Detection of antibodies against the CB9 to ICB10 region of merozoite surface protein-1 of Plasmodium vivax among the inh...

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Title:
Detection of antibodies against the CB9 to ICB10 region of merozoite surface protein-1 of Plasmodium vivax among the inhabitants in epidemic areas
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English
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Kim, Tong-Soo
Sohn, Youngjoo
Kim, Jung-Yeon
Lee, Won-Ja
Na, Byoung-kuk
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Bio-Med Central ( Malaria Journal)
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Abstract:
Background: The purpose of this study was to examine the usefulness of the conserved block 9 (CB9) to interspecies conserved block (ICB10) region of Plasmodium vivax merozoite surface protein-1 (MSP-1 (ICB910)) as a serodiagnostic tool for understanding malaria transmission. Methods: Antibody titre in the blood samples collected from the inhabitants of Gimpo city, Paju city and Yeoncheon county of Gyeonggi Province, as well as Cheorwon county of Gangwon Province, South Korea were determined by enzyme-linked immunosorbent assay (ELISA). Microscopic examination was performed to identify malarial parasites. Results: MSP-1(ICB910) is encoded by a 1,212-bp sequence, which produced a recombinant protein with a molecular weight of approximately 46 kDa. Antibody titres in 1,774 blood samples were determined with the help of ELISA using purified recombinant MSP-1(ICB910). The overall ELISA-positive rate was 8.08% (n = 146). The annual parasite incidences (APIs) in the regions where the blood sampling was carried out gradually decreased from 2004 to 2005 (1.09 and 0.80, respectively). Yeoncheon county had the highest ELISA-positive rate (10.20%, 46/451). Yeoncheon county also had the highest API both in 2004 and 2005, followed by Cheorwon county, Paju city and Gimpo city. Conclusions: The MSP-1 (ICB910)-ELISA-positive rates were closely related to API in the geographic areas studied. These results suggest that sero-epidemiological studies employing MSP-1 (ICB910)-ELISA may be helpful in estimating the prevalence of malaria in certain geographic areas. MSP-1(ICB910)-ELISA can be effectively used to establish and evaluate malaria control and eradication programmes in the affected areas.
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Kim et al. Malaria Journal 2014, 13:311 http://www.malariajournal.com/content/13/1/311; Pages 1-9
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doi:10.1186/1475-2875-13-311 Cite this article as: Kim et al.: Detection of antibodies against the CB9 to ICB10 region of merozoite surface protein-1 of Plasmodium vivax among the inhabitants in epidemic areas. Malaria Journal 2014 13:311.

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© 2014 Kim et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHOpenAccessDetectionofantibodiesagainsttheCB9toICB10 regionofmerozoitesurfaceprotein-1of Plasmodiumvivax amongtheinhabitantsin epidemicareasTong-SooKim1 †,YoungjooSohn2 †,Jung-YeonKim3,Won-JaLee3,Byoung-kukNa4,Yoon-JoongKang5*andHyeong-WooLee6*AbstractBackground: Thepurposeofthisstudywastoexaminetheusefulnessoftheconservedblock9(CB9)tointerspecies conservedblock(ICB10)regionof Plasmodiumvivax merozoitesurfaceprotein-1(MSP-1(ICB910))asaserodiagnostic toolforunderstandingmalariatransmission. Methods: AntibodytitreinthebloodsamplescollectedfromtheinhabitantsofGimpocity,PajucityandYeoncheon countyofGyeonggiProvince,aswellasCheorwoncountyofGangwonProvince,SouthKoreaweredeterminedby enzyme-linkedimmunosorbentassay(ELISA).Microscopicexaminationwasperformedtoidentifymalarialparasites. Results: MSP-1(ICB910)isencodedbya1,212-bpsequence,whichproducedarecombinantproteinwithamolecular weightofapproximately46kDa.Antibodytitresin1,774bloodsamplesweredeterminedwiththehelpofELISAusing purifiedrecombinantMSP-1(ICB910).TheoverallELISA-positiveratewas8.08%(n=146).Theannualparasiteincidences (APIs)intheregionswherethebloodsamplingwascarriedoutgraduallydecreasedfrom2004to2005(1.09and0.80, respectively).YeoncheoncountyhadthehighestELISA-positiverate(10.20%,46/451).Yeoncheoncountyalsohadthe highestAPIbothin2004and2005,followedbyCheorwoncounty,PajucityandGimpocity. Conclusions: TheMSP-1(ICB910)-ELISA-positiverateswerecloselyrelatedtoAPIinthegeographicareasstudied. Theseresultssuggestthatsero-epidemiologicalstudiesemployingMSP-1(ICB910)-ELISAmaybehelpfulinestimating theprevalenceofmalariaincertaingeographicareas.MSP-1(ICB910)-ELISAcanbeeffectivelyusedtoestablishand evaluatemalariacontrolanderadicationprogrammesintheaffectedareas.BackgroundPlasmodiumvivax causestherelapseofbenigntertian humanmalariathataffectsseveralhundredmillionsof individualsannually.Thisdiseaseisamajorpublichealth concerninmosttropicalandmanytemperateregions, includingNorthandSouthKorea[1].Thefirstscientific documentationofmalariaoccurrencewaspublishedin 1913[2].Anationalmalariaeradicationprogramme strengthenedbytheinvolvementoftheWorldHealth Organizationhassucceededinsignificantlyreducing theincidenceofmalariainSouthKorea[3,4].Malaria wasthoughttohavebeeneradicatedinSouthKoreain thelate1970suntiltwosporadiccasesweredetected inthe1980s[5].In1993,acasewasdiagnosedamong SouthKoreansoldiersservinginNorthernGyeonggi Province[6].Subsequently,Cho etal. reportedtwo instancesofinfectedcivilians[7].Thereafter,manynew caseshavebeenreportednearthedemilitarizedzone (DMZ):inPaju,Yeoncheon,Cheorwon,Gimpo,Ganghwa, Goyang,andDongducheon.Theincreasingnumberof newcasesraisestheconcernthatmalariawillbecome re-establishedintheregion[8,9]. *Correspondence: yjkang@jwu.ac.kr ; rainlee67@naver.com†Equalcontributors5DepartmentofBiomedicalScience,JungwonUniversity,Goesan,Chungbuk 367-805,RepublicofKorea6DepartmentofPathology,Immunology,andLaboratoryMedicine,College ofMedicine,UniversityofFlorida,Florida,FL32610,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Kimetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Kim etal.MalariaJournal 2014, 13 :311 http://www.malariajournal.com/content/13/1/311

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ThemalariaresearchteamoftheKoreaNationalInstitute ofHealth(KNIH)hasdevelopedanewdiagnosticmethod tosupportpathologicalexaminations.Thisantibodybaseddetectionmethodusesmerozoitesurfaceprotein-1 (MSP-1),anantigenandalarge(180 – 230kDa)glycoproteinthatissynthesizedasaprecursortoMSPduring schizogony[10].ComparisonsofthesequencesofMSP-1 from Plasmodiumvivax Plasmodiumfalciparum and Plasmodiumyoelii revealedtheexistenceofteninterspecies conservedblocks(ICBs)containingeightpolymorphic regions[11].Serologicalsurveyshaveprovidedvaluable epidemiologicalinformation,particularlyintheareasof lowendemicity[12].Estimationoftherateofparasitaemia istheclassicalmethodofmeasuringtheprevalenceof malaria.However,theincidenceofparasitaemiaalone mayfailtoadequatelydescribetheepidemiologyofmalariawithinagivenpopulation.Forinstance,whenthe incidenceofmalariaislow,massbloodsurveysdonot yieldresultscommensuratewiththeworkinvolved [13,14].Inthisstudy,theantiP.vivax MSP-1antibody levels(particularlyagainsttheCB9toICB10region) amongthepopulationsofGimpo,Paju,Yeoncheon,and Cheorwonweredeterminedtoevaluatetheusefulness oftherecombinantMSP-1(ICB910)antigenforassessing thelocalmalariaprevalence.MethodsBloodsamplesofinhabitantsToevaluatetheusefulnessoftherecombinantMSP-1 (ICB910)proteininserodiagnosis,bloodsampleswereobtainedfromtheKNIHoftheKoreanCenterforDisease ControlandPrevention(KCDC).Thesebloodsamples (from1,774individuals)werecollectedfromGimpoand Pajucities,YeoncheoncountyofGyeonggiProvince,and CheorwonofGangwonProvinceofSouthKorea,from NovembertoDecemberof2004(Figure1),andwere storedattheKNIH.Bloodsmearswerealsoobtained fromtheKNIHformicroscopicexamination.EthicsstatementThisstudywasconductedafterreceivingthewritten informedconsentfromallparticipantsandonlyafter receivingapprovalfromtheKNIH.Thestudyprocedures, potentialrisksandbenefitswereexplainedtoallofthem. Further,alldatawereanalysedanonymouslyandpatients werenotidentifiedbyname.Thisstudywasconducted strictlyadheringtotheprinciplesexpressedintheDeclarationofHelsinki.MicroscopicexaminationThinbloodfilmswerepreparedtodeterminetheinfectivityofbloodsamples.Thebloodfilmswerefixedwith methanolandstainedwithGiemsastaintorevealthe parasiteinclusionsintheredbloodcells(RBCs).Thin bloodfilmsareoftenpreferredforroutineestimationof parasitaemiabecausetheorganismscanbereadilyvisualizedandquantifiedwiththismethod[15].Toestimatethe densitiesofblood-stageparasitesbymicroscopy,thenumberofasexualparasitesobservedper200whitebloodcells (WBCs)wasdetermined,whichwasthenmultipliedby theassumednumberofWBCspermicrolitreofblood (8,000)[16].AmplificationoftheMSP-1geneToexpresstheCB9toICB10regionofthe P.vivax MSP-1gene,genomicDNAwasextractedfromthe wholebloodofapatientdiagnosedwithmalariausing aQIAampBloodKit(Qiagen,Hilden,Germany).The polymerasechainreaction(PCR)mixturecontained AccuPowerPCRPreMix(Bioneer,Daejeon,Korea),50ng ofpurifiedgenomicDNA,and40pmoleseachofforward (MSP-910F;5 -ggatccGAAGACCAAGTAACAACGGG AGAG-3 )andreverse(MSP-910R;5 -aagcttTTAAAGCT CCATGCACAGGAG-3 )primer(Figure2).Thetotal volumeofthereactionmixturewasadjustedto50 L withdistilledwater.Thethermalcyclingconditionswere asfollows:denaturationat94Cfor5min;35cyclesof 30secat94C,60secat55C,and45secat72C;anda finalincubationat72Cfor5min.AllPCRproductswere analysedbyagarosegelelectrophoresisona1%agarose gel,visualizedunderanultraviolettransilluminator,and purifiedusingaNucleoSpinExtractKit(Macherey-Nagel; Duren,Germany).DNAsequencingandanalysisTogenotypetheMSP-1(ICB910)geneof P.vivax ,the MSP-1(ICB910)genewasPCR-amplifiedandligated intoapCR2.1vector(Invitrogen,Carlsbad,CA,USA) andtransformedinto Escherichiacoli TOP10cells. The E.coli TOP10cellscontainingtherecombinant plasmidwereselectedinmediumcontainingampicillin [17].PlasmidswerepurifiedusingaQiagenplasmid isolationkitaccordingtothemanufacturer  sprotocols. Thetransformantswereconfirmedbyagarosegelelectrophoresisfollowingrestrictiondigestionwith Eco RI. TheMSP-1(ICB910)genesequencewasdeterminedusing anABIPRISMDyeTerminatorCycleSequencingReady ReactionKitFS(PerkinElmer,Cambridge,MA,USA) followingmanufacturer  sinstructions.M13reverseand forward( 20)primerswereusedforsequencing.Nucleotideanddeducedaminoacidsequenceswereanalysed usingEditSeqandClustalintheMegAlignprogram,a multiplealignmentprogramwithintheDNASTARpackage(DNASTAR,Madison,WI,USA).Theinternet-based BLASTsearchprogramoftheNationalCenterforBiotechnologyInformationwasusedtosearchprotein databases.Kim etal.MalariaJournal 2014, 13 :311 Page2of9 http://www.malariajournal.com/content/13/1/311

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Figure2 Nucleotideandaminoacidsequencesof Plasmodiumvivax merozoitesurfaceprotein-1(ICB910). Thenucleotidesequencewas depositedinGenBankBLAST(http://WWW.ncbi.nlm.nih.gov/nuccore,AccessionNo.KJ513462). A B C DDMZ South Korea North KoreaYellow Sea B PajuDMZNorth Korea North Korea Yeoncheon CheorwongunSouth KoreaSouth Korea2 km2 km ANorth Korea Paju-si Gimpo South Korea2 km Han river b c a d efghC DDMZ South Korea Cheorwon North Koreai j kl5 km Figure1 Bloodsamplecollectionareasaccordingtoadministrativedistricts.(A) Gimpo, (B) Paju, (C) Yeoncheon, (D) Cheorwon. a Haseongmyeon; b ,Wolgotmyeon; c ,Yangchonmyeon; d ,Papyeongmyeon; e ,Munsaneup; f ,Baekhakmyeon; g ,Wangjingmyeon; h ,Misanmyeon; i ,Gimhwaeup; j ,Seomyeon; k ,Cheorwoneup; l ,Geunnammyeon. Kim etal.MalariaJournal 2014, 13 :311 Page3of9 http://www.malariajournal.com/content/13/1/311

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ConstructionoftheMSP-1(ICB910)expressionvectorToexpresstheMSP-1genein E.coli DH5 cells,the CB9toICB10regionoftheMSP-1genefragmentwas amplifiedusingMSP-910FandMSP-910Rprimers (respectivelycontaining Bam HIand Hin dIIIsitesat their5 ends)fromabloodsamplethatwasconfirmedto beinfectedwiththedormanttypeof P.vivax (Figure2). Theamplified-PCRproductsweredigestedwith Bam HI and Hin dIII,theproductsweregel-purifiedusinga Qiagengelextractionkit,andwereligatedbetweenthe Bam HIand Hin dIIIcleavagesitesofthepQE30expressionvector(Qiagen).Theresultingplasmidwasusedfor theexpressionofaMSP-1(ICB910)-(His)6fusionprotein in E.coli cells.Thetransformantswerefirstconfirmedby agarosegelelectrophoresisfollowingrestrictiondigestion using Bam HIand Hin dIII.Finally,theinsertsequencewas confirmedbyDNAsequencing.ExpressionandpurificationofrecombinantMSP-1(ICB910)TheexpressionoftherecombinantMSP-1(ICB910)-(His)6fusionproteinin E.coli DH5 cellswasinducedwith isopropyl-1-thio-D-galactopyranoside(IPTG)[18].The MSP-1(ICB910)-(His)6fusionproteinwaspurifiedusing immobilizedmetalionaffinitychromatography[19]under nativeconditionsfollowingthemanufacturer  sprotocols (Qiagen).Proteinlevelswereanalysedbysodiumdodecyl sulphate-polyacrylamidegelelectrophoresis(SDS-PAGE) aftereachpurificationstep.WesternblotanalysisTherecombinantMSP-1(ICB910)-(His)6fusionprotein wasseparatedbySDS-PAGEona12%gelandtransferred ontoanitrocellulosemembrane.Afterthetransfer,the membranewascutintostripsandblockedfornonspecific bindingwith3%skimmilkfor12hrat4C.Themembranewasthenwashedthreetimesfor10mineachwith 0.15%Tween20inphosphate-bufferedsaline(PBS).Followingthis,thestripswereallowedtoreactfor4hrwith serafrompatientswithmalariaorfromuninfectedindividuals(diluted1:100,vol/vol).Themembraneswerethen washedthreetimesfor10mineachwith0.15%Tween20 inPBSandweresubsequentlyincubatedwithperoxidaseconjugatedgoatanti-humanIgGsecondaryantibody (1:1,000)(Sigma)for3hratroomtemperature.For colourdevelopment,asolutioncontaining0.2%diaminobenzidineand0.02%H2O2/PBSwasappliedtoeach well[20,21].Enzyme-linkedimmunosorbentassay(ELISA)AnELISAwasusedtodeterminewhethertheblood samplescontainedantibodiesagainstMSP-1(ICB910) antigens.Briefly,thecaptureantigensolution(50 L, 0.5 g/mL)wasplacedina96-wellplate(Corning,Lowell, MA,USA)andincubatedfor12hratroomtemperature. Theantigensolutionwasthenaspirated,blockingbuffer (1%bovineserumalbumin,0.05%PBS-Tween20)was addedtoeachwell,andtheplatewasincubatedfor1hrat roomtemperature.Afterthewellswerewashedthreetimes with0.05%Tween20/PBS,humanserumsamplesinblockingbufferatadilutionof1:100(vol/vol)wereaddedtothe wells.Fourpositiveandfourn egativecontrolserumsampleswerealsoincludedineachplate.Afterincubatingat roomtemperaturefor2hr,theplateswerewashedwith 0.05%Tween20/PBSthreetimes.Peroxidase-conjugated anti-humanIgG(Sigma,1:2,000,vol/vol)dilutedinblockingbufferwasthenaddedtoeachwellandtheplateswere incubatedfor1hratroomtemperature.Followingthis,the reactionwasstoppedbywashingtheplatesasdescribed above.Todevelopthecolour,100mLof2,2 -azino-di-(3ethyl-benzthiozoline-6-sulphonicacid)(ABTS)peroxidase substrate(Kirkegaard&Perry Laboratories,Gaithersburg, MD,USA)wasaddedtoeachwellandtheplateswereincubatedfor30min.Thiswasfollowedbymeasuringthe absorbanceofthesolutionat405nm.Cut-offvaluefor ELISA-positivitywasdefinedasthesumtotalofthemean valuesandtwotimesthestandarddeviationsofthenegativecontrolsamples.Estimationoftheannualparasiteincidence(API)Foreachofthestudysites,theannualparasiteincidence (API)wascalculatedasthenumberofmalaria-positive patientsper1,000inhabitants;API=(numberofpositive slides/totalnumberofslides)1,000.DataanalysisThedatawereanalysedusingGraphpadPrismsoftware version4.0(GraphPadSoftwareInc.,LaJolla,CA,USA). Pearson  scorrelationanalysiswasperformedtoexamine therelationshipbetweenseropositivityandtheAPIof P. vivax inagivenyear.ThedatawereanalysedusingSPSS software,version17.0(SPSSInc.,Chicago,IL,USA).A Pvalueof<0.05indicatedstatisticalsignificance.The correlationsizeswereinterpretedasnone(0.0-0.09),small (0.1-0.3),medium(0.3-0.5),orstrong(0.5-1.0)[22].ResultsBloodsamplingThestudylocationsareshownonthemapinFigure1. AllareaswereneartheDMZ,aknownhigh-riskarea. Bloodsampleswerecollectedfromparticipantsresiding in23villagesandthreecities(Gimpo,PajuandYeoncheon) locatedinGyeonggiProvince andsixvillagesinCheorwon ofGangwonProvince,SouthKorea.Atotalof1,774blood samples(1.92%)werecollected.Thetotalnumberof inhabitantsinthegeographicalareaswherethestudy wasconductedintheyear2004was92,246.Kim etal.MalariaJournal 2014, 13 :311 Page4of9 http://www.malariajournal.com/content/13/1/311

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DNAsequenceof Plasmodiumvivax MSP-1(ICB910) (Koreanisolate)TheregionfromCB9toICB10oftheMSP-1genethat wasPCR-amplifiedfromthegenomicDNAwasanalysed ona1.0%agarosegel.AmplificationoftheMSP-1(ICB910) geneyieldedanapproximately1,200-bpDNAfragment thatwasthenligatedtothepCR2.1cloningvector.RestrictionanalysisusingEcoRIconfirmedtheidentityof thetransformants.TheplasmidcontainingthePCR productwasnamedpMSP910andwassubjectedto DNAsequenceanalysis.DNAsequencingrevealedthat theclonedMSP-1(ICB910)genewas1,212-bpinlength andencoded403aminoacidsasidentifiedbyDNASIS (Figure2,GenbankaccessionNoKJ513462).ExpressionofMSP-1(ICB910)in Escherichiacoli andits antigenicityToconstructtheexpressionplasmid,theMSP-1(ICB910) genewasamplifiedfromthepatient  sgenomicDNA, digestedwith Bam HIand Hin dIII,andsubclonedintothe samerestrictionenzymesitesofthepQE30expression vectortoproducepMSPex910containinga(His)6-tag. TherecombinantpMSPex910plasmidwasthentransferred into E.coli DH5 cells.Forproteinexpression,1mMIPTG wasaddedto E.coli DH5 (pMSPex910)cellsgrownto logarithmicphaseinliquidLuria-Bertanimediumcontaining100 g/mLampicillinand50 g/mLkanamycin toinducetheexpression.SDS-PAGEfollowedbyCoomassiebluestainingshowedthatundernativepurification conditions,themolecularweightoftherecombinant MSP-1(ICB910)proteinwas~46kDa(Figure3A).The antigenicityoftheMSP-1(ICB910)recombinantprotein wasdeterminedbyWesternblot.Theserafrompatients withmalariareactedpositively(Figure3B,No1 – 3).One sample(Figure3B,No6),whichwascollectedfromanindividualwhohadnosymptomsofinfection,showedweak positivity.Duringthefollow-upstudy,thiscasebecameill fourmonthsafterthebloodsamplingandwasdiagnosed withvivaxinfection.Todeterminethesensitivityand specificityoftheMSP-1(ICB910)recombinantproteinby ELISA,theseraofpatientswithmalariawereused.Sera from68ofthe70patientswithmalaria(sensitivity,97.1%) wereELISA-positive,whereasallsamplesinthenormal controlgroup(n=8)wereELISA-negative(specificity, 100%)(Figure3C).OverviewofmalariatransmissioninfourlocationsOne-hundredandforty-sixofthe1,774studysubjects(8.23%)showedMSP-1(ICB910)-ELISApositivity. Yeoncheonpresentedthehighestpositiverate(Figure1C, 46/451,10.20%),followedbyPaju(Figure1B,35/372, 9.41%),Cheorwon(Figure1D,44/526,8.37%),andGimpo (Figure1A,21/425,4.94%).The2004APIwashigher thanthe2005API.APIdidnotvarywithinthesame geographiclocationduringtheyears2004and2005. YeoncheonpresentedthehighestAPIinbothyears, followedbyCheorwon,PajuandGimpo.Theseropositivityfortheyear2004showedastrongpositivelinear relationshipwiththeAPIsof2004and2005(r=0.691 andr=0.842,respectively) ,butwasstatisticallyinsignificant(Table1).LocalmalariatransmissioninGimpoTwenty-oneofthe425inhabitants(4.94%)presented apositiveresponseinMSP-1(ICB910)-ELISA.HaseongmyeonpresentedthehighestELI SA-positiverate(Figure1a, 6/53,11.32%),followedbyYangchonmyeon(Figure1c, 10/206,4.85%)andWolgotmyeon(Figure1b,5/166, 3.01%).WolgotmyeonpresentedthehighestAPIin 2004(1.42)and2005(0.71).Haseongmyeonpresented thesecondhighestAPIin2004(0.69),buttherewere nonewdiagnosesin2005.Yangchonmyeonwasthirdin APIin2004(0.50)andsecondintheyear2005(0.25). Theseropositivityin2004showedastrongnegativelinear relationshipwiththeAPIsoftheyears2004and2005 (r= 0.513,r= 0.887,respectively).However,these correlationswerenotstatist icallysignificant(Table2).LocalmalariatransmissioninPajuThirty-fiveofthe372inhabitants(9.41%)showedan ELISA-positiveresponse.Musaneuphadahigherpositive rate(Figure1e,28/232,12.07%)thanPapyeongmyeon (Figure1d,7/140,5.00%).Papyeongmyeonhada higherAPIthanMunsaneupintheyears2004and 2005.Theseropositivityin2004showedastrongnegativelinearrelationshipwiththeAPIsof2004and2005 (r= 1.000andr= 1.000,respectively).Thisresultwas statisticallysignificant( P =0.01foreachyear,Table3).LocalmalariatransmissioninYeoncheon-gunForty-sixofthe451inhabitants(10.20%)ofYeoncheon-gun showedanELISA-positiveresponse.Misanmyeonhad thehighestpositiverate (Figure1h,9/75,12.00%), followedbyBaekhakmyeon(Figure1f,28/265,10.57%)and Wangjingmyeon(Figure1g,9/111,8.11%).Baekhakmyeon hadthehighestAPIin2004(3.69)anddroppedtothird placein2005(1.34).MisanmyeonhadthesecondhighestAPIin2004(2.79)andrankedfirstin2005(2.79). WangjingmyeonhadthelowestAPIin2004(1.60)and rankedsecondin2005(2.40).Theseropositivityin2004 showedastrongpositivelinearrelationshipwiththe APIof2004(r=0.685)andaweaklinearrelationship withAPIof2005(r=0.111).However,theseresults werestatisticallyinsignificant(Table4).LocalmalariatransmissioninCheorwonForty-fourofthe526inhabitants(8.37%)ofCheorwon showedMSP-1(ICB910)-ELISApositivity.CheorwoneupKim etal.MalariaJournal 2014, 13 :311 Page5of9 http://www.malariajournal.com/content/13/1/311

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presentedthehighestpositiverate(Figure1k,17/142, 11.97%),followedbyGeunnammyeon(Figure1l,15/143, 10.49%),Gimhwaeup(Figure1i,12/115,10.43%)and Seomyeon(Figure1j,0/126,0.00%).Seomyeonhadthe highestAPIin2004(1.98),followedbyGimhwaeup (1.54),Cheorwoneup(0.86)andGeunnammyeon(0.45). However,GimhwaeuphadthehighestAPIin2005, followedbyGeunnammyeon(2.23),Seomyeon(0.49) andCheorwoneup(0.35).Theseropositivityin2004 showedastrongnegativelinearrelationshipwithanAPI of2004(r= 0.762)andastrongpositivelinearrelationshipwithAPIof2005(r=0.430).However,thesecorrelationswerestatisticallyinsignificant(Table5).DiscussionTheareassurveyed,Gimpo,Paju,Yeoncheon,and Cheorwon,whicharelocatedwithin10 – 15kmofthe southernDMZ,arepartofre-emergingmalarialoutbreak areasinSouthKorea[8].TheDMZisa4km-wideand 250km-longcorridorthatextendsacrossthemiddlepart oftheKoreanpeninsula.Naturallandscape,ecosystems Table1RatesofMSP-1(ICB910)-ELISApositivityand annual parasiteincidenceArea No.of seratested No.of positivesera Positive rate(%) APIa20042005 Gimpo425214.940.730.28 Paju372359.411.070.86 Yeoncheon4514610.202.991.99 Cheorwon526448.371.131.20 Total17741468.231.481.08aAPI ;Annualparasiteincidence. -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2004(r=0.691, P= 0.309). -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2005(r=0.842, P= 0.158). Table2RatesofMSP-1(ICB910)-ELISApositivityand annualparasiteincidenceinGimpoVillage No.of seratested No.of positivesera Positive rate(%) APIa20042005 Haseongmyeon53611.320.690.00 Yangchonmyeon206104.850.500.25 Wolgotmyeon16653.011.420.71 Total425214.940.870.49aAPI ;Annualparasiteincidence. -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2004(r= 0.513, P= 0.657). -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2005(r= 0.887, P= 0.306). Patient Normal 0.0 0.5 1.0 1.5 2.0 O.D at 450 nm1 2 3 4 5 6 7 PatientNormal MSP-1 (ICB910)kDa M 1 2 3 494.067.043.0 30.0 20.1 MSP-1 (ICB910)A BC Cut o Figure3 Characterizationofrecombinantmerozoitesurfaceprotein-1(ICB910).(A) PurificationofrecombinantMSP-1(ICB910)byNi-NTA agaroseaffinitychromatography.LaneM,molecularweightmarker;lane1, E.coli DH5 celllysateafterproteinexpressionwasinducedwithIPTG; lane2,flow-through;lane3,wash;lane4,elutedfraction. (B) WesternblotanalysisofrecombinantMSP-1(ICB910)protein.Lanes1 – 3,samples frompatientsinfectedwithmalaria;Lanes4 – 7,samplesfromuninfectedindividuals. (C) ImmuneresponsesofrecombinantMSP-1(ICB910)to vivaxmalariapatientgroupandnormalpersonbyEnzymelinkedimmunosorbentassay. Kim etal.MalariaJournal 2014, 13 :311 Page6of9 http://www.malariajournal.com/content/13/1/311

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andbiodiversityarehighlyconservedintheDMZ[23]. Theoutbreakareasexpandedyearlybothinthesouthern andeasterndirectionsfromtheDMZintheinitialstage ofre-emergence.Althoughtherehasbeenasharpdecline inthereportedcasesinrecentyears,are-emergenceof malariaintheseareascannotberuledout.Effectivecontrolprogrammeswillpreventthere-emergenceofmalaria. Sensitivediagnostictoolsthatallowrapidandaccurate diagnosiswillensuretheeffectivenessofmalariapreventionandcontrolprogrammes.MSP-1isconsideredasa usefulantigenforserodiagnosisandisanimportantvaccinecandidateagainstasexualbloodstages[24-27].The MSP-1proteinbindstothesurfaceoferythrocytesandis oneofthemerozoitesurfaceligandsinvolvedintheinvasionoftheerythrocytes.Comparisonofthesequencesof MSP-1proteinsfrom P.vivax P.falciparum ,and P.yoelii revealedsevenICBs(ICB1,ICB2,ICB4,ICB5,ICB6,ICB8, andICB10)andthreeCBs(CB3,CB7andCB9)[11].The immuneresponsestotheN-andC-terminalregionsof MSP-1havebeencharacterized[24].The11regionsof MSP-1expressedin E.coli wereglutathioneS-transferase (GST)regions.Itwasreportedthat83.3%ofmalaria patientshadIgGcomplexesagainstatleastoneofthe GST-fusionproteins.Inaddition,thefrequencyof patientswiththeIgGantibodiestorecombinantICB10 protein,whichcontainedonlythe111C-terminalamino acidsofMSP-1,increasedwiththenumberof P.vivax malariaexperiences,reaching83.3%afterfourexperiences.However,theresponsestorecombinantICB2-5, whichconsistedof506C-terminalaminoacidsofMSP-1, didnothavethesamefrequency.Thetitreoftheantibody againstrecombinantICB10proteinwasgreaterthanthat oftheantibodyagainstrecombinantICB2-5.Furthermore, ICB10helpsperipheralbloodmononuclearcells(PBMCs) tosecreteIFN,suggestingthatT-cellepitopesarepresent inthisregion.Soares etal. foundthattheC-terminalregion wasimmunogenictobothantibodiesandT-cellswere producedfollowinginfectionsinhumans[28].MSP-1 couldbeausefulvaccineagainst P.vivax malariabecause theC-terminalregionofMSP-1islesspolymorphic thantheN-terminalregion[29]andtheepitopestoBandT-cellshavespecifichumoralresponsesthatproduce longer-termstability[30].Therefore,theregionfromCB9 toICB10ofMSP-1wasselectedandexpressedasarecombinantproteinin E.coli foruseinsero-epidemiology. Inthepresentstudy,arecombinantMSP-1(ICB910) antigen-basedELISAdiagnosticmethodwasusedto evaluatetheantibodylevelsintheinhabitantsofhigh-risk areas.TheincidenceofmalariapeaksinAugustafterthe rainyseasonanddeclinestobaselinebymid-October. Therefore,bloodsamplingwascarriedoutbetweenlateOctoberandmid-December,whentheactiveanopheline populationhaddiminished.SinceELISA-basedscreening forthepresenceofanantibodycouldprovideusefulinformationregarding P.vivax infectioninapreviouslynave population,thesignificancebetweenthepositiverateand incidenceofmalariainhigh-riskareaswerecompared. TheMSP-1(ICB910)-ELISA-positiveratesandtheAPIs offourcitiesinbothyearsshowedstronglinearrelationships,butwerenotstatisticallysignificant( P =0.309and P =0.158,respectively)(Table1).Thelocaltransmission inGimposhowedastrongnegativerelationshipbetween APIandMSP-1(ICB910)-ELISA,butitwasnotsignificant (2004, P =0.657;2005, P =0.306).Localtransmissionin Pajushowedastrongnegativ erelationshipbetweenAPI andMSP-1(ICB910)-ELISAthatwasstatisticallysignificant Table4RatesofMSP-1(ICB910)-ELISApositivityand annualparasiteincidenceinYeoncheonVillage No.of seratested No.of positivesera Positive rate(%) APIa20042005 Wangjingmyeon11198.111.602.40 Baekhakmyeon2652810.573.691.34 Misanmyeon75912.002.792.79 Total4514610.202.692.18aAPI ;Annualparasiteincidence. -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2004(r=0.685, P= 0.519). -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2005(r=0.111, P= 0.929). Table5RatesofMSP-1(ICB910)-ELISApositivityand annual parasiteincidenceinCheorwonVillage No.of seratested No.of positivesera Positive rate(%) APIa20042005 Gimhwaeup1151210.431.542.46 Seomyeon12600.007.980.49 Geunnammyeon1431510.490.452.23 Cheorwoneup1421711.970.860.35 Total526448.371.211.38aAPI ;Annualparasiteincidence. -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2004(r= 0.762, P= 0.238). -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2005(r=0.420, P= 0.580). Table3RatesofMSP-1(ICB910)-ELISApositivityand annualparasiteincidenceinPajuVillage No.of seratested No.of positivesera Positive rate(%) APIa20042005 Munsaneup2322812.071.000.82 Papyeongmyeon14075.001.601.20 Total372359.411.311.01aAPI ;Annualparasiteincidence. -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2004(r= 1.000, P= 0.01). -CorrelationcoefficientbetweenMSP-1(ICB910)positiverateof2004andAPI of2005(r= 1.000, P= 0.01).Kim etal.MalariaJournal 2014, 13 :311 Page7of9 http://www.malariajournal.com/content/13/1/311

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(2004, P =0.01;2005, P =0.01).Localtransmissionin Yeoncheonshowedstrongrelationshipbetweenthe APIandMSP-1(ICB910)-ELISAof2004andaweak relationshipin2005(2004, P =0.519;2005, P =0.929). InCheorwon,theMSP-1(ICB910)-ELISAresponseand APIhadastrongnegativerelationshipin2004( P =0.238) andamediumpositiverelationshipin2005( P =0.580). Thesedifferencesmightberelatedtothedifferentmalaria controlprogrammesadoptedintheareas.Malariacontrol programmesareadministeredbyfourpublichealthcentres (PHCs).EachPHCusestheirownmanualthattakesthe localmalariaprevalenceandgeographicalcharacteristics intoaccount.Whileevaluatingthemalariatransmissionin agivengeographicalregion,f actorssuchastemperature, mosquitodensity,vectorcapacity,climate,rainfall,and humiditymustbeconsidered[31].However,collecting datarequiresmucheffort.Therefore,itisnecessaryto establishacost-effectivetooltoanalysethecurrentand futuretransmissionofmalariainspecificgeographic areasusingserodiagnosticmethods.Parasitaemiaprovides aclassicalmeansformeasuringmalariaendemicity,but patientincidencealoneisunlikelytoprovideacomplete understandingofmalariaprevalence.Factorsincluding thepopulationdensityofmosquitoes,vectorialcapacity, long-toshort-incubationpatientratio,symptomaticto asymptomaticpatientratio,differencesinrainfalland temperature,andimmunityofthecommunityallaffect malariaprevalenceinKorea.Theseresultssuggestthat antibodydetectionmethodsprovideresultsthatare morevaluablethanAPIvaluesobtainedbymicroscopic examinationincertaincircumstances.Suchmethodswill helptobetterunderstandmalariatransmissioninagiven geographicalarea.ConclusionsTheantibodydetectionmethodusingMSP-1(ICB910)ELISAprovidesusefulinformationregardingmalariaprevalenceinspecificgeographicalareasandindividuals. Thisserologicalmethodisusefulforidentifyingareas thatrequiremalariacontrol.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions TSK,YJKandHWLconceivedanddesignedthestudy,andcontributedtothe executionoftheresearch.HWLandTSKwrotethemanuscript.YSandYJK performedthestatisticalanalysis.TSK,YS,YJK,WJL,BKN,andHWLcollected bloodsamples.HWLpurifiedtheMSP-1(ICB910)recombinantprotein.TSK,WJL andHWLperformedMSP-1(ICB910)-ELISAandthediagnosisofmalariaby microscopicexamination.Allauthorshavereadandapprovedthefinal manuscript. Acknowledgements Wearegratefultoallblooddonorsandthestaffofthepublichealthcentres inGimpo,Paju,Yeoncheon,andCheorwon.Thisworkwassupportedbyan internalgrantoftheKoreanNationalInstituteofHealthandInhaUniversity ResearchFund(2012). Authordetails1DepartmentofParasitology,CollegeofMedicine,InhaUniversity,Incheon 405-751,RepublicofKorea.2DepartmentofAnatomy,CollegeofKorean Medicine,InstituteofKoreanMedicine,KyungHeeUniversity, Hoegi-dongDongdaemun-gu,Seoul130-701,RepublicofKorea.3Divisionof MalariaandParasiticDiseases,NationalInstituteofHealth,KoreaCentersfor DiseaseControlandPrevention,Osong363-951,RepublicofKorea.4DepartmentofParasitologyandInstituteofHealthSciences,Gyeongsang NationalUniversitySchoolofMedicine,Jinju660-751,Korea.5Departmentof BiomedicalScience,JungwonUniversity,Goesan,Chungbuk367-805, RepublicofKorea.6DepartmentofPathology,Immunology,andLaboratory Medicine,CollegeofMedicine,UniversityofFlorida,Florida,FL32610,USA. Received:13March2014Accepted:3August2014 Published:12August2014 References1.MendisK,SinaBJ,MarchesiniP,CarterR: Theneglectedburdenof Plasmodiumvivax malaria. AmJTropMedHyg 2001, 64: 97 – 106. 2.Hasegawa: MalariainKorea. 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