Seroprevalence study of Toscana virus and viruses belonging to the Sandfly fever Naples antigenic complex in central and southern Italy

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Pleasecitethisarticleinpressas:MarchiS,etal.SeroprevalencestudyofToscanavirusandvirusesbelongingtotheSandﬂyfever NaplesantigeniccomplexincentralandsouthernItaly.JInfectPublicHealth(2017),http://dx.doi.org/10.1016/j.jiph.2017.02.001

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JIPH-707; No.ofPages4

JournalofInfectionandPublicHealthxxx(2017)xxx–xxx

Contents lists available atScienceDirect

Journal

of

Infection

and

Public

Health

j o u r n a l h o m e p a g e :h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / j i p h

Seroprevalence

study

of

Toscana

virus

and

viruses

belonging

to

the

Sandﬂy

fever

Naples

antigenic

complex

in

central

and

southern

Italy

Serena

Marchi

a

_,

_Claudia

_M.

_Trombetta

a

_,

_Otfried

_Kistner

b,c

_,

_Emanuele

_Montomoli

a,c,∗

a_Department_of_Molecular_and_{Developmental}_Medicine,_University_of_Siena,₅₃₁₀₀_Siena,_Italy b_Independent_Consultant,_Vienna_1221,_Austria

c_VisMederi_Srl,_Enterprise_of_Service_in_Life_Sciences,₅₃₁₀₀_Siena,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1September2016

Receivedinrevisedform31January2017 Accepted5February2017 Keywords: Toscanavirus Sandﬂy Seroprevalence Phlebovirus Italy

a

b

s

t

r

a

c

t

Sandflyfevervirusesaretransmittedbythebiteofphlebotominesandflies;serotypessandflyfeverNaples virus,sandflyfeverSicilianvirusandsandflyfeverCyprusviruscausefebrileillness,whereasToscanavirus (TOSV)maycauseneuroinvasiveinfections.AlthoughTOSVisanimportantcauseofasepticmeningitis incentralandsouthernItaly,inmanycasestheinfectionisasymptomatic,leadingtounderestimationof theactualspreadofthevirus.ThisserosurveyaimedtoassesstheseroprevalenceofTOSVinarandom populationinSiena(Tuscany,centralItaly)in2003–2004and2013–2014andBari(Apulia,southern Italy)in2004and2015.2132serumsamplesweretestedforthepresenceofanti-TOSV/SFNVIgGby meansofELISAandIFAcommercialtests.Seroprevalencerateswerecomparedinthetwocitiesandover aten-yearperiodinthesamecity.SeroprevalenceresultsintheSienapopulation(22.95%in2003–2004 vs26.75%in2013–2014)confirmedtheendemiccirculationofTOSVandcloselyrelatedvirusesincentral Italy,withoutmajorchangesoverthelastdecade,whilenosignificantprevalencewasobservedinBari (2.90%in2004vs1.85%in2015).

Introduction

Sandflyfeverviruses(Bunyaviridaefamily,genusPhlebovirus) are widelydistributed in theMediterranean basin, where they are transmitted by thebite of infected phlebotomine sandflies belongingtothegenusPhlebotomus[1].Thefourmostcommon serotypesassociatedwithhumaninfectionsincludesandflyfever Naplesvirus(SFNV),sandflyfeverSicilianvirus(SFSV),sandflyfever Cyprusvirus(SFCV)andToscanavirus(TOSV)[2,3].Clinical dis-ease,knownas“pappatacifever”,usuallypresentsasaself-limiting febrileillness,characterizedbyfever,myalgiaandheadache.TOSV istheonlyserotypeassociatedwithneuroinvasiveinfections;in endemicregions,itisamongthemajorcausesofasepticmeningitis andmeningoencephalitisduringthesummerseason[4–6].

InItaly,themainvectorsofTOSVarethesandﬂies Phleboto-musperﬁliewiandPhlebotomusperniciosus,whicharefoundmainly incentralandsouthernregions.OneserosurveyfoundthatSFNV and SFSVinfectionshad disappearedfrom areaswhere malaria eradicationcampaignshadbeenimplemented[5,6];incontrast,

∗ Corresponding author at: Department of Molecular and Developmental Medicine,UniversityofSiena,53100Siena,Italy.

E-mailaddress:emanuele.montomoli@unisi.it(E.Montomoli).

neurologicaldiseaseduetoTOSVcontinuedtobeobserved annu-ally[7–12],suggestingthepossibilityofananimalreservoirforthis virus[5].Serologicalsurveyshaveshownthepresenceofanti-TOSV antibodiesinovinesandhorses[13];however,therearenoreliable dataindicatinghumansorothervertebratesasanaturalreservoir [5].

Afterreportsofinfectionintouristsfromnorth-centralEurope andtheUSA[14,15],severalstudieswereconductedonthe cir-culationof TOSVin theareaof Siena (Tuscany);most of these includedpopulationsofsubjectshospitalizedforneurological dis-orders[10,12].

Furtherserologicalanalyseswereperformedonpopulations liv-inginthesamehighlyendemicarea.Theﬁrstofthese,conducted onasymptomatichouseholdcontactsof centralnervoussystem (CNS)infectionpatients,revealedanti-ToscanavirusIgG seropos-itivityin22%ofsubjectsandIgM/IgGin6%[16].Aseroprevalence studyonapopulationofoccupationallyexposedsubjectsfromthe provincesofSiena,FlorenceandArezzorevealedpositiveanti-TOSV IgGin22.7%intheurbancontrolpopulation[17].These investi-gationsconﬁrmedthewidecirculation ofTOSVinSienaandits provinceandassociatedthiscirculationbothtoasymptomaticand symptomaticinfectionswithoutCNSinvolvementandtoaseptic meningitisandmeningoencephalitisduringthesummermonths. Arecentretrospectivestudyontheantibodyprevalenceratesof http://dx.doi.org/10.1016/j.jiph.2017.02.001

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2 S.Marchietal./JournalofInfectionandPublicHealthxxx(2017)xxx–xxx TOSVamongpatientsresidentinTuscanyandhospitalizedfor

rea-sonsotherthanneurologicalinfections,foundseroprevalencerates of19.8%inadultsand5.8%inchildren,indicatinganage-dependent increaseinTOSV-speciﬁcimmunity[18].

OthercasesofTOSVinfectionand highseroprevalencerates havebeenreportedinsouthernItalian regions,suchas Campa-nia[19]andSicily[20,21],thusexpandingthegeographicalarea ofcentralItalydeﬁnedintheﬁrststudies.

Theprimarypurposeofthepresentsurveywastobetter char-acterizethedistributionofTOSVinfectionintheterritoryofSiena overthelastdecade.Thesecondaimwastoinvestigatethe circu-lationofthevirusintheprovinceofBari(Apulia,SouthernItaly).In thislatterarea,epidemiologicaldataonTOSVarelacking,but cir-culationofthevirusissuggestedbypreviousstudiescarriedoutin otherregionsofsouthernItaly.Comparingtheseroprevalencerates inSienaandBariprovidesafullerdescriptionoftheepidemiology ofTOSVintwodifferentItalianregions.

Materialsandmethods

Testswereperformedonhumanserumsamplesfromthe inter-nal serum bank of the Laboratory of Molecular Epidemiology, DepartmentofMolecularandDevelopmentalMedicine,University ofSiena.Thesampleshadbeenanonymouslycollectedin compli-ancewithItalianethicslaw;theonlyinformationavailableonthese subjectswasageandgender.

Atotalof2132serumsamplesfromsubjectsofbothsexesandall ages,collectedinSienainthetwo-yearperiods2003–2004(n=903) and2013–2014(n=836)andinBariin2004(n=333)and2015 (n=60),weretestedforthepresenceofspeciﬁcanti-TOSV/SFNV IgGantibodies.

SerologicaltestswereperformedbymeansofEnzywellToscana virusIgG(DIESSE-Siena,Italy)commercialkitbasedonaprinciple ofenzyme-linkedimmunosorbentassay(ELISA)withrecombinant nucleoprotein[22],inaccordancewiththemanufacturer’s instruc-tions. Accordingto themanufacturer, thetest has a sensitivity of95%andspeciﬁcityof96.5%;furthermore,cross-reactionswith othercorrelatedserotypesofthePhlebovirusgenus,suchasSFSV andSFNV,cannotbeexcluded[22].

SerumsampleswithborderlineELISAresults(accordingtothe parametersoftheELISAkit)wereexcludedfromthestudy,while thosewithpositiveresultsunderwentconfirmatory immunoflu-orescenceassay (IFA)by meansof the commercialkit “Mosaic Sandflyfevervirus1”(Euroimmun,Germany)asindicatedbythe manufacturer.The IFA distinguishesIgGfor thefourserotypes: SFSV,SFNV,SFCVandTOSV.Consideringthatitisimpossiblewith ELISAandIFAteststodistinguishbetweenTOSVIgGandIgGraised after infection caused by another virus belonging to the same antigeniccomplex,samplespositive toTOSVIgGareconsidered positiveforTOSV/SFNVIgG[1].

Sex- and age-specific seroprevalence rates were calculated, alongwiththecorresponding95%confidenceintervals(CI). Sta-tisticalanalysiswasperformedbymeansoftheYatescorrected chi-squaretesttocompareprevalenceratesamongdifferentstudy groups.StatisticalsignificancewassetatP<0.05,two-tailed.

Results

Atotalof2132serumsamplesweretestedbyELISAassay,125 ofwhich yieldedborderlineresultsand2 werenotsuitable for IFA.These127sampleswereexcludedfromthestudy,while691 sampleswithELISApositiveresultsweretestedbyIFA.

SampleswithIFA negativeresultsfor TOSVwereconsidered negativealongwiththosewithELISAnegativeresults.Only

sam-Table1

Seroprevalenceofanti-TOSV/SFNVIgGbyIFAinSienapopulation2003–2004and 2013–2014,dividedbyage-group.

Agegroups Siena,2003–2004(%) Siena,2013–2014(%)

0–15 4/77(5.20) 0/17(0.00) 16–30 34/202(16.83) 11/121(9.09) 31–45 39/175(22.29) 41/200(20.50) 46k60 31/142(21.83) 71/230(30.87) 61–75 42/125(33.60) 66/173(38.15) >76 43/120(35.83) 25/59(42.37) Total 193/841(22.95) 214/800(26.75) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0-15 16-30 31-45 46-60 61-75 76+ Sero preva le nc e Age group TOT M F

Fig.1. Prevalenceofanti-TOSV/SFNVIgGbyIFAinSienapopulationin2013–2014, byage-groupandsex,with95%CI.

Table2

Prevalenceofanti-TOSV/SFNVIgGbyIFAinpopulationofBari2004and2015, dividedbyage-groups.

Agegroups Bari,2004(%) Bari,2015(%)

0–15 0/32(0.00) 0/6(0.00) 16–30 0/77(0.00) 1/12(8.33) 31–45 0/71(0.00) 0/13(0.00) 46–60 1/54(1.85) 0/9(0.00) 61–75 5/55(9.09) 0/8(0.00) >76 3/21(14.29) 0/6(0.00) Total 9/310(2.90) 1/54(1.85)

pleswithTOSVIFApositiveresultswereconsideredtobepositive

foranti-TOSV/SFNVIgG.

Samplesweredividedby genderand classiﬁedintosix

age-groups:0–15,16–30,31–45,46–60,61–75and>76yearsold.

Theresultsforanti-TOSV/SFNVIgGinsamplescollectedinSiena

in2003–2004and 2013–2014are reportedinTable1; 193/841

(22.95%,CI:20.11–25.79)samplescollectedinSienain2003–2004 werepositive for anti-TOSV/SFNVIgG (Table1), whilesamples collectedinthesameareain2013–2014dispilayedTOSV/SFNV seropositivityof26.75%(214/800,CI:23.68–29.82)(Table1).No signiﬁcantdifferenceinTOSV/SFNVseroprevalencewasobserved betweenthetwo-yearperiodsconsidered(P=0.085).

On considering the biennium 2013–2014, TOSV/SFNV sero-prevalencewassigniﬁcantlylower(P=0.011)infemales(23.11% CI:19.22–27.01)thaninmales(31.43%CI:26.56–36.29).On sub-dividing the age-groups by sex, the prevalence rate found in samplesof46–60yearsoldmales(38%,CI:28.49–47.51)collected in2013–2014wassigniﬁcantlyhigherthanthatofthesame age-groupinthe2003–2004population(20%,CI:9.88–30.12)(P=0.028) (Fig.1).

Moreover,theprevalenceofanti-TOSV/SFNVIgGsigniﬁcantly increased(P<0.001)withage.Indeed,seroprevalencedisplayeda linearage-relatedincrease:from0%inchildren<15years oldto 42.37%(CI:29.76–54.98)intheelderly(Fig.1).Noothersigniﬁcant differences,bysexoryearofcollection,wereobserved.

TOSV/SFNVseroprevalenceinsamplescollectedinBariin2004 and2015arereportedinTable2.Of310samplescollectedinBariin 2004,only9werepositiveforTOSV/SFNVIgG(2.90%,CI:1.03–4.77) (Table2).Asmallnumberofsamplescollectedinthesamearea

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S.Marchietal./JournalofInfectionandPublicHealthxxx(2017)xxx–xxx 3 Table3

Prevalenceofanti-TOSV/SFNVIgGbyIFAinpopulationof2004inBariandinSiena dividedbyage-groups.

Agegroups Bari,2004(%) Siena,2004(%)

0–15 0/32(0.00) 3/37(8.11) 16–30 0/77(0.00) 14/82(17.07) 31–45 0/71(0.00) 11/58(18.97) 46–60 1/54(1.85) 15/69(21.74) 61–75 5/55(9.09) 20/76(26.32) >76 3/21(14.29) 27/79(34.18) Total 9/310(2.90) 90/401(22.44) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0-15 16-30 31-45 46-60 61-75 76+ Seroprevale nce Age group BARI SIENA

Fig.2. Prevalenceofanti-TOSV/SFNVIgGbyIFAinSienaandBariin2004,by age-group,with95%CI.

Table4

SamplestestedbyIFAwithpositiveresultsforSFSVSFNVandSFCVinSienain 2003–2004and2013–2014andinBariin2004and2015.

Agegroups Siena 2003–2004 (%) Siena 2013–2014 (%) Bari2004 (%) Bari2015 (%) SFSV 0/299(0.00) 6/332(1.81) 4/60(6.67) 0/1(0.00) SFNV 8/299(2.68) 1/332(0.30) 4/60(6.67) 0/1(0.00) SFCV 1/299(0.33) 2/332(0.60) 0/60(0.00) 0/1(0.00)

in 2015weretested; 1/54(1.85%) showed anti-TOSV/SFNVIgG

(Table2).Noothersampleswereavailablefortesting;nosigniﬁcant differencewasfoundbetweenthetwoyears(P=1.00).

TheresultsobtainedfromsamplesfromBariin2004(n=310) werecomparedwiththoseofsamplescollectedinSienainthesame year(n=401)(Table3,Fig.2).ThedifferencebetweenTOSV/SFNV seroprevalence rates was highly signiﬁcant(P<0.001). Regard-ingthedistributionamongage-groups,nodifferencewasfound betweenchildrenaged0–15yearsinbothcities,whileavery sig-niﬁcantdifferencewasfoundinadults,especially inthoseaged 16–30and31–45years(P<0.001).

Of691samplestestedbyIFA,26sampleswerenegativefor anti-TOSV/SFNVIgG butshowedpositivitytooneoftheotherthree serotypesthatcouldberecognizedwithIFA(Table4).

Discussion

TOSVisanarbovirustransmittedbysandﬂiesofthegenus Phle-botomus,whicharewidespreadincentralandsouthern Italy.In manycases,theinfectionisasymptomaticorassociatedwith ﬂu-likesymptoms,resultinginunderestimationoftheactualspreadof thevirus.Nevertheless,inendemicareas,TOSVisamongthemajor causesofasepticmeningitisandmeningoencephalitisduringthe summerseason.InItaly,thevirusismainlyfoundinthecentral regions,butithasalsobeenobservedinthesouth[5,6].

Thisserologicalsurveywasconductedinordertoestimatethe prevalenceofanti-TOSVantibodiesinthegeneralpopulationsof Siena(CentralItaly)andBari(SouthernItaly)overthelastdecade,

andtomakeacomparisonbetweenthesetwodifferent geograph-icalareas.

Analysisof thedata obtainedfromthis survey confirms the resultsreportedbypreviousstudies[10,12,16,17].Inparticular,the resultsfromtheSienapopulationin2003–2004areconsistentwith thosereportedinaserologicalsurveyconductedinapopulation liv-inginTuscanyduringtheperiodof1999–2006[18],whichshowed aTOSVseroprevalenceof5.8%inchildrenand19.8%inadults.The TOSV/SFNVseroprevalenceobservedin2003–2004(22.95%)andin 2013–2014(26.75%)confirmsSienaasanendemicareaandthatno significantchangeshaveoccurredoverthedecade.

In the2013–2014studypopulationinSiena,31.43% ofmale and 23.11% of female subjects showed TOSV/SFNV immunity (P=0.011).Moreover,theprevalencerate(38%)foundinmale sub-jectsagedfrom46to60yearswassigniﬁcantlyhigherthanthat reportedfor46–60yearoldmalesin2003–2004(P=0.022). Unfor-tunately,asspecimenswerenotaccompaniedbyananamnestic questionnaire,itwasnotpossibletoassociateTOSV/SFNV seropos-itivitywithfactorsthatmayincreasetheriskofinfectioninthe subgroupconsidered.However,itcanbeassumedthatsome out-dooractivitiesrelatedtoagreaterriskofinfection,suchasforestry andfarming[23],aremorefrequentlypracticedbysome popula-tionsubgroups.

TheTOSV/SFNVseroprevalenceobservedalongage-groupswas significantlylowerinchildren(P=0.001)andshowedalinear age-related increase. Less exposuretothe vectorcould explain the virtualabsenceofTOSV/SFNVspecificimmunityinsubjectsless than 15years old. Considering only subjects over 15years old, inSienain2013–2014TOSV/SFNVseroprevalenceobservedwas 27.33%(23.53%in femalesand32.26%in males),indicatingthat theinfectionis morefrequentinadults.Specifically,insubjects aged61–75andover76years,thepercentageofpositivityreached 38.15%and42.37%,respectively.Confirmingtheresultsofprevious epidemiological studies[18],theincreasing seroprevalencerate concurrentwithagedemonstratesthatthepopulationis consis-tentlyexposedtoTOSVorcloselyrelatedvirusesthroughoutlife. Indeed,typicalhillside settlementsin theprovinceofSienaare exposedtogreatercontactwithsandflies,andthereforetoagreater riskofinfectioninallage-groups,assuggestedbyBraitoetal.[23]. StudiesconductedinsouthernItalyhaveindicatedTOSVasthe causeof 5.6%ofmeningitis casesin thesummertimein Naples (Campania)[19]andofhighseroprevalenceratesinSicily[20,21]. Incontrast,samplescollectedintheprovinceofBarididnotshow asignificantprevalenceofTOSV/SFNV,whichwas2.90%in2004 and1.85%in2015.Asnootherepidemiologicalstudieshavebeen conductedintheApuliaregion,itcanbeclaimedthatthisisthe firstsurveyofthepresenceofTOSVandcloselyrelatedvirusesin thisarea.

Moreover,theresultsobtainedfromsamplesfromBariinthe year2004werecomparedwiththoseofsamplescollectedinSiena in thesameyear.The differencebetweentheTOSV/SFNV sero-prevalencerates foundinSienaand Bariwashighly signiﬁcant (P<0.001).Thismaybeduetothedifferentbiologicalandclimatic nichesofthesegeographicalareas,whichcouldbedifferentially hospitabletothevector.Interestingly,almostallthesamples pos-itivetoanti-TOSV/SFNVIgGthatwerecollectedin theprovince ofBariwerefromsubjectsover61yearsold(8/10),suggestinga greatercirculationofthevirusesinpreviousdecades.

Inthisstudy,reactivitytootherserotypesrecognizablebyIFA was detected in 26 samples in total, 24 of them belonged to over-65-year-oldsubjects.EspeciallyforSFSVandSFNV,these ﬁnd-ingsareconsistentwithpreviousserologicalstudiesthatobserved thedecreaseordisappearanceoftheseviralinfectionsafterthe 1940smalariaeradicationcampaignsinItaly[8].Apossible cross-reactivitybetweenotherviralserotypesbelongingtothesandﬂy feverNaplesspeciescannotbefullyexcludedwithouttesting

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pos-Pleasecitethisarticleinpressas:MarchiS,etal.SeroprevalencestudyofToscanavirusandvirusesbelongingtotheSandﬂyfever NaplesantigeniccomplexincentralandsouthernItaly.JInfectPublicHealth(2017),http://dx.doi.org/10.1016/j.jiph.2017.02.001

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4 S.Marchietal./JournalofInfectionandPublicHealthxxx(2017)xxx–xxx itivesamplesalsobyvirusneutralization[24,25]buttakeninto

accountthegeographicalareaofinterestandtheresultsof pre-viousseroprevalencestudies[8,16–18]seropositiveresultsinthis studyclearlysupporttheclassiﬁcationasTOSV-positive.

Themainimportanceofthisstudyisthatthedetectionof anti-TOSV/SFNVantibodieswasconductedonsamplesthathadbeen collectedforother medicalpurposes, not necessarilyrelated to TOSVinfection.Indeed,randomlychoosing a populationallows characterizingthespread oftheviruseven amongmembers of thegeneralpopulationofan endemicarea. On theotherhand, thelackofinformation(e.g.employment,outdooractivities, extra-urbanresidence,petownership,neurologicaldisease,andprevious diagnosis of TOSV infection) on the subjects involved did not allowevaluatingtheinﬂuenceoffactorsthatmayberelatedtoan increasedriskofinfection.ThisstudyshowsthatinTuscanyisstill anendemicarea,andthatnosigniﬁcantchangeinprevalencerates hasoccurredinthelastdecade.InApulia,bycontrast,theviruses seemtobesomewhatrare.

Inconclusion,forresidentsinendemicareas,theprobabilityof beinginfectedbyTOSVandcloselyrelatedvirusesanddeveloping speciﬁcantibodiesincreasesduringtheirlifetime.Moreover,CNS infectionsappeartobemorefrequentinadultsthaninchildren [18];therefore,highseroprevalenceamongolderadultsisa con-cern.On theotherhand,populationsinendemicareascouldbe betterprotectedfromneurologicalinfections,owingtorepeated exposuretothevirus, which canboost immunity [26]. Indeed, pathogenicityseemstobehigherinsubjectswhocomefrom non-endemicareas[27];thismaybebecausetheyaremoresusceptible toinfectioninlaterlifeastheydonothavepreviouslyacquired immunityagainstTOSV.Thus,itisnecessarytocontinue monitor-ingthespreadofTOSVinendemicareas,inordertoassesstherisk forthehealthofbothresidentsandtourist.

Funding Nofundingsources. Competinginterests Nonedeclared. Ethicalapproval Notrequired. References

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