A large outbreak of Legionnaires Disease in an in

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde w w w . e l s e v i e r . p t / r p s p

Original

article

A

large

outbreak

of

Legionnaires’

Disease

in

an

industrial

town

in

Portugal

Francisco

George

,

Tara

Shivaji

,

Catia

Sousa

Pinto

,

Luis

Antonio

Oliveira

Serra

,

João

Valente

_,

_Maria

_João

_Albuquerque

_,

_Paula

_Cristina

_Olivenc¸a

_Vicêncio

_,

Ana

San-Bento

_,

_Paulo

_Diegues

_,

_Paulo

_Jorge

_Nogueira

_,

_Teresa

_Marques

_,

Helena

Rebelo

_,

_Filipa

_Costa

_,

_Raquel

_Rodrigues

_,

_Alexandra

_Nunes

_,

_Vitor

_Borges

_,

João

Paulo

Gomes

_,

_Daniel

_Sampaio

_,

_Paula

_Barreiro

_,

_Silvia

_Duarte

_,

Dina

Carpinteiro

_,

_Joana

_Mendonc¸a

_,

_Catarina

_Silva

_,

_Luís

_Vieira

_,

_Maria

_Joao

_Simões

_,

Paulo

Gonc¸alves

_,

_Baltazar

_Nunes

_,

_Carlos

_Dias

_,

_Jorge

_Machado

_,

_Fernando

_Almeida

_,

Elsa

A

Goncalves

_,

_Lucilia

_Carvalho

_,

_Pedro

_Viterbo

_,

_Dilia

_Jardim

_,

_Nuno

_Lacasta

_,

Filomena

Boavida

_,

_Ana

_Perez

_,

_Isabel

_Santana

_,

_Paula

_Matias

_,

Nuno

Banza

_,

_Carlos

_Rabacal

a_{Direc¸ãoGeraldaSaúde,MinistériodaSaúde,Lisboa,Portugal}

b_{InstitutoNacionaldeSaúdeDr.RicardoJorge,MinistériodaSaúde,Lisboa,Portugal} c_{CentroHospitalardeLisboaOriental,MinistériodaSaúde,Lisboa,Portugal}

d_{CentrodeEstudosAnglísticos,UniversidadedeLisboa,MinistériodaCiência,TecnologiaeEnsinoSuperior,Lisboa,Portugal} e_{InstitutoPortuguêsdoMaredaAtmosfera,MinistériodoAmbiente,Lisboa,Portugal}

f_{AgênciaPortuguesadoAmbiente,MinistériodoAmbiente,Amadora,Portugal}

g_{Inspec¸ão-GeraldaAgricultura,doMar,doAmbienteedoOrdenamentoTerritorial,MinistériodoAmbiente,MinistériodaAgricultura,}

DesenvolvimentoRuraleFlorestaleMinistériodoMar,Lisboa,Portugal

h_{HospitaldeVilaFrancadeXira,MinistériodaSaúde,VilaFrancadeXira,Portugal}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13October2016 Accepted14October2016 Availableonlinexxx

Keywords:

Legionnaires’disease

Legionellapneumophilafraseri

a

b

s

t

r

a

c

t

Background:WedescribetheinvestigationandcontrolofanoutbreakofLegionnaires’ dis-easeinPortugalinOctober,NovemberandDecember2014.

Methods:Confirmed cases were individuals with pneumonia, laboratory evidence of

Legionellapneumophilaserogroup1andexposure,byresidence,occupationalorleisureto theaffectedmunicipalities.49possiblesourceswerereducedtofourpotentialsources,all industrieswithwetcoolingsystem,followingriskassessment.Wegeo-referencedcases’ res-idencesandthelocationofcoolingtowersdefiningfourstudyareas10kmbuffercentered oneachcoolingtowersystem.Wecomparedthenumberofcaseswithexpectednumbers, calculatedfromtheoutbreak’sattackratesappliedto2011censuspopulation.UsingStones’ Test,wetestedobservedtoexpectedratiosfordeclineinrisk,withdistanceupto10kmfour directions.IsolatesofLegionellapneumophilawerecomparedusingmolecularmethods.

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](F.George).

http://dx.doi.org/10.1016/j.rpsp.2016.10.001

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde Results: Weidentified403cases,377ofwhichwereconfirmed,14patientsdied.Patients becameillbetween14Octoberand2December.ANEwindandthermalinversionwere recordedduringtheestimatedperiodofexposure.Diseaseriskwashighestinpeople liv-ingsouthwestfromalloftheindustriesidentifiedanddecreasedwithdistance(p<0.001). 71clinicalisolatesdemonstratedanidenticalSBTprofiletoanisolatefromacoolingtower. WholegenomesequencingidentifiedanunusualL.pneumophilasubsp.fraseriserogroup1 astheoutbreakcausativestrain,andconfirmedisolates’relatedness.

Conclusions: Industrialwetcoolingsystems,bacteriawithenhancedsurvivalcharacteristics andacombinationofclimaticconditionscontributedtothesecondlargestoutbreakof Legionnaires’diseaserecordedinternationally.

©2016TheAuthor(s).PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofEscolaNacionalde Sa ´udeP ´ublica.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Um

grande

surto

da

Doenc¸a

dos

Legionários

numa

cidade

industrial

em

Portugal

Palavras-chave:

Doenc¸adosLegionários

Legionellapneumophilafraseri

r

e

s

u

m

o

Contexto:Descrevemosainvestigac¸ãoepidemiológicaemedidasdecontrolodeumsurtode doenc¸adosLegionários,ocorridoemPortugalemoutubro,novembroedezembrode2014.

Métodos: Adefinic¸ãodecasoengloboudoentescomcritériosclínicosdepneumoniaaguda, comprovasimagiológicascompatíveiseconfirmac¸ãolaboratorialparaaidentificac¸ãode

Legionellapneumophila(L.pneumophila)serogrupo1,paraalémdocritérioepidemiológico de exposic¸ão,querpor motivosderesidência,ocupacionaloulazernasfreguesias sus-peitas.Quarentaenovepossíveisfontesdeinfec¸ãoforamreduzidasa4potenciaisfontes, apósavaliac¸ãode risco,todasasindústriascomsistemade torresdearrefecimento.A georreferenciac¸ãoporresidênciadoscasoselocalizac¸ãodetorrespermitiudefinir4áreasde investigac¸ãonumperímetrode10kmcentradoemcadaumadaquelas4torres. Comparou-seonúmerodecasosobservadoscomonúmerodecasosesperados,calculadosapartirde taxasdeataquedosurtoaplicadasàpopulac¸ão.UsandootestedeStones,testou-searazão entrecasosobservadosecasosesperadosedeclíniodoriscoemrelac¸ãoàdistânciadeaté 10kmem4direc¸ões.AsamostrasdeL.pneumophilaforamcomparadasutilizandométodos moleculares.

Resultados: Foramidentificados403casos,dosquais377foramconfirmados,tendo ocor-rido14óbitos.Osdoentesapresentaramsintomasentre14deoutubroe2dedezembro.Em termosmeteorológicos,foramregistadosventosNEeinversãotérmicaduranteoperíodo estimadode exposic¸ão.Oriscodedoenc¸afoimaiorempessoasquevivemasudoeste detodasasindústriasidentificadas,diminuindocomoaumentodadistância(p<0,001). Amostrasde71doscasosclínicosdemonstraramumperfilSBTidênticoàsamostras iso-ladasapartirdeumatorredearrefecimento.AsequênciadegenomadeL.pneumophila fraseriserogrupo1poucocomumcomoaestirpecausadoradosurtoconfirmouarelac¸ão dasamostrasisoladas.

Conclusões: Torresdearrefecimentoindustriais,agentesbacterianoscomcaracterísticas mais desenvolvidas para elevada sobrevivência e uma rara combinac¸ão de condic¸ões climáticas,contribuíramparaosegundomaiorsurtodedoenc¸adosLegionáriosregistado naliteratura.

©2016OAutor(s).PublicadoporElsevierEspa ˜na,S.L.U.emnomedeEscolaNacionalde Sa ´udeP ´ublica.Este ´eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

On 7November 2014,the Directorate-Generalof Health in Portugal was informed by a local hospital laboratory of 18 patients with Legionnaires’ disease,all admitted inthe

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde outbreakinvestigation,wepresentedareportthathasbeen

publishedasarapidcommunication, providingpreliminary dataoftheepidemiological,microbiologicaland environmen-talinvestigation.1_{Thisarticlepresentsthefinalresultsofthe} outbreakinvestigation.

Legionnaires’ disease, first described during the 1976 PhiladelphiaAmericanLegionconference2_isabacterial pneu-monicinfectionwithLegionellaespecies.Mostcasesattributed to L.pneumophila sg1.3 _{Disease characteristically develops} 2–10 days after the inhalation of aerosolized bacteria by susceptibleindividuals.Riskfactorsincludesmoking,older individualsandthosewithchroniccardiorespiratorydisease.4 Water,eitherinnaturalorartificialaquaticenvironments,is thereservoirforLegionellaespecies.5

From2008to2012,between88and140caseswerereported annually in Portugal of which the majority were sporadic communityacquiredcases.6_{Largecommunityoutbreakscan} resultinsignificantmorbidityandmortalityinashortspace oftime,5_{thelargesttodatewasreportedin2001inMurcia,} Spainwith449cases.7

Frequently identified as the source oflarge community outbreaks,industrialcoolingtowersystemssource8_areable todisseminatecontaminatedaerosolsoverlargedistances.9 Meteorologicalfactors,industrialoperationalconditionsand inadequatemaintenanceareriskfactorsforLegionella out-breaksassociatedwithtowers.8,10

Methods

Definitions

AconfirmedcaseofLegionnaires’diseasehadradiologically confirmedpneumoniawithsymptomonsetbetween12 Octo-beránd02December2014,wholivedorworkedwithin10km ofVila Franca de Xira and had laboratory confirmation of infection.Thelaboratorycriteriaforaconfirmedcaseincluded theisolationofLegionellaspp.fromrespiratorysecretions,the detectionofL.pneumophilasg1antigeninurine,asignificant riseinspecificantibodyleveltoL.pneumophilasg1inpaired serum samples. We defined a probable case differed only inthe laboratory detection of eitherLegionella spp. nucleic acidinrespiratorysecretionsorasinglehighlevelofspecific antibodytoL.pneumophilasg1.

Identificationofcasesandassessmentofexposure

Caseswere identifiedbyreviewingstatutorylaboratoryand electronic clinical notifications from 1 October 2014. Staff fromregionalhealthauthoritiesinterviewedallconfirmedand probablecasesapplyingastandardPortugueseLegionnaires’ diseasequestionnairewhichrecordeddemographic, epidemi-ologicalandclinicaldetails.Theresidentialaddressofeach casewasgeocodedinGoogleEarth.

Toidentifydeaths, weregularly cross-matchedthe out-break database with the Portuguese real time information systemfordeathcertification(SICO),fromNovember2014to March2015.

Sourceidentification

PossiblesourcesofLegionellacontamination,including indus-tries withwet coolingsystems,hospitals, mallsand public recreationfacilitieswereidentifiedand,asameasureof pre-cautioncloseduntilenvironmentalinvestigation.Operational andmaintenancereportswerecheckedandsamplestakento screenforthepresenceLegionellaspp.,resultsledtothe identi-ficationofpotentialsources.Thelocationsofpotentialsources weremappedusingvisualpinpointinginGoogleEarth.

Microbiological,phenotypicandgenotypiccharacterization

Detection of L. pneumophila sg 1 antigen was performed usingspecificurinaryantigentestsinhospitallaboratories. Respiratoryandbloodspecimenswereprocessedatthe ref-erencelaboratory,theNationalInstituteofHealthDrRicardo Jorge.Environmentalsampleswereobtainedduring inspec-tionscarriedoutbytheGeneralInspectorateforAgriculture, Sea, Environment and SpatialPlanninginaccordance with Portugueseregulations,andprocessedbytheatthereference laboratory,theNationalInstituteofHealthDrRicardoJorge.

Clinicalandenvironmentalsampleswereanalyzedusing cultureand/ormoleculartechniques.Clinicalspecimenswere cultured on BCYE-based media and environmental sam-ples were cultured in GVPC selective media. Isolates were identifiedbycommerciallatex-agglutinationkits(Legionella latexTest,Oxoid,UnitedKingdomandMicrogenLegionella, Microgen® _{Bioproducts,). Culture results for clinical} sam-ples were checked by real-time PCR targeted to the 16S

and/or mip genes (L. pneumophila Real-TM, Sacace Biotech-nologies,Italy;ArgeneLegiopneumo/Ccr-gene,bioMérieux, France).Isolates,orculture-negative/PCRpositivespecimens, werecharacterizedbysequence-basedtyping(SBT)in accor-dance with the guidance of the European Working Group forLegionellaInfections(EWGLI).11 _{Thesevenlociwere} sub-jectedtoSangersequencingbyusingBigDyev1.1chemistry on a3130XLGeneticAnalyzer(Applied Biosystems).Whole genomesequencing(WGS)wasperformedfor10clinicaland oneenvironmentalsampleonaMiSeqinstrument(Illumina Inc., San Diego,USA) using using MiSeq V2 flowcells and 150bppaired-endreads(depthofcoverage>100-fold)to ascer-tain their similarity. Illumina reads were assembled using Velvetversion1.2.10.12

PhenotypiccharacterizationofL.pneumophilasg1usedthe Dresdenpanelofmonoclonalantibodies(MAbs)consistingof sixMAbsplustheMAb3oftheInternationalPanelobtained fromtheAmericanTypeCultureCollection(ATCC).Analysis followedanestablishedalgorithm,endorsedbyEWGLIwhich cancharacterizeL.pneumophilaintooneofninesubgroups; Knoxville,Philadelphia,France/Allentown,Benidorm,OLDA, Oxford,Heysham,CamperdownandBellingham.13_Thepanel includedantibodiestoMAb3/1whichisconsideredasa viru-lencemarkerduetohighhydrophobicityofthesestrains.

Meteorologicalandairqualityaspects

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde

70

Estimated period of aerosol exposure

Date of symptom onset (2014)

Outbreak declared Environmental sampling and shutdown of cooling systems

Cases in workers/ contractors of two factories 60 50 40 30

Number of confir

med cases

20

10

0

14-out 15-out 16-out 17-out 18-out 19-out 20-out 21-out 22-out 23-out 24-out 25-out 26-out 27-out 28-out 29-out 30-out 31-out 1-no

v 2-no v 3-no v 4-no v 5-no v 6-no v 7-no v 8-no v 9-no v 10-no v 11-no v 12-no v 13-no v 14-no v 15-no v 16-no v 17-no v 18-no v 19-no v 20-no v 21-no v 22-no v 23-no v 24-no v 25-no v 26-no v 27-no v 28-no v 29-no v 30-no v 1-dec 2-dec

Fig.1–ConfirmedcasesofLegionnaires’diseasebydateofsymptomonset,VilaFrancadeXira,Portugal,14October–2 December2014(n=377).

recordingsoftemperatureandoncedailyrecordingsof humid-ity,airquality,windspeedanddirectionwerecollectedforthe periodof18October–1November2014.

Statisticalanalysis

Weconductedasourceproximityanalysisoffour potential sourcestotestthehypothesisthatriskofdiseasewasinversely relatedtodistancefromthesource.UsingQGISsoftware,a bufferof10kmradiusfurthersubdividedintoconcentric1km rings was placedon the towers of each industrial cooling system.Thebufferandringswerethendividedintofour quad-rants,NorthEast,SouthEast,SouthWestandNorthWest.We comparedthenumber ofcaseswithexpectednumbers.To calculateexpectedcases,ageand sexspecificratesforthe outbreakwere appliedtothesubsection populationcounts fromthe2011census.Subsectionswerecategorized accord-ingtothedistanceanddirectionoftheirgeographicalcentroid fromeachsource.Theexpectednumberofcasesforeachband quadrantwascalculatedasthesumoftheexpectedcasesfor thesubsectionswhosecentroidslaywithinit.UsingStones’ Test(ST),wetestedobservedtoexpectedratiosfordecline inrisk,withdistanceupto10km inthe NorthEast,North West,SouthEastand SouthWest14 _{with 9999MonteCarlo} simulationsandsetthecut-offforstatisticalsignificanceat 0.05.15

BackcalculationmodelsdevelopedbyEganetal.16 _were usedtoestimatetheperiodofaerosolizedreleaseatthestart oftheoutbreak.Themodelswererepeatedusingcasesnotified uptoMarch2015.StatisticalanalysiswasconductedusingR statisticalsoftware.

Results

Outbreakdescription

By2December2014,403casesofdiseasewereidentifiedof which377confirmedand26probablecases.Fourteenpeople died(casefatality3.5%).

Confirmedcasedevelopedsymptomsbetween14October and 2 December 2014. The number of cases peaked on 6 November and the outbreak was declared on 7 November (Fig.1).

ThehighestattackrateswereseenintheparishofPóvoa de Santa Iria/Fortede Casa (60 per 10,000population) and droppedwithincreasingdistancefromthisparish(Fig.2).

Therewere252confirmedcasesinmen(66%).Themedian age was 59 years, the attack rate was higher in men and increased with age in both sexes (25.1 per 10,000 men aged20–64years versus37.9 per 10,000menagedover 65) andwomen(9.0per10,000womenaged20–64yearsversus 26.1casesper10,000womenagedover65).

Twohundredandeight(55%)confirmedcasesalsoreported increaseddiseasesusceptibilitymostcommonlydueto smok-ingandchroniccardiorespiratorydisease.

Sourceidentification

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde Fig.2–MapshowingtheattackrateofLegionnaires’diseasebyplaceofresidence(parish),VilaFrancadeXira,Portugal.

Threeoutofthefourpotentialsourcesreportedsuspected casesamongemployees,ninecaseswereconfirmed.

Microbiologicalandphenotypicanalysisandgenetic characterization

The reference laboratory received 95 environmental sam-ples from 49 possible sources, of which, L. pneumophila

positive cultures were isolated in 8. These samples

originated from four industries with wet cooling sys-tems and one commercial premises. Six of the 8 positive cultures were L. pneumophila sg 2–15 (75%). L. pneumophila

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde

152 samples for further analysis including serology, respiratory secretions and isolated strains

130 serology/ respiratory/ bronchial secretions

22 strains isolated after culture in hospital laboratories

98 positive for Legionella pneumophila SG1

72 samples positive on PCR

14 samples positive on culture 12 samples

serology unique titre positive

108 samples underwent SBT analysis

75 samples complete SBT characterization

33 samples incomplete SBT characterization

1 sample ST23 1 sample ST62 1 sample ST94 1 sample ST1967

71 samples ST1905

≥4 alleles characterized 15 samples ST1905

≤4 alleles characterized 18 samples

10 isolates underwent Whole

Genome Sequencing (WGS)

Fig.3–Microbiolologicalprocessingandresultsofclinicalspecimens.

Laboratoryconfirmationofcaseswasdoneby;urinary anti-gentestingforL.pneumophilasg1(92%);seroconversion(2%); PCR(3%) and uniquetiter toL.pneumophila (3%). 152 clini-calspecimensweresenttothereferencelaboratory.Ofthese, 71revealedthepresenceofL.pneumophilasg1withidentical ST1905SBTprofile.

37isolatesofL.pneumophila sg1underwent phenotypic characterization,35 isolatesfrom clinical samplesand two from environmentalsamples. All 37 isolatespresentedthe same phenotypic characteristic; subtype France/Allentown andpossessedthevirulence-associatedepitoperecognizedby monoclonalantibodyMAb3/1(Fig.3).

To confirm the genetic relatedness of ST1905 isolates, 10 out of the 71 clinical isolates and one environmental positive isolateobtained from the industrial coolingtower werenominallyselectedforwholegenomesequencing.We were able to use about 99.8% of each draft sequence and foundnonucleotidedifferenceswithinthecompared3.47Mb ofthe genome. Phylogeneticanalysis involving multiple L. pneumophilaisolatesfromacrosstheworldrevealedthatthe ST1905 cluster clearly diverged from the branch enrolling themoststudiedL.pneumophilastrains(serogroups1,6and

12)17–19 _{and was more closely related with L. pneumophila} subsp.fraseristrains(serogroup4,5and15).20_WGS confirma-tionoftheST1905allelicprofilehighlightedabiasassociated with the in silico extraction of the allele sequence for themompSlocus.Thestudiedstraindisplayednon-matching

mompScopies,whichhamperedaproperSTattributionifSBT wasexclusivelydeterminedinsilico(Fig.4).

Meteorological,climate,andairqualityresults

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde Fig.4–Phylogenetictreecomparingthewholegenomesequenceofenvironmentalandclinicalisolates.

Periodofexposureassessment

Thebestfittingback-calculationmodelssuggestedalogistic growthoftheenvironmentalexposure,withgreatestintensity between16Octoberand4November(Fig.1).

Sourceproximityanalysisresults

When the source proximityanalysis was conducted, there were 365 confirmed cases, of which 328 (90%) were geo-referencedtowithin10kmofoneofthefourpotentialsources (Fig.5).

All four potential sources demonstrated a significant declineindisease riskwithincreasingdistance(clustering) intheSWQuadrant.Thehighestclustering(ST157,p<0.001) wasobservedintheSWquadrant ofindustry 1.Clustering wasalsoobservedwithindustry2(ST117,p<0.001),industry 3(ST126,p<0.001)andindustry4(ST66.1,p<0.001).Therisk ofdiseasefellconsistentlywithincreasingdistancefromthe sourcewithnoevidenceofpeaksortroughsinthisquadrant. NoclusteringwasobservedintheSEquadrantsofany indus-try,anareathatlargelycomprisedoftheriver.Industry1and2 demonstratedalowerdegreeofclusteringintheNEquadrant (ST5,p=0.01andST4.2,p=0.01respectively).Clusteringwas alsodemonstratedintheNWquadrantofindustry1(ST3.9,

p=0.01).

Discussion

We identified four industries with wet cooling system as the most likely sources of the second largest outbreak of Legionnaires’diseaserecordedwith403casesand14deaths. ElevatedconcentrationsofL.pneumophilasg1ST1905 demon-stratingthevirulentMAb3–1subgroupwereidentifiedinthe towers ofoneindustry. UsingSBT andWGS, wewere able toconfirmthegeneticrelatednessoftheoutbreak-associated clinicalandenvironmentalstrains.Theonlypositiveisolates forL.pneumophilasg1,werebothobtainedfromthecooling towerofoneindustry.SimilarconcentrationsofL.pneumophila

havebeen isolated inother coolingtowersassociatedwith outbreaks.5_{Itisprobable,thoughunproventhat cross} con-tamination of the four closely located towers contributed tothe magnitudeofthe outbreakand haspreviously been described.21

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde Fig.5–Legionnaires’diseasenearfourindustrieswithwetcoolingsystemsbydistanceofresidencefromthetower(s).

Theoutbreakoccurredinadenselypopulatedareacloseto thecapitalcityofPortugalcontainingheavilyusedcommuter routesfrom suburbanareas.Theactualnumbers ofpeople exposedwaslikelytohavebeenmuchhigherthanthe resi-dentpopulation.Standarddiagnostictestsmaynotdetectall cases.3_{ThefigurespresenteddonotcontaincasesofPontiac} feverorsuspectedcaseswho testednegativeandtherefore probablyunderestimates the morbidityexperienced bythe

Pleasecitethisarticleinpressas:GeorgeF,etal.AlargeoutbreakofLegionnaires’DiseaseinanindustrialtowninPortugal.RevPortSaúde (LpPhiladelphia-1,LpParis,LpLens,LpCorby,andLp2300/99

Alcoy).Itwas foundtobeclosely related toL.pneumophila

subsp. fraseri strainsfrom serogroupsother than sg1. The studiedstrain harbors an exclusive ∼38kb genomic region comparedwiththenextmostphylogeneticallyrelatedstrain. Thisregionwasfoundtobeintactandhighlysimilar(BLASTn, cover100%,identity99%,E-value0.0)onlyinonestrain(ATCC 33761=DSM 21215) of L. oakridgensis, a species that rarely causesLegionnaires’disease.25_{Wewouldexplainthelowcase} fatalityobservedin terms ofearlydiagnosis and appropri-ateand timelytreatment, whetherthe uniquetraitsofthe outbreak-associatedstrainunderlieimportantcharacteristics oflethalityremainsunderstudy.

Thesourceproximityanalysisestimatedexpected num-bers of cases from the resident population, which likely led to an underestimation of exposure. We reasoned that residentialinformationwas collectedsystematically,unlike displacementhistory,alsoconditionsforthehorizontal prop-agation of aerosols were more pronounced at night time when most residents would have been in their homes. Amorerobustapproachwould havetoperformthe analy-sisaspartofacasecontrolstudy,whichcouldalsoinclude exposuresinnonresidents.Althoughinitiallydiscussed,we didnotproceed withacase–control study due toresource and time constraints. The close geographical proximity of the towers meant that there was overlap of some of the studyareasrenderingitpossible,butunlikelythataclosely locatedsourcecouldleadtopresenceofadistancedecline relationshipforanothersource.Comparingthegeospatial dis-tributionofcaseswithplumemodelingofaerosolcouldbeof valuetoassesstheimpactofcloselylocatedsourcesoneach other.

Thisoutbreakwasthesecondlargestoutbreakof Legion-naires’ disease recorded to date and adds to the body of evidence regarding the impact of climatic conditions and bacterialphenotypesincoolingtowerassociatedcommunity outbreaksofLegionnaires’disease.

Conflicts

of

interest

Theauthorshavenoconflictsofinteresttodeclare.

Acknowledgements

OrganizationsandindividualsinvolvedintheTaskForcefor theLegionnaires’DiseaseoutbreakinVilaFrancadeXira;staff fromHospitaldeVilaFrancadeXira,HospitalCUF Descober-tas,CentroHospitalar deLisboa Central,CentroHospitalar deLisboaNorte;CentroHospitalardeLisboaOcidental;staff of the health authorities ofLisbon and Tagus Valley, cen-tral Portugal,northPortugal and the Algarve. Dr.Christian Lucke,TecnischeUniversitatDresden,forsupplyingtheMAbs reagents.MarionMuelhen,andPaulaVasconcelosforcritical review of the manuscript. Emmanuel Robesyn for techni-caladviceongeospatialanalysistechniquesinLegionnaires’ disease.

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