Italy, 2016 – 2018 the Acute Flaccid Paralyses (AFP) surveillance system inNorthern Emerging Non-Polio Enteroviruses recognized in the frameworkof International Journal of Infectious Diseases

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Emerging Non-Polio Enteroviruses recognized in the framework of the Acute Flaccid Paralyses (AFP) surveillance system in

Northern Italy, 2016 –2018

Laura Pellegrinelli

^a,

*, Cristina Galli

^a

, Valeria Primache

^a

, Laura Bubba

^a

, Gabriele Buttinelli

^b

, Paola Stefanelli

^b

, Elena Pariani

^a

, Sandro Binda

^a

aDepartmentofBiomedicalSciencesforHealth,UniversityofMilan,Milan,Italy

bDepartmentofInfectiousDiseases,ItalianNationalInstituteofHealth(IstitutoSuperiorediSanità),Rome,Italy

ARTICLE INFO

Articlehistory:

Received15December2020

Receivedinrevisedform16March2021 Accepted18March2021

Keywords:

Non-PolioEnterovirus

AcuteFlaccidParalysisSurveillanceSystem Molecularcharacterization

EV-D68

ABSTRACT

Background:AcuteFlaccidParalysesSurveillance(AFPS)monitorstheemergenceofpoliovirusesandcan trackNon-PolioEnteroviruses(NPEVs).WereportAFPSactivityintheLombardyregion(NorthernItaly) from2016to2018.

Methods:Fecalandrespiratorysampleswerecollectedfromchildren<15yearswhomettheWHO deﬁnitionofanAFPcase,analyzedbyvirusisolationincellcultures(RD/L20B)andbyaone-stepreal- timeRT-PCRassayspeciﬁcforthe5⁰-noncoding-regionofNPEV.NPEV-positivespecimenswerefurther analyzedbysequencingafragmentoftheVP1gene.

Results:36AFPcases(89stooland32respiratorysamples)werereportedwithanincidenceof1.1/

100’000,0.9/100’000,0.6/100’000children<15yearsin2016,2017,2018,respectively.Polioviruswasnot identiﬁed,whereasNPEVsweredetectedin19.4%(7/36)ofAFPcases.ThepresenceofoneEchovirus-25 (2016),twoEV-andD68(2016and2018),oneEV-A71(2016),andoneEchovirus-30(2016)sharinghigh nucleotideidentitywithNPEVsdetectedinEuropewasidentiﬁed.

Conclusion:Theabsenceofpoliowasconﬁrmed.TheunpredicteddetectionofemergingEV-D68,EV-A71, andE-30sharinghighsequencenucleotidesimilaritywithvirusesinvolvedinthelatestoutbreaks, provided valuable and up-to-date information, emphasizing the importance of monitoring NPEVs throughAFPS.

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

Introduction

TheWorldHealthOrganization(WHO)strategicplanforglobal poliomyelitis(polio)eradicationincludestheimplementationof infantimmunizationcoveragebyvaccinationcampaigns,national immunizationdays,andtargeted“mop-up”campaigns,alongwith theactivecommunity-basedPVinvestigation(WHO,1996,2021).

Recently, Poliovirus (PV) transmission decreased considerably

worldwide;however,AfghanistanandPakistanarestillreporting endemiccases,whichposeaglobalriskofPVreintroduction.

Following WHO procedures, an acuteflaccid paralysis (AFP) caseis confirmedthroughtheidentification ofallpatientswith polio-likesymptomsintheframeworkofAFPSurveillance(AFPS) systemandthesubsequentanalysisofbiologicalsamplestodetect PV strains (i.e., wild or vaccine-derived), allowing a rapid investigation of confirmed cases and their community (WHO, 2019).AFPSisa sensitivecase-basedschemethat,concurrently withPVdetection,canmonitortheintroductionandcirculationof Non-PolioEnteroviruses(NPEVs)inacommunity(WHO,2019).

Withover100serotypes,NPEVsareasigniﬁcantpublichealth concern,especiallysinceEV-D68,EV-A71,andEchovirus-30(E-30) emergedasacauseofneurologicalcomplications–includingAFP, acute ﬂaccid myelitis (AFM), and brainstem encephalitis - in children(Sureshetal.,2018;Sureshetal.,2020).

Generally,EV-D68,EV-A71,andE-30havebeenassociatedwith clinicalmanifestationssuchasrespiratorydisease(EV-D68),hand, Abbreviations: AFP, Acute Flaccid Paralysis; AFPS, AFP Surveillance; EV,

Enterovirus;GBS,Guillain-BarréSyndrome;IPV,Inactivatedpoliovaccine;NPEV, Non-PolioEnterovirus;NPRL,NationalPolioReferenceLaboratory;Polio,poliomyelitis;PV,Poliovirus;SNRL,Sub-NationalReferenceLaboratory;WHO,WorldHealth Organization.

*Corresponding authorat: Department of Biomedical Sciences for Health, UniversityofMilan,viaC.Pascal36,20133Milan,Italy.

E-mailaddress:laura.pellegrinelli@unimi.it(L.Pellegrinelli).

https://doi.org/10.1016/j.ijid.2021.03.057

1201-9712/©2021TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International Journal of Infectious Diseases

j o u r n a l h o m ep a g e : w w w . e l s e v i e r . c o m / l o c a te / i j i d

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foot, and mouth disease (HFMD,EV-71), and sporadic cases of asepticencephalitis(E-30).However,since2014,EV-D68hasalso beenreportedasacauseofAFMepidemics(Messacaretal.,2018), EV-A71 caused an outbreak of brainstem encephalitis in 2017 (Casas-Albaetal.,2017),andanupsurgeofE-30hasbeenobserved acrossEuropesince2018,alsoassociatedwithmeningitis-related outbreakswithlong-termsequelae(Brobergetal.,2018).

WeanalyzedthemolecularepidemiologyofNPEVsinAFPcases identiﬁedwithintheAFPSactivityfrom2016to2018inLombardy (Northern Italy) bymoleculardetection andcharacterization of NPEVsidentiﬁedinrespiratoryandstoolsamples.

MATERIALSANDMETHODS

ThisstudywasconductedfromJanuary1,2016,toDecember 31,2018,inLombardy,aregioninnorthernItaly,accountingfor nearly 1/6 of the national population with about ten million inhabitants.TheDepartmentofBiomedicalSciencesforHealth, UniversityofMilan,whichisoneofthesixItalianSub-National Reference Laboratories (SNRLs), performing virological inves- tigationsonbiologicalsamplescollectedintheframeworkofthe Italian AFPS, implemented at the national level since 1997 (Pellegrinelli et al., 2015). SNRLs strictly comply with WHO guidelines,methods,andrecommendationsforconductingAFPS (Pellegrinellietal.,2015),areannuallyevaluatedbytheNational Polio ReferenceLaboratory(NPRL)in theDepartment ofInfec- tious Diseases of the Istituto Superiore di Sanità (ISS) for proﬁciencyinisolation, detection,and typingof PVandNPEVs (Fioreetal.,1999).SNRLsshareregionalAFPSresultswithboth theNPRLandtheItalianMinistryofHealth.

AcuteFlaccidParalysisSurveillanceSystems

AccordingtoWHOcasedefinitions,anAFPcaseisachild<15 years witha clinically evident AFP of one or more limbswith decreased or absent deep tendon reflex or presenting with a sudden onset of bulbar paralysis and person of any age with paralyticillnesswithsuspicionofpoliovirusinfection(e.g.,travel- associated). (https://www.who.int/immunization/monitoring_- surveillance/burden/vpd/surveillance_type/active/poliomyelitis_- standards/en/).AFPcaseswerenotifiedbysendingan“AFPcase report,” according to “zero reporting” methodology, to the referenceSNRLtwiceamonth.Promptcollectionof(i)twostool specimensweretaken24–48hapartwithin14dayssincetheonset ofparalysis,and,ifavailable,(ii)arespiratorysamplewastaken withinsevendayssincetheonsetofparalysiswasrequestedfor virologicaltestsfromeachAFPcase.

Epidemiological (type of PV vaccine received and date of administration),demographical(age, gender,countryof origin), clinicalsymptoms(presenceoffeverattheonset,progressionof paralysis, whethersymmetric or asymmetricparalysis)and the date of onset, were collected in an ‘ad hoc’ questionnaire by physiciansorhealthcareworkers(https://www.who.int/immunization/monitoring_surveillance/burden/vpd/surveillance_type/

active/poliomyelitis_standards/en/).

Ethicalstatement

ThisstudywasperformedaccordingtotheInstitutionalReview Board guidelinesconcerningtheuseofbiologicalspecimens for scientiﬁcpurposesincompliancewithItalianlaw(art.13D.Lgs196/

2003).Patients’privacyandconﬁdentialityissuesweremanaged incompleteagreementwithnationallegislation.Approvalfroman ethicscommitteeandinformedconsentforvirusdetectionwere notrequiredsincedataandsampleswerecollectedandanalyzed anonymouslywithinthenationalAFPSsystem.

EVsidentiﬁcationandmolecularcharacterization

Respiratory (n=32) andstool (n=89)samples collected from each AFPcasewereanalyzedtodetectNPEVandPVwithviralisolationon cell cultures by using speciﬁc cells lines, following the WHO algorithm (https://www.who.int/immunization/monitoring_sur- veillance/burden/vpd/surveillance_type/active/poliomyelitis_stan- dards/en/).Inparticular,RDcells(humanrhabdomyosarcomacells), permissivetoallEVs,wereusedtodeterminethegrowthofany culturableEV,whereasL20B(murinetransgenicLcellsexpressing thegeneforthehumancellularreceptorforPV)wereusedtoidentify PVpositivesamples.

Contrary to respiratory samples, stool samples need to be processed before viral isolation, and the ﬁnal suspension was preparedwith10%ofstoolsamplesinphosphate-bufferedsaline andclariﬁedbymicro-tubecentrifugation(13,000rpmfor15min), asperWHOprotocol(https://www.who.int/immunization/mon- itoring_surveillance/burden/vpd/surveillance_type/active/polio- myelitis_standards/en/).

For rapid virusidentification,RNA was extractedfrom stool suspension,respiratorysamples,andthelysateofcellculturesthat showed a cytopathic effect (CPE), and a specific 5⁰ noncoding region(nucleotide[nt.]179-575)ofEVswasamplifiedbyanin- houseone-stepreal-timeRT-PCRassay(Pellegrinellietal.,2017a, 2017b).

The molecular characterization of EVs was performed by sequencingafragmentoftheVP1/2A(nt.2602-2977)gene(Nix et al., 2006). The nucleotide sequences were obtained by an automated DNA sequencing (Macrogen Inc, Korea); the study sequences were aligned with reference strains of previously publishedgenotypesretrievedfromaGenBank¹databaseonthe NCBIwebsite(https://www.ncbi.nlm.nih.gov/genbank/)byusing the ClustalW program implemented in the alignment editor BioEdit(Hall,1999).Nucleotidesequenceidentitywascalculated usingtheSequenceIdentityMatrixtoolofBioEditsoftware(Hall, 1999). The phylogenetic analysis was conducted by MEGA software,version6.0(Tamuraetal.,2013).

Results

Demographical,epidemiological,andclinicalcharacteristicsofAFP cases

FromJanuary1,2016,toDecember31,2018,36AFPcaseswere reportedin Lombardyfor 89 stool samples and 32 respiratory samplescollectedforvirologicalanalysis.

ThemedianageofAFPcaseswas5.2years(interquartilerange [IQR]:5.3years),and60.5%(23/36)ofAFPcaseswererecordedin children<5yearsold.Overall, 58.3%(21/36)of AFPcaseswere males.Allchildrenbutonereceivedinactivatedpoliovaccine(IPV) vaccinationaccordingtotheirage.

Nearly half (20/36; 55.6%) of AFP cases were identiﬁed by pediatricwards,followedbyneuropsychiatricunits(12/36;33.3%) andintensivecareunits(4/36;11.1%).

The vast majority of cases (83.3%; 30/36) presented with asymmetricparalysis,whilefeverover38Cwasobservedin38.9%

(14/36) of AFP cases and in 22.2% (8/36) of cases, paralysis progression occurred within four days from symptoms onset.

Paralysisofallfourlimbswasobservedin25%ofpatients(9/36), while13.8%ofcases(5/36)hadlimbparalysis.

Onfollow-upforms,“notconclusivediagnosis”wasreportedin sevencases(7/36;19.5%),ﬁveofthemhavinganinitialdiagnosisof GBSandtwocasesofacutemyelitis.Ofthe29clinicalfollow-ups (recorded 60-90 days from paralysis onset) with a conclusive diagnosis,51.7%(15/29)reportedGBS,17.2(5/29)wereAFM.Acute myelitis and encephalitis, including meningoencephalitis and

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brainstemencephalitis,werebothidentiﬁedin10.3%(3/29)ofAFP cases; also, hypotonia/asthenia or genetic/metabolic disorders werealsoreportedinthreeAFPcases(10.3%).

Figure 1 shows the temporal distribution of AFP cases by month:apeakofcaseswasobservedinNovemberandDecember when44.4%(16/36)ofcaseswerenotiﬁed(Figure1).

PerformanceindicatorsofAFPS

AccordingtotheLombardy’spopulationsizeagedlessthan15 years, 14 AFPcases per year were expected. During the study period,theannualAFPincidenceratewas1.1/100’000children<15 years in 2016, 0.9/100’000 in 2017, and 0.6/100’000 in 2018.

AccordingtotheWHOindicators,thesensitivityofAFPSfullymet the criteria of the non-polio AFP incidence (>1.00/100’000) in 2016. Completeness of case investigation (>80%) was always achieved,being93.3%(14/15)in2016,84.4%(11/13)in2017,and 100% (8/8) in 2018. Completeness of clinical follow-up was achieved duringthis 3-yearstudysince it wasavailable in 80%

(12/15),84.6%(11/13),and87.5%(7/8)ofAFPcasesidentiﬁedin 2016,2017,and2018,respectively.

Viralisolation,moleculardetection,andcharacterizationofEVs

Virological investigationsdidnot identifyany PV intheAFP casesfrom2016to2018.FollowingtheWHOalgorithm,samples fromtwocases(5.5%)showedCPEinRDcellsbutnotinL20bcells, allowing us to exclude the presence of PV but conﬁrming the presenceofcultivableNPEVsthatwerecharacterizedasE-25(in 2016)andE-30(in2018).

NPEVRNAwasdetectedinsevenoutof36AFPcases(19.4%),of whichthreewereidentiﬁedin2016,twoin2017,andtwoin2018.

NPEV-positiveAFPcasesweredetectedmainlyinJuly,irrespective oftheyearconsidered(Figure1).

In2016,thesequenceanalysisofthreeNPEV-positivesamples revealedthepresenceofE-25onfecalsamplesassociatedwitha GBS case observed in June, one EV-D68 in respiratorysamples associatedwithAFMinJuly(Parianietal.,2017),andoneEV-A71 on fecal and respiratory samples associated with brainstem encephalitis inDecember.TheEV-D68strain(accessionnumber [AN];MH118296)showedhighsimilarity(nt.identity:99.1-99.7%) withsequencesoftheB3sub-cladeidentiﬁedina2016-AFMDutch outbreak (Pariani et al., 2017).EV-A71 strain (AN; MH454234) sharedhighsimilarity(nt.identity: 98.4-98.7%)withsequences belonging tosubgenogroup C1also detected in an outbreak of brainstemencephalitisin2016inSpain(Casas-Albaetal.,2017).

Unfortunately, sequencinganalysisof thetwoNPEV-positive samples identiﬁed in 2017 was abortive; both samples were

collectedfromtwo GBScases identiﬁedin Mayand December, respectively.

In 2018, the molecular characterization of the NPEVs revealed the presence of EV-D68 and E-30, both associated with AFM identiﬁed in October and November, respectively.

TheEV-D68strain(AN;MK790106) wassimilar(nt.identity:

97.8-98.2%)tosequencesoftheB2sub-cladeidentiﬁedinthe USAduringanoutbreakofAFMin2016;whiletheE-30strain (AN; MK815069) showed high similarity (nt. identity: 98.2- 100.0%)tosequencesbelonging togroupG,whichhavebeen circulatinginEuropesince2016,dominatingin2018(Broberg etal.,2018).

Discussion

SincetheeradicationofPVisstillongoing,reintroductionsof this virus may pose a public health threat of major concern, especially for those countries within the current immigration routes,suchasItaly.Inthisscenario,itismandatorytostrengthen AFPSattheregionalandnationalleveltorapidlydetectanyvirus importation or emergence and enable a prompt public health response (Stefanelli et al., 2019). Although AFPS is the gold standardforPVcontrol,ItalybarelyachievestheWHOtargetfor AFPS performance indicators(WHO, 2016–2019).Thiscould be relatedtotheperceptionofAFPSasalowpublichealthpriority sinceItalyisapolio-freecountryand,consequently,PVbecamea neglectedinfectionthatisnotconsideredahealthhazardanymore (Stefanellietal.,2019).

The Lombardy region, according to theWHO indicators for AFPS sensitivity, fully met the criteria of the non-polio AFP incidence(>1.00/100’000)in2016 (1.1/100’000),whilein 2017 (0.9/100’000) and 2018 (0.6/100’000), this indicator was not achieved.Despitenotachievingtheindicators’goals,overall,the AFPSresultswereadequateinLombardyintermsofdataquality andtimeliness,completenessof caseinvestigation(>80%),and completeness of clinical follow-ups. Also, performances in Lombardy showed an overall improvement of AFPS compared tothe results obtained from 1997 to 2011 (Pellegrinelli et al., 2015) in the same surveyed area and those reached in other Italianregions(Fioreetal.,1999;Pattietal.,2000;Angelilloetal., 2001; D’Errico et al., 2008; Pellegrinelli et al., 2017a, 2017b;

Palandrietal.,2020).

TheAFPseriespresentedinthisstudyshowedepidemiological characteristicssimilartothosereportedinthecurrentscientiﬁc literature, withthe majority(60.5%) of patientsbeing younger thanﬁveyears.Itisworthyofunderliningthatnearly11%were hospitalized in intensive care units, and the remaining were reportedbyotherwards(Pattietal.,2000;Angelilloetal.,2001;

D’Errico et al., 2008; Pellegrinelli et al., 2017a, 2017b). The monthlydistributionofAFPcasesshoweda peakinNovember andDecemberwhen44.4%ofcases werenotiﬁed,in linewith what previously observed by others (Marx et al., 2000;

Pellegrinelli et al., 2017a, 2017b), even if NPEV-positive AFP cases are detected mainly in July when NPEV are generally detected(Pellegrinelli etal.,2017a,2017b;Brobergetal.,2018;

Bubbaetal.,2020).NowildPVwasdetectedduringthis3-year study, in line with what was observed at the national level, supportingtheresultthattheroutinePVimmunizationcontin- uedtoachieveitscoveragetargeteveryyear.

AftertheinterruptionofPVtransmission,GBSbecametheﬁrst causeofAFP(Angelilloetal.,2001;Barbadoroetal.,2020;Chen etal.,2020;D’Erricoetal.,2008;Marxetal.,2000;Pattiet al., 2000;Pellegrinellietal.,2017a,b),alongwithotherlessfrequently reportedclinical manifestations,suchas myopathy, unspeciﬁed causes of encephalitis, acute myelitis, and encephalomyelitis (Marxetal.,2000;Stefanellietal.,2019).Thiswasalsoobserved Figure1.TemporaldistributionofAFPcasesandNPEVsdetectedinLombardy

(NorthernItaly),fromJanuary1,2016,toDecember31,2018intheframeworkof AFPS.

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in our study, where GBS was the most common conclusive diagnosis,accountingfornearly42%ofAFPcases.

Unexpectedly,ﬁvechildrenpresentedAFM,a newlydeﬁned, raresyndromeclinicallydistinctfromAFP,thatgloballyemergedin recentyears,causingepidemics(Morensetal.,2019).Infact,since 2014 andin alternate years, anunexpectedand unprecedented upsurge of severe respiratory infections and AFM outbreaks temporally associated with re-emergences of NPEV has raised concern about NPEV infections. In particular, EV-D68 recently emergedasa causeof AFM(Messacaretal.,2018),EV-A71was implicated in outbreaks of brainstem encephalitis (Casas-Alba et al., 2017), and E-30 was associated with a wide meningitis- relatedoutbreakwithlong-termsequelaein2018(Brobergetal., 2018).

Overall,17.4% of AFP cases identified in this study resulted positivetoNPEV,thenmolecularlycharacterizedasE-25(2016;n= 1),EV-D68(2016;n=1–2018;n=1),EV-A71(2016;n=1),E-30 (2018;n=1)andEVuntypable(2017;n=2).Interestingly,both molecularand epidemiologicallinksobservedinourAFPseries overlapped what was observed in the same timeframe across Europe and the US. For instance, both EV-D68 and EV-A71 sequencesdetectedin2016inourstudyshowedhighsimilarities with theB3-clade identified in a DutchAFM outbreak in 2016 (Parianietal.,2017)andtothesubgenogroupC1,detectedin a Spanishbrainstemencephalitisepidemicin2016(Casas-Albaetal., 2017).This observation was also supported fromthe sequence analysesofNEPVsidentifiedin2018;infact,EV-D68identifiedin this studybelongedtotheB2sub-clade,sharinghighsimilarity withtheUSstrains(Morensetal.,2019),andE-30hadanucleotide identityof98.2–100%withsequencesbelongingtogroupG1that havebeencirculatinginEuropesince2016anddominatingin2018 (Brobergetal.,2018).

Todate,nosystematicNPEV hospital-and community-based surveillancesystemsaresetupinItaly;thus,informationonNPEV circulation is derived exclusively from observational studies, especially among childrenaffected bysevere conditions (Parisi etal.,2016;Pirallaetal.,2014;Pirallaetal.,2020).Nevertheless, withtheemergenceofseveralNPEVs-inparticularEV-D68,EV- A71, and E-30 - involved in severeneurological manifestations (Bubbaetal.,2020),AFPShasdemonstratedrenewedstrengthin continuing toprovideepidemiological evidenceto assistpublic healthefforts(Kimetal.,2014;Sureshetal.,2018;Bubbaetal., 2020; Roberts et al., 2020).Here, the unpredicted detection of emergingEV-D68,EV-A71,andE-30strainssharinghighnucleo- tide identitywithviralstrainsinvolvedintheEuropeanand US outbreaksidentiﬁedin thesameperiod,emphasizestheimpor- tance of monitoring NPEVs throughAFPS toassess appropriate publichealthresponses.

Inconclusion,AFPSprovedtobesuccessfulinPVsurveillance andrevealedwideNPEVscirculationlinkedwithotherconcurrent Europeanoutbreaks.Continuingthissurveillanceactivityiscrucial toensurethatPVwillberapidlyrecognizedifanewintroduction wouldoccurinpolio-freecountries;AFPSwasdemonstratedtobe ahelpfultooltomonitorNPEVscirculation,anditsenhancement should be encouraged as an early warning of future polio-like outbreaks.

Conﬂictofinterest

Theauthorshavenorelevantaffiliationsorfinancialinvolve- mentwithanyorganizationorentitywithafinancialinterestinor financialconflictwiththesubjectmatterormaterialsdiscussedin themanuscript.Thisincludesemployment,consultancies,hono- raria, stock ownership or options, expert testimony, grants or patentsreceivedorpending,orroyalties.

Fundingsource

Thisstudydidnotreceivefunding.

Ethicalapproval

ThisstudywasperformedaccordingtotheInstitutionalReview Boardguidelinesconcerningtheuseofbiologicalspecimensfor scientiﬁcpurposesincompliancewithItalianlaw(art.13D.Lgs196/

2003).Approvalfromanethicscommitteeandinformedconsent forvirusdetection anddatapublicationwas notrequiredsince data and samples from patients with AFP were collected and analyzed anonymously within the national AFP Surveillance Program.

Acknowledgments

Theauthorswouldliketothanksthephysiciansandhealthcare professionalsinvolvedinAFPsurveillance,allchildrenenrolledin thisstudyandtheirparents.

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