ContentslistsavailableatScienceDirect
Journal
of
Infection
and
Public
Health
jo u r n al ho me p ag 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
Demographic
characterization
and
spatial
cluster
analysis
of
human
Salmonella
1,4,[5],12:i:-
infections
in
Portugal:
A
10
year
study
R.
Seixas
a,∗,
T.
Nunes
a,
J.
Machado
b,
L.
Tavares
a,
S.P.
Owen
a,
F.
Bernardo
a,
M.
Oliveira
aaCIISA/FacultyofVeterinaryMedicineofUniversityofLisbon,AvenidadaUniversidadeTécnica,1300-477Lisboa,Portugal
bNationalReferenceLaboratoryofGastrointestinalInfections,NationalHealthInstituteDoutorRicardoJorge,AvenidaPadreCruz,1649-016Lisboa,
Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received27October2016 Receivedinrevisedform4June2017 Accepted9June2017
Keywords: Clusteranalysis
Geographicalinformationsystem(GIS) Portugal
Publichealth Salmonella
1,4,[5],12:i:-a
b
s
t
r
a
c
t
Salmonella 1,4,[5],12:i:- is presently consideredone of the majorserovars responsible for human
salmonellosisworldwide.Duetoitsrecentemergence,studiesassessingthedemographic
characteri-zationandspatialepidemiologyofsalmonellosis1,4,[5],12:i:-atlocal-orcountry-levelarelacking.In
thisstudy,aanalysiswasconductedovera10yearperiod,from2000tothefirstquarterof2011atthe
Por-tugueseNationalLaboratoryinPortugalmainland,withatotalof215Salmonella1,4,[5],12:i:-serotyped
isolatesobtainedfromhumaninfectionsbyapassivesurveillancesystem.Dataregardingsource,year
andmonthofsampling,gender,age,districtandmunicipalityofthepatientswereregistered.
Descrip-tivestatisticalanalysisandaspatialscanstatisticcombinedwithageographicinformationsystemwere
employedtocharacterizetheepidemiologyandidentifyspatialclusters.Resultsshowedthatmost
dis-trictshavereportsofSalmonella1,4,[5],12:i:-,withahighernumberofcasesatthePortuguesecoastland,
includingdistrictslikePorto(n=60,27.9%),Lisboa(n=29,13.5%)andAveiro(n=28,13.0%).Anincreased
incidencewasobservedintheperiodfrom2004to2011andmostinfectionsoccurredduringMayand
October.Spatialanalysisrevealed4clustersofhigherthanexpectedinfectionrates.Threewerelocated
inthenorthofPortugal,includingtwoatthecoastland(Cluster1[RR=3.58,p≤0.001]and4[RR=10.42
p≤0.230]),andoneatthecountryside(Cluster3[RR=17.76,p≤0.001]).Alargerclusterwasdetected
involvingthecenterandsouthofPortugal(Cluster2[RR=4.85,p≤0.001]).Thepresentstudywas
elab-oratedwithdataprovidedbyapassivesurveillancesystem,whichmayoriginateanunderestimationof
diseaseburden.However,thisisthefirstreportdescribingtheincidenceandthedistributionofareas
withhigherriskofinfectioninPortugal,revealingthatSalmonella1,4,[5],12:i:-displayedasignificant
geographicclusteringandtheseareasshouldbefurtherevaluatedtoidentifyriskfactorsinorderto
establishpreventionprograms.
©2017TheAuthors.PublishedbyElsevierLimitedonbehalfofKingSaudBinAbdulazizUniversity
forHealthSciences.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
TheprevalenceofSalmonellaserovarsareconstantlychanging
inmanyEuropeancountries[1]and,in2010,theEuropeanFood
SafetyAuthority(EFSA)PanelonBiologicalHazardspublisheda
scientificopinionalertingfortheincreasingnumberofoutbreaks
intheEuropeanUnionmemberstates promotedby“Salmonella
Typhimurium-like”strains[2].
∗ Correspondingauthor.
E-mailaddresses:[email protected](R.Seixas),
[email protected](T.Nunes),[email protected](J.Machado), [email protected](L.Tavares),[email protected](S.P.Owen),
[email protected](F.Bernardo),[email protected](M.Oliveira).
Salmonella1,4,[5],12:i:-isconsideredamonophasicvariantof
serovarTyphimurium(1,4,[5],12:i:1,2)duetogenotypic
similari-tiesbetweenthetwoserovars[3,4],beingcharacterisedbyalack
ofthefljBgeneexpression,whichencodesthesecondphase
flagel-larantigen[3].SerovarTyphimuriumisthesecondmostcommon
serovarassociatedwithhumancasesofSalmonellainfectioninthe
EuropeanUnion(EU),exceededonlybyserovarEnteritidis[1].On
theotherhand,serovar1,4,[5],12:i:-wasseldomisolatedbefore
themid-1990sbutisnowamongthetop3mostprevalentserovars
isolatedfromhumansinEU[4].
PublichealthmethodsappliedtothesurveillanceofSalmonella,
andinparticularofSalmonella1,4,[5],12:i:-strains,mayhelpto
monitordiseasedevelopment,reducemorbidityandmortalityand
improve health, avoiding unnecessary regulatorymeasures [5].
In the medical field, theapplication of geographic information
https://doi.org/10.1016/j.jiph.2017.06.002
1876-0341/©2017TheAuthors.PublishedbyElsevierLimitedonbehalfofKingSaudBinAbdulazizUniversityforHealthSciences.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
systems(GIS)hasbecomeextremelyusefulinunderstandingthe
bigger picture of disease’s dissemination. These methods
asso-ciated with others techniques, including spatial statistics, are
important tools for public health maintenance, as they allow
toidentifyriskareasrequiring fastintervention,promotingthe
rationalizationofprevention’sproceduresandalsothereduction
of costs[6]. Therefore, this studyaimedto performthe
demo-graphiccharacterizationofsalmonellosis1,4,[5],12:i:-casesandto
usespatialanalysiscombinedgeoprocessingtechniquesinorder
toidentifyspatialclustersforSalmonella 1,4,[5],12:i:-infection,
evaluating high-risk areas and providing useful information to
understandthespreadandepidemiologyofthisserovarinPortugal.
Methods
Datasource:studypopulationandarea
InPortugal,salmonellosisisanotifiablediseaseandisdefined
as the isolation of Salmonella spp. (excludingSalmonella Typhi
orParatyphi)fromanappropriate clinicalsample,namelystool,
urineandblood,collectedfrompatientswithorwithout
clinically-compatiblesignsandsymptoms.Medical doctorsareobligedto
reportbypostanyconfirmedorsuspectedcaseofsalmonellosis,
includingnontyphoidalsalmonellosis,tothelocalhealthauthority
ofthemunicipalitywherethecaselives[7].
Inthisstudy,215Salmonella1,4,[5],12:i:-isolateswereobtained
from cases reportedfrom 2001 to thefirst quarter of 2011 in
mainlandPortugalwereincluded.AllSalmonellawerepreviously
isolated at the National Health Institute Doutor Ricardo Jorge
(INSA),serotypedusingtheslideagglutinationmethodaccording
totheKauffmann–Whitescheme,andincludeddifferentsources,
namelyfeces,blood, peritonialfluid andurine [8].Additionally,
informationincluding,source,yearandmonthofsample,gender,
age,districtandmunicipalityofthepatientswereregistered.
Isolates were obtained from patients at Portugal mainland,
representinganareaof89.015km2,corresponding96.6%ofthe
Portuguesenationalterritory,with10.047.083inhabitants[9].For
administrativepurpose,thisterritoryisdividedinto18Districts,
asfollows: Aveiro,Beja, Braga,Braganc¸a, CasteloBranco,
Coim-bra,Évora,Faro,Guarda,Leiria,Lisboa,Portalegre,Porto,Santarém,
Setúbal,VianadoCastelo,VilaRealandViseu.TheEurostat-based
NomenclatureofTerritorialUnitsforStatistics(NUTS)system
sub-dividesPortugalmainlandin308municipalities[9].
Statisticalanalysis
Descriptivestatistics
DescriptivestatisticalanalyseswereperformedusingSPSS21.0
software(IBMCorporation,New York,USA).For statistical
pur-poses, age wasgrouped in three differentclasses accordingto
NationalStatisticsInstitute[9],namely:young(lessthan15years),
adult(16–64years)andelderly(higherthan65).Agemean,median,
mode,rangeandstandarddeviationwerealsodetermined.
Spatialanalysis
Salmonella1,4,[5],12:i:-reportsweregeocodedatthe
munici-palitylevel.Thecentroidsofeachmunicipalityweredetermined
usingtheopen-sourceQuantumGeographicInformationSystem
(QGIS)software.SpatialclusteringofSalmonella1,4,[5],12:i:-cases
wasanalyzedusingspatialscan statistics[10].Statistical
proce-dureswerecarriedoutinSaTScansoftwareusingapurelyspatial
Poissonmodel.Thefollowingdatawereconsideredforanalysis:
thenumberof positive casesin eachmunicipality,theresident
populationwithineachmunicipalityaccordingtothe2011
Por-tuguesecensus,andtheCartesiancoordinatesofthecentroidsof
eachmunicipalityincludedinthesurvey.Themodelwasfirstrun
Table1
DistributionofSalmonella1,4,[5],12:i:-clinicalcasesperdistrictandgenderin Portugalfrom2000andtothefirstquarterof2011.
District Gender
Notregistered F M Total
Aveiro Count 0 21 7 28 %oftotal 0,0% 9,8% 3,3% 13,0% Beja Count 0 3 4 7 %oftotal 0,0% 1,4% 1,9% 3,3% Braga Count 0 15 12 27 %oftotal 0,0% 7,0% 5,6% 12,6% Braganc¸a Count 0 2 0 2 %oftotal 0,0% 0,9% 0,0% 0,9%
CasteloBranco Count 0 2 6 8
%oftotal 0,0% 0,9% 2,8% 3,7% Évora Count 0 2 12 14 %oftotal 0,0% 0,9% 5,6% 6,5% Faro Count 0 0 1 1 %oftotal 0,0% 0,0% 0,5% 0,5% Leiria Count 1 1 0 2 %oftotal 0,5% 0,5% 0,0% 0,9% Lisboa Count 0 16 13 29 %oftotal 0,0% 7,4% 6,0% 13,5% Porto Count 2 24 34 60 %oftotal 0,9% 11,2% 15,8% 27,9% Santarém Count 0 2 3 5 %oftotal 0,0% 0,9% 1,4% 2,3% Setúbal Count 0 8 16 24 %oftotal 0,0% 3,7% 7,4% 11,2%
VilaReal Count 0 3 4 7
%oftotal 0,0% 1,4% 1,9% 3,3%
Viseu Count 0 0 1 1
%oftotal 0,0% 0,0% 0,5% 0,5%
Total Count 3 99 113 215
%oftotal 1,4% 46,0% 52,6% 100,0%
usingthedefaultmaximumspatialclustersizeof50%ofthetotal
studypopulationtoensurestatisticalpower.Themaximum-size
parameterwasthensetat10%tocheckforthepresenceofextreme
smallriskareas,possiblymaskedbythe50%scanningwindow.The
numberofMonteCarloreplicationstoestimatethestatistical
sig-nificanceofthemostlikelyclusterwassetat9999iterations.Ap
value<0.05wasconsideredstatisticallysignificant.
Results
ThemajorityofSalmonella1,4,[5],12:i:-isolateswereobtained
fromfeces(n=185, 86%),followed byunknownsources(n=16,
7.6%),blood(n=8,3.7%),bloodandfeces(n=3,1.4%),peritoneal
fluid(n=1,0.5%),bloodandurine(n=1,0.5%)andurine(n=1,0.5%).
Regardingthecases’location,themajoritywasreportedinPorto
(n=60,27.9%),followedbyLisboa(n=29,13.5%),Aveiro(n=28,
13.0%),Braga(n=27,12.6%)andSetúbal(n=24,11.2%).Ontheother
hand,singlecaseswerereportedinFaroat2010andViseuat2007,
aswellas,two-singlecasesinBraganc¸aat2008and2009andin
Leiriaat2002and2009(Table1).
ThedistributionthroughyearsofSalmonella1,4,[5],12:i:-
infec-tionsinPortugalfrom2001tothefirstquarterof2011revealthat
2010wastheyearwiththehighernumberofreports(n=53,24.6%),
withanincreasingtrendinthenumberofcasesfrom2004to2010
(Fig.1).Theseasonalvariationwasalsoevaluatedduringtheperiod
ofstudyandisshown inFig.2.Mostoftheinfectionsoccurred
22 6 0 7 14 12 17 28 40 53 16 0 10 20 30 40 50 60 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Fig.1.TrendinPortugalofthenumberofcasesperyearofSalmonella 1,4,[5],12:i:-from2001tothefirstquarterof2011.
14 7 11 10 31 16 11 20 32 43 12 8 0 5 10 15 20 25 30 35 40 45 50
Fig.2.NumberofclinicalcasespermonthofSalmonella1,4,[5],12:i:-from2001to thefirstquarterof2011.
Legend: Months were pooled into seasons, which were defined as Spring (March–May),Summer(June–August),Autumn(September–November)and Win-ter(December–February).
numberofcasesbeingdiagnosedinOctober(43infections)and
February(seveninfections),respectively.
Informationregardingthepatient’sgenderbydistrictisrevealed
inTable1.However,inthreepatients,onefromLeiriaandtwofrom
Porto,genderwasnotpossibletoregister.
Patient’sagewasregisteredfrom199individualsandrevealed
thatyoungindividualswerethemostaffectedwith69.3%(n=149)
ofSalmonella 1,4,[5],12:i:-cases, followed byadults with14.0%
(n=30)andelderlywith9.3%(n=20).Descriptivestatistical
analy-sesshowedthatmeanagewas17yearswithamedianandmodeof
3and1year,respectively.Additionally,standarddeviationwas25.9
yearswithaminimumageof0years,anewborn,andmaximum
ageof90years.
Spatialanalysis revealed 4 clusters of higher than expected
infectionrates,includingtwolocatedinthenorthcoastlandarea
involvingthedistrictsofPortoandAveiro(Cluster1and4,
respec-tively),one at thecountryside involving thedistricts Braganc¸a
VianaReal,ViseuandGuarda(Cluster3),andalargerclusterlocated
inthecenterandsouthofPortugalwhich includedthedistricts
CasteloBranco,Leiria,Santarém,Lisboa,Portalegre,Évora,Setúbal
andBeja(Cluster2)(Fig.3).Onlyclustersone,twoandthreewere
statisticallysignificant,attributingariskofinfectionof4,5and18
timeshigher,respectively,foraindividuallocatedinthedelimited
perimetercomparedtootherindividualoutsidetheseareas.The
numberofcases,observed/expectedratio,relativeriskandpvalue
foreachclustercanbefoundinTable2.
Discussion
The first worldwide report on the isolation of Salmonella
1,4,[5],12:i:-wasfromPortugalin1986inpoultry[11].Sincethis
firstreport,arapidincreasein Salmonella1,4,[5],12:i:-isolation
wasobservedonaninternationalscale,becomingthethirdmost
commonserovarrelatedwithhumansinfectionsin2015intheEU
[4].
Datapresentedinthisstudyrepresentsapassivemonitoring
basedonclinicalsamplessubmittedtothenationalreference
labo-ratory,whichoftenunderestimatesthenumberofcases,sincenot
everyindividualwithsalmonellosisgoestoaphysicianduetoits
self-limitingcourse[12,13].Inaddition,youngindividualsusually
presentmoreseveresymptomsofinfectionandbecauseofthat,
aremorelikelytobetestedthanadults[12,13].Thus,alimitation
tothisapproachistheprobableselectionbias[13].
Inthepresentstudy,asmanyotherreports[14,15],Salmonella
wascommonly isolatedfromstool samples.Otherssources are
scarcelydocumentedand toourknowledge,this reportinclude
thefirstisolationofSalmonella1,4,[5],12:i:-fromperitonealfluid,
highlightinganunusualsourceofisolation,particularlyimportant
inclinicalsettings[16].
Itisimportanttoreferthatpositivestoolsamplesforenteric
pathogenscanbefoundinasymptomaticallypatients,manyweeks
aftertheacuteinfectionepisode[17].Somepatientswith
nonty-phoidalSalmonellainfectionsmaydevelopachroniccarrierstate,
characterizedasa positive stoolor urineculturefor Salmonella
at12 monthsfollowingthediarrhoealillness.Althoughchronic
carriageofnontyphoidalSalmonellaoccursinfrequently,
approxi-mately0.15%inhealthyadultsand3.9%inchildren,itrepresentsan
importantmechanismofperson-to-persontransmission.
Addition-ally,antimicrobialadministrationhasnotbeenproventoenhance
theclearanceofinfectionandmayactuallyincreasetheduration
ofasymptomaticshedding[18].
TheagedistributionofindividualswithSalmonella
1,4,[5],12:i:-infectionsinthepresentstudyisinagreementwithothersreports
[13,14,19], where most infections caused by this serovar are
observedinyoungindividuals.WhilethisstudydetectedSalmonella
1,4,[5],12:i:-infectionsmorefrequentlyinmen,othersreportsalso
identifiedbothmenorwomenfrequentlyaffected[14,19].
Althoughintheperiodofthisstudyanincreasingannual
inci-dencewasobserved,thenumberofreportedisolatesislow,which
maybeattributedtoadecreasingintheamountofsalmonellosis
casesreportedintheEUduringthisperiod,includingPortugal[2].
In this country, most districts have reports of Salmonella
1,4,[5],12:i:-,whichsuggestsawidedistributionfromthenorthto
southofthecountry.AhigherprevalenceinthePortuguese
coast-landwasobserved,whichthehigherhumanpopulationdensityin
theseareasmayexplainandbecausepatientsaremorelikelytogo
toadoctorifgastrointestinalsymptomsarepresented.Itisworth
notingthatsomedistrictsareclassifiedwithoutreports,whichdo
not necessarily representtheabsence ofthe disease,especially
whenthereisevidenceofunderreportingduetoitsself-limiting
course.
RatesofhumanSalmonella1,4,[5],12:i:-infectionappeartobe
higherinsummerand earlyautumn,withmostcasesoccurring
betweenMayand October, beingthis last month theone with
thehighernumberofcases.Inseveralstudies,Salmonella
infec-tionsamonghumansgenerallypeakinsummermonths[20,21].
Thereasonsoftheseseasonaldifferencesarenotentirelyknown
andcouldberelatedtocombinationoffactors,includingseasonal
humanbehaviors[20],theparallelSalmonellasheddingtrendsby
animalreservoirs[22] andenvironmentalvariationsinfluencing
thepathogenvirulenceorpersistence[20].
ClusteranalysisofSalmonella1,4,[5],12:i:-infectionsshowed
Fig.3. PrevalenceandspatialclustersofSalmonella1,4,[5],12:i:-casesinPortugal,2000tothefirstquarterof2011.
Legend:PrevalenceofSalmonella1,4,[5],12:i:-per100,000inhabitants,bymunicipalitiesaccordingtotheEurostat-basedNomenclatureofTerritorialUnitsforStatistics system.SpatialclustersresultsbasedonapurelyspatialPoissonmodelusingtheSatScanTMsoftware.
Table2
SpatialclusterofhighSalmonella1,4,[5],12:i:-infectionratesinmainlandPortugalfrom2000tothefirstquarterof2011. Cluster Numberof cases Numberof municipalities involved Annualcases per100,000 habitants
Expectedcases Overlap Observed/expected Relativerisk Loglikelihoodratio pValue
1 109 22 4.8 48.02 4 2.27 3.58 41.188036 ≤0.001
2 84 109 4.9 46.70 No 1.80 4.85 27.495199 ≤0.001
3 10 18 49.8 0.99 No 10.14 17.76 16.440983 ≤0.001
districtslocatedatthenorthwest,northeast,centerandsoutheast
of Portugal. This observation suggests an occurrence of
non-random cases, confirmed by the representation of three high
rateclusters,whichmayrevealanincreasedexposuretohuman
Salmonella1,4,[5],12:i:-infectionintheseareas.
Interestingly,cluster1locatedatnorthwestofPortugalpresents
thehighestnumberofhumanSalmonella1,4,[5],12:i:-infections.
Thiscluster contains two districts,Porto and Aveiro, withonly
22municipalities involved.Individualsin theseareas present a
riskabout4timeshighertoacquirethisinfectionthatinhabitants
ofothermunicipalities.Althoughitmayexistseveralreasonsto
explainthisspatialcluster,onepossibleexplanationisbecausepig
farmsareprevalentintheselocations,especiallyinAveiro [23].
Sincepigscanbereservoirsforthisserovar,thismayjustifythe
spatialclusterandthehighfrequencyofcases[24,25].Thisreason
mayalsobevalidforcluster2,assomeofthedistrictsincludedlike
Beja,LeiriaandSantarém,arealsotheimportantlocationsforpig
productioninPortugal[23].
Inthepublichealthperspective,spatialclustersanalyzesmay
revealthespacedimensionsofthediseasesprocesses.
Identifica-tionofSalmonella1,4,[5],12:i:-casesthatareconcentratedinan
specificlocationinPortugalmaybeessentialfortheefficient
distri-butionofresourcesforpreventionandtreatmentofthisnotifiable
disease.
Conclusions
Asfarasweknow,thisisthefirstreportdescribingtheincidence
andthepresenceofareaswithahigherriskforhumanSalmonella
1,4,[5],12:i:-infectionsinPortugal.Althoughpassivesurveillance
mayrepresentanunderestimationofdiseaseburden,theyprovide
valuableinformationonincidenceandtrendsthatcouldaidpublic
healthauthoritiesindevelopingpreventionandcontrolprograms.
Thereisaneedtobetterunderstandthedemographic,geographic,
and seasonalfactors associated withtheincrease of Salmonella
1,4,[5],12:i:-infections and toprovide evidence-based
informa-tionforpolicymakerstoprioritizefutureeffortsinaddressingthe
increasingnumberofinfections.
Funding
This study was conducted with the financial support of
the “Centro de Investigac¸ão Interdisciplinar em Sanidade
Ani-mal, Faculdade de Medicina Veterinária da Universidade de
Lisboa”—ProjectUID/CVT/00276/2013(fundedbyFoundationfor
ScienceandTechnology—FCT).RuiSeixasholdsaPhDfellowship
(SFRH/BD/75836/2011)fromFCT,Portugal. Competinginterests Nonedeclared. Ethicsapproval Notrequired. Acknowledgements
Theauthorswouldliketoacknowledgethedataprovidedby
Portuguesenationalreferencelaboratory,theNationalHealth
Insti-tuteDoutorRicardoJorge(INSA).
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