Respiratory infections in elderly people

Respiratory

infections

in

elderly

people:

Viral

role

in

a

resident

population

of

elderly

care

centers

in

Lisbon,

winter

2013

–2014

Maria-Jesus

Chasqueira

*

,

Paulo

Paixão

,

Maria-Lúcia

Rodrigues

,

Cátia

Piedade

,

Iolanda

Caires

,

Teresa

Palmeiro

,

Maria-Amalia

Botelho

,

Madalena

Santos

,

Martin

Curran

,

Raquel

Guiomar

,

Pedro

Pechirra

,

Inês

Costa

,

Ana

Papoila

,

Marta

Alves

,

Nuno

Neuparth

a

NOVAMedicalSchool-FaculdadedeCiênciasMédicas,CampoMártiresdaPátria,130,1169-056Lisboa,Portugal

b

CentrodeEstudosdeDoençasCrónicas,CEDOC,NOVAMedicalSchool-FaculdadedeCiênciasMédicas,CEDOCI,RuadoInstitutoBacteriológico,n.
_s5,5-Ae

5-B,1150-190Lisboa,Portugal

c

HospitalCurryCabral,CentroHospitalardeLisboaCentral,RuadaBeneficêncian.
8,1069-166Lisboa,Portugal

d

ClinicalMicrobiologyandPublicHealthLaboratory,Addenbrooke’sHospital,HillsRd,CambridgeCB20QQ,UK

e_{LaboratórioNacionaldeReferênciaparaoVírusdaGripe,InstitutoNacionaldeSaúdeRicardoJorge,Av.PadreCruz,1600Lisboa,Portugal} f_{EpidemiologyandStatistics,ResearchUnit,CentroHospitalardeLisboaCentral,EPE,RuaJoséAntónioSerrano,1150-199Lisboa,Portugal} g

CEAUL,DepartamentodeBioestatísticaeInformática,NOVAMedicalSchool-FaculdadedeCiênciasMédicas,CEDOCI,RuadoInstitutoBacteriológico,n.
_s5,

5-Ae5-B,1150-190Lisboa,Portugal

ARTICLE INFO

Articlehistory: Received25July2017

Receivedinrevisedform15January2018 Accepted17January2018

CorrespondingEditor:EskildPetersen, Aar-hus,Denmark. Keywords: Elderly Respiratoryinfections Respiratoryviruses Legionellapneumophila Elderlycarecenters RealtimePCR

ABSTRACT

Objective:Theaimofthisstudywastoanalyzetheetiologyandclinicalconsequencesofviralrespiratory infectionsin18elderlycarecenters(ECC)inLisbon,whichhousedatotalof1022residents. Methods:Nasopharyngealswabswerecollectedwheneveranelderlyhadsymptomsofacuterespiratory infections(ARI).PCRandRT-PCRwereperformedforinfluenzaA/B,humanparainfluenzavirus1–4, adenovirus,humanmetapneumovirus(HMPV),respiratorysyncytialvirus(RSV),rhinovirus,enterovirus, humancoronavirusandhumanBocavirus(HBoV).Arraycardsforatypicalbacteriawerealsousedin severecases.

Results:In total,188episodesofARIwerereported,beingrhinovirusthemostfrequentlydetected (n=53),followedbyinfluenzaA(H3)(n=19)andHBoV(n=14).Severeinfectionswerereportedin19 patients,11ofwhichwerefatal,Legionelapneumophila,rhinovirus,HMPVandRSVassociatedwiththese fatalities.Nineinfluenzastrainswereanalyzed,allantigenicallydissimilarfromvaccinestrain2013/14. “Age”,“HMPV”and“Respiratorydisease”showedanassociationwithsevereinfection.

Conclusions:Inthisstudyanetiologicagentcouldbefoundin60%oftheacuterespiratoryepisodes.These dataprovidesinformationaboutthecirculatingvirusesinECCandhighlightstheimportanceofsearching bothvirusesandatypicalbacteriainsevereARI.

©2018TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(

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

Introduction

Ageingisanaturalprocessthatimpairsseveralphysiological systems,includingtheimmune systemand resultsin increased

susceptibilitytoinfectious,autoimmuneandneoplasticdiseases. Thenumberofelderlyisexpectedtoincreasefrom901millionto an estimated 1.4 billion between 2015 and 2030 (UN, 2015). Therefore, emphasis on the overall quality of life of the older

* Correspondingauthorat:NOVAMedicalSchool/FaculdadedeCiênciasMédicas,UniversidadeNovadeLisboa,CampodosMártiresdaPátria,130,1169-056Lisboa, Portugal.

E-mailaddresses:mjchasqueira@nms.unl.pt(M.-J.Chasqueira),paulo.paixao@nms.unl.pt(P.Paixão),lucia.rodrigues@nms.unl.pt(M.-L.Rodrigues),

catia.piedade@labco.eu(C.Piedade),iolanda.caires@nms.unl.pt(I.Caires),teresapalmeiro@hotmail.com(T.Palmeiro),amalia.botelho@nms.unl.pt(M.-A.Botelho),

maria.santos22@chlc.min-saude.pt(M.Santos),martin.curran@addenbrookes.nhs.uk(M.Curran),raquel.guiomar@insa.min-saude.pt(R.Guiomar),

Pedro.Pechirra@insa.min-saude.pt(P.Pechirra),ines.costa@insa.min-saude.pt(I.Costa),ana.papoila@nms.unl.pt(A.Papoila),marta.l.alves@gmail.com(M.Alves),

nuno.neuparth@nms.unl.pt(N.Neuparth).

https://doi.org/10.1016/j.ijid.2018.01.012

1201-9712/©2018TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

personrepresentsamajorpublichealthchallengeandisnowa globalissue.

Pneumoniaisacommonillnessthatcontinuestobeamajor cause of death in young childrenin developing countries and elderlypeopleindevelopedcountries.About200millioncasesof viralcommunity-acquiredpneumonia occureveryyear, equally distributedbetweenchildrenandadults,withtheelderlybeingthe mostaffectedgroupamongtheadultpopulation(Ruuskanenetal., 2011).However,despitethepotentialseverityofviralrespiratory infections(VRI),thespectrumofclinicalpresentationsinelderly patientsis extremelybroad, andmany ofthem haveonly mild upperrespiratoryinfections(Nicholsonetal.,1997).

In Portugal, 65.3% of hospital admissions for respiratory diseases in 2013 were due to bacterial or viral pneumonias (DGS,2014).

ThelistofvirusesresponsibleforARIinelderlypeopleincludes influenzaAandBvirus,adenovirus,RSV,rhinovirus,enterovirus, humancoronavirus(HCoVs), HMPV,humanparainfluenza virus (HPIVs),HBoV and varicella,among otherlesscommon viruses (Ruuskanen et al., 2011). However, the attribution of the pneumoniatovirusescansometimesbedifficult,becauseviruses maybefoundinrespiratorysecretionsduetoasymptomaticviral shedding, and co-infection with two organisms may occur (Cesario,2012).

Thecurrentarticlereports themicrobiologyresultsfromthe GERIAProject“GeriatricStudyonHealthEffectsofAirQualityin ECC” conducted in Portugal, the country with the 5th oldest populationintheworld(UN,2015)andthe4tholdestinEurope, with20%ofthepopulationolderthan65years(Eurostat,2016). Resultsofchronicrespiratorydiseasesandqualityoflifefromthis projecthavealreadybeenpublished(Carreiro-Martinsetal.,2016). Theteaminvolvedwasthesameofapreviousonethatstudiedthe health impact of indoor air environment in Children Day Care Centers(ENVIRH)(Araújo-Martinsetal.,2014;Paixãoetal.,2014). Here,weaimedtostudytheviralroleinARIinresidentsofECCof Lisbon,duringthe2013/14winter.Inaddition,inordertobetter clarify the role of the respiratory viruses in severe infections, atypicalbacteriawerealsosearchedinasubsetofpatients.

Methods Studydesign

Withinthescopeofthisstudy,33ECCwereselectedthrough proportionalstratifiedrandomsampling(byparish)fromthe95 includedinthePortugueseSocialCharterofLisbon.Ofthese33,18 agreedtoparticipate.

Considering theparticular characteristicsof thesurrounding environment,these18ECCwereclassifiedasurbanandhouseda totalof1022residents,witharangeof5to334occupants.The majorityoftheseelderlyspendalloftheirtimeindoors.

Beforestartingthestudy,opensessionswereperformedinall ECC,conductedbyavirologistoftheGERIAteam.Intheseopen sessions,abriefreviewoftheimportanceoftheARIinelderlywas

providedtothelocalstaff,alongwithadescriptionoftheGERIA project.

Participants’ epidemiological data (sociodemographic and clinicalinformationincludingdataonunderlyingconditionsand co-morbidities)werecollectedbyaninterviewerwithadedicated questionnaire, which included the burden of obstructive lung diseasequestionnaire(BOLD).Clinicalfileswerereviewedusing InternationalClassificationofPrimaryCare,2ndedition(ICPC-2). Theseinterviewswereperformedbyhealthtechnicianswhowere trainedandcertifiedbytheGERIAteam.

TheviralstudyincludedphonecontactbytheECCstafftothe researchteameachtimeanelderlypersonhadsymptomsofARI (ECDCcriteria):suddenonsetofsymptoms,atleastonerespiratory symptom(cough,sorethroat,shortnessofbreathandcoryza),and aclinician’sjudgementthattheillnesswasduetoaninfection(OJ, 2012).

SamplecollectionswereperformedbetweenNovember2013 andApril2014attheECC,bymembersoftheresearchteamwithin thefirst48hafterthephonecall.Twoswabswerecollectedfrom each patient (nasopharyngeal and oropharyngeal)and immedi-ately pooled into viral transport medium (Vircell's Transport Mediumforvirus,ChlamydiaandMycoplasma).

The clinical outcome of each patient was assessed by completing a structured questionnaire, two weeks after the specimencollection,conductedforaphonecalltotherespective ECCstaff.Inthisfollow-upquestionnaire,infectionswereclassified as severe if there was a worsening in the initial symptoms, resultinginhospitalizationand/ordeath.

Inordertoencourageparticipation,everyweektheresearch teamcalledtheECCthatdidnotreportanycases.

The study was approved by the Ethics Committee of Nova MedicalSchool(nr.08/2013/CEFCM)andNationalDataProtection Commission (nr. 1279/2013). The participants, or their legal representatives, were informed aboutthe study objectives and their signed consents were obtained previously tothe sample collection.

Laboratorymethods

Viralnucleic acidwasextractedusing theQIAamp MinElute VirusSpinKit(Qiagen),accordingtothemanufacturer’s instruc-tions.Astartingvolumeof200

m

m

m

m

PCR for respiratory viruseswas performed using “inhouse” real-timeTaqmanPCRandRT-PCR.Briefly,fourpanelswereused,a pentaplex andthree additional multiplex real-timePCR assays: panel1(influenzaA,A(H1),A(H3),B,internalcontrol),panel2(RSV A/B, HPIVs types 1/3, adenovirus), panel 3 (HPIVs types 2/4, enterovirus, rhinovirus, HMPV),panel4 (HCoVs group1 (229E, NL63),HCoVsgroup2(OC43,HKU1))(EllisandCurran,2011;Clark et al., 2014, 2015). SuperscriptTM_{III Platinum one-step enzyme}

(Invitrogen) was used for the reverse transcription. All assays sharedidenticalampli_ficationconditions(50
_{Cfor30min,95
C}

Table1

PrimersandprobeusedforBocavirusreal-timePCR.

Target Sequence(50_!30_{) Function Productsize Primersource}

HBoV CACKCCCAGGAARTGACGTAT Forwardprimer 118bp Thisstudy

30non-codingregion CCAGAGATGTTCACTCGCCGGA Reverseprimer TCAGACTGCATCCGGTCTa _Probe

Platinum1QuantitativePCRSuperMix-UDGInvitrogenenzyme(Cat.No.11730-005)wasusedforPCRamplificationoftheabovetargetsinamonoplexformatinafinal25ul volumereaction(containing20ulofmastermixand5ulofnucleicacidextract)with3mMMgCl2,400nMprimersand120nMprobeconcentration.

Cyclingconditionswere50
_{Cfor2min,95
Cfor2min,40cyclesat95
Cfor15sand60
Cfor1minandacquiringonFAMforbothassays.} a

for2min,45cyclesat95
Cfor15s,and60
Cfor1min),allowing allfourpanelstorunsimultaneouslyonthesameinstrument.An additionalmonoplexassayspecificforHBoVwasalsoincludedin thisstudy(Table1).AmplificationanddetectionofPCRproducts were performed using the RotorgeneTM PCR system (Qiagen) acquiringthefluorescenceonFAM,JOE,CY5,andROXchannelsat eachcycle.Samplesandnegativecontrol(moleculargradewater) wereindividuallyspikedwithMS2bacteriophageinternalcontrol (4,600pfu per extraction) prior to nucleic acid extraction to identifyanyinhibitors.Positivecontrolsforallpanelsweremade froma combination of recombinant plasmids and from known positivespecimens,dilutedtogiveacyclethresholdvalueof20_– 25.

Inasecondphaseofthestudy,“inhouse”TaqManarraycards (Steenselsetal.,2015)wereusedfor severalrespiratoryviruses and atypical bacteria in severe cases. The cards included adenovirus,HBoV,influenzaA(H1,H3,H7,H9)andB,HPIVs1/4, RSV,enterovirus,rhinovirus,HMPV,HCoVs,Legionellapneumophila (L.pneumophila),Mycoplasmapneumoniae,Chlamydophila pneumo-niae and Coxiellaburnetii. To confirm the L. pneumophila cards results,an“inhouse”real-timePCRwas alsoused(Weltietal., 2003).

InfluenzapositivesamplesweresenttotheNationalInfluenza ReferenceLaboratoryforvirusisolationandcharacterization.Virus isolation was performed in MDCK-Siat1 cell line (WHO, 2011). Antigeniccharacterizationwasaccomplishedbyhemagglutination inhibition assay (HIA) using reference viruses and monoclonal antiserums gently provided by WHO (2014) collaborating for Europeanregion.HIAwas performedwithguineapigredblood cells.Isolatedviruseswerecomparedwith2013/14and2014/15A (H3) vaccine strains, genetic groups reference strains and circulatingviruses(ECDC,2014,2015),geneticanalysiswasbased ontheHA1subunitoftheviralhemagglutiningene.Nucleotide sequenceassembly was carried outusing theSeqmanprogram (Lasergene99package,DNASTAR)andformultiplealignmentsof nucleotideanddeducedaminoacidsequencestheMEGASoftware 6.0.Module(Tamuraetal.,2013).

Statisticalanalysis

Categoricaldatawerepresentedasfrequencies(percentages), andcontinuous variablesas meanand standard deviation(SD). ProportionswerecomparedusingFisher’sexacttest.Tostudythe association between severe infection and socio-demographic characteristics, clinical information and co-morbidities, logistic regression models were used. The variables attaining a p-value<0.25intheunivariableanalysiswereselectedascandidates forthemultivariablemodel.A levelofsignificance

a

Results

BetweenNovember2013andApril2014,188episodesofARI from163patientswerereportedtotheresearchteam(49menand 114women),withameanageof83years(SD=9.2;range:59–101) for men and 85 years (SD=7.2; range: 62–101) for women (Table 2). These patients were from 14 institutions, as four institutionsdidnotreportanyepisode(Table3).

Overall, there was a significant difference between ECC (p<0.001) concerning the ratio of the number of episodes to thenumberofresidents.

OnehundredandthirteenepisodeswerepositivebyPCRand/or arrayforatleastonerespiratoryvirus,co-infectionswithtwoand three viruses were detected in multiple episodes (Table 4). Rhinoviruswasdetectedin53samples,followedbyinfluenzaA (H3),HBoV,HCoVsgroup1,HMPV,RSV,HCoVsgroup2,andHPIVs types 1/3, respectively with 26, 19,14,11, 5, 3 and 1 positive samples(Table3).

Regardingtheseverityof theinfections, mostwereclinically mild, although some degree of dyspnea was reported in 50 episodesatthetimeofcollection.Theclinicalsituation deterio-ratedin19(11.7%)patients(severeinfection:hospitalizationand/ or death).Of these,14hospitalizations werereported, and6 of them died. Five additional deaths in non-hospitalized patients werereported,givingatotalof11(6.7%)patientsdyingwithARI duringthisstudy.

Allofthesamplesfromthesepatientswithsevereinfections werelatertestedbythearraycardtechniqueandbyamonoplex PCRtechniqueforL.pneumophila.Resultsofthesetwoapproaches inseverepatientsarepresentedinTable5.

Tostudytheassociationbetweensevereinfectionand socio-demographiccharacteristics,clinicalinformationand co-morbid-ities, a univariable analysiswas performed(see Supplementary TableS1).

Inthefinalmultivariablemodel,variables“age”,“HMPV”and “Respiratorydisease”showedanassociationwithsevereinfection. Foreach5yearsincreaseinagetherewasa49%increaseinthe oddsofsevereinfection(OR=1.49;95%CI:1.02–2.17;p=0.042). TheresidentsinfectedwithHMPVhadaseven-foldincreaseinthe oddsofsevereinfection(OR=6.99;95%CI:1.96–24.95;p=0.003). Also,theoddsofsevereinfectionwasfourtimeshigherinresidents with “Respiratory disease” (OR=4.09; 95% CI: 1.51–11.06; p=0.006).

Informationaboutvaccinationstatusforinfluenzaviruseswas obtained from 159 patients (159 from 163 with reported infections). The rates of influenza A(H3) infections among vaccinated and unvaccinated patients were quite similar (p=0.682)(Table6).Moreover,influenzawasnotassociatedwith seriousinfections(p=0.215andp=0.357forhospitalizationand death,respectively).

Table2

Distributionofglobalattackbyagegroupsandgender.

Agegroups Gender Totala

Male Female

Positivecases Negativecases Positivecases Negativecases

<65 1 1 2 0 3/4 65<75 2 1 5 1 7/9 75<85 15 7 27 16 42/65 85<95 12 6 27 29 39/74 95 0 4 6 1 6/11 TOTAL 30 19 67 47 97/163 a

Concerning the geneticcharacterization of in_fluenza strains, typingwasobtainedonlyfor9strainsA(H3),frompatientslivingin 3differentECC(ECC1,ECC2and ECC6).Eightviruseswerefrom vaccinatedpatientsand1fromanunvaccinatedpatient.Allthe strainsbelongtothegeneticgroup3C,which includesalsothe strainA(H3)ofthevaccine2013/14season.However,they were

divided into different subgroups: samples of ECC1 (n=1) and ECC06(n=5)belongtothegeneticsubgroup3C.3andsamplesof theECC02(n=3)belongtothe3C.2(thevaccinestrainbelongsto subgroup 3C.1). Thevirus detected in theunvaccinated patient showsthesamesequenceofstrainsdetectedinvaccinatedpatients ofthesameECC(ECC02)(Figure1).

Relatingtotheaminoacidsubstitutions,9aminoacidposition changeswereidentifiedintheHA1subunitoftheinfluenzastrains fromthisstudy,whencomparedwiththestrainincludedinthe 2013/14influenzavaccine,A/Texas/50/2012.SamplesoftheECC02 share the same substitutions of amino acids present in the reference strains of the 3C.2 subgroup (N128T, N145S, V186G, P198S and F219S), which distinguishes them from the vaccine strain.Thevirusesdetectedin ECC01andECC06 share4of the substitutionsin3C.2subgroup(N145S,V186G,P198SandF219S) andacquirethreenewreplacements(N128A,R142GandL157S). Regarding viruses from ECC06, all accumulate the R261Q substitution. All these mentioned amino acid substitutions occurredin thehemagglutininantigenicsites,andhavealready beenobservedinotherstrainsthatcirculatedintheseason2013/ 14.

Discussion

ECCrepresentanincreasingformofcaringforolderpersonsin developedcountries.However,theseinstitutionscanalsoprovide the requisite ingredients for outbreaks of infectious diseases. Prolonged lengths of stay of residents with multiple chronic diseases and functional impairments, limited capacities for diagnosisandinadequateinfection-controlprograms,oftenallow outbreaks ofrespiratory viruses topropagatein thesefacilities (Strausbaughetal.,2003).

Thepresentstudyaimedtoidentifythevirusesresponsiblefor ARIinaresidentsampleofECCinthecityofLisbonduringthe winter of 2013/14. One of the strengths of this studywas the relativelylargesamplesizeofelderlypeople.Eighteenofthe95 ECCregisteredintheLisbonarea(19%ofthetotalECC)agreedto participate, resulting in a total sample size of 1022 residents. Moreover,everyresidentwhowasabletoanswerthe question-nairewasinvitedtoparticipateinthesurvey.Ontheotherhand, wecouldnotinterviewmanyresidents,astheyweretoodisabled to participate in the study. For this reason, our sample is not entirelyrepresentativeoftheelderlywhoattendedtheECC.

Table3

Numberofresidents,episodesanddistributionoftherespiratoryvirusesbyECC.

ECC Numberofresidents Numberofepisodes Rhinovirus InfluenzaA(H3) HBoV HCoVsgroup1 HMPV RSVA/B HCoVsgroup2 HPIVs1/3

1 45 34 4 11 5 5 1 1 2 148 27 8 7 3 3 1 3 21 1 1 4 51 0 5 44 1 6 44 13 7 3 3 1 7 14 2 1 8 39 1 1 1 9 33 0 10 334 76 30 1 5 5 2 1 1 1 11 16 3 2 1 12 47 0 13 48 20 6 1 1 2 14 5 1 15 38 5 1 1 16 72 3 1 3 17 9 1 18 14 0 Total 1022 188 53 26 19 14 11 5 3 1 Table4

Singleandmixedviralinfections.

Viruses Singleinfection Doubleco-infection Tripleco-infection

Rhinovirus 46 7 0 InfluenzaA(H3) 20 5 1 HBoV 8 11 0 HCoVsgroup1 7 6 1 HMPV 10 1 0 RSVA/B 3 2 0 HCoVsgroup2 1 1 1 HPIVs1/3 0 1 0 Table5

Resultsofpatientswithsevereinfections(PCRandarraycards). Patients(ECC/Ep) Hospitalization Death Pathogens

2/3 Yes Yes Negative

2/32 Yes No Rhinovirus

2/44 No Yes HMPV,L.pneumophila 2/45 No Yes HMPV,L.pneumophila 2/67 No Yes L.pneumophila

2/76 Yes Yes Negative

2/133 Yes Yes Rhinovirus 6/134 Yes No HMPV,HBoVb 10/40 No Yes Rhinovirusa

,L.pneumophila

10/60 Yes No HMPV

10/125 Yes No Negative 10/126 Yes Yes Rhinovirus 10/137 No Yes L.pneumophila 10/142 Yes No Rhinovirus 10/163 Yes Yes L.pneumophila 10/164 Yes No Negative 10/169 Yes No Negative 10/171 Yes No Negative 15/159 Yes Yes RSVa Ep:Episodenumber.

a

Detectedonlybyarraycard.

b

Anotherweaknessofourstudywasthelowstaffcompliance withtheprojectforsomeoftheECC,despiteanintensivecampaign ofinformationandcontactswiththem.Infact,fourofthemdidnot report a singleevent, which cannotbe explainedby thelower numberofresidents(ECC01andECC04aregoodexamples,with rates of 34/45 and 0/51, respectively). Therefore, any study or campaign including ECC should take into consideration the

Table6

DistributionofinfluenzaA(H3) infectionsamongvaccinatedandunvaccinated residents.

InfluenzaA(H3)positive InfluenzaAnegative

Vaccinated 20 107

Non-vaccinated 6 26

Figure1.PhylogeneticcomparisonbasedonthesimilarityofHA1subunitofinfluenzastrainsfromthisstudyandcirculating/vaccineviralstrainsin2013/14and2014/15. NucleotidesequenceswereassembledwiththeSeqmanprogram(Lasergene99package,DNASTAR).ThephylogenetictreewascreatedwithMEGAsoftwareversion6,using themaximumlikelihoodmethod,Hasegawa–Kishino–Yanonucleotidedistancesandbootstrappedwith500replicates.

variabilityofstaffcompliancebetweencenters,whichcanaffect significantlytheresultsobtained.

Onehundredandthirteenof188episodesofreportedARIwere positiveforatleastonerespiratoryvirus.Mostoftherespiratory virusessearchedforinthisprojectweredetected,confirmingthe diversityontheaetiologyofARIinelderlypatients.Infact,only adenovirus,enterovirus,andHPIVstypes2/4wereneverdetected inthisstudy,whichisinaccordancewithreportsthatsuggestthat thesevirusesarenotfrequentlydetectedinthisagegroup(Falsey and Walsh, 2006), although adenovirus can occasionally be responsiblefor severeinfections inlong-term carefacilities for theelderly(Kandeletal.,2010).

Rhinoviruses werethemostfrequentlydetected viruses,but werealsodetected in 5severe infections, 3of them withfatal outcomes(althoughoneofthesewasassociatedwithL. pneumo-phila). Therefore our results corroborate the notion that these virusescanberesponsibleforsevereARIinelderlypatients( Pop-VicasandGravenstein,2011).

Influenzaremainsaleadingcauseofinfectiousdeathamongthe elderly(Pop-VicasandGravenstein, 2011).Influenzavaccination ratesof80%ofresidentsatagiveninstitutionhavebeendescribed tobeassociatedwithadecreasedrisk ofanoutbreak,although someoutbreakscanoccurevenwhenresidentvaccinationrates are90%(Strausbaughetal.,2003).Inourstudy,thevaccination rate within the group of patients with episodes was 78% (all vaccinationswereperformedatleastonemonthbeforethefirst reportedepisode).Thevaccinationrate oftheentirepopulation couldnot bedetermined,becausetheinitialquestionnairethat was made for the 1022 residents (BOLD) did not include the questionaboutthevaccinationstatus, andthereforeonlyinthe follow-up questionnairefor thegroup of infected patients this questionwasaddressed.

InfluenzaA(H3)wasthesecondmostprevalentvirusdetected. Interestingly,A(H1)wasnotdetectedinthisstudy,althoughthis viruscirculatedinthePortuguesepopulationduringthiswinter andinaproportionequivalenttothatofA(H3)(Cristovãoetal., 2014).TheratesofinfluenzaA(H3)infectionsbetweenvaccinated andunvaccinatedresidentswerequitesimilar,suggestingalow effectiveness of the vaccine. As a possible explanation, we observedthat all analyzedstrainswere antigenically dissimilar from vaccine strain 2013/14. Interestingly, influenza cases in vaccinatedelderlywerenotassociatedwithseriousinfectionsin thisstudy,whichisinaccordancewiththeinformationfromPublic Healthauthoritiesthatvaccinationforinfluenzaisaimedmainlyto prevent complications (CDC, 2016). Nevertheless, it remains unclearifthis wastheresultofvaccineprotection, becausethe six unvaccinated residents with influenza infection did not experienceseriousillnessaswell.

As expected, the samples of the same ECC share identical sequencesofinfluenzavirus,indicatingaprobablecommonsource ofinfectionandsustainedtransmissionbetweenresidents.

In thepresentstudy, HBoVwasdetected in19episodesand rankedthirdin prevalence,in contrastwithanotherstudythat presentedvery low virusprevalence in the respiratory tractof adults(Schildgenetal.,2008).OtherreportsuggeststhatHBoV mightbeassociatedwithupperandlowerrespiratorydiseasein elderlypatients(Liuetal.,2011), althoughinourstudymostof thesedetectionswereco-infections(11/19)withotherviruses,and onlyin 8episodesHBoV was detected alone.Therefore, results suggestthatHBoVdetectionbyPCR canbeafrequenteventin elderlypatients,butthepathogenicroleofthesevirusesinmanyof theseinfectionsisstillunclear.

Among theother respiratory viruses detected in this study, HCoVsgroup1/2werecommonlydetected,particularlythefirst, butnotassociatedwithsevereinfections.HMPVwasdetectedin11 patients,with4ofthesehavingsevereinfections.However,the2

fatalities associated with this virus had mixed infection and thereforethecontributionoftheHMPVisnotclear.Nevertheless, these results suggest that HMPV can beresponsible for severe infectionsinolderpopulations,asdescribedbyothers(Pandaetal., 2014).

RSV was positive only in 5 patients, but associated with 1 fatality,beingtheonlypathogendetectedinthiscase,butwitha lateCTvalue.Noconclusionscanbedrawnduetothesmallsample size, although the associationwith severeinfections in elderly patientshasbeenwellknownforseveralyears(WalshandFalsey, 2012).

In the current study, 40% of thesamples were negativefor respiratoryviruses.FalsenegativeresultswiththePCRandRT-PCR, orinfectionsbyotherrespiratoryvirusesnotsearched,arepossible explanationsforsomeofthenegativeresults.Anotherpossibilityis thepresenceofbacterialinfections,consideringtheirimportant roleinelderlywithpneumonia.

Toconfirmthislastpossibility,patientswithsevereinfections werealsotestedforatypicalbacteria,aspreviouslydescribed.The Taqmanarrayscardsallowedustodetect2morevirusesinthese severe infections, suggesting that if they were used in all the patients,therateofviraldetectioncouldhavebeenhigherthan usingPCRalone.Butthemajorcontributionofthistechniquewas fortheatypicalbacteria:L.pneumophilawasdetectedin6patients (in 2 different ECC), and all of them subsequently died. When comparingTaqmanarraycardsandmultiplexPCRresultsforviral detection,somedifferencesweredetected(Table5), butonlyin samples with low viral load. Therefore, sensitivity of these techniques can be compromised when studying samples with lowviralorbacterialload.

L.pneumophila isa well-knowncauseof severeARIand the population in this study belongs to the group with high susceptibilityin acquiringinfectionswiththisbacterium. More-over,themortalityrate,usuallybetween8–12%,ishigherinelderly people(Phinetal.,2014).Therefore,despitethefactthat3ofthe fatalcaseshadmixedinfectionswithviruses(2withHMPVand1 withrhinovirus)thereisahighprobabilitythatthisbacteriacould have contributed to these fatalities. Nevertheless, we cannot excludethatvirusescouldhavehadanimportantcontributionas well;autopsywasnotorderedinanyofthefatalities,and post-mortem lung specimens were not collected, which could have helpedtoclarifythissituation.InfectionscausedbyL.pneumophila havebeendescribedinECCbutoftengoundetected(Seenivasan etal.,2005),mostfacilitiesdonothavethelevelofmicrobiology laboratorysupportthat is availabletoacutecarehospitals,and onlyiftheresidentistransferredtothehospitalwillhe/shehave access to different methods to detect this bacterium. Another importantpointisthefactthattheL.pneumophilainfectionrate was probably underestimated, for the following reasons: L. pneumophila was searched only in severe infections, and more casescouldhavebeenfoundifthisbacteriumweresearchedinall patients;L.pneumophilainfectionsshowaseasonalpattern,with peakactivityinlatesummertoautumn,andthisstudywasheld duringthewinter(Phinetal.,2014);and_finally,upperrespiratory samples are not the best samples for the diagnosis of these infections(Choetal.,2012).Allofthedetectedcaseswerenotified to the National surveillance scheme so that environmental research could be triggered. At the end of the study a recommendationfortheuseofaurinaryantigentestforLegionella wasmadetotheECCmedicalteamsinvolved,butalsoapublic discussionofthesefindingswasconductedbyourteam.

Interestingly, the variables “Age”, “HMPV” and “Respiratory disease”showedanassociationwithsevereinfectioninthefinal multivariablemodel.Theseassociationsarenotunexpected,since increased ageand previous respiratory disease are knownrisk factors for respiratory infections (Ruuskanen et al., 2011), and

HMPV has been associated with severe disease, as previously mentioned (Panda et al., 2014). Despite the suggestive results obtainedwithL.pneumophila,noconclusionscanbedrawnfrom this study becausethis bacteriumwas searched only in severe infections.

Conclusion

This is the first study performed in Portugal, with elderly populationsinECC,aimedtostudytheviralaetiologyoftheARI.An aetiologicalagentcouldbefoundin60%oftheacuterespiratory episodes,aratehigherthanusually reported(Ruuskanenetal., 2011).Mostoftheinfectionswereinitiallyconsideredmild,but worseningwas observedin asignificantproportion ofpatients. “Age”, “HMPV” and “Respiratory disease” wereassociates with severeinfection.Thedatahighlighttheimportanceofsearching bothvirusesandatypicalbacteriainsevereARIinelderlypatients, inparticularL.pneumophila,whichwasassociatedwith6fatalities inthe studiedpopulation. Thestudyalsoprovided information aboutthecirculatinginfluenzavirusesinthisseason.

Financialsupport

This work was supported by Foundation for Science and Technology (Fundação paraa Ciência eTecnologia –FCT, grant numberPTDC/SAU-SAP/116563/2010)throughOperational Com-petitiveness Programme (COMPETE) as part of the National StrategicReferenceFramework.

Conflictofinterest None.

Ethicalapproval

The study was approved by the Ethics Committee of Nova MedicalSchool,LisbonandtheNationalDataProtection Commis-sion.

Acknowledgements

Theauthorsexpresstheirdeepestgratitudetoalltheelderly residentsfortheirimportantcontribution.Wealsothankallthe otherECCstaffandauthoritiesinvolvedinthestudy.

AppendixA.Supplementarydata

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