Potential serotype coverage of three pneumococcal conjugate vaccines against invasive pneumococcal infection in Italian children.

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ContentslistsavailableatSciVerseScienceDirect

Vaccine

j o ur na l ho me p ag e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Potential

serotype

coverage

of

three

pneumococcal

conjugate

vaccines

against

invasive

pneumococcal

infection

in

Italian

children

Chiara

Azzari

a,b,∗

,

Maria

Moriondo

a,b

,

Martina

Cortimiglia

a,b

,

Claudia

Valleriani

a,b

,

Clementina

Canessa

a,b

,

Giuseppe

Indolﬁ

a,b

,

Silvia

Ricci

a,b

,

Francesco

Nieddu

a,b

,

Maurizio

de

Martino

a,b

,

Massimo

Resti

a,b

,

the

Italian

group

for

the

study

of

Invasive

Pneumococcal

Disease

1

a_Department_of_Science_for_Women_and_Child_Health,_University_of_Florence,_Viale_Pieraccini_24,₅₀₁₃₂_Firenze,_Italy

b_Anna_Meyer_Children_Hospital,_Florence,_Viale_Pieraccini_24,₅₀₁₃₂_Firenze,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30August2011

Receivedinrevisedform9November2011

Accepted3December2011

Available online 14 December 2011 Keywords: Streptococcuspneumoniae Potentialcoverage PCV7 PCV10 PCV13 Serotype RealtimePCR

a

b

s

t

r

a

c

t

Backgroundandaimofthework:Sincetheintroductionofthe7-valentvaccine,invasivepneumococcal diseasehavegreatlydecreased;however,changesinthedistributionofpneumococcalserotypeshave recentlyhighlightedtheneedforvaccineswithwidercoverage.Theaimoftheworkwastoassessthe potentialserotypecoverageofthreepneumococcalconjugatevaccines(7-,10-and13-valent)against bacteremicpneumococcalpneumoniaandmeningitis/sepsisinItalianchildren.

Patientsandmethods:Wedeterminedpneumococcalserotypesinimmunocompetentpatientswhohad beenadmittedtohospitalwithsuspicionofinvasivebacterialdiseaseandhadconﬁrmedbacteremic pneumococcalpneumoniaormeningitis/sepsisdeterminedbymoleculardetectionofStreptococcus pneu-moniaeinanormallysterilesite.PositivesampleswereserotypedusingRealtime-PCR.

Results:BetweenApril2008andMarch2011,atotalof144patients(agemedian4.1years;Interquartile range1.8–5.6)withpneumococcalmeningitis/sepsis(n=43)orpneumonia(n=101)from83 participat-ingcenterslocatedin19of20Italianregionswereserotyped.The10mostprevalentserotypeswere1 (29.9%),3(16.0%),19A(13.2%),7F(8.3%),5(4.2%),14(4.2%),6A(3.5%),6B(3.5%),18C(3.5%),19F(3.5%). Overall,serotypecoverageforPCV-7,-10and-13wererespectively19.4%,61.8%and94.4%withno sta-tisticaldifferencebetweenpneumoniaandmeningitis/sepsis.Potentialcoveragewassimilarforchildren 0–2or2–5yearsofage.Culturesresultedpositivein35/99(35.4%)samplessimultaneouslyobtainedfor bothcultureandRT-PCR.

Conclusion:ThesefindingsindicatethatincreasingthepotentialserotypecoveragebyintroducingPCV13 inthevaccinationscheduleforinfancycouldprovidesubstantialaddedbenefitforprotectionfrom pneu-mococcalpneumoniaormeningitis/sepsisinItalyinchildrenbelow2yearsaswellinolderchildren.The importanceofmolecularmethodsfordiagnosisandserotypingofinvasivepneumococcaldiseasewas confirmed.

1. Introduction

Streptococcuspneumoniaeis theleadingcauseof community acquiredinvasivebacterialinfectionsinyoungchildrenandalso

Abbreviations: IPD,Invasivepneumococcaldisease;PCV,Pneumococcal

conju-gatevaccine;IQR,Interquartilerange;RT-PCR,RealtimePCR.

∗ Correspondingauthorat:ImmunologyDepartment,AnnaMeyerChildren

Hos-pital,VialePieraccini24,50132Firenze,Italy.Tel.:+390555662542;

fax:+390554221012.

E-mailaddresses:c.azzari@meyer.it(C.Azzari),m.moriondo@meyer.it

(M.Moriondo),m.cortimiglia@meyer.it(M.Cortimiglia),c.valleriani@meyer.it

(C.Valleriani),clecle81@hotmail.com(C.Canessa),g.indolﬁ@meyer.it(G.Indolﬁ),

silviaricci-85@hotmail.it(S.Ricci),maurizio.demartino@uniﬁ.it(M.deMartino),

m.resti@meyer.it(M.Resti).

1 _See_Appendix_A_.

causessubstantialmorbidityandmortalityinelderlypatients[1]. Morethan90serotypesexist,butonlyasubsetisassociatedwith invasivedisease[2].

Vaccinesbasedonpneumococcalcapsulepolysaccharidesalone arenoteffectiveforinfantsunder2yearsofage[3]andtheir efﬁ-cacyonolderpeopleisunderdebate[4].Severalpneumococcal conjugatevaccines(PCVs)weredevelopedoverthelastdecadeto provideeffectivevaccineprotectionespeciallyinyoungchildren. Theconjugatevaccineshaveproveneffectiveforreducingcarriage andprotectingagainstinvasivedisease[5,6].However,thereis evi-dencethatserotypesnotcoveredbythevaccineareincreasingin incidence[7,8],eitherforserotypeshiftfacilitatedbyvaccination orforthewellknownseculartrendofserotypes[9,10].

In2000aPCVformulatedwithpolysaccharideantigensfromthe sevenserotypesisolatedmostfrequentlyinnorthAmerica(PCV7) wasinitiallyusedforroutineimmunizationofchildrenintheUSA.

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Fig.1. Vaccination(PCV7)coverageindifferentItalianregions.Black:high

cover-age(>60%vaccinatedchildren);darkgrey:mediumcoverage(30–60%vaccinated

children),lightgrey:lowcoverage(<30%vaccinatedchildren);white:unknown

coverage.

Todatemorethan100milliondoseshavebeendistributed world-wide.TheEuropeanMedicinesAgency(EMEA)grantedalicense forPCV7in2001.TheItalianMinistryofHealthinitially recom-mendedPCV7forchildrenathighrisk,subsequentlyincludingitin theNationalVaccinationPlanfor2005–2007.

HoweverPCV7hadbeenprogressivelyintroduced,since2003, insomeItalianregions,withdifferentstrategiesaccordingto epi-demiological,organizational,andﬁnancialcriteria.

InLiguria,forinstance,PCV7wasrecommendedfree-of-charge toallchildren intheﬁrstyearoflife alreadyin 2003,and vac-cinecoveragerapidlyincreasedtoover80%in2004[11].Onthe contrary,other Administrative Regions, such as Lombardia did notrecommendmassvaccinationtillJuly2010.Thisscenariohas undoubtedlycontributedtojeopardizevaccinationcoveragerates ofthedifferent Italiangeographical areas [12] whichwould be expectedtoinﬂuencethenumberofthevaccine-preventablecases andcosteffectiveness.PCV7uptakeinItalywas50–60%overthe period2005–2007basedoninternationalreportsandreportsfrom theNationalDepartmentof Health(ICONA2008) [13,14]; how-ever,accordingtotheICONA2008survey,agreat differencein PVC7coveragewaspresentamongdifferentItalianregions,ranging between20and27%respectivelyforCampania(South)and Lom-bardia(North)upto94.3–95.2%respectivelyforBasilicata(South) andEmilia-Romagna(North)[14](Fig.1).

TwonewPCVscontainingadditionalserotypeshavebeen devel-oped: the 10-valent PCV conjugated to Haemophilus inﬂuenzae glycoprotein-d,whichaddsserotypes1,5,and7F(PCV10);anda CRM197-conjugated13-valentPCV,whichaddsserotypes1,3,5, 6A,7F,and19A(PCV13).InItaly,PCV13willbedeployedinmany RegionsinsubstitutionofPCV7[15,16].

Theplanningofasuccessfulvaccinationprogramrequires infor-mationaboutpotentialcoverage.Thelimitedcoverageprovided byavailablePCVswithrespecttothetotalnumberofserotypes emphasizes the importance of monitoring for emergence of

non-vaccineserotypes.Differencesinthepathologicpotentialof variousserotypesshouldalsobeconsidered[2].

Theaimofthepresentworkwastoassessthepotentialserotype coverageofthreepneumococcalconjugatevaccines(PCV7,PCV10 andPCV13)againstpneumococcalpneumoniaandmeningitisin Italianchildren.

2. Patientsandmethods

2.1. Patients

Thestudywasdesignedtoinclude,withinanactive surveil-lanceprogram,allchildren0–16yearswithaconﬁrmeddiagnosis ofmeningitis/sepsis(themostsevereIPD)orbacteremic pneumo-nia(themostcommonIPD).OnlypatientsadmittedtoPaediatric Hospitals orPaediatric wards ofgeneralhospitals inItaly from April2008throughMarch2011wereconsidered.Hospitalsfrom allItalianregionswereinvitedtoparticipate.

IPDwasdefinedasclinicalsuspicionofbacterialdisease (pneu-monia,meningitis/sepsis)[17–21]andlaboratory-confirmationof thepresenceof S.pneumoniae ina normallysterilesite (blood, cerebrospinalfluidorpleuralfluid)aspreviouslydescribed[20].

Patients with severe concomitant disease (neoplasia, immunodepression) or nosocomial acquired infections were excluded. In order to select for community acquired disease, childrenwhohadbeenadmittedtohospital,hadbeenoutpatients orhadattendedanemergencyroomintheprevious14dayswere excludedfromthestudy.Informedwrittenconsentwasobtained fromallparentsorguardians beforeinitiationofthestudy.The studywasapprovedbythelocalethicscommittee.

Werecordeddataonanti-pneumococcalvaccinestatus, includ-ingthenumberofdosesreceived.Childrenwereconsideredfully vaccinatediftheyhadcompletedthenationalvaccination sched-ule,including3dosesofthe7-valentconjugateanti-pneumococcal vaccine(PCV7)at3,5and12monthsofage,ortwodosesbetween theﬁrstandthesecondyearoflife,orasingledoseafterthe sec-ondyearoflife.Childrenwereconsideredincompletelyvaccinated iftheyhadstartedbutnotcompletedthevaccineschedule.

AccordinglytothecoveragerateforPCV7reportedinICONA 2008 surveillance report [14] Italian regions were divided in: low-coverageregions(0–33%coverage),mediumcoverageregions (31–60%coverage)andhighcoverageregions(61–100%coverage). ThedistributionoverthecountryisshowninFig.1.

2.2. Samplehandling

Wholebloodwasobtainedfromallchildrenassoonas possi-bleafterhospitaladmission.Samplesformoleculartestsweresent atroomtemperaturetothecentralLaboratory(Immunology Labo-ratory,AnnaMeyerChildren’sUniversityHospital,Florence,Italy) usinganovernightfreepostcarrierandmoleculartestswere per-formedwithin2hofdelivery;200_␮lofwholebloodwereusedfor bothdiagnosisandserotypingbyRealtime-PCR(RT-PCR).Clinicians wereallowedtochoosewhentoalsorequestculturesfrombloodor cerebrospinalﬂuid(CSF).Forculturepurposes,bloodorCSF sam-pleswereimmediatelysenttothelocallaboratoryandprocedures establishedbyeachhospitalforthesetestswereused.

2.3. DiagnosisandserotypingofIPD

Diagnosisof laboratory-confirmedIPDwasbasedonRT-PCR resultsforthelytAgeneaspreviouslydescribed[20,22].A sam-plewasconsiderednegativeiftherewasnoincreaseinfluorescent signalbeforeRT-PCRcycle45.Wehadpreviouslyconfirmedthe specificityofPCRusingwholebloodsamplesdrawnfromcarrieror non-carrierhealthycontrols[20,23].

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Table1

PrimerandprobesetsforpneumococcalserotypingbyRealtimePCR.

Serotype Forwardprimer Reverseprimer Probe

2 TTATGGACTGGCTGATGGTTCTC AAATCCTGACCCAATAATAGCCTTT AGGTCAACGTATTGGAACTCTTAGAAATTGGGAAA

11AD AGCTATCCTTTAGGCATTCCGTTA TCCCGTTGGCTTAGATATGTGTT TTGAAACACTAGATGAACTGGCAAACCT

16F CAGGCGAAAAGCGAGCAT TGGGTTCCCCTCATCTACGTT TGCTTTGGTAGCTTGTATGAGTGC

17F CTTAGCGTACGTTCTTCGTATGCTA CCCGTACTCGGAAGCAAAAC TCTAAGAGAGCTACTGAAACACTTTGTGC

21 GGTTTAAATATCGCTCCGGGTAT CAAAAAAAGGGCTTGTAGACGAA TGTGAATTGGACACGTTATGGAGC

23A GGGAATTGGCACTCTTCTGAAT GATCGGCAAATGTTGAAACCA TTGGCGGTAAACAATTAAGGCGT

23B TTGAAGAAATTGCTCCAGAAACAT CCAAAAGACTAGCCTCAACCACTAA TAGAGCTATTTATCTTTCGTGGTTTT

29 TTCGAGTTGTGCCGTTTTTACA GGCGTACCCACCTCTAAAATTTT AGGAGTACGCAGAGAAAAGACTAGGATTCAA

All samples were serotyped using RT-PCR as previously

described [20,22]. Twenty-nineprimer/probesets targeting

dif-ferent regionsof theCpsAgene wereused,each specificfor 29 different serotypes. Twenty-one primer-probe sets were previ-ouslypublished[20,22];thesequencesofthe8additionalprimer probesetsareshowninTable1.Ifnoincreaseinfluorescent sig-nalwasobservedafter45cyclesforanyoftheserotype-specific primer/probesetsinspiteofapositiveresultwithbothRT-PCR(lytA gene)andend-pointPCR(CpsAgene)[22],thesamplewas consid-erednon-typeablewiththeserotype-specificprimersinReal-time PCR.PneumococcalserotypeswereclassifiedascoveredbyPCV-7, PCV-10orPCV-13vaccinesiftheywereincludedinthe7-valent(4, 6B,9V,14,18C,19F,23F),10-valent(7-valentwiththeadditionof 1,5and7F)or13-valent(10-valentwithfurtheradditionof3,6A and19A)conjugatevaccines,respectively.Thepotentialcoverage ofthe3availableconjugatevaccineshasbeenevaluatedaccording tothedisease;moreover,sinceacampaignforvaccinationof chil-dren2–5yearsoldhasbeenproposedinsomeItalianregions,the potentialcoveragehasbeenevaluatedaccordingtodifferentage. 2.4. Statisticalanalysis

Twotailedpvalueswereusedandpvalues<0.05were con-sideredstatisticallysigniﬁcant.Resultswereexpressedasmeans andstandarddeviations(SD)orasmedianandinterquartilerange (IQR)asappropriate.DatawereprocessedwiththeSPSSXstatistical package(SPSS11.0,SPSSInc,Chicago,IL).

3. Results

3.1. DiagnosisofIPD

Weidentiﬁedatotalof153patientswithbacteremic pneumo-coccalpneumonia(n=104)ormeningitis/sepsis(n=49)from83 participatingcenterslocatedamong19ofthe20Italianregions, representing99.8%oftheItalianpopulationbelowtheageof16 years.

Amongthe20Italianregions,4regionshadlow-coveragefor PCV7,7regionshadmediumcoverageforPCV7,6regionsandone autonomousprovincehadhighcoverageforPCV7.DataonPCV7 coveragewerenot availablefor2 regionsand oneautonomous province.Geographical distribution wasnotuniform(Fig.1) so that PCV7high-coverage regionsare present both in thenorth andinthesouthofItaly.Twenty-ﬁvesampleswereobtainedfrom low-coverageregions,80samplesfrommedium-coverageregions and 48 fromhighcoverage regions.The onlyregion which did notinclude anypatienthas18,807residentsbelow 16years of agethatis0.2%ofItalianpopulation(9,044,793)0–16years (Ital-ianNationalInstituteofStatistic,ISTAT,2010http://demo.istat.it/; accessed6.11.11).

Median(IQR)agewas3.3(1.7–5.6)years.ThegenderratioM/F was1.12.Vaccinationrecordsindicatedthat40.5%(62/153)had completedthe vaccinationscheduleand were fullyvaccinated; amongthem1childhadcompletedthevaccinationschedulewith

Fig.2. Incidenceofvariousserotypesasacumulativepercentageaccordingtotype

ofinvasivepneumococcaldiseaseinItalianchildren.

onedoseofPCV13;7.2%(11/153)hadreceivedatleastonedosebut notthefullcourse(2ofthemhadreceived1doseofPCV13)and 43.8%(67/153)hadnotbeenvaccinated.Dataregardingvaccination wasnotavailablefor8.5%(13/153)patients.

In99samplessimultaneouslyobtainedforbothcultureand RT-PCRandpositiveforS.pneumoniaebyRT-PCR,culturesresulted positivein 35/99(35.4%)children.Molecularmethodsappeared 2.8timesmoresensitivethanculturalmethods(p<10−6;ORand 95%conﬁdencelimitsnotcalculable).PathogensdifferentfromS. pneumoniaewereneverfound,inanyofthesamplespositiveforS. pneumoniaebyRT-PCRwiththeexceptionofonesample,positive forStaphilococcusepidermidisbycultureandreportedas contami-natedbythelaboratorymicrobiologist.

3.2. Serotypedistribution

Individuation of pneumococcal serotype was obtained in 144/153(94.1%) patients(101pneumonia,43meningitis/sepsis; medianage3.3,IQR1.8–5.6).IPDremainednon-typeablein9/153 (5.9%)patientsbecauseofsamplepaucity(1sample)orbecause negativitytoallavailableprimer/probesets(8/153,5.2%samples). The10mostprevalentserotypeswere1(43/144,29.9%),3(23/144, 16.0%),19A(19/144,13.2%),7F(12/144,8.3%),5(6/144,4.2%),14 (6/144,4.2%),6A(5/144,3.5%),6B(5/144,3.5%),18C(5/144,3.5%), 19F(5/144,3.5%).Thedistributionoftheserotypesidentiﬁedin thesepatientsaccordingtounderlyingdiseaseisshowninFig.2. Overall,serotypecoverageforPCV-7,-10and-13were19.4%,61.8% and94.4%.Inordertodeterminetheimpactofageonpotential cov-eragewehavepresentedserotypedistributionaccordingtopatient age(Fig.3):serotypedistributionwassimilarinthetwogroupsof patients.

Thepercentageofvaccinated(fullyorpartiallyvaccinated) chil-drendecreased,asexpected,withage,goingfrom59.1%inchildren below2yearsofage,to56.9%inchildren2–5yearsofage,to12.5% inchildrenover9yearsofage.

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Fig.3. Incidenceofvariousserotypesasacumulativepercentageaccordingtoage

inItalianchildrenwithinvasivepneumococcaldisease.

PCV7serotypeswerefoundin7/25(28.0%)serotypedsamples fromlow-coverageregions,in 15/71(21.1%) serotypedsamples frommedium-coverageregionsandin5/45(11.1%)serotyped sam-plesfromhighcoverageregions.

NocaseofIPDduetoPCV7serotypeswasrecordedinchildren whohadbeenfullyorpartiallyvaccinated.

4. Discussion

Knowingthepresent distributionofpneumococcalserotypes isessentialtoplanandmonitormassvaccination.Ourstudy, per-formedonalargepopulationofchildrendistributedthroughout Italyshowsthat,atpresent,lessthan20%IPDareduetoserotypes includedinPCV7,and thepotentialcoverage obtainableby 10-valentor13-valentformulationisrespectively61.8%and94.4%.

ThecomparisonbetweenRealtimePCRandculturesensitivity wasbeyondtheaimofthestudy;howeverthedataobtainedinthis studyconﬁrm[19,20]thehighersensitivityofmolecularmethods comparedtoculturemethodsandunderlinetheessentialroleof molecularmethodsinsurveillancestudies.

Oursurveyrevealsachangeinthedistributionofpneumococcal serotypesinItalysincethe7-valentvaccinewasintroduced. Sur-veysconductedbeforePCV7wasintroducedinItalyhadrevealed higherpotentialcoverageforthatvaccine,bothinchildren<2years and<5years[12,24–28].Actually,priortotheintroductionofPCV7, activesurveillancecarriedoutin10hospitalsdemonstratedthat PCV-7,PCV-10andPCV-13couldhaveprevented,respectively,70, 80and87%casesinchildren<2yearsold[24].Regardingchildren <5yearsold,publishedresultsdemonstratedthatPCV-7,PCV-10 andPCV-13couldhavepreventedrespectively67–80%,78–88%and 87–100%casesintheperiodbetween1997and2003[12,24–27]. Unfortunately,sinceinItalysurveillanceofinvasivebacterial dis-easewas,upto2008,onlyfocusedonmeningitis,largerstudies includedonlythatdisease,andfewinformationonotherIPD dif-ferentfrommeningitiswereavailable.

Thepresentworkshowsthat,inthelast3years,mostfrequent serotypesare1,3and19AinIPDpatientswithpneumoniaand 7F,3and6Ainpatientswithmeningitis(noneofthoseserotypes isincludedinthePCV7formulation).Over50%ofmeningitisand over80%ofallpneumoniacasesareduetoserotypeswhichare notincludedinthePCV7but areincludedinthe13-valent for-mulation.Asexpected,thereisa trendtoa lowerrateofPCV7 serotypesinregionswithhighPCVcoveragerate;evenifdatadoes notreachsigniﬁcantdifferences,therateofinfectionsduetoPCV7 serotypesisalmostdoubleinregionswithmediumPCV7-coverage andalmostthreetimesinlowcoverageregionswhencomparedto highPCV7-coverageregions.

Similarﬁndingshave beenreportedacrossEurope.Themost commonserotypesassociatedwithIPDbeforetheintroductionof PCV7were14,6B,19F,and23F,whichareallcoveredbyPCV7. Atthattime,themeanincreasesinpotentialcoverageforPCV10 andPCV13were7%and16%,respectively,forchildren<2yearsof age[29].Currently,datafromcountriesconstitutingapproximately 60%oftheEuropeanpopulationrevealthatthemostcommonIPD serotypes aftertheintroductionof PCV7are 1, 19A,3, 6A, and 7F[29].InparticularinPortugal[30]andFrance[31]the3 lead-ingserotypesare1,19Aand7F,withsimilarpercentagesinthe twocountries(24–26%,20–25%and12–14%respectively).Different fromthosedata,ourresultsshowahigherprevalenceofserotype 3;thisismaybeduetothefactthatPortugueseandFrenchdata wereobtainedbetween2006and2009,whileourdataaremore recent.Actually,inourItalianpatients,theprevalenceofserotype 3wasabout8%in2008butitincreasedupto22.0%inthelastyear. Amildincreaseofnon-vaccineserotypeshasbeendescribed aftermassvaccination[32]andisassociatedwithadecreaseinthe numberofcasesduetovaccineserotypes[5,11].

HoweverthisdoesnotseemtobethecaseinItalywhere vacci-nationhasbeenlimitedtosomeregionsandonlyfromJuly2010is offeredfreeofchargetoallchildrenbelow1yearofage.Actually therateofvaccinatedchildrenseemstobelessthan50%inthelast 3yearsandundoubtedlylowerinthepreviousyears.Therefore,the highfrequencyofserotypes1,3and19Ashowninthepresentwork andtheincreaseofthoseserotypescomparedto5–10yearsago [12,24–27],cannotbecompletelyascribedtotheimmunological pressurecausedbythePCV7vaccination.Aspontaneousincrease ofsomeserotypes,asdemonstratedintherecentpre-vaccination era[33]andtheseculartrenddemonstratedfordifferentserotypes inthelast50years[9,10],mayprobablycontributetothetrend observedinItaly.

Ourdataindicatesthata shifttovaccinesthat includemore serotypesisnecessaryinItaly.PCV10wouldprovidepotential cov-erageforabout62%oftheserotypesidentiﬁed,whilePCV13would providecoverageofmorethan94%(86%formeningitis/sepsisand nearly100%forpneumonia).

Thereisnodoubtthatmassvaccinationofchildrenbelowone yearofageisworthy;decisionregardingvaccinationofchildren between1and5yearswillbetakenattheregionallevel.Similarly, catch-upofchildrenpreviouslyvaccinatedwithPCV7inorderto expandtheirprotectionneedstobeevaluated.Firstofall,the eval-uationofserotypedistributioninthisageclassismandatory,in ordertoconsiderwhichcoveragecouldbeobtainedwith differ-entvaccineformulations.Ourdatademonstratethatpneumococcal serotypedistributioninchildrenbetween2and5yearsdoesnot differfromepidemiologydemonstratedinyoungerchildrenand thatPCV13couldpreventover90%IPDinthatcohortasitdoes inchildrenbelow2years.Otherstudies[34]evaluatingthecost beneﬁtanalysisforthatagearenowinprogressinourlaboratory.

5. Conclusions

OurresultsstronglysuggestthatintroductionofPCV13could haveasigniﬁcantaddedbeneﬁtinreducingtheburdenof pneu-mococcaldiseaseinItaly.Molecularmethodsareessentialtoolsin themonitoringofpneumococcalserotypedistribution.Continuous surveillanceofpneumococcalserotypedistributionismandatory toplanandmonitortheeffectofpneumococcalvaccinationand promptlycatchserotypeshift.

Acknowledgments

WearesincerelygratefultoFrancescaRomanoandGiusy Man-gonefortheirtechnicalsupport.Theresearchhasbeenpartially

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supportedbytheItalianDepartmentofHealth;ItalianCenterfor DiseaseControlandPrevention(grant117-19.01.07-#6728toC.A.). Theauthorsdonothaveanyconﬂictofinterest.

AppendixA.

ItaliangroupforthestudyofInvasivePneumococcalDisease: AgostinianiR.,Pistoia;AllieviP.,GarbagnateMilanese;AllùG., Ragusa;AmigoniA.,Ravenna;BernardiP.,Bologna;BernardiniR., Empoli;BibanP.,Borgotrento; BigiM., Rimini;BoldriniA.,Pisa; BossiG.,Pavia;BottoneU.,Massa;CardinaleA.,Montevarchi; Car-donaA.,Foligno;CastronariR.,Perugia;CelandroniA.,Pontedera; ChiossiM.,ColleselliP.,Vicenza;Cuneo;CorreraA.,Napoli;D’Ascola G,Arezzo;D’AquinoA.;Udine;DeBenedictisF.M.,Ancona;DiniE., Firenze;DollfusL.,Terni;DomeniciR.,Lucca;FlaccoV.,FurbettaM., Perugia;GaettiM.T.,Jesi;GagliardiL.,Viareggio;GianiI., Montepul-ciano;GiglioP.,Gubbio;GualaA.,Verbania;Lanciano;LanariM., Imola;LippiF.,Firenze;LizzoliC.,Magenta;LombardiE.,Firenze; MacchiaPA,Pisa;MagniniM.,Esine;MemminiG.,Carrara;Mesirca P.,Montebelluna; MichelettiE.,Livorno;MigliozziL., Senigallia; NunziataF.,Solofra;PecileP.,Udine;PepeG.,Gallipoli;PerferiG., Firenze;PerisA.,Firenze;PerriP.F.,Macerata;PescollderunggL., Bolzano;PezzatiM.,Firenze;PoggiG.M.,Firenze;PratoR.,Foggia; PrincipiN.,Milano;RapisardiG.,Firenze;RegoliM.,Firenze;Riva A.,Brescia;RizzoL.,Portoferraio;RomanB.,Vimercate;ToffoloA., Oderzo;StranoM.,BorgoSanLorenzo;TrapaniS.,Firenze;Vasarri P.,Prato;VascottoM.,Siena;VecchiV.,Rimini;VenturaA.,Trieste; VeriniM.,Chieti;ZorziC.,Camposanpiero.

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