Age-specific malaria seroprevalence rates: a cross-sectional analysis of malaria transmission in the Ouest and Sud-Est d...

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Title:
Age-specific malaria seroprevalence rates: a cross-sectional analysis of malaria transmission in the Ouest and Sud-Est departments of Haiti
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Fricken, Michael E von
Weppelmann, Thomas A.
Lam, Brandon
Eaton, Will T.
Schick, Laura
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Bio Med Central (Malaria Journal)
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Abstract:
Background: Malaria transmission continues to occur in Haiti, with 25,423 confirmed cases of Plasmodium falciparum and 161,236 suspected infections reported in 2012. At low prevalence levels, passive surveillance measures, which rely primarily on reports from health systems, becomes less appropriate for capturing annual malaria incidence. To improve understanding of malaria transmission in Haiti, participants from the Ouest and Sud-Est departments were screened using a highly sensitive enzyme-linked immunosorbent assay (ELISA). Methods: Between February and May 2013, samples were collected from four different sites including a rural community, two schools, and a clinic located in the Ouest and Sud-Est departments of Haiti. A total of 815 serum samples were screened for malaria antibodies using an indirect ELISA coated with vaccine candidates apical membrane antigen (AMA-1) and merozoite surface protein-1 (MSP-119). The classification of previous exposure was established by using a threshold value that fell three standard deviations above the mean absorbance for suspected seronegative population members (OD of 0.32 and 0.26 for AMA-1 and MSP-1, respectively). The observed seroprevalence values were used to fit a modified reverse catalytic model to yield estimates of seroconversion rates. Results: Of the samples screened, 172 of 815 (21.1%) were AMA-1 positive, 179 of 759 (23.6%) were MSP-119 positive, and 247 of 815 (30.3%) were positive for either AMA-1 or MSP-1; indicating rates of previous infections between 21.1% and 30.3%. Not surprisingly, age was highly associated with the likelihood of previous infection (p-value <0.001). After stratification by age, the estimated seroconversion rate indicated that the annual malaria transmission in the Ouest and Sud-Est department is approximately 2.5% (95% CI SCR: 2.2%, 2.8%). Conclusions: These findings suggest that despite the absence of sustained malaria control efforts in Haiti, transmission has remained relatively low over multiple decades. Elimination in Haiti appears to be feasible; however, surveillance must continue to be strengthened in order to respond to areas with high transmission and measure the impact of future interventions. Keywords: Malaria, Haiti, Plasmodium falciparum, AMA-1,MSP-119, Serology, SCR
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von Fricken et al. Malaria Journal 2014, 13:361 http://www.malariajournal.com/content/13/1/361; Pages 1-8
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doi:10.1186/1475-2875-13-361 Cite this article as: von Fricken et al.: Age-specific malaria seroprevalence rates: a cross-sectional analysis of malaria transmission in the Ouest and Sud-Est departments of Haiti. Malaria Journal 2014 13:361.

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© 2014 von Fricken et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHOpenAccessAge-specificmalariaseroprevalencerates: across-sectionalanalysisofmalariatransmission intheOuestandSud-EstdepartmentsofHaitiMichaelEvonFricken1,2*,ThomasAWeppelmann1,2,BrandonLam1,3,WillTEaton1,LauraSchick4,RoselineMasse5, MadsenVBeauDeRochars1,6,AlexandreExiste7,JosephLarkinIII3andBernardAOkech1,2AbstractBackground: MalariatransmissioncontinuestooccurinHaiti,with25,423confirmedcasesof Plasmodiumfalciparum and161,236suspectedinfectionsreportedin2012.Atlowprevalencelevels,passivesurveillancemeasures,whichrely primarilyonreportsfromhealthsystems,becomeslessappropriateforcapturingannualmalariaincidence.Toimprove understandingofmalariatransmissioninHaiti,participantsfromtheOuestandSud-Estdepartmentswerescreened usingahighlysensitiveenzyme-linkedimmunosorbentassay(ELISA). Methods: BetweenFebruaryandMay2013,sampleswerecollectedfromfourdifferentsitesincludingarural community,twoschools,andacliniclocatedintheOuestandSud-EstdepartmentsofHaiti.Atotalof815serum sampleswerescreenedformalariaantibodiesusinganindirectELISAcoatedwithvaccinecandidatesapicalmembrane antigen(AMA-1)andmerozoitesurfaceprotein-1(MSP-119).Theclassificationofpreviousexposurewasestablishedby usingathresholdvaluethatfellthreestandarddeviationsabovethemeanabsorbanceforsuspectedseronegative populationmembers(ODof0.32and0.26forAMA-1andMSP-1,respectively).Theobservedseroprevalencevalues wereusedtofitamodifiedreversecatalyticmodeltoyieldestimatesofseroconversionrates. Results: Ofthesamplesscreened,172of815(21.1%)wereAMA-1positive,179of759(23.6%)wereMSP-119positive, and247of815(30.3%)werepositiveforeitherAMA-1orMSP-1;indicatingratesofpreviousinfectionsbetween21.1% and30.3%.Notsurprisingly,agewashighlyassociatedwiththelikelihoodofpreviousinfection(p-value<0.001).After stratificationbyage,theestimatedseroconversionrateindicatedthattheannualmalariatransmissionintheOuestand Sud-Estdepartmentisapproximately2.5%(95%CISCR:2.2%,2.8%). Conclusions: ThesefindingssuggestthatdespitetheabsenceofsustainedmalariacontroleffortsinHaiti,transmission hasremainedrelativelylowovermultipledecades.EliminationinHaitiappearstobefeasible;however,surveillance mustcontinuetobestrengthenedinordertorespondtoareaswithhightransmissionandmeasuretheimpactof futureinterventions. Keywords: Malaria,Haiti, Plasmodiumfalciparum ,AMA-1,MSP-119,Serology,SCR *Correspondence: Michaelvonfricken@epi.ufl.edu1EmergingPathogensInstitute,UniversityofFlorida,2055MowryRoad, Gainesville,FL32611,USA2DepartmentofEnvironmentalandGlobalHealth,UniversityofFlorida, Gainesville,FL32610,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014vonFrickenetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated.vonFricken etal.MalariaJournal 2014, 13 :361 http://www.malariajournal.com/content/13/1/361

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BackgroundOverthepastdecadetherehasbeenarenewedinterest ineliminatingmalariafromtheislandofHispaniola, withabi-nationalstrategyrecentlyadoptedbetweenthe DominicanRepublicandHaititoeliminatemalariaby 2020[1].RecentreportsofemergingchloroquineresistanceinHaiti[2],coupledwithincreasedinternational aid,presentatimesensitivewindowinwhichmalaria controleffortsshouldbescaledup,beforetreatment strategiesmustswitchtomoreexpensivecombination therapies[3].Furthermore,onlyonespeciesofmalaria parasiteispresentinHaiti, Plasmodiumfalciparum, and theprincipalmosquitoresponsibleformalariatransmission, Anophelesalbimanus, isprimarilyzoophilicmakingitapoorvectorofdisease[1].Finallythereislittle chanceofmalariabeingreintroducedintoHaitionceit hasbeensuccessfullyeliminated[4]. AlthoughtransmissioncontinuestooccurinHaiti,with 25,423confirmedcasesand161,236suspectedinfections reportedin2012[5],findingsfroma2012countrywide cross-sectionalsurveyadministeredbyPopulationServices Internationalsuggestparasiteprevalenceratestobe<1% [1].However,focaltransmissionhasbeendocumentedby otherstudies,withparasiteratesintheArtiboniteValleyof 3.1%[6],andparasiteratesrangingfrom0-34%inthe Sud-EstDepartment[7],indicatingpersistentandheterogeneousmalariatransmission. AsHaitigearsupformalariaelimination,obtaining sensitivemeasurementsofmalariatransmissionwillbe crucialtomonitoringtheimpactofcontroleffortsadopted toachievethisgoal[8].Inlowtransmissionsettings,there isatendencytorelyonpassivemalariasurveillanceover activesurveillanceduetobudgetaryconstraints;however, passivesurveillanceisnotassensitiveataccuratelycapturingmalariaincidence,especiallyinareaswithpoorhealth infrastructurelikeHaiti.Toovercomethisdifficultly,serologicalmarkersofmalariahavebeenusedtodetermine malariaexposureratesinlowtransmissionsettings,allowingresearcherstoestimateseroconversionrates(SCR)by modellingtheagespecificseroprevalence[9-15].Recently, astudybyArnold etal. examinedcross-sectionaland longitudinaldatafrom1991-1998usingmerozoitesurface protein-119(MSP-1),andfoundtheSCRtoberoughly 2.3%inLeogane,whichislocatedintheOuestdepartment ofHaiti[12].Estimatingmalariatransmissionbymeasuringlong-lastingantibodyresponsesgeneratedfrom previousmalariainfectionsalsoallowstheinvestigation oflong-termtrendswithouttheestimatedseroconversionratesbeingskewedbyseasonaltransmission, whichisappropriateinthissettingsincetheendemicepidemicstateofmalariacoincidescloselywithrainfall patternsinHaiti[10,16]. Thepurposeofthisstudywastoprovidevaluableinformationoncurrenttrendsinmalariatransmissionin theOuestandSud-EstdepartmentsofHaitibyanalysing datacollectedin2013withELISAtechniquesemploying morethanone P.falciparum specificantigen.Thisdata addstothecurrentbodyofliteratureonmalariain Haiti,whileprovidingpolicy-makersbaselineinformationonmalariatransmissionratesintheseregionsthat supporttherationaleformalariaeliminationinHaiti.MethodsStudylocationandenrollmentThesamplesanalysedinthisstudywerecollectedfrom foursiteslocatedintheOuestandSud-Estdepartmentof HaitiinthecommunesofGressierandJacmel,between FebruaryandMay2013.AmapofHaitiincludingtheenrollmentlocations,studycommunes,anddepartmentsis presentedinFigure1.Enrollmentwasbasedonconveniencesamplingfrombothclinicalandnon-clinicalsettings, aspartofalargerstudyonhostprotectivegeneticfactors [17].Studysitesincludedaruralcommunity,twoschools, andacliniclocatedintheOuestandSud-Estdepartments ofHaiti,withparticipationopentoallindividualsattendingeachsite.Healthychildrenwereenrolledfromthe ChristianvilleSchoolinGressierandfromtheHossana BaptistSchoolinJacmel.Patientsandhealthyfamily membersattendingPortailLeoganeClinicinJacmelwere enrolledonavoluntarybasis.IndividualsfromChabin wereenrolledfromcommunitymembersseekinggeneral healthservicesatamobileclinic.Participantsateach locationweregivenopportunitiestoasktheenrolling physiciansquestionsduringinformationsessionspriorto consent.Afterobtainingconsentfromparticipantsortheir guardian,localclinicianscollectedapproximately3mlof bloodbyvenipunctureintoserumseparationtubesfrom participants,whichwerecentrifugedimmediatelyat6,000 RPMfortwominutesaftercollectionwascomplete.All serumsampleswerestoredat-80CintheUniversityof FloridafieldlaboratoryinGressier,untilshipmenttothe EmergingPathogensInstitute,inGainesville,FL,for analysisandstorage.Serumsampleswerecollectedfroma totalof823participantsbetweentheagesoftwoand80. Malariainfectedparticipants(5/823),determinedviarapid diagnostictest(RDT)wereexcludedfromanalysisto reducetheeffectofpositiveindividualsseekingtreatment forcurrentinfections.Participantsmissingagedata(3/ 823)werealsoexcluded,resultinginafinalsamplesizeof 815(483femalesand332males).Ofthe815samples analysed,allwerescreenedforpreviousexposureusing theantigenAMA-1,butonly759/815sampleswere screenedusingtheMSP-1,duetolimitedamountsof serumfromsomeparticipants.EthicalapprovalEthicalapprovaltoconductthisresearchwasobtained fromtheHaitian-basedEthicalReviewCommittee,thevonFricken etal.MalariaJournal 2014, 13 :361 Page2of8 http://www.malariajournal.com/content/13/1/361

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UniversityofFloridaInstitutionalReviewBoard,andthe OfficeofResearchProtections,UnitedStatesArmy MedicalResearchandMaterialsCommand.ELISAprotocolandproceduresSerumsampleswerescreenedforantibodiesagainstAMA1andMSP-119usinganindirectenzyme-linkedimmunosorbentassay(ELISA).Serumsamplesofsubjectswere dilutedin5%non-fatskimmilkinphosphatebufferedsaline(NFSM-PBS).ELISAplateswerecoatedinduplicate withtherespectiveantigendilutedin5%NFSM-PBStoa finalconcentrationof1ml/mlforAMA-1and0.5ml/ml forMSP-1,beforeovernightincubationat4C.Thenext day,antigenwasremovedandplateswerewashedfive timeswith0.05%tween-20inPBS-Kandthenblockedfor onehourwith5%NFSM-PBStoreducenon-selectivebinding.Followingadditionalwash,dilutedserumsamplesas wellaspositiveandnegativecontrolserawereplatedinduplicateandincubatedfortwohoursat4C.Horseradish peroxidaseconjugatedrabbitanti-humanIgGsecondary antibodywasdiluted1:1,000in5%NFSM-PBSandadded totheplate.Afteronehour,plateswerewashedseventimes andtreatedwith3,3 ’ ,5,5 ’ -tetramethylbenz idine(TMB)substratesolutioninthedarkfor20minutestoallowsufficient colourdevelopmentandstoppedwith2Msulfuricacid.Determinationofseropositiveandseronegative populationmembersAftermeasurementofthesamplesinduplicate,theaverageabsorbanceat450nmwasusedtodeterminethe thresholdsfortheclassificationofasampleasseropositive orseronegative.Giventhelowincidenceandrelatively largesamplesizeforeachantigen(n=815or759),the seronegativepopulationresponsesforAMA-1andMSP-1 shouldfollowanormaldistributionfunctionwithsample mean ^ andsamplestandarddeviation ^ .Thisbehaviour ispresentedinFigure2,wherethesuspectedseronegativepopulationsapproximatednormaldistributionswith ^ 0 : 191 ; 0 : 140 and ^ 0 : 0427 ; 0 : 04 forAMA-1and MSP-1,respectively.ThresholdsforpositiveAMA-1 andMSP-1responseswereclassifiedbytheadditionof three,four,andfivesamplestandarddeviationstothe samplemean,suchthat 99.7%ofseronegativepopulationmemberswouldnotbeclassifiedasseropositive. Theresultingthresholdsusingthree,four,andfive, sampledeviationsforAMA-1were0.319,0.362,0.405 and0.260,0.30,0.340forMSP-1.Responsesabovethree standarddeviationsofthemeansuspectedaveragewere consideredseropositive,giventheminimalimpactusing moreconservativethresholdshadontheestimatedprevalence(seeDiscussion). Figure1 MapofHaitiwiththelocationofthestudyenrollmentsites. ThefourstudyenrollmentsiteslocatedinsidetheOuestandSud-Est DepartmentsofHaitiinthecommunesofGressierandJacmel.ParticipantswereenrolledfromChristianvilleSchoolinGressierandfromHosana BaptistSchoolandPortailLeoganeClinic,andtheruralcommunityofChabininJacmel.AlongwithaninsetoftheentirecountryofHaiti,the enrollmentsites(redcircles)appearrelativetothestudycommune(yellow),thenationalcapital(star)andnationalhighwaysystems(pinklines) vonFricken etal.MalariaJournal 2014, 13 :361 Page3of8 http://www.malariajournal.com/content/13/1/361

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Estimationofseroconversionra tesfromcross-sectionaldataTheobservedcross-sectionalseroprevalencewasusedto estimatetheseroconversionrateusingamethodsimilarto thosepreviouslydescribed[ 11,18].Briefly,anagespecific seroconversionmodelwasfittotheprevalenceofAMA-1, MSP-1,andAMA-1orMSP-1seropositivepopulation membersusingallparticipants(aged2to80)andseparately forparticipantslessthan20yearsofagetoestimatetherate ofseroconversion( ).Participantswereseparatedinto “ age classes ” todepictaggregatedseroprevalence,withwiderage rangesusedinoldergroups,duetothelowernumberof participantsovertheageof20.SincetheAMA-1and MSP-1responsesarelong-lastingandtheestimationof reversionrateshasbeensuggestedtobeunreliablewith cross-sectionaldata,areversionrate( )ofzerowasused forthefinalanalysis[13],howevernonzeroreversionrates werealsoexplored.Withoutseroreversion,theprobability ofinfection(prevalence)atage x wasmodeledusingthe equation P ( x )=[ 1 exp ( Š x )] Whenseroreversionwas includedinthemodel,theprobabilityatage x wasmodeled usingtheequation P ( x )= /( + )[ 1 exp ( Š ( + ) x )] The functionswereoptimized(usingR)togiveestimatedseroconversion/reversionrates ^ and ^ ,aswellastheirstandard errorsforthecalculationof95%confidenceintervals for ^ and ^ .Oddsratiosfortheprobabilityofhavinga previousexposure,asdeterminedbyapositiveELISA response,werecalculatedusi ngasimplelogisticregressionbyagecategory.ResultsEstimationofseroprevalenceusingAMA-1andMSP-1Characteristicsoftheresultingstudypopulationcanbe foundinTable1. Usingtheaverageabsorbancefromeachserumsample andathresholdofthreestandarddeviationsfromthe samplemeantoindicatethepresenceofAMA-1or MSP-1antibodies,172of815(21.1%)hadthepresence ofAMA-1antibodies,179of759(23.6%)hadthepresenceofMSP-1antibodies,and247of815(30.3%)had thepresenceofeitherAMA-1orMSP-1antibodies. Usingthresholdsfromthreetofivestandarddeviations gavesimilarestimatesofseroprevalencethatranged from16.3%to21.1%forAMA-1,17.4%to23.6%for MSP-1and24.0%to30.3%foreitherAMA-1orMSP-1. Theseroprevalencebyagegroup(rangingfrom2to 80years)ispresentedinTable2andFigure3. Asexpected,theprevalenceofseropositiveparticipantsincreaseswithparticipantage.Theprevalenceof Figure2 DistributionofELISAresponsesinabsorbanceunits( =450nm). ThedistributionofELISAresponsesfromthestudyparticipantsin absorbanceunits(at450nm)appearforAMA-1andMSP-1ontheleftandrightp anelsofFigure2,respectively.Theupperpanelsshowthehistogramsof thesuspectedseronegativeELISAresultsoverlaidwithanormaldistributi onfunction.Thesamplemean(thickblackline)andsamplestandarddevia tionof thesefunctionswereusedtodetermineminimumab sorbancevalues(thresholds)fortheclassificationofasampleasseropositiveusingthesampleme an forthesuspectednegativepopulationmembersplusthreetofivesa mplestandarddeviations(gold,orange,andreddashedlines). vonFricken etal.MalariaJournal 2014, 13 :361 Page4of8 http://www.malariajournal.com/content/13/1/361

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previousinfectionsdeterminebyAMA-1responsewas notsignificantlydifferent(pvalue>0.05)betweenage classesunder20yearsofage.Howevercomparedto participantsyoungerthan20yearsofage,thelikelihoodof previousinfectioninthoseover20yearsofagewas6.0 timeshigher(95%CIOR4.03,8.99).ForMSP-1responses,differencesinprevalencecomparedtotheyoungestageclassweresignificant(pvalue=0.023)beginning atthethirdageclass(9to13yearsofage),withthoseover 20yearsofagehaving3.7timesthelikelihoodofprevious infection(O.R. … 3.7;95%CI=2.48,5.53).Ofparticipants whoweretestedwithbothAMA-1andMSP-1(n=759), 104wereclassifiedasseropositiveusingbothantigens (13.7%),75wereMSP-1positiveandAMA-1negative (9.9%),57wereAMA-1positiveandMSP-1negative (7.5%),and523werenegativeforbothAMA-1andMSP1(68.9%).ThedistributionofpositiveELISAresponses forAMA-1orMSP-1antigensappearsgraphicallyin Figure4,withregionsI,II,III,andIVrepresentingthe averageabsorbancevaluefortheresponsetotheAMA-1 andMSP-1antigensforsampleswithAMA-1(+)/MSP-1 (+)results,AMA-1(-)/MSP-1(+)results,AMA-1(+)/MSP1(-)results,andAMA-1(-)/MSP-1(-)results,respectively.EstimationofseroconversionratesforAMA-1andMSP-1Theprevalenceandtheestimatedprobabilityofprevious infectionasdeterminedbyAMA-1andMSP-1antigen responsearepresentedinFigure5fortheentirestudy population(2to80years)andthoselessthan20years ofage.TheestimatedSCRforAMA-1(toppanel)from theentirestudypopulationwas0.016(SCR … 0.016;95% CI=0.013,0.018)and0.014(SCR … 0.014;95%CI= 0.012,0.017)whenfittodatafromparticipants20years oryounger.ForMSP-1(middlepanel),theestimated SCRfromtheentirestudypopulationwas0.018(SCR … 0.018;95%CI=0.016,0.021)and0.019(SCR … 0.019; 95%CI=0.015,0.022)forparticipants20yearsoryounger.UsingAMA-1orMSP-1todefineseropositive participants(bottompanel),theestimatedSCRfromthe entirestudypopulationwas0.025(SCR … 0.025;95%CI= 0.022,0.028)and0.026(SCR … 0.026;95%CI=0.022, 0.302)forparticipants20yearsoryounger.Whenanonzeroseroreversionrate(SRR)wasusedtomodeltheseroprevalencedatafromallparticipantsaged2to80yearsof age,thefollowingconversionandreversionrateswerederived:0.014(95%CI ^ 0.011,0.017)and-0.006(SRR … 0.006;95%CI=-0.016,0.003)forAMA-1responses, Table1StudypopulationcharacteristicsbysiteofenrollmentDemographicfactorsParticipantsgivenELISA SiteofenrollmentSizeAgeGenderAMA-1MSP-1Combined (N)Years%MaleNo.testsNo.testsNo.tests Christianvilleschool51012.241.4510461510 HosanaBaptistschool1028.2952102102102 PortailLeoganeclinic7228.929.2727272 Chabincommunity13129.835.9131124131 Total81516.140.7815759815Thetotalnumberoftotalsamples,averageageinyears,percentageofmaleparticipants,andthenumberofparticipantswhowerescreenedusinganELI SAwith AMA-1,MSP-1,oreitherAMA-1orMSP-1antigensarelistedbystudyenrollmentsite. Table2NumberandprevalenceofseropositiveparticipantsbyageclassAgeclassAMA-1MSP-1Combined (years)nNo.Pos.%Pos.nNo.Pos.%Pos.NNo.Pos.%Pos. 2to56146.66158.26169.8 6to91902613.7189157.91903518.4 9to132163315.31934221.82165826.9 14to171542516.21293627.91544629.9 18to20631625.4602236.7632946.0 21to29401435.0401537.5401537.5 30to49502652.0482347.9503060.0 over50412868.3392153.8412868.3 Total81517221.175917923.681524730.3Thenumberoftotalsamples,positiveresults,andprevalenceforeachageclassappearforparticipantswithpositiveELISAresultstowardAMA-1,MS P-1,oreither AMA-1orMSP-1antigens.vonFricken etal.MalariaJournal 2014, 13 :361 Page5of8 http://www.malariajournal.com/content/13/1/361

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0.0204(SCR … 0.204;95%CI=0.016,0.025)and0.008 (SRR … 0.008;95%CI=-0.005,0.022)forMSP-1responses,and0.0273(SCR … 0.0273;95%CI=0.022,0.032) and0.007(SRR … 0.007;95%CI=-0.003,0.018)foreither anAMA-1orMSP-1response.DiscussionInanefforttomeettheislandwidegoalofmalariaeliminationby2020,thegametocidaldrugprimaquine(PQ), wasaddedtothemalarianationaltreatmentpolicyfor Haitiin2010[1].Thistreatmentpolicychangeplaces Haitiinauniquepositiontomonitorandquantifythe impactsingledosePQadministrationhason P.falciparum transmission,whichcouldholdvaluableinformationonPQtoleranceandmalariaeliminationstrategies abroad.Findingssuggestthattheseregionshaveexperiencearelativelylowandconstantstateof P.falciparum transmission,giventhestableincreaseinseroprevalence byageobservedinthisstudy.InsamplesthathadpositiveresponsestoeitherAMA-1orMSP-1,theestimated SCRof2.5%(95%CI ^ 2.2%,2.8%)fromthisstudyis slightlyhigherthanthe<1%prevalencerateestimateby PSIin2012[3].However,whentheseroconversionrates weredeterminedindividuallyfromapositiveAMA-1or MSP-1response,theestimatedSCRdecreasedto1.6% (95%CI ^ 1.3%,1.8%)and1.8%(95%CI ^ 1.6%,2.1%) forAMA-1andMSP-1,respectively.Thedifferencesin SCRestimatescouldbearesultinvariationinindividual antibodyresponses,assuggestedbyFigure4,wheresome seropositiverespondentshavestrongresponsestoonlya singleantigen(regionsIIandIII).Trendswerealso observedintheantibodyresponsesafterstratificationfor age,whichcouldindicatethatthedurationofAMA-1and MSP-1antibodytitersaredifferent,aspreviouslysuggested[19].InFigure3,in4outof5agegroupsbelow 20yearsofagetheseroprevalenceofMSP-1washigher thantheseroprevalenceofAMA-1,whereas2of3ofthe agegroupsabove20showhigherseroprevalenceofAMA1comparedtoMSP-1.However,inthissamplethe likelihoodofparticipantshavingastrongAMA-1response (>0.5AU)andaMSP-1responsebelowthethreshold (Figure4,regionIII)wasnotsignificantlydifferentby age(p>0.1). Theinclusionofaseroreversionrateinthemodelalso slightlyincreasedtheestimatedseroconversionrates.Due tothecross-sectionalnatureofthisstudyandthelong durationofantibodydetectionforAMA-1andMSP-1,it wasappropriatetosettheseroreversionratetozero.When seroreversionwasincludedinthemodel,theseroreversion rateswere-0.006(95%CI ^ -0.016,0.003)and0.008(95% CI ^ -0.005,0.022)forAMA-1andMSP-1respectively. Sincebothoftheconfidenceintervalsfortheseroreversion ratesincludezeroandtheinclusionofaseroreversionrate inthemodelhadlittleeffectontheestimatesofseroconversion,therefore apriori exclusionofaseroreversionrate fromthefinalmodelwasjustifiedinthiscircumstance. Whencomparingtheestimatedseroconversionratesfrom studyparticipantsunder20yea rsofage,thecontinuityin theage-specificseroprevale ncecurvecouldindicatethat overmultipledecades,arelativelyconstantstateoflow Figure4 ComparisonofsampleswithpositiveAMA-1orMSP-1 responses. ThescatterplotofAMA-1andMSP-1responses(average absorbanceat450nm)isshownalongwiththereferencelines denotingpositiveAMA-1orMSP-1responses(dottedlines)using thethresholdabsorbancevaluesforpreviousinfectionof0.319and 0.260absorbanceunits,respectively.Sampleswerepositivefor AMA-1andMSP-1(regionI),MSP-1positiveandAMA-1negative (regionII),AMA-1positiveandMSP-1negative(regionIII),or negativeforbothAMA-1andMSP-1(regionIV).Forclarity,samples withanabsorbancevalueabove1.8AUareshownashavinga maximumvalueof1.8AUandthosewithnopositiveresponseto eitherantigen(regionIV)arenotshown. Figure3 Seroprevalencebyageclassforparticipantsranging from2to80yearsofageasdeterminedbyELISAusingAMA-1 andMSP-1antigens. Theprevalenceofsamplesthathadan responsetotheAMA-1orMSP-1antigensarepresentedbyage classforthoseclassifiedasbeingseropositivewithAMA-1(white), MSP-1(charcoal),oreither(blackdotted)antibodies. vonFricken etal.MalariaJournal 2014, 13 :361 Page6of8 http://www.malariajournal.com/content/13/1/361

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malariatransmissionhasoccurredintheseregions,evenin theabsenceofsustainedmalariacontrolefforts.EntomologicalstudiesinvestigatingthevectorcompetencyofA. albimanusmosquito,maybette rexplainthisphenomenon, ofstablelowtransmission.LimitationsOneoftheprimarylimitationsofthisstudywasthat serumsampleswerecollectedusingaconveniencesample,whichlimitedourabilitytoinferhowthissample populationrepresentsHaitiasawhole.Findingsmay havebeenskewedbypotentiallyenrollingparticipants fromclinics(n=203),however,thispotentialsampling biaswasadequatelyaddressedbyexcludingallmalaria RDTpositiveindividuals(5/815)fromfinalanalysis. Thisstudyalsoonlyscreenedforpreviousexposureto P.falciparum ,althoughthelikelihoodoffindingother speciesormixedinfectionsremainslow,givenrecentreports,andthepresenceofhostprotectivefactors[5,20]. AswithotherELISAprotocols,settingathresholdforthe classificationofasampleasseropositiveissubjecttointerpretation.Tovalidatethemethodusedinthisstudy, thresholdsusingabsorbancevaluesoffourandfivestandarddeviationsabovethesuspectedseronegativepopulation meanwerealsoevaluatedandfellwithinthecalculated confidenceintervalsforseroprevalenceandSCRusing onlythreestandarddeviations.ConclusionAsreportedcasesofmalariaintheDominicanRepublic havereacheda15-yearlowof952cases[1],malariacontinuestobeamajorpublichealthconcerninHaiti.Findingsfromthisstudyfurthersupportthenotionofsustained lowlow-leveltransmissioninHaiti,whileusingahighly sensitivetechniquethatcouldbeusedtodeterminemalaria transmissionelsewhereinHaiti.Thesedatasuggeststhat anyeffortstoadvancemalariacontrollocallyhavenothad muchimpactoverthelastfivedecades,yetneitherhavethe pastpoliticalupheavalsornaturaldisastersfromrecent decadesresultedinmajormalariaepidemics. Figure5 SeroprevalenceestimatesforthepresenceofAMA-1orMSP-1antibodiesbyagefortheentirestudypopulation. Theactual seroprevalenceforAMA-1andMSP-1antibodies(circles)appearbyagealongwiththeprobabilityofinfectionineachageclassforAMA-1and MSP-1(blacklines)andtherespective95%confidencelimits(dottedlines),derivedfromthemodelestimatedseroconversionrate( ).Thetop, middle,andlowerpanelsshowtheincrementalincreasesinseroprevalenceforAMA-1,MSP-1,andeitherAMA-1orMSP-1withage,respectively. Theleftpanelsshowthemodelfitsusingdatafromparticipants2yearsto20yearsofage,whiletherightpanelsshowthemodelfitsusingthe entiredatasetincludingparticipantsfrom2to80years. vonFricken etal.MalariaJournal 2014, 13 :361 Page7of8 http://www.malariajournal.com/content/13/1/361

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Futurestudiesshouldexpandseroprevalencemethodologiestootherdepartmentsinor dertoestablishcountrywide trends.Researchexaminingb arrierstoaccess,protective hostcharacteristics,theextentofheterogeneousmalaria transmissioninotherdepartments,andvectorproficiency couldalsoenhanceeliminationmodelsinHaiti.Elimination inHaitiappearstobefeasible;however,surveillancemust continuetobestrengthenedinordertorespondtoareas withhightransmission,whilemeasuringtheimpactof futureinterventions.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterestintheoutcomes orinterpretationofthisdata. Authors ’ contributions MEVandBAOcontributedtotheconceptanddesignofthestudy.MEV, TAW,BL,WTE,LS,RM,JLandVMBcontributedtothecollectionandanalysis ofdata.TAWandMEVanalysedandinterpretedtheresults.Allauthors contributedtothedraftingandrevisingofthispaper.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements Theauthorswouldliketoextendaspecialthankstothededicatedstaffat CommunityCoalitionforHaitiandtheChristianvilleFoundationforwithout theirsupportthisstudywouldnotbepossible.Theauthorswouldliketo thankBenjaminD.AndersonfortechnicalassistancewiththeELISAprotocol andAlexanderKirpichforhisassistancewiththeseroconversionmodel. TheantigensusedintheELISAcamefromthefollowingsources: Plasmodiumfalciparum yP30P2PfMSP-119(Q-KNG)FVO/VK1,MRA-53deposited byDCKaslow,obtainedthroughtheMR4aspartoftheBEIResources Repository,NIAID,NIHandRecombinantPfAMA-1mixtureof3D7andFVO& Anti-PfAMA-1rabbitserumand/orpurifiedIgG,providedbyDavidNarum, obtainedfromtheNationalInstituteofHealthLaboratoryofMalariaand VectorResearch(LMVR),NIAID,NIH,DHHS.Thisstudywasfundedbythe ArmedForcesHealthSurveillanceCenter,GlobalEmergingInfections SurveillanceandResponseDivisiontoB.A.O.andbyUniversityofFlorida, CollegeofPublicHealthandHealthProfessionfundstoM.E.V. Authordetails1EmergingPathogensInstitute,UniversityofFlorida,2055MowryRoad, Gainesville,FL32611,USA.2DepartmentofEnvironmentalandGlobalHealth, UniversityofFlorida,Gainesville,FL32610,USA.3DepartmentofMicrobiology andCellScience,UniversityofFlorida,Gainesville,FL32611,USA.4CommunityCoalitionforHaiti,PortailLeoganeClinic,Jacmel,Haiti.5ChristianvilleFoundation,ChristianvilleBoulevardMareshall,Gressier,Haiti.6DepartmentofHealthServicesResearchManagementandPolicy,University ofFlorida,Gainesville,FL32611,USA.7LaboratoireNationaldeSantPublique, MinistredelaSantPubliqueetdelaPopulation,Port-au-Prince,Haiti. Received:3June2014Accepted:26August2014 Published:14September2014 References1.ClintonHealthAccessInitiative: Thefeasibilityofmalariaelimination ontheislandofHispaniola,withafocusonHaiti. 2013.Availableat http://globalhealthsciences.ucsf.edu/eliminating-malaria-on-the-island-ofhispaniola%20. 2.KeatingJ,KrogstadDJ,EiseleTP: MalariaeliminationonHispaniola. 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MalarJ 2013, 12: 30.doi:10.1186/1475-2875-13-361 Citethisarticleas: vonFricken etal. : Age-specificmalaria seroprevalencerates:across-sectionalanalysisofmalariatransmission intheOuestandSud-EstdepartmentsofHaiti. MalariaJournal 2014 13 :361.vonFricken etal.MalariaJournal 2014, 13 :361 Page8of8 http://www.malariajournal.com/content/13/1/361