Evaluation of circumsporozoite protein of Plasmodium vivax to estimate its prevalence in the Republic of Korea: an obser...

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Title:
Evaluation of circumsporozoite protein of Plasmodium vivax to estimate its prevalence in the Republic of Korea: an observational study of incidence
Series Title:
Malaria Journal
Physical Description:
Mixed Material
Creator:
Pyo-Yun Cho
Sang-Wook Lee
Seong Kyu Ahn
Jin Su Kim
Seok Ho Cha
Byoung-Kuk Na
Yun-Kyu Park
Sung Keun Lee
Won-Ja Lee
Ho-Woo Nam
Sung-Jong Hong
Jhang Ho Pak
Yoon-Joong Kang
Youngjoo Sohn
Young-Yil Bahk
Han-Ik Cho
Tong-Soo Kim
Hyeong-Woo Lee
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Malaria Journal
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Abstract:
Background: Plasmodium vivax re-emerged in 1993. Although the number of infections has been steadily decreasing, it is likely to continue to affect public health until it is eradicated. The aim of this study is to measure anti-circumsporozoite protein (CSP) antibody and compare malaria prevalence. As to understand the prevalence, an epidemiology study has to be conducted in the Republic of Korea. Methods: A total of 1,825 and 1,959 blood samples were collected in 2010 and 2011, respectively, from the inhabitants of Ganghwa and Cheorwon counties. The antibody titers of the inhabitants were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant protein purified from Escherichia coli transformed with a CSP gene-inserted pET-28a(+) expression vector. Microscopic examination was performed to identify malaria parasites. Results: The annual parasite incidence (API) in Ganghwa decreased from 4.28 in 2010 to 2.23 in 2011, and that in Cheorwon decreased from 1.88 in 2010 to 1.15 in 2011. The antibody-positive CSP rate in these areas also decreased from 18.14% (331/1825) in 2010 to 15.36% (301/1959) in 2011. Pearson analysis showed a strong correlation between the API and the antibody-positive CSP rate in these areas (r = 1.000, P < 0.01). The intensity of the immune responses of the inhabitants of Cheorwon, as measured by the mean optical density, decreased from 0.9186 ± 0.0472 in 2010 to 0.7035 ± 0.0457 in 2011 (P = 0.034), but increased in Ganghwa from 0.7649 ± 0.0192 in 2010 to 0.8237 ± 0.1970 in 2011 (P = 0.006). The immune response increased according to age (r = 0.686, P = 0.041). Conclusions: The positive CSP-ELISA rate was closely related to the API in the study areas. This suggests that seroepidemiological studies based on CSP-ELISA may be helpful in estimating the malaria prevalence. Moreover, such studies can be used to establish and evaluate malaria control and eradication programmes in high-risk areas in Korea.

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RESEARCHOpenAccessEvaluationofcircumsporozoiteproteinof Plasmodiumvivax toestimateitsprevalencein theRepublicofKorea:anobservationalstudyof incidencePyo-YunCho1 †,Sang-WookLee2 †,SeongKyuAhn1,JinSuKim1,SeokHoCha3,Byoung-KukNa4,Yun-KyuPark1, SungKeunLee5,Won-JaLee6,Ho-WooNam7,Sung-JongHong8,JhangHoPak9,Yoon-JoongKang10, YoungjooSohn11,Young-YilBahk12,Han-IkCho13,Tong-SooKim1*andHyeong-WooLee2*AbstractBackground: Plasmodiumvivax re-emergedin1993.Althoughthenumberofinfectionshasbeensteadily decreasing,itislikelytocontinuetoaffectpublichealthuntilitiseradicated.Theaimofthisstudyistomeasure anti-circumsporozoiteprotein(CSP)antibodyandcomparemalariaprevalence.Astounderstandtheprevalence,an epidemiologystudyhastobeconductedintheRepublicofKorea. Methods: Atotalof1,825and1,959bloodsampleswerecollectedin2010and2011,respectively,fromthe inhabitantsofGanghwaandCheorwoncounties.Theantibodytitersoftheinhabitantsweremeasuredby enzyme-linkedimmunosorbentassay(ELISA)usingrecombinantproteinpurifiedfrom Escherichiacoli transformed withaCSPgene-insertedpET-28a(+)expressionvector.Microscopicexaminationwasperformedtoidentifymalaria parasites. Results: Theannualparasiteincidence(API)inGanghwadecreasedfrom4.28in2010to2.23in2011,andthatin Cheorwondecreasedfrom1.88in2010to1.15in2011.Theantibody-positiveCSPrateintheseareasalsodecreased from18.14%(331/1825)in2010to15.36%(301/1959)in2011.Pearsonanalysisshowedastrongcorrelation betweentheAPIandtheantibody-positiveCSPrateintheseareas(r=1.000, P <0.01).Theintensityoftheimmune responsesoftheinhabitantsofCheorwon,asmeasuredbythemeanopticaldensity,decreasedfrom0.91860.0472in 2010to0.70350.0457in2011( P =0.034),butincreasedinGanghwafrom0.76490.0192in2010to0.82370.1970 in2011( P =0.006).Theimmuneresponseincreasedaccordingtoage(r=0.686, P =0.041). Conclusions: ThepositiveCSP-ELISAratewascloselyrelatedtotheAPIinthestudyareas.Thissuggeststhat seroepidemiologicalstudiesbasedonCSP-ELISAmaybeh elpfulinestimatingthemal ariaprevalence.Moreover, suchstudiescanbeusedtoestablishandevaluatemalariacont rolanderadicationprogrammesinhigh-riskareasinKorea. *Correspondence: tongsookim@inha.ac.kr ; rainlee67@naver.com†Equalcontributors1DepartmentsofParasitology,InhaUniversitySchoolofMedicine,Incheon 400-712,RepublicofKorea3DepartmentofParasitologyandInstituteofHealthSciences,Gyeongsang UniversitySchoolofMedicine,Jinju660-751,RepublicofKorea Fulllistofauthorinformationisavailableattheendofthearticle 2013Choetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublicDomainDedication waiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwise stated.Cho etal.MalariaJournal 2013, 12 :448 http://www.malariajournal.com/content/12/1/448

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BackgroundPlasmodiumvivax isthecausativeagentofrelapsingbenigntertianhumanmalaria,thesecondmostcommon typeofhumanmalaria,anditafflictsseveralhundred millionindividualsannually.Thisdiseaseisamajorpublichealthprobleminmosttropicalregionsandmany temperatecountries,includingtheDemocraticPeople ’ s RepublicofKorea(DPRK)andtheRepublicofKorea (ROK)[1]. Thefirstscientificdocumentationofmalariaoccurrence waspublishedin1913,althoughmalariahadbeenprevalentthroughouttheKoreanpeninsulaforseveralcenturies[2].Asaresultofanationalmalariaeradication programmeconductedincooperationwiththeWorld HealthOrganization,thein cidenceofvivaxmalariain ROKhasrapidlydecreased[3,4].Infact,vivaxmalaria wasthoughttohavebeeneradicatedinROKinthelate 1970suntiltwosporadiccasesweredetectedinthe1980s [5].In1993,onecasewasdiagnosedinanROKsoldier servinginnorthernGyeonggiprovince[6],andCho etal. subsequentlyreportedcasesintwoinfectedcivilians[7]. Thereafter,manycaseswerereportednearthedemilitarizedzone(DMZ),whichcentresonPaju-si,Yeoncheongun,Cheorwon-gun,Gimpo-si,Ganghwa-gun,Goyang-si, andDongducheon-si.Thereisnowconsiderableconcern thatmalariawillbecomere-establishedintheregionand thenexpandtoothergeographicalareas[8]. Parasitaemiaprovidesthebasisfortheclassicalmethod ofmeasuringtheendemicityofmalaria.However,using dataononlytheincidenceofparasitaemiamaybeinsufficienttoadequatelydeterminetheepidemiologyofmalaria inagivenpopulation.Forexample,whentheincidenceof malariaislow,massbloodsurveysdonotyieldresults commensuratewiththeworkinvolved[9,10].Serological surveyshaveprovidedvaluableepidemiologicalinformation,especiallyinareasoflowendemicity[11,12].Interestingly,mostpatientswithmalariainROKshowalong incubationperiod[13].Itisthoughtthatalongincubation periodisassociatedwithsporogonicparasites;thus,circumsporozoiteprotein(CSP)wasselectedforthepresent seroepidemiologicalstudy.CSPisasporogonicantigen andsurfacemembraneproteinthatisexpressedinall Plasmodiumsporozoites.CSPhasacentralimmunodominantregioncomprisingashorttandemrepeatof aminoacidsequencescontainingmultiplecopiesofthe immunodominantB-cellepitope[14].CSPisclassified intotwoserotypes,VK210[thedominantform,with GDRA(D/A)GQPArepeats]andVK247[thevariantform, withANGA(G/D)(N/D)QPGrepeats],whichhavedifferentsequencesintherepeatedregionoftheCSPgene.Itis knownthatthevivaxmalariaprevalentinKoreaisthe VK210type[15]. Theannualparasiteincidence(API)inGanghwa county(Figure1A)fluctuatedfrom2001to2012(from 0.76to3.36)andpeakedin2007(Figure2A).Two islands,GyodongmyeonandSamsanmyeon,whichcontain24.5%(15.1% – 30.9%)ofpatientswithmalaria amongallpatientsinGanghwawithmalaria,wereselectedfortheseroepidemiologicalstudytoevaluatethe CSPantigen.InCheorwon(Figure1B),fouradministrativeareaswereselectedforbloodcollection.TheAPIof Cheorwonalsofluctuatedduringthesametimeperiod (from0.12to4.07)andpeakedin2001(Figure2B).The numberofpatientswithmalariawas9.9%(2.9% – 22.5%) inCheorwoneup,33.5%(20.4% – 44.7%)inDongsongeup, 12.6%(8.1% – 16.7%)inGimhwaeup,and7.1%(0.0% – 12.7%)inSeomyeonamongallpatientswithmalariain Cheorwoncounty(Unpublisheddata,KoreaCenterfor DiseaseControlandPrevention). Inthisstudy,thedormantformofCSPwasexpressed andpurifiedfrom Escherichiacoli toproduceantigenfor thedetectionofantiP.vivax CSPantibodylevelsintheinhabitantsofGanghwaandCheorwoncountiestoevaluate CSPantigenincomparingthelocalmalariaprevalence.MethodsStudyareaThestudywasconductedinGanghwacountyof IncheonMetropolitancityandCheorwoncountyof Gangwonprovince,ROK.Ganghwacounty(3731 -45N ’ 12533 -1262 E)contains179villages(administrative village,-ri)inanareaof411km2thatiscoveredwith forestedhillymountains(181km2,44%),agricultural land(164km2,40%),buildingsite(13km2,3%),and others(52km2,13%).ThetotalpopulationofGanghwa countywas67,668persons(male,33,725;female, 38,943)livingin29,055householdsin2010[16].Cheorwoncounty(3805 -24N ’ ,12656 -12724 E)contains 109villages(administrativevillage,-ri)inanareaof 889km2thatiscoveredwithforestedhillymountains (601km2,67%),agriculturalland(209km2,24%),buildingsite(8km2,1%),andothers(71km2,8%).Thetotal populationofGanghwacountywas49,463persons (male,25,820;female,23,643)livingin20,316householdsin2010[17].BloodsamplecollectionThestudylocationsareshownonthemapinFigure1.All ofthestudyareaswereneartheDMZ,whichislocated alongtheborderlineofDPRK.Bloodsampleswerecollectedfromparticipantsresidingin32villagesamongtwo administrativeareasinGanghwacountyofGyeonggi provinceandin10villagesamongfouradministrative areasinCheorwoncountyofGangwonprovince.Itwas conductedinNovemberandDecemberof2010andin NovemberandDecemberof2011intwoareasofROK: GanghwacountyofIncheoncity,whichcontainstwo islands,GyodongmyeonandSamsanmyeon(Figure1A),Cho etal.MalariaJournal 2013, 12 :448 Page2of11 http://www.malariajournal.com/content/12/1/448

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andCheorwoncountyofGangwonprovince,whichcontains3 – eupand1 – myeon,Cheorwoneup,Dongsongeup, Gimhwaeup,andSeomyeonwhichisconnectedtoDPRK byland(Figure1B).ToevaluateCSPrecombinantprotein asantigenforserodiagnosis,atotalof1,825(4.77%)and 1,959(5.12%)bloodsampleswerecollectedin2010and 2011fromwhowantedbeavolunteer,respectively,from 38,288totalinhabitantsinthestudyareas. Bloodsmearswerepreparedformicroscopicexamination.Serawereseparatedandstoredat 20Cforantibodyanalysis.Informedconsentwasobtainedfromall individuals.Allsampleswerecollectedusinghuman A BDMZ ROK DPRK Yellow Sea Cheorwon5 km 5 kmYellow Sea DMZ DPRK ROK ROK DPRKA BDMZ Ganghwa Gimpo Paju Hwacheon Yeoncheona b c d e f Figure1 Studyareas.A ,Ganghwacounty; B ,Cheorwoncounty;a,Gyodongmyeon;b,Samsanmyeon;c,Cheorwoneup;d,Dongsongeup;e, Gimhwaeup;f,Seomyeon. 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 0 5 10 15 YearAnnual parasite incidence 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 0 5 10 15 YearAnnual parasite incidenceAB; Total ; Gyodongmyeon ; Samsanmyeon ; Total ; Cheorwoneup ; Dongsongeup ; Gimhwaeup ; Seomyeon Figure2 Annualparasiteincidenceofstudyareasfrom2001to2012.A ,Ganghwacounty; B ,Cheorwoncounty. Cho etal.MalariaJournal 2013, 12 :448 Page3of11 http://www.malariajournal.com/content/12/1/448

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protocolsthatwerereviewedandapprovedbythe HumanEthicsCommitteeofInhaUniversityMicroscopicexaminationThinbloodfilmswerepreparedtodeterminetheinfectivityofbloodsamples.Thebloodfilmswerefixedwith methanolandstainedwithGiemsastaindilutedwith bufferedwateratpH7.2toemphasizetheparasiteinclusionsintheredbloodcells(RBCs).Thefixedmonolayer ofRBCsinthisproceduremakesthemorphological identificationoftheparasitetothespecieslevelmuch easierandprovidesgreaterspecificitythanthatobtained withthick-filmexamination.Thinbloodfilmsareoften preferredforroutineestimationofparasitaemiabecause theorganismsareeasiertoseeandcountwiththis method[18].Toestimatethedensitiesofblood-stage parasitesbymicroscopy,itwascountedthenumberof asexualparasitesobservedrelativeto200whiteblood cells(WBCs)andthenmultipliedtheparasite:WBCratio by8,000,thatis,theassumednumberofWBCsper microlitreofblood[19].AmplificationoftheCSPgeneToexpresstheCSPgene,genomicDNAwasextracted fromthewholebloodofapatientwithmalariausinga QIAampBloodKit(Qiagen,Hilden,Germany).Polymerasechainreaction(PCR)wasperformedusingAccuPowerPCRPreMix(Bioneer,Daejeon,Korea),50ngof purifiedgenomicDNA,and40pmoleseachofforward (F1;5 -CACGTAGGACAAAGTGCTAGCCG-3 ) andreverseprimer(R1;5ATGGACTCCATGCAG TGTAA-3 ).Thetotalvolumewasadjustedto50 l withdistilledwater.Thethermocyclerconditionswere asfollows:denaturationat94Cfor5min;35cyclesof 30sat94C,30sat55C,and45sat72C;andfinally, incubationat72Cfor5min.AllPCRproductswereanalyzedona1%agarosegel,confirmedunderaUVtransilluminator,andpurifiedwithaNucleoSpinExtractKit (Macherey-Nagel,Duren,Germany).DNAsequencingandanalysisTogenotypetheCSPgeneof P.vivax ,thePCRproduct oftheCSPgenewasligatedintoapGEM-TEasyVector (Promega,Madison,WI,USA)andtransformedinto E.coli DH5 .ThePCRproductcontaining E.coli DH5 wasselectedonampicillin-containingmedium[20].To confirmthetransformants,gelelectrophoresiswasperformedwith Eco RIdigestionproductsaftertheplasmid waspreparedwithaQiagenplasmidisolationkitaccordingtotheprotocolsuppliedbythemanufacturer.The CSPgenesequencewasdeterminedusinganABI PRISMDyeTerminatorCycleSequencingReadyReactionKitFS(PerkinElmer,Cambridge,MA,USA)accordingtothemanualsuppliedbythemanufacturer. M13reverseandM13forward( 20)primerswereused inthesequencing.Nucleotideanddeducedaminoacid sequenceswereanalyzedusingEditSeqandClustalin theMegAlignprogram,amultiplealignmentprogramin theDNASTARpackage(DNASTAR,Madison,WI, USA).Theinternet-basedBLASTsearchprogramofthe NationalCenterforBiotechnologyInformationwasused tosearchproteindatabases.ConstructionoftheCSPexpressionvectorToexpresstheCSPgenein E.coli DH5 ,aCSPgene fragmentwasamplifiedfromabloodsamplethatwas confirmedtobeinfectedwiththedormanttypeof P.vivax asdescribedabovewiththeexceptionofthe additionoftheFex2(5 -ggatccAAAAAGGATGGAAA GAAAG-3 )andRex2(5 -aagcttGAC TTTTCATTTGG GGCA-3 )primers,whichcontain Bam HIand Hin dIII sitesontheir5 ends,respectively.TheamplifiedPCR productsweredigestedwith Bam HIand Hin dIII,purifiedwithaQiagengelextractionkitafterbeingrunon anagarosegel,andthenintegratedintothe Bam HIand Hin dIIIcleavagesitesofthepET-28a(+)expressionvector(Novagen,5,369bp).TheresultingplasmidwassubsequentlyusedfortheexpressionofaCSP-(His)6fusion proteinin E.coli .Thetransformantswereconfirmedby bothgelelectrophoresisofplasmidDNAafterrestrictionenzymedigestionwith Bam HIand Hin dIIIand DNAsequencing.ExpressionandpurificationofrecombinantCSPExpressionoftherecombinantproteinwasinducedin E.coli DH5 withisopropyl-1-thio-D-galactopyranoside (IPTG)[10].TheCSP-(His)6fusionproteinwaspurified usingimmobilizedmetalionaffinitychromatography [21].Thepurificationwasperformedundernativeconditionsaccordingtothesupplier ’ sprotocol(Novagen). Proteinswereanalyzedbysodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE)aftereach purificationstep.WesternblotanalysisTherecombinantCSP-(His)6fusionproteinwasseparated ona12%SDS-PAGEgelandtransferredtoanitrocellulosemembrane.Afterthetransfer,themembranewascut intostripsandblockedfornonspecificbindingwith3% skimmilkfor12hat4C.Themembranewasthen washedthreetimesfor10mineachwith0.15%Tween 20-PBS.Thestripswereallowedtoreactwithserafrom patientswithmalariaorfromuninfectedindividuals(diluted1:100,vol/vol)for4h;theywerethenwashedusing theproceduredescribedabove.Themembranewassubsequentlyincubatedwithdilutedperoxidase-conjugatedgoat anti-humanIgGsecondaryantibody(1:1,000,v/v)(Sigma) for3hatroomtemperature.Forcolourdevelopment,aCho etal.MalariaJournal 2013, 12 :448 Page4of11 http://www.malariajournal.com/content/12/1/448

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solutioncontaining0.2%diaminobenzidineand0.02% H2O2/PBSwasappliedtoeachwell[22,23].Enzyme-linkedimmunosorbentassayEnzyme-linkedimmunosorbentassays(ELISAs)were usedtodeterminewhetherthebloodsamplescontained antibodiesagainsttheCSPVK210of P.vivax .Briefly, thecaptureantigensolution(50ml,0.5 g/ml)was placedona96-wellplate(Corning,Lowell,MA,USA) andincubatedfor12hatroomtemperature.Thewells wereaspiratedandfilledwithblockingbuffer(1%BSA, 0.05%PBS-Tween20)andincubatedfor1hatroom temperature.Afterthewellswerewashedwith0.05% PBS-Tween20threetimes,humanserumsamplesin blockingbufferatadilutionof1:100(vol/vol)were addedtothewells.Fourpositiveandfournegative controlserumsampleswerealsoaddedtoeachplate. After2hofincubationatroomtemperature,theplates werewashedwith0.05%PBS-Tween20threetimes,and peroxidase-conjugatedanti-humanIgG(Sigma,1:2,000, vol/vol)dilutedinblockingbufferwasthenadded.The plateswerere-incubatedfor1hatroomtemperature. Thereactionwasstoppedbywashingtheplatesas describedabove.Todevelopthecolour,100 lof 2.2 -azino-di-(3-ethyl-benzthiozoline-6-sulfonicacid)peroxidasesubstrate(Kirkegaard&PerryLaboratories, Gaithersburg,MD,USA)wasadded,andtheplateswere incubatedfor30min.Absorbancewasmeasuredat 405nm,andthecut-offvalueforpositivitywasdefined asthemean+3standarddeviationsofthenegative controlsamples.NegativeserawerecollectedfromvolunteersamongthestaffoftheKoreaNationalInstituteof Health(KNIH).CalculationoftheannualparasiteincidenceTheannualparasiteincidence(API)wascalculated asthenumberofmalaria-positivepatientsper1,000 inhabitantsforeachofthestudysitesusingmicroscopy:API=(numberofpositiveslides/totalnumberof slides)1,000.DataanalysisThedifferencesinthepositiveCSPratesbetween2010 and2011weredeterminedbyaMann – Whitneytest. DataanalyseswereperformedusingGraphPadsoftware (GraphPadSoftwareInc.,LaJolla,CA,USA).Pearson ’ s correlationanalysiswasperformedtoexaminethe relationshipbetweenseropositivityandtheAPIof P.vivax inagivenyear.Thedatawereanalyzedusing SPSSsoftware,version17.0(SPSSInc.,Chicago,IL, USA).A P valueof<0.05wasconsideredstatistically significant.Thecorrelationsizeswereinterpretedas none(0.0 – 0.09),small(0.1 – 0.3),medium(0.3 – 0.5),or strong(0.5 – 1.0)[24].ResultsDNAsequenceoftheVK210KoreanisolateTheCSPgenethatwasamplifiedbyPCRfromgenomic DNAwasanalysedona1.0%agarosegel.Amplificationof theCSPgeneyieldedanapproximately750-bpDNAfragment(Figure3A)that,afterpurification,wasligatedinto thepGEM-TEasyvector.ThetransformantswereconfirmedtocontainPCRinsertsby Eco RIdigestion.The plasmidcontainingthePCRproductwasnamedpCS210 andwasusedforDNAsequenceanalysis.DNAsequencingrevealedthattheclonedCSPgenewas717bplong andcomprised239aminoacidsthatwereidentifiedby DNASIS.Thenonapeptiderepeatunit,locatedbetween RegionI(KLKOP)andRegionII(PCSVT),showedthesequenceGD(N)R(G)AD(G/A)GQP(A)Aandwasrepeated 18times.ExpressionandantigenicityofCSPVK210typein E.coliTogeneratetheexpressionplasmid,therepeatregionof theCSPgenewasamplifiedfrompatientgenomicDNA, digestedwith Bam HIand Hin dIII,andsubclonedinto thesamerestrictionenzymesitesofexpressionvector pET-28a(+)(Figure3B).Theresultantplasmid,pCS210, containedtherepeatregionoftheCSPgenefusedtoa (His)6-tag.TherecombinantplasmidpCS210wasthen transferredinto E.coli DH5 .Next,1mMIPTGwas addedtoculturesof E.coli DH5 (pCS210)grownto logarithmicphaseinliquidLBplus100g/mlampicillin toinduceexpressionofthetargetprotein.SDS-PAGE followedbyCoomassiebluestainingshowedthatthe molecularweightoftheCSPrecombinantproteinwas 45kDaundernativepurificationconditions(Figure4A). Themolecularweightofthetargetproteinwastwiceas largeastheexpectedmolecularweight(24kDa),which hadbeendeterminedbyDNASIS,itmayaffectedbythe repeatedregionofCSP. TheantigenicityoftheCSPrecombinantproteinwas determinedbywesternblot.Theseraofpatientswith malariareactedpositively(Figure4B).Todeterminethe sensitivityandspecificityoftheCSPrecombinantproteinbyELISA,theseraofpatientswithmalaria,which hadbeenreservedinKNIHaftercollectionbetween 2009and2010,wereused.Serafrom15of51patients withmalaria(sensitivity,29.4%)werepositive,whileone serumsamplefromthenormalcontrolgroup(n=10), whohadneverbeenexposedtomalaria,waspositive (specificity,90.0%)(Figure5).LocalmalariatransmissioninGanghwaTwolargeislandslocatedwestofGanghwa,Gyodongdo island(Gyodongmyeon)andSeokmodoisland(Samsanmyeon),werealsosurveyed(Figure1A).Atotalof230 of1,235(18.62%)and256of1,348studysubjects (18.99%)showedapositiveresponseonCSP-ELISAinCho etal.MalariaJournal 2013, 12 :448 Page5of11 http://www.malariajournal.com/content/12/1/448

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2010and2011,respectively(Table1).Buttherewasno positiveinmicroscopicexaminations. Gyodongmyeondisplayedahigherpositiveratein 2010(21.01%,196/933)thanin2011(18.53%,179/966). However,Samsanmyeonhadahigherpositiveratein 2011(20.12%,77/382)thanin2010(11.26%,34/302). TheAPIfor2010(4.28)washigherthanthatfor2011 (2.23).TheAPIsfor2010(4.86)and2011(3.56)inGyodongmyeonwerehigherthanthosefor2010(3.50)and 2011(0.44)inSamsanmyeon.TheseropositivityofCSP in2010showedastrongpositivelinearrelationshipwith theAPIsof2010and2011(r=1.000, P <0.01).However, theseropositivityofCSPin2011showedastrongnegativelinearrelationshipwiththeAPIsof2010and2011 (r=1.000, P <0.01)(Table1).LocalmalariatransmissioninCheorwonAtotalof101of590(17.12%)and45of611(7.36%) studysubjectsinCheorwon(Figure1B)showedapositiveresponseonCSP-ELISAin2010and2011,respectively(Table2).Buttherewasnopositiveinmicroscopic examinations.Interestingly,Gimhwaeupexhibiteda50% (35/70)CSP-ELISApositiveratein2010,butthisrate droppedto3.08%(2/65)in2011.In2010,thesecond bp M 1 CSPbp M 1 2 CSP pET-28a(+)B A Figure3 Geneticcloningofcircumsporozoiteproteingene.(A) ConformationofthePCRproductofthecircumsporozoiteproteingeneof Plasmodiumvivax Koreanisolate M,Molecularsizemarker;lane1,CSPgene. (B) Confirmationofcircumsporozoiteproteingenein Escherichiacoli DH5 byrestrictionenzymedigestionwith Bam HIand Hin dIII.M,Molecularsizemarker;lane1; Bam HIand Hin dIIIdigestedplasmid;lane2, undigestedplasmid. kDa M 1 2 3 4 CSPkDa M 1 2 3 CSPB A Figure4 Expressionofcircumsporozoiteproteingenein E.coli .(A) PurificationofrecombinantcircumsporozoiteproteinwithNi-NTA agaroseaffinitychromatography LaneM,Molecularweightproteinmarker;lane1,induced E.coli DH5 celllysatewithIPTG;lane2, flow-through;lane3,wash;lane4,elute. (B) Westernblotanalysisofrecombinantcircumsporozoiteprotein.M,Molecularweightprotein marker;lanes1 – 3,patientswithmalaria. Cho etal.MalariaJournal 2013, 12 :448 Page6of11 http://www.malariajournal.com/content/12/1/448

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highestpositiverate(31.51%,23/73)occurredinSeomyeon,adjacenttotheeastsideofGimhwaeup;thislocationwasrankedhighestin2011(15.00%,6/40).The thirdhighestpositiverateinboth2010(10.85%,28/258) and2011(6.08%,20/329)wasfoundinDongsongeup, adjacenttothewestsideofGimhwaeup.Thelowest positiverate(7.94%,15/189)occurredinCheorwoneup, tothefarwestofCheorwon,in2010;however,thislocationhadthesecondhighestratein2011.Inaddition, GimhwaeupshowedthehighestAPIsinboth2010and 2011(3.24and2.36,respectively).Theseresultssuggest thatGimhwaeup,locatedinthecentreofCheorwon, wasthefocusofmalariatransmissionduringthestudy period.TheseropositivityofCSPin2010showeda moderatelypositivelinearrelationshipwiththeAPIof 2010(r=0.435)andstrongpositiverelationshipwiththe APIof2011(r=0.509),butwithoutstatisticalsignificance( P =0.565, P =0.491,respectively).However,the seropositivityofCSPin2011showedastrongnegative linearrelationshipwiththeAPIsof2010(r= 0.926) and2011(r= 0.931),butagainwithoutstatisticalsignificance( P =0.071, P =0.069,respectively)(Table2).IntensityofimmuneresponsesThemeanresponseintensityamong101positivesamples obtainedfrom590inhabitantsofCheorwonin2010was 0.91860.0472.Thisratedroppedto0.70350.0457 among45positivesamplesobtainedfrom611inhabitants ofCheorwonin2011( P =0.006)(Figure6A).However, thisratedidnotchangesignificantlyinGanghwa,increasinglyslightlyfrom0.76490.0192among230positive samplesobtainedfrom1,235inhabitantsin2010to 0.82370.1970among256positivesamplesobtained from1,348inhabitantsin2011( P =0.034)(Figure6B).It shouldbenotedthatthestudyareasinGanghwawere islands,andmalariatransmissionthusmightnothave beenaffectedbyforeignfactors,whereasCheorwonmay havebeenaffectedbytransmissionfromDPRK. Atotalof632of3,784inhabitantsexhibitedapositive CSPresponse,and625of632positivecasescould becategorizedbyage.GroupAcomprisedCSP-positive inhabitantsunder10yearsofagewhoexhibitedan immuneresponseintensityof0.78400.0044.Group Bcomprisedindividualsbetween11and20yearsof agewhoexhibitedanimmuneresponseintensityof 0.69740.0435.GroupCcomprisedindividualsbetween 21and30yearsofagewhoexhibitedanimmuneresponseintensityof0.70280.0300.GroupDcomprised individualsbetween31and40yearsofagewhoexhibitedanimmuneresponseintensityof0.77300.0375. GroupEcomprisedindividualsbetween41and50years ofagewhoexhibitedanimmuneresponseintensityof 0.72700.0347.GroupFcomprisedindividualsbetween 51and60yearsofagewhoexhibitedanimmuneresponse intensityof0.82720.0379.GroupGcomprisedindividualsbetween61and70yearsofagewhoexhibitedan immuneresponseintensityof0.79530.0253.GroupH comprisedindividualsbetween71and80yearsofagewho exhibitedanimmuneresponseintensityof0.84350.0264. Finally,GroupIcomprisedindividualsover81yearsof agewhoexhibitedanimmuneresponseintensityof 0.81660.0409.Astrongpositivecorrelationwasshown betweenimmuneresponseandage(r=0.686, P =0.041) (Table3).DiscussionOurmalariaresearchteamisinterestedindiagnosing vivaxmalariabasedonantibodydetection.Although Pa t ient Normal 0.0 0.5 1.0 1.5 O.D at 450 nm Cut off Figure5 Antigenicityofrecombinantcircumsporozoiteprotein. Patient,individualinfectedwith Plasmodiumvivax ;Normal, healthyvolunteers. Table1PositiveratesofcircumsporozoiteproteinandannualparasiteincidenceinGanghwaAreaNo.ofseratestedNo.ofpositiveseraPositiverate(%)APIa20102011201020112010201120102011 Gyodongmyeon93396619617921.0118.534.863.56 Samsanmyeon302382347711.2620.123.500.44 Total1235134823025618.6218.994.282.23aAPI;Annualparasiteincidence.Cho etal.MalariaJournal 2013, 12 :448 Page7of11 http://www.malariajournal.com/content/12/1/448

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microscopicexaminationisthegoldstandardmethod formalariadiagnosisworldwide,ithasdisadvantages,includingtheneedfortrainedexperts,itstime-consuming nature,anditstendencytomisscasesoflowparasitaemialevels.However,antibodydetectioncancompensateforthedisadvantagesofmicroscopicexamination.If anantibodydetectionmethodisappliedinthefield usingarapiddiagnostictestkit,themethodcanbeused withoutrigoroustrainingandthetimerequiredfordiagnosiswillbereduced.Furthermore,mostpatientswith malariahaveantibodiesagainstmalariaparasites,even thosewithlowparasitaemialevels,andthismethod canbeappliedonalargeseroepidemiologicscale.In thisstudy,theCSPofthevivaxmalariaantigenwas usedtoseroepidemiologicallyevaluateitsusefulnessin understandingmalariatransmission.Thesurveyareas, GanghwaandCheorwon,aretwore-emergingmalarial outbreakareasinSouthKorea,andbothareasare locatedwithin10to15kmofthesouthernDMZ[8]. TheincidenceofmalariapeaksinAugustaftertherainy seasonanddeclinestobaselinebytheendofOctober. Therefore,bloodcollectionwascarriedoutbetween NovemberandDecember,whentheactiveanopheline populationwasdiminished.TheDMZisa4-km-wide, 250-km-longcorridorthatextendsacrossthemiddle partoftheKoreanpeninsula.Nocivilianshavebeen allowedtoentertheDMZformorethan50years;therefore,naturalecosystemsandbiodiversityarehighly conservedintheDMZ[25].Outbreakareashavebeen expandingyearlybothsouthandeastoftheDMZ. Outbreaksintheseareasarebelievedtohaveoriginated fromthenorthernpartoftheDMZ.There-emergence ofmalariaispresumedtohaveoriginatednotfrom theimmigrationofinfectedpeoplefromthenorth,but frommosquitoesinfectedwith P.vivax thatflewfrom thenorthbecausehumanpassagethroughtheDMZis almostimpossible(althoughtherearesomeexceptions intheGaeseongIndustrialZone).Thecorridorisheavily fortifiedonbothsidesofthebufferzoneswithland minesandbarbedwirefences.Therefore,itisbelieved thattheseareasareexposedtomosquitoes.Toestimate theprevalenceofmalariaexposureinthesehigh-risk areasinKorea,CSPrecombinantproteinswereapplied. Itisasporogony-stageproteinthatexistsonthe surfacemembraneofallplasmodiumsporozoites.CSP hasacentralimmunodominantregioncomprising shorttandemrepeataminoacidsequencesthatcontain multiplecopiesoftheimmunodominantB-cellepitope [14].Becauseitishighlyimmunogenicandcaninduce aprotectiveresponseinsporozoite-immunizedexperimentalanimalsandhumans,C SPisbeinginvestigated asacandidateforahumanmalariavaccine.The Table2PositiveratesofcircumsporozoiteproteinandannualparasiteincidenceinCheorwonAreaNo.ofseratestedNo.ofpositiveseraPositiverate(%)APIa20102011201020112010201120102011 Cheorwoneup18917715177.949.600.680.34 Dongsongeup258329282010.856.082.581.58 Gimhwaeup706535250.003.083.242.36 Seomyeon734023631.5115.000.460.15 Total5906111014517.127.361.881.15aAPI;Annualparasiteincidence. 2 0 10 2 0 11 0.00 1.00 2.00 3.00 P < 0.0001Cheorwon ***O.D at 450 nmA Ganghwa 201 0 2011 0.00 1.00 2.00 3.00 P = 0.0020 **O.D at 450 nmB Figure6 ComparisonofimmuneresponsestoCSPbetween2010and2011. Inhabitantsof (A) Cheorwonand (B) Ganghwa. Cho etal.MalariaJournal 2013, 12 :448 Page8of11 http://www.malariajournal.com/content/12/1/448

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immunodominantB-cellepitopesofCSPfromalarge numberof P.falciparum isolatesofdiversegeographicaloriginsandofasmallernumberof P.vivax isolates havebeenconservedwithinthespecies[26].Interestingly,thelifespanoftheCSPantibodyinhumanbeings iswithin27daysininhabitantsofThailandandisnot boostedbyadditionalexposuretoCSPantigen,i.e., additionalinfectionbyanophelinemosquitoes[27]. Thesefindingsledustoconsiderpatientswithmalaria withlongincubationperiodswhousuallyshowmalaria onsettheyearfollowingafi ve-month-longwinterseasonwithoutmosquitoes.Furthermore,themeanincubationperiodof P.vivax hasbeenreportedtobeas longas27941days(range,153 – 452days)[28].This findingsuggeststhatpatientswiththeselongincubationperiodscoulddisplayeitheranabsentorreduced antibodylevelagainsttheCSPantigen.Thepercentages ofpatientswithshortandlongincubationperiodswere 25%and75%,respectively[8].However,itispossible thatthepatientswithshortincubationperiodsandan onsetwithin1monthafterexposuretotheCSPantigen deliveredviainfectiveanophelinemosquitoeshadan elevatedanti-CSPantibodylevel.ThisiswhytheCSP antigenwasselectedamongthemanypossiblemalaria antigens.Thehypothesissu ggestedbyourmalariateam isthattheAPIoftherelevan tyearincludespatients withshortincubationperiodswhowereinfectedinthe relevantyearandpatientswithlongincubationperiods whowereinfectedinthepreviousyear.Therefore,the positiveanti-CSPantibodyrateswerecomparedwith theAPIsfortherelevantandsubsequentyears.The positiveCSP-ELISAratein2010wasrelatedtothe 2010malariaprevalenceinGanghwaandCheorwon. Thisratemightbeinfluencedbyprevalenceinpatients withshortincubationperiods.ThepositiveCSP-ELISA ratein2010wassignificantlyrelatedtothe2011malariaincidenceinGanghwaandCheorwon.Thisfinding suggeststhatthemalariasituationinthesubsequent yearcanbepredictedbytherelevantpositiveCSPrate, andaportionofthisratemightinfluencetheincidence inpatientswithlongincubationperiods.Inother words,thepositiveCSPratecouldbeconsideredtobe theinoculationrateofsporozoitesbyinfectivevector mosquitoes.Ifapersonisbittenbyinfectivemosquitoes,heorshewillshowapositiveresponseonCSP ELISA.However,itisverydifficulttocalculatehow manyindividualsbecomepatientsafterhavingbeen bittenbyinfectivemosquitoes.Thisnumberishighly dependentonindividualimmunecompetency.Unfortunately,itwasfailedtoidentifyparasite-positiveinhabitantsbymicroscopicexaminationof632CSP antibody-positiveinhabitants.Inaddition,noneofthe CSP-positiveindividualsin2010becomepatientsin 2011.However,thepositiveCSPantibodyrateapparentlyaffectedtheoutbreakinthefollowingyear.This observationmayindicatethattheantibody-positive CSPrateiscloselyrelatedtothecommunityresponse ratherthantheindividualr esponse.However,inapreliminarystudy,theuseofabloodantigentodetectan antibodywithanindirectfluorescentantibodytest (IFAT)showedthat16.67%(4 /24)ofindividualswith IFATseropositivityamonginhabitantsofGanghwa becamepatientswithmalariainthefollowingyear (authors ’ unpublisheddata).InanotherstudyperformedinGimpoin1999,16of125individualswith IFATseropositivity(12.80%)werealsopositiveformalariabyPCRdetection.Bloodsampleswithanantibody titerof>1:256hadahighpositiveratebyPCRanalysis [29].Evenifitisneededtoobtainmoredata,thedifferencesinPCRpositiveratesbetweenCSPandIFAT amongantibody-positivecasesareattributabletodifferentantigens,onefromthesporogonystage(CSP) andtheotherfromtheschizogonystage(IFAT).These differencessuggestthatthestage-specificantigenand antibodydetectionmethodsshouldbematched.Therefore,itisbettertouseliverbiopsysamplestodetect parasitesthatarepresentinindividualswithantibodypositiveresponses.BecausetheCSPantigenisa sporogony-stageantigen,itcanbeobservedintheearly developingstageofhypnozoites,whichareliver-stage parasites.However,thereareethicalandeconomic problemsinobtainingtheliversamplesnecessaryto detecthypnozoitesbyPCR.Therefore,thepositiveresponsesonCSPantibodyassaysdidnotcoincidewith thePCRresponsesforantigendetectionthatusedonly bloodsamples. Toevaluatemalariatransmissioninagivengeographical region,manyfactors,includingtemperature,mosquito density,vectorcapacity,climate,rainfall,andhumidity, shouldbeconsidered[30].Parasitaemiaprovidesa classicalmeansofmeasuringmalariaendemicity.However,patientincidencealonecannotprovideacomplete Table3TheimmuneresponsestoCSPininhabitants accordingtoageGroupAgeOpticaldensityStandarddeviation A<100.78400.0044 B11-200.69740.0435 C21-300.70280.0300 D31-400.77300.0375 E41-500.72700.0347 F51-600.82720.0379 G61-700.79530.0253 H71-800.84350.0264 I80>0.81660.0409 Cho etal.MalariaJournal 2013, 12 :448 Page9of11 http://www.malariajournal.com/content/12/1/448

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understandingofmalariaprevalencebecausemanyfactors affectthemalariaprevalenceinROK,includingthepopulationdensityofmosquitoes,vectorialcapacity,long-to short-incubation-patientratio,symptomatictoasymptomaticpatientratio,differencesinrainfallandtemperature, andimmunityofthecommunity.ConclusionsAntibodydetectionusingCSP-ELISAmayprovideuseful informationregardingmalariaprevalenceincertain areasandindividuals.Theseserologicalmethodsare usefulinidentifyingareasthatrequiremalariacontrol andevaluatingthesurveillancesystemincertainareas.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions TSK,SHC,YS,HIC,BKN,andHWLconceivedanddesignedthestudyand contributedtotheexecutionoftheresearch.HWLandTSKwrotethe manuscript.YS,BKN,andWONperformedthestatisticalanalysis.SHC,JHP, WON,SJH,WJL,SKL,YKP,PYC,SKA,JSK,andYYBcollectedtheblood samples.SHC,YJK,PYC,SJH,WJL,andJHPperformedCSP-ELISA.SWL (EastsideHighSchool)whohasbeenworkingaytheUniversityofFlorida involvedinexpressionofCSPrecombitantproteinandperformedELISA.HIC alsoprovidedmostofresearchfundingforthisstudy.Allauthorshaveread andapprovedthefinalmanuscript. Acknowledgments WearegratefultoallblooddonorsandthestaffofthePublicHealth CentresinGanghwaandCheorwon.ThisworkwassupportedbytheKorea AssociationofHealthPromotionandInhaUniversityResearchFund,2012. Authordetails1DepartmentsofParasitology,InhaUniversitySchoolofMedicine,Incheon 400-712,RepublicofKorea.2DepartmentofPathology,Immunology,& LaboratoryMedicine,CollegeofMedicine,UniversityofFlorida,J-566,1275 CenterDrive,GainesvilleFL32610,USA.3DepartmentofBiomedical Technology,InhaUniversitySchoolofMedicine,Incheon400-712,Republic ofKorea.4DepartmentofParasitologyandInstituteofHealthSciences, GyeongsangUniversitySchoolofMedicine,Jinju660-751,RepublicofKorea.5DepartmentofPharmacology,InhaUniversitySchoolofMedicine,Incheon 400-712,RepublicofKorea.6DepartmentofParasitology,NationalInstituteof Health,Osong363-951,RepublicofKorea.7DepartmentofParasitologyand CatholicInstituteofParasiticDiseases,CollegeofMedicine,Catholic UniversityofKorea,Seoul137-701,RepublicofKorea.8Departmentof MedicalEnvironmentalBiology,CollegeofMedicine,Chung-AngUniversity, Seoul156-756,RepublicofKorea.9AsanInstituteforLifeSciences,University ofUlsanCollegeofMedicine,AsanMedicalCenter,Seoul138-736,Republic ofKorea.10DepartmentofBiomedicalScience,JungwonUniversity,Goesan Chungbuk367-805,RepublicofKorea.11DepartmentofAnatomy,Collegeof KoreanMedicine,InstituteofKoreanMedicine,KyungHeeUniversity,Seoul 130-701,RepublicofKorea.12DepartmentofBiotechnology,Collegeof BiomedicalandHealthSciences,KonkukUniversity,Chungju380-701, RepublicofKorea.13KoreaAssociationofHealthPromotion,Seoul157-928, RepublicofKorea. 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antibodiestothefalciparumsporozoitevaccine1antigen(R32tet32). JClinMicrobiol 1987, 25: 1002 – 1008. 28.KhoWG,JangJY,HongST,LeeHW,LeeWJ,LeeJS: Bordermalaria charactersofreemergingvivaxmalariaintheRepublicofKorea. KoreanJParasitol 1999, 37: 71 – 76. 29.LeeWJ,KimHH,HwangSM,ParkMY,KimNR,ChoSH,InTS,KimJY, SattabongkotJ,SohnY,KimH,LeeJK,LeeHW: Detectionofanantibody against Plasmodiumvivax inresidentsofGimpo-si,SouthKorea,using anindirectfluorescentantibodytest. MalarJ 2011, 31: 10 – 19. 30.SnowRW,GillesHM: The Anopheles vector .In EssentialMalariology. 4th edition.EditedbyWarrellDA,GillesHM.London:Arnold;2002:59 – 84.doi:10.1186/1475-2875-12-448 Citethisarticleas: Cho etal. : Evaluationofcircumsporozoiteproteinof Plasmodiumvivax toestimateitsprevalenceintheRepublicofKorea: anobservationalstudyofincidence. MalariaJournal 2013 12 :448. 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 Cho etal.MalariaJournal 2013, 12 :448 Page11of11 http://www.malariajournal.com/content/12/1/448