Frequency of viral etiology in symptomatic adult upper respiratory tract infections

The

Brazilian

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INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Original

article

Frequency

of

viral

etiology

in

symptomatic

adult

upper

respiratory

tract

infections

Raquel

Cirlene

da

Silva

a

_,

_Gabriella

_da

_Silva

_Mendes

a

_,

_Miguel

_Angel

_Rojas

a

_,

Ariane

Ribeiro

Amorim

a

_,

_José

_Nelson

_Couceiro

a

_,

_Omar

_Lupi

b

_,

_José

_Elabras

b

_,

Gisele

Pires

b

_,

_Solange

_Valle

b

_,

_Norma

_Santos

a,∗

a_{InstitutodeMicrobiologiaPaulodeGóes,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil} b_{FaculdadedeMedicina,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil}

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Articlehistory:

Received12July2014 Accepted21August2014 Availableonline13October2014

Keywords: Respiratoryinfection Viralinfection Viraldiagnosis Epidemiology

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Aims:Todeterminethefrequencyofviralpathogenscausingupperrespiratorytract infec-tionsinnon-hospitalized,symptomaticadultsinthecityofRiodeJaneiro.

Methods:Respiratorysamples(nasal/throatswabs)werecollectedbetweenAugust2010and November2012andrealtimePCRwasusedtodetectdifferentviralpathogens.

Results:Virusesweredetectedin32.1%(43/134)ofsamplesfrom101patients.Specifically, 9%(12/134)werepositiveforHBoV,8.2%(11/134)werepositiveforHAdV,5.2%(7/134)were positiveforHRV,and1.5%(2/134)werepositiveforFLUBVorHMPV,assingleinfections. HRSV-A,HPIV-3,andHCoV-HKU1weredetectedinone(0.75%)sampleeach.Co-infections weredetectedin4.8%(6/134)ofthesamples.Peaksofviralinfectionswereobservedin March,April,May,August,andOctober.However,positivesamplesweredetectedallyear round.Only23.3%(10/43)ofthepositivesampleswerecollectedfrompatientswithfebrile illness.

Conclusion:Resultspresentedin thisreportsuggestthatrespiratory viralinfections are largelyunderdiagnosedinimmunocompetentadults.Althoughthemajorityofyoungadult infectionsarenotlife-threateningtheymayimposeasignificantburden,especiallyin devel-opingcountriessincetheseindividualsrepresentalargefractionoftheworkingforce.

©2014ElsevierEditoraLtda.Allrightsreserved.

Introduction

Acute respiratory infections represent a significant mor-bidity and mortality burden worldwide and are caused

∗ _{Correspondingauthorat:DepartamentodeVirologia,InstitutodeMicrobiologiaPaulodeGóes,UniversidadeFederaldoRiodeJaneiro,}

CCS–Bl.I–CidadeUniversitária,IlhadoFundão,RiodeJaneiro,RJ21.941-972,Brazil. E-mailaddresses:nsantos@micro.ufrj.br,nsosantos@gmail.com(N.Santos).

primarilybyviralinfections.1–3_{Thehighmorbidityand}

mor-tality rates due to respiratory viruses have made these infections a global health concern. Since 1977, the World HealthOrganizationhasadvocatedthesurveillanceofacute viralrespiratorydiseaseprogramsworldwide.4_However,the

http://dx.doi.org/10.1016/j.bjid.2014.08.005

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majority ofthe data collected regarding infections in chil-dren and the epidemiology of community-acquired viral respiratoryinfections amongadultsisinsufficient,perhaps with the exception of infections caused by the influenza virus. Most data available focuses on specific populations such as immunosuppressed,5–9 _homeless,10,11 _{or elderly}

individuals.5,12,13

Virusesaretheleadingcausesofacuterespiratorydisease throughouttheworld.Causativeagentsofrespiratorydisease inhumansincludehumanrespiratorysyncytialvirus(HRSV), humanparainfluenzavirus(HPIV),influenzaAvirus(FLUAV) and influenza B virus (FLUBV), human adenovirus (HAdV), humancoronavirus(HCoV),humanrhinovirus(HRV),human metapneumovirus(HMPV),andhumanbocavirus(HBoV).1–3

Inaddition, twohumanpolyomaviruses(HPyV), KIPyVand WUPyV, have been detected in patients with respiratory infections.1,2

In2000,theBrazilianMinistryofHealthestablisheda coun-trywidesurveillancesystem forrespiratoryviral infections. Thissystemcomprisesanetworkofsentinelunitsthatinclude outpatientclinics,emergencycaredepartments,andgeneral hospitalsthatissueaweeklyreport(usinganonlinesystem) informingthetotalnumberofvisitsandthenumberofvisits associatedwithinfluenza-likeillness(ILI)(definedasacase offeveraccompaniedbycoughorsorethroatwithnoother diagnosis).14,15_{Thesystemisdesignedtodetectonlyasmall}

numberofvirusspeciesincludingFLUAV,FLUBV,HRSV,HAdV, andHPIV-1,-2,and-3.14_{Infectionscausedbyotherimportant}

viralpathogenssuchasHPMV,HRV,HBoV,andHCoVarenot monitored.

Theaimofthisstudywastodeterminethefrequencyand typeofupperrespiratoryviralinfectionsinnon-hospitalized adultsinthecityofRiodeJaneiro,Braziloveraperiodoftwo yearsusingmoleculardiagnosticmethods.

Patients

and

methods

Immunocompetent patients (n=134) who attended the ImmunologyServiceoftheHospitalUniversitárioClementino FragaFilho(HUCFF)/FederalUniversityofRiodeJaneiro(UFRJ) betweenAugust2010and November2012(medianage50.5 years;rangingfrom19to80years)wereenrolledinthepresent study.Theagedistributionofthesubjectswasasfollows:six were19–24yearsold,21were25–35yearsold,17were36–45 yearsold,33were46–55yearsold,37were56–65yearsold,and 20were>65yearsold(Table1).

Respiratory samples(nasal/throat swabs) were obtained fromallparticipants.Followingcollectionswabswereplaced intoviraltransportmediaandstoredat−70◦_{Cuntilprocessed.} Relevantclinical information including age, sex, and clini-calsymptomswerecollectedduringthemedicalvisitusing astandardizedquestionnaire.Respiratoryillnesswasdefined bythepresenceofrhinorrheaand/orcoughand/orrespiratory distressand/orsorethroat,withorwithoutfever.

Nucleicacid was extracted from 200␮L ofthe collected samples using the Wizard Genomic DNA Purification KIT (Promega,Madison, WI)and the Totally RNA® _Kit(Applied Biosystems/Ambion, Grand Island, NY) according to the manufacturer’sinstructions. Specimensweretestedforthe

presenceofFLUAV andFLUBV,16 _{HRSV(variantAand B),}17

HPIVspecies1–4,18_{HRVspeciesAandB(HRV-A/B),}19_HMPV,20

HAdV,21_{HBoVspecies1–4,}22,23_{WUPyVandKIPyV,}24_andHCoV

speciesOC43,229E,NL63,andHKU125_{byrealtimePCRassays.}

Genomic RNAsofFLUV,HRSV,HPIV,HRV,HMPVand HCoV were subjected to onecycle ofreverse transcription (5min at25◦Cfollowed by45minat42◦C)using 50pmol random primer(hexamerpd[N]6,LifeTechnologies,Carlsbad,CA,USA) inaVeriti96well(AppliedBiosystems,FosterCity,CA,USA) thermocycler,priortoPCRamplification.Real-timePCR ampli-ficationswereperformedforeachvirusseparately,inanABI StepOne Real-time PCR System (Applied Biosystems). The amplificationconditionsconsistedof10minat95◦C,followed by45cycles of10sat95◦Cand 60sat60◦C.Amplification wascarriedoutin24␮Lreactionvolumes,including5␮Lof DNA,specificprimerforeachvirusand12␮LofMaxima®_SYBR GreenqPCRMasterMix(Fermentas/ThermoFischerScientific, Canada).EachPCRassayforHMPV,HRV-A/B,HAdV,KIPyVor WUPyVusedasinglepairofprimers.Multiplexassayswere usedtodetectanddiscriminatethedistinctspeciesofFLUV, HRSV,HBoV,HIPVandHCoV.

AconventionalRT-PCRprotocolwasusedfordetectionof HRVspeciesC(HRV-C).26_{Afterthereversetranscriptionstep,}

the viralcDNA wassubjected toonestepof8minat94◦C followedby35cyclesofPCReachconsistingof45sat94◦C, 45sat60◦Cand45sat72◦C,and afinalextensionstepof 8minat72◦CinaVeriti96wellthermocycler.ThePCR prod-uctswereanalyzedby1.2%(w/v)agarosegelelectrophoresis andvisualizedbystainingwithethidiumbromide.

Positiveandnegativecontrolswereincludedineachrun. InfectedcellcultureswereusedaspositivecontrolsforHRSV (HEp-2 cells),HPIV (Verocells), HMPV(LLC-MK2 cells), HRV (MRC-5 cells) and HAdV (A549 cells). Positive controls of FLUAV and FLUBV consisted of allantoic fluid from FLUV infected embryonic eggs.Clinical samples ofHRV-C, HCoV, KIPyV,WUPyVandHBoV,confirmedbyPCRamplificationand sequencinganalysis,wereobtainedfrompatientswith respi-ratoryillnessandusedaspositivecontrols.Negativecontrols consistedofviraltransportmedium.

Results

Patients (n=101) (16 male and 85 female) presenting with respiratory illnesssymptoms were enrolled in the present study.Respiratorysamples(n=134)werecollectedand ana-lyzed for the presence of viruses. The most common symptomsobservedwerecough(88.4%),sneezing(67.4%),and nasalcongestion(58.1%).Feverwasreportedin22.4%(30/134) ofrespiratoryinfectionepisodes(Fig.1).

Patientswerestratifiedintosixagegroups(Table1).The overalldetectionfrequencyofanyviralrespiratoryinfections was33.3%(2/6)inindividuals<24yearsold,33.3%(7/21)among those25–35yearsold,41.2(7/17)amongpatients36–45years old,24.2(8/33)amongpatients46–55yearsold,35.1(13/37) amongpatients56–65yearsold,and30.0%amongpatients>65 (6/20)yearsold.Theidentificationratesofrespiratoryviruses didnotdiffersignificantlybetweenagegroups.

Co-infectionsoccurredinsix(4.5%)specimens,ofwhich five were double infections (HAdV+HBoV1; HAdV+HBoV2;

b r a z j i n f e c t d i s . 2 0 1 5; 1 9(1) :30–35

Table1–Agedistributionofpatientspresentingwithrespiratoryvirusinfections.

Agegroup(years) Numberofsubjects Numberofpositivesamples

HAdV HBoV-1 HBoV-2 HRV-A/B HRV-C FLUBV HMPV HRSV-A HCoV-HKU1 HPIV-3 Co-infections Total(%)

<24 6 1 1 2(33.3) 25–35 21 1 1 3 1 1 7(33.3) 36–45 17 2 1 1 1 1 1 7(41.2) 46–55 33 1 1 1 1 1 3(HAdV+HBoV-1; HAdV+HBoV-2, HAdV+KIPyV+ HRV-A/B) 8(24.2) 56–65 37 3 4 1 1 1 3 (HAdV+HCoV-229E; HBoV-2+HRV-A/B; HBoV-2+KIPyV) 13(35.1) >65 20 3 1 1 1 6(30.0) Total 134 11 4 8 4 3 2 2 1 1 1 6 43(32.1)

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Sore throat Watering eyes Rhinorrhea Fever Myalgia Sneezing Nasal Congestion Cough 0 20 40 60 80 100 Frequency of symptoms (%)

Fig.1–Frequencyofrespiratorysymptomsinpatientswith

acuterespiratoryviralinfections.

HAdV+HCoV-229E;HBoV2+HRV-A/B;HBoV2+KIPyV)andone tripleinfection(HAdV+HRV-A/B+KIPyV).Co-infectionswere onlydetectedamongpatientsover45yearsofageand symp-tomswerenotdifferentfromsingleinfectedindividuals.

Forty-threesamples(32.1%)collectedfrom40patientswere positiveforatleastoneoftheviralpathogensscreenedfor, namely11HAdV,12 HBoV(fourHBoV-1andeightHBoV-2), seven HRV (4HRV-A/B and three HRV-C), two HMPV posi-tive,twoFLUVBpositive,andoneHRSV-A,HCoV(HKU1),and HPIV-3.Co-infectionswiththeseviruseswereobservedinsix samplesincludingonesamplepositiveforKIPyV.FLUVA, HPIV-1,2and4,HBoV-3and4,and WUPyVwerenotdetectedin anyofthesamplesexamined(Table1).Only23.3%(10/43)of thevirus-positivesampleswerecollectedfrompatientswith febrileillness.

Oftheseven(5.52%,n=134)patientspresentingonlywith HRV (four positive for HRV-A/B and three for HRV-C) the most pronounced symptoms were cough (100%), sneezing (71.4%),nasalcongestion(57.1%),wateryeyes(57.1%), rhinor-rhea(42.9%),andfever(42.9%)(Fig.2).Symptomsweresimilar forinfectionscausedbyHRV-A/BorHRV-C.

HAdV was detected in samplesfrom 11 (8.2%) patients presentingwithasingleinfectionandinsamplesfromfour patients co-infected with HBoV-1, HBoV-2, HCoV-229E, or

HRV HAdV HBoV Sore throat Watering eyes Rhinorrhea Fever Myalgia Sneezing Nasal congestion Frequency of symptoms (%) Cough 0 20 40 60 80 100

Fig.2–FrequencyofsymptomsassociatedwithHAdV,

HRV,orHBoVinfections.

KIPyV+HRV-A/B.Themostfrequentsymptomspresentedby patientsinfectedwithHAdVwerecough(91%),sneezing(82%), nasalcongestion(54.4%),andmyalgia(36.4%)(Fig.2).

Ofthe16(11.9%)patientsinfectedwithHBoV (consider-ingbothsingleandco-infections),fivepatientswereinfected withHBoV-1(foursingleinfectionsandoneco-infectionwith HAdV)and11wereinfectedwithHBoV-2.Eightpatientshad single infectionsand three were co-infectedwithHRV-A/B, KIPyV, and HAdV. The most common symptoms observed among HBoV single-infected patients were cough (66.7%), sneezing(50%),nasalcongestion(50%),andrhinorrhea(33.3%) (Fig.2).ClinicalsymptomsweresimilarforHBoV-1andHBoV-2 infections.

FLUVBwasdetectedintwo(1.5%)patients.A45-yearold femalepresentingwithcoughandmyalgiaanda48-yearold femalepresentingwithcough,sneezing,wateringeyes,nasal congestion,andsorethroat.

Two(1.5%)HMPVinfectionsweredetected.Thefirstcase wasa69-yearoldfemalewithcough,sneezing,nasal conges-tion,andmyalgia,andthesecondwasa25-yearoldfemale withcough,rhinorrhea,nasalcongestion,andsorethroat.

HCoV-HKU1 was detected as a single infection in a 63 year-oldmalepresentingwithcoughandnasalcongestion. Co-infectionwithHCoV-229EandHAdVwasdetectedina 57-yearoldfemalewithcough,sorethroat,rhinorrhea,sneezing, nasalcongestion,andmyalgia.

A31-yearoldmalepresentingwithfever,cough,andsore throat wasinfectedwith HRSV-Aand a45-year old female patient infectedwithHPIV-3presentedwithcough, rhinor-rhea,sneezing,nasalcongestion,andasorethroat.KIPyVwas onlydetectedinco-infections(Table1).

Peaks ofviral infections wereobservedprimarilyduring March(33.3%;n=6),April(58.8%positivity;n=17),May(33.3%;

n=9),August(30.6%positivity;n=49),andOctober(41.2% pos-itivity;n=17).However,virusesweredetectedinsamplesall yearround.Therewasnosamplecollectionduringthemonths ofJanuaryandDecemberandonlyonesamplewascollected duringNovember(Fig.3).

Discussion

Thefrequencyofrespiratoryviralinfections(RVIs)in immuno-competentadultsisoftenoverlooked;however,availabledata suggestthatRVIsareresponsibleforasignificantnumberof respiratoryinfectionsaffectingnotonlytheelderly(persons aged≥65years)butalsoyoungadults.12,13,27–32_Althoughthe

majorityofinfectionsinyoungadultsarenotlife-threatening theycanimposeasignificantburdenoncommunitiessince theaffectedpopulationrepresentsalargefractionofthework forceindevelopingcountries.Data presentedinthisreport indicate that 65.1% (28/43) of virus-positive samples were detectedinpatients<60yearsofage.

InBrazil,mostoftheavailabledataonadultRVIisrestricted toimmunocompromisedor elderlypatientswithlittledata collectedonRVIspresentinginimmunocompetentadults.It hasbeenpreviouslydemonstratedthatHAdVinfectionswere detectedin8–24.1%,HPIV-3in32%,HRSVin20%,HBoVin12%, HMPVin12%,FLUAVin4%,andFLUBVin4%oftransplant patients.8,33HRVandHMPVweredetectedin28.6%and2%,

50 45 40 35 30 25 20 15 10 5 0 N umber of samples

Jan Fev Mar Abr May Jun Jul Ago Sep Oct Nov Dec

Virus positive Tested samples

Fig.3–Monthlydistributionofacuterespiratoryviral

infectionsamongadultsinRiodeJaneirobetweenAugust

2010andNovember2012.

respectively,inelderlypatients.29_{Evenso,astudyperformed}

inPortoAlegre(SouthernBrazil)betweenNovember2008and October2010reportedthat22%ofinfectionsin immunocom-petentadultpatients(medianage50.6yearsofage)admitted toemergenceroomswithILIorsevereacuterespiratory infec-tion(SARI)werecausedbyrespiratoryviruses,mostlyFLUAV and HRV-A/B. FLUVB,HAdV, HPIV, HRSV,HMPV, and HCoV werelessfrequentlydetected.34_{Inthepresentstudy,the}

over-allfrequencyofvirusdetectionwas32.1%.Thediscrepancy indetection ratescould be attributedtodifferences inthe methodologiesused,forexample,intheformerstudysome sampleswere testedusinganindirectimmunofluorescence assay(IFA)andothersamplesweretestedusingconventional PCRassayscomparedtotheuseofbothconventionalandreal timePCRassaystodetectvirusisolatesinthepresentstudy.

ThesentinelsurveillancesysteminBrazilfocuseson mon-itoring infections caused by FLUAV and FLUBV and other respiratoryvirusessuchasHAdV,HPIV,HRSV,andHRV-A/B. Samplesaresenttoandtestedinpublichealthlaboratoriesin eachstatebyIFAandallpositive,inconclusive,and10%of neg-ativesamplesareforwardedtooneofthreenationalreference laboratorieswheretheyare retestedbyPCRforrespiratory viruses.14,15_{Between2000and2010theBraziliansurveillance}

systemrecorded3,291,946casesofILI.Ofthesecases,37,120 (1%oftotalILIvisits)hadnasopharyngealsamplescollected andtestedforviralinfections.Atotalof16,962(45.7%)tested sampleswerefrompatientsover 15yearsofagewith2604 (15.4%) positive for respiratory viruses. Overall FLUAV and HPIV-3werethemostcommonvirusesdetected.However,the detectionrateofFLUAVdeclinedfrom41%to18%duringthis period.15_{Inourstudy,HAdV,HBoV,andHRVwerethemost}

commonvirusesdetected.FLUBVwasdetectedinonly1.5% (2/134)oftestedsamplesandFLUAVwasnotdetected.

It should be underscored that FLUV was not detected amongpatientsover60yearsofage.Theinfluenzavaccine inBrazilisfreelydistributedforatriskindividualsincluding

pregnantwomenofallgestationalages,womenupto45days after childbirth,childrenfrom six monthsup totwo years of age, people over 60 years of age, persons with chronic non-communicablediseases,nativeIndians,andhealthcare workers.14_{AccordingtotheMinistryofHealth,influenza}

vac-cinecoverageinBrazilbetween2010and2013reached80–88% ofthetargetpopulationandmayexplainthedeclineofFLUV detectionovertheyearsandthelowfrequencyofFLUV infec-tions found in our study. Lima et al.33 _{collected samples}

betweenAugust2011andAugust 2012anddetectedFLUAV andFLUBVinonly4%ofthesamplesusingrealtimePCR.

Acute viral respiratory disease surveillance programs around the world monitor cases of febrile ILI, therefore patients presenting with respiratory symptoms but with-outfeverareusuallynotincludedinsurveys.15,28,35,36_Inthe

presentstudyweusedabroaderdefinitionforrespiratory ill-nessthatincludedpatientswithandwithoutfever.Afebrile patients accounted for76.3% ofthe virus-positive samples identified, suggesting that respiratory virus infections are largelyundiagnosed,particularlyamongimmunocompetent adults.

Funding

ThisstudywassupportedinpartbytheConselhoNacional de Desenvolvimento Científico e Tecnológico (CNPq) grant number471063/2012-6,Coordenac¸ãodeAperfeic¸oamentode PessoaldeNívelSuperior(CAPES), andtheFundac¸ãoCarlos Chagas de Amparo àPesquisa doEstadodoRiode Janeiro (FAPERJ)grantnumberE-26/103.113/2011,Brazil.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

WewouldliketothankSoluzadosSantosGonc¸alvesfor tech-nicalassistance.

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