombinantPHISTbantigensaspotentialtargetsofnaturally-acquiredimmunityagainstmalariainhumansandpossibleserologicalmarkerstoP.falciparuminfectionaimedatcontributingtomalariacontrolthroughvaccinedevelopment.

PP-29TheEffectofMalaria/HIV/TBTripleInfectiononMalariaParasitemiaamongpatientsattendingtheLimbeRegionalHospital

MbiAliceEnekegbe1,2,SamjeMoses2,EmmanuelNTufon3,CheAmadineLem3

1DepartmentofMicrobiologyandParasitology,UniversityofBuea,Cameroon,2DepartmentofBiomedicalScience,FacultyofHealthScience,UniversityofBamenda,Cameroon,3St.LouisUniversityInstituteofHealthandBiomedicalScience,Bamenda,CameroonMalaria,Tuberculosis(TB)andHumanImmunedeficiencyvirus/AcquiredImmuneDeficiencySyndrome(HIV/AIDS)are3oftheworld’smostcommonandseriousinfectiousdiseasesthatunderlinedevelopmentinlowandmiddle-incomecountries.Theseinfectionsarenotonlyassociatedwithpoverty,butalsooccurinthesamegeographiczoneandhavemajorpublichealthimplications.Hencethereislikelihoodforco-infectionsofthese3infections.Thiswasahospitalbasedcrosssectionalstudyinwhich400participantswererandomlyselected.Venousbloodsampleswerecollectedfromthestudyparticipants.Malariadiagnosiswascarriedoutusingpolymerasechainreactionandpositivesampleswerequantifiedusingmicroscopy,HIVdiagnosiswasdoneusingUni-GoldrapidtestkitsandSDBioline.TBwasdiagnosedusingculture.Theprevalenceofmalariaoccurringasmono,coandtripleinfectionswas23.3%(malaria),(1.5%)TB/malaria,(21.3%)HIV/malariaandHIV/TB/malaria(2.5%).Themeanparasitedensityinsinglemalariainfectionwas410±2515.5parasites/µl.Whenthiswascomparedwiththeparasitedensityincoandtripleinfections,thosewithTB/HIV/malariatripleinfectionhadthehighestmeanparasitemia(461.1±295.0parasites/µl).Thelowestmeanparasitedensity(271.0±198.7parasites/µl)wasobservedinpatientsco-infectedwithTB/malaria.Howeverthedifferenceinthemeanparasitedensitywasnotstatisticallysignificant(p=0.329).Hence,theremaybeneedtosetupguidelinesforthemanagementofmalariainpeoplewithco-infectionsofHIVandTB.

PP-30Polymorphismofexon-20inthevoltage-gatedsodiumchannelgeneofAnophelesgambiaes.s.populationsfromwetlandsacrosstheCameroonvolcanicline NathalieAmvongo-Adjia1,2,3,4,JacobM.Riveron5,WinstonP.ChounnaNdongmo2,6,FlobertNjiokou1,4,SamuelWanji2,6,CharlesS.Wondji4,5

1ParasitologyandEcologyLaboratory,DepartmentofAnimalBiologyandPhysiology,UniversityofYaoundé,Cameroon,2ResearchFoundationforTropicalDiseasesandtheEnvironment,Cameroon,3InstituteofMedicalResearchandMedicinalPlantsStudies,Cameroon,4CentreforResearchinInfectiousDiseases,LSTMResearchUnit,Cameroon,5VectorBiologyDepartment,LiverpoolSchoolofTropicalMedicine,Liverpool,UnitedKingdom,6ParasiteandVectorBiology



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ResearchUnit,DepartmentofMicrobiologyandParasitology,UniversityofBuea,Cameroon

Establishingtheextent,geographicaldistributionandmechanismsofinsecticideresistanceinmalariavectorsisaprerequisiteforresistancemanagement.Here,werereportawidespreaddistributionofknockdownresistance(kdr)inthemajorvectorofhumanmalaria,Anophelesgambiaesensustricto(s.s)acrosswetlandsoftheCameroonvolcanicline(CVL).FemaleAnophelesmosquitoeswerecollectedthroughoutsevenwetlandslocatedinfivevolcanicmassifsinCameroon.Thespeciescomposition,Plasmodiuminfectionrateandmolecularbasesofthekdrresistancewerethenanalyzed.MosquitocollectionrevealedapredominanceofAn.gambiaes.s.(includingAn.coluzziiandAn.gambiae)mosquitoeswithalowhybridrate(1.1%)suggestingrareoccurrenceofhybridization.Overall,An.gambiaes.s.specimenswerethesecondPlasmodiumvector(0.72%infection)andexhibitedanentomologicalinoculationraterangedbetween0.7to2.24infectedbitesperhumanpermonth.Both1014Sand1014FkdralleleswerefoundinAn.gambiaes.s.withoverallfrequenciesof8.9%and97%respectively.Analysisofa500bpregionofexon-20downstreamthekdrlocusrevealedatotalof18polymorphicsites.Thenumberofhaplotypesvariesfrom4to11perwetlands,withanoverall0.802haplotypediversity.Themeannumberofnucleotidedifferenceswasestimatedat2.30.Additionally,nosignatureofselectionwasobservedonthevoltage-gatedsodiumchannelgene.TheextensivedistributionofknockdownresistanceinCVLAn.gambiaes.s.populationsrepresentsachallengetovectorcontrol.Moreover,themosaicofgeneticeventsfoundinwetlandsacrosstheCVLofthesituationthroughoutthecountry.Thishighlightstheimportanceofevaluatingthespatialandtemporalevolutionofkdrallelesforabettermanagementofinsecticideresistance.

PP-31ThrombocytopeniaandwholebloodtransfusioninchildrenwithseverefalciparummalariaMatthewM.Ippolito1,2,Jean-BertinKabuya3,ManuelaHauser4,BenjaminKussin-Shoptaw2,AustinPeer1,MarcoRuegg,4AlbertBaschong,4LucK.Kamavu,5ThomasA.Louis,2andWilliamJ.Moss1fortheSouthernandCentralAfricaInternationalCentersofExcellenceforMalariaResearch1JohnsHopkinsUniversitySchoolofMedicine,2JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,3TropicalDiseasesResearchCentre,Ndola,Zambia,4UniversityChildren’sHospital,Zurich,Switzerland,5SaintPaul’sGeneralHospital,Nchelenge,ZambiaSeveremalarialanemiaduetoPlasmodiumfalciparumisoftenaccompaniedbythrombocytopenia.Treatment

includestransfusionofwholeblood,whichcontainserythrocytes,platelets,andotherbloodcomponents.Toassesstheeffectofwholebloodtransfusiononsurvivalinchildrenwithseverefalciparummalariaandtoexaminethepotentialassociationofthrombocytopeniawithmalariamortalityandtransfusionresponse,weanalyzedaretrospectivecohortof842hospitalizedchildreninZambiawithseveremalarialanemia(703transfused,139nottransfusedduetostock-outorotherreason).Severemalarialanemiawasdefinedasapositiverapiddiagnostictestorbloodsmearincombinationwithanadmissionhemoglobinconcentration≤5g/dL.Mortalitywas13%(94/703)inthetransfusedgroupand24%(34/139)inthenon-transfusedgroup.Kaplan-Meiersurvivalestimatesstratifiedbytransfusionstatusandthrombocytopenia(150,000/μLthreshold)showedincreasedmortalityinchildrenwiththrombocytopeniawhodidnotundergotransfusion,withnodifferencesinmortalityamongtheothertransfusedandnon-transfusedgroups(log-ranktestP=0.0001).EffectmodificationanalysisbyCoxproportionalhazardsregressionadjustedforage,sex,hemoglobinconcentration,bloodgrouptype,andeosinophiliashowedasignificantinteractionbetweenplateletcountandtransfusionstatus(P=0.028).Childrenwiththrombocytopeniawhoweretransfusedanddiedhadlittleornopost-transfusionincreaseinplatelets,incontrasttothosewhosurvived.Freshnessoftransfusedwholeblood,construedfromexpirationdates,correlatedwithgreaterplateletrecoveryandimprovedsurvival.Theroleofplateletsinmalariapathophysiologyiscomplexandincompletelyunderstood;priorstudiesdescribepreferentialbindingofplateletstoparasitizederythrocytesanddirectparasitocidalactivity,whereasothersdetaileddeleteriouseffectsinmalariainvolvingthecentralnervoussystemvasculature.Thesefindingspointtoapotentialclinicalroleforplatelet-directedtransfusionstrategiestoimprovesurvivalinchildrenwithseverefalciparummalaria,whichshouldbefurtherassessedinrandomizedinterventionalstudies.

PP-32DeterminingspatialheterogeneityofpotentialAnophelesbreedingandblood-mealsitesinChomaDistrict,Zambiathroughimageminingofremotesensingdata BrendanF.Fries1,DouglasE.Norris2,KellyM.Searle3,TimothyM.Shields1,WilliamJ.Moss,1,2

1DepartmentofEpidemiology,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2W.HarryFeinstoneDepartmentofMolecularMicrobiologyandImmunology,JohnsHopkinsMalariaResearchInstitute,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,3DivisionofEpidemiologyandCommunityHealth,UniversityofMinnesotaSchoolofPublicHealth,Minneapolis,Minnesota



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Modelinglocaltransmissionandconductinggeostatisticalanalysisofmalariatransmissiondynamicsrequirelocationdataforseveraltypesofsites:aquatichabitatspotentiallyhostingAnophelesmosquitolarvae,andhouseholdswherehumanhostsresidearebothtoonumerousandexpensivetomanuallygeolocate.Thus,wesoughttominesatelliteimageryofMacha,ZambiaandthesurroundingareaforthesesitesofinterestwithGIStechniquesandsoftware.WeutilizedArcGISsoftwaretoconductouranalysesanddevelopourmodelandforthisrequiredtwodifferentapproachestoharvestourtwotypesofspatialcoordinatesbecauseofthehigh-resolutionnecessarytoresolvesmallfeatures.Theclassificationstageoftheaquatic-habitats/breedingsitemodelconsistedofaPrincipleComponentsAnalysis(PCA),manualtrainingsetsupervisedMaximumLikelihoodClassification(MLC).Theclassificationstageofthehousehold/blood-mealmodelappliedpansharpeningtothepanchromaticrasterfollowedbya25-classunsupervisedISODATAclassifier,whichwasreclassifiedtoabinaryrasterwithanalystgeneratedclassselection.Bothmodelssharedtechniquesforfeatureextractiononcewehadreachedabinaryraster;whichconsistedofatransformationfromrastertopolygon,thenfilteringthepolygonalshapebasedonempiricaldata.Aftertransformationandgeolocationofcoordinates,anaccuracyassessmentofourmodelsresultedinenumerationandcoordinatesfor1,702aquatichabitatlocationsand27,548hosthouseholdlocations.Anaccuracyassessmentusingat350km2testregiongavetheaquatichabitatgeolocationmodelanF1score:0.9181,andtheblood-meal/householdgeolocationmodelanF1score:0.8547.Wewereabletomaptheobservedhouseholdandaquatichabitatdistributionanddensitiesacrossthe2400km2studyareaaswellasinferspatialrelationshiptopotentialmalariatransmissionsitesfromthesedata.ThesefindingssupporttheuseofGISsoftwareandspatialmodelingtoinformnewmicroscaleagent-basedmodelsforpolicyorinterventionassessment.

PP-33ManualdissectionofmosquitosalivaryglandswithshortenedtrainingtimelinesAlyssaArnheim,UrvashiRai,HajarHazime,StephenL.Hoffman,KimLeeSim,SumanaChakravartySanariaInc.,Rockville,MarylandSanaria’sproductportfolioisbasedontheonlyprovenapproachinhumans,ofinducingrobustandlong-lastingprotectiveefficacyagainstPlasmodiumfalciparum(Pf)malariabysporozoite(SPZ)-basedvaccines.Allproductsusethestageofmalariaparasitethatistransmittedfrommosquitoestohumans.Therefore,inSanaria’smanufacturingprocess,themosquitobehavesasabioreactor,fromwhichparasitesarethenextractedby

microdissectionoftheinsects’salivaryglandsensuringseveralthousand-foldpurificationawayfromirrelevantmosquitoconstituents.Theextractionofsalivaryglandsbymicrodissectionofmosquitoesinvolvesfourdistinctstepsperformedbyskilledpersonnela)mosquitoalignmentb)decapitationc)glandextrusionandd)glandcollection.Inearlieriterationsallstepsofdissectionwereperformedbyindividualtraineddissectors,whereasinlate2015,alteredconfigurationsledtoa2to3-foldincreaseinefficiencyandthroughputoffullymanualdissection.Inaddition,ergonomicsetupoftheworkstationsreduceddissectorfatigueandallowedlongerworkinghours.SanariaiscurrentlyintheprocessofscalingupproductiontomeettheneedsofupcomingPhase3clinicaltrials.Overthecourseoftenmonths,wehaveimplementedanewtrainingprogramthatbringsonpart-timedissectorstoparticipateinGMPdissectionformanufacturing,selecting22dissectorsfromapoolof85applicants.Inthepasteightmonths,wehavetrainedfivecohortsofdissectors,fullyqualifyingtwocohorts.Aconcentratedtrainingscheduleandconsistenteffortbythepart-timedissectorshasresultedinasignificantdecreaseintrainingtime,fromseventothreemonths.Wewilldiscussthestructureofthetrainingprogramanditsimpactontheefficiencyandflowofourmanufacturingprocess.

PP-34Non-PlasmodiumfalciparummalariaparasiteinfectionsinsymptomaticandasymptomaticadolescentsinNigeriaMedardErnest1,2,AbdulraheemMuhydeen1,IfeomaEzugbo-nwobi1,2,AdebolaE.Orimadegun3,AkiraKaneko4,5&RichardCulleton1 1MalariaUnit,DepartmentofPathology,InstituteofTropicalMedicine,NagasakiUniversity,2GraduateschoolofBiomedicalSciences,NagasakiUniversity,Japan,3InstituteofChildHealthCollegeofMedicineUniversityofIbadan,Nigeria,4IslandMalariaGroup,DepartmentofMicrobiology,TumorandCellBiology,KarolinskaInstitutet,Stockholm,Sweden,5DepartmentofParasitology,GraduateSchoolofMedicine,OsakaCityUniversity,Osaka,JapanMalariaiscausedbyspeciesofthegenusPlasmodium.Therangesofthesespeciesoverlap,andmixedspeciesinfectionsofthesamehostarecommon.Thereiscurrentlylittleknownconcerningtheextenttowhichinteractionbetweencoinfectingparasitespeciesimpactonthepathogenicityandtransmissiondynamics.Inmalariaendemicregionsacertainpercentageofthepopulationwillharborparasitesintheirbloodwithoutsuccumbingtothesymptoms.Theseasymptomaticmalariaparasitecarriersdonotseekantimalarialtreatmentandsoinfectionsarelikelytopersistforlongperiods,moreoverindividualsmaystilltransmittheparasitetomosquitoes,thusconstitutingasilentinfectiousreservoir.Inordertogaugethelevelofasymptomatic



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carriageamongstadolescentsinahighlyendemicarea,andtoidentifytheriskfactorsassociatedwithsuchcarriage,weconductedacrosssectionalsurveyof1032adolescents(aged11-17)fromeightschoolslocatedinIbadanregion,SouthWestNigeria.BloodfilmandfilterpapersampleswerepreparedformicroscopicexaminationandforDNA.Theprevalenceofasymptomaticandsymptomaticmalariawasdeterminedusingmicroscopy,RapiddiagnostictestsandPCR.SpeciestypingwasperformedusingPCRtargetingthemitochondriaCOXIIIgene.WefoundahigherrateofmixedinfectionsandahigherprevalenceofP.ovaleandP.malariaethanusuallyobservedinasymptomaticindividuals.Interestingly,thesymptomaticagematesinhabitedsingleinfectionspredominantlyP.falciparum.Wearecurrentlyinvestigatingthehypothesisthatthepresenceofnon-falciparumspeciesinmixedinfectionsisresponsibleforprotectionagainstsymptomaticP.falciparuminfection.

PP-35A7-yeartrendofmalariaatprimaryhealthfacilitiesinNorthwestEthiopiaAyenewAddisuUniversityofGondar,EthiopiaMalariaisasevereparasiticdiseasethatcanprogresstocomplicationsofthenervoussystem,respiratorydistress,renalproblems,metabolicacidosisandhypoglycemiawhichcanresultindeathincaseofdelayorabsenceofappropriatetreatment.Theaimofthistrendanalysiswastoassesstheprevalenceandtheimpactofmalariaovertheseasonsandyears.Across-sectionalretrospectivestudywasconductedattwohealthcentresGorgoraandChuahitinDembiadistrict.Thedatawascollectedfromlablogbooksroutinelydiagnosedandregisteredforsevenyears.Asystematicsamplingtechniquewasusedbytakingpatientresultsfromlablogbooksduringthefirsttendaysofeverymonth.DatawasentereddirectlyintoEpiDataEntrysoftwareversion3.1andanalysedwithSPSSsoftwareversion20.Moreover,achi-squaretestwithalevelofsignificancesetatlessthan5%wasused.Inthestudyatotalof11879clients6724(56.6)ofwhomweremalesparticipated.Theoverallmalariaprevalenceinthelastsevenyearswas21.8%andthedominantparasitewasPlasmodiumfalciparumwhichacounted16.5%ofthecases.Intheanalysisofthesevenyears,OctoberandSeptemberinwhichtheprevalenceofmalariawas32.6%and27.2%respectively,constitutedthepeakmonths.Highmalariaprevalencewasobservedonautumn(SeptembertoNovember)seasonandtheleastwasobservedinwinter(DecembertoFebruary)with17.8%cumulativeprevalence(p≤0.001).Malariaattackshowedasignificantvariabilityamongdifferentagegroups,andtheagegroup15-29wasthemostaffected(p≤0.001).Therewasalsoasignificantdifferencesinmalariaprevalencebetweenthetwosexesandmalesweremoreaffectedthanfemales

(p≤0.001).Inthisstudy,malariatransmissionremainedhigh,whichaffectedmalesmorethanfemales.Thetrendoverthesevenyearsvariedwiththehighestprevalenceinautumn.Malaria,whichhighlyaffectedproductiveagegroup,wasmoreprevalentatGorgorahealthcenterthanatChuahit.Thus,appropriateparasitedetection,effectivetreatmentandcontrolmethodsareneededtoaalleviatetheproblem.

PP-36AntimalarialeffectsofnanocapsulesofsyntheticpeptidesandbovineLactoferrincombinationinin-vivomodelofPlasmodiumberghei SunilKumar1,JagatRKanwar2,RakeshSehgal3,BalanLouis4,RakeshK.Vashishta5,AsifkhanShanavas6,KamranZaman1ICMR-RegionalMedicalCenter,BRDMedicalCollegeCampusGorakhpurUP,2NanomedicineLaboratoryofImmunology&MolecularBiomedicalResearchDeakinUniversity,AustraliaSchoolofMedicine,3DepartmentofMedicalParasitology,PGIMER,Chandigarh,4NextGenPathDiagnosisMDPathology,Chandigarh,DepartmentofHistopathology,NallasiriyarNagar,Thottipalayam,Coimbatore,5DepartmentofHistopathologyPGIMER,Chandigarh,6InstituteofNanoScienceandTechnology,Mohali,Punjab,IndiaDrugresistanceagainstmalariaparasiteisincreasingdaybyday.Tokeepthisbackgroundinthemindwefindouttheformulationwhichcanhelpfultoreducethedrugresistanceagainstmalariaparasite.SothestudywasdesignedtoprepareacombinationtherapyformalariatreatmentininvivomodelofmalariaparasitePlasmodiumberghei,thestudyhasbeencarriedoutintheDepartmentofMedicalParasitologyPostGraduataeInstituteofMedicalEducationandResearchChandigarhIndia.Total36micewereusedinthisstudy.Totalsixgroupslivertissueswereprocessedfortheimmunohistochemistry.PlasmodiumbergheiinfectedmiceweretreatedwithdifferentformulationsinmicemodelBALB/c.Parasitemia,Histopathologyand,Immunohistochemistry(IHC),Flowcytometryanalysis(FACS)wasassessedandTransmissionElectronmicroscopy(TEM)ofdifferentorganwasdone.AntimalarialeffectsofnanocapsulesincombinationwithsyntheticpeptideandthebLfloadednaonocapsuleswereassessed.ThecomparisonwasdonewiththehelpofDrugChloroquine.ThetissueswerepreparedintheHistopathologydepartmentwiththehelpoftechnicalstaffsectioningwasdonebymicrotome.ThetissuespreparationforTEMwasperformedintheTEMtissueprocessinglab.Thetissuesofintestine,liver,kidneywereprocessed.AntigenandantibodycomplexwasobservedinthePositivecontrolgroupsaswellastreatedwiththedrugs.TheinternalizationoftheNanocapsuleshasbeenobservedwiththehelpofTEMofIntestinaltissues.ThegrouptreatedwiththehelpofcombinationtherapyofbLfandsyntheticpeptideshasshownmaximuminhibitionofparasite.Thefurthertheresultswereconfirmedwiththe



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helpofFACSanalysisandDAPIstainingtheoverallparasitereductionwasnoticed.FurthertheresultswereconfirmedbytheFACSanalysisandDAPIstainingoftheinfectedRBCsfromallthegroups.SothepresentstudyrevealedantimalarialeffectsofthecombinationtherapyofSyntheticpeptidesandbLfloadednanocapsules.Thisisthefirststudywhichconfersthebestcombinationtherapyofproteinandsyntheticpeptidesformalariaparasiteinhibitionintherodentmodel.Thestudyrevealsthatthecombinationtherapyalongwiththepeptidesandproteinisprovidingthebesttreatmentfortherodentmalariainfection.Thiswillbethefuturemedicineforthetreatmentofhumanmalariaparasite.Forthebestresultsinthehuman’sparasitemorestudiesarerequiredinthehighervertebratehostandincelllinesalsotocheckthebestmechanismoftheseformulations.Moreoverthematerialusedinthisformulationisbiodegradable.Itissafeanddon’trequiredanytoxicitystudy.Itwillbethefutureofantimalarialcompounds.Withthehelpofthisstudywecandesigntheothermoleculesforthebestresults.

PP-37FactorsAssociatedwithAsymptomaticMalariaamongPeopleLivingwithHIVFollowingDiscontinuationofCotrimoxazoleProphylaxisatKitgumHospital,NorthernUgandaPhilipOrishaba1,PaulineByakika2,ThomasKatairo1,WaniMuzeyi1,JoanNKalyango1,3,MosesRKamya2,JoaniterNankabirwa1

1ClinicalEpidemiologyUnit,CollegeofHealthSciences,MakerereUniversity,2DepartmentofInternalMedicine,CollegeofHealthSciences,MakerereUniversity,3DepartmentofPharmacy,CollegeofHealthSciences,MakerereUniversity,UgandaTheeffectofdiscontinuationofcotrimoxazoleprophylaxisonmalariaparasitaemiainpatientsonantiretroviraltherapy(ART)whohaveregainedimmunecompetenceandlivinginamalariaendemicarearemainsunknown.WedeterminedtheprevalenceandfactorsassociatedwithmalariaparasitaemiaamongpeoplelivingwithHIVfollowingdiscontinuationofcotrimoxazolepreventivetherapyattheKitgumhospitalHIVclinic.ThiswasacrosssectionalstudyconductedbetweenMarchandApril2019attheKitgumhospitalHIVclinic.Weconsecutivelyenrolled599participantsaged18yearsandaboveandlivingwithHIVattendingtheclinic.Apre-testedquestionnairewasadministeredtotheparticipants,andastandardizedphysicalexamwasconducted.Allparticipantsprovidedafinger-prickthickbloodsmearwhichwasstainedwithGiemsaandusedtoassessforthepresenceofmalariaparasites.Factorsassociatedwithmalariaparasitaemiaamongthosewhohadbeendiscontinuedoncotrimoxazolewereassessedusingbinarylogisticregression.Ofthe599participantsenrolled,452(75.5%)hadstoppedcotrimoxazole

prophylaxisforatleast3months.Theoverallprevalenceofmalariaparasitaemiawas4.5%(95%confidenceinterval[CI]3.0%-6.5%).Therewasasignificantdifferenceinmalariaparasitaemiaprevalenceamongparticipantswhohadstoppedcotrimoxazole(5.5%[95%CI3.6%-8.1%])comparedtoparticipantsoncotrimoxazoleprophylaxis(1.4%[95%CI0.17-4.83],p=0.034).Factorsassociatedwithmalariaparasitaemiaincludedincreasingdurationfollowingthediscontinuationofprophylaxis(adjustedoddsratio[aOR]1.79,95%CI1.225-2.621,p=0.003),CD4countgreaterthan250cells/ul(aOR0.17,95%CI0.071-0.426)andbednetusethenightpriortosurvey(aOR0.31,95%CI0.105-0.906)werefoundtobesignificantlyassociatedwithmalariaparasitaemia.PeoplelivingwithHIVwhohadstoppedcotrimoxazoleprophylaxisforatleast3monthshadasignificantlyhigherprevalenceofmalariaparasitaemiacomparedtothoseonprophylaxis.IncreaseddurationwasassociatedwithincreasingoddsofhavingparasiteswhileIRS,bed-netuseandhighCD4countwereassociatedwithreducedoddsofhavingmalariaparasites.TheseresultssuggestthatmalariapreventivemethodsincludingIRSandbed-netuseshouldbepromotedinHIVpositivepatientsinwhomcotrimoxazoleprophylaxisisbeingdiscontinuedsothattheyareprotectedfrommalaria.

PP-38ScreeningPathogenBoxCompoundsforActivityAgainstPlasmodiumfalciparumMotilitySachieKanatani,NatashaVartak,SukanatKanchanabhogin,RubayetElahi,AbhaiTripathi,SeanT.Prigge,PhotiniSinnisW.HarryFeinstoneDepartmentofMolecularMicrobiologyandImmunology,TheJohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,MarylandAsthemalariaparasitebecomesresistanttoeverydrugthatwedevelop,identificationanddevelopmentofnoveldrugcandidatesisessential.Manystudieshavescreenedcompoundsdesignedtotargettheclinicallyimportantbloodstages.However,toeradicatemalaria,newdrugsthatblocktransmissionoftheparasitebetweenthemammalianhostandthemosquitovectorareessential.Malariainfectionisinitiatedwhenmosquitoesinoculate10to100sporozoitesastheyprobeforblood.Sporozoitesactivelymoveintheskininordertofindandenterthebloodcirculationandbecarriedtotheliver.Assporozoiteinoculumissmallandmotilityiscriticaltotheirabilitytoexittheinoculationsiteandestablishinfection,wehaveinitiatedastudytoscreenthePathogenBoxcompoundsfortheiractivityonsporozoitemotility.Tothisend,wehaveestablishedamoderatethroughputassayusingsporozoitesofthehumanmalariaparasite,Plasmodiumfalciparum.SporozoitesisolatedfrominfectedAnophelesmosquitoes,wereincubatedwith400drug-likecompoundsfromthePathogenboxprovidedbyMedicinesforMalariaVenture.Compoundsexhibiting



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inhibitoryeffectsonsporozoitemotilitywerefurtherassessedagainstasexualstagegrowthandtransmission-blockingactivity.SixcompoundshadasignificantinhibitoryeffectoninvitroP.falciparumsporozoiteglidingat1µMconcentration.Ofthese6compounds,5compoundsshowedsignificantinhibitiononasexualparasitegrowthand3compoundsdemonstratedsignificantinhibitoryactivityonbothasexualstageandtransmissionstageinmosquitoes.Insummary,wehaveidentified3compoundsthathavesignificantinhibitoryeffectsonmultiplestagesofP.falciparum,includingbothtransmissionstages.Furtherstudiesarerequiredtorevealthemechanismofactionofthesecompounds.Ourfindingsprovidenewantimalarialdrugcandidatesforcombinationtherapyaswellasprophylaxis.

PP-39UsingAntonovsky’sSalutogenicModelofHealthasnewlensesformalariaresearchValerieMakoge1,HarroMaat²,LennekeVaandrager²,MariaKoelen²1InsituteforMedicalResearchandMedicinalPlantsStudies,Yaoundé,Cameroon,²WageningenUniversityandResearch,TheNetherlandsMalariaasamajorpublichealthproblemremainsuncontesteddespiteglobalefforts.Malariaeliminationandcontrolhasbeenafeaturepointinthe2005millenniumdevelopmentgoalsaswellasthe2016sustainabledevelopmentgoalsbutthatnotwithstandingtheproblemremains.Sofar,thefightagainstmalariaandotherpoverty-relateddiseases(PRDs)havebeendiseasefocusedandhashardlyhighlightedeffortspeopleputinplacetocombatinfectiousdiseases.Thefutureofmalariaresearchthereforeneedstoseemalariaeradicationthroughdifferentlenses.Weproposeasalutogenicapproachtothefightagainstmalaria.Salutogenesisasksthequestion:whatcreateshealth?Insteadoffocusingonriskfactorsandpreventionstrategies,thesalutogenicmodelfocusesonhowpeopleidentifyandusegeneralisedandspecificresistanceresourcesaroundthemtonotonlyunderstandthestressorcalledmalariabutalsotoovercome.Inthispaper,wewouldpresentatwo-casemixedmethodstudyinvolvingstudentsoftwouniversitiesinCameroonandCameroonDevelopmentCorporationcampdwellerstohighlighthowthesalutogenicmodelofhealth(SMH)revealscopingstrategieswithmalaria.Thesalutogenicperspectivelaysemphasisoneffortspeopleundertaketostayintheeaseendoftheease-dis(ease)continuum.OurresultsconfirmedmalariaasthemostcommonlyperceivedPRDbyover90%ofrespondentsinbothsettings.Thepresenceofmalariawasattributedtopoorhygienicconditionsoftheenvironment.Thestudyrevealedthatpeoplewithahighersenseofcoherence(SOC)weremostlikelytoidentifyanduseresourcesintheir

environmentusingvariedmechanismsinordertocopewithmalaria.TheresultsofthisstudycallfortheconsiderationandinclusionoftheSMHinfuturemalariaresearchforbetterandmoresustainableoutcomesandthepromotionofhealthinaffectedpopulations.

PP-40AHierarchicalBayesianModelforBackgroundCorrectionofProteinMicroarraysSophieBérubé1,TamakiKobayashi2,AmyWesolowski2,WilliamJ.Moss2,,3,IngoRuczinski1ThomasA.Louis1

1DepartmentofBiostatistics,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2DepartmentofEpidemiology,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,3W.HarryFeinstoneDepartmentofMolecularMicrobiologyandImmunology,JohnsHopkinsMalariaResearchInstitute,JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland

ProteinmicroarraysallowuserstocharacterizelargenumbersofproteinsinparallelandhavethepotentialtoprovideinformationaboutcumulativeexposuretoPlasmodiumantigenswithacross-sectionalstudydesign.However,toextractthisinformationfromaproteinmicroarray,attentionmustbepaidtoremovingnoisewhileretainingthebiologicallyrelevantsignal.WhilemethodshavebeendevelopedtocorrectforbackgroundonDNAmicroarrays,theuniquepropertiesofproteinsincludingvariablesizeandcharges,structuralcomplexityandbindingaffinitywarrantsbackgroundcorrectionmethodsoptimizedforproteinmicroarrays.WeproposeahierarchicalBayesianmodelthatincludesmeasuredforegroundandbackgroundsignalsateachprobe.MarkovchainMonteCarloproducestheposteriordistributionforthetruesignal,andtheposteriormeanandothersummariesareavailable,orthefulldistributioncaninputtothenextphaseofanalysis.Weshowthatthismodelleaveshighintensitysignals,forwhichbackgroundnoisehaslowimpact,closetothedirectlycomputedestimate.Lowintensitysignalsreceiveasubstantialandbeneficialadjustment,whilekeepingthemassoftheposteriordistributiononpositivevalues.Moreover,ourproposedmodelingapproachimpactstherankofthefluorescentintensityofseveralantigensinPlasmodiumfalciparum-specificantibodyarraysrunwithserumsamplesfromhighandlowmalariatransmissionsettingsinZambiaandZimbabwe.

PP-41IdentificationofExpressedVargenesinWholeBloodClinicalSampleswithaCustomCaptureArrayVersusRNAEnrichmentMethods



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EmilyM.Stucke1,AntoineDara1,AnkitDwivedi2,TheresaHodges2,DrissaCoulibaly3,AbdoulayeK.Koné3,KarimTraoré3,BouréimaGuindo3,BouramaM.Tangara3,AmadouNiangaly3,ModiboDaou3†,IssaDiarra3,YoussoufTolo3,ModySissoko3†,AlbertE.Zhou,MatthewB.Laurens1,AmedOuattara1,BouremaKouriba3,OgobaraK.Doumbo3†,ShannonTakala-Harrison1,DavidSerre2,MahamadouA.Thera3,ChristopherV.Plowe4,MarkA.Travassos1,JoanaC.Silva2,51MalariaResearchProgram,CenterforVaccineDevelopmentandGlobalHealth,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,2InstituteforGenomeSciences,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,3MalariaResearchandTrainingCenter,UniversityofScience,TechniquesandTechnologies,Bamako,Mali,4DukeGlobalHealthInstitute,DukeUniversity,Durham,NorthCarolina,5DepartmentofMicrobiologyandImmunology,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,†Deceased ThevargenefamilyencodesPlasmodiumfalciparumerythrocytemembraneprotein-1(PfEMP1)antigens.Thesehighlydiverseantigensaredisplayedonthesurfaceofinfectederythrocytesandplayacriticalroleinimmuneevasionandsequestration.Studiesofvarexpressionusingnon-leukocyte-depletedbloodarechallengingduetothepredominanceofhostgeneticmaterialandlackofconservedvarsegments.Toaddressthesebarriers,wecomparedtwoenrichmentmethodsforparasiteRNAextractedfromwholebloodclinicalsamples—globinandrRNAdepletionfollowedbypolyAselectionvs.acustomcapturearraybasedonRoche’sSeqCapEZEnrichmentSystem.Thecapturearraywasdesignedwithprobescoveringthe3D7referencegenomeandanadditional>4,000full-lengthvargenesequences.WetestedeachmethodonthesamesamplesfromMalianchildrenwithsevereoruncomplicatedmalariainfections,andsequencedusingIllumina.Var-liketranscriptswereidentifiedfromthedenovoassemblyofnon-humanreadsandannotated.Foreachsample,wecomparedtranscriptlengthandnumberofuniquetranscriptsgeneratedfromeachenrichmentmethod.Todetermineifeachmethodyieldedidenticalvarsequences,wecomparedsequencesgeneratedbyeachmethod.Wethenquantifiedvarexpressiontodetermineifexpressioncorrelatedbetweenmethods.DepletionofthemostabundanthumanRNAsfollowedbypolyAselectionproducedtranscriptswithgreatermedianlengthinsampleswiththehighestparasitemiascomparedtothecapturemethod.Thecapturearrayproducedthelongestmaximumlengthandlargestnumbersoftranscriptsforeachsample,particularlyforsampleswithlowparasitemia(<2,000parasites/µL).Thecapturemethodproducedmoreuniquefragments,includingupto20distinctacidicterminalsequencedomainspersample.Furtherevaluationwillincludeexpressionanalysesofsampleswithknownvarrepertoirestodeterminethe

methodbestsuitedtoanalyzevarexpressioninstudieswithbothlowandhighparasitemiasamples.

PP-43AroboticsystemforextractingsalivaryglandsfromanophelesmosquitoesformalariavaccineproductionHenryPhalen1,PrasadVagdargi1,HongtaoWu1,MichaelPozin1,AndrewShaughnessy1,SumanaChakravarty2,GregoryS.Chirikjian1,IulianIordachita1,RussellH.Taylor1

1LaboratoryforComputationalSensingandRobotics,JohnsHopkinsUniversity,2Sanaria,Rockville,Maryland

Thistalkreportsprogresstowardthedevelopmentofanautomatedsystemforextractingsalivaryglandsfromanophelesmosquitoes,aspartanongoingcollaborationbetweenJohnsHopkinsandSanaria,Inc.(RockvilleMaryland)tofacilitatetheproductionofaliveorganismvaccineformalariausingPlasmodiumfalcimarumsporozoites(PfSPZ).Inpreviouswork,wehavedevelopeda“semi-automated”system,inwhichmosquitoesaresortedmanuallyintocartridgessothattheirheadsextendbetweencutterblades.Thenthebladesareactuatedtodecapitateallthemosquitoesinacartridge,andacomb-likedeviceisusedtoextrudeallthesalivaryglands,whicharethencollectedviaasuctiondevice.Basedonthisexperience,wehavebegundevelopmentofafullyautomatedsystem.Thistalkfocusesonakeystepinourautomatedprocess,thepickingandplacingofmosquitoesfromastagingapparatusintoadissectionassemblyusinga4degree-of-freedomrobotundertheguidanceofacomputervisionsystem.Mosquitoesareautonomouslygraspedfromameshplatformandpulledtoapairofnotcheddissectionbladestoremovetheheadofthemosquito,allowingaccesstothesalivaryglands.Placementintothesebladesisadaptedbasedonoutputfromcomputervisiontoaccommodatefortheuniqueanatomyandorientationofeachgraspedmosquito.Inthispilottestofthesystemon50mosquitoes,wedemonstratea100%graspingaccuracyanda90%accuracyinplacingthemosquitowithitsneckwithinthebladenotchessuchthattheheadcanberemoved.Thisisapromisingresultforthisdifficultandnon-standardpick-and-placetask.Ananalysisofthefailurecasesprovidesinsightsforimprovementstobeimplementedasthisroboticpick-and-placesystemisintegratedintoalargerautomatedmosquitodissectionsystemunderdevelopment.

PP-44MappingtheAedesaegyptiantennallobesShrutiShankar,ConorJ.McMenimanJohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland



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Theantennallobes(ALs)aretheprimaryolfactorycentreofthebrainoftheyellowfevermosquito,Aedesaegypti.Inmosquitoes,theALsmediateprocessingofadiversityofvolatileodorantsthatdriveinnatebehaviorssuchashostseeking,egglayingandforaging.Anatomically,theALsarecomposedoffunctionalunitsofneuropilknownasglomeruli.Anindividualglomerulusisthepointatwhichtheaxonalprojectionsofolfactorysensoryneuronsexpressingasingletypeofchemoreceptor,orcomplementofchemoreceptorsconvergeandsynapsewiththedendriticarborsofprojectionneuronsandlocalinterneurons.Wepresent3DmodelsbasedonmanualimagesegmentationandreconstructionoftheleftandrightALsfrom10maleand10femalemosquitobrains.WeobservethattheALisentirelycomposedof77-80olfactoryglomeruli,andbasedondepthandspatialposition,alongtheanterior-posterior,ventral-dorsalandmedial-lateralaxes,weclassify13glomerularspatialgroupsinthemaleandfemaleALs.AlthoughwedidnotidentifyanysexuallydimorphicglomeruliintheAe.aegyptiALs,wefoundoverallthatthevolumeofthefemaleantennallobe,wasapproximately1.5timeslargerthatofthemalemosquitoes.Furthermore,usingthebinaryQF2-QUASbinaryexpressionsystem,wetracetheneuronalprojectionsofthreeclassesofchemosensoryneurons,tofindnon-overlappingsubsetsof62Orco,14Ir8aand1GR1positiveglomeruliineachAL.ThesestudiesdescribearevisedmodeloftheAedesaegyptiALs.Thehigh-resolutionanatomicalmappresentedherewillserveasausefulreferenceforfuturefunctionalimagingstudiesthataimtoidentifyglomeruliactivatedbyhumanodorstodecodethemolecularandcellularbasisofmosquitoattractiontohumans.

PP-45IdentifyingproteinsthataredifferentiallyexpressedinAsaiabogorensisunderbloodmeal-likeconditions MarisaGuido,DavidJ.Lampe

DepartmentofBiologicalSciences,DuquesneUniversity,Pittsburgh,Pennsylvania Malariaisoneofthedeadliestvector-bornediseasesandclaimsthousandsoflivesyearly.Boththecausativeagentofmalaria,Plasmodiumsp,andtheAnophelesmosquitothattransmitsitaregainingresistancetomanycurrentmethodsofcontrol.Bygeneticallyengineeringsymbioticbacteriawithinthemosquitomidgut,aprocesstermedparatransgenesis,antiplasmodialeffectorscanbesecretedtoreducePlasmodiumtransmission.AsaiabogorensisisaGram-negative,rodshapedbacteriathatnaturallycolonizesthemidgut,ovaries,andsalivaryglandsofAnophelesmosquitoes,makingitanidealcandidateformodification.However,thereareconcernsthatintroducingforeignDNAsequenceswillcausethetransgenicA.bogorensistobe

outcompetedbywildtype.InsertingantiplasmodialeffectorsdownstreamofsecretedproteinsthatareupregulatedinthepresenceofbloodwouldintroducetheleastamountofforeignDNA,andmaybetheleastdisruptivesystemfortransgenicA.bogorensis.FASTAsequencesforproteinsbothupstreamanddownstreamofpromotersknowntobebloodmealinducible(BMI)werecollected,andthePSORTprogramwasusedtopredictthelocalizationofthecandidateproteins.FivecandidateswereidentifiedaroundtheHeminpromoter;onewasapredictedextracellularproteindownstream,threewerepredictedoutermembraneproteinsupstream,andonewasapredictedextracellularproteinupstream.Primerswerethendesignedforthecandidateproteinstoobserveexpressionwithorwithoutblood.A.bogorensiswasgrownonbothminimalmediaandchocolateagarcontaininglysedredbloodcells.TheRNAwasthenharvestedandusedinRT-qPCRtodetermineiftherewasdifferentialexpressionoftheseproteinsinbloodmeal-likeconditions.Proteinsthatappearedtobeupregulatedinbloodmealconditionswerethenselectedforfurthermodification.

PP-46AssessingRapidDiagnosticTestandBloodSmearMicroscopyoutcomeswithclinicaldiagnosesformalariainKano,NigeriaMichaelVera1,NirmalRavi2

1TheGeorgeWashingtonUniversityMilkenInstituteSchoolofPublicHealth,2eHealthAfrica,Washington,DC

Malariaisaleadingglobalhealthissuethatdisproportionate-lyeffectssub-SaharanAfricamorethananyotherregionintheworld.In2017,Nigeriaaccountedforthegreatestmalariaburdenatapproximatelyonequarteroftheestimated219millionglobalcases.RapidDiagnosticTest(RDT)andBloodSmearMicroscopy(BSM)aretheprincipaldiagnosticmethodsusedbyclinicianstosupplementclinicalevaluation.Duetotheiraffordability,robustness,andhighsensitivityandspecificity,cliniciansrelyontheseparasitologicaltestsforcaseconfirmation.ThepurposeofthisstudywastoinvestigateRDTandBSMtestoutcomesreportedfromaprivatehealthfacilityinNorthcentralNigeriatoinformtheclinic’smanagementoftestpositiveratesandlaboratoryadherencetotestingpolicy.Asecondaryanalysistodeterminetheassociationsbetweenfinaldiagnosticoutcomesandpatientvisitcharacteristicswasperformed.Anexaminationof386patientvisitrecords(60.3%visitsmale,28.5years(SD=11.9)averagepatientvisitage,248individualpatients)fromJuly2018toMarch2019revealedRDTandBSMtestpositiveratesof8.8%(n=385)and24.3%(n=322),respectively.MalariaprevalenceinKanowaspreviouslyreportedat60.6%(n=551)in2016.TestpositiveratesreportedforRDTandBSMinnearbyKadunawere11.9%and10.5%(n=295),respectively.Adiseaseoutcomestatus



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algorithmreliantonRDT,BSM,Loop-MediatedIsothermalAmplification,prescribedanti-malarialmedication,andclinicpolicywasdevelopedandfoundamalariapatientvisitcase-positiveperiodprevalenceof13.5%(n=385).Univariateanalysisoftheassociationbetweenmalariacase-positiveoutcomeandpatientvisitcharacteristicsfoundthatsubjectivereportingoffever(OR=8.3,95%CI=2.5-27.1),pain(OR=2.1,95%CI=1.2-3.8),andlossofappetite(OR=2.8,95%CI=1.2-6.5)werestatisticallysignificant.Presumedselectionbiasduetothehigherpatientsocioeconomicstatusrequiredforclinicservicesanddiscrepanciesbetweenthisinvestigation’sfindingsandpublishedfindingssuggestsfurtherbiasanalysis.

PP-47Theeffectofanartificialbloodmeal,SkitoSnack,onvectorcompetenceofAedesaegyptiandAnophelesstephensiforPlasmodiumgallinaceumandP.falciparum. KristinaK.Gonzales-Wartz1,JulianaM.Sá1,KevinLee1,YonasGebremicale1,AndreLaughinghouse1,SamuelMoretz1,AnaM.Ortega-Villa2,MichaelFay2,ThomasE.Wellems1 1LaboratoryofMalariaandVectorResearch,NationalInstituteofAllergyandInfectiousDisease,NationalInstitutesofHealth,Bethesda,Maryland,2BiostatisticsResearchBranch,DivisionofClinicalResearch,NationalInstituteofAllergyandInfectiousDisease,NationalInstitutesofHealth,Bethesda,MarylandTheAedesaegyptimosquitoisavectorfortheyellowfever,dengue,chikungunya,andzikaviruses,andtheAnophelesstephensimosquitoisavectorforthemalariaparasites,PlasmodiumfalciparumandP.vivax.WithurbanexpansionofAe.aegyptiandAn.stephensi,innovativevectorcontrolstrategiesareneededtoreducethespreadofmosquito-bornedisease.Forresearchandsomecontrolapproaches,massrearingofmosquitoesininsectariesrequireslargeamountsofvertebrateblood,whichisexpensiveandmayintroducepathogens.SeveralartificialbloodmealshavethereforebeencreatedandtestedonAedesaegyptimosquitoesand,morerecently,Anophelinemosquitoes,butlittleisknownaboutpotentialinfluencesofthesemealsonvectorcompetence.Wehavetestedoneofthesemeals,SkitoSnack,onthecompetenceofAe.aegyptifortheavianmalariaparasite,Plasmodiumgallinaceum,andofAn.stephensiforthehumanmalariaparasite,P.falciparum.MosquitoeggsfromNIH-establishedAe.aegyptiandAn.stephensicolonieswerehatchedandmaintainedunderstandardinsectaryconditions.Adultfemalemosquitoes,3-7daysold,werefedabovinebloodorSkitoSnackmealviaanartificialmembranefeedingsystemandwerebredonthesemealsthroughseveralconsecutivegenerations.Todeterminevectorcompetence,Ae.aegyptifemaleswerefedonananesthetizedP.gallinaceum-infectedchicken,whileAn.stephensifemaleswerefedthroughanartificialfeeder

containingP.falciparum-infectedblood.Mosquitomidgutsweredissected7dayspostinfectionandoocystswerestainedandcountedunderalightmicroscope.WefoundtheP.gallinaceumparasiteloadsofAe.aegyptiraisedonbovinebloodvs.SkitoSnackfor1,3,and5generationsweresimilar.ResultsfromSkitoSnack-raisedAn.stephensiarepending.SkitoSnackasabloodsubstitutewillsupportpathogen-freemosquitolinesandmayhelptoreducecostsoflarge-scalemosquitoproductionininsectaryfacilities.

PP-48ArecombinationsystemforthecreationofstableantiplasmodialparatransgenicAsaiasp. ThomasKelly,SumerJasmine,DavidLampe DuquesneUniversity,Pittsburgh,Pennsylvania Malaria,adiseasecausedbytheparasitePlasmodiumsp.,isresponsibleforhundredsofthousandsofdeathseachyear.Reductionsininfectionandlethalityofmalariahavestalledinrecentyears,sonewcontrolstrategiesmustbesought.Onesuchstrategyincludesthetransgenicmodificationofsymbioticbacteriawithinthemosquitomidguttoproduceantiplasmodialeffectorsinaprocessknownasparatransgenesis.Asaiasp.,aGram-negativerod-shapedbacteriumwhichcolonizesthemidgut,ovariesandsalivaryglandsofAnophelesmosquitoes,hasbeenidentifiedasanidealcandidateforantiplasmodialparatransgenesis.PreviousworkdevelopedparatransgenicstrainsofAsaiawhichwereshowntobeeffectiveatreducingoocystprevalencewithininfectedmosquitoes.However,thesestrainsareplasmidbasedandrelyondrug-selectionforstability,whichisunsuitableforfield-release.OnewaytodevelopstablestrainsofparatransgenicAsaiaistoinserttheantiplasmodialeffectorsintothechromosome.Site-specificrecombinationviasacBcounterselectionwasusedtoinsertthegeneralantimicrobialpeptidescorpinedownstreamofthenativeconditionalbloodmealinducedpromoterHlyCwithinthechromosomeofAsaiaSF2.1.Thisstrainwasgrowninbloodmeal-likeconditionstoinduceproductionofscorpine.Westernblotswereperformedonbothcelllysateandsupernatantfractionsandcomparedtouninducedtransgeniccontrols.FurthertestingwillconfirmtheefficacyofthisstrainagainstPlasmodiuminarodentmodel.

PP-49Comparativeanalysesofparasiteswithacomprehensivedatabaseofgenome-scalemetabolicmodelsMaureenA.Carey1,2,GregoryL.Medlock3,4,MichałStolarczyk5,6,WilliamA.Petri,Jr.2,JenniferL.Guler2,5,JasonA.Papin2,3



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1DepartmentofMicrobiology,Immunology,andCancerBiology,2DivisionofInfectiousDiseasesandInternationalHealth,DepartmentofMedicine,3DepartmentofBiomedicalEngineering,4DivisionofGastroenterology,Hepatology,andNutrition,DepartmentofPediatrics,5DepartmentofBiology,6CenterforPublicHealthGenomics,UniversityofVirginiaSchoolofMedicine,Charlottesville,VirginiaProtozoanparasitescausediversediseaseswithlargeglobalimpacts.Researchonthepathogenesisandbiologyoftheseorganismsislimitedbyeconomicandexperimentalconstraints.Accordingly,studiesofoneparasitearefrequentlyextrapolatedtoinferknowledgeaboutanotherparasite,acrossandwithingenera.Modelinvitroorinvivosystemsarefrequentlyusedtoenhanceexperimentalmanipulability,butthesesystemsgenerallyusespeciesrelatedto,yetdistinctfrom,theclinicallyrelevantcausalpathogen.Characterizationoffunctionaldifferencesamongparasitespeciesisconfinedtoposthocorsingletargetstudies,limitingtheutilityofthisextrapolationapproach.Toaddressthischallengeandtoaccelerateparasitologyresearchbroadly,wepresentafunctionalcomparativeanalysisof192genomes,representingeveryhigh-quality,publicly-availableprotozoanparasitegenomeincluding44genomesfor20Plasmodiumspecies,aswellasToxoplasma,Cryptosporidium,Entamoeba,Trypanosoma,Leishmania,Giardia,andotherspecies.Wegeneratedanautomatedmetabolicnetworkreconstructionpipelineoptimizedforeukaryoticorganismstoserveasbiochemicalknowledgebasesforeachparasite,enablingqualitativeandquantitativecomparisonsofmetabolicbehavioracrossparasites.Weidentifiedputativedifferencesinenzymeessentialityandpathwayutilizationtofacilitatethecomparisonofexperimentalfindings.WepredictthatToxoplasmagondiiisabettermodelsystemforCryptosporidiumspeciesthanforPlasmodiumspecies.Moreover,theenzymeessentialityofP.falciparum3D7ismoresimilartoP.vivaxSal1thantoP.bergheiANKA.Wealsodiscoveredthatphylogenyisnotthesolepredictorofmetabolicsimilarity.Morebroadly,thisknowledgebaserepresentsthelargestcollectionofgenome-scalemetabolicmodelsforbothpathogensandeukaryotes;withthisresource,wecanpredictspeciesorstrain-specificfunctions,contextualizeexperimentalresults,andoptimizeselectionofexperimentalsystemsforfastidiousspecies.

PP-50Cholesterol-dependentenrichmentofunderstudiederythrocyticstagesofhumanPlasmodiumparasitesAudreyC.Brown1,ChristopherC.Moore2,JenniferL.Guler1,2

1DepartmentofBiology,UniversityofVirginia,Charlottesville,2DivisionofInfectiousDiseasesandInternationalHealth,UniversityofVirginia,Charlottesville,Virginia

Plasmodiumparasitesundergoroundsofasexualreplicationinsidehumanerythrocytes,progressingfromringstage,totrophozoitesandschizonts,beforeegressandreinvasion.Giventhediscoveryofring-specificartemisinintoleranceandquiescenceinPlasmodiumfalciparum,thereisgreaturgencytobetterunderstandringstagebiology.However,thelackofaneffectiveenrichmentmethodhasleftringsandrelatedparasitestagesunderstudiedcomparedtotheirlatestagecounterparts,whichcanbeeasilyisolatedduetotheirparamagneticproperties.Here,amethodforseparatingallPlasmodiuminfectederythrocytesfromuninfectederythrocytesispresented.Thisapproachtakesadvantageofstreptolysin-O(SLO)topreferentiallylyseuninfectederythrocytesaspreviouslyshownbyJackson,etal.Followinglytictreatment,Percollgradientcentrifugationremoveslysedcells,leavinganintactcellpopulationenrichedininfectederythrocytes.ThisSLO-Percoll(SLOPE)methodiseffectiveonstagesfromtheentireerythrocyticcycle,includingpreviouslyinaccessibleformssuchascirculatingringsfrommalaria-infectedpatientsandartemisinin-inducedquiescentparasites.Furthermore,theutilityofSLOPEisextendedtomultiplemediaformulationsusedforthepropagationoftwohumanPlasmodiumspecies.ThealterationofexternalcholesterollevelsmodulatesSLOPEeffectiveness,demonstratingtheroleoferythrocytemembranecholesterolinlyticdiscrimination.Importantly,enrichmentdoesnotimpactparasiteviability,whichestablishesthenon-toxicnatureofSLOPE.TargetedmetabolomicsofSLOPE-enrichedringstagesamplesconfirmstheimpactontreatedsamples;parasite-derivedmetabolitesareincreasedandcontaminatinghostmaterialisreducedcomparedtonon-enrichedsamples.TheSLOPEmethodisanaccessible,scalable,rapid(30-40min),andnon-toxicenrichmentmethodthatisbroadlyeffectiveonmanyerythrocyticstages.Thismethodisidealforuseupstreamofavarietyofsensitiveanalyses,whichwillincreaseexperimentalqualityinvirtuallyallareasofasexualPlasmodiumparasiteresearch.

PP-51Invitroevolutionofhigh-levelresistancetoP.falciparumcytochromeBinhibitorsKristinD.Lane1,JianbingMu1,JinghuaLu2,AnnaLiu1,SeanT.Windle1,T.ParksRemcho1,ThomasE.Wellems11LaboratoryofMalariaandVectorResearch,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,Maryland,2LaboratoryofImmunogenetics,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,Bethesda,MarylandThemajorenergyproductionpathwayinPlasmodiumfalciparumiscytosolicglycolysis,however,themitochondrialmembranepotentialisvitalinallstages(erythrocytic,liver,mosquito).Thus,electrontransportchain(ETC)enzymes



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remainattractivedrugdevelopmenttargets.ThesolelicensedETCinhibitor,atovaquone(ATQ),bindsthecytochromeB(CytB)quinoloxidation(Qo)pocket;resistanceisconferredbymutationsofmethionine133(invitro)ortyrosine268(invivo).ResistancetoCytBinhibitorsATQorRYL-552in106/1mappedQositemutationstyrosine268toserine(Y268S,ATQ)orvaline259toleucine(V259L,RYL-552).Therewasnocross-resistancetoATQorRYL-552,althoughV259Lwascross-resistanttotheCytBinhibitorCK-2-68.AllthreesmallmoleculesbindtheQopocketinasimilarlocation.WeaskedifselectiononY268SorV259Lwithasecondcompoundwouldresultinfurthermutationsanddifferentialdrugresponsephenotypes.SecondaryselectionswereY268S(RYL-552),V259L(ATQ),andbothY268SandV259L(CK-2-68).NoadditionalCytBmutationswereidentifiedafterY268Ssecondarydrugpressure,howeverthelineswerehighlyATQresistantandmarkedlysusceptibilitytoplumbagin(PL),asuccinatedehydrogenaseinhibitor.V259L(ATQ)resistantlinesgainedtheadditionalCytBsubstitutionsL144SorV284A.BothL144S/V259LandV259L/V284Ademonstratedmid-levelATQresistance,high-levelresistancetoantimycinA(AMA,CytBinhibitorofthequinonereductionpocket),anddecreasedPLsensitivity.V259L(CK-2-68)evolvedaY126Csubstitution,conferringhigh-levelCK-2-68andAMAresistance.DockingstudieswithCytBmodelsdemonstratedY126Cmayconferhigh-levelCK-2-68resistanceontheV259Lbackground.Givenadwindlingpipelineofeffectivechemotherapeutics,theemergenceofmulti-drugresistantparasitesisanactivethreattoeradicationefforts.ThesedatasuggestthatCytBinhibitorstargetingthesameenzymaticdomainareeffectivechemotherapeuticsinvitro,lackingsubstantivecross-resistance.CombinationsoftheseinhibitorsmayproveusefulagainstfutureP.falciparuminfections.

PP-52Trackingmosquitoesovertime:testingtheroleofaestivationindryseasonpersistence

RoyFaiman1,AdamaDao2,AlphaSeydouYaro2,MoussaDiallo2,SamakeDjibril2,ZanaLamissaSanogo2,YossiOusmane2,MargerySullivan1,LauraVeru1,BenjaminJ.Krajacich1,ChristineA.M.France3,andToviLehmann11LaboratoryofMalariaandVectorResearch,NationalInstituteofAllergiesandInfectiousDiseases,theNationalInstitutesofHealth,Rockville,Maryland,2MalariaResearchandTrainingCenter,FacultyofMedicine,PharmacyandOdonto-stomatology,Bamako,Mali,3SmithsonianInstitutionMuseumSupportCenter,Suitland,MarylandDespiteitsrecognizedimportance,trackingmosquitosoverextendedtimehasbeenbeyondmedicalentomology’stoolkit.Stableisotopeenrichmentofmosquitobreedingsitesenablesmarkingofmosquitoeswithouthumanhandling.Larvaedevelopingin[2H]-enrichedwaterare

structurallymarkedforlife,providingauniqueopportunitytotestthehypothesisthatAnophelescoluzzii,butnotA.gambiaes.sandA.arabiensis,locallypersistsintheSahelthroughthedryseasonviaaestivation.Alarge-scaleexperimenttotestthishypothesisbeganinSeptember2017intwoSahelianvillagesinMali.Weaimedtoestimatethecontributionofaestivationtopersistenceofmosquitoesthroughthe7-monthlongdryseasonbymarkingatleast10%oftheA.gambiaes.l.adultsbytheendofthewetseasonandassesstheproportionofmarkedadultsthroughthedryseason,andimmediatelyafterthefirstraininJune2018.IfaestivationistheonlywayA.coluzziipersists,thefrequencyofmarkedmosquitoesshouldbesimilarthroughout.Findingnomarkedmosquitoeswouldbeevidenceagainstaestivation.Twenty-fournaturallarvalsiteswereenrichedfromlateSeptember.Themarkedadultproportionbytheendofthewetseasonwasabove60%.Fivemonthslaterasimilarfrequencyofmarkedadultswasdetected.Notably,nearly8monthsafterenrichment,6%showedclearmarking.AllthemarkedmosquitoesdetectedaftertheonsetoftheDSwereA.coluzzii,inaccordwithaestivationinthisspeciesalone.Because2H-markinginmosquitoeshassteadilyweakenedovertime,wesuspectsomeoftheseeminglyun-enrichedmosquitoeshadeitherlosttheirmarkingordispersedtoneighboringvillages.Accordingly,wefindthatthedistributionof2Hinmosquitoesafterthefirstrainexhibitsanexceptionallyheavyrighttail,indicatingthatinadditiontonaturallyun-enrichedmosquitoestherearemanyenrichedmosquitoesthatlostmuchoftheirenrichmentbutareclumpedinthatzone.Ourresultsprovide,forthefirsttime,hardevidenceofpopulation-wideaestivationinA.coluzzii.

PP-53Manufactureofaseptic,purified,cryopreservedPlasmodiumvivaxsporozoitesSumanaChakravarty,NatashaKC,StephenL.Hoffman

SanariaInc.,Rockville,Maryland, Plasmodiumvivax(Pv),thesecondmostimportanthumanmalariaparasite,causesmorethan80millioncasesannuallyincludingsevere,fataldisease.Preventionandcontrolarechallengedbyemergingdrugresistanceandrelapsesfromdormantliverstageparasitescalledhypnozoites.Theonlytherapyagainstrelapse,primaquine,causeslifethreateningacutehemolyticanemiainpatientswithG6PDdeficiency,themostprevalenthumangeneticdisorder,affecting8%ofpeopleinmalaria-endemicnations.ThisbarriertotreatmentresultsinrepeatedPvattacks,aggravatingtheproblemofcontrol.Effortstodevelopbetterdrugsorproduceamuch-neededvaccinearefurtherhamperedbytheinabilitytopropagatebloodstagesofPvparasitesinvitro,unlikePf.Therefore,generatinginfectedmosquitoesforcontrolledhumanmalariainfection(CHMI)asameanstoassessanti-Pv



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drugsandvaccines,isentirelyreliantonfeedingofmosquitoesonfresh,Pv-infectedbloodfrompatientswithPvmalaria.Together,thesebottlenecksmakethetaskofdevelopingandtestingrobustinterventionsagainstPvmalariamorechallengingcomparedtoPf.WehavenowovercomethismajorlimitationbyusingPvgametocyte-infectedSaimiriboliviensis(Sb)non-humanprimates(NHPs)toproducePvSPZ.InfactwearetheonlylaboratorywithaninventoryofvialedPvSPZmadefromNHP-infectedblood,havingproducedasmuchas80millionPvSPZvialedin1dayfrom2,000mosquitoes.ThesecryopreservedPvSPZare1)infectioustohepatocytecelllinesinvitrointraditionalmonolayerformatsover3-6daysandinmicro-patternedco-culturedprimaryhumanhepatocytesover12-21days,and2)infectioustoNHPsinvivo.Inoureffortstoincreaseregulatorycompliancetomanufactureaproductforhumanuse,wenextattemptedtoproduceaseptic,purified,cryopreserved,infectiousPvSPZ(PvSPZChallenge)byusingaspecificgerm-free(SPF)colonyofthepermissiveSbasthesourceforPv-infectedblood.UnderanNIHphaseISBIRfundingwehavenowsuccessfullyvialedaseptic,purifiedPlasmodiumvivax(Pv)sporozoites(SPZ)thatweregeneratedinasepticmosquitoesusinginfectedbloodfromSPFSb.Thevialedproductmetasepticitycriteriainallin-processstepsaswellasreleaseassays,similartoanalogousPfSPZ-basedproducts.ThisnewlyestablishednovelpipelineisintendedtogeneratecGMP-compliant,controlledbatchesofPvSPZ,aninnovationbySanariathatwillofferaconsistent,quality-controlledstockofcryopreservedPvSPZtopromotewell-controlled,reproducibleinvitroandinvivostudiesinPvincludingCHMI.Thisenablingtechnologywillsupportthedevelopmentandtestingofanti-PvdrugsandvaccinesinCHMIsworld-wide,justasPfSPZChallengehasdoneforPfCHMIs.ItwillalsoformthebasisofapowerfulvaccineapproachtopreventingPvmalariawhenadministeredwithanti-malarialchemoprophylaxis,thePvSPZchemoprophylaxisvaccine(PvSPZ-CVac).

PP-54Whole-genomeanalysisofPlasmodiumfalciparumtounderstandclinicalimmunitytomalariaZalakShah1,AlexisBoleda2,KaraA.Moser3,MatthewAdams1,AndreaBuchwald1,KarlSeydel4,5,DonMathanga6,DavidSerre3,MiriamK.Laufer1,MichaelP.Cummings2,JoanaC.Silva3,andShannonTakala-Harrison1

1MalariaResearchProgram,CenterforVaccineDevelopmentandGlobalHealth,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,2CenterforBioinformaticsandComputationalBiology,UniversityofMarylandCollegePark,CollegePark,Maryland,3InstituteforGenomeSciences,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,4BlantyreMalariaProject,UniversityofMalawiCollegeofMedicine,Blantyre,Malawi,5DepartmentofOsteopathicMedicalSpecialties,CollegeofOsteopathic

Medicine,MichiganStateUniversity,EastLansing,Michigan,6MalariaAlertCenter,UniversityofMalawiCollegeofMedicine,Blantyre,MalawiAfterrepeatedP.falciparuminfections,individualsinhigh-transmissionareasacquireclinicalimmunitytomalaria.However,themechanismsimportantindeterminingclinicalimmunityarenotentirelyknown.Herewetakeawhole-genomeapproachtoidentifygenesthatmaybeinvolvedinacquisitionofclinicalimmunitytomalariabysequencingandanalyzingparasitegenomescollectedfrominfectedindividualsfollowedovertimeaspartofalongitudinalstudyinMalawi.Wecomparedparasitegenomesfromindividualswithvaryinglevelsofclinicalimmunity,definedusinganindividual’sproportionofsymptomaticinfections.Wealsoexaminedpairsofparasitescollectedatdifferenttimepointsfromthesameindividuals,hypothesizingthattheseparasiteswillbemoredifferentfromeachotherthanexpectedbychance,atlociimportantforclinicalimmunity.UsingFSTasameasureofgeneticdifferentiation,weidentifiedseveralSNPs,includingSNPsinvaccinecandidateantigenssuchasAMA1,LSAP2andRESA,thataresignificantlygeneticallydifferentiatedbetweenindividualswithvaryinglevelsofclinicalimmunity.Analysisofinfectionsfromthesameindividualsshowedthatthereisloweridentity-by-descentbetweenparasitesfromthesameindividualcomparedtoparasitesfromdifferentindividuals,highlightingtheroleofallele-specificimmunity.Wealsofoundthatseveralvaccinecandidateantigens,suchasSERA5,MSP6,AMA1,etc.,differmorethanexpectedbychanceinparasitesfromthesameindividualcomparedtoparasitesfromdifferentindividuals.SNPsingenessuchasama1,andclag2,aswellasseveralgenesencodingproteinsofunknownfunction,wereidentifiedbybothanalyses,lendingfurthersupportfortheirinvolvementinthedevelopmentofimmunity.WewillanalyzeinsilicopredictedT/B-cellepitoperegionsinlocithesegenestounderstandwhichregionsoftheseproteinsmightbeimmunologicallyimportant.Identifyingandfurtheranalyzingthesegenomicregionswillprovideinsightsintomechanismsinvolvedinacquiredimmunityandantigenicescape.

PP-55EvaluationofPfCRTmutationsassociatedwithpiperaquineresistanceinCambodiaBirajShrestha1,ZalakShah1,AndrewP.Morgan2,MatthewAdams1,PiyapornSaingam3,ChaiyapornChaisatit3,PaphaveeL.Ketwalha3,ChristianParobek2,HuyRekol4,SoklydaChann3,MicheleD.Spring3,MariuszWojnarski3,MarkM.Fukuda3,BrianA.Vesely3,DavidL.Saunders1,PhilipL.Smith3,ChanthapLon3,JessicaT.Lin2,NormanC.Waters1,ShannonTakala-Harrison1

1UniversityofMarylandBaltimore,Baltimore,Maryland,2UniversityofNorthCarolinachapelHill,ChapelHill,North



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Carolina,3ArmedForcesResearchInstituteofMedicalSciences,Bangkok,Thailand,4NationalCentreforParasitology,EntomologyandMalariaControl,PhnomPenh,CambodiaResistancetoartemisininsandkeypartnerdrugssuchaspiperaquine(PPQ)havebecomewell‑establishedinregionsofCambodiaandneighboringcountries.In2016,Cambodiareplaceddihydroartemisinin‑piperaquinewithartesunate‑mefloquineasthefirst‑linetherapy.WeandothershavepreviouslyshownthatmutationswithinthePlasmodiumfalciparumchloroquineresistancetransporter(PfCRT)areassociatedwithreducedsusceptibilitytoPPQ.Subsequentgene‑editingstudieshaveshownthatthesemutationscanindependentlyconferPPQresistanceinparasiteswithsinglecopyplasmepsinII.TobetterunderstandtheemergenceanddistributionofPfCRTmutationsassociatedwithPPQresistance,weareexaminingparasitesfrom478clinicalinfectionscollectedfromeightprovincesinCambodiafrom2009‑2017.WeareusingPacBioampliconsequencingorwholegenomesequencingdatatoidentifyPfCRTmutationsandquantitativePCR(orreadcoverageforsampleswithwholegenomesequencesavailable)toestimateplasmepsinII(pfpm2)copynumber.Inthisdataset,amplifiedpfpm2isobservedin<1%ofsamplesin2009,doesnotreachhighfrequencyuntil2013(62%)and2014(79%),thendecreasesinfrequencyin2016(48%)and2017(55%).OurpreliminarydatafromasiteinnorthernCambodiaidentifymultiplePfCRTmutations,includingmutationsH97Y,F145I,A195V,I218F,andG353Y.F145Iisfirstobservedin2013(24%),peaksin2014(34%),thendecreasesinprevalencein2016(24%)and2017(13%).However,H97Y,I218F,andG353Veachincreaseinprevalenceto24%in2017from10%,13%,and20%in2016,respectively.Whole‑genomesequencingwillbeperformedonparasiteswithPfCRTmutationsofinteresttodetermineparasiteorigins.Thisworkprovidesinsightintotheemergence,dynamics,andoriginsofPfCRTmutationscontributingtoPPQresistance.

PP-57InsilicoreadcaptureandassemblyenablesallelereconstructionofhighlyvariablelociTheresaK.Hodges1§,AnkitDwivedi1§,JamesMatsumura1,KaraA.Moser1,AndreaA.Berry2,ShannonTakala-Harrison3,JonathanCrabtree1,JoanaCarneiroDaSilva1

1InstituteforGenomeSciences,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,2UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,3MalariaResearchProgram,CenterforVaccineDevelopmentandGlobalHealth,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,§SharedfirstauthorshipThereconstructionofhighlyvariableprotein-codinggenesinPlasmodiumfalciparumremainschallenging,despiterecent

advancesinDNAsequencingtechnologiesandwholegenomeassemblyalgorithms.Inparticular,geneswithlengthpolymorphismsandlowcomplexityregionsaredifficulttoassemble.Manyofthesegenescontributetoantigenicvariabilityintheparasiteandarepotentialtargetsforvaccinedesign.Wepresentanovelpipelinetoreconstructthesequenceoftargetgene(s)fromwholegenomesequencing(WGS)datafromnewpathogenisolates.TheWGSreadsfromtargetedgenesarerecruitedbyusingGSNAPtomapreaddatatooneormorereferenceallelicsequencesfromthelociofinterestandretainingsuccessfullymapreadsandtheirmatepairs.Foreachtargetedgene,denovoassemblyofallrecruitedreadsisperformedusingSPAdesandtheresultingassemblyisassessedbyaligningitbacktothereferencegenome.AssemblyisfurtherimprovedbydenovoassemblingtherecruitedreadsusingHierarchicalGenomeAssembly(HGA)andScaffoldBuilder,basedonuser-definedthresholds.Thesestepsarerepeatedforunassembledrecruitedreads,usingtheSMALTalignerand/oriterativeroundswithdifferentassemblyparametervalues,untilnofurtherimprovementscanbemadetotheassembly,atwhichpointtheassembledfragment(s)isconsideredfinal.ThepipelinewasvalidatedusingshortreadWGSdatafromfourP.falciparumstrainsfromdifferentcontinents,including3D7,NF166,7G8andNF135.PacBio-basedreferenceforeachstrainwereusedaspositivecontrolandconfirmedtheaccuratereconstructionof>95%ofallprotein-codinglociover90%oftheirlength.Subsequently,228malariavaccineantigenswerereconstructedfrom459P.falciparumisolatesfromsevengeographicareas,basedonshortreadWGSdata.Ofthese,210lociwerefullyreconstructedfrom>350isolates.TotalamountofstartingDNAmaterialisacriticalfactorinthesuccessfulreconstructionofallelicsequencesfortargetloci.

PP-58WillmosquitoesdevelopresistancetotheChromobacteriumspCsp_pbiopesticide?CeciliaS.Engdahl1,EricCaragata1,RaulSaraiva1,HannahMacLeod1,LuisaM.Otero1,2,GeorgeDimopoulos1

1JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2CentersforDiseaseControlandPrevention,SanJuan,PuertoRicoVectorcontrolplaysakeyroleinreducingthenumberofmosquito-bornediseases.Todaysvectorcontrolstrategiesheavilyrelyonsyntheticinsecticidesthatstrugglewithresistantmosquitopopulations.Analternativeandpromisingapproachinvolvestheimplementationofnaturalproduct-basedinsecticides(biopesticides)intovectorcontrolprograms.Mechanismofresistancerelatedtosyntheticinsecticidesiscommonlygroupedintothreecategories;metabolicresistance,targetsiteresistanceandbehavioral



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resistance.Theseallposeaseriousthreattothepublichealtheffortsofvectorcontrolandarethereforewellstudied,whileverylittleisknownaboutthepathwaysaffectedbyselectionwithbioinsecticides.ArecentlyisolatedChromobacteriumspecimenfromPanama(Csp_p)hasbecomeinterestingforitspotentialuseasabioinsecticide.Thisgramnegativesoilbacteriumdisplaymosquitocidalactivityagainstbothlarvaeandadultsofmultiplevectorspecies;Anopheles,AedesandCulex.Inthisstudyweaimtoinvestigateif,(whenandhow)ourbiopesticidecandidateCsp_pwillcausemosquitoestodevelopresistance,andhowcostlythiswouldbe.WewillselectforsurvivaltoadoseinitiallycorrespondingtoLD70intwoseparatelinesofAedesaegyptioverseveralgenerations.Thechangeoflifehistorytraitssuchasdevelopmenttimewillberecordedandoncethesurvivalrateisequaltothatfornon-treatedmosquitoes,genome-widechangesintranscriptionlevelsintheselectedlinesrelativetounselectedcontrolswillbeexplored.

PP-59Re-designingmalariacontrolandsurveillance:anengineeringcasestudyinsub-SaharanAfricaSophiaDiaz1,2,3,4,MonetSlinowsky1,2,KileyGersch1,2,TristanFord1,2,KarinaFrank1,2,EbenezerArmah1,2,ZacharyBuono1,2,AdamGoodwin1,2,MargaretGlancey1,2,SoumyadiptaAcharya1,2

1JohnsHopkinsWhitingSchoolofEngineering,2JohnsHopkinsCenterforBioengineering,Innovation&Design,3JohnsHopkinsInstituteforClinicalandTranslationalResearch,4NationalCenterforAdvancingTranslationalSciences,NationalInstitutesofHealthHigh-qualitymosquitosurveillanceiscrucialtothecontrolofmalaria,afataldiseasethatdisproportionatelyaffectssub-SaharanAfrica.Despiteasignificantscale-upofinterventionsoverthelastdecade,malariacasescontinuetorise.Thisstudyaimedtoreinvigoratemalariaeliminationeffortsbyidentifyinginnovationopportunitiesinsub-SaharanAfrica.Toachievethisaim,aninterdisciplinaryteamofengineersspentAugust2019observingandparticipatingincontrolandsurveillanceactivitiesinZambiaandUganda.Theteamidentifiedthreecriticalopportunitiesforinnovation:first,thereisaneedformorereliablequalitycontrolofimplementedinterventions.Currently,interventionsareassessedusingHumanLandingCatches(HLCs).Volunteersexposethemselvestomalaria-carryingmosquitoes,collectingdataonbitingrates,peakbitingtimes,andvectorpopulations.Thismethodishighlysusceptibletohumanerror,asHLCsmayfallasleeporfailtoaccuratelyfollowcollectioninstructions.Astandardizedmethodofcollectingvectordatawouldproducemorerepresentativeinformation,therebyimprovingqualitycontrol.Secondly,therewasaneedforinexpensiveoutdoorcontrolmethods.Currently,Long-LastingInsecticideTreated

Nets(LLINs)andIndoorResidualSpraying(IRS)aretheprimarycontrolmechanismsused.Thesemethodsfailtoprovidecoverageduringpeakmosquitobitinghours,timesatwhichruralcitizensareoftenoutsidefrequentingthenumerousoutbuildingsthatmakeuptheirhomes.Finally,insecticideresistancemonitoringisinsufficient.Currentpracticesarelabor-intensive,measureonlyonevariable,andoftenleadtogeographicallyunrepresentativedata.Ascalablemethodofmonitoringregionalinsecticideresistancewouldallowforthetimely,bespokedeploymentofcontrolinterventionsinagivenregion.Itwouldalsoshedlightontheimpactresistancehasondeployedinterventions.Identifyingchallengesguidesthedevelopmentofnewtechnologiestoimprovecurrentpractices.Futureworkwillutilizethesefindingstodevelopatechnicalsolutioninhopesofadvancingtheroadtoelimination.

PP-60Semi-fieldmeasurementsofmosquitoolfactorypreferenceDiegoGiraldo,GenevieveTauxe,ConorMcMeniman DepartmentofMolecularMicrobiologyandImmunology,JohnsHopkinsBloombergSchoolofPublicHealthFemalemosquitoesdetectvolatilechemicalsemittedbytheirhoststolocatethem.Differencesinthechemicalcompositionofhumanbodyodormayinfluencemosquitoolfactorypreferenceforcertainhumanswithindisease-endemiccommunities,withimportantimplicationsformalariaepidemiology.However,themolecularandcellularbasisofmosquitoolfactorypreferenceforparticularhumansremainsunknown.Laboratoryexperimentshavebeenusefultostudymosquitohostpreference,however,theyoftenfailtocapturethefullcompositionofodorantsemittedbywholehumans.Semi-fieldexperimentsallowustoovercomethislimitationandtostudymosquitobehaviorinamorenaturalcontext.Westudiedmosquitoolfactorypreferencesinalarge20mx20msemi-fieldcageinMacha,Zambiausinganodorguidedthermotaxisassay(OGTA)andvideotracking.TheOGTAconsistedofalandingarenaheatedto35°CandilluminatedwithinfraredLEDs,andavideocameraontop.8tentsaroundthecagewereconnectedtoitwithairconditioningductingthatendednexttoeacharena.Airfromthetentswaspumpedintothecage.EachtentwasbaitedwitheitherCO2orhumans,andcontroltentswereempty.100An.gambiaefemaleswerestarvedandreleasedintotheflightcageandtheirpreferencewasassessedbyrecordinglandingsbetween22:00-04:00usingvideotrackingforanalysis.TheOGTAallowedustosuccessfullyassessolfactorypreferencesofAn.gambiaeinsemi-fieldconditions.MosquitoespreferredlandingonCO2baitedarenasvscleanair.Additionally,mosquitoesweremoreattractedtoarenasbaitedwithhumansvsCO2.Thispilotdataindicatesthatthismulti-choiceassaymaybeapromisingsystemtoscreenfor



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highlyattractivehumansinthefuture.Animprovedunderstandingoftheodorprofileofhighlyattractivehumansmayfacilitatedevelopmentofpotentchemicalluresthatimprovemosquitotrappingformalariasurveillanceandcontrol.

PP-61GutmicrobiomepredictslumefantrinepharmacokineticsinhealthymiceMatthewM.Ippolito1,2,JoshuaE.Denny3*,ElizabethNenortas1,TheresaA.Shapiro1,NathanialW.Schmidt31JohnsHopkinsUniversitySchoolofMedicine,2JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,3UniversityofPennsylvania,Philadelphia,Pennsylvania,4IndianaUniversitySchoolofMedicine,Indianapolis,Indiana,*equalcontributorsTheantimalarialdruglumefantrineexhibitshighlyvariablepharmacokinetics—absorption,distribution,andclearance—betweenindividualswithupto16-folddifferencesindrugexposure.Differencesaredue,inpart,topoorabsorptionwhichismitigatedbyadministrationwithfattyfood.Wehypothesizedthattheintestinalmicrobiomemayalsoplayanimportantroleinlumefantrinedisposition.Fourcohortsofisogenicmicefromdifferentvendors(n=24)previouslyshowntoharbordistinctiveenterotypeswereadministeredahumanizeddoseoflumefantrine(150μg/g)bygavage.Gutmicrobiomewascharacterizedby16srRNAampliconsequencingoffecalpelletsandenterotypeswereclassifiedaccordingtoβ-diversityusingweightedUniFracdistance.Plasmawascollectedat0,0.5,1.5,10,and24hpostdosefordruganalytequantitationbyliquidchromatographytandemmassspectrometry.Drugexposuremeasuredasmaximalconcentration(Cmax)andareaunderthedrugconcentration-timecurve(AUC0-24)wasestimatedbythelinear-logtrapezoidalmethodinnon-compartmentalanalyses.Oralclearance(Cl/F)andapparentvolumeofdistribution(Vd/F)wereestimatedusingcovariateanalysisofenterotypewithstepwiseselectioninpopulation-basedcompartmentalmodels.Fourdiscreteenterotypeswereidentified.Enterotypes1and2hadsignificantlygreatertaxonomicabundancethan3and4,andβ-diversityplotsshowedclusteringofthesamepairs.Micewithenterotypes1and2hadhigherlumefantrineexposurethanenterotypes3and4(Cmax=542and768vs.392and380ng/ml,P≤0.04forpairwisecomparisons;AUC0-24=511,000and657,000vs.344,000and333,000ng�h/ml,P≤0.02).Resultsofcompartmentalanalysessuggestthatenterotype-relateddifferencesinCl/FandnotVd/Fcorrelatedwithdrugexposure.Thegutmicrobiomehaspreviouslybeenshowntocontributetointer-individualvariationinmetabolismoforallyadministereddrugs.Here,wedemonstratethatgutmicrobiomedifferencesmaypartiallyaccountforvariationinlumefantrineexposure.

PP-62

PlasmodiumfalciparumHRP2andnucleicacidclearancepatternsfollowingACTtreatmentinMyanmarChristineF.Markwalter,1OriannaPoteete,1ZayYarHan,1,2KayThweHan,2ChrisV.Plowe,1MyaingM.Nyunt11DukeGlobalHealthInstitute,DukeUniversity,2DepartmentofMedicalResearch,MyanmarMinistryofHealthandSportsHistidine-richprotein2(HRP2)isthemostcommonantigentargetinrapiddiagnostictests(RDTs)usedgloballyforthediagnosisofPlasmodiumfalciparum.ItisknownthatstandardRDTsremainpositiveforweeksafterparasiteclearance,asdeterminedbymicroscopy,followingmalariatreatment.ThisislikelyduetopersistenceofHRP2inthebloodandposesariskoffalse-positiveRDTresults.Asmalariatransmissiondeclines,detectingmalariainfectionsbecomesmorecomplexanddifficultduelowerparasitedensities.Newhigh-sensitivityHRP2-basedRDTsarecurrentlyunderdevelopmentandfieldtesting.Thesetestsaretypicallyevaluatedagainstsensitivemolecularmethods,suchasultrasensitivePCR(usPCR).ThefalsepositiveratesofnewRDTs,againstusPCR,maybeashighas20-30%.ThepersistenceoftheHRP2biomarkerrelativetousPCRpositivityaftertreatmenthasnotbeenwell-characterized.Inthisstudy,HRP2clearancepatternsrelativetousPCRpositivityweremeasuredinagroupof68individualsfromMyawaddy,Myanmar,whohadbeentreatedforuncomplicatedP.falciparumwithdihydroartemisinin-piperaquine,andfollowedfor42days.Driedbloodspots(DBS)from8timepointsover42dayswereanalyzedbyusPCRandanHRP2enzyme-linkedimmunosorbentassay(ELISA),providingcontextforevaluationofHRP2-baseddiagnosticsinmalariaeliminationsettings.

PP-63DiscrepancyinEfficacyofWR99210fromDifferentManufactures T.ParksRemcho1,GlennNardone2,AnnaLiu1,ThomasE.Wellems1,KristinD.Lane1

1LaboratoryofMalariaandVectorResearch,NIAID,NationalInstitutesofHealth,2ResearchTechnologiesBranch,NIAID,NationalInstitutesofHealth,Bethesda,MarylandPrior to the 1997 report of its use for selection of Plasmodium falciparum transformants, WR99210 had entered pre-clinical trials as a potent antimalarial compound with nanomolar efficacy. Because of its specificity in targeting dihydrofolate reductase (DHFR) of P. falciparum and not the human enzyme, WR99210 was soon widely



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employed for the selection of parasite lines transformed with plasmid constructs expressing human DHFR. Early studies sourced WR99210 from Walter Reed Army Institute of Research directly, and subsequent studies obtained the compound from Jacobus Pharmaceutical (JP) or, more recently, Sigma Aldrich (SA). Effective compound concentrations (EC50s) can vary depending on source. Indeed, following relatively ineffective selections of transformants with WR99210 from SA, we established that P. falciparum DD2 parasites survived constant drug pressure of 2nm WR99210 from SA, but not from JP. Further testing indicated greatly different EC50s of 0.62nM vs. 547nM for the JP- and SA-sourced WR99210, respectively. Potential causes of these discrepancies have been investigated. Assured and effective sources of WR99210 will be vital to the continued utility of plasmid constructs.

PP-64Cas9-mediatedgene-editinginthemalariamosquitoAnophelesstephensibyReMOTControlVanessaM.Macias1,SageMcKeand1,DuverneyChaverra-Rodriguez1,2,GrantL.Hughes3,AnikoFazekas4,SujitPujhari1,NijoleJasinskiene4,AnthonyA.James4,5,JasonL.Rasgon1,61DepartmentofEntomology,PennsylvaniaStateUniversity,UniversityPark,Pennsylvania,2DivisionofBiologicalSciences,UniversityofCalifornia,SanDiego,LaJolla,California,3LiverpoolSchoolofTropicalMedicine,Liverpool,UnitedKingdom,4DepartmentofMolecularBiologyandBiochemistry,UniversityofCalifornia,Irvine,Irvine,California,5DepartmentofMicrobiologyandMolecularGenetics,UniversityofCaliforniaIrvineSchoolofMedicine,Irvine,California,6CenterforInfectionDiseaseDynamics,HuckInstitutesfortheLifeSciences,PennsylvaniaStateUniversity,UniversityPark,PennsylvaniaInnovativetooldevelopmentisessentialforcontinuedadvancementinmalariacontrolanddependsonadeeperunderstandingofthemolecularmechanismsthatgoverntransmissionofmalariaparasitesbyAnophelesmosquitoes.Targeteddisruptionofgenesinmosquitovectorsisapowerfulmethodtouncovertheunderlyingbiologyofvector-pathogeninteractions,andgenomemanipulationtechnologiescanthemselvesformthebasesofmosquitoandpathogencontrolstrategies.However,theembryoinjectionmethodsusedtogeneticallymanipulatemosquitoes,andinparticularAnophelesspecies,aredifficultandinefficient,particularlyfornon-specialistlaboratories.WehaveadaptedastrategycalledReMOTControl(Receptor-mediatedOvaryTranslocationofCargo)todelivertheCas9

ribonucleoproteincomplextoadultmosquitoovariesandgeneratetargetedandheritablemutationsinthemalariavectorAnophelesstephensi.WefoundthatgeneeditingbyReMOTControlinAnophelesmosquitoeswascomparabletothetechniqueinAe.aegyptiandasefficientineditingasstandardembryoinjections.TheadaptationofthistechnologytoAnophelesmosquitoesopensupthepowerofreversegeneticstomalariavectorlabsthatdonothavetheequipmentortechnicalexpertisetoperformembryoinjectionsandestablishestheflexibilityofReMOTControlforgene-editinginnon-Aedesspecies.

PP-65Post-ArtemisininDelayedHemolysispresentingwithfever:acasereportEilraynaGelyana,JessicaS.Lin

JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MarylandPost-artemisinindelayedhemolysisisararelyobservedsideeffectofparenteralartesunate,thefirst-linetreatmentforsevereP.falciparummalaria,andhasbeenfoundtooccur1-3weeksafterinitiationoftreatment.Wedescribethecaseofa22-year-oldfemalewithrecenttraveltoAfricaandtheMiddleEast,whocompletedartesunatetherapyoneweekago,andpresentedwithfever,lightheadednessanddyspnea.Physicalexamrevealedmildscleralicterus,jaundice,andcracklesatbilaterallungbases.Labsshowedhemolyticanemiawithahemoglobinof6.5g/dL,LDHof1680U/L,andnegativeDAT.InitialmalariarapidscreenwaspositiveforP.falciparumantigen,butnobloodparasiteswereseenonGiemsasmears.Shecontinuedtobefebrileoverthenexttwodays(Tmax38.8°C),andLDHpeakedonday5at3315U/L.Noantibioticsorsteroidswerestarted.InfectiousworkupincludingRMSF,Typhus,CMV,Dengue,Toxoplasma,EBV,andInfluenzawereallnegative.Thepatientrequired6unitsofbloodduringheradmission,andwasdischargedonday11withstablehemoglobinof8.2g/dLandLDHof1294U/L.Thiscaseillustratesanuncommonpresentationofpost-artemisinindelayedhemolysiswithrecurrentfever.Althoughthecaseisconsistentwithpreviousreportsof≥10%decreaseinhemoglobinand≥10%increaseinLDHatleast7daysafterartesunatetreatmentinitiation,fewreportshavenotedarecurrentfeverwiththeabsenceofleukocytosisornewinfection.Associatedfevermaywarrantfurtherinvestigationintotheproposedhemolyticmechanismofdelayedsplenicclearanceofonce-infectederythrocytes.Furthermore,althoughrecentcasereportshavediscussedcorticotherapytotreatpost-artemisinindelayedhemolysis,thiscasedemonstratesresolutionofsymptomswithtransfusionsandwithouttheuseofsteroids,particularlyimportantconsideringtheriskofneutropeniafollowingartesunatetherapy.



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PP-66Theimpactofglyphosateonthemelanin-basedimmunesystemofAnophelesgambiaeDanielF.Q.Smith1,2,EmmaCamacho1,RavirajThakur3,ArturoCasadevall1,2

1CellularandMolecularMedicineProgram,2DepartmentofMolecularMicrobiologyandImmunology,TheJohnsHopkinsUniversitySchoolofPublicHealth,Baltimore,Maryland,3DepartmentofOHNSurgery,JohnsHopkinsSchoolofMedicine,Baltimore,MarylandDespiteglobalcampaigneffortstoeradicatemalaria,over200millionpeoplefallsickfromthemosquito-bornediseaseannually.Amajorstrategytocombatmalariaincludesmanipulatingthemosquito’simmunesysteminordertoreducesusceptibilitytodiseasesthatcouldthenbetransmittedtohumans.Duetothegrowthofagricultureinmalaria-endemicregions,andtheaccompaniedincreaseduseofherbicides,weexaminedtheeffectofglyphosate,theactiveingredientinthewidelyusedherbicideRoundup,onmosquitoes.Wefoundthatglyphosateistoxicwhenchronicallygiventothemalariavector,An.gambiaefemalesatconcentrationsconsistentwithagriculturalglyphosateapplicationoncrops.Interestingly,lowdosesofglyphosateappeartoprolongmosquitosurvivalcomparedtonon-druggedadults.WefoundthatglyphosateinhibitstheabilityofAn.gambiaetoproducemelanin.MelaninpigmentencapsulationofpathogensisacrucialcomponentoftheinsectimmuneresponseagainstPlasmodiuminfection.Withoutit,theinsectscouldhaveahigherdiseaseburden.Weseethatglyphosate-druggedmosquitoeshaveanincreasedparasiteburdenwhentheyareinfectedwithPlasmodiumfalciparum,theetiologicalagentofmostlethalcasesofmalaria.These“immune-compromised”mosquitoeswithadampenedmelanizationresponsemaythereforeposeanincreasedthreattohumanhealth.WefurthervalidatedtheseexperimentsinGalleriamellonellawaxmothlarvae,anotherinsectmodelofinfection.InG.mellonellalarvae,weseeadosedependentinhibitionofmelanizationwithglyphosate,andanincreasedsusceptibilitytoCryptococcusneoformansfungalinfectionwhenfirsttreatedwithadoseofglyphosate.OurdatasuggeststhatglyphosatemayincreaseAnophelesabilitytoserveasavectorbyposingadoublethreat:elongatinglifespanatsomeenvironmentallyrelevantconcentrations,andmakingtheselonger-livedindividualsmoresusceptibletopathogensthatcouldbetransmittedtopeople.

PP-67ComparativegenomeanalysisidentifiesconservedgeneamplificationfeaturesbetweenPlasmodiumspecies

AdamC.Huckaby1,ClaireGranum1,MaureenA.Carey2,KarolSzlachta3,BaselAl-Barghouthi3,Yuh-hwaWang3,andJenniferL.Guler1,4 1DepartmentofBiology,UniversityofVirginia,2DepartmentofMicrobiology,Immunology,andCancerBiology,UniversityofVirginiaHealthSystem,3DepartmentofBiochemistryandMolecularGenetics,UniversityofVirginiaHealthSystem,4DivisionofInfectiousDiseaseandInternationalHealth,UniversityofVirginiaHealthSystemGeneamplifications,atypeofDNAcopynumbervariation(CNV),facilitateoverexpressionofdrugtargetsandcontributetoadaptation.LongmonomericA/TtracksarefoundatthebreakpointsofmanyP.falciparumresistance-conferringCNVs.Wehaveappliedanovelsequencinganalysispipelinetoobtainsinglenucleotidebreakpointresolutionofuniqueamplificationsin35P.falciparumcloneswith19previouslyidentifieduniqueamplifications,7P.vivaxclinicalsampleswith5confirmedamplifications,andonehuman-adaptedP.knowlesiclonewithapreviouslyconfirmedamplification.VerylongA/Ttracks(32bponaverage)werefoundtobethebreakpointforvirtuallyallP.falciparumsamplesandstablehairpinswerefoundnearallknownfuturebreakpointlocations.WethereforehypothesizedthatCNVformation“triggersites”areformedbyA/Ttracksnearotherproximalsequencefeatures,suchasDNAhairpins.Fivesharedbreakpointswereinproximitytoextremelystablehairpins(top0.2%genome-wide)whichaddsfurthersupport.This“triggersite”modelisthusfarcorroboratedbytheP.vivaxandknowlesisamples.BreakpointsforthepreviouslyidentifiedamplificationsinP.vivaxandknowlesiwereallA/Ttracksandwere18bponaverageforeach.Preliminarycomparisonsbetweenspeciesindicatethattriggersitesareenrichedinallthreespecies.Asexpected,basedonoverallgenomeA/TcontenttherearefewerlongATtracks(>9bp)invivaxandknowlesigenomesbutverylongtracks(>20bp)are95%ofthosefoundinthefalciparumgenome.CurrentinvestigationsincludeexpandingourdatasetwithmoreP.vivaxandknowlesiamplifications,refiningcomparisonsbetweenPlasmodiumspeciesgenome-wide,andinvestigatingtriggersitesinsyntenicblocksofsequence.

PP-68ImpactoftargetedIRSonvectorcountsandmalariacasesinahightransmissionsettinginNchelengeDistrict,Zambia JessicaL.Schue1,MbangaMuleba2MarisaA.Hast3,MikeChaponda2,Jean-BertinKabuya2,JamesLupiya2,TamakiKobayash1,ModestMulenga2,DouglasE.Norris1,WilliamJ.Moss1andJenniferC.Stevenson1,4fortheSouthernandCentralAfricaInternationalCentersofExcellenceforMalariaResearch



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1JohnsHopkinsBloombergSchoolofPublicHealth,Baltimore,Maryland,2TropicalDiseasesResearchCentre,Ndola,Zambia,3CentersforDiseaseControlandPrevention,Atlanta,Georgia,4MachaResearchTrust,Macha,ZambiaLuapulaProvinceinnorthernZambiaisanareaofhighmalariatransmissionwithaparasiteprevalenceof32.5%inchildrenunder5years.Inthisprovince,targetedindoorresidualspraying(IRS)andLLINsareusedasvectorcontroltopreventmalaria.TheobjectiveistodetermineiftargetedIRScanlowertheriskofmosquitosinahousehold,whichinturncanreducetheriskofmalariainfectioninthebroaderpopulation.NchelengeDistrictinLuapulaProvinceisastudysitefortheSouthernandCentralAfricaInternationalCentersofExcellenceforMalariaResearch.TargetedIRScampaignsareconductedannually.Dataarefromcross-sectionalhouseholdsurveysandmosquitocountsaswellaspassivesurveillanceathealthcentersfrom2012through2019.ClimatedatawereextractedfromtheAfricaFloodandDroughtMonitor.TheassociationofmosquitocountsperhouseholdandIRSwasmodeledusingquasi-Poissonregression,stratifiedbymosquitospecies.Theassociationofmosquitocountsperhouseholdandmalariainfectionsweremodeledusingalog-linearregressionmodel.Allmodelsincludedaflexibletimefunctiontoaccountforseasonality.Householdsself-reportingIRShada52%reductioninrelativeriskofA.funestusintheirhousehold(RR:0.48,95%CI:0.34-0.67).However,therewasnoassociationwithself-reportedIRSandtheriskofhavingA.gambiaecapturedintheirhousehold.TherewasalsonoassociationbetweenthenumberofmosquitosofeitherspeciesinthehouseholdandPlasmodiumfalciparuminfection.HouseholdstargetedforIRShadalowerriskofmosquitosintheirhomeforonlyoneofthetwodominantmalariatransmittingspecies.Givendifferencesinthesetwospecies,itispossiblethatonceayearsprayingisinsufficienttolastthroughbothpopulationexpansions.Vectorcountswithinahouseholdwerenotassociatedwiththenumberofparasiteinfectionsinahousehold.

PP-69Useofmolecularinversionprobestoelucidatewithin-countrypopulationstructureanddrugresistancepatternsofPlasmodiumfalciparuminfectionsinmainlandTanzaniaKaraA.Moser1,RashidMadebe2,OzkanAydemir3,MercyG.Chiduo2,CelineI.Mandara2,SusanF.Rumisha4,FrankChaky5,MadelineDenton1,PatrickMarsh3,RobertVerity6,OliverJ.Watson6,BillyNgasala7,SigsbertMkude5,FabrizioMolteni5,RithaNjau8,MarianWarsame9,10,RenataMandike5,AbdunoorM.Kabanywanyi11,MuhidinK.Mahende11,ErasmusKamugisha12,MaimunaAhmed12,ReginaldA.Kavishe13,GeorgeGreer14,ChongeA.Kitojo14,ErikJ.Reaves14,LindaMlunde15,DanstunBishanga15,AllyMohamed5,JonathanJ.Juliano1,11,12,DeusS.Ishengoma2,JeffreyA.Bailey3

1InstituteforGlobalHealthandInfectiousDiseases,UniversityofNorthCarolinaChapelHill,ChapelHill,NorthCarolina,2NationalInstituteforMedicalResearch,Tanga,Tanzania,3DepartmentofPathologyandLaboratoryMedicine,BrownUniversity,RhodeIsland,4NationalInstituteforMedicalResearch,Headquarters,DaresSalaam,Tanzania,5NationalMalariaControlProgram,Dodoma,Tanzania.6ImperialCollege,London,UnitedKingdom,7MuhimbiliUniversityofHealthandAlliedSciences,DaresSalaam,Tanzania,8WorldHealthOrganizationCountryOffice,DaresSalaam,Tanzania,9GothenburgUniversity,Gothenburg,Sweden,10GlobalMalariaProgramme,WorldHealthOrganization,Geneva,Switzerland,11IfakaraHealthInstitute,DaresSalaam,Tanzania,12CatholicUniversityofHealthandAlliedSciences/BugandoMedicalCentre,13KilimanjaroChristianMedicalCentre/KilimanjaroChristianMedicalUniversityCollege,Moshi,Tanzania,14U.S.President'sMalariaInitiative,U.S.AgencyforInternationalDevelopment,U.S.Embassy,DaresSalaam,Tanzania,15Jhpiego/BoreshaAfyaProject,DaresSalaamTanzania,16CurriculuminGeneticsandMolecularBiology,UniversityofNorthCarolinaChapelHill,17DepartmentofEpidemiology,GillingsSchoolofGlobalPublicHealth,ChapelHill,NorthCarolina,18FacultyofPharmaceuticalSciences,MonashUniversity,Melbourne,AustraliaHigh-throughputparasitegenomicsisincreasinglyusefulinassessingprevalenceofclinicallyimportantmutations,parasitetransmissionpatterns,andtheimpactofinterventions.Asmalariaeliminationeffortsshifttohightransmissionregions,afullerunderstandingoflocalandregionalparasitediversityandpopulationstructureisneededsothatgenomicsurveyscanhelpinforminterventionefforts.However,manygenotypingmethods,suchaswholegenomesequencing,canbecost-prohibitiveforlargesurveysneededforsuchdiscrimination.Here,weusemolecularinversionprobe(MIP)panelstoinvestigateP.falciparumpopulationstructureusingnearly1,000Tanzaniaclinicalinfectionscollectedfrom13districtsacrossthecountry.WefoundthatparasitesinTanzaniaarecomprisedoftwomainpopulations,separatingparasitesfromthenorthernandsouthernregionsofthecountry.Therewasalsoarelationshipbetweengeneticandgeographicdistance,withisolatesfromdistrictsclosetooneanothermorelikelytobegeneticallyrelated(byidentity-by-descent)comparedtoparasitessampledfrommoredistantdistricts.Drugresistancemutationsinthepfdhpsandpfcrtgenesalsoshoweddifferentialfrequenciesbygeographiclocation;severalotherSNPsnotknowntodirectlycontributetoantimalarialresistancealsoappearedtosegregatebypopulationstructure.Inaddition,demographicdatafromthe2017MalariaIndicatorSurveycorrespondedwithgeneticfindingsfromtheMIPdata,includingapositivecorrelationwithaveragecomplexityofinfectionestimateswithmalariaprevalenceestimatesbydistrict.TheseanalyseswillprovideinformationonthegeneticdiversityandpopulationstructureofTanzanian



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parasitestotheNationalMalariaControlProgram,onwhichassessmentsofpublichealthinterventionscanbemade.

PP-70OntheHuntforHemozoin:Hello,Archaeomagnetism.MeetMalaria.MalloryCoxYaleUniversitySchoolofMedicine,NewHaven,Connecticut In2018,undertheauspicesoftheYaleUniversityArchaeologyLaboratoryMalariaProject,Ibeganexploringtheuseofmagneticmethodologiestoidentifyhemozoininancientskeletalremains.HereIpresentpreliminarydatatoasserttheusefulnessofArchaeomagnetismtoancientmalariaresearch.Iscrapedallsamplesfromthemarrowcavitiesoflongbones,wherehemozoinisknowntosequester.First,100mgsampleswerefixedonaborosilicateglassplate,mountedtoanamberprobe,andinsertedintotheAlternatingGradientForceMagnetometer(AGM).InsamplesfromPanama(2500BC-1600AD)andPuertoRico(0AD-1400AD),Iobservedsuperandparamagneticbehavior.Interestingly,bothmagneticstatedomainsarereportedintheliteratureascharacteristicofhemozoin,withsomegroupsobservingparamagneticbehaviorwhileothersreportsuperparamagnetism.Then,IutilizedanMPMS2CryogenicSusceptometer(InstituteforRockMagnetism-IRM)toanalyzesamplesatlowtemperatures(2K)andhightemperatures(400K)toinducecrystallographictransitionsthatarediagnosticofthemagneticmineralcomposition.Inadditiontomaghemite,weidentifiedmagnetotacticbacteriatoexplainthepresenceofiron-bearingcrystals<50nminsomesamples.Notably,as50%ofthemagneticsignatureinsomesampleswasunidentifiablebyleadinggeologistsandarchaeomagneticscientistsattheIRM.Toourknowledge,hemozoinfromskeletalremainshasnotbeenanalyzedusingmagnetometry.Withthehelpofcolleagues,YUALMPisbuildingmagneticprofilesformalarialhemozoinsandotheriron-bearingmineralsthatoccurnaturallyinsoils,toenrichthemethodologiesavailableforstudyingmalariainthearchaeologicalrecord.

PP-71ComparingluciferaseexpressionintransfectedstagesofPlasmodiumfalciparumandP.knowlesiAngelaC.Ellis1,JulianaM.Sá2,JianbingMu1,RobertoR.MoraesBarros2,ThomasE.Wellems11LaboratoryofMalariaandVectorResearch,NationalInstitutesofAllergyandInfectiousDiseases,Bethesda,Maryland,2DisciplinadeBiologiaCelular,DepartamentodeMicrobiologia,ImunologiaeParasitologia,EscolaPaulistadeMedicina,UniversidadeFederaldeSãoPaulo,SãoPaulo,Brasil

Transfectionofmalariaparasitesprovidesanimportantmeanstoinvestigatetherolesofspecificgenesinparasitebiologyandpathogenesis.PlasmodiumfalciparumandP.knowlesiaretwospeciesresponsibleforextensivemalariamorbidityandmortality;unlikeotherwidespreadhumanparasitesincludingP.vivax,P.falciparumandP.knowlesicanbereadilycultivatedandtransfectedunderlaboratoryinvitroconditions.Herewedescribetheefficienciesofdirect-electroporationonbothP.falciparumandP.knowlesiusingafireflyluciferasereporterconstructatdifferentintra-erythrocyticstages.PreliminarydatashowvariationsinluciferaseexpressionamongdifferentP.falciparumstages:signalsinthesestagesarehigherfromthereporterconstructintroducedbydirect-electroporationofringsthanbydirect-electroporationofschizonts.Incontrast,previousliteraturehasreportedthatdirect-electroporationofmatureP.knowlesischizontstages(“segmenters”)withluciferaseplasmidsyieldedgreaterexpressionthandiddirect-electroporationoflatetrophozoitesoryoungdevelopingschizontstages.ThesedifferencesmayreflectarelativerobustnessofP.knowlesirelativetoP.falciparumsegmentersandmerozoites,sothatP.knowlesisegmenterswithdevelopedmerozoitescanwithstanddirect-electroporationbetterthancorrespondingstagesofP.falciparum.Experimentstodirectlycompareluciferaseexpressionfromelectroporatedlate-schizont/segmenterstagesofP.falciparumandP.knowlesiareunderway.

INDEXBYAUTHORFirstand/orpresentingauthor Abstractno.Addisu,Ayenew PP-35Akter,Romana PP-08Amvongo-Adjia,Nathalie PP-30Andronescu,Liana* OP-07Arnheim,Alyssa PP-33Balta,Victoria§ PP-12Bérubé,Sophie PP-40BirajShrestha PP-55Bishnoi,Ritika* OP-08Bogale,AgajieLikie PP-05Brashear,Awtum* OP-05Brown,Audrey PP-50Carey,Maureen§ PP-49Chakravarty,Sumana PP-53Cheng,Breagh PP-19Chiu,Michelle PP-18Connelly,Sean PP-14Cox,Mallory PP-70DelCarmenMiluskaVargasRodriguez,Rosa PP-13Denny,Joshua PP-61Diaz,Sophia PP-59



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Dwivedi,Ankit PP-57Ekoka,Elodie PP-07Ellis,Angela PP-71Enekegbe,MbiAlice PP-29Engdahl,CeciliaSpringer PP-58Ernest,Medard PP-34Faiman,Roy§ PP-52Foquet,Lander PP-11Fries,Brendan PP-32Gebhardt,Mary* OP-04Gelyana,Eilrayna§ PP-65Giraldo,Diego PP-60Gonzalez-Wartz,Kristina PP-47Guido,Marisa PP-45Huang,Wei PP-17Huckaby,Adam PP-67Ippolito,Matthew PP-31Isebe,Tony PP-28Kabuya,Jean-Bertin PP-09Kanatani,Sachie PP-38KC,Natasha PP-53Kelly,Tom PP-48Kobayashi,Tamaki PP-21Koepfli,Cristian* OP-06Kumar,Sunil PP-36Kussin-Shoptaw,Benjamin PP-22Lane,Kristin PP-51Levine,Zoe PP-14Lin,Jessica§ PP-65Lukwa,Nzira PP-26Ma,Wenchuan PP-12Macias,Vanessa§ PP-64Makoge(Maat),Valerie PP-39Markwalter,Christine* OP-09Moser,Kara PP-69Mosunda,Moses PP-01Mylin,Lawrence PP-23Nair,Sethu PP-06Obayemi,OlubunmiSharon PP-02Okuneye,Kamaldeen* OP-01Orishaba,Philip PP-37Pandey,Rajan PP-10Poteete,Orianna PP-62Remcho,T.Parks PP-63Sahu,Tejram PP-20Schue,Jessica PP-68Sekar,Padmapriya* OP-02Shah,Zalak PP-54Shankar,Shruti PP-44Siddiqui,Ghizal* OP-03Smith,Daniel§ PP-66Stasic,Andrew PP-04

Stiffler,Deborah PP-25Stucke,Emily PP-41Taylor,Russ PP-43Tchongwe,Lizzie PP-03Vanheer,Leen PP-24Vera,Michael PP-46West,Rachel PP-16Zhou,Albert PP-15

*Oralpresentation§ Turbotalk

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5th FMR Abstract Book - malaria.jhsph.edu FMR Abstr… · THE JOHNS HOPKINS MALARIA RESEARCH INSTITUTE 5TH ANNUAL FUTURE OF MALARIA RESEARCH SYMPOSIUM, NOVEMBER 18TH, 2019 3 López-Uribe1,