Survey of Plasmodium falciparum multidrug resistance-1 and chloroquine resistance transporter alleles in Haiti

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Title:
Survey of Plasmodium falciparum multidrug resistance-1 and chloroquine resistance transporter alleles in Haiti
Series Title:
Malaria Journal
Physical Description:
Mixed Material
Creator:
Maha A ElBadry
Alexandre Existe
Yves S Victor
Gladys Memnon
Mark Fukuda
John B Dame
Charles A Yowell
Bernard A Okech
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Malaria Journal
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Subjects / Keywords:
Hispaniola
Chloroquine
Mefloquine
Anti-malarial drug resistance
Genotyping

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Abstract:
Background: In Haiti where chloroquine (CQ) is widely used for malaria treatment, reports of resistance are scarce. However, recent identification of CQ resistance genotypes in one site is suggestive of an emerging problem. Additional studies are needed to evaluate genetic mutations associated with CQ resistance, especially in the Plasmodium falciparum multi-drug resistance-1 gene (pfmdr1) while expanding the already available information on P. falciparum CQ transporter gene (pfcrt) in Haiti. Methods: Blood samples were collected on Whatman filter cards (FTA) from eight clinics spread across Haiti. Following the confirmation of P. falciparum in the samples, PCR protocols were used to amplify regions of pfmdr1and pfcrt codons of interest, (86, 184, 1034, 1042, and 1246) and (72-76), respectively. Sequencing and site-specific restriction enzyme digestions were used to analyse these DNA fragments for the presence of single nucleotide polymorphisms (SNPs) known to confer resistance to anti-malarial drugs. Results: P. falciparum infection was confirmed in160 samples by amplifying a segment of the P. falciparum 18S small subunit ribosomal RNA gene (pfssurrna). The sequence of pfmdr1 in 54 of these samples was determined between codons 86,184 codons 1034, 1042 and 1246. No sequence differences from that of the NF54 clone 3D7 were found among the 54 samples except at codon 184, where a non-silent mutation was found in all samples predicted to alter the amino acid sequence replacing tyrosine with phenylalanine (Y184F). This altered sequence was also confirmed by restriction enzyme digestion. The sequence of pfmdr1 at codons 86, 184, 1034 and 1042 encoded the NFSN haplotype. The sequence of pfcrt codons 72-76 from 79 samples was determined and found to encode CVMNK, consistent with a CQ sensitive genotype. Conclusion: The presence of the Y184F mutation in pfmdr1 of P. falciparum parasites in Haiti may have implications for resistance to antimalarial drugs. The absence of mutation in pfcrt at codon 76 among 79 isolates tested suggests that sensitivity to CQ in Haiti remains common. Wide-spread screening of the pfmdr1 and pfcrt especially among patients experiencing treatment failure may be a useful tool in early detection of the emergence of antimalarial drug resistance in Haiti.

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University of Florida
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University of Florida
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RESEARCHOpenAccessSurveyof Plasmodiumfalciparum multidrug resistance-1andchloroquineresistancetransporter allelesinHaitiMahaAElBadry1,AlexandreExiste2,YvesSVictor3,GladysMemnon4,MarkFukuda5,JohnBDame6, CharlesAYowell6andBernardAOkech1,7*AbstractBackground: InHaitiwherechloroquine(CQ)iswidelyusedformalariatreatment,reportsofresistancearescarce. However,recentidentificationofCQresistancegenotypesinonesiteissuggestiveofanemergingproblem. AdditionalstudiesareneededtoevaluategeneticmutationsassociatedwithCQresistance,especiallyinthe Plasmodiumfalciparum multi-drugresistance 1gene( pfmdr 1)whileexpandingthealreadyavailableinformation on P.falciparum CQtransportergene( pfcrt )inHaiti. Methods: BloodsampleswerecollectedonWhatmanfiltercards(FTA)fromeightclinicsspreadacrossHaiti. Followingtheconfirmationof P.falciparum inthesamples,PCRprotocolswereusedtoamplifyregionsof pfmdr 1and pfcrt codonsofinterest,(86,184,1034,1042,and1246)and(72-76),respectively.Sequencingandsite-specificrestriction enzymedigestionswereusedtoanalysetheseDNAfragmentsforthepresenceofsinglenucleotidepolymorphisms (SNPs)knowntoconferresistancetoanti-malarialdrugs. Results: P.falciparum infectionwasconfirmedin160samplesbyamplifyingasegmentofthe P.falciparum 18Ssmall subunitribosomalRNAgene( pfssurrn a).Thesequenceof pfmdr1 in54ofthesesampleswasdeterminedbetween codons86,184codons1034,1042and1246.NosequencedifferencesfromthatoftheNF54clone3D7werefound amongthe54samplesexceptatcodon184,whereanon-silentmutationwasfoundinallsamplespredictedtoalter theaminoacidsequencereplacingtyrosinewithphenylalanine(Y184F).Thisalteredsequencewasalsoconfirmedby restrictionenzymedigestion.Thesequenceof pfmdr 1atcodons86,184,1034and1042encodedtheN F SNhaplotype. Thesequenceof pfcrt codons72-76from79sampleswasdeterminedandfoundtoencodeCVMNK,consistentwitha CQsensitivegenotype. Conclusion: ThepresenceoftheY184Fmutationinp fmdr1 of P.falciparum parasitesinHaitimayhaveimplicationsfor resistancetoantimalarialdrugs.Theabsenceofmutationin pfcrt atcodon76among79isolatestestedsuggeststhat sensitivitytoCQinHaitiremainscommon.Wide-spreadscreeningofthe pfmdr1 and pfcrt especiallyamongpatients experiencingtreatmentfailuremaybeausefultoolinearlydetectionoftheemergenceofantimalarialdrugresistance inHaiti. Keywords: Hispaniola,Chloroquine,Mefloquine,Anti-malarialdrugresistance,Genotyping *Correspondence: bokech@ufl.edu1DepartmentofEnvironmentalandGlobalHealth,UniversityofFlorida, Gainesville,FL32610,USA7EmergingPathogensInstitute,UniversityofFlorida,Gainesville,FL32610,USA Fulllistofauthorinformationisavailableattheendofthearticle 2013ElBadryetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.ElBadry etal.MalariaJournal 2013, 12 :426 http://www.malariajournal.com/content/12/1/426

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BackgroundAnestimated30,000casesofmalariainfectionsare reportedannuallyinHaiti[1,2],wheretransmissionis hypo-endemicwithsporadicepidemicsfueledbyheavy rainfall.MalariainfectionsinHaitiaredominantlycaused by Plasmodiumfalciparum [3,4].Chloroquine(CQ)has beeninuseasananti-malarialsincethe1950s[5],andis currentlyusedextensivelyinthetreatmentofmalaria inHaiti[1].Recently,anationalmalariatreatmentpolicy revisionaddedsingledoseprimaquinetotargetgametocytes[1].Despitelong-termuse,therearescarcereports ofCQ-resistant P.falciparum malariainHaiti[6-8]. However,therecentreportsofCQ-resistant P.falciparum parasitesharbouringknownresistantallelesof pfcrt [9,10] arealarmingandcouldpresentseriouschallengesto clinicalmanagementofmalariainHaitianddiminish prospectsforitseliminationfromHispaniola(the CaribbeanislandwherebothHaitiandtheDominican Republicarelocated). Researchstudiesconductedmorethanthreedecades agofoundonlyCQsensitive P.falciparum strains[6,7], butveryfewfollow-upstudieshavebeenconducted duringtheinterveningyears.Intherecentpast,afew studiesidentifiedtheCQ-resistance-associatedK76T mutationinthe pfcrt ataverylowprevalenceinsamples fromtheArtibonitevalley,fromLeogane,andfrom travellersreturningfromHaiti[8-10].Thepotential roleofresistantallelesof pfmdr1 inCQresistancein Haitihasyettobeinvestigated.Inaddition,thereisa needtoscreenfortheCQresistance-associated pfcrt and pfmdr1 allelesinotherpartsofHaiti. Singlenucleotidepolymorphisms(SNPs)inthe pfmdr1 havebeenassociatedwithresistancetoCQandother anti-malarialdrugsincludingamodiaquine(AQ)and artemether-lumefantrine(AL).The P.falciparum glycoproteinhomologue-1foundinthedigestivevacuoleof themalariaparasitesisencodedby pfmdr1 gene[11]. CQdiffusestoparasitefoodvacuole(FV)membrane neutrallywhereitprotonatesbyacidspresent,which preventsfreediffusionoutthevacuoleandresultsin accumulationofCQinsensitiveparasites.InCQresistant parasites,changesinFVmembraneproteinsencoded by pfcrt and pfmdr1 genesarethoughttoplayarolein reducingaccumulationofdruginsidethevacuole[11-14]. Theroleplayedby pfmdr1 mutations(N86Y,Y184F, S1034CandD1246Y)inmediating invivo and invitro CQresistancehasreceivedalotofresearchinterest [13,15-23].Nopreviousreportshavedescribedalleles of pfmdr1 thatmaybeassociatedwithanti-malarial drugresistanceinHaiti.Therefore,thisresearchstudy evaluatedinHaitithemutationsin pfmdr1 thatinother regionsoftheworldareassociatedwithresistanceto CQandexpandedthesurveillancelocationsinHaitifor CQresistancemutationsin pfcrt .Thisstudycomplements theanti-malarialdrugresistancesurveillanceeffortsofthe Haitiangovernment.MethodsStudysitesThesiteswherethesamplesoriginatedarelocatedin Port-au-Prince,Artibonite,NorthCapHaitian,Leogane, HincheandJacmel(Figure1).AllsitesareruralcommunitiesexceptthePort-au-Princesite,whichisnear theinternationalairport.Alllocationssufferdrastically frompoorinfrastructuralfacilities,whichiscommonin Haiti.Theaveragetemperatureinthesesitesranges from(22C-35C)allyearround[24]whichfavorsmalaria vectorbreedingandconsequentlymalariatransmission. PeakrainfalloccursbetweenNovemberandJanuary[25], andthemajorityofmalariacasesweresampledduring thisperiod.SamplecollectionPlasmodiumfalciparum malariainfectedbloodsamples werecollectedbyfingerprickandpreservedasdriedblood spots(DBS)onFTAcards(WhatmanGEHealthcare,MA, USA)betweenMay2010andJune2012.Sampleswere obtainedfrompatientsattendingstudyclinics,meeting theinclusioncriteria(anypatientabovetheageoftwo yearoldwithmalaria-likesymptoms)whotestedpositivewithrapiddiagnostictest(CareStat ™ FirstResponse MalariaAgpLDH/HRP2Combocard,PremierMedical Corporation,India)andmicroscopyfor P.falciparum andagreedtosignaconsentform.Allpatientsreceived treatmentaccordingtotheHaitianapprovedstandard ofcareavailableattheclinics.TheHaitiEthicalReview Board,UniversityofFloridaInstitutionalReviewBoard andtheOfficeofResearchProtections,USArmyMedical ResearchandMaterialCommandapprovedthisstudy.DNAextractionPlasmodiumfalciparum DNAwasextractedfromtheDBS withamethanolextractionwashprotocolaspreviously described[26,27].Asingledisc3mmindiameterwas punchedfromtheDBSandplacedina0.2mlmicrocentrifugetube.Avolumeof100 lofabsolutemethanol wasaddedandthetubeincubatedatroomtemperaturefor 15min.Methanolwasthendecantedandthe3mmdisc lefttoairdryfor30min.Sixty-fivemicrolitresofsterile DNA-freegradewaterwasthenaddedtothedrieddisc,agitatedslightlyandthenheat edto97Cfor30min.Thetube wasthencentrifugedatlows peedandthesupernatant storedat 20CforuseinthePCRamplificationprotocol.Detectionof Plasmodiumfalciparum andamplificationof pfmdr1 byPCRAllsampleswerePCRtestedtoconfirmthepresenceof P.falciparum byamplifyingthesmallsubunitribosomalElBadry etal.MalariaJournal 2013, 12 :426 Page2of6 http://www.malariajournal.com/content/12/1/426

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RNA( pfssurrna )gene[GenBank:KC906718.1]of P. falciparum withaone-stepPCRaspreviouslydescribed [10].Confirmed P.falciparum positivesampleswere subsequentlyusedforamplificationofsegmentsof pfmdr1 encodingfivecodonsofinterest;86,184,1034,1042and 1246usinganestedPCRprotocolaspreviouslydescribed [28].Allprimersequencesusedtoamplifysegmentsof pfmdr1 arelistedinTable1.AllPCRreactionsincluded thefollowingreagents:1xTaqPCRMasterMix,0.2mM dNTPs,0.75mMMgCl2,and0.2 Mofeachprimer. Thetemperatureprotocolusedforeachreactionwereas follows:onecycleat94Cfor2minutes,anamplification for35cycles(94Cfor30sec,45Cfor1min,and72C for1min)andafinalextensionat72Cfor5min.Two microlitresoftheproductoftheinitialPCRwasusedasa templateforthenestedsecondPCRreactioninallregions. TheproductsofthenestedPCRwereelectrophoresedin 2%agarosegels,stainedwithethidiumbromide(EtBr)and visualizedwithaBioRadTransluminator(UniversalHood II).Amplificationproductswereusedinrestrictiondigest analysisandsequencedasdescribedbelow.Restrictionenzymedigestionfor pfmdr1 mutationsThe pfmdr1 amplificationproductsweresubjectedto restrictionenzymedigestion.Therestrictionenzymesused includedApoI/AflIII,DraI,DdeI,AseIandEcoRVfor analysisofsequencesatcodons86,184,1034,1042and 1246,respectively.AllenzymeswerepurchasedfromNew EnglandBiolabs(Ipswich,MA,USA).Techniquewas performedaspreviouslydescribed[28,29].Digestionwas carriedoutaccordingtomanufacturerinstructions.The productofdigestionwasresolvedona2.5%agarosegel andvisualizedwithBioRadTransilluminator(Universal HoodII). Figure1 MapofHaitishowingsamplingsites. Table1ThePCRamplificationprimerslistfor pfmr1 and pfcrt usedinthestudyGenePCRstepPrimerSequenceNucleotidelocation pfmdr1 Nest1dr25-AGATGGTAACCTCAGTAT-327 … 44 dr35-AGTCTTTTCTCCACAATA-3756 … 739 Nest2A45-TGTTGAAAGATGGGTAAAGAGCAGAAAGAG-324 … 54 A25-GTCAAACGTGCATTTTTTATTAATGACCAttTA-3584 … 552 Nest1dr55-GAAATGTTTAAAGATCCAAG-32929 … 2949 dr65-CAGCAAACTTACTAACAC-33864 … 3848 Nest21034f5-AGAATTATTGTAAATGCAGCTTTATGGGGAcTC-33067 … 3099 1042r5-AATGGATAATATTTCTCAAATGATAAcTTAGCA-33299 … 3267 1246f5-ATGATCACATTATATTAAAAAATGATATGACAAAT-33545 … 3589 pfcrt PfcrtF15-CCGTTAATAATAAATACACGCAG-325 … 47 PfcrtR25-ATGTTTTATATTGGTAGGTGGAAT-3694 … 716 ElBadry etal.MalariaJournal 2013, 12 :426 Page3of6 http://www.malariajournal.com/content/12/1/426

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Amplificationof pfcrtSamplespositivefor pfssurrna wereusedtoamplifycodons 72 – 76of pfcrt geneaspreviouslydescribed[10,30].A one-stepPCRwascarriedoutusingPhusionflashhigh fidelitymastermixfrom(NewEnglandBiolabs,Ipswich, MA,USA)witha25 lfinalvolumeofPCRreaction composedof1xPCRMasterMixcontaining0.2mM dNTPs,0.5 Meachprimer,2.5mMMgCl2,and1 l ofDNAextract.Thecyclingtemperatureprotocolused was:twocyclesat95Cfor5min,amplificationfor 40cycles(94Cfor30sec,60Cfor30sec,and72Cfor 30sec)andafinalextensionat72Cfor10min.Products ofamplificationwerethenelectrophoresedon2%agarosegel,stainedwithEtBrandvisualizedwithBioRad Transilluminator(UniversalHoodII).SequencingandalignmentAmplifiedsegmentsof pfmdr1 and pfcrt weresequenced bytheSangerchain-terminationmethodtodetermine polymorphicsitesassociatedwithCQorotherantimalarialresistance.SequencingwasdoneonaBiosystem 3730GeneticAnalyzerattheUniversityofFlorida ’ s InterdisciplinaryCenterforBiotechnologyResearchDNA SequencingCoreLaboratory.Allsequencesresultswere alignedto P.falciparum 3D7 pfmdr1 and pfcrt sequences. [GenBank:NC_004326.1andNC_004328.2]respectively.ResultsAtotalof375microscopy-confirmedpositivesamples for P.falciparum werecollectedforanalysisbyPCR.A segmentofthe pfssurrna genewassuccessfullyamplified from160ofthesesamples.Oneormoresegmentsof interestfromthe pfmdr1 geneweresuccessfullyamplified forsequenceanalysisfrom54samples.Nomutationswere detectedincodons86,1034,1042and1246amongthe samplesinwhichcodonsweresuccessfullyamplified. However,mutationswerefoundatcodon184(Y F)in allthesamplesinwhichthecodonsuccessfullyamplified whichwasfurtherconfirmedbyrestrictionenzyme digestion.Thenumberofsamplesthatsuccessfully amplifiedforeachcodonpersiteisshowninTable2. Thirty-eightsamplesthatamplifiedatcodons86,184, 1034and1042wereallNFSNhaplotype.Intheanalysis of pfcrt ,thepolymorphicregionassociatedwithCQresistancealleles,encompassingcodons72 – 76,wassuccessfully amplifiedfrom79samples.Nomutationsweredetected withallsamplesexpressingthewildtypesequence CVMNK.Theintersectionofsamplesfromwhichboth alleles – pfcrt and pfmdr1 -successfullyamplifiedwere50.DiscussionAlthoughthisstudywaslimitedbysmallsamplesize, itprovidespreliminaryinformationaboutthe pfmdr1 allelesinHaitiandaddsadditionaldataon pfcrt alleles inparasitesfrombothpreviouslysampledregionsof Haitiandfromregionsnotpreviouslysampled.This studyisthefirsttoexamineinHaitithe pfmdr1 genetic mutationslinkedtoanti-malarialdrugresistanceinother regionsoftheworld.ThisstudyfoundneithertheK76T mutationin pfcrt northeN86Y,S1034C,N1042Dor D1246Ymutationsin pfmdr1 ,thathavebeenreported toconferresistancetoCQ[22,23,31-33].Theabsenceof mutationsatcodons1034and1042in pfmdr1 suggests thatthe P.falciparum parasitesanalysedinthisstudy likelyremainsensitivetomefloquine[34]andAL[35]. Interestingly,allthesamplestestedhadaY >Fmutation incodon184in pfmdr1 .Thewidespreadoccurrence ofthissinglemutationin pfmdr1 isnotuniqueinthe literature[36,37].Previousstudiesofclinicalisolatesof P. falciparum inCambodiareportedasimilarwidespread occurrenceofsinglepointmutations,andthemutation resultingintheY184Fmutationwaspredominantover other pfmdr1 mutations[38]. AlthoughtheY184Fmutationin pfmdr1 hasbeen associatedmainlywithlowlevelresistancetoAL[39], thereisnoconsensusintheliteratureforitsrolein CQresistance[28,34,40].Inapreviousstudy[15],the Y184FpolymorphismwasshowntoplayaroleinCQ resistanceincloseassociat ionwiththeK76Tmutation in pfcrt .TheY184Fmutationisthoughttoalterthe Table2PresentssummaryofPCRamplificationresultsandgeographicdistributionofsamplescollectedinthestudy, numberofsamplesamplifiedfordifferentgenes pfssurrna pfcrt ,anddifferentialdataonno.ofsamplesamplifiedfor differentcodonsof pfmdr1SiteNo.ofsamplespositive formicroscopy Positivefor pfssurrna Positive for pfcrt Codon86-184Codon1034-1042Codon1246No.ofsamplesamplified forcodons86-1042 Port-au-Prince25170392927823 Leogane40391781114 CapHaitian10230100 L ’ CayJacmel17161513908 Artibonite502632201 Nippes7722302 Total375160795453938 ElBadry etal.MalariaJournal 2013, 12 :426 Page4of6 http://www.malariajournal.com/content/12/1/426

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kineticsofanti-malarialdrugtransport[32,41],andthus efficacy.TheabsenceoftheK76Tmutationsinthestudy sampleindicatesthattheY184Fmutationin pfmdr1 is rarelyfoundinHaitiincombinationwithK76Tin pfcrt Althoughthis pfmdr1 allelemaynotaloneincreaseCQ resistance,it ’ shighprevalencecouldhaveanimpactif thefrequencyofthe pfcrt K76Talleleweretoincrease inHaiti[8].Otherstudieshaveshownamodification ofresistancetoALin P.falciparum withtheY184F mutationunderaN86YandD1246Ybackground[36]. TheabsenceofN86Y,S1034C,N1042DorD1246Yin pfmdr1 allelesinthisstudysustainsCQorALsensitivity inHaiti.Therefore,thefindingswouldseemtoindicate thatthepresenceoftheY184Fmutationalonewould notcontributegreatlytoanti-malarialresistanceinHaiti intheabsenceofwidespreadK76Tmutationsin pfcrt oradditionalmutationsin pfmdr1 .Routinemolecular surveillanceoftheanti-malarialdrugresistancegenes inHaiticombinedwith invitro drugsensitivitystudiesare neededtoascertainthesensitivityofHaitian P.falciparum strainstothemajoranti-malarialmedicationsinusein Haitiandotherpartsofworld. TherecentreportsofCQresistancegenotypesinHaiti arealarmingforpublicheal thofficialsinHaitiand concertedeffortsareneededtomonitortheanti-malaria drugresistancegenotypesinHaiti.Theresultsofthis studyhaveprovidedbackgrounddataon pfmdr1 ,which maybereferencedindiscussionsoffuture P.falciparum anti-malarialresistancepatternsinHaiti.Thisstudyalso providesadditionaldataontheprevalenceofthe pfcrt K76TalleleinHaiti,butadditionalstudiesareneededto increasethesamplesizeandprovideanaccurateestimate forK76Talleleprevalenceanddistribution.Nonetheless, thefindingsinthisstudyhaveincreasedtheknowledge oftwoimportantanti-malariadrugresistancemarkers inHaiti,wherewidespreaddrugresistancehasnotyet beenobserved.StudylimitationsThesmallsamplesizeisindeedthelargestlimitationof thisstudy,howeverduetothedifficultyofthestudy initiation;propersamplestoragefortheFTAcardswas lackingwhichaffectedthequalityofDNA.Thestudy lackadequatesampledistribution,weresomesites failedtoenrollsufficientnumberofpatients,thiscan beattributedtochangeinsiteadministrationwhich affectedthestudyenrollment.Competinginterests Theauthorsdeclaretheyhavenocompetinginterest. Authors ’ contributions MAEgeneratedthemoleculardataandinterpretation,contributedtowriting ofthemanuscript.AEorganizedsamplecollectioninHaiti.YVorganized samplecollectionatBlanchardclinic.GMorganizedsamplecollectionin HospitalSaintCroix.MFrevieweddataandwritingofmanuscript.JD participatedinwritingmanuscript.CYworkedongenotypingofsamples.BO designedthestudy,draftedmanuscript,coordinatedthesamplecollection, anddataanalysis.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements TheauthorsthanktheHaitiMinistryofSanitationandPublicPractice(MSPP), ChristianvilleFoundationHaiti,FamilyHealthMinistries,Haiti,HospitalSainte CroixandBlanchardClinicinHaitifortheirsupportintheexecutionofthis work.TheworkinthispaperwassupportedbyagrantfromtheDepartment ofDefense(DoD)GlobalEmergingInfectionsSurveillance&Response System(GEIS)Grant#P0103_13__UNawardedtoBAO. Authordetails1DepartmentofEnvironmentalandGlobalHealth,UniversityofFlorida, Gainesville,FL32610,USA.2NationalPublicHealthLaboratory,Ministryof PublicHealthandPopulation,PortauPrince,Haiti.3BlanchardClinic,Family HealthMinistriesHaiti,TerreNoire,PortauPrince,Haiti.4HospitalSaintCroix, Leogane,Haiti.5ArmedForcesHealthSciencesSurveillanceCenter,Silver Spring,MD,USA.6DepartmentofInfectiousDiseasesandPathology, UniversityofFloridaGainesville,Gainesville,FL32610,USA.7Emerging PathogensInstitute,UniversityofFlorida,Gainesville,FL32610,USA. Received:17August2013Accepted:25October2013 Published:19November2013 References1.WHO: WorldMalariaReport2012. Geneva:WorldHealthOrganization;2012. 2.RobertsL: EliminationmeetsrealityinHispaniola. Science 2010, 328: 850 – 851. 3.EiseleTP,KeatingJ,BennettA,LondonoB,JohnsonD,LafontantC, KrogstadDJ: Prevalenceof Plasmodiumfalciparum infectioninrainy season,ArtiboniteValley,Haiti,2006. 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AntimicrobAgentsChemother 2005, 49: 840 – 842.doi:10.1186/1475-2875-12-426 Citethisarticleas: ElBadry etal. : Surveyof Plasmodiumfalciparum multidrugresistance-1andchloroquineresistancetransporterallelesinHaiti. MalariaJournal 2013 12 :426. 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 ElBadry etal.MalariaJournal 2013, 12 :426 Page6of6 http://www.malariajournal.com/content/12/1/426