Distribution of invasive meningococcal B disease in Italian pediatric population: implications for vaccination timing

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ContentslistsavailableatScienceDirect

Vaccine

jo u rn al h om ep a g 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

Distribution

of

invasive

meningococcal

B

disease

in

Italian

pediatric

population:

Implications

for

vaccination

timing

夽

Chiara

Azzari

a,∗

,

Clementina

Canessa

a

_,

_Francesca

_Lippi

a

_,

_Maria

_Moriondo

a

_,

Giuseppe

Indolﬁ

a

_,

_Francesco

_Nieddu

a

_,

_Marco

_Martini

c

_,

_Maurizio

_de

_Martino

a

_,

Paolo

Castiglia

b

_,

_Vincenzo

_Baldo

d

_,

_Massimo

_Resti

a

_,

_the

_Italian

_group

_for

_the

_study

_of

Invasive

Bacterial

Disease

1

a_Pediatric_Section,_Department_of_Health_Sciences,_University_of_Florence_and_Anna_Meyer_Children’s_University_Hospital,_Florence,_Italy b_Department_of_Biomedical_Science,_Hygiene_and_Preventive_Medicine,_University_of_Sassari,_Sassari,_Italy

c_San_Donato_Hospital,_Pediatric_Division,_Arezzo,_Italy

d_Department_of_Molecular_Medicine,_University_of_Padua,_Padua,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received4June2013 Receivedinrevisedform 16September2013 Accepted24September2013 Availableonline8October2013 Keywords:

NeisseriameningitidisgroupB Children Incidence RealtimePCR Sensitivity Vaccine

a

b

s

t

r

a

c

t

NeisseriameningitidisgroupB(MenB)isaleadingcauseofmeningitisandsepsis.Anewvaccinehas beenrecentlylicensed.TheaimofthepresentstudywastoevaluatetheepidemiologyofMenBdisease inpediatricageanddefinetheoptimalageforvaccination.Allpatientsaged0–18yearsadmittedwith adiagnosisofmeningitisorsepsistothe83participatingItalianpediatrichospitalswereincludedin thestudy.Bloodand/orcerebrospinalfluid(CSF)samplesweretestedbyRealtime-PCRand/orculture. Onehundredandthirty-sixcases(meanage5.0years,median2.7)ofMenBdiseasewerefound.Among these,96/136(70.6%)werebetween0and5years,61/136(44.9%)werebetween0and2years.Among thelatter,39/61(63.9%)occurredduringthefirstyearoflifewithhighestincidencebetween4and8 months.Acase-fatalityrateof13.2%wasfound,with27.8%casesbelow12months.Sepsislethalitywas 24.4%.RT-PCRwassignificantlymoresensitivethanculture:82patientsweretestedatthesametime bybothmethods,eitherinbloodorinCSF;MenBwasfoundbyRT-PCRinbloodorCSFin81/82cases (98.8%),cultureidentified27/82(32.9%)infections(Cohen’sKappa0.3;McNemar’s:p<10−5).Thestudy showsthatthehighestincidenceofdiseaseoccursinthefirstyearofage,withapeakbetween4and8 monthsoflife;30%ofdeathsoccurbefore12months.Theresultssuggestthatthegreatestprevention couldbeobtainedstartingMenBvaccinationinthefirstmonthsoflife;acatch-upstrategyuptothefifth yearoflifecouldbeconsidered.OurresultsalsoconfirmthatRealtimePCRissignificantlymoresensitive thanculture.Inthosecountrieswhereonlyisolatepositiveinfectionsarecountedascases,theincidence ofMenBinfectionresultshighlyunderestimated.

1. Introduction

Neisseria meningitidis is one of the most frequent causes of bacterialmeningitisandsepticemiaworldwide[1,2]. Meningococ-caldiseaseisassociatedwithsigniﬁcantmortality(10–20%)and

夽 Thisisanopen-accessarticledistributedunderthetermsoftheCreative Com-monsAttribution-NonCommercial-NoDerivativeWorksLicense,whichpermits non-commercialuse,distribution,andreproductioninanymedium,providedthe originalauthorandsourcearecredited.

∗ Correspondingauthorat:ImmunologyDepartment,AnnaMeyerChildren Hos-pital,VialePieraccini24,50132Firenze,Italy.Tel.:+390555662542;

fax:+390554221012.

E-mailaddress:c.azzari@meyer.it(C.Azzari).

1 _The_members_of_Italian_group_for_the_study_of_Invasive_Bacterial_Disease_are

listedinAppendixsection.

long-term morbidity[1], especially in pediatricage and 20%of affected people have permanent neurological disabilities [3–5]. Lethality of sepsis is over 20% in children [6,7]. Prevention is thereforeapriority.

Thirteendifferentserotypesareknown,but,asknown,most invasivemeningococcaldiseaseiscausedbyoneofsixcapsular groupsA,B,C,W135,XandY.Excellentconjugatevaccineshave beenlicensedsofar.InItaly,sincetheintroductionofconjugate meningococcalCvaccine(MenC),arapidandsustainedreduction intheincidenceofinvasive MenCdiseaseacrossallagegroups occurred[8,9]. Asa consequence,capsular groupB(MenB) has becomeresponsibleformostcases[7,9].AvaccineagainstgroupB hasrecentlybeenlicensedinEuropeandothervaccinesareunder study; preliminary data regarding immunogenicity and safety arepromisingboth ininfantsand adolescentsoradults[10,11]. http://dx.doi.org/10.1016/j.vaccine.2013.09.055

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Withtheaimtoprovidebroadercross-protection,vaccinesunder developmentincludehighlyconservedsubcapsularproteinssuch asPorA,variantsoffactorHbindingprotein(fHbp),Neisserial Hep-arinbindingAntigen(NHBA)andNeisserialadhesinA(NadA)[1]. Inordertoplananeffectivevaccinationschedule,itisimportantto knowwhenthegreatestburdenofmeningococcalBdiseaseoccurs andifvaccinepreventionshouldbedoneduringtheﬁrstyearof lifeorlater.Theaimofthepresentstudyisthereforetodescribe the epidemiology of invasive meningococcal B disease across pediatricagegroupssotodeﬁnetheoptimalageforvaccination.

2. Methods

2.1. Studydesign

Thisobservational,retrospective,cohortstudywasdesignedto evaluatethedistributionofmeningococcalBinvasivediseasecases acrossagegroupsinchildrenadmittedwithaclinicalsuspicionof community-acquiredmeningitisorsepsistoPediatricHospitalsor PediatricwardsofgeneralhospitalsinItalyfromDecember2006 toDecember2012.Thisstudywasa partofaprospectivestudy aimedatobtainingepidemiologicalandclinicaldataofItalian chil-drenwithinvasivebacterialdiseases[12].HospitalsfromallItalian regionswereinvitedtoparticipate(seeTableA,providedas supple-mentaryﬁle,forthecharacteristicoftheparticipatinghospitals).

Supplementarymaterialrelated tothis article canbefound, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2013.09.055.

2.2. Casedeﬁnition

Bacterialmeningitiswassuspectedinthepresenceofatleast twoofthefollowingclinicalsigns:bulgingfontanelle,drowsiness orirritability,opisthotonus,neckstiffness,vomitorseizures[13] Abacterialmeningitiscasewasdefinedwhenclinicalsignswere associatedtothepositivityofRT-PCR(RealtimePolymeraseChain Reaction)and/orbloodorCSF(CerebralSpinalFluid)culturefora bacterium.Meningococcalmeningitiswasdefinedbythepresence ofclinicalsuspiciontogetherwithchemicalCSFtestsandthe posi-tivityofcultureorRT-PCRonCSFforN.meningitidis.Meningococcal meningitiswasdefinedassociatedtosepsiswhenRT-PCRwas pos-itiveforN.meningitidisinblood,too.Sepsiswasclinicallysuspected inthepresenceofpreviouslydescribedsigns[14,15]andconfirmed bycultureorRT-PCRforN.meningitidis.

2.3. Patients

Allpatientsaged0–18yearsadmittedwithadiagnosisof menin-gitisorsepsistotheparticipatingcentersduringthestudyperiod wereincludedinthestudy.Dataregardingage,sex,clinical pre-sentation,blood tests, radiologic exams and vaccination status werecollected.Biologicalsampleswereobtainedaspartof rou-tineexamsforetiologicdeﬁnition.Thestudy,partiallyfundedby theItalianCenterforDiseaseControl(CCM),wasapprovedbythe localinstitutionalreviewboard.

2.4. Clinicalsamples

Samplesofbloodand/orCSF,accordingtotheclinical presen-tation,wereobtainedfromallchildrenincludedinthestudyas soonaspossibleafterhospitaladmissionandwereusedfor molec-ulartestingbyRT-PCRand/orculture.Allsamplesforculturaltests wereimmediatelysenttothelocal laboratoryusing the proce-duresestablishedbyeachhospitalforculturetests.Allsamplesfor moleculartestsweresenttothecentralLaboratory(Immunology Laboratory,AnnaMeyerChildrenHospital,Florence,Italy)using

afree-postcarrier,deliveredwithinthefollowingdayandtested within2hafterdelivery.Allthesamplesformoleculartestswere accompaniedbyaformcollectingdemographicandlaboratorydata andthemainclinicalﬁndingsofthepatient.

Forculturepurposes,4–6mlofblood samples(upto3sets) wereused.AllcasesinwhichRT-PCRorculturedemonstratedthe presenceofN.meningitidiswereserogroupedusingmolecular tech-niques;inthecentralLaboratory200␮lofwholebloodwereused forbothdiagnosisandserogroupingbyRT-PCR.

2.5. DNAextraction

BacterialgenomicDNAwasextractedfrom200␮lof biologi-calsamplesusingtheQIAmpDneasyBlood&Tissuekit(Qiagen), accordingtothemanufacturer’sinstructions.

2.6. RT-PCR

RT-PCRamplificationwasperformedin25␮lreactionvolumes containing2×TaqManUniversalMasterMix(AppliedBiosystem, Foster City,CA,USA);primers wereused at a concentrationof 400nM;FAMlabeledprobesataconcentrationof200nM.Six␮l ofDNAextractwasusedforeachreaction.Allreactionswere per-formedintriplicate.Anegativecontrol(no-template)andapositive controlwereincludedineveryrun.DNAwasamplifiedinanABI 7500sequencedetectionsystem(AppliedBiosystem,FosterCity, CA,USA)using,foralltheprimerscouples,thesamecycling param-etersasfollows:50◦for2minforUNGdigestion95◦Cfor10min followedby45cyclesofatwo-stagetemperatureprofileof95◦C for15sand60◦Cfor1min.Ifnoincreaseinfluorescentsignalwas observedafter40cycles,thesamplewasassumedtobenegative. 2.7. Meningococcalserogrouping

AllsampleswhichwerepositiveinRealtime-PCRforctragene wereincludedinserogroupinganalysis.Thefollowingserogroups weretested:A,B,C,W135,Yusingprimersandprobesasdescribed inTable1.

2.8. Statisticalanalysis

Data wasprocessedwiththeSPSSX11.0 statisticalpackage; p values<0.05 were considered statistically signiﬁcant. Results wereexpressedas meanlevelsandstandard deviations(SD)or asmedianandinterquartilerangeasappropriate.2_was_used_to assessgroupdifferencesincategoricalvariables.Oddratio(OR)and 95%conﬁdencelimits(95%CL),whenpossible,werecalculated.For continuousvariables,thet-testwasusedwithLogarithmic trans-formationofnon-normaldistributedvariables.

3. Results

3.1. Casedistributionaccordingtoage

Inthestudyperiod,136casesofinvasivemeningococcalB dis-easewerereported.Themeanagewas5.0years,median2.7years, interquartilerange10.2months–6.4years.

Amongthese,96/136(70.6%)patientswerebetween0and5 years,61/136(45.2%)patientswerebetween0and2years.Among casesunder2yearsofage,39/61(63.9%)occurredduringtheﬁrst yearoflife.DistributionofcasesaccordingtoageisshowninFig.1. Withintheﬁrstyearofagethehighestincidencewasobserved betweenthe4thandthe8thmonthofage,where20/39(51.3%) casesoccurred. Case distributionaccordingtomonthsof ageis showninFig.2.

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Table1

Primersandprobesusedfordiagnosisandserotypingofmeningococcalinfection.

Target Gene Forwardprimer Reverseprimer Probe

N.meningitidis ctra gctgcggtaggtggttcaa gctgcggtaggtggttcaa FAMcattgccacgtgtcagctgcacat SerogroupA sacB cccccagcatggctagattt agggcactttgtggcataattt FAMaccctaaaattcaatgggtatatcacga SerogroupB siaDB ttggacttggttaagctgacctaa gttgacaacatctccattttatcttacc FAMttagatatgacaaataaattgttacgtggg SerogroupC siaDC agggaaccgcaacctatgc cacaaaacgttgctcaaattttg FAMccactcttagaatcatttacatacaaaccc SerogroupW135/Y siaDW135/Y agggaaccgcaacctatgc cacaaaacgttgctcaaattttg FAM accctaaaattcaatgggtatatcacga

0 5 10 15 20 25 30 35 40 cases

Fig.1. Distribution,accordingtoage,ofcasesofinvasivemeningococcalinfection(meningitisorsepsis).

3.2. CultureandRT-PCRsensitivityinbloodorCSF

Fifty-twobloodsamplesweretestedbothbycultureand

RT-PCR. MenB wasfound in 43/52 (82.7%); the9 (17.3%) patients

who were negative for both tests in blood were positive by

RT-PCR in CSF. MenB wasidentiﬁed by RT-PCR alone in 32/43

(74.4%)patientsandbybothRT-PCRandculturein11/43(25.6%)

patients(McNemar’sp<10−3);nosamplewasidentiﬁedbyculture

alone.

Fifty-nineCSFsamplesweretestedbothbycultureandRT-PCR.

MenBwasfoundin57/59(96.6%);the2(3.4%)patientswhowere

negativeforbothtestsinCSFwerepositivebyRT-PCRinblood.

MenBwasidentiﬁedbyRT-PCRalonein35/57(61.4%)patients;

byculturealonein1/57(1.8%)andbybothRT-PCRandculturein

21/57(36.8%)patients(McNemar’sp<10−3).

Overall, 82 patients were tested at the same time by both

molecular and cultural tests either in blood or in CSF or in

both and a Neisseria meningitidis infection was found by

RT-PCRinbloodorCSFin81/82cases(98.8%).Inthesamepatients

culture could identify 27/82 (32.9%) infections. RT-PCR was

0 2 4 6 8 10 12 14 16 18 20 0-4 >4-8 >8-12 >12-16 >16-20 >21-24 >24-28 >28-32 >32-36

Fig.2. DistributionofmeningococcalBcaseswithintheﬁrst36monthsofage.

signiﬁcantly more sensitive than culture in achieving

labora-tory diagnosisof meningococcal infection (Cohen’s Kappa: 0.3;

McNemarp<10−5).

Sensitivityaccordingtoclinicalpresentationwasevaluated.In

44patientswhowereadmittedtohospitalwiththediagnosisof

sepsiswithorwithoutmeningitis,RT-PCRwasperformedinthe

bloodof29/44andinCSFof15/44andwaspositivein29/29(100%)

bloodandin13/15(86.7%)CSF.Culturewasperformedintheblood

of24/44andintheCSFof10/44andwaspositivein6/24blood

(25.0%)andin2/10(20.0%)CSF.Asformeningitis,in90patients

withthediagnosisofmeningitiswithnosignofsepsis,RT-PCRwas

performedin39bloodsamplesandin61CSFsamplesandwas

posi-tivein29/39(74.4%)bloodsamplesand60/61(98.4%)CSFsamples.

Culturewasperformedin31bloodsamplesand50CSFsamples

andwaspositivein5/31(16.1%)bloodsamplesand21/50(42.0%)

CSFsamples.Asexpected,CSFisthemostsuitablesamplefor

diag-nosisofmeningococcalmeningitisandbloodisthemostsuitable

sampleinmeningococcalsepsis.RT-PCRhasalwaysagreater

sen-sitivity(2–8timeshigher)whencomparedtoculture,rangingfrom

2.3timesintheCSFofpatientswithmeningitis,to8.7timesinCSF

ofpatientswithsepsis.

3.3. Case-fatalityrate,follow-upandoutcome

Overthestudyperiod therewere18 deaths,constitutingan

overall case fatalityratio(CFR)of 13.2%.Five out of18 (27.8%)

deathsoccurredintheﬁrstyearofage,9outof18(50.0%)occurred

betweenthesecondandtheﬁfthyearofage;3casesoccurredin

adolescents(13–17yearsofage).Onecaseoccurredat6.2years.

CFRwas24.4%(11/45cases)inchildrenadmittedwithadiagnosis

ofsepsis,and7.7%(7/91cases)inchildrenadmittedfor

menin-gitisandinwhomsepsiswasnotmentionedatadmission.Twelve

patients(8.9%)hadcomplicationsduringtheacutephaseofdisease

(cutaneousorsubcutaneousnecrosis,acuterenalfailure,seizures).

During thefollow-up, severesequelae suchasabnormalities in

NuclearMagneticResonanceofbrain(gliosis,idrocephalus)

asso-ciatedwithneurologicsymptoms,mentalretardation,amputation

of bothhandand footﬁngershave beenreportedin4 patients

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4. Discussion

Theresults,obtainedinalargepediatricpopulationofItalian

patients,demonstratethatinvasivemeningococcalinfectionhas

thehighestincidenceintheﬁrst5years oflifewhere over70%

casesoccurandinparticularintheﬁrstyearofage,whereover

20%ofallcasesfoundinpediatricagearefound.Theincidence

peak,similarlytowhatreportedinothercountries[16],isbetween

the4thandthe8thmonthoflife.

InparallelwiththeintroductionofroutineMenCvaccinationin differentItalianregions,theincidenceofmeningococcalinfection duetoserogroupChasprogressivelydecreasedininfantsand ado-lescents[8,9,13,17].However,invasivemeningococcaldiseaseis stilltheﬁrstcauseofmeningitisandissecondonlyto pneumococ-calinfectionforcasesofsepsis.Themostcommoncauseofinvasive meningococcaldisease,accountingforover80%ofcasesfoundin patientsyoungerthan24yearsofage[9,17]isnowMenB.

Culture has been, so far, the most used technique for meningococcalsurveillance; however,bacterial cultureleadsto an important underestimation of disease burden. Conﬁrming previousresults,[16,18,19]onceagainRealtimePCRresults signif-icantlymoresensitivethancultureinidentifyingmeningococcal infection,independentofthebiologicalsampleusedandthe clini-calpresentation.Infact,inourdataobtainedinpatienttestedatthe sametimewithbothmethods,sensitivityofculturewaslessthan onethirdthatofRealtimePCR.Thisdataisparticularlyimportant andsuggeststhatinthosecountries(asinItaly)[9]whereonly isolatepositivesamplesarecountedasmeningococcalcases,the incidenceresultslargelyunderestimated.Furthermore,itiswell knownthatculture-based methods haveeven lower sensitivity comparedtomolecularmethodswhenthepatienthasbeentreated withantibiotics[13].

Realtime-PCRhastheadvantageofprovidingadiagnosisinthe presenceofculture-negativesamples[12,13,20,21];andcanalso determinethecapsulargroupand even thecomplete sequence ofbacterialgeneswhenneeded.Therefore,somecountrieshave includedPCR-basedapproachesinsurveillanceprocedures,while performing cultural tests too. In the United Kingdom, 58% of laboratory-confirmedmeningococcalcaseswereidentifiedbyPCR alone[22];thatpercentageisevenhigherincountrieswithlower healthresources,wheresampletransportandstoragenegatively influencetheresults;amongthemBrazil,wheretheuseofPCRhas almostdoubledthefiguresobtainedbyculturetests[19].

RT-PCRhastheadditionaladvantageofprovidingresultsinless than2h[12]soallowingtostartprophylaxisofcontactsverysoon andonlywhenneeded.

Casefatalityratiohasbeenrecentlydescribedtobeabout5%for MenBinpatientsofanyage[16].Ourdata,obtainedinapediatric population,showahigherfatalityrateof13.2%withalmost30% casesintheﬁrstyearofageandover75%intheﬁrst5yearsofage. TheCFRisevenhigherforpatientspresentingwithsepsis,where itreaches24.4%.

Asreportedinotherwesterncountries[16,23,24]thenumber ofcasesfoundinourstudyrapidlyincreasedintheﬁrstmonthsof life,withapeakbetweenthe4thand8thmonthofage.Therefore, inordertoobtainthehighesteffectiveness,thevaccineshouldbe offeredtoallinfantsintheﬁrstmonthsoflife.

Ithasbeenrecentlydemonstratedthattherecentlylicensed 4CMenBishighlyimmunogenicininfantsafter3dosesgivenat 2,3,4or2,4,6monthsoflife[10].

Howeverasdemonstratedforothervaccines(eithermadeof polysaccharidesconjugatedtoproteinsorofproteins)inorderto establishgoodimmunememoryandlongtermprotectionadose inthesecondyearofageisalwaysrecommended[25].

Itcannotbeexcludedthat asingledosegivenaftertheﬁrst yearofagecouldprotectalsoinfantsthroughamechanismofherd

protection,butthishypothesishasnotbeendemonstrated,sofar. Reductionincarriageisconsideredanimportantdeterminantofthe MenCvaccinationsuccess[25]andwasobtainedvaccinatingatthe sametimebothinfantsandadolescentandyoungadults;classes, thelatter,inwhichthecarriagestateismorefrequent.Theeffect ofMenBvaccinesoncarriageisstillunderstudy,but,ifundergoing studieswilldemonstratecarriagecanbeeliminatedbyvaccination, inclusionofadolescentsinvaccinationprogramswouldhavealso anadvantageonprotectionofinfants.

Otherapproachesforprotectionsofinfantsintheﬁrstyearoflife couldevaluateacocoonstrategy,inwhichmothersarevaccinated duringpregnancyinordertoofferthenewbornsapassiveantibody protection.Nodataareavailableonthisprocedurewhichhasnot beenprovenveryeffectivewithothervaccines[26]forthepresence offrequentnon-householdsourcesofinfections.

Thepresentworkprovidedcountryspeciﬁcdatawhichcanbe animportantkeypoint,assuggestedbyinternational recommen-dations[1]tomakesustainabledecisions,giventhegreatregional variabilityinMenBincidenceandserogroupdistribution.Sincethe availablevaccineismadeofamixof4subcapsularproteinofMenB, whichcanbeabsentindifferentMenBisolates,itwillbemandatory togoonwithepidemiologicalstudiestoevaluatewhether,under theimmunepressureinducedbyvaccination,newmutantswhich donotexpressthe4proteinstargetofthevaccinewillescapethe immunesystem[27].

Largeepidemiological studies willcontinue to beneededto monitorandevaluatetheintroductionofthisnewvaccine,andto measuretheimpactofvaccinationonachievingthegoalof elimi-natingmeningococcaldiseaseandRT-PCRshouldbeincludedinall surveillanceprogramsinordertoobtainmorepreciseevaluation ofincidence,casefatalityrateandserogroupdistribution.

Acknowledgments

TheresearchwaspartiallysupportedbytheItalianDepartment ofHealth(CCM),bytheAnnaMeyerChildren’sUniversityHospital andbytheUniversityofFlorence.

Conﬂictofinterest statement:Theauthorshave noconﬂictof interest.

Appendix. ItaliangroupforthestudyofInvasiveBacterial Disease

Themember of theItalian groupare: Agostiniani R,Pistoia; Allievi P., Garbagnate Milanese; Allù G., Ragusa; Amigoni A., Ravenna;BartoliniE.,Firenze;BernardiF.,Bologna;BernardiniR., Empoli;BibanP.,Verona;BigiM.,Rimini;BossiG.,Pavia;BottoneU., Massa;CardinaleA.,Montevarchi;CardonaA.,Foligno;Castronari R.,Perugia;CelandroniA.,Pontedera;ChiossiM.,Cuneo;Colleselli P.,Vicenza;CorreraA.,Napoli;CortimigliaM.,Firenze;D’AscolaG, Arezzo;DeBenedictisF.M.,Ancona;deMartinoM.,Firenze;Dini E.,Firenze;DollfusL.,Terni; DomeniciR.,Lucca; Flacco V., Lan-ciano;VerrottiA.,Perugia;GaettiMT,Jesi;GagliardiL.,Viareggio; GalliL.,Firenze;GiglioP.,Gubbio;GualaA.,Verbania;LanariM., Imola;LasagniD.,Firenze;LizzoliC.,Magenta;LombardiE.,Firenze; MagniniM.,Esine;MatteiR.,Lucca;MemminiG.,Carrara;Mesirca P., Montebelluna; Gragnani S., Livorno; MigliozziL., Senigallia; NunziataF., Solofra; Pecile P., Udine; PepeG., Gallipoli;Perferi G.,Firenze;PerisA.,Firenze;PerriP.F.,Macerata;Pescollderungg L.,Bolzano;PezzatiM.,Firenze;PoggiG.M.,Firenze;PoggiolesiC., Firenze;RapisardiG.,Firenze;RattaL.,Rimini;RicciS.,Firenze;Ridi F.,Firenze;RivaA.,Brescia;RizzoL.,Portoferraio;RomanB., Vimer-cate;RomanoF.,Firenze;ToffoloA.,Oderzo;StranoM.,BorgoSan Lorenzo;TrapaniS.,Firenze;VallerianiC.,Firenze;VasarriP.,Prato;

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VascottoM.,Siena;VergineG.,Rimini;VeriniM.,Chieti;ZorziC., Camposanpiero.

References

[1]TheGlobalMeningococcalInitiative.Recommendationsforreducingtheglobal burdenofmeningococcaldisease.Vaccine2011;29:3363–71.

[2]SáfadiMA,González-AyalaS,JäkelA,WiefferH,MorenoC,VyseA.The epidemi-ologyofmeningococcaldiseaseinLatinAmerica1945–2010:anunpredictable andchanginglandscape.EpidemiolInfect2012Aug;9:1–12.

[3]BedfordH,deLouvoisJ,HalketS,PeckhamC,HurleyR,HarveyD. Menin-gitisininfancyinEnglandandWales:followupatage5years.BrMedJ 2001;323(7312):533–6.

[4]Halket S, de Louvois J, Holt DE, Harvey D. Long term follow up after meningitisininfancy:behaviourofteenagers.Arch DisChild2003;88(5): 395–8.

[5]deLouvoisJ,HalketS,HarveyD.Effectofmeningitisininfancyon school-leavingexaminationresults.ArchDisChild2007;92(11):959–62.

[6]AngusDC,Linde-ZwirbleWT,LidickerJ,ClermontG,CarcilloJ,PinskyMR. Epi-demiologyofseveresepsisintheUnitedStates:analysisofincidence,outcome, andassociatedcostsofcare.CritCareMed2001;29(7):1303–10.

[7]WatsonRS,CarcilloJA,Linde-ZwirbleWT,ClermontG,LidickerJ,AngusDC.The epidemiologyofseveresepsisinchildrenintheUnitedStates.AmJRespirCrit CareMed2003;167(5):695–701.

[8]Bechini A,LeviM,Boccalini S,TiscioneE,BalocchiniE,Canessa C, etal. Impact on disease incidence of a routineuniversal and catch-up vacci-nationstrategy againstNeisseriameningitidis Cin Tuscany,Italy.Vaccine 2012;30(45):6396–401.

[9]http://www.simi.iss.it/ﬁles/ReportMBI.pdf[accessed27.03.13].

[10]GossgerN,SnapeMD,YuLM,FinnA,BonaG,EspositoS,etal.European MenBVaccineStudyGroup.Immunogenicityandtolerabilityofrecombinant serogroup Bmeningococcalvaccineadministeredwithorwithoutroutine infantvaccinationsaccordingtodifferentimmunizationschedules:a random-izedcontrolledtrial.JAmMedAssoc2012;307(6):573–82.

[11]SantolayaME,O’RyanML,ValenzuelaMT,PradoV,VergaraR,Mu ˜nozA,etal. V72P10MeningococcalBAdolescentVaccineStudygroup.Immunogenicity andtolerabilityofamulticomponentmeningococcalserogroupB(4CMenB) vaccineinhealthyadolescentsinChile:aphase2b/3randomised, observer-blind,placebo-controlledstudy.Lancet2012;379(9816):617–24.

[12]RestiM,MoriondoM,CortimigliaM,IndolﬁG,CanessaC,BeccioliniL,etal. ItalianGroupfortheStudyofInvasivePneumococcalDisease, Community-acquiredbacteremicpneumococcalpneumonia inchildren:diagnosisand serotypingbyreal-timepolymerasechainreactionusingbloodsamples.Clin InfectDis2010;51(9):1042–9.

[13]RestiM,MicheliA,MoriondoM,BeccioliniL,CortimigliaM,CanessaC,etal. Comparisonoftheeffectofantibiotictreatmentonthepossibilityofdiagnosing

invasivepneumococcaldiseasebycultureormolecularmethods:aprospective, observationalstudyofchildrenandadolescentswithprovenpneumococcal infection.ClinTher2009;31(6):1266–73.

[14]AzzariC,MoriondoM,IndolﬁG,MassaiC,BeccioliniL,deMartinoM,etal. Moleculardetectionandserotypingonclinicalsamplesimprovediagnostic sensitivityandrevealincreasedincidenceofinvasivediseasebyStreptococcus pneumoniaeinItalianchildren.JMedMicrobiol2008;57(Pt10):1205–12.

[15]RussellJA.Managementofsepsis.NEnglJMed2006;355:1699–713.

[16]LadhaniSN,FloodJS,RamsayME,CampbellH,GraySJ,KaczmarskiEB,etal. InvasivemeningococcaldiseaseinEnglandandWales:implicationsforthe introductionofnewvaccines.Vaccine2012;30:3710–6.

[17]http://www.simi.iss.it/ﬁles/Report MBI.pdf[accessed27.03.13].

[18]GrayS,CampbellH,MarshJ,CarrA,NewboldL,MallardR,etal.The epidemiol-ogyandsurveillanceofmeningococcaldiseaseinEnglandandWales.In:Poster P041.17thInternationalpathogenicconference(IPNC).2010.Availableat:

http://neisseria.org/ipnc/2010/IPNC2010abstracts.pdf[accessed27.03.13]. [19]SacchiCT,FukasawaLO,GoncalvesMG,SalgadoMM,ShuttKA,Carvalhanas

TR,etal. IncorporationofRT-PCRinto routinepublic healthsurveillance formeningococcalmeningitisin SãoPaulo State,Brazil. In:PosterP042. 17thInternationalpathogenicNeisseriaconference(IPNC).2010.Availableat:

http://neisseria.org/ipnc/2010/IPNC2010abstracts.pdf[accessed27.03.13]. [20]CorlessCE,GuiverM,BorrowR,Edwards-JonesV,FoxAJ,KaczmarskiEB.

SimultaneousdetectionofNeisseriameningitidis,Haemophilusinﬂuenzae,and Streptococcuspneumoniaeinsuspectedcasesofmeningitisandsepticemia usingreal-timePCR.JClinMicrobiol2001;39:1553–8.

[21]GurleyES,HossainMJ,MontgomerySP,PetersenLR,SejvarJJ,MayerLW,etal. EtiologiesofbacterialmeningitisinBangladesh:resultsfromahospital-based study.AmJTropMedHyg2009;81:475–83.

[22]GrayS,CampbellH,MarshJ,CarrA,NewboldL,MallardR,etal.The epidemiol-ogyandsurveillanceofmeningococcaldiseaseinEnglandandWales.In:Poster P041.17thInternationalpathogenicconference(IPNC).2010.Availableat:

http://neisseria.org/ipnc/2010/IPNC2010abstracts.pdf[accessed27.03.13]. [23]LevyC,TahaMK,WeilOC,QuinetB,LecuyerA,AlonsoJM,etal.Association

ofmeningococcalphenotypesandgenotypeswithclinicalcharacteristicsand mortalityofmeningitisinchildren.PediatrInfectDisJ2010;29:618–23.

[24]HersheyJH,HitchcockW.Epidemiologyandmeningococcalserogroup distri-butionintheUnitedStates.ClinPediatr(Phila)2010;49:519–24.

[25]Trotter CL, Edmunds WJ. Modelling costeffectiveness of meningococcal serogroupCconjugatevaccinationcampaigninEnglandandWales.BrMed J2002;324(7341):809.

[26]WileyKE,ZuoY,MacartneyKK,McIntyrePB.Sourcesofpertussisinfection inyounginfants:areviewofkeyevidenceinformingtargetingofthecocoon strategy.Vaccine2013;31(4):618–25.

[27]VogelU,TahaMK,VazquezJA,FindlowJ,ClausH,StefanelliP,etal. Pre-dictedstraincoverageofameningococcalmulticomponentvaccine(4CMenB) inEurope:aqualitativeandquantitativeassessment.LancetInfectDis2013 May;13(5):416–25.