The bacterial lysate Lantigen B reduces the number of acute episodes in patients with recurrent infections of the respiratory tract: the results of a double blind, placebo controlled, multicenter clinical trial

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24 July 2021

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Original Citation:

The bacterial lysate Lantigen B reduces the number of acute episodes in patients with recurrent infections of the

respiratory tract: the results of a double blind, placebo controlled, multicenter clinical trial

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ContentslistsavailableatScienceDirect

Immunology

Letters

jo u r n 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 / i m m l e t

The

bacterial

lysate

Lantigen

B

reduces

the

number

of

acute

episodes

in

patients

with

recurrent

infections

of

the

respiratory

tract:

The

results

of

a

double

blind,

placebo

controlled,

multicenter

clinical

trial

Fulvio

Braido

a

_,

_Giovanni

_Melioli

a,∗

_,

_Piero

_Candoli

b

_,

_Andrea

_Cavalot

c

_,

Mario

Di

Gioacchino

d

_,

_Vittorio

_Ferrero

e

_,

_Cristoforo

_Incorvaia

f

_,

_Carlo

_Mereu

g

_,

Erminia

Ridolo

h

_,

_Giovanni

_Rolla

i

_,

_Oliviero

_Rossi

j

_,

_Eleonora

_Savi

k

_,

_Libero

_Tubino

l

_,

Giorgio

Reggiardo

m

_,

_Ilaria

_Baiardini

a

_,

_Eddi

_di

_Marco

n

_,

_Gilberto

_Rinaldi

o

_,

Giorgio

Walter

Canonica

a

_,

Lantigen

Study

Group

,

Carlo

Accorsi

c

_,

_Claudia

_Bossilino

i

_,

_Laura

_Bonzano

h

_,

_Michela

_DiLizia

d

_,

_Barbara

_Fedrighini

c

_,

Valentina

Garelli

a

_,

_Vincenzo

_Gerace

l

_,

_Sara

_Maniscalco

g

_,

_Ilaria

_Massaro

j

_,

Alessandro

Messi

b

_,

_Manlio

_Milanese

g

_,

_Silvia

_Peveri

k

_,

_Arminio

_Penno

c

_,

Stefano

Pizzimenti

i

_,

_Tiziana

_Pozzo

e

_,

_Alberto

_Raie

i

_,

_Sergio

_Regina

e

_,

_Francesca

_Sclifò

a

a_Allergy_&_Respiratory_Diseases_Department,_University_of_Genoa,_IRCCS_A.O.U._San_Martino_–_IST,_Lg._Benzi,_10,₁₆₁₃₂_Genova,_Italy b_Divisione_{Pneumologica,}_Ospedale_Lugo_di_Romagna_Viale_Dante,_10,₄₈₀₂₂_Lugo,_RA,_Italy

c_Ospedale_S._Croce_Moncalieri_(TO),_P.zza_A._Ferdinando,_3,₁₀₀₂₄_Moncalieri,_TO,_Italy

d_Dipartimento_di_Medicina_e_Scienza_{dell’Invecchiamento,}_Immunologia_e_medicina_del_lavoro,_Università_G._{D’Annunzio,}_Chieti_Via_dei_Vestini,_31,₆₆₁₀₀

Chieti,Italy

e_Ospedale_Gradenigo_Torino,_C.so_Regina_Margherita,_8,₁₀₁₅₃_Torino,_Italy f_Istituti_Clinici_di_{perfezionamento,}_Milano_Via_Bignami,_1,₂₀₁₂₆_Milano,_Italy

g_Centro_Asma,_Struttura_Complessa_Pneumologia,_Ospedale_Santa_Corona,_Via_XXV_Aprile,_38,_Pietra_Ligure,_SV,_Italy

h_Ambulatorio_di_Allergologia_Padiglione_Barbieri₂◦_piano,_Dipartimento_di_Scienze_Cliniche,_Università_degli_Studi_di_Parma,_Via_Gramsci,_14,₄₃₁₂₅_Parma,

Italy

i_Allergologia_e_Immunologia_Clinica,_Dipartimento_di_Scienze_Mediche_{dell’Università}_di_Torino_&_AO_Ordine_Mauriziano,_Largo_Turati,_62,₁₀₁₂₈_Torino,

Italy

j_Azienda_Ospedaliera_{Universitaria}_Careggi,_D.A.I._Biomedicina_–_S.O.D._{Immunoallergia}_PAD,_13,_Largo_Brambilla_3,_Firenze,_Italy k_UO_di_{Allergologia,}_AUSL_di_Piacenza,_Via_Taverna,_49,₂₉₁₀₀_Piacenza,_Italy

l_Ospedale_di_Chivasso_(TO),_C.so_Galileo_Ferraris,_3,₁₀₀₃₄_Chivasso,_Italy m_Biometry_unit,_Mediservice_srl,_Milano,_Italy

n_Laboratorio_di_Analisi,_Istituto_G._Gaslini,_Via_G._Gaslini_5,₁₆₁₄₇_Genova,_Italy o_Direzione_Medica,_Bruschettini_srl,_Via_Isonzo_5,₁₆₁₄₇_Genova,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Availableonline3November2014 Keywords:

Bacteriallysates

Recurrentrespiratorytractinfections Allergy

Prophylactictreatment Immune-stimulantdrugs

a

b

s

t

r

a

c

t

Studiesinthe1970sand1980sreportedthatbacteriallysates(BL)hadaprophylacticeffectonrecurrent respiratorytractinfections(RRTI).However,controlledclinicalstudyprocedureshaveevolved substan-tiallysincethen.WeperformedatrialusingupdatedmethodstoevaluatetheefﬁcacyofLantigenB®_,_a chemicalBL.Thisdoubleblind,placebocontrolled,multi-centerclinicaltrialhadtheprimaryobjective ofassessingthecapacityofLantigenBtosigniﬁcantlyreducethetotalnumberofinfectiousepisodesin patientswithRRTI.SecondaryaimsweretheRRTIduration,thefrequencyandtheseverityoftheacute episodes,theuseofdrugsandthenumberofmissedworkdays.Inthesubgroupofallergicpatientswith RRTI,thenumberofallergicepisodes(AE)andtheuseofanti-allergicdrugswerealsoevaluated.One hundredandsixtypatients,79allocatedtothetreatedgroup(TG)and81totheplacebogroup(PG),were enrolled;30werelostduringthestudyand120(79femalesand38males)wereevaluated.ThePGhad 1.43episodesinthe8-monthsoffollow-upwhiletheTGhad0.86episodes(p=0.036).Asimilarresultwas observedintheallergicpatients(1.80and0.86episodesforthePGandtheTG,respectively,p=0.047). ∗ Correspondingauthor.Tel.:+393465168826.

E-mailaddress:giovannimelioli@gmail.com(G.Melioli). http://dx.doi.org/10.1016/j.imlet.2014.10.026

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186 F.Braidoetal./ImmunologyLetters162(2014)185–193

Theuseofantibioticswasreduced(mean1.24and2.83daysoftreatmentfortheTGandthePG).Logistic regressionanalysisindicatedthattheestimatedriskofneedingantibioticsandNSAIDswasreducedby 52.1and30.6%,respectively.WithregardtothenumberofAE,nosignificantdifferencewasobserved betweenthetwogroups,butbronchodilators,antihistaminesandlocalcorticosteroidswerereducedby 25.7%,56.2%and41.6%,respectively,intheTG.LantigenBsignificantlyreducedthenumberofinfectious episodesinpatientswithRRTI.Thisfindingsuggestsafirstlineuseofthisdrugfortheprophylaxisof infectiousepisodesinthesepatients.

(http://creativecommons.org/licenses/by/3.0/).

1. Introduction

Recurrentrespiratorytractinfections(RRTI)arelessfrequentin adultsthaninthechildren,butthefrequencyofpatientsaffected remainssignificant[1].Thetherapyisbasedonanti-inflammatory drugsandantibiotics,andonlyrarelyisinfectionprophylaxis pro-vided.Bacteriallysates(BL)areafamilyof“biological”drugsthat wereintroducedin therapy manydecades ago,but only inthe last10yearshastheirmechanismofactionhasbeenelucidated. Sincethefirstinvivostudies,itwashypothesizedthattheclinical effectsobservedwererelatedtothecapacityofBLtoelicita spe-cificimmune-responseagainstbacterialantigens.Alongthisline, sincethemid1970s,specificIgAdirectedagainstbacterialantigens havebeenobservedinthesalivaryfluidoftreatedpatients[1–4]

followingtheoraladministrationofbacteriallysates.Duringthe 1990s,thedescriptionofthetoll-likereceptor(TLR)familyandits functions[5]withinthelargercontextofthepatternrecognition receptor(PRR)systemexplainedhowtheinnateimmunesystem wasalertedbythepresenceofdifferentstimuli,mainlyrepresented bybacterialderivedstructures.Inthatsameperiod,thecentralrole ofdendriticcells(DC)inthegenerationofanefficient immune-responsewasdescribed[6].Startingfromtheselinesofevidence, differentbacteriallysateswereshowntobeabletoinduce signif-icantactivationandmaturationofDCinvitro[7,8].Morerecently, usingaspecificmemberofthebacteriallysatefamily(the Poly-valentMechanicalBacterialLysate–PMBL),otherrelevanteffects wereobservedonBcells, Tcells andNK cells [9,10]and these effectswerealsocorrelatedwiththeclinicalresults[11,12]. Bacte-riallysatesareobtainedbyphysicalorchemicallysis.Theformer areingeneralcomposedoffragmentsofbacterialbodies.Thelatter maybeconstitutedbyeitheronlylowmolecularweightproteins oramixtureofbacterialbodiesandsolubleproteins.LantigenB belongstothelatterfamily,beingasuspensionofbacterial anti-gensobtainedfromStreptococcuspneumoniaetype3,Streptococcus pyogenesGroupA,Branhamellacatarrhalis,Staphylococcusaureus, HemophilusinfluenzaetypebandKlebsiellapneumoniae.Theinvivo effectsofLantigenBwerefirstdescribedinthemid1970s[11–13]. Sincethattime,fewstudieshavebeenperformedonthisspecific drug.Althoughthefirststudieswereconsideredobsoletefroma proceduralpointofview[14–16],thedrugremainedinthe phar-macologicalarmamentariumofbothgeneralpractitionersand spe-cialists.Inthiscontext,otherstudiesevenifnottotallyinlinewith modernclinicaltrialpracticehavebeenpublished[17–19],further demonstratingtheclinicalefficacyofLantigenB.Inparticular,the maineffectobservedwasthereductionofinfectionsduringthe observationperiod.Indeed,byanalyzingalltheabovementioned studies,itcanbecalculatedthatameanof1.73episodes/yearwere observedintreated patientsvs. 2.63/yearintheplacebogroup, correspondingtoareductionof34%ofthenumberofinfectious episodes.Inaddition,inthesamestudies,thereductionofcough, oflostdaysandantibioticsusewasalsoobserved.Ofnote,onlyone non-positivestudyonthecommoncold[20]waspublished,while alltheothersdemonstratedtheefficacyofthetreatment.

In this study, using modern and effective clinical practice tools, we re-evaluated the efﬁcacy of Lantigen B in a double

blind randomized placebo-controlled clinical trial in inducing a signiﬁcant reduction in the number of infectious episodes in an unselected (real life-like) population of patients with RRTI. This type of study is needed not only to verify previous results using an up-dated methodology but also for the avail-ability of drugs (such as third generation anti-histamine,local corticosteroidsand modern bronchodilators)that haverecently demonstrated a deep impact on the management of allergic patients who represent a signiﬁcant fraction of patients with RRTI.

2. Materialandmethods

2.1. Deﬁnitionofacuteinfectiousepisodes

Acuteepisodesoftheupperrespiratoryairwayswere identi-fiedbythecontinuouspresenceofrhinorrhea(bothsero-mucous andpurulent),pharyngitis,andcouch,lastingatleast48h,withor withoutfever.Acuteepisodesofthelowerrespiratoryairwayswere definedas thecontinuouspresenceof stridor, wheeze,crackles andrhonchi,anindrawingrespiratoryfrequency>50cycles/min,or cyanosislastingatleast48h,withorwithoutfever.Acuteepisodes ofotitisweredefinedasthecontinuouspresenceofpain,erythema andareductioninorlossoftympanicmembranemobility.Finally, anacuteinfectiousepisodewasdefinedasnewifatleast72hhad passedwithacompleteabsenceofsymptomsfromtheresolution ofthepreviousepisode.Theidentificationofaninfectiousevent wasbasedonpatient’sreport(feverorcoughordyspneaoruse ofdrugs)aswellasondoctor’sreport(visitsandtherapeutic indi-cations).Theprotocolprovidedthatwhenaninfectiousepisode occurs,thepatientmightcontacttotheprimarycarephysicianor thestudycenterthatenrolledher/him.Inbothcases,thediagnosis andthetherapywerereportedinthepatient’sdiaryandintheCase ReportForm.

2.2. Studydesign

Thiswasanationalmulti-centerlongitudinal,prospective ran-domized double blind versus placebo controlled clinical phase IV study, conductedon two parallel groups for an observation period of 8 months. The protocol EudraCT code was 2011-00-3229-76.Theprimarygoalwastheevaluationoftheefﬁcacyof theLantigen Btreatmentonthenumber of infectiousepisodes (IE) in thegroup of treated patients. Secondary goalswere (a) thetotalnumber ofdays withIE(ina6 monthfollow-up); (b) thefrequencyandseverityofepisodesofallergy(AE),in partic-ularasthmaandrhino-conjunctivitis;(c)thediseasefreeperiod evaluatedasthetimeofoccurrenceofanIEaftertheendofone ofthetreatmentcycles;(d)anyotherdrugconsumptioninthe 8monthperiod,includinganti-infectious,anti-inﬂammatoryand anti-allergicdrugs;(e)theenumerationofdaysofabsencefrom thecommunity(school,work);(f)thesafetyandtolerabilityofthe treatment.

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2.3. Studyapproval

The study was conducted in accordance with good clinical practicewasalsoapprovedbytheEthicalCommittee(EC)ofthe UniversityofGenovaandtheAOUSanMartino,referenceofthe InstitutionofthePrincipalInvestigator. Theother10ECsofthe remaining11centersalsoapprovedtheprotocol.

2.4. Studytiming

ThestudyrecruitmentstartedinSeptember2012andﬁnished attheendofDecember2012.Thefollow-upﬁnishedandthestudy wasclosedinSeptember2013.Theclinicalandstatisticaldatawere availablefromDecember2013.

2.5. Patientselection

Thestudy wascarriedout at 12different centers, represen-tativeof thedifferentItalian climates.Thisstudy enrolledmen and woman between the ages of 18 and 65 years. Inclusion criteria were: (a) patients complaining two or more infections of theupper respiratory in theyear precedingthis study, pos-siblyassociatedwithrespiratory allergy,(b)theabsenceofany type of malformation or other significant diseases (i.e., cancer, immunologicaldisorders),and(c)thecapacitytounderstandthe study and collaborate withthe investigators. Exclusion criteria were:(a)recurrentrespiratoryinfectionsinthelast7daysbefore enrollment;(b)acutediseases(eitherinfectiousornotinfectious) requiring hospitalization; (c) gastro-esophageal reflux; (d) cys-tic fibrosis, ␣1-antitrypsin defects or ciliar diskynesis; (e) any chronicsystemicdisease;(f)bodymass<3thpercentileforage; (g)autoimmunediseases;(h)anydrugssuchasimmunoglobulins, immunostimulants, antineoplastic drugs, cytokines, interferons, interleukins,immunosuppressors,orsystemiccorticosteroids;(i) knownallergyorintolerancetothestudyproductoritsexcipients; (k)patientswhocouldnotbecontactedbytelephoneduringthe study;(l)patientsrecruitedintoanyotherclinicalstudyinthelast monthbeforetheenrollmentorduringthestudy.

2.6. Randomizationprotocol

Therandomizedlistwasbasedonthe“RANUNI”random num-bergeneratoroftheSASsoftware(SASInstitute,Cary,NC,USA). Thetypewasa1:1randomizationoftwogroups.

2.7. Treatment

ThetreatedgroupreceivedLantigenB(BruschettiniSrl,)inoral drops.Lantigen Bis asuspensionofbacterialantigensobtained fromS.pneumoniaetype3,S.pyogenesgroupA,B.catarrhalis,S. aureus,H.inﬂuenzaetypebandK.pneumoniaeindistilledwater, pH7.30,containingundetectabledosesofchlorhexidinediacetate

(0.02mg/mL). Theplacebo control had thesame characteristics (namely, aspect, pH,and chlorhexidine),but the bacterial anti-genswerenotpresentinthebottles.A4-weekcycleoftreatment representedbyatotalof30dropsintwodifferentdaily admin-istrations(15+15),morningandevening,fasting,wasscheduled, followedbyanintervalof2weeksandanother4weeksof treat-ment.Aftera6-weekobservationperiod,twoadditional4-week treatmentswerescheduled,withanintervalof2weeks.Attheend ofthesecondtreatmentcycle,thepatientswerefollowed-upfor another6weeks.Table1showsthetreatmentschedule.During theperiodofthestudy,immune-stimulantdrugs,cytokines, inter-ferons,immune-suppressorsand anti-neoplasticdrugswerenot allowed.Systemicsteroids,ifusedcontinuouslyformorethan2 weeks,werealsonotallowed.Theuseofforbiddendrugscausedthe exclusionofpatientsfromthestudy.Anyotherdrugwasallowed andrecordedinthepatient’spersonaldiary.

2.8. Visits

Threevisitswerescheduledduringthetreatment:afirstvisit, whichincludedtheenrollment,asecondvisitbeforethebeginning ofthesecond4+4weekcycleandathirdvisitattheendofthe secondfollow-upperiod.Duringthefirstvisit,thepatientswere evaluatedtodefinewhethertheinclusionandtheexclusioncriteria weremet.Inpositivecases,thepatientswereaskedtobeenrolled intheclinicaltrial.Ifenrolled,inthesamevisit,thepatients under-wentaphysicalexaminationfortheevaluationofvitalsignsand they werefurther evaluated for theirlife style, clinical history and smoking habits.The enrolledpatientssigned theinformed consent and wereincluded in therandomization procedureby receivingthestudydrug.Allthepatientswerethentrainedinthe useofthedropdispenserandwereinstructedbytheinvestigator regardingwhenand howtotakethedailytreatment,according totheprovidedschedule.Finally,thepatientswereexhaustively instructedtocompileadailydiaryandwereaskedtorecordany relevanteventsthatoccurred.Thus,inthepresenceofadeclineina patient’sconditions,thesymptomswererecordedandtheintensity wasevaluatedusinga4-pointscalefrom0(absence)to3 (pres-enceofseveresymptoms).Theadministereddrugs,medicalvisits andpossiblehospitalizationwerealsorecordedinthediary.The patientswerealsoaskedtocarefullyrecordtheuseofthestudy drugaccordingtotheprovidedschedule.Theywerealsorequired toreturnthebottlesandthecontainingboxestotheinvestigatorat theirnextvisit.Duringthesecondvisit,theinvestigatorsrecorded anyadversereactions,hospitalizationperiods,modificationsoflife style,workorschoolhabitsanddruguse(inparticularantibiotics, anti-inflammatorydrugsetc.).Atthesamevisit,thefirstphasediary wasretiredandbothinfectiousandallergicepisodeswererecorded intheCaseReportForm(CRF).Theremainingdrugwasalsoretired andthedrugforthesecondcyclewasgiventogetherwiththe sec-ondpartofthediary.Thethirdandfinalvisit(attheendofthe secondperiodofobservation)wasusedtorecordthesame

param-Table1 Studyplan. Firstcycle Month 0 1 2 3 4 Week 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Treatment * * * * * * * * Visit X X Secondcycle Month 5 6 7 8 End Week 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Treatment * * * * * * * * Visit X

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188 F.Braidoetal./ImmunologyLetters162(2014)185–193 etersasthesecondvisitandtoretiretheremainingdrugandthe

diary.

2.9. Patientconsentwithdrawal

Patientswereallowedtowithdrawfromthetreatmentforany reason.Whenpossible,theadversereactionsandtheuseofdrugs wererecordedfortheretiredpatientsandthepersonaldiarywas retired.AnyrelevantinformationwasrecordedintheCRF. 2.10. Patientcompliancecontrol

Thecompliancetothetreatmentwas evaluatedby compar-ingtheregistereddosesandtheamountofremainingdruginthe bottles.

2.11. Safetyassessments

The patients and the investigators were asked to monitor thepossibleoccurrence of adverse reactions(ARs).These were classifiedascertain,likely, possible,unlikely, unrelatedand not evaluable,accordingtothedefinitionoftheprotocol.ARswerealso classifiedasslight(ifnotinterferingwithdailyactivities), interme-diate(iftheARinterferedwiththedayactivities)andsevere(ifthe ARinhibiteddailyactivities).

2.12. Exclusionofpatientsfromthestudy

Patientswere excludedfromthe studyin thepresence of a severeARincompatiblewiththecontinuationofparticipationin thestudy.

2.13. Viro-epidemiologicalsurveyofthestudyperiod

Thefrequencyofviralinfectionsinthegeneralpopulation dur-ingtheperiodofthestudyandthepreviousyearwasevaluated.The resultsofmolecularbiology-basedresearchoninﬂuenzaviruses, para-inﬂuenzaviruses,bocaviruses,coronaviruses.Rhinoviruses, enteroviruses,adenovirusesandRespiratorySyncytialviruswere recorded.Thefrequencyofviralinfectionswasbasedonthe per-centageofpositivesamplesasdescribed[10].Thus,anincreasein thepercentagesofpositivesampleswasconsideredrepresentative ofanincreasedcirculationofpathogensinagivenperiod. 2.14. Statisticalanalysis

For the sample size calculation, a total of 190 patients (85 patientspergroup)wererequiredtoberecruited,byconsidering anexpecteddropoutof15%ofthepatientsenrolledwiththe inten-tiontotreat(ITT).Thus,atotalof160patientswereexpectedtobe evaluable.TheprimaryefficacyanalysiswasbasedontheITT.The efficacyofthetreatmentonthetwogroupswasevaluatedbyan analysisofcovariance(ANCOVA).Forthisanalysis,thetotal num-berofinfectiveepisodeswasthedependentvariable,thetreatment groupwasthefactorand thenumberofpreviousyearinfective episodeswasthecovariate.IntheANCOVAmodel,theadjustment forbaselinecovariateswasperformed.Thesubgroupanalyseswere performedincompliancewiththerulessuggestedintheEuropean MedicineAgencyGuidelineontheinvestigationofsubgroupsin confirmatoryclinicaltrials.Binaryvariablesmeasuringtheeffects oftreatmentonthesecondaryobjectiveswereanalyzedbya2

test.Finally,alogisticregression(multivariate)analysiswasused toevaluatetheprobabilityofusingdrugs:antibioticsandNSAID, forthewholepopulationandlocalcorticosteroids,antihistamine drugsandbronchodilatorsforthesubsetofallergicpatients.All

Table2

Clinicalcharacteristicsofthepatients,numberofpatientswithagivendiseasesand numberofepisodesofdiseaseintheperiodofthestudy(2012–2013).

Patientswithdisease Numberofdisease episodes

Placebo LantigenB Placebo LantigenB

Otitis 2 1 2 1 Tonsillitis 5 2 7 2 Sinusitis 9 3 18 6 Pharyngotonsillitis 17 11 23 15 Rhinopharyngitis 20 18 32 21 Bronchitis 15 8 23 12 Pneumonia 0 1 0 1 Total 68 44 105 58

thesestatisticswerecarriedoutbyusingSPSSondataextracted fromtheCRFs.

3. Results

3.1. Studypopulation

Atotalof160patients(79inthetreatedgroupand81inthe placebogroup)wereenrolled.Theageoftheplacebogroupwas 42.4±15.14(mean±standarddeviation)andtheageofthetreated groupwas42.4±13.94years.Onehundredandseventeenpatients (58inthetreatedand59intheplacebogroup)completedthestudy (Fig.1).ThedropoutwasobservedbothintheLantigenBgroup (17patients)andintheplacebogroup(23patients).Somepatients werelostforinadequatecomplianceorviolationoftheprotocol (sevenpatients),threeforadversereactions,ﬁvewerelostinthe follow-upand23(correspondingtothe57.5%)forconsent with-drawal.Finally,twowerelostforotherreasons.Inpatientswho completedthestudy,thegenderswereequilibratedbetweenthe placebo(20malesand38females)andinthetreatedgroup(18and 41,respectively)(2₌_0.21,_p₌_0.65)._Table₂_,_in_the_lanes_related_to

thereferenceperiod(2011–2012)showstheclinicalcharacteristics ofthesepatients,inlinewiththeeligibilitycriteria.

3.2. Post-hocpower

Thenumberofpatientsrecruitedandthenumberofdrop-outs couldhave impactedthe powerof the studyresults. However, becausethedifferencesbetweentheplaceboandtreatmentwere significantandtheefficacyofthetreatmentwashigherthancould havebeenexpectedonthebasis ofthepublishedliterature,the post-hocpowerofthestudywasconfirmed.Indeed,asamplesize of58ineachgrouphadan84%powertodetectaprobabilityof0.34 thatanobservationintheLantigenBgroupwaslessthanan obser-vationintheplacebogroup(WilcoxonRank-sumtestwitha0.05 twosidedsignificancelevel).Ofnote,apowerof80%isconsidered acceptablebythescientificcommunityforclinicalstudies.Thus,the originaldesignofthisstudyhadapowersuitabletoachieve com-pletestatisticalsignificanceeveninthepresenceofaconspicuous drop-outoftheenrolledpatients.

3.3. Theprimaryobjective

Theprimaryobjective ofthestudywasthereductionofthe numberofIEduringan8monthsfollow-upperiod.Inthewhole groupofenrolledpatients,themeanfrequencyofIEintheyear precedingthestudywas3.66/year(C.I.3.38–3.93).Thefrequency was3.34/yearformalesandslightlyhigher,3.81/yearforfemales, correspondingtoamean 2.56IEin 8months(2.5and 2.9 in8 months,respectively).ThedistributionofIEbetweentheplacebo

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Fig.1.TheConsort2010ﬂowchart.

and treatedgroups was3.78and 3.54, respectively. Duringthe periodofthestudy,theplacebogroup(58patients)experienced a mean of 1.43(C.I. 1.01–1.86)episodes in the8 month study period,while0.86(C.I.0.54–1.19)episodeswererecordedinthe 59patientsoftheactivegroup(t-test=2.129,p=0.036).Tofurther supportthisdifference,anon-parametricanalysisofthenumber ofinfectiousepisodesshowedamedianvalueof0inthetreated groupand 1 in theplacebo group. Tobetter deﬁnethis result, thedifferenceinepisodesin thesinglepatientswasalso evalu-ated.For this, thefrequencyof thepatientswith0,1,2 etc.IE wasanalyzed.Fig.2showsthefrequencyofIEin thepre-study population, inthe LantigenBtreated groupand in theplacebo group.TheplacebogrouphadmoreIE/patients(2₌_14.7,_p₌_0.023)

whencomparedwiththeactivegroup.Withregardtothediseases sufferedbythepatients,thefrequencywas16%for pharyngoton-sillitis(PT),36%forrhinopharyngitis(RP)and22%forbronchitis (B);theremaining26%wereotitis,tonsillitis,sinusitisand,rarely, pneumonia.AfterthetreatmentwithLantigenB,thetotal num-berofinfections wassignificantlyreduced,asstatedbefore,but therelativefrequencyofdifferentclinicalpicturesremained vir-tuallyunmodified(25%forPT,41%forRP,18%forB),whileothers wereslightlyreducedto16%(Table2).Asubgroupanalysisofthose patientssufferingfromrespiratoryallergy(22intheplaceboand 22inthetreatedgroup,accordingtotheevidencecollectedduring theeligibilityvisit)wasalsoperformed.Ofthesepatients,only24 hadallergicepisodesduringthestudyperiod:9hadoculorhinits, 11hadasthmaand4hadboth.Ofnote,thefrequencyofIEinthose patientswithallergywasslightlyhigher(3.80IE/8months)thatin thenon-allergicpatients(3.58IE/8months)evenifnotsignificant (t=−0.81,p=0.42).Liketheoriginalpopulation,non-significantly differenteffectswereobservedwhenthegender(12malesand32

A

B

C

Fig.2. PercentagesofIEepisodes(Yaxis)distributedpernumberofepisodes(from 0to9)(Xaxis)duringthestudyperiod.Panel(A)Pre-studyresults.Panel(B)Placebo groupresults.Panel(C)LantigenBgroupresults.Thearrowsindicatethetwozero valuesrepresentingtheeligibilitycriteriaofthestudy(i.e.,eligiblepatientshad >2episodesofrespiratoryairwaysinfectionsinthepreviousyear).Both0and 1episodeswereabsentinthe“pre-study”periodbecauseofthestricteligibility criteria.Duringthetreatment,thesetwogroupsappeared.

females)oftheallergicpatientswasevaluated.Similarly,no sig-niﬁcanteffectsonthetreatmentresultswereobservedwhenthe smokinghabitsandthedifferentclinicalcenterswereevaluated (ANCOVA,F=1.473,p=0.227andF=1.817,p=0.087,respectively).

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Table3a

Adversereactionintheactivegroup.

Patient Adversereaction Seriousness Intensity Action Relationshipswiththedrug

2 Headache Notserious Light None None

2 Dryandburningmouth Notserious Intermediate None Possible

31 Trauma,leftankle Notserious Light None None

32 Nasopharynxbiopsy Serious Light None None

35 Persistentcough Notserious Light None None

57 Hypothyroidism Notserious Light None None

85 Vaginalcandidiasis Notserious Light None None

85 Dermatitis Notserious Intermediate None None

85 Urinarytractinfection Notserious Intermediate None None

94 Arthralgia Notserious Intermediate None None

112 Lungcancer Serious Severe Discontinuationoftherapy None

123 Headache Notserious Light None None

123 Backache Notserious Light None None

123 Nausea Notserious Light None None

123 Gastro-esophagealreﬂux Notserious Light None None

123 Backache Notserious Intermediate None None

3.4. Secondaryobjectives

ThedurationoftheIEinthetwogroupswasdifferent(10.14 daysintheplaceboand5.83intheLantigenBgroup),buta statisti-calsignificancewasnotachieved(F=2.724,p=0.102).Theintervals betweenIEwereevaluatedattheendofthefirstandsecond treat-mentcycle.Inthefirstinterval,thedifferencesinthenumberof events(eightintheplaceboandfiveinthetreatedgroup)aswell asthemeanintervals(indays)werenotsignificant.However,the cumulativehazardofsufferingfromanIEwas15%fortheplacebo and9%fortheLantigenBgroup.Similarresults,evenifnot sta-tisticallysignificant,wereobservedforthesecondinterval(sixEI fortheplaceboand twofortheLantigenBgroup).Therelevant cumulativehazardsofdevelopinganIEinthesecondintervalwere 0.11and0.035forplaceboandLantigenBgroups.Theconcomitant druguseinthetreatedandinthecontrolgroupswasalso eval-uatedbyalogisticregressionanalysis.Forthis,threefamiliesof drugstheuseofantibioticsandNSAIDswasevaluatedinthewhole population,whileanti-allergicdrugs,whichincluded bronchodila-tors,local corticosteroidsand anti-histaminewereevaluated in the subset of allergic patients. No significant differences were observedbetweenthetreatedandcontrolgroups,evenifaclear trendwasevident:indeed,thetreatedpatientswerealwaystreated less. Despite the logistic regression analysisdemonstrated that thedifferenceswerenotsignificant(p=0.421,p=0.134,p=0.283, p=0.519andp=0.667forantibiotics,NSAID,anti-histamine,local corticosteroidsandbronchodilators,respectively),thesame statis-ticsshowedthatprobability(Exp(B),alsocalledtheodds-ratio)of usingantibioticswasreducedby52.1%(beingExp(B)=0.479)in thetreatedpatients,ofNSAIDby30.2%(Exp(B)=0.698),of anti-allergicdrugsby56.2%(Exp(B)=0.438),ofcorticosteroidsby41.2% (Exp(B)=0.588)andofbronchodilatorsby25.7%(Exp(B)=0.734)in thetreatedgroups.Thus,notonlythelogisticregressionshowed thatforallthedrugsthetrendwastowardareduction,but fur-therconfirmedthesignificanceofthereductionofthenumberofIE (p=0.021andp=0.044,forthewholegroupandthesubsetof aller-gicpatients).Finally,inbothgroups,themeandaysofabsencefrom workwereverylow(0.79forplaceboand0.74daysforLantigenB, p=0.925).

3.5. Adversereactions(ARs)

WithregardtotheAR,21wererecordedforLantigenBand23for placebo.TheARsaredescribedindetailinTables3aand3b.Brieﬂy,

theARoftheLantigenBgroupoccurredin10differentpatients, whileto17differentpatientsintheplacebogroup.SomeARcould berelatedtotheadministration ofLantigen Band theplacebo, suchasa dryand burningmouth (twoepisodesin onepatient of thegroup),stomatitis, odinophagia, andoropharynx burning (fiveepisodesintheplacebogroup). ThelastthreeARrequired thesuspensionofthetherapy.OtherARincludedhypothyroidism, distortionoftheleftankle,arthralgia,lungcancer,headache, uri-narytractinfection,vaginalcandidiasis,viralgastroenteritis,oral herpes,hypertension,nauseaandgastro-entericrefluxandwere notrelatedtothetreatment.Onlylungcancerrequiredthe sus-pensionofthedrug.TheARswerealsoclassifiedaccordingtothe severityandtheneedfortherapyinterruption(TI).Intheplacebo group,the23ARsweredividedinto11slight(1requiringTI),10 intermediate(requiringTI)and2severe,bothinterrupted.Inthe LantigenBgroupwere12,7and1(requiringTI),respectively. 3.6. Epidemiologicalsurveysofthestudyperiod

Duringthestudyperiod(namely,September2012–June2013), thecirculationofrespiratoryviruses(bothinqualitativeand quan-titativeterms)waslargelysuperimposableonthatoftheprevious year,usedasthereferenceintervalforthecalculationofthe num-berofIEforpatientrecruitment.Fig.3showstheresultsofthis survey:theRSVandinﬂuenzaviruseswereclearlypresentinthe December–Aprilintervalofthetwodifferentperiods,whileother viruses,suchasadenovirusesandrhinovirusesweredetectedin theremainingperiod.

4. Discussion

Inthisreport,theefﬁcacyofachemicalbacteriallysate, Lanti-genB,inthereductionofthenumberofIEinpatientswithRRTIhas beenclearlydemonstratedusingarigorousscientiﬁcandclinical approach.Indeed,thisphaseIVstudywasconductedat12different centers,usingadoubleblind,placebocontrolledstudyprotocol. Theauthorsareawarethatfewstudiesontheeffectsofbacterial lysateshavebeenconductedinthepast,usingcontrolledclinical trials in GoodClinical Practice (GPC) trained hospitals. For this study,theforeseennumberofpatientstoberecruitedwas190and 30wereexpectedtobelostduringthestudy.However,theclinical centers,eventhoughstronglycommitted,wereabletorecruit160 patients.Ofthese,morethan25%werelostandonly117patients couldbeevaluatedattheendofthestudy.Thelossofone-fourthof

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Table3b

Adversereactionintheplacebogroup.

Patient Adversereaction Seriousness Intensity Action Relationshipswiththedrug

4 Dryandburningmouth Notserious Light None None

5 Dryandburningmouth Notserious Light None None

25 Candidiasis Notserious Intermediate None None

25 Surgery(lipomaremoval) Notserious Intermediate None None

26 Hypothyroidism Notserious Intermediate None None

26 Pre-menopausalsyndrome Notserious Light None None

42 Dentalabscess Notserious Intermediate None None

43 Constipation Notserious Light Discontinuationoftherapy Unlikely

65 Backache Notserious Light None None

65 Odinophagia Notserious Severe Discontinuationoftherapy Possible

81 Dyspepsia Notserious Intermediate None None

81 Viralgastro-enteritis Notserious Intermediate None None

81 Dyspepsia Notserious Light None None

89 Hypertension Notserious Intermediate None None

97 Fiparthroplasty Serious Severe Discontinuationoftherapy None

100 Oropharynxburning Notserious Intermediate Discontinuationoftherapy Possible 100 Oropharynxburning Notserious Intermediate Discontinuationoftherapy Possible 122 Stomatitis Notserious Intermediate Discontinuationoftherapy Possible

133 Oralherpes Notserious Light None None

161 Thoracoalgia Notserious Light None None

165 Migraine Notserious Light None None

168 Conjunctivitis Notserious Light None None

Fig.3. Thecumulativefrequenciesofvirusisolationinthereferenceperiod(2011–2012)andinthestudyperiod(2012–2013)arerepresentedashistograms.Itisevident theincreasedfrequencyofvirusinfectioninthelatewinter–earlyspringperiods.

thepatientcohortisarelevantphenomenon.Despitethis reduc-tion,thepost-hocanalysisshowedthatthenumberofevaluable patientswasstillsufficienttomaintainthestatisticalrelevanceof thestudyandthusthevalidityoftheobservedresults.Moreover, theadverse reactionsrecordedaswellasthecausesofdropout wereextremelyheterogeneousandallvirtuallyunrelatedtothe treatment.AsignificantreductioninthenumberofIEwasobserved inthetreatedgroup:anaverageof0.86episodesinthe8month studyperiodwasobservedintheLantigenBtreatedpatients,while intheplacebogroupthemeanwas1.43episodes.Inthiscontext, itshouldbenotedthat,forthepopulationrecruited,theaverage number of previousIE was 5.34/year (corresponding to 3.56/8 months).Asstatedbefore, intheperiodofthestudy,themean numberofIEintheplacebogroupwas1.43/8months(females1.24 IE,males1.80IE).Thisdifferencemayhavedifferentexplanations. Differentweatherandepidemicconditionsinthetwosubsequent years,differencesinwinterandspringclimatessuchasawarmer temperatureora lessaggressivefluvirusepidemicmayreduce thenumberofrespiratory tractinfections, allowingtheplacebo

tomimicapharmacologicallypositiveeffect.Moreover,acertain susceptibilitytotheeffectsofaplacebo(moreevidentinfemales thaninmales)andacertaintendencytoincreasethenumberof IEinbothgroupswhendescribingtheseverityoftheirconditions mayhave alsoinfluenced thestudyresults. Withregard tothe differentperiodepidemiology,itshouldbenotedthatthesurvey ofvirusinfectionsinthestudyseason,whencomparedwiththat oftheprevious“cold”season,showedvirtuallyidenticalresults, withthemainviruses(influenza,rhinoviruses,adenovirusesand RSV)havingthesameprevalenceinthetwoperiod.Therefore,this findingdid notsupport thehypothesis of different“infectious” environments causing a different number of respiratory tract infectionsinthestudypopulation.Thereductioninthenumber ofIEintheplacebogroupmayhavedependedonawell-known bias,theHawthorneeffect.Thisphenomenon,originallydefined in anindustrial setting[23] hasbeenextended totherealmof medicalresearch[24,25]andsuggeststhatthesubjects’behavior or thestudyresultsmaybe alteredby thesubjects’awareness thattheyarebeingstudiedoriftheyreceivedadditionalattention.

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Itshouldalsobenotedthatrecruitmentwasbasedonthe num-berof respiratory tract infections in the previousyear.Even if notgeneralized, a certain tendencyof patientsto(involuntary) increasetheirreportednumberofinfectiousepisodesmighthave occurred,andthisfactshouldatleastinpartexplainthedifferences observed. However, toovercome these problems, double blind placebocontrolledstudies(likethepresentone)aremandatory tomeasurethedifferences betweenthetreatmentand placebo inthesameperiodoftheyearandinthesamelocations.Inthis context,theﬁndingthatneitherthedoctorsnorthepatientsreally recognizedthesuperioractivityofthetreatmentisalsointriguing. Ofcourse,thepositiveeffectsoftheplacebo,asdiscussedabove, hadacertainrole.But,moreimportantly,thedifferencesobserved were homogenously distributed between the placebo and the treated groups. This clearly means that the positive effects of the administration of bacterial lysates could be observed at a populationlevelwhiletheywerelessevidentatindividuallevel. Inaddition,thiscanalsoexplainwhythemedicalcommunityis sometimesskeptical aboutthe useof these drugs. Indeed,just using personal daily experience and feelings, it is difﬁcult to establishwhetherthisfamilyofdrugsisactive.

Someinteresting,ifinconclusivedatacanbederivedfromthe analysisoftheuseofthedrugsinthetwogroups.Nostatistically significantdifferences wereobserved betweenthe placebo and thetreatedgroupsforantibiotics,NSAIDs,bronchodilators, anti-histamineandlocalsteroids.However,acleartrendwasobserved inthetreatedgroups;theuseofdrugswasalwaysreducedinthis group.Withregardtothesmallgroupofallergicpatients,thefirst observationwasthatinthepopulationofRRTIpatients,patients withallergysymptomsrepresented40%ofthetotal.Itwasevident thatthetreatmentwithLantigenBdidnotsignificantlyalterthe numberofallergicepisodesorthenumberofdayswithallergy.This findingwasnotexpectedevenifthestrictcorrelationbetweenthe allergysymptomsandrespiratoryinfectiousdiseasescouldsuggest apotentialpositiveeffect[20].Acertaineffectwasobserved,asa trend,intheuseofanti-allergydrugs:indeed,inallergicpatients, logisticregressionshowedthattheprobabilityofusingdrugs spe-cificforallergywasreduced.Somecriticismcanberaisedonthis study:(a)thenumberofpatientsrecruited,evenifstatistically cor-rect,isnotverylarge;(b)duringpatient’srecruitmentandfollow up,functionalandlaboratorytestswereperformedonlyifsomeIE oradversereactionoccurred;(c)concomitantdiseases,potentially mimickingsymptomsoftherespiratorytract(suchasunknown primaryimmunedeficiency–undiagnoseddefecthoweverrarein astudycohortagedbetween18and65years)werenotspecifically considered;(d)thenon-perfectsynchronismof therecruitment (startedin SeptemberandclosedinDecember2012) associated tothe8-monthfollow-up, thatcouldatleastin partdilutethe effectsoftypicalinfectionsofthewinter–springperiod.However, strengthsofthestudyareseveral.In particular,(a)therigorous randomization homogeneously distributed theabovementioned biasesinthetwocohorts;(b)thestatisticalanalyses(ANCOVAand logisticregression)notonlydocumentedtheeffects ofLantigen Bonthenumber ofIE,butalsoidentified acleartrend toward reductionoftheuseofconcomitanttherapiesinLantigenBtreated patients;(c)thereallife-likeenrollmentofpatients(asstatedby theheterogeneityofadversereactionsnotrelatedtothe admin-istrationofthedrug)isrepresentativeofthepopulationthathas dailyaccesstotheofficesofgeneralpractitionersandspecialists: thus,theobservedresultsarereliable.

Inconclusion, theadministrationofLantigen B, evaluatedin adoubleblindcontrolledclinicalstudy,resultedinasigniﬁcant reductioninthenumberofIEinthetreatedgroup.Inparticular, notonlywasthefrequencyofIEreducedatthepopulationlevel but alsoat thelevel of theindividual, thenumber of episodes waslowerinthetreatedthanintheplacebogroup.Thecontrol

of respiratory tract infections, at least in the familial or work environment,resultsinareducedspreadofthepathogenandina reduceduseofdrugssuchasantibiotics,withevidentadvantages forthepatientandthecommunity.Inconclusion,thedescribed results, together withthe significant reduction in concomitant therapiesandthevirtualabsenceofanyadversereactions, indi-catesthat this drugmaybeused asan effectivefirst linedrug fortheprophylaxisofinfectiousepisodesinpatientswithRRTI. Thecapacityofcontrollingrespiratorytractinfectionsisafinding particularlyrelevantinthisperiod,whennotonlyaglobalincrease ofrespiratoryinfectionsisrecorded[24],butalsonewantibiotics willnotbeavailableonthemarketforthenextyears[25].

Conﬂictsofinterest

G.Rinaldi,MDdeclaresthatheisfull-timeScientiﬁcDirector ofBruschettiniS.r.l.;G.Melioli,MDisanadvisorofBruschettini S.r.l.forResearchandDevelopment.Theauthorsreportnoother conﬂictsofinterestinthiswork.

Acknowledgment

This study was ﬁnancially supported by Bruschettini S.r.l., Genova,Italy.

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