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Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar
Marcello Sato, Armand Rafalimanantsoa, Charles Ramarokoto, Alain Rahetilahy, Pascaline Ravoniarimbinina, Satoru Kawai, Toshifumi Minamoto,
Megumi Sato, Masashi Kirinoki, Voahangy Rasolofo, et al.
To cite this version:
Marcello Sato, Armand Rafalimanantsoa, Charles Ramarokoto, Alain Rahetilahy, Pascaline Ravo- niarimbinina, et al.. Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar. International Journal of Infectious Diseases, Elsevier, 2018, 76, pp.130 - 136.
�10.1016/j.ijid.2018.08.018�. �pasteur-01926161�
Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar
Marcello Otake Sato
a,*, Armand Rafalimanantsoa
b,c, Charles Ramarokoto
b, Alain Marcel Rahetilahy
c, Pascaline Ravoniarimbinina
b, Satoru Kawai
a,
Toshifumi Minamoto
d, Megumi Sato
e, Masashi Kirinoki
a, Voahangy Rasolofo
b, Mathilde De Calan
b, Yuichi Chigusa
aaDepartmentofTropicalMedicineandParasitology,DokkyoMedicalUniversity,Mibu,Japan
bUnitédeRecherchesurlesHelminthiases,InstitutPasteurdeMadagascar,Antananarivo,Madagascar
cMinistryofPublicHealth,Antananarivo,Madagascar
dGraduateSchoolofHumanDevelopmentandEnvironment,KobeUniversity,Kobe,Japan
eGraduateSchoolofHealthSciences,NiigataUniversity,Niigata,Japan
ARTICLE INFO
Articlehistory:
Received20July2018
Receivedinrevisedform27August2018 Accepted30August2018
CorrespondingEditor:EskildPetersen,Aar- hus,Denmark
Keywords:
Eco-epidemiology EcoHealth OneHealth Environment Schistosomiasis Africa Madagascar eDNA
ABSTRACT
Objectives:SchistosomiasisisanimportantdiseaseinMadagascar,andseveralstudiesonthedisease havefocusedontheoccurrenceoftheparasiteinhumans.However,therangeofthepathogeninthe environmentanditsimpactonhumaninfectionisdifficulttopredict.AnenvironmentalDNA(eDNA) detectionsystemforSchistosomamansoniwasdevelopedtoimproveschistosomiasiseco-epidemiology studies.
Methods:Primersandprobesweredesignedandtestedinexperimentalbiotopes.Thefieldstudywas conductedinMaevatananaDistrictofMadagascar.Sevenwatersourceswithhumanuseweresampled, witha totalof 21watersamplescollected.Snails werecollected,and patientswereexaminedby ultrasoundtodeterminetheoccurrenceofschistosomiasisinthestudyarea.
Results:OnewatersourcewithactivetransmissionwasidentifiedthroughthedetectionofS.mansoni eDNAinthewaterandtheintermediatehostBiomphalariapfeiffericollectedfromthesamewatersource.
Peoplewithclinicalschistosomiasiswerefoundinthearea,reinforcingthefindings.
Conclusions:TheapplicationofeDNAineco-epidemiologyenablesthedeterminationofhotspotsand safespotsinendemicareas,constitutinganalternativeecologicaltoolforfollow-upandmonitoringof controlprogramsforschistosomiasis,andcontributinginformationonwatersafetyforimprovingthe standardoflivingofthepeopleinendemicareas.
©2018TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.
ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by- nc-nd/4.0/).
Introduction
Schistosomiasisisawaterbornebloodflukeinfection,andover 90%ofaffectedpeopleliveinthecountriesofSub-SaharanAfrica, wherethereispooraccesstocleanwaterandsanitaryfacilities (Chitsuloetal.,2000;Adenowoetal.,2015).Itisoneoftheleading causesofimpairedhealthandsocio-economicdevelopmentinthe world,and thecontrolmeasurescurrently inuseare based on large-scale preventive chemotherapy, as recommended by the WorldHealthOrganization(WHO)(WHO,2012,2013).
Schistosomiasisis consideredendemic in Madagascarand it constitutesamajorpublichealthissuein107ofthe114districtsof the country(Madagascar, 2016a). Thenorth part and thewest slope of the island have high transmission of Schistosoma haematobium(urogenitalform);theeastcoast,centralhighland, andsouthpartofMadagascarareendemicforSchistosomamansoni (intestinalform) (Doumenge et al.,1987; Madagascar, 2016a,b).
Maevatanana, one of three districts of Betsiboka Region, is particularlyendemicfor bothintestinalandurogenitalformsof schistosomiasis (Amat-Roze, 1978). Maevatanana has already benefitedfromthreeroundsoflarge-scalechemotherapycombin- ingpraziquantelandmebendazole,targetedatschool-agechildren since2011.Furthermore,arecentstudythroughbaselinepreva- lenceofneglectedtropicaldiseases(NTDs)sentinelsitesshowed that there is limited access to adequate water, sanitation, and
*Correspondingauthorat:DepartmentofTropicalMedicineandParasitology, DokkyoMedicalUniversity,880Kitakobayashi,Mibu,Tochigi321-0293,Japan.
E-mailaddress:marcello@dokkyomed.ac.jp(M.O.Sato).
https://doi.org/10.1016/j.ijid.2018.08.018
1201-9712/©2018TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
ContentslistsavailableatScienceDirect
International Journal of Infectious Diseases
j o u r n a lh o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i j i d
hygiene facilities in the schools of western Madagascar (Madagascar,2016a,b).
Thepreventionofschistosomeinfectionofindividualscanbe directlyrelatedtothepopulation’sawarenessofsafewatersources (Chitsuloetal.,2000;Adenowoetal.,2015;FenwickandJourdan, 2016).However,determiningthesafetyofwaterisadifficulttask.
This has been done in ecological surveys by searching for intermediate hosts snails and performing shedding tests for cercariaateachcollectionspottodeterminetheriskofinfection (Wolmaransetal.,2002;Kingetal.,2006).
TheanalysisofenvironmentalDNA(eDNA)asatechniquefor thedetectionofspecificorganismshasbeenperformedpreviously instudiesontheecology,distribution,andphylogenyofcercozoa usingsoilsamples(BassandCavalier-Smith,2004).Furthermore, toimprovesurveyingmethods,eDNAhasbeenusedtodetectthe presenceofamphibiansandfishthroughthedirectdetectionof specificDNAin watersamples(Ficetolaet al.,2008; Minamoto etal.,2012).Morerecently,theeDNAtechniquewassuccessfully appliedforthedetectionoftheliverflukeOpisthorchisviverriniand itsintermediatefishhostsinwaterofendemicareas(Hashizume etal., 2017).Withregardtoschistosomiasis, studieshave been doneforthedetectionofSchistosomajaponicumcercariaDNAin watersamples(HungandRemais,2008;Worrelletal.,2011).
Inthisstudy,wedevelopedareal-timePCRdetectionsystemby designing species-specific short-amplicon primers and a probe targetingthemitochondrialcytochromecoxidasesubunitI(COI) regionofS.mansoni.Afterconfirmingthespecificityandsensitivity of the assay, it was possible to detect S. mansoni eDNA in an endemicareaofmixedinfectioninMadagascar,indicatingthatthis systemcouldbeusedasanalternativedetectionmethodineco- epidemiology studies and surveillance in areas endemic for schistosomiasis.
Materialsandmethods
ObtainingeDNAfromwatersamples
PurificationofeDNAwas performedusingtheDNeasy Blood andTissueKit(Qiagen),asdescribedpreviously(Uchiietal.,2016), with some modifications. Briefly, water samples were filtered throughglassfiberfilterpapers(GF/F,0.7
m
m;Whatman,UK)andfixed in 70% ethanol (Minamoto et al., 2016). Filter papers containing the filteredmaterial wereplaced in Salivette tubes (Sarstedt, Germany), and 200
m
l of Buffer AL and 20m
l ofproteinaseK wereadded.The tubeswereincubatedfor 30min at56Candcentrifugedat5000gfor3min.Onerinsestepwas done,adding200
m
lofTris-ethylenediaminetetraaceticacid(TE) buffer directly onto the filter papers. After 1min at room temperature,thetubeswerecentrifugedat5000gfor3min,after which300m
lofethanolwasaddedtothefiltratedsolution.The bindingstepwasdonebytransferring650m
ltoaDNeasycolumn,followedbycentrifugationfor1minat6000g.Thebindingstep wasrepeatedfortheentirefiltratedsolution.Thesubsequentsteps wereperformedasrecommendedinthemanufacturer’sprotocol, withanelutionvolumeof110
m
lofBufferAE.ThepurifiedeDNA wasstoredinafreezerat25Cuntilfurtheruse.Primers,probes,andreal-timePCR
The mitochondrial COI DNA sequences of 22 Schistosoma specieswereobtainedfromNCBIGenBankandaprimersetand probe were designed for species-specific eDNA detection of S.
mansoniusingPrimerExpressSoftware3.0(AppliedBiosystems, USA)with defaultsettings. Theforwardprimer, Sma-COI-F(50- CAGGGGTTTCAAGTCTAATTGGAT-30),andthereverseprimer,Sma- COI-R(50-CAAATAATAACATCGTTATTCCTCTGG-30), weredesigned
for partial amplification of theS.mansoniCOI withapredicted designedampliconsizeof162bp.TheTaqManMGBprobe(Thermo FisherScientific,Waltham,MA,USA)wasdesignedasSma-COI-P (50-FAM-TTCAAATGTTCGATAATA-NFQ-MGB-30).Thespecificityof theprimers/probe wasthentested insilicoincomparisonwith otherorganismsusingBLAST.
To determine specificity, the newly designed real-time PCR assay (Sma-COI) was conducted to specifically distinguish S.
mansoni fromS.japonicumand Schistosomamekongi usingDNA obtainedfromadultworms.Real-timePCRwasperformedwitha StepOnePlusthermocycler(ThermoFisherScientific).Thereaction was performed in a 20-
m
l final volume containing 10m
l of2Taqman Environmental Master Mix 2.0 (Thermo Fisher Scientific),0.1
m
lofAmpEraseUracilN-Glycosylase(ThermoFisher Scientific),2m
lof DNAtemplate(including 100pgof totalDNAderivedfromworms),900nMeachof theSma-COI-F/Rprimers, and 125nM of theSma-COI-P probe. The PCR conditions were 2minat50Cand10minat95Casinitialsteps,followedby55 cycles of 15s at 95C and 60s at 60C. All reactions were conductedwiththreereplications.
PCRandsequencing
PCR reactions were done as described previously in 20-
m
lvolume reactionswithSMA-COI-FandSMA-COI-R primersand using 2
m
l of sample DNA as template. One positive control(tissue-derived DNA) and a negative control were included in each batch of tests. The PCR was performedin a StepOnePlus thermocycler (Thermo Fisher Scientific) with the following conditions:50Cfor2min,followedby95Cfor10minand55 cyclesof95C for15s,60Cfor1min.PCRforotherspeciesof Schistosoma wasperformed asdescribed byKato-Hayashiet al.
(2010). After PCR, electrophoresis was performed at 100V for 60minusing2%agarosegel.ThePCRproductswerepurifiedand directly sequenced using an ABI3730XL sequencer (Applied Biosystems,USA)atMacrogenInc.(Korea)andatDokkyoMedical University(Japan).
DetectionofeDNAinS.mansoniexperimentalinfectionwater samples
Water was collected from aquariums (30cm length20cm width20cm height) containing Biomphalaria glabrata snails infectedandnotinfectedwithS.mansoni.Briefly,S.mansonieggs wererecoveredfromexperimentallyinfectedmiceandhatchedas described by Standen (1951). Snails were placed in 24-well microplatesand eachsnailwasinfectedovernightat25Cwith five miracidia collected by pipetting under stereomicroscope observation. Non-infected snails werefrom the same breeding batchasthesnailsusedforS.mansoniinfection.
Twoaquariumscontaining10litersofwaterand20infected snailseachwereusedtodeterminethecapabilityofdetectionof S. mansoni eDNA in water samples. Water samples were also collected from two aquariums containing non-infected snails under the same conditions. The water in all aquariums was changedweekly,andthecollectionforthisstudywasdone24h afterawaterchange,60daysafterinfectionofthesnails.
Atotalof250mlofwaterwascollectedfromeachaquarium,to which250
m
l(1:1000v/v)of10%benzalkoniumchloridesolution (w/v) was added to each sample to prevent DNA degradation (Yamanakaetal.2017).Allsampleswerefilteredusingaglassfiber filter(GF/F,0.7m
m;Whatman,UK).Afterwaterfiltration,10mlof 70%ethanol was passedforfixation,and thefilterpaperswere dried,wrappedinaluminumfoil,andstoredinafreezerat25C.DNA trapped on the filters was extracted using the method describedabove.
FieldtrialofSchistosomaeDNAdetectioninMadagascar Locationofthestudy
ThisstudywasperformedinMaevatananaDistrict(Figure1).
With a totalarea of 10752.2km2, Maevatanana District is composedof 17communes, whichrange inareafrom33.1km2 to1670.5km2.Communesareatanaveragealtitudeof282.1m, withaloweraveragealtitudeof30minAmbalajiaandahighest averagealtitudeof918.9minMahatsinjo.Theaveragetempera- tureinMaevatananaDistrictis35.6Cduringthedayand20.7Cat night,witha minimumaverage readingof 16.0C. Theaverage precipitationinthis districtis 1215mmperyear,varyingfrom 1065mmto1337mm(IUNC/UNEP/WWF,1987;WMO,1996;IRI, 2006).According tothelatestdata,the precipitationtrend has remainedconstant despiteminorfluctuationsfromyeartoyear (IRI,2006).
ConsideringMaevatananaDistrictasamixedinfectionendemic areawithreportedcasesofS.mansoniandS.haematobium(Amat- Roze,1978;Madagascar,2016a,b),thisstudyfocusedonthetwo differentcommunesofAmbalanjanakombyandAmpisavankara- tra, mixed endemic areas for S. haematobium and S. mansoni, respectively,accordingtolocalhealthcenterdata.
EnvironmentalsamplinginMadagascar
Water samples were collected from seven different water sourcesinAmbalanjanakombyandAmpisavankaratracommunes inJune2016.ThewatercollectionspotsareindicatedinFigure1
and are located at the following coordinates: 1641033.9100S 473057.1900E, 1641030.7700S 47 300.1500E, and 1641031.9000S 47 3023.5800E (collection spots1, 2, and 3, respectively, fromthree watersourcesinAmbalanjanakombyvillage,withatotalofnine samples), and 1727024.9900S 4658028.6700E, 1727026.7800S 4658027.2200E, 1727010.5400S 4657047.0800E, and 1727011.1000S 4657046.5900E(collection spots4,5, 6,and7, respectively,from fourwatersourcesinAmpisavankaratravillage,withatotalof12 watersamples).Thesamplinglocationsweredeterminedusinga wirelessHolux M-241GPS logger(Holux,Taiwan) andaneTrex VistaHCxGPSreceiver(Garmin,USA).WatertemperatureandpH were estimated using a portable LAQUAact D-74 multimeter (Horiba,Japan). Foreachsample,500ml ofwaterwascollected fromatleastthreedifferentpointsofthewatersource.Allsamples werefilteredusingaglassfiberfilter(GF/F,0.7
m
m;Whatman,UK),and10mlof70%ethanolwaspassedforfixationafterfiltration.
Asnailsurveywasperformedatthesamepointsasthewater collection,usingthefractionaltechnique,foralimitedtime;snail scoopswereusedandthesamplingtimewasfixedat30min.The collected snails were identified in the Helminthology Unit of InstitutPasteurdeMadagascar.
Clinicalandultrasoundexamination
To confirm active schistosomiasis cases occurring in the samplingarea, a total of six patientsfrom Ambalanjanakomby, four from Maevatanana, and seven from Ampisavankaratra presented voluntarily at each correspondinghealth center and
Figure1.Madagascarandadetailedmapofthewatersamplingspots.TheupperleftmapshowsMadagascar,dividedpoliticallyintosixprovinces(thinblacklines).Thearea surroundedbythedottedgraylinerepresentsMaevatananaDistrict,wherethisstudywasperformed.Antananarivo,thecapitalcity,isrepresentedasablackstarintheupper leftandrightmaps.Intheupperrightmap,thegraylinescorrespondtothemainroadnetwork,andthecityofMaevatananaisrepresentedasaclosedwhitecircle.Thedotted linesaretheprovincelimits,withthecapitalcitiesToamasinaandMahajangarepresentedwithdonutmarksintherespectiveprovinces.Lowerleftandrightmaps(dropA andB)showthewaterbodies,thevillages(houses)withthehealthcenters(representedasacrossinacircle),andthepointsofcollectioninthisstudy,numberedfrom1to7.
were examined. A full explanation of the objectives and methodologyofthisstudyandtheuseofinformation,including theopt-outpossibilityatanystageofthestudy,wasgiveninthe Malagasylanguageforacompleteunderstanding.Theultrasonog- raphyexaminationwasperformedusingaportableVscandevice (GEHealthcare,USA).Thehistoryandgradeofthediseasewere recordedandpairedwiththeobservedimagingdata.Theimage diagnosis was performed according to the clinical morbidity:
acute, chronic, urinary, intestinal, hepatosplenic, and ectopic schistosomiasis,followingthelateststandardizationandreviewed classification(Richteretal.,2000;Sahetal.,2015).
Dataanalysis
Alldatacollectedonpaperformsweretabulatedandanalyzed inExcel2016(Microsoft)andEpiInfoversion7.1.5.0(CDC,Atlanta, USA).GPSdatawerecollectedandanalyzedusingHolux logger utilitysoftwareandGoogleEarthProversion7.1.5.1557.
Results
SchistosomeeDNAdetectioninexperimentallyinfectedsnail aquariumwaterandtheecologicalsurveyinMadagascar
Eachdesignedprimer/probeset(Sma-COI-F,Sma-COI-R,Sma- COI-P)wastestedinPCRusingS.mansonigenomicDNAsamples.
DNA amplification was performed in three repetitions. The expectedampliconof162bpwassuccessfullyobtained.Confirma- tioncertifyingspecies-specificamplificationwasdoneusingnon- targetSchistosomasppDNAsastemplatesornegativecontrolswith noamplification,asshowninFigure2A.eDNAwasobtainedfrom snailaquariumwatersamples,andthedetectionofS.mansoniwas testedforeachaquarium.Real-timePCRcouldcorrectlydiscrimi- natetheaquariumswithsnailsinfectedwithS.mansonifromthe aquariums containing non-infected snails, which presented no signal,asshowninFigure2B.
WatercollectioninMadagascarwasperformedatsevenpoints (Figure1)foratotalof21watersamples.Allcollectionspotswere confirmed to be water sources with human use. The pH and temperatureof thecollectedwaterwere7.7/24.8C, 9.8/25.9C, and9.0/26Catpoints1,2,and3,respectively,ofAmbalanjana- komby,and8.5/19.7C,8.25/19.4C,8.28/19.6C,and8.43/19.9C atpoints4–7inAmpisavankaratra.
Oneoutof14environmentalwatersampleswaspositiveinthe Sma-COI real-time PCR assay. The positive sample was from
sampling site number 5 (Figure 1), a small river behind some community houses. This water sourcewas used as a place for washing and cleaning domestic utensils and clothes, and for leisure. The amplicon was sequenced and confirmed to be S.
mansoni.
Schistosomiasisandthesnailhostsinthestudyarea
All 17 patients examined were residents of Maevatanana Districtanddescribedahistorycompatiblewithschistosomiasis, confirmingtheinformationprovidedbythelocalhealthagentsof the occurrenceof the diseasein thearea. The ultrasonography examinations revealedtypicallesionsofchronicschistosomiasis infectioninthecasesexamined.Itwasseenthatadvancedstagesof schistosomiasisarepresentinMaevatananaDistrict.
Thesnailsurveywasperformedatthewatersourcesusedby thecommunityforwashing,cooking,anddrinking.Thepresence ofBiomphalariapfeifferisnailswasdetectedatsamplespotnumber 5inAmpisavankaratra(Figure1B).Therewerenosnailsatanyof thesamplespotsofAmbalanjanakombyCommunewatersources (Figure1A).
Discussion
UsefulnessofeDNAinschistosomiasissurveillance
Theprovisionofasafewatersupplyandeducationofpeopleon behavioralchangesarethekeyfactorsinschistosomiasiscontrol (Rollinson et al., 2013; Adenowo et al., 2015). Additionally, ecologicalsurveysarenecessarytodeterminethesafety(orrisk) of water sources. Ecological surveys of trematodes in the environment are based onthe collection of intermediate hosts and performance of shedding tests with snails to determine locations with transmission of the disease (Phongsasakulchoti etal.,2005;Kingetal.,2006;Kiatsopitetal.,2012).Inthisstudy, thesamecharacteristicofhostdependence/diseasewasobserved using the eDNA detection survey. The positive sample was collectedatthesameplacewhereB.pfeifferi,thelocalintermediate hostforS.mansoni,wasfound.
Climate change, urbanization, and the shift in land use (extractivism, tocropping, topasture,and soon) have a direct impactonthehabitatof thesnails–theintermediate hosts of schistosomiasis (Tanaka and Tsuji, 1997). These changes are happening faster and it is important to determinehow this is interferinginthepatternsofdiseaseslikeschistosomiasis.Thus,
Figure2.ThedeterminationofspecificitywasdoneusingSchistosomamansoniandotherSchistosomaspecies,usingthedesignedprimers/probeontheleftside(A)wherea specificsignalwasobserved.TestsofaquariumwaterbyqPCR,ontherightside(B),showthecapabilityoftheSma-COIreal-timePCRsystemforuseoneDNAfromwater samples,withaspecificsignalsuccessfullydetectedintheinfectedsnailaquarium(positivetank).
rapidandlow-costsurveymethodsarerequiredtoprovidetimely informationinendemicareasforanintegrateddiseaseprevention system(Hashizumeetal.,2017;Satoetal.,2018).Assuch,eDNA analysiscouldbeaninterestingalternative.Thismethodcanbe appliedforecologicalsurveysofdifferentorganismsandhasbeen reportedtobeausefulecologicaltoolfordetectingtargetpathogen DNA,reducingtimeandcosts(Reesetal.,2014;Hashizumeetal., 2017;Minamotoetal.,2009,2012,2016;Pitulaetal.,2012;Hyman andCollins,2012;Huveretal.,2015;Halletal.,2015).Theuseof thismethodforthedetectionofcercariaDNAinwatersamplesfor schistosomiasisstudieshasalsobeenreported(HungandRemais, 2008;Worrelletal.,2011).ThedetectionofeDNA constitutesa feasibletechnique inendemic areas todetermine thesafety of watersourcesforhumanuse.
The primersforthedetectionofSchistosoma sppinenviron- mentalsamples developedin this studytarget theCOIgene of mitochondrialDNA.Thisgeneisoftenusedforgenebarcodingand withdiagnosticobjectives(Kato-Hayashietal.,2010,2015),with productsfrom254bpto614bpinPCRs.ForeDNAsamples,because of the degradation occurring under environmental conditions, primersaregenerallydesignedtoyieldshortampliconstargeting highcopy number genes, enhancingthe detection level of the assays(Huveretal.,2015;Strickleretal.,2015).Theprimerset developed in this study can be considered highly versatile for application on different sources of material, including non- environmental samples.The system was able todetect species ofschistosomesusinggenomicDNAandfieldenvironmentalwater samples,demonstratingitspracticalvalue,anditwassuccessfulin detectingS.mansonifromeDNAofwatersourcesinanendemic areaofMadagascar(Figure1B).Itisimportanttonotethatpositive samplesweredetected onlyatthespotwhereB.pfeifferi snails werefound,whereasschistosomiasispatientswereconfirmedin both Ambalanjanakomby and Ampisavankaratra communes.
Interestingly,despitethepresenceofpatientswithanultrasound diagnosisofS.haematobium,noBulinussppsnailswerefoundin thefield.Also,thePCRtestscouldnotdetectS.haematobiumeDNA inthewatersamples.
This study is the first field trial using eDNA detection for schistosomiasis.Thetechniquepresentedpromisingresults,andit couldbeusedforaccuratediscriminationofdangerouswatersand moreovertodeterminesafewaterforhumanuse.Itcouldalsobe usedasa tooltofollowuptheimpactof alleventual effortsat communitybehaviorchange.However,toensurethatitsapplica- tionis effective,the system needstobe tested in furtherfield studies,inwiderareas,andcomparedwiththecurrentconven- tionalmethods.
SchistosomiasisinMadagascarandthesituationinMaevatanana District
InMadagascar,theMinistryofHealthestablishedtheNational Program against Bilharzia in the mid-1980s, with the aim of reducingtheprevalencetolessthan20%ineachhyper-endemic village,prevent theoccurrence of severe complications,and to reducereinfection (Madagascar, 2002).However,arecentstudy showed persisting hyper-endemic areas for S. mansoni and S.
haematobium (Madagascar, 2016b).Starting in 2012, free mass drug treatment (praziquantel and mebendazole) of school-age childrenwasstartedinMaevatananawiththeworldwideinitiative onan integratedapproachtoNTDs;however,there isa lackof information on the results of each session of mass drug administration (Dr Rafalimanantsoa, personal communication).
We observed chronic and acute schistosomiasis cases in the patientsinthisstudy,showingthattheproblempersistsunderthe currentadoptedcontrolstrategies.
Ultrasonographyisanimportantimagingtoolinthediagnosis of schistosomiasis, providing information on thelesions in the target tissues and their pattern, and delineating the possible prognosisaftertreatment;moreoveritisalow-cost,non-invasive, andportablesystem(Sahetal.,2015;Abdel-Wahabetal.,1992).
Withaportableultrasound,itwaspossibletoexaminepatientsin areaswithoutelectricity,constitutingagreatsupportfordiagnosis.
Allofthepatientsexaminedpresentedtypicallesionsofchronic schistosomiasisinfection,showingadvancedstagesofcomplicated casesofschistosomiasisinMaevatananaDistrict,despiteyearsof massdrugadministration.
In Maevatanana District, monitoring of freshwater snails (intermediate hosts) and their environment has not yet been conducted.Thereisnoinformationontheseasonality,transmis- sion, or habitats of Schistosoma transmission in this specific region.Thisinitialstudyrepresentsarecommencementofeco- epidemiology in endemic areas in Madagascar using a newly developed eDNA technique. The eDNA results can help to determinesafe/unsafewatersources,asdemonstratedinprevi- ousfood/waterbornepathogenstudies(Hashizumeetal.,2017;
Jonesetal.,2018).ConsideringthatBetsibokaisafarmingregion andthatmorethan80%ofmenandwomeninMaevatananawork inagriculture,theimminentriskofinfectionexistsintheirdaily life.Thepreventionoffood/waterbornediseasesrequireseduca- tionandsanitation(Tomokawaetal.,2018;Takeuchietal.,2013;
UNICEF, 2009). However, according to EDSMD 2008–2009 EDSMD, 2010EDSMD 2008–2009, 23.6%of peoplein Betsiboka regionhavenoaccesstoaschooleducationand58%cannotfinish primaryschool.InruralareasofMaevatananaregion,only1%of householdsareequippedwithasanitarytoiletand48%haveno kind of toilet (EDSMD 2008–2009EDSMD, 2010EDSMD 2008– 2009),indicatingthatenvironmentalcontaminationbyschisto- somesorotherNTDsoccurscontinuously.Ascale-upfieldsurvey shouldbeconductedtomapareasofoccurrenceoftheparasite, make people aware of the infection, and generate positive informationtoimprovethehealthofthepeople,notonlythose livingintheendemicareas,butalsotourists,workers,andpeople passingthroughtheseareas(Satoetal.,2003,2006;Hashizume etal.,2017).
Over50%ofthepopulationof Madagascarisinfectedwith intestinalorurinaryschistosomiasis(Rollinsonetal.,2013).The diseaseslowlydebilitatesinfectedpersons,whoarefrequently children,resultinginlowerproductivityandreducedlearning inschool-agechildren.Thissituationleadstoaviciouscycle, perpetuated by several environmental, psychological, and socialfactors.A multifacetedapproach,involving masstreat- ment,biologicalcontrol,environmentalcontrol,education,and diseasesurveillance,couldleadtoschistosomiasiscontroland eliminationinMadagascar. Pathogen-specificeDNAdetection, as shown in this study, could contribute as an accurate surveillance tool to be applied to NTDs inthe field, helping to establish intelligent control programs for NTDs and contributingtofoodand watersafetyfor thosepeople living inendemicareas.
Conclusions
Inthisstudy,wesucceededindevelopingspecificandshorter amplification length primers targeting the COI region of mitochondrial DNA of S. mansoni. The detection of S. mansoni DNAinenvironmentalwaterbyapplyinganenvironmentalDNA analysiswasperformedsuccessfully.Thus,anewmethodforeco- epidemiology studies is presented, which could be used as an additionaltoolincontrolprogramsforschistosomiasisinendemic areas.
Acknowledgments
The authors are thankful for the help in logistics and field planning provided by the staff of the basic health centers at Ambalanjanakombyand Ampisavankaratra,theheads ofdecen- tralizedstructuresatMaevatananaDistrictandBetsibokaregional level, staff members of the Ministryof Public Health, and the officials of the embassies of France and Japan in Madagascar.
Thanks tothe peopleof Maevatanana for theirhospitality and specialthankstothepatientswhovoluntarilyjoinedthisstudy.
Ethicalapproval
This study was approved by the Ministry of Public Health Madagascar(OfficialReferencedLetterNo.220MSANP/SG/DGS– 2016).Theanimalexperimentsinthisstudywereapprovedbythe Ethics Committee of Dokkyo Medical University (Document Number0006/2001).
Funding
ThisstudywassupportedinpartbytheEmbassyofFrancein Japan(GrantNo.98/2015).
Conflictofinterest
Nocompetinginterestdeclared.
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