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Malaria Treatment Policy, Management and Epidemiology in Haiti

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Title:
Malaria Treatment Policy, Management and Epidemiology in Haiti
Creator:
Von Fricken, Michael E
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (5 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Public Health
Environmental and Global Health
Committee Chair:
OKECH,BERNARD ACHERO
Committee Co-Chair:
SABO-ATTWOOD,TARA L
Committee Members:
LEDNICKY,JOHN
LAUZARDO,MICHAEL
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Antigens ( jstor )
Dehydrogenases ( jstor )
Dosage ( jstor )
Infections ( jstor )
Malaria ( jstor )
Phosphates ( jstor )
Population estimates ( jstor )
Seroepidemiologic studies ( jstor )
Trinity ( jstor )
Environmental and Global Health -- Dissertations, Academic -- UF
elimination -- g6pd -- haiti -- malaria
Genre:
Electronic Thesis or Dissertation
bibliography ( marcgt )
theses ( marcgt )
Public Health thesis, Ph.D.

Notes

Abstract:
Malaria remains a significant public health issue in Haiti, with 25,423 confirmed cases in 2012. However, reliable data on malaria in Haiti is not available. To reduce transmission of gametocytes, a single dose of primaquine (PQ) was added to the previous treatment policy of chloroquine monotherapy in 2010. The use of PQ is contraindicated in people with glucose 6 phosphate dehydrogenase (G6PD) deficiency due to the risk of drug induced hemolysis. Unfortunately there is no information in Haiti on the prevalence of G6PD deficiency. These concerns will be introduced in Chapter 1 and addressed in the subsequent chapters of this dissertation. Briefly, chapter 2 documents the history of treatment policies in Haiti from 1955 to 2012, and provides details on the rationale behind the adoption of PQ into current treatment strategies. This section discusses studies that have previously occurred in Haiti, while identifying the absence of data for much of this period. Chapter 3 and 4 present the results of two G6PD deficiency studies implemented in Haiti. Chapter 3 focuses on determining the prevalence of G6PD deficiency in our sample population, which was found to be 22.8% (14.9% to 24.7%). Chapter 4 examines the performance of the CareStart G6PD deficiency rapid diagnostic test in a school based population. Results suggest that the test detects severe G6PD deficiency correctly 90% of the time, indicating that this test could be incorporated into current malaria treatment strategies that rely on PQ therapy Chapter 5 presents serological findings that were used to calculate an annual transmission rate in the Ouest and Sud Est departments of Haiti. An age adjusted seroconversion rate of 2.5% suggests that despite the absence of sustained malaria control efforts in Haiti, transmission has remained low over multiple decades. Chapter 6 provides a general discussion on future prospects for malaria elimination in Haiti and Hispaniola. Policy makers should consider the risks of PQ treatment in G6PD deficient individuals, and what low transmission of malaria in Haiti entails for the future. These findings provide valuable information that can be used to guide future elimination efforts and strategies for combating malaria in Haiti. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
General Note:
Adviser: OKECH,BERNARD ACHERO.
General Note:
Co-adviser: SABO-ATTWOOD,TARA L.
Statement of Responsibility:
by Michael E Von Fricken.

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UFRGP
Rights Management:
Copyright Micheal Von Fricken. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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LD1780 2014 ( lcc )

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REVIEWOpenAccessMalariatreatmentpoliciesanddrugefficacyin Haitifrom1955-2012MichaelEvonFricken1,2*,ThomasAWeppelmann1,2,JenniferDHosford2,AlexanderExiste3andBernardAOkech1,2AbstractObjectives: Chloroquine(CQ),after67yearsofuseinHaiti,isstillpartoftheofficialtreatmentpolicyformalaria. SeveralcountriesaroundtheworldhaveusedCQinthepastduetoitslowincidenceofadverseevents, therapeuticefficacy,andaffordability,butwereforcedtoswitchtreatmentpolicyduetothedevelopmentof widespreadCQresistance.Thepurposeofthispaperwastocompileliteratureonmalariatreatmentpoliciesand antimalarialdrugefficacyinHaitiover67-yearperiod. Methods: AsystematicreviewofPubMed,WebofScience,andtheArmedForcesPestManagementBoard,was conductedtofindpertinentdocumentsonnationalmalariatreatmentpoliciesandantimalarialdrugefficacy studiesinHaitibetween1955and2012.Atotalof329citationsandabstractswerereviewedindependentlybytwo researchers,ofwhichthirtythreemetthefinalinclusioncriteriaofstudiesoccurringinHaitibetween1955and 2012whichspecificallydiscussmalariatreatmentpoliciesanddrugefficacy. Results: ResultssuggestthatCQhasbeenthepredominantantimalarialdruginusefrom1955to2012.In2010 singledoseprimaquine(PQ)wasaddedtothenationaltreatmentpolicy,howeveritisnotclearwhetherthisnew policyhasbeenputintopractice. Conclusions: AlthoughnowidespreadCQresistancehasbeenreported,somestudieshavedetectedlowlevelsof CQresistance.IncreasedsurveillanceandmonitoringforCQresistanceshouldbeimplementedinHaiti. Keywords: Hispaniola,Haiti,Malariatreatmentpolicy,Chemotherapy,Chloroquine,Anti-malarialdrugresistance, Plasmodiumfalciparum ,PyrimethamineIntroductionMalariapersistsinHispanioladespiteitseliminationfrom otherCaribbeancountries[1].It ’ sestimatedthatover99% ofmalariacasesinHaitiarecausedby Plasmodiumfalciparum with Anophelesalbimanus servingastheprincipal mosquitovector[2-4].Theburdenofmalariaishighin Haiti,relativetoitspopulationof10millionpeople,with 80%oftheHaitianpopulation livinginareaswheremalaria isendemic[5,6].Historically,Haitihasunder-reportedthe numberofmalariacases,largelyduetolimitedresources, inadequatesurveillanceandashortageoftrainedpersonnel [7-9].Theantimalarialchloroquine(CQ)hasbeenrelied onheavilyinHaiti,duetoitslowincidenceofadverse events,affordability,andperceivedtherapeuticefficacy,despitetheemergenceofCQresistanceglobally. TobetterunderstandHaiti ’ sprolongedcommitment toCQ,wecompiledreportsandpublicationsonmalaria chemotherapeuticpoliciesandantimalarialresistancein Haitifrom1955to2012basedonasystematicsearchof historicalliterature.Priorto1955,quininewasused intermittentlyduringperiodsofUSoccupation[10].The purposeofthispaperistodocumentthehistoryofHaiti ’ s malariatreatmentpolicies,whileprovidingausefulreferenceforantimalarialdrugresistancestudiesinHaiti.MethodsDatasourcesPublishedstudiesandreportsaboutmalariainHaiti wereidentifiedfromanelectronicsearchofMEDLINE®/ *Correspondence: Michaelvonfricken@epi.ufl.edu1DepartmentofEnvironmentalandGlobalHealth,UniversityofFlorida,P.O. Box100188,Gainesville,FL32610,USA2EmergingPathogensInstitute,UniversityofFlorida,P.O.Box100009, Gainesville,FL32610,USA Fulllistofauthorinformationisavailableattheendofthearticle ©2013vonFrickenetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated.vonFricken etal.JournalofPharmaceuticalPolicyandPractice 2013, 6 :10 http://www.joppp.org/content/6/1/10

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PubMed®(1955-2012)andWebofScience(1955-2012). AsearchofgrayliteraturewascarriedoutontheArmed ForcesPestManagementBoard(AFPMB)database[11], whichcontainspublicationsandreportsfromthe CentersforDiseaseControlandPrevention(CDC), WorldHealthOrganization(WHO),andPanAmerican HealthOrganization(PAHO).Searchtermswerechosen tocapturemalariatreatmentpolicyanddrugefficacy studiesinHaitionly,usingacombinationofsimple subjectheadingsandtermcombinations,focusingon treatment,malaria,andHaiti.StudyselectionTheselectioncriteriaofthedatasourcesincludedare;1) reportsfromtheperiodof1955to2012,2)allstudies carriedoutinHaiti,3)andstudiesthatdiscussmalaria. Fulltextmanuscriptsandallcitationswhichmetthe predefinedselectioncriteriawereobtainedandexaminedbytwoindependentresearchers.Finalinclusion andexclusiondecisionsweremadewith100%agreementbetweentheexaminersbasedonstudiesthatreportedmalariatreatmentpoliciesanddrugefficacyfor finalinclusion.Whereduplicationswereobservedthe mostrecentversionofthemanuscriptwasselected.The year1955wasusedasastartdateforanalysis,becauseit coincidedwiththeGlobalMalariaEliminationProgram campaigninHaiti.ResultsAtotalof329citationsandabstractsintheelectronic searcheswerefound.Ofthese,85citationsandabstracts metthepreliminarycriteriaandtheirfulltextswere reviewed.ThirtythreestudiesdescribedmalariatreatmentpoliciesorantimalarialdrugefficacyinHaitifrom 1955-2012[1,2,4,6-9,12-37].CharacteristicsofincludedstudiesChloroquinewasfirstintroducedinHaitiin1955,when TheEighthWorldHealthAssemblyrecommendeditsuse incombinationwithpyrimethamine(P)fortheeliminationofmalaria.Thechloroquine/pyrimethamine(CQ/P) combinationstrategywasthefirsttreatmentpolicyswitch inHaitireplacingthepreviousacceptedpracticeof quinineforthetreatmentofmalaria[38-40].Accordingto thedatasources,from1955to1970,CQ/Pwasadministeredinaprophylacticmannerthroughmassdrugadministration(MDA)campaigns,aspartoftheGlobalMalaria EradicationPrograminHaiti[13,15,16,40].From1970to 2010,thetreatmentformalariaswitchedfromacombinationtherapyofCQ/PtherapytoCQmonotherapyfor uncomplicatedmalariacases[16].In2010aCDCreport mentionedtheadditionofthetransmissionblockingdrug primaquine(PQ)atasingledoseof0.75mg/kg,suggestingamajorchangeinpolicy[36].Allfinalarticles(N=33) thatweevaluatedreportedCQasorpartofthestandard treatmentpolicyinHaiti.Abriefsummaryoftreatment policiescanbeseeninthetimelineprovided(Figure1).EvidencefortreatmentresistanceinHaitiOnlyfourteenstudiesfromthedatasourcesexamined [6,9,12-19,21,26,32,35]discussedchemotherapeuticefficacyorpatienttreatmentoutcomedata(Table1).Sixof thesefourteenstudiescontaineddocumentationofresistancetoCQorP[6,16,18,19,21,32].Thefirstyearof reportedofanti-malarialresistance,where P.falciparum haddocumentedpyrimethamineresistancewas1971[16]. In1982,areportofpossibleresistancetoCQtreatment wassuggested[6]afterapatientonCQmonotherapyhad resurgenceinparasitemiaonday28oftreatment.A follow-up invitro testfound4/16 P.falciparum cultures includingthe P.falciparum strainsfromthepatientwith resurgentparasitemia,requiredCQdoseslargeenough thatsuggestedpossibleresistant P.falciparum parasites. Thisreportprovidedthefirstcredible invitro evidenceof P.falciparum resistancetoCQinHaiti[18].In1984,an invivo and invitro study,testedfor P.falciparum parasite resistancetosulfadoxine/pyrimethamine(S/P)[19]toprovidebaselinedataonsusceptibilityintheeventthatCQ resistancedevelopedinHaiti.Theresultsindicatedparasiteresistancetopyrimethaminealone,butnotto sulfadoxine.Despite invitro evidenceofresistancetopyrimethamine,all invivo infectionsweresusceptibletoa combinationofS/P[19].In1985,another invivo studyto complementtheprevious invitro assayfoundresistance topyrimethaminealone[21].Morerecently,areportby Londonoetal,hasdocumentedthedetectionofgenetic Before 19551955 1970 1970 2010 2010 – 2012 CQ monotherapy becomes treatment policy, due to pyremithamine resistance Intermittent quinine use during periods of US Navy occupation CQ + pyremithamine during the Global Malaria Elimination Program CQ + single dose PQ, toreduce transmission by targeting gametocytes Figure1 TimelineofHaitianmalariatreatmentpolicies. vonFricken etal.JournalofPharmaceuticalPolicyandPractice 2013, 6 :10Page2of5 http://www.joppp.org/content/6/1/10

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markersviaPCR,forCQresistanceinfiveof79patients (6%)fromtheArtiboniteValley[32].However,astudyby Neubergeretal,foundzeroof49infectedpatientstohave mutationssuggestiveofCQresistance[36].Thesestudies presentconflictingevidenceaboutthepresenceorabsence ofCQdrugresistanceinthepopulationsstudied,withno definitivedocumentationof invivo resistancereported.DiscussionFollowingthewidespreaduseofCQ/Pfrom1955-1968, resistancetopyrimethaminewasreportedinHaitiin 1971[16].Itwassuggestedthatparasiteadaptationto pyrimethaminehadoccurredduetoselectivedrugpressurefromtheMDAcampaign,whichendedin1968, resultinginitsremovalfromthenationaltreatment policy[16,19]. HaiticontinuedtorelypredominantlyonCQaspart ofthetreatmentformalariainHaitiupuntil2010,when singledosePQwasaddedtotheofficialpolicyforthe treatmentofuncomplicatedmalariainHaiti[2,37].Haiti representsauniquescenariowherePQhasnotbeen usedpreviouslyontheislandduetotheabsenceof Plasmodiumvivax, butisbeingintroducedtospecifically blockmalariatransmissionbytargetingtheadultstage Plasmodiumfalciparum parasites.Itremainsunclear whetherornotPQhasbeenimplementedinpractice, norhaveanystudiesexaminedpopulationratesof G6PDdeficiencyinHaiti,ageneticmutationthatplaces deficientindividualsatriskofacutehemolyticanemia whenexposedtoPQ. Overall,wefoundtheuseofCQhasfeaturedprominentlyinthemanagementofmalariainHaitifordecades. WhileCQresistanceinmanySouthandCentralAmerican countrieshasforcedthesecou ntriestochangepolicies,the HaitianMinistryofHealth(MSPP)hascontinuedtorely almostentirelyonCQastheprincipaltreatmentformalaria.Despitesuchprolonge duse,ourresultsfoundno conclusiveevidenceofthepresenceorabsenceofCQresistanceinHaiti(Table1).However,findingsfromthestudiesthatreportontreatmentse nsitivityarelackingdueto smallsamplesizes[6,18,19,21,32,36],localizedenrollment [6,15,18,19,21,32,36],andmissinginformationonpatient treatmentoutcomes,significantlylimitingtheirabilitytoinfluencenationaltreatmentpolicies(Table1).Tenofthe fourteenstudiesthatmakementionofdrugefficacy,relied solelyonsecondarysourcesofdata,oftenonlycontaining briefcommentsontreatmenteffectiveness,whichfurther suggestanabsenceofdesign eddrugsensitivitystudies Table1ReportsoftreatmentefficacyformalariainHaitiPeriodSample size SiteinHaitiDrug combination Sourceof dataused ResistantSusceptibleReference 19601966 N/ACountryWideCQ/PSecondaryN/ACQPAHO.1967[ 14 ] 1961N/ACountryWideCQSecondaryN/ACQWHO.1965[ 13 ] 19621965 N/ACountryWideCQ/PSecondaryN/ACQ/PMasonJandPhilippeA,1967 [ 12 ] 19621965 N/APetitieGoaveCQ&CQ/PSecondaryN/ACQ/PWHO,1968[ 15 ] 1971N/ACountryWideCQSecondaryVV,P& CG CQWHO,1972[ 16 ] 1976N/ACountryWideN/ASecondaryN/ACQWHO,1978[ 17 ] 1980N/ACountryWideCQSecondaryN/AVVCQPAHO,1980[ 9 ] 19811983 92LesCayes,Port-au-Prince,Limbe, Gros-MorneandJacmel CQSecondaryVT&VV CQ CQDuverseauYT,MagloireR,etal., 1986[ 6 ] 198219Port-au-PrinceCQSecondaryVT&VV CQ N/AMagloireRandNguyen-dinhP, 1983[ 18 ] 198218Port-au-PrinceP&S/PPrimaryVT&VV P VVS/PNguyen-DinhP,Zevallos-Ipenza A,etal.,1984[ 19 ] 198522Port-au-PrinceP&S/PPrimaryVTPVV&VTS/PNguyen-DinhP,PayneD,etal., 1985[ 21 ] 1995N/ACountryWideN/APrimaryN/AVVCQDrabickJJ,GambelJM,etal., 1997[ 26 ] 20062007 79ArtiboniteValleyCQSecondaryPCRCQN/ALondonoBL,EiseleTP,etal., 2009[ 32 ] 20102011 49LeoganeCQPrimaryN/APCRCQNeubergerZhongK,etal.,2012 [ 36 ]Key:Chloroquine(CQ),pyrimethamine(P),chloroquinewithpyrimethamine(CQ/P),sulfadoxinewithpyrimethamine(S/P), invivo susceptibility(VV), invitro susceptibility(VT),DNAexaminedformutationsviapolymerasechainreaction(PCR).vonFricken etal.JournalofPharmaceuticalPolicyandPractice 2013, 6 :10Page3of5 http://www.joppp.org/content/6/1/10

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occurringinHaiti[6,9,12-18 ,32].ThetwomostrecentstudiesexaminingCQresistancebyLondonoandNeuberger gavedifferentresultsaboutthepresenceofgeneticmarkers ofCQresistanceinHaiti[32,36].However,bothstudieshad smallsamplesizesof79and48respectivelyandwerelocated indifferentregionsofthecountry.CQresistancemutations areemerginginHaiti,buttowhatextentremainsunknown, suggestinganeedforincreasedsurveillanceforparasite resistance.LimitationsWewereunabletoexaminegrayliteraturefromthe MSPPasmostdocumentswerelostduringthe2010 earthquake.Althoughweexcludednon-Englishdatabases,webelievethisomissiontonotbeverysignificant, sincewereliedheavilyonWHOandPAHOreports, whicharebasedonbothEnglishandnon-Englishdatabasesandreports.Therewerelimiteddatapertaining totreatmentfailuresandparasiteresistance,inaddition toanoveralllackofreportingbetween1985-2005dueto politicalunrest[8,32].ConclusionsAsof2012,thefewdocumentedreportsofCQresistanceinHaitididnotexceedtheWHOrecommended thresholdtreatmentfailurerateof 10%[41].However, duetolimitedsurveillanceondrugefficacyandbarriers topatientfollowup,furtherstudiesarenecessarytodetermineifratesofCQresistancefallbelowthisthresholdinHaiti.Meanwhile,Non-governmentorganizations, foreignaidagencies,andtheHaitianMSPPshouldconsiderimplementingacomprehensivemalariacontrol programwhileCQremainsaviabletreatmentoption [32,35,36].Otherdrugregimens,suchasartemisininbasedcombinationtherapies,arepartofanarsenalof availabletreatmentoptions,intheeventofCQresistance.TheaffordablecostofCQ,andits ’ lowincidence ofadverseeventsmakeitanidealtreatmentoptionona largescale.ItisourrecommendationthatincreasedsurveillanceandmonitoringforCQresistancebeimplemented,duetosignificantgapsindataonCQtreatment failureandresistanceinHaiti.Regardingtherecent additionofPQtothenationalpolicy,moreinformation isneededonhowPQistoleratedinthispopulation, giventheabsenceofinformationonG6PDprevalence rates,andwhetherornotthispolicyhasbeenputinto practiceinHaiti.Futurestudiesexaminingtransmission ratesinHaitimaygeneratevaluableinformationonthe impactsingledosePQhasonmalariarates,potentially providingatemplateforeliminationinotherlowtransmissionsettingsglobally.Abbreviations CQ: Chloroquine;P:Pyrimethamine;CQ/P:Combinedchloroquinepyrimethamine;PQ:Primaquine;S:Sulfadoxine;S/P:Combinedsulfadoxinepyrimethamine;ACT:Artemisinin-basedcombinationtherapy;WHO:World HealthOrganization;PAHO:PanAmericanHealthOrganization; MSPP:MinistryofHealthandPopulation;MDA:MassDrugAdministration. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authordetails1DepartmentofEnvironmentalandGlobalHealth,UniversityofFlorida,P.O. Box100188,Gainesville,FL32610,USA.2EmergingPathogensInstitute, UniversityofFlorida,P.O.Box100009,Gainesville,FL32610,USA.3National LaboratoryofPublicHealth,MinistryofPublicHealthandSanitation,Angle Delmas33etRueCharbonnieres,Haiti. Received:4September2013Accepted:7November2013 Published:11November2013 References1.RobertsL: Tuberculosisandmalariaeliminationmeetsrealityin Hispaniola. Science 2010, 328: 850 – 851. 2.WorldHealthOrganization: WorldMalariaReport2010. Geneva:WHO;2011. 3.HobbsJHSJ,StJeanY,JacquesJR: Thebitingandrestingbehaviorof AnophelealbimanusinnorthernHaiti. JAmMosqControlAssoc 1986, 2: 150 – 153. 4.KrogstadDJ,JosephVR,NewtonLH: Aprospectivestudyoftheeffectsof ultralowvolume(ULV)aerialapplicationofMalathiononEpidemic PlasmodiumfalciparumMalariaIVEpidemiologicalaspects. 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23.DeloronP,DuverseauYT,Zevallos-IpenzaA,MagloireR, etal : Antibodiesto Pf155,amajorAntigenofPlasmodiumFalciparum:Seroepidemiological studiesinHaiti. BullWorldHealthOrgan 1987, 65: 339 – 344. 24.PanAmericanHealthOrganization: MalariaintheAmericas.Washington, D.C. BullPanAmHealthOrgan 1992, 13: 1 – 6. 25.BonnlanderH,RossignolAM,RossignolPA: MalariaincentralHaiti:a hospital-basedretrospectivestudy,1982-1986and1988-1991. BullPan AmHealthOrgan 1994, 28: 9 – 16. 26.DrabickJJ,GambelJM,HuckE,DeYoungS, etal : Microbiological laboratoryresultsfromHaiti:June-October1995. BullWorldHealthOrgan 1997, 75: 109 – 115. 27.PanAmericanHealthOrganization: VollumeII:healthintheAmericas 1998.Washington,D.C. BullPanAmHealthOrgan 1998, 2: 316 – 330. 28.VanderwalT,PaultonR: MalariaintheLimbéRivervalleyofnorthernHaiti:a hospital-basedretrospectivestudy,1975 – 1997. AmJPublicHealth 2000, 7: 162 – 167. 29.PanAmericanHealthOrganization: Situationofmalariaprogramsinthe Americas.Washington,D.C. BullPanAmHealthOrgan 2001, 22: 10 – 14. 30.EiseleTP,KeatingJ,BennettA,LondonoB, etal : Prevalenceof Plasmodiumfalciparuminfectioninrainyseason,ArtiboniteValley,Haiti, 2006. EmergInfectDis 2007, 13: 1494 – 1496. 31.KeatingJ,EiseleTP,BennettA,JohnsonD, etal : Adescriptionof malaria-relatedknowledge,perceptions,andpracticesintheArtibonite ValleyofHaiti:implicationsformalariacontrol. AmJTropMedHyg 2008, 78: 262 – 269. 32.LondonoBL,EiseleTP,KeatingJ,BennetA, etal : Chloroquine-resistant HaplotypePlasmodiumfalciparumparasitesHaiti. EmergInfectDis 2009, 15:735 – 740. 33.AdamsP: RainyseasoncouldhamperHaiti'srecovery. Lancet 2010, 375: 1067 – 1069. 34.CenterforDiseaseControlandPrevention: MalariaAcquiredinHaiti — 2010. Atlanta,GA:CDCMMWR;2010:217 – 219. 35.TownesD,ExisteA,BoncyJ,MagloireR, etal : MalariasurveyinpostearthquakeHaiti – 2010. AmJTropMedHyg 2012, 86: 29 – 31. 36.NeubergerA,ZhongK,KainKC,SchwartzE: Lackofevidencefor chloroquine-resistantPlasmodiumfalciparummalaria,LeoganeHaiti. EmergInfectDis 2012, 18 (9):1487 – 1489. 37.CentersforDiseaseControlandPrevention: MalariainPost-EarthquakeHaiti: CDC'sRecommendationsforPreventionandTreatment. Atlanta,GA:CDC Alerts;2010. 38.CentersforDiseaseControlandPrevention: TheHistoryofMalaria,an AncientDisease. Atlanta,GA:CDC;2010. 39.InternationalCooperationAdministration: MalariaManual,ForU.S.Technical CooperationPrograms ,PublicHealthDivision.WashingtonD.C:ICA;1956. 40.WorldHealthOrganization: TheEighthWorldHealthAssembly. Geneva:WHO; 1955. 41.WorldHealthOrganization: Guidelinesforthetreatmentofmalaria ,Volume 2.WHO;2010.Availableathttp://www.who.int/malaria/publications/atoz/ 9789241547925/en/index.html.doi:10.1186/2052-3211-6-10 Citethisarticleas: vonFricken etal. : Malariatreatmentpoliciesand drugefficacyinHaitifrom1955-2012. JournalofPharmaceuticalPolicy andPractice 2013 6 :10. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit vonFricken etal.JournalofPharmaceuticalPolicyandPractice 2013, 6 :10Page5of5 http://www.joppp.org/content/6/1/10



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MALARIA TREATMENT POL ICY , MANAGEMENT AND EPIDEMIOLOGY IN HAITI By MICHAEL E. VON FRICKEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGRE E OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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© 2014 Michael E. von Fricken

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To my Mother and Father, for never losing faith in me

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4 ACKNOWLEDGMENTS I would like to acknowledge and thank the many collaborators , both in the United States and in Haiti, without whom this study would not have been possible. I would like to especially thank my committee members for their continued guidance and mentorship, allowing me the opportunity to develop as a res earcher, scholar, and human being. I am deeply thankful and appreciative for the assistance Dr. Bernard Okec h has provided me as my committee chair, sacrificing countless hours mentoring me, helping me edit my manuscripts, and brainstorming future protoco ls. Dr. Okech has played a key role in shaping my habits and goals, preparing me for a career in research and academia. I thank Dr. Tara Sabo Attwood for giving me my first job as a teaching assistant at the University of Florida. I have based many of m y teaching philosophies on her education style. Dr. Sabo Attwood has been a wonderful mentor and a great friend and her support and sense of humor has been truly appreciated . I thank Dr. Michael Lazuardo for his continued support and advice throughout th is dissertation process. His kind words of encouragement have helped keep me focused and energized. I thank Dr. John Lednicky for his constructive criticism and the wealth of knowledge that he has brought to this project. I thank Dr. Paul Psychas for br inging his experience in malaria control to my committee; I greatly enjoyed our many hours of d iscussing various malaria projects. To my Haitian colleagues and collaborators; In Jacmel, I would like to thank the Community Coalition for Haiti (CCH) staff; including Karen Carr, Laura S c hick, Rubinste St. Louis, Dr. Mary Schmidt, Jude Lafle ur, Dr. Jean Baptists Calix, Dr. Ant oine Pierre Dilene , and the nursing staff Natasha and Judy. In Christianville I would like to thank Dr. Roseline Masse, Jean Mo ise Fleuranvil, Dr. Taina Philippe , Tricia Morrissey , Jeff

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5 Fortune, Edsel Redden , Dr. Madsen Beau De Rochars, Marsha and Ken DeVore, and our driver Jean Maken zie Luma . In Leogane I would like to extend my thanks to Dr. Jean Roosevald Romain, Dr. Gladys Me mnon, and our nursing staff Carole and Ph ara. To the others who have supported this project, who were not mentioned, you have my deepest gratitude Mesi Anpil. This dissertation is the result of all of your efforts and I thank you all for your cr itical assistance. Many thanks for the support from my colleagues at the University of Florida, I would like to thank Dr. Gregory Gray, Maha Elbadry, Will Eaton, Meer Alam, Brandon Lam, Tamar Carter, Dr. Glenn Morris, Dr. Kevin Fennelly, Dr. Juliet Puliam, the Mulligan Lab, the Dame Lab, and the wonderful departmental staff at the EGH; Jenn Wert, Ashley Condell, and Christa Roberts. I would especially like to thank Thomas A. Weppelmann individually for hi s support and assistance on this project . H is friend ship , counsel , and collaboration we re much needed and truly appreciated throughout this program . To my friends and family : w ithout your love and support, I would not be where I am today. I thank my brothers, Alex and Matt, for their continued love and feigned interest in my research. I thank my sister Natalie and her husband Hank for their enthusiasm and unwavering support. A very special thanks to Brooke Eckman, for the help and support she has provided ; her confidence in me has truly been a blessin g. Finally, I am grateful to my parents, Linda and Manfred von Fricken, for inspiring in me a spirit of wanderlust and adventure. They have taught me the importance of humility, hard work, and respect, which I will continue to incorporate into my life. The ir example

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6 has given me much to aspire to and I can only hope to repay them one day for all they have done. Thank you. This dissertation research was funded by a grant from the Armed Forces Health Surveillance Center, Global Emerging Infections S urveillance and Response Division to Bernard A. Okech, and by the University of Florida, College of Public Health and Health Profession funds to Michael E. von Fricken. The following reagents were obtained through the MR4 as part of the BEI Resources Repos itory, NIAID, NIH: Plasmodium falciparum yP30P2 PfMSP1 19(Q KNG)FVO/VK1 , MRA 53, deposited by DC Kaslow The following reagents were obtained from the National Institute of Health Laboratory of Malaria and Vector Research (LMVR), NIAID, NIH, DHHS: Recombi nant PfAMA1 mixture of 3D7 and FVO, provided by David Narum Anti PfAMA1 rabbit serum and/or purified IgG, provided by David Narum Competing Interest The author declares that he has no competing interest or stakes with the success or failure of any of the outcomes discussed in this dissertatio n and he assumes responsibility for the collection and analysis of all data.

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7 TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Malaria in Haiti ................................ ................................ ................................ ........ 16 Focus of Research ................................ ................................ ................................ .. 19 Study 1) Examine the History of Mal aria Treatment Policies and Practice over the Past 65 Years in Haiti ................................ ................................ ...... 19 Study 2) Determine the Prevalence of Glucose 6 Phosphate Dehydrogenase (G6PD) Deficiency in Haiti ................................ .................. 20 Diagnostic Test ................................ ................................ ............................. 20 Study 4) Measure the Rates of Malaria Exposure in Haiti by Serological Surveys of Asymptomatic and Symptomatic Populations ............................. 21 Approach to Fieldwork (Studies 2 4) ................................ ................................ ....... 22 Ethical Approval ................................ ................................ ............................... 22 ................................ ............ 22 Study Sites ................................ ................................ ................................ .............. 22 Participant Enrollment ................................ ................................ ............................. 23 2 MALARIA TREATMENT POLICIES AND DRUG EFFICACY IN HAITI FROM 1955 2012 ................................ ................................ ................................ ............... 29 Chapter Summary ................................ ................................ ................................ ... 29 Background ................................ ................................ ................................ ............. 30 Materials & Methods ................................ ................................ ............................... 30 Data Sources ................................ ................................ ................................ .... 30 Study Selection ................................ ................................ ................................ 31 Results ................................ ................................ ................................ .................... 31 Characteristics of Included Studies ................................ ................................ .. 32 Evidence for Treatment Resistance in Haiti: ................................ ..................... 32 Discussion ................................ ................................ ................................ .............. 33 Limitations ................................ ................................ ................................ ........ 35 Chapter Conclusions ................................ ................................ ........................ 35

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8 3 PREVALENCE OF GLUCOSE 6 PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY IN THE OUEST AND SUD EST DEPARTMENTS OF HAITI ........... 40 Chapter Summary ................................ ................................ ................................ ... 40 Background ................................ ................................ ................................ ............. 41 Materials and Methods ................................ ................................ ............................ 42 Study Location ................................ ................................ ................................ .. 42 Quality Assurance ................................ ................................ ............................ 42 Study Enrollment and Sample Collection ................................ ......................... 43 Determination of G6PD Activity ................................ ................................ ........ 43 Establishing Cutoff Values in our Population ................................ .................... 44 Statistical Analysis ................................ ................................ ............................ 45 Resul ts ................................ ................................ ................................ .................... 45 Discussion ................................ ................................ ................................ .............. 46 Limitations ................................ ................................ ................................ ........ 47 Chapter Conclusions ................................ ................................ ........................ 47 4 PERFOMANCE OF THE CARESTART G6PD RAPID DIAGNOSTIC TEST (RDT) IN GRESSIER, HAITI ................................ ................................ ................... 55 Chapter Summary ................................ ................................ ................................ ... 55 Background ................................ ................................ ................................ ............. 56 Methods and Materials ................................ ................................ ............................ 57 Study Location, Informed Consen t, and Sample Collection ............................. 57 The CareStart Qualitative G6PD Deficiency RDT ................................ ............ 58 Trinity Biotech Quantitative G6PD Deficiency Assay ................................ ........ 59 Quality Assurance ................................ ................................ ............................ 59 Statistical Analyses of RDT Performance ................................ ......................... 60 Results ................................ ................................ ................................ .................... 60 Discussion ................................ ................................ ................................ .............. 61 Limitations ................................ ................................ ................................ ........ 62 Chapter Conclusions ................................ ................................ ........................ 63 5 AGE SPECIFIC MALARIA SERO CONVERS ION RATES: AN ANALYSIS OF MALARIA TRANSMISSION IN THE OUEST AND SUD EST DEPARTMENTS OF HAITI ................................ ................................ ................................ ................. 67 Chapter Summary ................................ ................................ ................................ ... 67 Background ................................ ................................ ................................ ............. 68 Methods ................................ ................................ ................................ .................. 69 Study Location & Enrollment ................................ ................................ ............ 69 ELISA Protocols and Procedures ................................ ................................ ..... 70 Determination of Seropositive and Seronegative Population Members ..... 71 Estimation of Sero Conversion Rates from C ross Sectional Data ............. 71 Estimation of Seroprevalence Using AMA and MSP ................................ ........ 72 Estimation of Sero Conversion Rates for AMA and MSP ................................ . 73 Discu ssion ................................ ................................ ................................ .............. 74

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9 Limitations ................................ ................................ ................................ ........ 75 Chapter Conclusions ................................ ................................ ........................ 76 6 GENERAL DISCUSSION AND CONCLUSION S ................................ .................... 85 Summary of Findings ................................ ................................ .............................. 85 Future Research ................................ ................................ ................................ ..... 87 APPENDIX: PUBLICATIONS ................................ ................................ ....................... 89 REFERENCES ................................ ................................ ................................ .............. 90 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 97

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10 LIST OF TA BLES Table P age 1 1 Description of sites by study ................................ ................................ ............... 28 2 1 Reports and publication discussing malaria treatment policy in Haiti ................. 37 2 2 Reports of Treatment Efficacy for Malaria in Haiti. ................................ ............. 39 3 1 Summary statistics of study participants by gender and location ........................ 51 3 2 G6PD deficiency by gender and enrollment locations ................................ ........ 52 4 1 Performance measures and test results of the G6PD RDT compared to the spectrophotometric method ................................ ................................ ................ 65 5 1 Study population characteristics by site of enrollment ................................ ........ 79 5 2 Number and pr evalence of seropositive participants by age class ..................... 81

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11 LIST OF FIGURES Figure page 1 1 Malaria Risk in Haiti ................................ ................................ ............................ 26 1 2 Study locations in the Ouest and Sud Est departments of Haiti. ........................ 27 2 1 Selection process for systematic review of studies examining malaria treatment policies in Ha iti between 1955 and 2012. ................................ ........... 38 2 2 Timeline of Haitian malaria treatment policies ................................ .................... 38 3 1 Global distribution of G6PD deficiency. ................................ .............................. 49 3 2 Study area in the Ouest and Sud Est departments of Haiti ................................ 50 3 3 Histogram of G6PD activity (U/g Hgb) for the entire study pop ulation. ............... 53 3 4 Histogram of G6PD activity (U/g Hgb) by gender ................................ ............... 54 4 1 Access Bio CareStart G6PD rapid test results. ................................ .................... 64 4 2 Performance of the CareS tart G6PD RDT ................................ .......................... 66 5 1 Seroprevalence study area in the Ouest and Sud Est departments of Haiti. ...... 78 5 2 ...................... 80 5 3 Seroprevalence by age class for participants ranging in age fro m 2 to 80 for AMA and MSP antibodies ................................ ................................ ................... 82 5 4 Comparison of samples with positive AMA 1 or MSP 1 responses .................... 83 5 5 Seroprevalen ce estimates for the presence of AMA 1 or MSP 1 antibodies by age class for children and adults and for the entire study population. ................ 84

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12 LIST OF ABBREVIATIONS ACT A rtemisinin based combination therapies AMA 1 A pical membr ane antigen CDC Centers for Disease Control and Prevention CQ C hloroquine CQ/P C hloroquine with pyrimethamine DIH D rug induced hemolysis G6PD G lucose 6 phosphate dehydrogenase GMEP Global Malaria Eradication Program EDTA E thylenediaminetetraacetic Acid ELISA Enzyme Linked Immun osorbent Assay MSP 1 19 M erozoite surface protein MSPP Ministère de la Santé Publique et de la Population NADPH N icotinamide adenine dinucleotide phosphate oxidase P Pyrimethamine Pf Plasmodium falciparum PQ Primaquine RDT R apid diagnostic test SCR S ero conversion rate S/P S ulfadoxine with pyrimethamine WHO World Health Organization

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MALARIA TREATMENT POLIC Y , M ANAG E MENT AND EPIDEMIOLOGY IN HAITI By Michael von Fricken August 2014 Chair: Bernard A. Okech , Major: Public Health Malaria remains a significant public health issue in Haiti, with 25,423 confirmed cases in 2012. However, rel iable data on malaria in Haiti is not available. T o reduce transmission of gametocytes , a single dose of primaquine (PQ) was added to the previous treatment policy of chloroquine monotherapy in 20 10 . T he use of PQ is contraindicated in people with glucose 6 phosphate dehydrogenase (G6PD) deficiency due to the risk of drug induced hemolysis. Unfortunately there is no information in Haiti on the prevalence of G6PD deficiency . These concern s will be introduced in Chapter 1 an d addressed in the subsequent chapters of this dissertation. Briefly, C hapter 2 documents the history of treatment polic ies in Haiti from 1955 to 2012 , and provides details on the rationale behind the adoption of PQ into curr ent treatment strategies . This section discusses studies that have previously occurred in Haiti, while identifying the absence of data for much of this period . Chapter 3 and 4 p resent the results of two G6PD deficiency studies implemented in Haiti. Chapt er 3 focuses on determining the prevalence of G6PD deficiency in our sample population, which was found to be 22.8% ( 14.9% to 24.7%). Chapter 4 examines the performance of the CareStart G6PD de ficiency rapid diagnostic test in a

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14 school based population . R esults suggest that the test detects severe G6PD deficiency correctly 90% of the time , indicating that this test could be incorporated into current malaria treatment strategies that rely on PQ t herapy Chapter 5 presents serological findings that were us ed to calculate an annual transmission rate in the Ouest and Sud E st departments of Haiti . A n age adjusted seroconversion rat e of 2.5% suggest s that despite the absence of sustained malaria control efforts in Haiti, transmission has remained low over mult iple decades. Chapter 6 provides a general discussion on future prospects for malaria elimination in Haiti and Hispaniola. Policy makers should consider the risks of PQ treatment in G6PD deficien t individuals , and what low transmission of malaria in Haiti entails for the future. These findings provide valuable information that can be used to guide future elimination efforts and strategies for combating malaria in Haiti.

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15 CHAPTER 1 INTRODUCTION The World Health Organization ( WHO ) estimates th at 207 million (135 million to 287 million) cases of malaria occurred globally in 2012, caused by the four species of malaria Plasmodium falciparum , P. vivax, P. ovale, and P. malariae, with the Africa Region accounting for 80% of malaria cases and 90% of malaria deaths worldwide. Malaria continues to be responsible for an estimated 627,000 (CI 473,000 789,000) deaths globally each year, of which a majority are among African children under 5 years of age [ 1 , 2 ] . In 2013, there were a total of 97 countries and territories with ongoing malaria transmission, and 7 countries in the p revention of reintroduction phase, with roughly 3.4 billion people at risk of malaria globally [ 1 ] . Malaria remains one of the top five causes of infectious disease mortality globally, ranking number four behind lower respiratory infections, HIV/ AIDS, and diarrheal diseases. Th e region of the Americas as a whole accounts for less than 1 % of all cases globally, however there is a disproport ionate risk of malaria by country with a majority of cases occurring in Haiti, the Dominican Republic, and B razil [ 1 ] . The WHO criteria for malaria elimination are based on the malaria epidemiological situation, case management practices, and the state of the surveillance system for each individual country. Currently seven countries from the region of the Americas have entered the : Argentina, Belize, Costa Rica, Ecuador, El Salvador, Mexico, and Paraguay . Malaria persists in Hispaniola despite its elimination from other Caribbean countries [ 3 ] , with t he Dominican Republic on track to achieve a 50% decrease in malaria incidence by 2015. N oticeably absent from th e WHO list of pre elimination countries is Haiti , [ 1 ] .

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16 Limited surveillance and ba seline data on malaria in Haiti pose a major obstacle to internal and external agencies att empting to document trends, quantify the impact of interventions, and monitor elimination benchmarks . O ver the past decade there has been a renewed interest in eliminating malaria from the island of Hispaniola, with a bi national stra tegy to eliminate malaria by 2020 recently adopted between the Dominican Republic and Haiti [ 4 ] . As reported cases of malaria in the Dominican Republic have reached a 15 year low of 952 reported cases in 2012 , malaria continues to burden Haiti with 25,4 23 confirmed cases and 161,236 suspected cases reported in 2012 [ 1 , 4 ] . However, Haiti has historically under reported the number of malaria cases, largely due to limited resources, inadequate surveillance and a shortage of trained personnel [ 5 7 ] . Until Haiti can adequately address malaria transmission, import ation of cases from Haiti to the Dominican Republic across the porous 275km long border, will continue to occur, making malaria control in Haiti key to sustainable malaria elimination for the island of Hispaniola. T his dissertation focuses on the history of malari a treatment policy in Haiti , the prevalence of G6PD deficiency and what it means for current treatment strategies , and modeling previous exposure to malaria as a tool for increased surveillance. Malaria in Haiti M alaria was first introduced to the Caribbea n from Africa through the slave trade. Historical records dating back to the 1780s indicate that Haiti was primarily populated by West Africans taken to the New World through the trans Atlantic slave trade [ 8 ] who were brought to the island to work in the sugar cane fields. As th e proportion of slaves and freed blacks ( g ens de coul eur ) increased compared to French colonists, stricter rules and discriminatory laws were put into effect. This eventually resulted in the 1791 Haitian Revolution, whe re Haitian slaves , le d by General Toussaint Louverture, revo lted

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17 against Fr ench rule , lea ding to eventual victory over in 1802 was heavily attributed to over 24,000 French soldiers dying of yellow fever and malaria [ 9 ] . Two hundred and ten years later , m alaria continues to present a complex public health challenge in Haiti , with roughly 80% of Haiti population of 10 million liv ing in areas where malaria is endemic [ 10 , 11 ] . I in Haiti are caused by Plasmodium falciparum transmitted by Anopheles albimanus , the principal mosquito vector in Haiti [ 12 14 ] . A. albimanus is primarily a coastal mosquito and is largely a non domestic zoophilic feeder, but will opportunis tically bite human s [ 15 ] . However, A. a lbimanus feeding behaviors may influence the effectiveness of bed net campaigns in Haiti, as they prefer feeding outside of houses at night [ 16 ] . Malaria in Haiti has been characterized as meso and hyop endemic, w here t ransmission follo ws a seasonal pattern, with peak transmission occu rring during the rainy seasons, November to February and July to August [ 17 ] . In Haiti, the number of reported malaria cases increased between 2000 and 2012, however it is unclear if this is the result o f increased surveillance or an actual rise in rates. A similar spike occurred after the January 12, 2010 earthquake, where cases increased from 32,000 to 84,000 followed by a decrease to 25,423 confirmed cases in 2012 [ 1 , 18 ] . This has also been attributed to increased surveillance and health services immediately following the 2 010 disaster. Additionally, Haiti has seen an increase in Non Governmental Organizations (NGOs) and foreign aid agency activity, especially in the health sector over the past decade [ 19 ] . Although transmission continues to occur in Haiti, findings from a 2012 country wide cross sec tional survey administered by Population Services International

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18 suggest parasite prevalence rates to be <1% [ 20 ] . However, focal transm ission has been documented elsewhere, with reported parasite prevalence ranging from 0 up to 34% in the Sud Est department and 3.1% [ 21 ] in the Artibonite valley [ 22 ] , suggesting the possibility of heterogeneous ma laria transmission in Haiti and disproportionate risk of exposure (Figure 1 1) . In an effort to reduce malaria transmission a nd address concerns of developing drug resistance, the gametocidal drug primaquine (PQ) , was added to the Haitian national treatment policy in 2010. Prior to this change, t he principal treatment for malaria in Haiti had relied primarily on chloroquine (CQ) for over 65 years, despite the loss of efficacy of CQ due to wide spread drug resistance in several countries around the globe , which will be discussed in further detail in Chapter 2 . However, PQ , like other 8 aminoquinoline drugs, can cause severe and acute hemolytic anemia in people with glucose 6 phosphate dehydrogenase (G6PD) deficiency [ 23 ] . Further complicating the matter , there is a n absence of data on the prevalence of G6PD enzyme deficiency in both malaria infected patients and the general population in Haiti . I nf ormation on G6PD deficiency rates in Haiti and whether there are tools available to accurately screen for G6PD deficiency in the field at minimal costs, is much needed and will be useful in guiding current malaria treatment policies in Haiti . Additionally , there is a n absence of clear data o n malaria transmission in Haiti . Accurate d ata must be collected if malaria transmission dynamics in Haiti are to be correctly modeled . Using sound serological methods this dissertation will provide information o n r at es of previous malaria exposure . Establishing the magnitude of malaria exposure can assist in identifying locations in need of targeted malaria control

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19 interventions, while also providing broader information on the feasibility of malaria elimination in Ha iti . As Haiti continues to work towards malaria elimination, findings from this dissertation offer valuable data on the prevalence of G6PD deficiency, the utility of a possible G6PD rapid diagnostic test (RDT) , and a malaria transmission model based on se roprevalence data. Focus of Research T his dissertation covers four closely related studies, each of which has been published or submitted to a peer review journal. Specifically, it examine s the history of treatment policy in Haiti, the prevalence of G6PD deficiency, the performacnce of a G6PD RDT , and previous malaria exposure through the detection of malaria antibodies in Haiti. An overall lack of infrastructure in Haiti presents daunting barriers to passive surveillance, which can be addressed by r obust data. Th e lack of baseline data on G6PD deficiency and serological markers presents an opportunity for this research to lay a foundation for future studies examining malaria and its potential for elimination in Haiti. Study 1 ) Examine the History o f Malaria Treatment P olicies and P ractice over the P ast 65 Y ears in Haiti Chloroquine, after 69 years of use in Haiti, is still part of the official treatment policy for malaria. A majority of malaria endemic countries globally have used CQ in the past du e to its low incidence of adverse events, therapeutic efficacy, and affordability, but were forced to switch treatment policy due to the development of widespread CQ resistance [ 1 ] . The dissertation will discuss findings from a systematic review of publications from PubMed , Web of Science, and the Armed Forces Pest Management Board, that focus on malaria treatment policies and antimalarial drug efficacy studies in

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20 Haiti between 1955 and 2012, providing a useful reference for an timalarial drug resistance studies in Haiti. After the January 2010 earthquake in Haiti, most documentation pertaining to the previous half The purpose of this review i s to create a reference that details the history of trea tment policy used by the Haitian government to treat malaria over the past several years based on published papers and reports. This study represents the first comprehensive report on historical treatment policies of malaria in Haiti, while providing a cl ear documented reference on CQ drug resistance studies in Haiti. Study 2 ) Determine the P revalence of Glucose 6 P hosphate Dehydrogenase (G6PD) D eficiency in Haiti G6PD deficiency is a hereditary enzyme abnormality in which individuals with the disorder are more susceptible to oxidative stress due to medications or certain infections. The identification of G6PD deficiency is of clinical importance especially in malaria endemic regions where severely deficient individuals are at risk of drug induced hemolysi s (DIH) when treated with the gametocytocidal drug, PQ. Due to the absence of available literature on G6PD deficiency rates in Haiti, coupled with the recent change in malaria treatment policy incorporating PQ , there is an urgent need for information on G6 PD deficiency rates in Haiti. This dissertation provide s relevant data on the rates of G6PD deficiency in Haiti, which may assist in shaping future malaria policy decisions. Chapter 3 details the first study to ever examine G6PD deficiency in Haiti. S tudy 3) Field Test the P erf ormance of the CareStart T est Administering PQ to treat malaria patients with glucose 6 phosphate G6PD deficiency can pose a serious risk of drug induced hemolysis (DIH). The current "gold standard" is to measure G6PD enzyme activity using a spectrophotometric assay that

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21 often precludes its use in low resource settings such Haiti. This dissertation evaluate s the use of a new point of care RDT , which were purchased from AccessBio (Somerset, NJ) , to identify patients with moderate and severe G6PD deficiency that would be most at risk for DIH. Since the application of the CareStart test is easier to implement than the labor intensive spectrophotometric method, and much less expensive than other G6PD RDTs [ 24 ] , we aim to discuss its feasibility and appropriateness as a point of care diagnostic tool in Haiti. Study 4) Measure the R ates of M alaria E xposure in Haiti by S erologica l S ur veys of A symptomatic and S ymptomatic P opulation s Unfortunately there is a lack of solid data that can accurately estimate the risk of malaria exposure in Haiti , which is a key metric used to determine elimination progress . Serological surveys that ar e more sensitive in dete rmining rates of exposure th a n traditional parasite prevalence rates in low transmission settings were conducted to better characterize risk of malaria exposure in the general population. These findings may help tailor future strat egies in Haiti that are geared towards total elimination of malaria from the island of Hispaniola. This chapter documents the first time that both Apical Membrane Antigen and Merozoite surface protein (MSP) , ha ve been used to determine previous exposure o f malaria in Haiti . By u sing multiple antigens to screen for previous malaria exposure, we were able to increase the sensitivity of detection. This data in turn can be used to calculate age specific malaria seroconversion rates, which can then be used to model annual incidence.

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22 Approach to Fieldwork (Studies 2 4) Ethical Approval Ethical approval was obtained from the Comité national de Bioéthique d'Haïti ( National Bioethics Committee of Haiti) , the University of Florida Institutional Review Board (I RB), and the Office of Research Protections, United States Army Medical Research and Materials Command for all research involving humans in this dissertation . All ethical guidelines provided by the University of Florida IRB have been followed, w ith our st udies receiving annual full board review. All studies were minimal risk, with participant information thoroughly de identified . Participants were given opportunities to ask our enrolling physicians questions during informational sessions prior to consenti ng. All informed consents were obtained by physicians/health care workers on site from adult participants and from the parents or legal guardians of minors. The research facilities provided by the Emergin g Pathogens Institute at the University of Florida and with our partners in Christianville, Haiti, combined with the well established infrastructure of the Community Coalition for Haiti (CCH) , were all essential to the success of these studies. In additio n to having access to adequate resources, our malaria research group has strong ties with the Ministère de la Santé Publique et de la Population ( MSPP ) and numerous non governmental agencies active in Haiti. Study Sites For this research, study enrollmen t and data collection occurred at five different sites throughout 2012 and 2013, ( F igure 1 2 ) . Study sites include Hospital Sainte Croix in Leogane, Portail Leogane Clinic & Hosanna Baptist School in Jacmel, a mobile clinic

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23 in the rura l mountain community of Chabin and the Christianville School in Gressier. Further detail on study sample sizes and population characteristics can be found in Table 1 1. Participant Enrollment Leogane Hospital Sainte Croix : From July 17 to 19 , 2012, we passively enrolle d 66 febrile patients attending Hospital Sainte Croix in Leogane Haiti. Febrile status was determined based on an axillary temperature reading of >99°F . Patients were informed of the study and consented on site by Sainte Croix staff, under the supervisio n of Dr. Gladys Memnon and Dr. Jean Rooseveld Romain. Blood samples were drawn by venipuncture into 4m L purple top collection tubes coated with ethylenediaminetetraacetic acid (EDTA), by trained phys ician and nursing staff from Hospital Sainte Croix. Part icipants were de identified by Sainte Croix nursing staff prior to analysis. Data from this location was only included in Study 2 , examining the prevalence of G6PD deficiency , as there were no serum sam ples collected at this location and enrollment occurr ed before CareStart G6PD RDTs were obtained. Jacmel Portail Leogane Clinic : From February 22 to March 10 , 2013, we passively enrolled 78 febrile patients at the Portail Leogane Clinic in Jacmel , Haiti. Febrile status was determined based on an axillary temperature reading of >99°F. Patients were informed of the study and consented on site by our collaborators at Comm unity Coalition for Haiti (CCH). Blood samples were drawn by venipuncture into 4m L serum separating collection tubes (Red Top) and 4m L EDTA collection tubes by trained physician and nursing staff from CCH. All participant information was managed and de identified by CCH collaborators prior to analysis by University of Florida personnel. Data from this location were only included in Study 2 , examining the

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24 prevalence of G6PD deficiency and Study 4 , measuring previous malaria exposure , since enrollment occurred before CareStart G6PD RDTs were obtained. Jacmel Hosanna Baptist School : February 22 to March 10 , 2013, we enrolled 16 6 asymptomatic school aged children participants from Hosana Baptist School in Jacmel, Haiti. Febrile status was determined based on an axillary temperature reading of >99°F. Community sensitization was done on two separate occasions, where parents of sch ool children were given the opportunity to ask questions pertaining to the study goals, procedures implemented, and the right to refuse participation. Parents were asked to send their signed consent forms in with their child if they wanted to participate. Blood samples were drawn by venipuncture into 4m L serum separating collection tubes (Red Top) and 4m L EDTA collection tubes by trained physician s and nursing staff from CCH. All participant information was managed and de identified by CCH collaborators p rior to analysis by University of Florida personnel. Data from this location were only included in Study 2 , examining the prevalence of G6PD deficiency and Study 4 , measuring previous malaria exposure , since enrollment occurred before CareStart G6PD RDTs were obtained. Chabin CCH Mobile Clinic Community Outreach : From March 17 to March 27 , 2013, we enrolled 228 participants in the rural community of Chabin, through mobile outreach services provided by CCH. Study enrollment was based on community based sampling, with individuals attending the mobile clinic given the option of participating in into 4m L ser um separating collection tubes and 4m L purple EDTA collection tubes by trained physician and nursing staff from CCH. All participant information was managed

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25 and de identified by CCH collaborators prior to analysis by University of Florida personnel. Data from this location was only used in Study 4 , measuring previous ma laria exposure. Data was not included in Study 2 because entire families were enrolled, potentially skewing prevalence rates. Gressier Christianville School : From May 10 to May 27 , 2013 , we enrolled a total of 573 participants from the Christianville School. With the help of our collaborators at the Christianville F oundation, I was able to sensitize and inform the community about our study, giving the parents of school children the opportunity to ask questions pertaining to the study goals, procedures implemented, and the right to refuse participation. Parents were asked to send their signed consent forms in with their child if they wanted to participate. Blood samples were drawn by venipuncture into 4m L serum separating collection tubes (Red Top) an d 4m L purple EDTA collection tubes by trained visiting nursing staff from Hospital Sainte Croix. All participant information was managed and de identified by Christianville collaborators prior to analysis by University of Florida personnel. Data from thi s location was used in Study 2 , examining the prevalence of G6PD deficiency, Study 3 , testing the performance of the CareStart G6PD RDT , and Study 4 , measuring previous malaria exposure .

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26 Figure 1 1 Malaria Risk in Haiti. Predicted malaria risk in Haiti based on MSPP data collected in 2011. Retrieved from http://globalhealthsciences.ucsf.edu/sites/default/files/content/ghg/ mei malaria elimination haiti.pdf [ 4 ]

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27 Figure 1 2 . S ites of enrollment in the Ouest and Sud Est departments of Haiti for Study 2, 3, and 4 . The inset shows the overall study area (shaded) relative to the country of Haiti, covered in this diss ertation. The capital of Port au Prince, is located approximately 16 miles due East of Gressier. The five sites of enrollment include: Gressier (Christianville school), Leogane (Hospital St. Croix), Chabin (rural community), and Jacmel (Hosana Baptist sch ool and Portail Leogane clinic).

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28 Table 1 1. Description of sites by s tud y Site City Date Population (age range) Enrolled Study 2 G6PD Prevalence Study 3 G6PD RDT Study 4 Malaria Seroprevalence Hospital Saint e Croix Leogane (u rban) Jul 12 Febrile* (ages 2 80) 66 YES (n=66) NO NO*** Portail Leogane Clinic Jacmel (urban) Feb March 13 Febrile* & Family members (ages 2 80) 78 YES (n=74) NO YES (n=72) Hosana Baptist School Jacmel (urban) Feb Marc h 13 School children (ages 4 15) 166 YES (n=137) NO YES (n=102) CCH Mobile Clinic Chabin (rural) Mar 13 Febrile* & community members (2 80) 228 NO** NO YES (n=131) Christianville School Gressier (suburban) May 13 School children (4 23 ) 573 YES (n=523) YES (n=456) YES (n=510) Total N/A N/A N/A 1111 800 456 815 *axillary temperature >99 **samples excluded, due to e nrollment of entire households with related family members ***No serum collected from site

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29 CHAPTE R 2 MALARIA TREATMENT POLICIES AND DRUG EFFICACY IN HAITI FROM 1955 2012 Chapter Summary Chloroquine (CQ), after first being introduced in 1955 , is still part of the official treatment policy for malaria. Several countries around the world have used CQ i n the past due to its low incidence of adverse events, therapeutic efficacy, and affordability, but were forced to switch treatment policy due to the development of widespread CQ resistance. The purpose of this paper was to compile literature on malaria tr eatment policies and antimalarial drug efficacy in Haiti over a 67 year period. A systematic review of PubMed, Web of Science, and the Armed Forces Pest Management Board, was conducted to find pertinent documents on national malaria treatment policies and antimalarial drug efficacy studies in Haiti between 1955 and 2012. A total of 329 citations and abstracts were reviewed independently by two researchers, of which thirty three met the final inclusion criteria of studies occurring in Haiti between 1955 and 2012 which specifically discuss malaria treatment policies and drug efficacy. Results suggest that CQ has been the predominant antimalarial drug in use from 1955 to 2012. In 2010 single dose primaquine (PQ) was added to the national treatment policy, howev er it is not clear whether this new policy has been put into practice. Although no widespread CQ resistance has been reported, some studies have detected low levels of CQ P ublished: Journal of Pharmaceutical Polic y and Practice 2013, 6:10 Available at http://www.joppp.org/content/pdf/2052 3211 6 10.pdf doi:10.1186/2052 3211 6 10

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30 resistance. Increased surveillance and monitoring for CQ resistance should be impleme nted in Haiti. Background The antimalarial chloroquine (CQ) w as first introduced into Haiti in 1955 as part of the Global Malaria Eradication Program (GMEP), where it was relied on heavily as the, due to its low incidence of adverse events, affordability, and perceived therapeutic efficacy . Prior to 1955, the only antimalarial treatment in Haiti was quinine , which was used intermittently during periods of US occupation [ 25 ] . However, the wide spread emergence of CQ resistance throughout malaria endemic countries in Africa, South Eas t Asia, and much of South America, forced changes in national treatment policies elsewhere. Haiti appears to have remained committed to using CQ, despite resistance elsewhere; however documentation of previous official Haitian treatment policies is limited . and publications on malaria chemotherapeutic policies and antimalarial resistance in Haiti from 1955 to 2012 based on a systematic search of historical literature. The purpose while providing a useful reference for antimalarial drug resistance studies in Haiti. Materials & Methods Data Sources Published studies and reports about malaria in Haiti we re identified from an electronic search of MEDLINE®/PubMed ® and Web of Science, with the search years confined to 1955 2012. A search of gray literature was carried out on the Armed Forces Pest Management Board (AFPMB) [ 26 ] , which contains publications and reports from

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31 the Centers for Disease Control and Prevention (CDC), World Health Organization (WHO), and Pan American Health Organization (PAHO). Search terms were chosen to capture malaria treatment policy and drug efficacy studies in Haiti only, using a combination of simple sub ject headings and term combinations, focusing on treatment, malaria, and Haiti. Study Selection The selection criteria of the data sources included are : 1) reports from the period of 1955 to 2012, 2) studies carried out in Haiti, 3) and studies that di scuss malaria. Full text manuscripts and all citations which met the predefined selection criteria were obtained and examined by t hree independent researchers , Michael von Fricken, Thomas Weppelmann, and Jennifer Hosford . Final inclusion and exclusion deci sions were made with 100% agreement between the examiners based on studies that reported malaria treatment policies and drug efficacy for final inclusion. Where duplications were observed the most recent version of the manuscript was selected. The year 1 955 was used as a start date for analysis, because it coincided with the Global Malaria E radication Program (GMEP) campaign in Haiti. A full list of studies examined can be found in Table 2 1. Results A detailed description of study selection can be found in F igure 2 1. A total of 329 citations and abstracts in the electronic searches were found, of these, 85 citations and abstracts met the preliminary criteria and had their full texts reviewed. Thirty three studies described malaria treatment policies or antimalarial drug efficacy in Haiti from 1955 2012 [ 3 , 5 7 , 10 , 12 15 , 17 , 22 , 27 52 ] .

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32 Characteristics of I ncluded S tudies Chloroquine was first introduced in Haiti in 1955, when The Eighth World Health Assembly recommended its use in combination with pyrimethamine (P) for the elimination of malaria. The chloroquine/p yrimethamine (CQ/P) combination strategy was the first treatment policy switch in Haiti replacing the previous accepted practice of quinine for the treatment of malaria [ 27 , 28 , 53 ] . According to the data sources, from 1955 to 1970, CQ/P was administered in a prophylactic manner through mass drug administration (MDA) campai gns, as part of the GMEP in Haiti [ 27 , 29 , 32 , 33 ] . From 1970 to 2010, the treatment for malaria switched from a combination therapy of CQ/P therapy to CQ monotherapy for uncomplicated malaria cases [ 33 ] . In 2010 a CDC report mentioned the addition of the transmission blocking drug primaquine (PQ) at a single dose of 0.75 mg/kg, sugg est ing a major change in policy [ 52 ] . This policy change was based on a PQ tolerance study that was implemented by the CDC and the Carter Center in the city Ouanaminthe, which lies directly on the Haiti DR boarder [ 4 ] . All final articles (N = 33) that we evaluated reported CQ as or part of the standard treatment policy in Haiti. A brief summary of treatment policies can be seen i n the timeline provided (Figure 2 2 ). E vidence for Treatment R esistance in Haiti: Only fourteen studies from the data sources examined [ 10 , 15 , 29 , 30 , 32 36 , 38 , 39 , 44 , 47 , 51 ] discussed chemotherapeutic efficacy or patien t treatment outcome data(Table 2 2 ). Six of these fourteen studies contained documentation of resistance to CQ or P [ 10 , 33 , 35 , 36 , 38 , 47 ] . The first year of reported of anti malarial resistance, where P. falciparum had documented pyrimethamine resistance was 1971 [ 33 ] . In 1982, a report of possible resista nce to CQ treatment was suggested [ 10 ] after a patient on CQ

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33 monotherapy had resurgence in parasitemia on day 28 of treatment. A follow up in vitro test found 4/16 P. falciparum cultures including the P. falciparum strains from the patient with resurgent parasitemia, required CQ doses large enough that suggested possible resistant P. falciparum parasites. This report provided the first credible in vitro evidence of P. falciparum resistance to CQ in Haiti [ 35 ] . In 1984, an in vivo and in vitro study, tested for P. falciparum parasite resistance to sulfadoxine/pyr imethamine (S/P) to provide baseline data on susceptibility in the event that CQ resistance developed in Haiti. The results indicated parasite resistance to pyrimethamine alone, but not to sul fadoxine. Despite in vitro evidence of resistance to pyrimethamine, all in vivo infections were susceptible to a combination of S/P [ 36 ] . In 1985, another in vivo study to complement the previous in vitro assay found resistance to pyrimethamine alone [ 38 ] . More recently, a report by Londono et al, documented the detection of genetic markers via PCR, for CQ resistance in five of 79 patients (6%) from the Artibonite V alley [ 47 ] . However, a study by Neuberger et al, found zero of 49 infected patients to have muta tion s suggestive of CQ resistance [ 52 ] . These studies present conflicting evidence about the extent of CQ drug resistance in the p opulations studied, with no definitive documentation of in vivo resistance reported. Discussion Following the widespread use of CQ/P from 1955 1968, resistance to pyrimethamine was reported in Haiti in 1971 [ 33 ] . It was suggested that parasite ada ptation to pyrimethamine had occurred due to selective drug pressure from the MDA campaign, which ended in 1968, resulting in its removal from the national treatment polic y [ 33 , 36 ] .

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34 Haiti continued to rely predominantly on CQ as part of the treatment for malaria in Haiti up until 2010, when single dose PQ was added to the official policy for the treatment of uncom plicated malaria in Haiti [ 14 , 50 ] . Haiti represents a unique scenario where PQ has not been used previously on the island due to the absence of Plasmodium vivax, but is being introduced to specifically block malaria transmission by targeting the adult stage Plasmodium falciparum parasites. It remains unclear whether PQ has been implemented in practice, nor have any studies examined population rates of G6PD deficiency in Haiti, a genetic mutation that places deficient individuals at r isk of acute hemolytic anemia when exposed to PQ. Overall, we found the use of CQ has featured prominently in the management of malaria in Haiti for decades. While CQ resistance in all of Africa, South East Asia, and much of South and Central American c ountries has forced these countries to change policies, the Haitian Ministry of Health (MSPP) has continued to rely almost entirely on CQ as the principal treatment for malaria. Despite such prolonged use, our results found no conclusive evidence of CQ re sistance rates exceeding the WHO [ 54 ] in Haiti (Table 2 2 ). However, findings from the studies that report on treatment sensitivity are lacking d ue to small sample sizes , localized enrollment , and miss ing information on patient treatment outcomes, significantly limiting their ability to influence nati onal treatment policies . Ten of the fourteen studies that make mention of drug efficacy, relied solely on secondary sources of data, often only containing brief comments on treatment effectiveness, which further suggest an absence of designed drug sensitivity studies occurring in Haiti [ 6 , 10 , 15 , 29 , 30 , 32 35 , 47 ] . The two most recent studies examining CQ resistance by

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35 Londono and Neuberger gave different results about the presence of genetic marke rs of CQ resistance in Haiti [ 47 , 52 ] . London found 5 /79 ( 6 %) carried genetic markers for resistance, but had no information on treatment failure, which is the me tric used by the WHO to warrant a change in national treatment policy. Neuberger found no markers for CQ resistant parasites, but was limited by a small sample size of 48. It would appear CQ resistance mutations are emerging in Haiti, but to what extent remains unknown, suggesting a need for increased surveillance for parasite resistance. Limitations We were unable to examine gray literature from the MSPP as most documents were lost during the 2010 earthquake. Although we excluded non English databases, we believe this omission to not be very significant, since we relied heavily on WHO and PAHO reports, which are based on both English and non English databases and reports. There were limited d ata pertaining to treatment failures and parasite resistance, in addition to an overall lack of reporting between 1985 2005 due to political unrest [ 5 , 47 ] . Chapter Conclusions As of 2012, t he few documented reports of CQ resistance in Haiti d id not exceed [ 54 ] . However, due to limited surveillance on drug efficacy and barriers to patient follow up, further studies are necessary to determine if rates of CQ resistance fall below this threshold in Haiti. Meanwhile, Non government organizations, foreign aid agencies, and the Haitian MSPP should consider implementing a comprehensive malaria control program while CQ remains a viable treatment option [ 47 , 51 , 52 ] . Other drug regimens, such as artemisinin based combination therapies, are part of an arsenal of available treatment options, in

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36 adverse events make it an ideal treatment option on a large scale. It is our recommendation that increased surveill ance and monitoring for CQ resistance be implemented, due to significant gaps in data on CQ treatment failure and resistance in Haiti. Regarding the recent addition of PQ to the national policy, more information is needed on how PQ is tolerated in this po pulation, given the absence of information on G6PD prevalence rates, and whether or not this policy has been put into practice in Haiti. Future studies examining transmission rates in Haiti may generate valuable information on the impact single dose PQ ha s on malaria rates, potentially providing a template for elimination in other low transmission settings globally.

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37 Table 2 1 R eports and publication discussing malaria treatment policy in Haiti . Publications and reference material that document trea tment and policy for malaria management in Haiti summarized by period, site in Haiti and anti malarial medication Period Site Haiti Treatment P olicy Ref 1955 Country Wide Mass treatment of CQ/P [ 27 ] 1955 Country Wide Mass treatment of CQ/P [ 28 ] 1960 1966 Country Wide Mass treatment of CQ/P [ 30 ] 1963 Country Wide CQ [ 15 ] 1962 1965 Country Wide Mass treatment of CQ/P [ 29 ] 1962 1965 Petit Goave Mass treatment of CQ/P [ 32 ] 1971 Country Wide CQ [ 33 ] 1972 75 Miragoane Valley CQ [ 13 ] 1976 Country Wide CQ [ 34 ] 1976 1979 Country Wide CQ [ 6 ] 1982 Port au Prince CQ [ 35 ] 1982 Port au Prince CQ [ 36 ] 1980 1983 Country Wide CQ [ 37 ] 1972 1983 Country Wide CQ [ 39 ] 1985 Port au Prince CQ [ 38 ] 1982 1986, 1988 1991 Artibonite Valley CQ [ 43 ] 1981 1983 Country Wide CQ [ 10 ] 1985 Les Cayes and Jeremie CQ [ 40 ] 1991 Country Wide CQ [ 42 ] 1995 Country Wide CQ [ 5 ] 1995 Country Wide CQ [ 44 ] 1995 Country Wide CQ [ 45 ] 1975 1997 Limbe River Valley CQ [ 17 ] 1996 1999 Country Wide CQ [ 46 ] 2006 Artibonite Valley CQ [ 22 ] 2006 Artibonite Valley CQ [ 55 ] 2006 2007 Country Wide CQ [ 47 ] 2010 Country Wide CQ [ 50 ] 2010 Country Wide CQ [ 48 ] 2010 Hispaniola CQ [ 3 ] 2000 2010 Country Wide CQ+PQ [ 14 ] 2010 2011 Leogane CQ [ 52 ]

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38 Figure 2 1. Selection process for systematic review of studies examining malaria treatment policies in Haiti between 1955 and 2012. Figure 2 2. Timeline of Haitian malaria treatment policies

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39 Table 2 2. Reports of Treatment Efficac y for Malaria in Haiti . Chloroquine (CQ), pyrimethamine (P), chloroquine with pyrimethamine (CQ/P),sulfadoxine with pyrimethamine (S/P), in vivo susceptibility (VV), in vitro susceptibility (VT), DNA analysis for mutations polymerase chain reaction (PCR) . Period Sample Size Location Drug Data Method Resistance reported Susceptible Ref 1960 1966 Unknown Country Wide CQ/P Secondary Unknown No CQ [ 30 ] 1961 Unknown Country Wide CQ Secondary Unknown No CQ [ 29 ] 1962 1965 Unknown Country Wide CQ/P Secondary Unknown No CQ/P [ 15 ] 1962 1965 Unknown Petitie Goave CQ & CQ/P Secondary Unknown No CQ/P [ 32 ] 1971 Unknown Country Wide CQ Secondary VV P & CG CQ [ 33 ] 1976 Unknown Country Wide N/A Secondary Unknown N o CQ [ 34 ] 1980 Unknown Country Wide CQ Secondary Unknown No VV CQ [ 6 ] 1981 1 983 92 Country Wide CQ Secondary VT & VV CQ CQ [ 10 ] 1982 19 Port au Prince CQ Secondary VT & VV CQ No [ 35 ] 1982 18 Port au Prince P & S/P Primary VT & VV P S/P [ 36 ] 1985 22 Port au Prince P & S/P Primary VT & VV P S/P [ 38 ] 1995 Unknown Country Wide N/A Primary VV N o CQ [ 44 ] 2006 2007 79 Artibonite Valley CQ Secondary PCR CQ N o [ 47 ] 2010 2011 49 Leogane CQ Prima ry PCR N o CQ [ 52 ]

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40 CHAPTER 3 PREVALENCE OF GLUCOSE 6 PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY IN THE OUEST AND SUD EST DEPARTM ENTS OF HAITI Chapter Summary Malaria remains a significant public health issue in Haiti, with chloroquine (CQ) used almost exclusively for the treatment of uncomplicated infections. Recently, single dose primaquine (PQ) was added to the Haitian national malaria treatment policy, despite a lack of information on the prevalence of glucose 6 phosphate dehydrogenase (G6PD) deficiency within the population. G6PD deficient individuals who take PQ are at risk of developing drug induced hemolysis (DIH). In this first study to examine G6PD deficiency rates in Haiti, 22.8% (range 14.9% 24.7%) of participants were found to be G6PD deficient (class I, II, or III) with 2.0% (16/800) of participants having severe deficiency (class I and II). Differences in deficiency were observed by gender, with males having a much higher prevalence of severe deficiency (4.3% vs. 0.4%) compared to females. Male participants were 1.6 times more likely to be classified as deficient and 10.6 times more likely to be classified as severely deficient compared to females, as expected. Finally, 10.6% (85/800) of the participants were considered to be at risk for DIH. Males also had much higher rates than females (19.3% vs. 4.6%) with 4.9 times greater likelihood (p value 0.000) of having an a ctivity level that could lead to DIH. These findings provide useful information to policymakers and clinicians who are responsible for the implementation of PQ to control and manage malaria in Haiti. Published: Acta Tropica 2014, 135 ;62 66 Available at http://www.sciencedirect.com/science/article/pii/S0001706X14000941 doi: 10.1016/j.actatropica.2014.03.011.

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41 Background nationa l treatment policy for malaria has relied heavily on chloroquine (CQ) as the principal treatment for many years, with limited evidence of widespread CQ resistance, [ 47 , 52 ] despite its prolonged use [ 56 ] . In an effort to decrease the risk of chloroquine resistance developing and reduce malaria transmission, the Haitian Ministry of Health (MSPP) recently adopted the anti malarial drug primaquine (PQ) to its national treatm ent policy . This decision was based on a small PQ tolerance study by the CDC and the Carter Center in the city Ouanaminthe , Haiti, which lies directly on the Haiti DR boarder [ 4 , 50 , 57 ] . Of particular concern are drug induced adverse reactions in individuals that carry X linked mutations for the enzyme glucose 6 phospha te dehydrogenase (G6PD ) [ 58 60 ] . Globally, G6PD deficiency is estimated to affect approximately 400 million people, with higher rates observed in West Africa (Figure 3 1). The metabolic activities of G6PD maintain the level of nicotinamide adenine dinucleotide phosphate oxidase (NADPH), which in turn sustains glutathione levels in red blood cells. Since red blood cells lack mitochondria, this pathway is the only source of NADPH, thus G6PD deficient individuals are unable to produce adequate amounts of glutathione to neutralize reactive oxygen species (ROS) predisposing them to non immune hemolytic anemia [ 61 ] . Affected individuals, many of whom are unaware of their G6PD status [ 23 , 61 ] , may suffer oxidativ e stress triggered by PQ leading to drug induced hemolysis (DIH), which can be fatal in severely deficient individuals. A recent study in 2014 by Eziefula et al. examined Plasmodium falciparum gametocyte clearance post treatment with single dose PQ at 0.1 mg/kg, 0.4 mg/kg and 0.75 mg/kg, in G6PD normal individuals [ 62 ] . Findings suggest that 0.4 mg/kg has similar gametocytocidal

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42 activity as 0.75 mg/kg and that further efficacy and safety trials are necessa ry to examine dosage between 0.1 mg/kg, 0.25 mg/kg, and 0.4mg/kg. While these results are promising, a key issue with the study design is that it provides no information on PQ tolerance in G6PD deficient individuals, most likely due to ethical constraints . Given the risks of DIH and lack of data on G6PD deficiency prevalence in Haiti, there are serious concerns about administering PQ to malaria infected patients with unknown G6PD status. Risks associated with administering PQ are compounded by a weak Hea lthcare system in Haiti , where patients are rarely seen after their initial appointment, limiting the ability of health care workers to monitor drug induced adverse events [ 4 ] . Thus, there is a great need to determine G6PD deficiency rates in Haiti befor e this policy can be safely implemented. This study is the first of its kind to investigate the prevalence of G6PD deficiency in Haiti with the goal of providing decision makers with information that will be a useful basis upon which to implement a PQ ba sed treatment policy. Materials and Methods Study Location This study was conducted in the Ouest and Sud Est departments of Haiti (Figure 3 2 ) between July, 2012 and May, 2013. Along the northern coast of the Ouest department, participants were recruited f rom Hospital Saint e Croix in Leogane and the Christianville School in Gressier . In the Sud Est department, participants were recruited from the coastal city of Jacmel at the Portail Leogane Clinic and Hosana Baptist School. Quality Assurance It was decided to exclude the collection site of Chabin (n=142) from analysis due to a flaw in our sampling methodology. At Chabin, e ntire households were enrolled in

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43 the study, potentially skewing results due to the hereditary nature of G6PD deficiency. We felt this necessary and prudent to avoid any misinterpretation of our findings. It has been suggested that G6PD deficiency grants some protection against malaria infection, potentially causing G6PD normal individuals to be over represented in our clinical pop ulations [ 63 ] . However, the protection conferred by G6PD deficiency is minimal and malaria is thought to be hypoendemic in Haiti, limiting the influence our sampling methods had on our prevalence rates. Study Enrollment and Sample Collect ion A total of 828 participants (337 males and 491 females) were enrolled between the ages of 2 and 87. Enrollment was based on sampling from both clinical and non clinical settings. Patients attending Hospital Sainte Croix in Leogane, and Portail Leogane Clinic in Jacmel, were enrolled on a voluntary basis. Healthy children were enrolled from the Christianville School in Gressier and from the Hosana Baptist School in Jacmel. Participants were given opportunities to ask our enrolling physicians questions during informational sessions prior to consenting. After obtaining consent from participants or their guardian, local clinicians collected ~ 3 m L of blood by venipuncture, into EDTA tubes from participants. G6PD activity was measured within 3 hours of col lection. Determination of G6PD Activity A commercially available quantitative G6PD deficiency diagnostic kit (No. 345B, Trinity Biotech, St. Louis, MO, USA) was used to measure G6PD activity in blood samples. Briefly, aliquots of blood in purple top vac uum tubes were added to cuvettes containing glucose 6 phosphate, buffer, magnesium salt, and maleimide. The rate of

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44 minutes incubation at 25°C at two time points five minutes apart. Hemoglobin levels were determined by a digital hemoglobin meter (HemoCue Incorporated, Cypress, CA, USA). Dehydrated blood samples supplied by the manufacturer for normal (No. G6888, Trinity Biotech, St. Louis, MO, USA) and deficient (No. G5888, Trinity Bi otech, St. Louis, MO, USA) levels of G6PD enzyme activity were used as controls. A few samples (28/828 (3.4%) with detected G6PD activity levels above the manufacturer specified activity limit for the assay (19.5 U/ g Hgb) were not considered in this stud y. Establishing Cutoff Values in our P opulation G6PD deficiency was classified as previously described by Kim et al., 2011. Briefly, samples from 299 healthy participants with a G6PD activity above 4.6 U/g Hgb and a hemoglobin concentration equal to or a bove 12 g/d L were used to define the population mean. G6PD deficiency was classified using the WHO classifications of enzyme activity as a percentage of the population [ 41 ] . For this study with a population mean of 9.40 U/ g Hgb the classifications were as follows: (i) Class I: very severely 10% residual activity, >0.094 to 0.940 U/g Hgb, (iii) Class II I: mildly to moderately deficient, >10 to 60% residual activity, >0.940 to 5.64 U/g Hgb, (iv) Class IV: normal activity, >60 to 150% residual activity, >5.64 to 14.1 U/g Hgb, and (v) Class V: increased activity, >150% residual activity, >14.1 U/g Hgb. For further comparison of Individuals with moderate and severe G6PD activity lev els (< 30% residual activity) are considered at risk for DIH [ 64 ] .

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45 Statistical Analysis Histograms and tables of descriptive statistics of hemoglobin concentration (g Hgb/d L ), G6PD activity, and G6PD deficie ncy classification were generated by gender, and study location (Table 3 1) . Logistic regression (Stata ® v 12, StataCorp, College Station, TX, USA) was performed to determine the association between gender and G6PD deficiency as well as the study location on the likelihood of being deficient (class I, II, or III), severely deficient (class I and II only), or at risk of DIH (<30% residual activity). To address concerns of sampling bias, G6PD deficiency was also analyzed by method of study enrollment, i.e. c linical versus school based enrollment, using logistic regression and adjusting for gender (Table 3 2). Results A total of 800 participants (326 males and 474 females) were included in the study. Overall 22.7% of participants were found to be G6PD defic ient (range: 14.9% 24.7%), with 10.63% of participants considered at risk for DIH (range: 6.8% 12.0%), and 2.0% of participants classified as having severe deficiency (range: 0.0% 3.0%). According to the WHO classification system 0.13% had very seve re deficiency (class I), 2.0% had severe deficiency (class II), 20.7 had moderate and mild deficiency (class III), 70.3% had normal activity (class IV), and 7.0% had increased activity (class V). Class III deficiency was further differentiated by moderate (8.6%) and mild (12.1%) deficiency. Summary statistics of the age, G6PD activity (U/g Hgb), and G6PD classification appear in Table 3 1. The distribution of G6PD activity l evels is presented in Figure 3 3 . Differences in deficiency were observed by gende r, with males having much higher prevalence of deficiency (27.6% vs. 19.4%), DIH (19.3% vs. 4.6%), and severe deficiency (4.3% vs. 0.4%) compared to females. This can be seen in the histograms by

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46 gender (Figure 3 4 ), where a bimodal distribution of G6PD ac tivity is observed in males, but not in females, which is expected given the X linked nature of G6PD deficiency, thus placing males at higher risk for DIH than females. The statistical associations between gender, location, or enrollment type and the prese nce of mild, moderate, or severe deficiency is presented in Table 3 2. Compared to females, male respondents were 1.6 times more likely to be classified as deficient (95% Confidence Interval OR: 1.14 2.21), 4.9 times more likely to be at risk for DIH (95 % Confidence Interval OR: 2.96 8.18) and 10.6 times more likely to be classified as severely deficient (95% Confidence Interval OR: 2.40 46.91). The prevalence of deficiency was not significantly different by the enrollment location (p value = 0.21) or between clinical versus school based enrollment (p value = 0.161). Discussion Our results indicate that G6PD deficiency is quite prevalent in the Ouest and Sud Est departments of Haiti. We found that 22.7% (14.9% 24.7%) of the sample population had s ome form of G6PD deficiency, with 10.63 % (range 6.8% 12.0%) of our sample having moderate or severe G6PD deficiency. This finding is not surprising given the ancestry of native Haitians, who mostly descended directly from West Africa, where similar G6PD deficiency rat es (10 27%) have been reported [ 31 , 65 ] . Males were much more likely to be severely d eficient than females as is consistent with other G6PD deficiency studies, and not surprising due to the X link ed pattern of G6PD inheritance [ 66 , 67 ] . Given that over 10% of the population are at risk of DIH (<30% residual activity), and the relatively low transmission of malaria in Haiti; point of care G6PD screening may be feasible and practical to prevent th e risk of PQ induced adverse reactions in malaria patients. Ideally, confirmation of G6PD status would precede

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47 administration of PQ; however this can be difficult in resource limited settings. Therefore, i f G6PD deficiency is unable to be measured, it ma y be appropriate to reduce the current dosage of PQ from a single dose at 0.75 mg/kg to a more conservative single dose of 0.25 mg/kg treatment policy for individuals with unknow n status , as recently suggested [ 68 ] . Limitations These findings on G6PD deficiency are limited by the sites of enrollment, and may not be representative of the general Haitian population. Sampling individuals from both clinical and non clinical sites represents a potential sampling bias; however statistical analyses did not indicate significant differences in the rates of G6PD deficiency by place or method of enrollment , as mentioned . Chapter Conclusions This study provides valuable preliminary information on the presence of G6PD deficiency in the Ouest and Sud Est departments of Haiti. We recommend t esting should be expanded to other departments in Haiti to provide the government and other policy implementer s with more information on G6PD deficiency prevalence rates, which may be used to fine tune future malaria treatment policies that include PQ. Further research needs to be conducted to examine the magnitude of DIH associated with single dose PQ in G6PD de ficient individuals before this policy can be implemented safely. Globally, PQ has been used for the radical cure of Plasmodium vivax malaria infections, providing policy makers with historic data on PQ treatment outcomes [ 69 ] . However, there is no prior documentation of PQ ever being used in Haiti at scale. Despite the uncertainty of how PQ would be tolerated in this population, Haiti presents a unique opportunity to study the impact and adverse effects associated with PQ

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48 administration. Due to the li mited potential for future reintroduction of malaria into Haiti and the circulation of only Plasmodium falciparum parasite species, one could hypothetically monitor the impact PQ has on malaria elimination in this island set ting. However, caution would be advisable, given the relatively high rates of G6PD deficiency observed in this study.

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49 Figure 3 1 World map distribution of G6PD deficiency. Retrieved from doi: 10.1016/S0140 6736(08)60073 2 . Reprinted from The Lancet , Vol. 371, MD Cappellini, G Fiorelli. Glucose 6 phosphate dehydrogenase deficiency, Pages 64 74, Copyright (2008), with permission from Elsevier [ 61 ]

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50 Figure 3 2 . Study area in the Ou e st and Sud Est departments of Haiti . The inset shows the study area (shaded) relative to the country of Haiti, and the capital of Port au Prince, located approximately 16 miles due East of Gressier. The enlarged study area contains the four sites of enrollment: Christianville S chool in Gressier , Hospit al Saint e Croix in Leogane , and the Hosana Baptist S chool and Portail Leogane both of which are located in the city of Jacmel .

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51 Table 3 1 Summary statistics of study participants by gender and location . The summary statistics of the age, hemogl obin concentration, G6PD activity, and classification of G6PD function appear with the number of samples, averages, and standard deviations for males and females and from the five study locations. (S) denotes participants enrolled from schools, (C) repres ents participants enrolled from clinics. Age G6PD activity G6PD classification Units Obs Years (U/g Hgb) I, II, or III III III I, II (n) Avg. (Sd.) Avg (Sd) Deficient Mild Moderate Severe < 60% 60 30% 30 10% <10% Gender Female 474 14. 4 (9.6) 8.6 (3.5) 19.4 14.8 4.2 0.4 Male 326 13.4 (9.0) 8.0 (4.3) 27.6 8.3 15.0 4.3 Location Christianville (S) 523 12.1 (4.3) 8.4 (3.8) 24.7 12.6 9.4 2.7 Hosana Baptist (S) 137 12.1 (10.4) 8.4 (3.8) 20.4 13.1 7.3 0.0 Portail Leogane (C) 74 29. 1 (17.4) 8.5 (3.2) 14.9 8.1 6.8 0.0 Hospital S t. Croix (C) 66 23.4 (1.3) 8.4 (4.3) 21.2 10.6 7.5 3.0 Total 800 14.0 (9.4) 8.37 (3.9) 22.8 12.1 8.6 2.0

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52 Table 3 2. The statistical relationships of G6PD deficiency between gender and enrol lment locations . The statistical relationships between gender and locations are presented (compared to those not in the group) for the likelihood of having moderate to severe deficiency (less than 30% residual G6PD activity) and severe deficiency (less tha n 10% residual G6PD activity). The odds ratios and confidence intervals for the likelihood of having moderate to severe deficiency, or being severely deficient have been adjusted for the gender of the participants. Predictor Mild to severe deficiency Se vere deficiency Residual activity < 60% Residual activity < 10% OR p value 95% Conf. int. OR p value 95% Conf. int. Gender Female 0.632 0.007 0.453 0.881 0.094 0.002 0.021 0.418 Male 1.583 0.007 1.135 2.209 10.59 0.002 2.390 46.91 L ocations Gressier (S) 1.384 0.076 0.966 1.982 3.782 0.080 0.853 16.76 Jacmel (S) 0.849 0.479 0.540 1.335 omitted Jacmel (C) 0.567 0.093 0.292 1.100 omitted Leogane (C) 0.907 0.756 0.491 1.677 1.607 0.536 0.357 7.228 Enrollment T ype School 1.400 0.161 0.875 2.240 1.326 0.713 0.294 5.975 Clinic 0.714 0.161 0.446 1.143 0.754 0.713 0.167 3.399 adjusted for gender

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53 Figure 3 3 . Histogram of G6PD activity (U/g Hgb) for the entire study population . The distribution of G6PD activity (U/g Hgb) appear with a normal distribution (orange dashed line) as well as two reference lines denoting 10% residual activity and 60% residual activity (red dashed lines), and a dotted blue line for 30% residual activity (risk of drug se nsitivity).

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54 Figure 3 4 . Histogram of G6PD activity (U/g Hgb) by gender . The distribution of G6PD activity (U/g Hgb) appears by gender with two reference lines denoting 10% residual activity and 60% residual activity (red dashed lines), and dotted blue line for 30% residual activity (risk of drug sensitivity).

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55 CHAPTER 4 PERFOMANCE OF THE CARESTART G6PD RAPID DIAGNOSTIC TEST (RDT) IN GRESSIER, HAITI Chapter Summary Administering primaquine (PQ) to treat malaria patients with G 6PD deficiency can p ose a serious risk of drug induced hemolysis (DIH). Thus, it is important to test patients for G6PD deficiency before administration of PQ. However, current methods are labor intensive and expensive. New easy to use point of care rapid diagnostic tests (R DTs) are being developed as an alternative to these labor intensive spectrophotometric methods, but require field testing before the can be used at scale. To assess the specificity and sensitivity of a new rapid G6PD deficiency test, 456 participants were screened i n Gressier, Haiti, using the new Access Bio CareStart qualitative G6PD RDT compared against the lab based Trinity Biotech quantitative spectrophotometric assay , which has been used as a gold standard in similar studies . Findings suggest that the test was 90% sensitive for detecting individuals with severe deficiency and 84.8% sensitive for detecting individuals with moderate and severe deficiency when compared against the Trinity Biotech assay. A high negative predictive value of 98.2% indicates excellent performance in determining those able to take PQ safely. The G6PD test holds much value for screening malaria patients to determine eligibility for PQ therapy. Published: American Journal of Tropical Medicine and Hygiene (in press) Avail able at http://www.ajtmh.org/content/early/2014/04/24/ajtmh.14 0100 doi:10.4269/ajtmh.14 0100

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56 Background As mentioned in Chapter 3, ssociation with P Q induced hemolysis, also called primaquine sensitivity, poses a major obstacle for malaria control strategies that attempt to block malaria transmission by targeting gametocytes [ 59 ] . A ffected individuals, many of whom are unaware of their G6PD activi ty, may suffer oxidative stress triggered by PQ leading to drug induced hemolysis (DIH), which can be fatal in severely deficient individuals [ 23 , 61 ] . The ability to rapidly identify this deficiency has become more important in recent years, as malaria endemic countries around the world adopt PQ into their standard treatme nt regimen [ 18 ] . Conce rns of G6PD deficient individuals developing DIH after treatment with PQ have also delayed the use of this antimalarial drug in many countries [ 70 ] . The determination of G6PD deficiency in patients is particularly relevant to the treatment of malaria in Haiti, due to the recent addition of single dose PQ to the national malaria treatment policy [ 18 , 50 ] . As malaria transmission continues to occur in Haiti, wi th 25,423 confirmed cases and 161,236 suspected cases reported in 2012 [ 1 ] , this new policy could represent a major population level risk of drug induced hemolysis, given the likely similarities between G6PD rates in modern Hai tians and their ancestral West Africa ns [ 50 ] . As described in Chapter 3 , G6PD deficiency rates range d from 1 4.9% to 24.7% of the population in a sample of 800 Haitians from the Ouest and Sud Est Departments [ 71 ] . The current quantitative standard for detecting G6PD activity levels is the spectrophotometric assay, which measures the enzymatic activity of G6PD. This test requires a consistent source of electricity, refrigeration for reagents and substrates, information on patient hemoglobin levels, a spectrophotometer to measure the change

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57 of absorbance, and skilled personnel to perform the test, making it difficult to use in resource poor settings. To increase the ability to perform point of care asse ssment of G6PD activity, Access Bio has recently developed the G6PD qualitative rapid diagnostic test (RDT). The G6PD Deficiency Test is a qualitative test that uses a filter strip coated with reagents and substrates that changes color from white to purple in the presence of adequate G6PD concentrations. T he manufacturer of the RDT stated that the test has at least a sensitivity of 95% for detecting Class I and II deficiency classified by the World Health Organization (WHO), diagnosing those most at risk of experiencing DIH upon treatment with PQ (available at http://www.accessbio.net/pr/down/Acessbio_Brochure_eng.pdf ) . To explore the feasibility of a less labor intensive diagnostic test, we examined the performance of the colorimetric test against the Trinity Biotech spectrophotometric assay, which has been used as a comparative standard for testing the performance of other G6PD qualit ative tests [ 7 2 , 73 ] . Methods and Materials Study L ocation, Informed C onsent, and S ample C ollection The validation of the RDT was conducted as part of a larger study (n=1007) in Ouest and Sud Est departments of Haiti , which was described in Chapter 1 . A total of 456 primary school children (267 females and 189 males) in Gressier, Haiti were tested using both methods during the month of May 2013. After informed consent by parent or legal guardian was received, local health care providers collected approximately 3m L of blood EDTA treated tubes until laboratory analysis of G6PD activity. Sample s were screened with the Access Bio RDT and the Trinity Biotech quantitative assay at the

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58 University of Florida field laboratory at constant temperature (25 °C) within 48 hours of collection. As described in Chapter 3, c lassification of G6PD activity was made using the World Health Organizat ion recommendations of residual enzyme activity as a percentage of the mean G6PD activity [ 41 ] . Using a method similar to that previously described, the mean of this population was set using a subsample (n=168) of healthy, non anemic participants that had G6PD activity levels greater than or equal to 4.6 U/g Hgb and hemoglobin concentrations greater than or equal to 12 g Hgb/d L . This gave a population mean of 9.5 U/g Hgb that was used to define the mild, moderate, and severe G6PD deficiency. They are as follows: (i) severe deficiency (Class I and II) with residual activity less than 10% of the mean ( less than 0.95 U/g Hgb), (ii) moderate deficiency (Class III) with residual activity between 10% and 30% of the mean (0.95 to 2.84 U/g Hgb), and (iii) mild deficiency (Class III) with residual activity between 30% and 60% of the mean (2.84 to 5.68 U/g Hgb). Individuals with moderate and severe G6PD activity levels (< 30% residual activity) are conside red at risk for DIH [ 64 ] . The performance measures (sensitivity, specificity, negative predictive value, positive predictive value, and accuracy) of the Access Bio RDT were calculated (Stata ® 12, S tataCorp, College Station, Texas, USA), using the Trinity Biotech assay to determine The CareStart Qualitative G6PD D eficiency RDT The G6PD Defi ciency Test (Cat# G0223, Access Bio, Somerset, NJ , USA) was used according to manufacturer instructions. Briefly, two microliters (µ L ) of blood were pipetted from EDTA vacutainers and added to the sample wells using the pipet provided, followed by the addition of two drops of assay buffer supplied by the

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59 manufacturer. All results were read within 10 minutes of administering the test according to manufacturer guidelines. The AccessBio RDT was performed prior to running the Trinity Biotech quantitative assay to blind the researchers from potentia l bias in interpreting the color change of the RDT. (Figure 4 1). Trinity Biotech Quantitative G6PD Deficiency A ssay The Trinity Biotech G6PD assay (kit no. 345B, Trinity Biotech, St. Louis, MO, USA) utilizes a spectrophotometric method to quantify G6PD enzyme activity by measuring the production of nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of substrate glucose 6 phosphate (G6P) and G6PD. The enzyme activity was after a five minute incubation period at a temperature of 25°C. Control blood samples with normal (No. G6888, Trinity Biotech, St. Louis, MO, USA) and deficient (No. G5888, Tri nity Biotech, St. Louis, MO, USA) levels of G6PD enzyme activity were used as quality controls. The kit also requires that the activity be standardized to the amount of hemoglobin present, which was measured by a digital hemoglobin meter (HemoCue Hb 201 pl us, Hemo C ue Incorporated, Cypress, CA, USA). As per manufacturer recommendations, G6PD activity levels that were measured above 19.5 U/g Hgb were not included in this study. Quality Assurance To ensure accuracy and the high quality of the data the status o f each RDTs results was interpreted by two separate researchers . For a ny results where there was discordance between the researchers , sample measurements were repeated . All samples were run within 48 hours of collection in the temperature controlled sett ing of

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60 the University of Florida Haiti Lab. All research materials were stored, shipped under conditions that ensured no deterioration in quality , and used appropriately. Statistical Analyses of RDT P erformance The performance measures (sensitivity, speci ficity, negative predictive value, positive predictive value, and accuracy) of the Access Bio RDT were Sensitivity measures the tests ability to identify a condition correctly, while specificity measures the tests ability to exclude a condition correctly. Results The test results and the performance measures of the AccessBio RDT as compared to the spectrophotometric method to id entify the presence of moderate to severe G6PD deficiency are included in the Table 4 1. Compared to the Trinity Biotech assay, the RDT was 90% sensitive and 87.4% specific for detecting individuals with severe deficiency (<10 % residual G6PD ac tivity); with a predictive value of 99.7% for a negative RDT result. Similarly, the RDT was 84.8% sensitive and 93.7% specific for detecting individuals with moderate or severe deficiency (< 30% residual G6PD activity); with a predictive value o f 98.2% for a negative RDT result. As illustrated in the Figure 4 2 , the ability of the rapid test to correctly identify G6PD deficiency is highest when the G6PD activity is the lowest. The positive predictive values (13.8% and 60%) in this study are affec ted by the relatively low prevalence of severe (2.19%) and moderate or severe (10.09%) participants; however the negative predictive values remain robust across different G6PD activity levels due to the constantly high specificity. Gender did not significa ntly influence the performance of the test in both severe and moderately deficient individuals.

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61 Ambiguous test results were conservatively called deficient, which would be the norm in a clinical setting, resulting in a decrease in spe cificity. Discussion The test was 90% sensitive in determining individuals with severe deficiency and 84.8% sensitive in determining individuals with moderate or severe deficiency. This test was also effective at detecting G6PD normal populatio n members, with a negative predictive value of 98.2%. These findings indicate a highly sensitive test, which would prove quite valuable in mitigating risks associated with drug induced hemolysis in G6PD deficient individuals. Given the low costs of the Car test compared to other G6PD diagnostic test s , and its ability to be used for point of care testing with limited training required, screening malaria patients for G6PD deficiency at scale remains a feasible and practical strategy in Haiti. T he cost per G6PD test was approximately $1.50 US compared with $2.14 US for the Trinity Biotech assay. Our data suggest that this rapid test is effective in identifying moderate to severe G6PD deficiency (less than 30% residual activity), who represent those most at risk of experiencing DIH resulting from PQ therapy. The high negative predictive value of 98.2% for participants with severe and moderate G6PD activity translate into meaningful clinical information, where negative test results (purple color) are a strong indicator for normal G6PD enzyme levels. The low positive predictive values were a consequence of the prevalence of G6PD deficiency in this sample population and should not represent an impediment to the use of this RDT. Identified w eaknesse s in this study include the possibility of operator error due to the subjective nature of qualitative colorimetric results (similarity of dark red and purple colors), and a reduction in test performance in determining Class III G6PD deficiency .

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62 Since G6PD enzyme concentrations in patients with mild deficiency are sufficient to generate a purple color change (negative result), this test is unable to accurately determine mild forms of G6PD deficiency, as noted in a similar study conducted in Cambodia [ 24 ] . Additionally, the magnitude of DIH in mild G6PD deficient individuals compared to severely G6PD deficient individuals, post treatment with PQ, has yet to be clearly quantified, a nd warrants further investigation before acceptable risk thresholds can be established. U sing alternative antimalarial drugs to treat patients with ambiguous test results has little impact on their clinical management and represents a viable strategy to f urther decrease the risk of DIH. The development of a quantitative or semi quantitative rapid test would be helpful to avoid misinterpretation of G6PD RDT results. Limitations This study used the Trinity Biotech quantitative assay as a comparative assay to determine G6PD activity levels, it is still subject to error. Our small sample size of severely G6PD deficient samples represents an additional limitation, as a si ngle false positive drastically reduced test sensitivity for Class I and II G6PD deficiency. Because only blood collected from venipuncture was used in this study, we were unable to compare the performance of the rapid test with blood from a sterile fing er prick. Although we believe that blood obtained by venipuncture and finger prick would have similar G6PD enzyme concentrations and hence, test performance, we were unable to evaluate the differences betwee n the two sampling techniques.

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63 Chapter Conclusio n s The results from this study suggest that the G6PD RDT is highly specific and effective at determining population members with normal levels of G6PD activity. The G6PD test is an attractive tool for point of care G6PD deficiency tes ting given that it is relatively inexpensive and requires significantly less time and training to implement than other methods. In countries that have adopted PQ into their malaria treatment guidelines, this test could be used before treatment of malaria p atients, which would allow clinicians to administer PQ while substantially reducing the risk of adverse treatment outcomes in G6PD deficient patients. Findings suggest the G6PD RDT represents a practical option for the identification of G6PD def icient individuals in Haiti and should be incorporated into current malaria elimination strategies that use PQ.

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64 Figure 4 1. Access Bio G6PD rapid test results . The test on the left is deficient, the test on the right with purple stains is n ormal.

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65 Table 4 1. Performance measures and test results of the G6PD RDT compared to the spectrophotometric method . Test performance measures are based on the ability to detect severe (less than 10% residual G6PD activity) and moderate to severe (less tha n 30% residual G6PD activity) deficiency in study participants, when compared to the spectrophotometric method. Severe G6PD deficiency Performance measures (%) Trinity (+) Trinity ( ) Total Sensitivity 90.0 RDT (+) 9 56 65 Specificity 87.4 RDT ( ) 1 390 391 Accuracy 87.5 Total 10 446 456 Positive predictive value 13.8 Prevalence of deficiency (%) 2.19 Negative predictive value 99.7 Moderate and severe G6PD deficiency Performance measures (%) Trinity (+) Trinity ( ) Total Sensitivity 84. 8 RDT (+) 39 26 65 Specificity 93.7 RDT ( ) 7 384 391 Accuracy 92.8 Total 46 410 456 Positive predictive value 60.0 Prevalence of deficiency (%) 10.09 Negative predictive value 98.2

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66 Figure 4 2. Performance of the G6PD RDT compared to the s Hgb . The sensitivity, specificity, as well as the positive and negative predictive values of the G6PD RDT to classify severe, moderate, or mildly deficient individuals from the sample population is presented with respect to the G6PD activity level (U/g Hgb) measured by the spectrophotometric assay.

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67 CHAPTER 5 AG E SPECIFIC MALARIA SERO CONVERSION RATES: AN ANALYSIS OF MALARIA TRANSMISSION IN THE OUEST AND SUD EST DEPARTMENTS OF HAITI Chapter Summary At low prevalence levels, passive surveillance measures become less appropriate for capturing malaria transmission intensity. To improve understanding of malaria transmission in Haiti, participants from the Ouest and Sud Est depa rtments were screened using a highly sensitive enzyme linked immunosorbant assay (ELISA). Between February and May 2013, samples were collected from four different enrollment sites including a rural community, two schools, and a clinic located in the Oues t 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 1 19 ). The classification of p revious 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 va lues were used to fit a modified reverse catalytic model to yield estimates of seroconversion rates. Of the samples screened, 172 of 815 (21.1%) were AMA 1 positive, 179 of 759 (23.6%) were MSP 1 19 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 indica ted that the annual malaria transmission in the Ouest and Submitted to Malaria Journal on 06/3/2014

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68 Sud Est department is approximately 2.5% (95% CI SCR: 2.2%, 2.8%). Our findings suggest that despite the absence of sustained malaria control efforts in Haiti, transmission has remained relatively low over multiple decades. These findings provide valuable information on malaria epidemiology, which can be used to make a case for the elimination of malaria in Haiti Background Over the past decade there has been a renewed interest in eliminating mal aria from the island of Hispaniola, with a bi national strategy recently adopted between the Dominican Republic and Haiti to eliminate malaria by 2020 [ 4 ] . As Haiti progresses towards elimination, obtaining valid measurements of malaria transmission wil l be crucial to monitoring the impact of control efforts adopted to achieve this goal [ 74 ] . In low transmission settings, passive malaria surveillance measures become less sensitive at capturing transmission intensity. To address this, serological markers have been used in other low transmission settings, allowing researchers to estimate seroconversion rates (SCR) by modelling the a ge specific seroprevalence [ 75 81 ] . Recently a study by Arnold et al. examined cross sectional and longitudinal data from 1991 1998 using merozoite surface protein 1 19 (MSP 1), and found the SCR to be roughly 2.3% in Leogane, which is located in t he Ouest department of Haiti [ 81 ] . Estimating malaria transmission by measuring long lasting antibody responses generated from previous malaria infections also allows the investigation of long term trends without the estimated seroconversion rates being sk ewed by seasonal transmission [ 79 ] . The purpose of this study was to provide information on current trends in malaria transmission in the Ouest and Sud Est departments of Haiti . Th e data obtained will be

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69 used to address gaps in malaria surveillance, while providing policy makers baseline information that can help make a case for future elimination prospects in Haiti. Methods Study Location & Enrollment The samples analyz ed in this study were collected from four enrollment sites located in the Ouest and Sud Est department of Haiti in the communes of Gressier and Jacmel, between February and May 2013. A map of Haiti including the enrollment locations, study communes, and d epartments is presented in Figure 5 1. Enrollment was based on sampling from both clinical and non clinical settings, as part of a larger study described in Chapter 1 [ 71 ] . Samples were collected from four different enrollment sites including a rural community, two schools, and a clinic located in the Ouest and Sud Est depar tments of Haiti. Healthy children were enrolled from the Christianville School in Gressier and from the Hosana Baptist School in Jacmel. Patients and healthy family members attending Portail Leogane Clinic in Jacmel were enrolled on a voluntary basis. Ind ividuals from Chabin were enrolled from community members seeking general health services at a mobile clinic. Participants at each location were given opportunities to ask our enrolling physicians questions during information sessions prior to consent. Aft er obtaining consent from participants or their guardian, local clinicians collected approximately 3 m L of blood by venipuncture into serum separation tubes from participants, which were centrifuged immediately at 6000 RPM for 2 minutes after collection wa s complete. All serum samples were stored at 80°C in the University of Florida field laboratory in Gressier, until shipment to the Emerging Pathogens Institute, in Gainesville, FL, for analysis and storage. Serum samples were collected from a total of 82 3 participants between the ages of 2 and 80. Malaria infected participants (5/823),

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70 determined via rapid diagnostic test (RDT) were excluded from analysis, to reduce the influence clinical enrollment has on seroprevalence rates. Participants missing age d ata (3/823) were also excluded, resulting in a final sample size of 815 (483 females and 332 males). Of the 815 samples analyzed, all were screened for previous exposure using the antigen AMA 1, but only 759/815 samples were screened using the MSP 1, due to limited amounts of serum from some participants. The characteristics of the resulting study population are presented in Table 5 1. ELISA P ro tocols and P rocedures Serum samples were screened for antibodies against AMA 1 and MSP 1 19 using an indirect enz yme linked immunosorbant assay (ELISA). Serum samples of subjects were diluted in 5% non fat skin milk in phosphate buffered saline (NFSM PBS). ELISA plates were coated in duplicate with the respective antigen diluted in 5% NFSM PBS to a final concentrati on of 1 µL /m L for AMA 1 and 0.5 µL /m L for MSP 1, before overnight incubation at 4°C. The next day, antigen was removed and plates were washed 5 times with 0.05% tween 20 in PBS K and then blocked for 1 hour with 5% NFSM PBS to reduce non selective binding . Following additional wash, diluted serum samples as well as positive and negative control sera were plated in duplicate and incubated for 2 hours at 4° C. Horseradish peroxidase conjugated rabbit anti human IgG secondary antibody was diluted 1:1000 in 5% NFSM PBS and added to the plate. After one hour, plates Tetramethylbenzidine (TMB) substrate solution in the dark for 20 minutes to allow sufficient color development and stopped with 2M sulfuric acid.

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71 Deter mination of Seropositive and Seronegative P opulation M embers After measurement of the samples in duplicate, the average absorbance at 450 nm was used to determine the thresholds for the classification of a sample as seropositive or seronegative. Given the low incidence and relatively large sample size for each antigen (n=815 or 759), the seronegative population responses for AMA 1 and MSP 1 should follow a normal distribution function with sample mean and sample standard deviation . This behavior is presented in Figure 5 2, where the suspected seronegative populations approximated normal distributions with and for AMA 1 and MSP 1 respectively. Thresholds for positive AMA 1 and MSP 1 responses were classified by the addition of three, four, and five sample standard deviations to the sample mean, such that 99.7% of seronegative population members would not be classified as seropositive. The resulting threshol ds using three, four, and five, sample deviations for AMA 1 were 0.319, 0.362, 0.405 and 0.260, 0.30, 0.340 for MSP 1. Responses above three standard deviations of the mean suspected average were considered seropositive, given the minimal impact using mor e conservative thresholds had on the estim ated prevalence . Estimation of Sero C onversion R ates from C ross S ectional D ata The observed cross sectional seroprevalence was used to estimate the seroconversion rate using a method similar to th ose previously de scribed [ 75 , 82 ] . Briefly, an age specific seroconversion model was fit to the prevalence of AMA 1, MSP 1, an d AMA 1 or MSP 1 seropositive population members using all participants (aged 2 to 80) and separately for participants less than 20 years of age to estimate the rate of 1 and MSP 1 responses are long lasting and the estima tion of reversion rates has been suggested to be unreliable with cross sectional

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72 s used for the final analysis [ 76 ] , however non zero reversion rates were also explored. Without seroreversion, the probability of infection (prevalence) at age was modeled using the equation . When seroreversion was included in the model, the probabi lity at age was modeled using the equation . The functions were optimized (using R) to give estimated seroconversion/reversion rates and , as well as their standard errors for the calculation of 95% co nfidence intervals for and . Results Estimation of S eroprevalence U sing AMA and MSP Using the average absorbance from each serum sample and a threshold of three standard deviations from the sample mean to indicate the presence of AMA 1 or MSP 1 antibodies, 172 of 815 (21.1%) had the presence of AMA 1 antibodies, 179 of 759 (23.6%) had the presence of MSP 1 antibodies, and 247 of 815 (30.3%) had the presence of either AMA 1 or MSP 1 antibodies. Using thresholds from three to five standard deviatio ns gave similar estimates of seroprevalence that ranged from 16.3% to 21.1% for AMA 1, 17.4% to 23.6% for MSP 1 and 24.0 % to 30.3 % for either AMA 1 or MSP 1. The seroprevalence by age group (ranging from 2 to 80 years) is presented in Table 5 2 and Figur e 5 3. As expected, the prevalence of seropositive participants increases with participant age. The prevalence of previous infections determine by AMA 1 response was not significantly different (p value > 0.05) between age classes under 20 years of age . However compared to participants younger than 20 years of age, the likelihood of previous infection in those over 20 years of age was 6.0 times higher (95% CI OR 4.03,

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73 8.99). For MSP 1 responses, differences in prevalence compared to the youngest age c lass were significant (p value = 0.023) beginning at the third age class (9 to 13 years of age), with those over 20 years of age having 3.7 times the likelihood of previous infection (95% CI OR 2.48, 5.53). Of participants who were tested with both AMA 1 a nd MSP 1 (n=759), 104 were classified as seropositive using both antigens (13.7%), 75 were MSP 1 positive and AMA 1 negative (9.9%), 57 were AMA 1 positive and MSP 1 negative (7.5%), and 523 were negative for both AMA 1 and MSP 1 (68.9%). The distribution of positive ELISA responses for AMA 1 or MSP 1 antigens appears graphically in Figure 5 4, with regions I, II, III, and IV representing the average absorbance value for the response to the AMA 1 and MSP 1 antigens for samples with AMA 1(+)/MSP 1(+) result s, AMA 1( )/MSP 1 (+) results, AMA 1(+)/MSP 1( ) results, and AMA 1 ( )/MSP 1( ) results, respectively. Estimation of S ero C onversion R ates for AMA and MSP The prevalence and the estimated probability of previous infection as determined by AMA 1 and MSP 1 antigen response are presented in Figure 5 5 for the entire study population (2 to 80 years) and those less than 20 years of age. The estimated SCR for AMA 1 (top panel) from the entire study population was 0.016 (95% CI 0.013, 0.018) and 0.014 (95% CI 0.012, 0.017) when fit to data from participants 20 years or younger. For MSP 1 (middle panel), the estimated SCR from the entire study population was 0.018 (95% CI 0.016, 0.021) and 0.019 (95% CI 0.015, 0.022) for participants 20 years o r younger. Using AMA 1 or MSP 1 to define seropositive participants (bottom panel), the estimated SCR from the entire study population was 0.025 (95% CI 0.022, 0.028) and 0.026 (95% CI 0.022, 0.302) for participants 20 years or younger. When a

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74 no n zero seroreversion rate was used to model the seroprevalence data from all participants aged 2 to 80 years of age (data not shown), the following conversion and reversion rates were derived: 0.014 (95% CI 0.011, 0.017) and 0.006 (95% CI 0.016 , 0.003) for AMA 1 responses, 0.0204 (95% CI 0.016, 0.025) and 0.008 (95% CI 0.005, 0.022) for MSP 1 responses, and 0.0273 (95% CI 0.022, 0.032) and 0.007 (95% CI 0.003, 0.018) for either an AMA 1 or MSP 1 response. Discussion Findi ngs suggest that these locations have experience d a relatively low and constant state of P . falciparum transmission, given the stable increase in seroprevalence by age observed in this study. In samples that had positive responses to either AMA 1 or MSP 1, the estimated SCR of 2.5% (95% CI 2.2%, 2.8%) from this study is slightly higher than the <1% prevalenc e rate estimate by PSI in 2012 [ 20 ] . However, when the seroconversion rates were determined individually from a positive AMA 1 or MSP 1 response, the estimated SCR decreased to 1.6% (95% CI 1.3%, 1.8%) and 1.8% (95% CI 1.6%, 2.1%) for AMA 1 and MSP 1, respectively. The differences in SCR estimates could be a result in variation in individual antib ody responses, as suggested by F igure 5 4, where some seropositive respondents have stro ng responses to only a single antigen (regions II and III). Trends were also observed in the antibody responses after stratification for age, which could indicate that the duration of AMA 1 and MSP 1 antibody titers are different, as previously sug gested [ 83 ] . In F igure 5 3, in 4 out of 5 age groups below 20 years of age the seroprevalence of MSP 1 was higher than the seroprevalence of AMA 1, whereas 2 of 3 of the age gro ups above 20 show higher seroprevalence of AMA 1 compared to MSP 1. However, in this

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75 sample the likelihood of participants having a strong AMA 1 response ( > 0.5 AU) and a MSP 1 response below the threshold ( F igure 5 4, region III) was not significantly di fferent by age (p > 0.1) The inclusion of a seroreversion rate in the model also slightly increased the estimated seroconversion rates. Due to the cross sectional nature of this study and the long duration of antibody detection for AMA 1 and MSP 1, we fel t it was appropriate to set the seroreversion rate to zero. When seroreversion was included in the model, we found the seroreversion rates to be 0.006 (95% CI 0.016, 0.003) and 0.008 (95% CI 0.005, 0.022) for AMA 1 and MSP 1 respectively. Si nce both of the confidence intervals for the seroreversion rates include zero and the inclusion of a seroreversion rate in the model had little effect on the estimates of seroconversion, we feel the a priori exclusion of a seroreversion rate from the final model was justified. When comparing the estimated seroconversion rates from study participants under 20 years of age, the continuity in the age specific seroprevalence curve could indicate that over multiple decades, a relatively constant state of low ma laria transmission has occurred in these areas , even in the absence of sustained malaria control efforts. Limitations One of the primary limitations of this study was that serum samples were collected using sample s collected from only three si tes , which limited our ability to infer how this sample population represents Haiti as a whole. Findings may have been skewed by potentially enrolling participants from clinics (n=203), however, we felt this potential sampling bias was adequately addresse d by excluding all malaria RDT positive individuals (5/815) from final analysis , nor was method of enrollment a significant risk factor for likelihood of previous exposure, after adjusting for age . This

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76 study also only screened for previous exposure to P. falciparum , although the likelihood of finding other species or mixed infections remains low, given recent reports, and the prese nce of host protective factors [ 1 , 84 ] . As with other ELISA protocols, setting a threshold for the classification of a sample as seropositive is subject to interpretation. To validate the method used in this study, threshold s using absorbance values of four and five standard deviations above the suspected seronegative population mean were also evaluated and fell within the calculated confidence intervals for seroprevalence and SCR using only three standard deviations. Moreov er, we felt it was more appropriate to use a normal distribution of suspected negatives from the native population than aggregate data from negative control serum provided by the manufacturer or from controls derived from other populations with no travel h istory to malaria endemic countries. Chapter Conclusions As reported cases of malaria in the Dominican Republic have reached a 15 year low of 952 cases , malaria continues to be a major public health concern in Haiti , with over 160,000 suspected cases in 2012 . Currently there are seven countries from the Costa Rica, Ecuador, El Salvador, Mexico, and Paraguay, with the Dominican Republic, on track to achieve a 50% decr ease in malaria incidence by 2015. Noticeably absent from this WHO istent data to assess [ 1 ] . Findings from this study further support the notion of sustained low level transmission in Haiti [ 81 ] , while demonstrating a relatively simple technique that could be used to determine malaria transmission elsewhere in Haiti. This data suggests that any efforts to advance malaria control locally have not had much impact over the five

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77 decades, yet neither have the past political upheavals or natural disas ters from recent decades resulted in major malaria epidemics. Investigating the vector competency of A. albimanus mosquito, may better explain this phenomenon, of stable low transmission. Future studies should expand seroprevalence methodologies to o ther departments so country wide trends can be estimated. Until Haiti can adequately address malaria transmission, imported cases from Haiti to the Dominican Republic will likely continue to occur, making malaria surveillance a key component to achieving sustainable elimination for the island of Hispaniola. In an effort to meet the island wide goal of malaria elimination by 2020, the gametocidal drug primaquine (PQ), was added to the malaria national treatment policy for Haiti in 2010 [ 50 ] . This treatment policy change places Haiti in a unique position to monitor and quantify the impact single dose 0.75 mg/kg PQ administration has on P. falciparum transmission, which could hold valuable information on PQ tolerance and malaria elimination strategies abroad. Further research examining barriers to access, protective host characteristics, the extent of heterogeneo us malaria transmission in other departments, and vector proficiency would enhance elimination models in Haiti. Elimination in Haiti appears to be feasible; however, surveillance must continue to be strengthened in order to respond to areas with high tran smission foci and measure the impact of future interventions .

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78 Figure 5 1. Seroprevalence study area in the Ou e st and Sud Est departments of Haiti . The four study enrollment sites located inside the Ouest and Sud Est Departments of Haiti in the communes of Gressier and Jacmel. Participants were enrolled from Christianville School in Gressier and from Hosana Baptist School and Portail Leogane Clinic, and the rural community of Chabin in Jacmel. Along with an inset of the entire country of Haiti, the enrollment sites (red circles) appear relative to the study commune ( yellow ), the national capital (star) and national highway systems (pink lines).

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79 Table 5 1. Study population characteristics by site of enrollment . The total number of total s amples, average age in years, percentage of male participants, and the number of participants who were screened using an ELISA with AMA, MSP, or either AMA or MSP antigens are listed by study enrollment site . (S) denotes participants enrolled from schools, (C) represents participants enrolled from clinics. . Demographic factors Participants given ELISA Site of enrollment Size Age Gender AMA MSP Combined (N) Years % Male No. tests No. tests No. tests Christianville (S) 510 12.2 41.4 510 461 510 Hosana B aptist (S) 102 8.29 52 102 102 102 Portail Leogane Clinic (C) 72 28.9 29.2 72 72 72 Chabin community (C) 131 29.8 35.9 131 124 131 Total 815 16.1 40.7 815 759 815

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80 Figure 5 . The distr ibution of ELISA responses from the study participants in absorbance units (at 450 nm) appear for AMA 1 and MSP 1 on the left and right panels of F igure 5 2, respectively. The upper panels show the histograms of the suspected seronegative ELISA results ov erlaid with a normal distribution function. The sample mean (thick black line) and sample standard deviation of these functions were used to determine minimum absorbance values (thresholds) for the classification of a sample as seropositive using the sampl e mean for the suspected negative population members plus three to five sample standard deviations (gold, orange, and red dashed lines).

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81 Table 5 2. Number and prevalence of seropositive participants by age class . The number of total samples, positi ve results, and prevalence for each age class appear for participants with positive ELISA results toward AMA 1, MSP 1, or either AMA 1 or MSP 1 antigens. Age AMA 1 MSP 1 Combined (years) n No. Pos. % Pos. n No. Pos. % Pos. n No. Pos. % Pos. 2 to 5 61 4 6.6 61 5 8.2 61 6 9.8 6 to 9 190 26 13.7 189 15 7.9 190 35 18.4 9 to 13 216 33 15.3 193 42 21.8 216 58 26.9 14 to 17 154 25 16.2 129 36 27.9 154 46 29.9 18 to 20 63 16 25.4 60 22 36.7 63 29 46.0 21 to 29 40 14 35.0 40 15 37.5 40 15 37.5 30 to 49 50 2 6 52.0 48 23 47.9 50 30 60.0 over 50 41 28 68.3 39 21 53.8 41 28 68.3 Total 815 172 21.1 759 179 23.6 815 247 30.3

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82 Figure 5 3. Seroprevalence by age class for participants ranging in age from 2 to 80 for AMA and MSP antibodies. The pr evalence of samples that had an response to the AMA or MSP antigens are presented by age class for those classified as being seropositive with AMA (white), MSP (charcoal), or either (black dotted) antibodies.

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83 Figure 5 4. Comparison of samples w ith positive AMA 1 or MSP 1 responses . The scatterplot of AMA 1 and MSP 1 responses (average absorbance at 450 nm) is shown along with the reference lines denoting positive AMA 1 or MSP 1 responses (dotted lines) using the threshold absorbance values for previous infection of 0.319 and 0.260 absorbance units, respectively. Samples were positive for AMA 1 and MSP 1 (region I), MSP 1 positive and AMA 1 negative (region II), AMA 1 positive and MSP 1 negative (region III), or negative for both AMA 1 and MSP 1 (region IV). For clarity, samples with an absorbance value above 1.8 AU are shown as having a maximum value of 1.8 AU and those with no positive response to either antigen (region IV) are not shown.

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84 Figure 5 5 Seroprevalence estimates for the presenc e of AMA 1 or MSP 1 antibodies by age class for children and adults and for the entire study population. The actual seroprevalence for AMA 1 and MSP 1 antibodies (circles) appear by age class along with the probability of infection in each age class for AM A 1 and MSP 1 (black lines) and the respective 95% confidence limits (dotted lines), derived from the model estimated seroconversion rate ( ). The top, middle, and lower panels show the incremental increases in seroprevalence for AMA 1, MSP 1, and either AMA 1 or MSP 1 with age, respectively. The left panels show the model fits using data from participants less than 5 years to 20 years of a ge, while the right panels show the model fits using the entire data set including participants from 2 to 80 years.

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85 CHAPTER 6 GENERAL DISCUSSION AND CONCLUSION S Summary of Findings As Haiti continues to progress towards malaria elimination, findings from t his dissertation offer valuable information on the prevalence of G6PD deficiency, the utility of a possible G6PD RDT, and transmission rates based on a malaria seroprevalence model. It is the goal of this research to inform policy makers about the relative risks and benefits of PQ administration and G6PD deficiency and to provide information on transmission dynamics, contribut ing to the overall malaria elimination efforts taking place in Haiti. Despite the continued use of CQ malaria trea tment policy since 1955 , there is no evidence of widespread CQ resistance. The few drug resistance studies that have been implemented in Haiti were limited by small sample sizes [ 47 , 52 ] . . T he addition of PQ duce transmission rates and slow the spread of dru g resistant parasites [ 85 ] . T his policy , while sound in theory, is no t without controversy, because of c oncerns regarding PQ induced hemolysis in G6PD deficient individuals , which was found in 22.8% (range 14.9% 24.7%) of our sample population. According to the World Malaria Report 2013, there is no plan to adopt G6PD defic iency screening into current malaria diagnostic guidelines in Ha i ti , but instead, the WHO has suggested the dosage of PQ be lowered from 0.75mg/kg 0.25 mg/kg [ 1 ] . T he extent this drop in do sage influences drug efficacy is still being explored elsewhere as indicated by the Eziefula et al. study , which reported a single dose of 0.4 mg/kg had similar gametocyte clearance as a dose of 0.75 mg/kg [ 6 2 ] . While these results are

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86 promising, a key issue with th e study design is that it provides no information on PQ tolerance in G6PD deficient individuals. I t is important for Haiti an policymakers to consider incorporating G6PD deficiency screening pr ior to PQ administration in malaria infected patients, given the high prevalence observed of G6PD deficiency reported in Chapter 3. However, it will be necessary to strengthen the overall capacity of health centers in Haiti to implement G6PD screening, wh ich in turn should increasing the ability of health care workers to follow and monitor patients for adverse events associated with PQ. T he G6PD deficiency RDT represents a sensitive tool that could be used in tandem with malaria diagnostics tes ts . At a market cost of approximately $1.50 US per test, policy makers should consider the feasibility and cost benefit of adopting this RDT, into current malaria management strategies. This may facilitate the adoption of PQ into practice as physicians a re otherwise hesitant to administer PQ to patients with unknown G6PD status. To meet their 2020 elimination goal, policy makers and clinicians in Haiti will likely have to use PQ to reduce malaria transm ission . Distributing b ed nets may not be as effect ive at preventing malaria transmitted by A. albimanus, which prefers feeding at dusk and dark outside of homes [ 16 ] , nor is there an established vector control branch in Haiti that could be used to reduce mosquito populations. . Findings fro m the seroprevalence study confirm that malaria transmission is hypoendemic in this setting and that Haiti [ 81 ] remained low in Haiti, despite an absence of control programs, indicating that additional variables may be at play in this setting, including host genetic fa ctors [ 86 , 87 ] and vector

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87 competency [ 88 ] . A n SCR of 2.5% (95% CI 2.2%, 2.8%) presents similar findings to the Arnold et al study, which estima ted that the SCR was 2.3% in the Ouest Department [ 81 ] . If this rate were applied t o the entire country, the annual prevalence would be 250,000 cases (220,000 280,000) , which is 90,000 cases higher than the 160,000 suspected cases reported in 2013 [ 1 ] . However, t his is likely an overestimation of prevalence given the heterogeneous transmission rates observed in other malaria studies in Haiti [ 4 , 17 , 21 , 22 ] , hence the need for expanded serological studies across multiple departments. T aken together, the chapters of this dissertation can be used to inform decis ion makers about current treatment policies and the risks associated between G6PD deficiency and PQ administration, while providing valuable baseline data on malaria transmission in Haiti. Future Research Moving forward, malaria research in Haiti should focus on elimination strategies . Pharmacovigil a nce , which ensures proper drug quality, distribution, and use , is currently neglected in Haiti , especially in the context of discord between treatment polic y and practice. While PQ may be the official nation al treatment policy in writing, t he extent at which it has been adapted in health facilities around Haiti is unknown. T here is a need for information regarding the tolerance of single dose PQ in G6PD deficient individuals , which will help determine the ca pacity PQ should be used in Haiti. If research suggests that t he risk of PQ induced hemolysis is minimal, mass drug administration may become a viable tactic to eliminate malaria in Haiti, which c ould be carried out in spite of weaknesses in current surve illance systems and in the absence of vector control programs . Malaria drug distributions could be linked to the current lymphatic Filariasis

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88 program, wh ere at least a single dose of albendazole and diethylcarbamazine was administered to roughly 71% of th e population in 2012 [ 89 ] . Using the serological methods described in Chapter 5 , transmission rates c ould be measured at a minimal cost elsewhere on the island, which could be used to determine th e magnitude of current interventions. If monitored correctly, the impact single dose PQ has on malaria rate s in Haiti could potentially provide a template for elimination i n other malaria endemic settings globally. However, many gaps in our knowledge need to be addressed before a n MDA strategy could be implemented with confidence. In the meantime , a major push from both internal and external stakeholders is needed, if Haiti aims to meet current 2020 elimination benchmarks.

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89 APPENDIX PUBLICATIONS Carter TA, von Fricken ME, Romain JR, Memnon G, St. Victor Y, Schick L, Okech BA, Mulligan CJ. Detection of Sickle Cell Hemoglobin in Haiti by Genotyping and Hemoglobin Solubility Tests . American Journal of Tropical Medicine and Hygiene, 2014 (in press). von Fricken ME, Weppelmann TA, Eaton WT, Alam MT, Carter TE, Schick L, Masse R, Romain JR, Okech BA. Prevalenc e of glucose 6 phosphate dehydrogenase (G6PD) deficiency in the Ouest and Sud Est departments of Haiti . Acta Tropica , 2014 135: 62 66. Carter TE, Maloy H, von Fricken ME, St. Victor Y, Romain JR, Okech BA, Mulligan CJ. Glucose 6 phosphate dehydrogenase deficiency A variant in febrile patients in Haiti . American Journal of Tropical Medicine and Hygiene, 2014 (in press). von Fricken ME, Weppelmann TA, Eaton WT, Masse R, Beau De Rochars MV, Okech BA. Performance of the CareStart glucose 6 phosphate dehydrogenase (G6PD) rapid diagnostic test, in Gressier, Haiti . American Journal of Tropical Medicine and Hygiene, 2014 von Fricken ME, Weppelmann TA, Hosford JD, Existe A, Okech BA. Malaria Treatment Policies and drug efficacy in Haiti from 1955 2012 . Journal of Pharmaceutical Policy and Practice 2013 6 :10 Weppelmann TA, Carter TE, Zhongsheng C, von Fricken ME , Memnon G, Victor YS, Existe A, Okech BA. Lack of Plasmodium vivax infections and high frequency of the erythroid silent Duffy antigen geno type in Haiti . Malaria Journal 2013 12: 30

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91 15. Mason J and Cavalie P, Malaria Epidemic in Haiti Following a Hurricane. American Journal of Tropical Medicine and Hygiene, 1965. 14 (4): p. 533 &. 16. Sinka ME, Rubio Palis Y, Manguin S, Patil AP, et al., The dominant Anopheles vectors of human malaria in the Americas: occurence data, distribution maps and bionomic precis. Parasite Vector, 2010. 3 (72). 17. Vanderwal T and Paulton R, Malaria in the Limbe River valley of northern Haiti: a hospital based retrospective study, 1975 1997. Revista panamericana de salud publica = Pan American journal of public health, 2000. 7 (3): p. 162 7. 18. WHO, World Malaria Report 2012 , 2012, World Health Organization: Geneva 19. Ramachandran V and Walz J, Haiti: W here Has All the Money gone? , 2012 Center for Global Development: Washington D.C. 20. P opulation S ervices I nternational , Haiti: TRaC Malaria: Étude TRaC sur la prévalence du Palu disme en Haiti. , 2011, Population Services International: Port au Prince 21. Raccurt CP, Ciceron M, Dossil R, and Boncy J, Prevalence of Plasmodium falciparum during the rainy season (June December) in the southeast district of Haiti. Médecine Santé Tropi cales, 2012. 22 (4). 22. Eisele TP, Keating J, Bennett A, Londono B, et al., Prevalence of Plasmodium falciparum infection in rainy season, Artibonite Valley, Haiti, 2006. Emerging Infectious Diseases, 2007. 13 (10): p. 1494 1496. 23. Beutler E, G6PD: Popula tion genetics and clinical manifestations. . Blood rev., 1996. 10 (1): p. 45 52. 24. Kim S, Nguon C, Guillard B, Duong S, et al., Performance of the CareStart G6PD deficiency screening test, a point of care diagnostic for primaquine therapy screening. PloS One, 2011. 6 (e28357). 25. Cook SS, Malaria Control in Haiti. South Med J, 1930. 23 (5): p. 454 459. 26. A rmed F orces P est M anagement B oard , Literature Retrieval System 2012: Washington D.C. 27. WHO, The Eighth World Health Assembly , 1955, World Health Orga nization: Geneva 28. I nternational Cooperation Administration , Malaria Manual, For U.S. Technical Cooperation Programs , 1956, Washington D.C.

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92 29. WHO, Malaria Eradication in 1965 , 196 5, World Health Organization: Gen eva p. 286 300. 30. PAHO, Report on the Status of Malaria Eradication in the Americas. PAHO Epidemiol Bull., 1967: p. 1 120. 31. WHO, Standardization of procedures for the study of glucose 6 phosphate dehydrogenase: report of a WHO Scientific Group , in WHO Techinical Report Series 1967, World H ealth Organization. 32. WHO, Development of the Haiti Malaria Eradication Programme , 1968, World Health Organization: Geneva p. 4 20. 33. WHO, Malaria Eradicatioin in 1971 , in WHO Technical Report Series 1972, World Health Organization: Geneva p. 485 496. 3 4. WHO, The Malaria situation 1976 , in WHO Chron. 1976: World Health Organization: Geneva p. 9 17. 35. Magloire R and Nguyendinh P, Chloroquine Susceptibility of Plasmodium Falciparum in Haiti. Bull World Health Organ, 1983. 61 (6): p. 1017 1020. 36. Nguyen Dinh P, Zevallos Ipenza A, and Magloire R, Plasmodium falciparum in Haiti susceptibility to pyrimethamine and sulfadoxine pyrimethamine. Bull World Health Organ, 1984. 62 (4): p. 623 626. 37. PAHO, Status of Malaria Programs in the Americas. PAHO Epidemiol Bull., 1984: p. 1 51. 38. Nguyen Dinh P, Payne D, Teklehaimanot A, Zevallos Ipenza A, et al., Development of an in vitro microtest for determining the susceptibility of Plasmodium falciparum to sulfadoxine pyrimethamine: laboratory investigations and field studies in Port au Prince, Haiti. Bull World Health Organ, 1985. 63 (3): p. 585 92. 39. PAHO, Malaria Control in the Americas: A Critical Analysis. Bull Pan Am Health Organ, 1986(20): p. 9 17. 40. Deloron P, Duverseau YT, Zevallosipenza A, Magloire R, et a l., Antibodies to Pf155 a major antigen of Plasmodium falciparum seroepidemiological studies in Haiti. Bull World Health Organ, 1987. 65 (3): p. 339 344. 41. WHO, Glucose 6 phosphate dehydrogenase deficiency Bulletin of the World Health Organization 1989. 67 (6): p. 601 611. 42. PAHO, Malaria in the Americas. Bull Pan Am Health Organ, 1992(13): p. 1 6.

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94 56. von Fricken ME, Weppelmann TA, Hosford JD, Existe Alexandre, et al., Malaria Treatment policies and drug efficacy in Haiti from 1955 2012. Journal of Pharmaceutical Policy and Practice, 2013. 6 (10). 57. Bray PG, Deed S, Fox E, Kalkanidis M, et al., Primaquine synergises the activity of chloroquine against chloroquine resistant P. flaciparum. Biochemical Pharmacology, 2005. 70 : p. 1158 1166. 58. Ruwende C and Hill A, Glucose 6 phosphate dehydrogenase deficiency and malaria. J ournal of Molecular Medicine (Berlin), 1998. 76 (8): p. 581 8. 59. Beutler E, Glucose 6 phosphate dehydrogenase deficiency: a historical perspective. Blood 2008. 111 (1): p. 16 24. 60. Tavazzi D, Taher A, and Cappellini MD, Red Blood Cell Enzyme Disorders: A n Overview. Pediatric Annals., 2008. 37 (5): p. 303 310. 61. Cappellini MD and Fiorelli G, Glucose 6 phosphate dehydrogenase deficiency. Lancet, 2008. 371 : p. 64 74. 62. Eziefula AC, Bousema T, Yeung S, Kamya M, et al., Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double blind, dose ranging trial. Lancet Infectious Diseases, 2014. 14 (2): p. 130 139. 63. Louicharoen C, Patin E, Paul R, Nunchprayoon I, et al., Positively selected G6PD Mahidol mutation reduces Plasmodium vivax density in Southeast Asians Science, 2009. 326 : p. 1546 1549. 64. Kuwahata M, Wijesinghe R, Ho MF, Pelecanos A, et al., Populatio n screening for glucose 6 phosphate dehydrogenase deficiencies in Isabel Province, Solomon Islands, using a modified enzyme assay on filter paper dried bloodspots. Malar J, 2010. 9 (223). 65. Scriver C, Glucose 6 Phospahte Dehydrogenase Deficiency . 7th ed19 95, New York: McGraw Hill. 66. Khim N, Benedet C, Kim S, Siv S, et al., G6PD deficiency in Plasmodium falciparum and Plasmodium vivax malaria infected Cambodian patients Malar J, 2013. 12 : p. 171. 67. Leslie T, Moiz B, Mohammad N, Amanzai O, et al., Preval ence and molecular basis of glucose 6 phosphate dehydrogenase deficiency in Afgan populations: implications for treatment policy in the region. Malar J, 2013. 9 : p. 223. 68. White NJ, Qiao LG, Qi G, and Luzzatto L, Rationale for recommending a lower dose o f primaquine as a Plasmodium falciparum gametocytocide in populations where G6PD deficiency is common. Malar J, 2012. 11 : p. 418.

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95 69. Fernando D, Rodrigo C, and Rajapakse S, Primaquine in vivax malaria: an update and review on management issues. Malar J, 2 011. 10 : p. 351. 70. von Seidlein L, Auburn S, Espino F, Shanks D, et al., Review of key knowledge gaps in glucose 6 phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8 aminoquinoline treatment regimens: a workshop report. . Malar J, 2013. 12 : p. 112. 71. von Fricken ME, Weppelmann TA, Eaton WT, Masser R, et al., Prevalence of glucose 6 phosphate dehydrogenase (G6PD) deficiency in the Ouest and Sud Est departments of Haiti. Acta Trop, 2014. 135 : p. 62 66. 72. Nadara jan V, Shanmugam H, Sthanesshwar P, Jayaranee S, et al., Modification to reporting of qualitative fluorescent spot test results improves detection of glucose 6 phosphate dehydrogenase (G6PD) deficient heterozygote female newborns. Int. Jnl. Lab. Hem., 201 1. 33 : p. 463 470. 73. De Niz M, Eziefula AC, Othieno L, Mbabazi E, et al., Tools for mass screening of G6PD deficiency: validation of the WST8/1 methoxy PMS enzymatic assay in Uganda. Malar J, 2013. 12 : p. 210. 74. Greenwood BM, Control to elimination: im plications for malaria research. Trends Parasitol, 2008. 24 (10): p. 449 454. 75. Drakeley CJ, Corran PH, Coleman PG, Tongren JE, et al., Estimating Medium and Long term trends in malaria transmission using serological markers of malaria exposure. PNAS, 200 5. 102 : p. 5108 5113. 76. Wilson S, Booth M, Jones FM, Mwaatha JK, et al., Age adjusted Plasmodium falciparum antibody levels in school aged children are a stable marker of microgeographical variations in exposure to Plasmodium infection. BMC Infect Dis, 2 007. 7 : p. 67. 77. Stewart L, Gosling R, Griffin J, Gesase S, et al., Rapid Assessment of Malaria Transmission Using Age Specific Sero Conversion Rates. PLoS One, 2009. 4 (6): p. e6083. 78. Bousema T, Youssef RM, Cook J, Cox J, et al., Serologic Markers for Detecting Malaria in Areas of Low Endemicity, Somalia, 2008. Emerg Infect Dis, 2010. 13 (3). 79. Cook J, Reid H, Iavro J, Kuwahata M, et al., Using serological measures to monitor changes in malaria transmission in Vanuatu. Malar J, 2010. 9 : p. 169. 80. Co ok J, Kleinschmidt I, Schwabe C, Nseng G, et al., Serological Markers Suggest Heterogeneity of Effectiveness of Malaria Control Interventions on Bioko Island, Equatorial Guinea. PLoS One, 2011. 6 (9): p. e25137.

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96 81. Arnold BF, Priest JW, Hamlin KL, Moss DM, et al., Serological Measures of Malaria Transmission in Haiti: Comparison of Longitudinal and Cross Sectional Methods. PLoS One, 2014. 9 (4): p. e93684. 82. Corran P, Coleman P, Riley E, and Drakeley C, Serology: a robust indicator of malaria transmission intensity? Trends Parasitol, 2007. 23 (12): p. 575 582. 83. Tongren JE, Drakeley CJ, McDonald SL, Reyburn HG, et al., Target Antigen, Age, and Duration of Antigen Exposure Independently Regulate Immunoglobulin G Subclass Switching in Malaria. Infect Immun, 2006. 74 (1): p. 257 264. 84. Weppelmann TA, Carter TE, Chen Z, von fricken ME, et al., High frequency of the erythroid silent Duffy antigen genotype and lack of Plasmodium vivax infections in Haiti. Malar J, 2013. 12 : p. 30. 85. Maude RJ, Socheat D, S.P. N guon C, Dara P, et al., Optimising Strategies for Plasmodium falciparum Malaria Elimination in Cambodia: Primaquine, Mass Drug Administration and Artemisinin Resistance. PLos One, 2012. 7 (5): p. e37166. 86. Howes R, Piel FB, Patil AP, Nyangiri OA, et al., G6PD deficiency prevalence and estimates of affected populations in malaria endemic countries: A geostatistical model based map. PLoS Med, 2012. 9 (11): p. e1001339. 87. Taylor SM, Cerami C, and Fairhurst RM, Hemoglobinopathies: Slicing the Gordian Knot of Plasmodium falciparum Malaria Pathogenesis. PLoS Pathog, 2013. 9 (5): p. e1003327. 88. Lefevre T, Vantaux A, Dabire KR, Mouline K, et al., Non Genetic Determinants of Mosquito Competence for Malaria Parasites PLoS Pathog, 2013. 9 (6): p. e1003365. 89. CDC, M ass Drug Administration for the Elimination of Lymphatic Filariasis Port au Prince, Haiti, 2011 2012. MMWR 2013. 62 (23): p. 466 468.

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97 BIOGRAPHICAL SKETCH Michael is a Virginian who was raised in Great F alls, Virginia, near Washington DC . He i s a first generation American , with his father Manfred emigrating to the United States from Germany in 1958 at the age of nine . Michael completed his secondary education at Gonzaga College High School in Washington DC, where he learne Throughout his education, Michael has focused o n the field of public health Science from James Madison University and a Masters of Public Health from the University of Florida. It was during his time at the University of Florida that he had the opportunity to interact extensively with Dr. Bernard Okech, through his course, Environmental Management of Vector Borne Disease , which helped spark his interest in malaria and vector borne d isease . After graduating with his MPH in 2011, he maintained contact with Bernard, who agreed to take him on as a doctoral student with the Department of Environmental and Global Health at the University of Florida. While working on his PhD, Michael took multiple trips to Haiti, where he was study coordinator for the University of Florida M alaria Surveillance P roject, gaining valuable experience in navigating the complex channels of international research and policy. During his time at University of Flo rida, Michael held various teaching and research assistant positions giving him the experience necessary to teach the undergraduate class Global Public Health in 2013 and 2014 . After receiving his PhD in August 2014, Michael aims to obtain a post doctoria l teaching & research position, while shifting his attention to malaria control and elimination in Africa, which will allow him to incorporate his love of teaching with his skills acquired in the field.