Little, M. Manuscript · 2017. 12. 13. · Clostridium difficile infection (CDI) is the result of...

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©TheAuthor2014.PublishedbyOxfordUniversityPressonbehalfoftheInfectiousDiseasesSocietyofAmerica.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.

Serummannose‐bindinglectinconcentration,butnotgenotype,is

associatedwithClostridiumdifficileinfectionrecurrence:a

prospectivecohortstudy

SwaleA.1,2,#,MiyajimaF.1,2,#,Kolamunnage‐DonaR.3,Roberts,P.2,Little,M.1,2,

Beeching,N.J.2,4,5,Beadsworth,M.B.J.2,Liloglou,T.6,PirmohamedM1,2

1TheWolfsonCentreforPersonalisedMedicine,UniversityofLiverpool,Liverpool,UK

2TheRoyalLiverpoolandBroadgreenUniversityHospitalsNHSTrust,Liverpool,UK

3DepartmentofBiostatistics,UniversityofLiverpool,Liverpool,UK

4LiverpoolSchoolofTropicalMedicine,Liverpool,UK

5NIHRHealthProtectionUnitinGastrointestinalInfections,Liverpool,UK

6CancerResearchCentre,UniversityofLiverpool,Liverpool,UK

Correspondence:[email protected]

#Theseauthorsequallycontributedtothisstudy

Summary

Lowmannose‐bindinglectinconcentration,butnotgenotype,wasassociatedwith

diseaserecurrenceinalargeprospectivecohortofpatientswithClostridiumdifficile

infection(CDI).

Clinical Infectious Diseases Advance Access published August 28, 2014 at L

iverpool University L

ibrary on August 29, 2014

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Abstract

Background:Mannose‐bindinglectin(MBL)playsakeyroleintheactivationofthe

lectin‐complementpathwayofinnateimmunityanditsdeficiencyhasbeenlinkedwith

severalacuteinfections.However,itsroleinpredisposingto,ormodulatingdisease

severityin,Clostridiumdifficileinfection(CDI)hasnotbeeninvestigated.

Methods:Weprospectivelyrecruited308CDIcasesand145controlspatientswith

antibiotic‐associateddiarrhea(AAD).CDIoutcomemeasureswerediseaseseverity,

durationofsymptoms,30‐daymortalityand90‐dayrecurrence.Serumconcentrations

ofMBLweredeterminedusingacommercialELISAassaytransferredtoan

electrochemiluminescence(ECL)‐basedplatform.MBL2polymorphismsweretyped

usingacombinationofpyrosequencingandTaqmangenotypingassays.

Results:ThefrequencyoftheMBL2geneticvariantswassimilartothatreportedin

otherCaucasianpopulations.MBLserumconcentrationsinCDIandAADsubjectswere

determinedbyMBL2exonicvariantsB,CandDandthehaplotypes(LYPB,LYQCand

HYPD).TherewasnodifferenceineitherMBLconcentrationsorgenotypesbetween

CDIcasesandAADcontrols.MBLconcentration,butnotgenotype,wasadeterminantof

CDIrecurrence(oddsratiosof3.18(95%CI1.40‐7.24)and2.61(95%CI1.35‐5.04))at

the<50ng/mland<100ng/mlcut‐offpoints,respectively;p<0.001).However,neither

MBLconcentrationnorMBL2genotypewaslinkedwiththeotherCDIoutcomes.

Conclusion:SerumMBLconcentrationdidnotdifferentiatebetweenCDIcasesandAAD

controls,butamongstCDIcases,MBLconcentration,butnotgenotype,wasassociated

withCDIrecurrence,indicatingMBLactsasamodulatorofdisease,ratherthana

predisposingfactor.

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Introduction

Theinitiationandpropagationofinflammatorycascadesisanessentialhousekeeping

propertyoftheinnateimmuneresponseduringinfections.Mannose‐bindinglectin

(MBL)activatesthelectin‐complementpathwayofinnateimmunitythroughbindingto

repetitivesugararraysonmicrobialsurfaces[1].MBLisalsoapotentregulatorof

inflammatorypathways:itcanmodulatephagocyteinteractionwithmucosalorganisms

atthesiteofinfection[2],andinteractswithothercomponentsoftheinnateimmune

systemsuchastoll‐likereceptors[3].

LowMBLconcentrationshavebeenassociatedwithincreasedsusceptibilityto

infectionsinbothanimalmodelsandhumans[4,5],aswellaswithpoordisease

prognosis[1].Themodulationofdiseaseseverityispartlythoughttobethrougha

complex,dose‐dependentinfluenceoncytokineproduction[6].SerumMBL

concentrationsrangefromnegligibletoashighas10,000ng/ml[7‐9];thisvarieswith

ethnicityandwiththescreeningmethodadopted[10].

MBLsecretioninhumansisdependentontheMBL2geneticarchitecture[11,12].To

date,57geneticvariantshavebeenidentifiedwithintheentireMBL2gene(SNP

database,Build140),withonlysixofthemknowntoaffectsecretionand/orfunctionof

theencodedprotein(Figure1)[8,13].ThemutatedallelesB,CorDarecollectively

termedOandtheircorrespondentwild‐typeallelesarejointlyreferredtoasvariantA,

withthepresenceofanygivenOvariant(ineithertheheterozygousorhomozygous

state)resultinginMBLdeficiency[8,13].Theexistenceofstronglinkagedisequilibrium

(LD)betweenthepromoterandstructuralgenevariantsmeansthatonlysevencommon

haplotypes(outofapossible64),whichmayaffectserumconcentrations,havebeen

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described:HYPA,LYQA,LYPA,LXPA,HYPD,LYPBandLYQC[14,15].HYPD,LYPBand

LYQCleadtotheproductionofunstableligandswithshorterhalf‐livesthatareeasily

degradedtoloweroligomericforms.Studiesthathaveevaluatedbothgeneticmutations

andserumconcentrationsinWhitesaresummarizedinSupplementaryTable1.

Clostridiumdifficileisanopportunisticspore‐formingbacteriumthatcaneffectively

colonisetheintestinaltractfollowingantibiotic‐drivendysbiosis.Clostridiumdifficile

infection(CDI)istheresultofintensecolonicinflammationcausedbythereleaseof

potententerotoxins.ResearchintohostbiomarkersforCDIhasfocusedonmediatorsof

inflammationinthegutsuchasfaecalinterleukin‐8[16],lactoferrin[16]and

calprotectin[17],andlinkedthemwithdiseaseseverity[16,18].Morerecently,both

seruminterleukin‐23andprocalcitoninhavealsobeenproposedaspotential

biomarkersforCDIseverity[19,20].However,theroleofthesebiomarkersinthe

stratificationofproblematicCDIpatientsremainsunclear,andthusremainsan

importantareaofresearch.Additionally,severalclinicalpredictionruleshavebeen

proposedfortheevaluationofCDIoutcomes[21‐23],butnonehavegainedwidespread

clinicalacceptance.

Todate,therehavebeennostudiesontheroleofeitherMBLlevelsorMBL2genetic

variantswithCDI,possiblybecauseMBLisnotthoughttobindtothesurfaceofC.

difficile[24].However,thereisgrowingevidenceforanassociationbetweenMBLand

majormodulatorsofinflammation,suchastoll‐likereceptorsandCRP,bothofwhich

havebeenassociatedwithCDI[25,26].Therefore,wesoughttoinvestigatetheroleof

MBLinaprospectivecohortofCDIcasesandinpatientcontrols.

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Methods

Cohort

Acohortof453inpatientswasconsecutivelyrecruitedfromalargehospitalsettingin

Merseyside,UK.Patientswereeligibleforinclusioniftheyhadhealthcare‐associated

diarrhea,definedas≥3liquidstoolspassedinthe24hoursprecedingassessment,an

onsetafterbeinginhospitalforover48hoursandrecentexposuretoeither

antimicrobialsand/orprotonpumpinhibitors.Usingcriteriapreviouslydescribed[27],

308CDIcasesand145controlswithantibiotic‐associateddiarrhea(AAD)were

classifiedbasedontoxinELISAtest(TOXA/BII,Techlab,Blacksburg,USA),

microbiologicalcultureandclinicaldiagnosismadebyindependentclinicians.PCR

ribotypingandmultiplexPCRwereperformedtodeterminestrainstypesandthe

toxigenicnatureoftheisolates[28].

Bloodandfecalspecimenswerecollectedfrompatientsatstudyentry,ofwhom98%

wereWhites.Relevantinformationondemographics,admissionandclinicalhistorywas

collectedforeachpatient.EthicalapprovalwasobtainedfromtheLiverpoolResearch

EthicsCommittee(referencenumber08/H1005/32)andeachpatientprovidedwritten

informedconsentpriortorecruitment.

Definitionofoutcomes

Casesandcontrolsweredefinedasdescribedabove.TheseverityofCDIsymptomswas

assessedatbaselinebyresearchnursesusingtheguidelinesproposedbyPublicHealth

England[29],whichweadjustedtoincorporateamorestringentwhitebloodcellcount

cut‐offof>20x109/LwhilstalsoreplacingacuterisingcreatininewithaGlomerular

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FiltrationRate(eGFR)of<30ml/minatthetimeofdiagnosis.Durationofsymptoms

fromCDIdiagnosiswasrecordedfromthedatepatientstestedpositiveandthen

dichotomisedintoepisodeslastingmoreorlessthan10days.Mortalitywasactively

monitoredforaperiodof30daysandrecurrentCDIwasdefinedasthedevelopmentof

subsequentCDIepisodesuptoaperiodof90daysfollowingtreatmentoftheinitial

episode.Ifthepatientwasdischargedfromhospitalpriortofinalfollow‐up,we

attemptedineverycasetoobtaindatafromthehospital,generalpractitionerorthe

patient(thelatterbyatelephonecall).

DeterminationofMBLserumconcentrations

Acommercially‐availableinvitrodiagnosticELISAkit(SanquinBloodSupply;

Amsterdam,Netherlands)wastransferredontotheMesoScaleDiscovery

electrochemiluminescence(ECL)‐basedplatform,undergoingappropriateoptimization

priortouse.TheMBLkitcontrolwasusedacrossallplatestodetermineinter‐plate

variabilityandasubsequentcorrectionfactorusedforeachplate.Finalminimum

detectionlevel(lowerlimitofdetection;LLOD)andminimumquantificationlevel

(lowerlimitofquantification;LLOQ)werecalculatedbytakingthemeanvaluesacross

allplates.ThemeanLLODandLLOQacrossallplateswere11.3and11.0ng/μl,

respectively,withoverallmedianvaluesof491.9ng/mlamongstcontrolsand361.8

ng/mlincases.Signalvaluesrangedfromonly50‐500ECLunits,whichdenotesa

compressedsignalrangeinherentwiththeassay.Sincethismayhavepotentiallylimited

discriminationofthequantitativevalues,dataweresubjecttobinarycategorisation

basedonthreepreviouslyuseddeficiencycut‐offs:50,100and500ng/ml[30‐32].

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DeterminationofMBL2variants

Atotalofninevariantslyinginthepromoterandexon1weretyped(Figure1)byeither

pyrosequencing(PyroMarkQ96customassays,Qiagen;rs36014597,rs7084554,

rs1800451,rs1800450,rs5030737andrs10556764)orTaqmanSNPgenotyping

(AppliedBiosystems;rs7096206,rs11003125andrs11003123).Thevariants

rs1800451(C),rs1800450(B),rs5030737(D),rs7096206(X/Y)andrs11003125(H/L)

wereusedforhaplotypedetermination,whilstrs10556764,a6bpIns/Delincomplete

linkagedisequilibriumwithrs7095891(P/Q),wasusedasaproxy.Anotherrecognized

taggingmarkerforP/Q(rs11003123)wasindependentlytypedtoevaluatetheaccuracy

ofthepyrosequencingassays.

Pyrosequencing

PCRoptimizationwasconductedusing20nggenomicDNAandtemperaturegradients

followingstandardguidelines.OptimizedproductswererunonaPyroMarkQ96ID

followingtherecommendedassayprotocol.Repeatsamplesandblankswereincluded

forqualitycontrol(QC)purposesanddatawereanalyzedusingPyroMarkQ96v.2.5.8

software.

Taqmangenotyping

Reactionsconsistedof20nggenomicDNA,1xTaqmanSNPgenotypingassays,runonan

AppliedBiosystemsHT7900FastReal‐TimePCRsystem(AppliedBiosystems,USA)

usingstandardcyclingconditions.RepeatsamplesandblankswereincorporatedforQC

purposes,andresultsanalyzedusingSDSsoftware(version2.2).

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Statisticalanalysis

MedianMBLserumconcentrationswerecomparedforindividualSNPsandhaplotypes

bytheMann‐WhitneyU‐test,andthensubjectedtostratificationbaseduponpreviously

usedtwo‐markergroupingprofilestermedhigh‐(YA/YA&XA/YA),intermediate‐

(XA/XA&YA/YO)andlow‐expressing(XA/YO&YO/YO)genotypes[32,33].

TheeffectofbothMBL2genetics(basedonstratifiedexpressiongenotypes)andserum

MBLconcentrations(basedupondeficiencycut‐offs)wereindividuallytakenforward

forcase‐controlcomparisonandsub‐groupanalysisofcases.Forthelatter,thisincluded

logisticregressionforthefollowingoutcomemeasures:A)severityofdisease,B)

durationofsymptomslongerthan10days,C)90‐dayrecurrence,andD)30‐day

mortality.Covariatesincludingdemographicvariables,thepresenceofPCRribotype

027/NAP/BI1andpotentialconfounders(immunosuppressivetherapy,renaldisease

anddiabetes,scoreonCharlsonComorbidityIndexandtimedelaybetweensample

testingpositiveandrecruitment)wereindividuallyassessed.Severityofdiseasewas

assessedbothasaCDIoutcomeandasabaselinepredictorfortheotheroutcome

measures.Statisticallysignificantcovariateswereaddedtothefinalregressionmodelto

produceadjustedP‐values,oddsratiosand95%confidenceintervals.Allanalyseswere

carriedoutusingSPSSv.20.

PowercalculationsweresimulatedusingnQueryAdvisor+nTerim2.0.Thisshowed

thatthepoweraposterioriwas≥99%forthemajorityofanalyses.However,foranalysis

of30‐daymortalityanddiseaseseverityatbaselinepowerwaslower(67&75%,

respectively;SupplementaryTable2).

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Results

Patientdemographics

CDIcasesandAADcontrolsweredemographicallycomparable(Table1).However,

mortalityat1year(35%versus18%;P<0.001)anddurationofdiarrheasymptoms

(≥10days60%versus24%;P<0.0001)weresignificantlygreateramongstCDIcases.In

relationtomedicationhistory,9%(28/308)and1%(2/145)ofCDIcasesandAAD

controlshadpriorexposuretoPPIsbutnotantibioticswithin90daysofthe

developmentofCDI,respectively,with58%(180/308)and54%(79/145)exposedto

bothanantibioticandaPPI.OfCDIcases,41%(127/308)hadseverediseaseand38%

(83/220)experiencedrecurrencewithin90days.Twenty‐eightCDIcases,whohadnot

experiencedanyrecurrenceofsymptomsbutdiedwithinthe90dayfollow‐upperiod,

couldnotbeincludedinouranalysisofrecurrence.

RelationshipofgenotypewithserumMBLconcentrations

Ofthe9variantstypedintheCDIcasesandAADcontrols,3wereexcluded:1SNP

(rs7084554)deviatedfromHardy‐WeinbergEquilibrium(HWE<0.001);rs11003123

wasdeemedredundantduetocompleteLDwiththeINS/DELpolymorphism

(rs10556764);andrs36014597wasalsoincompleteLDwithbothrs10556764and

rs11003123.Ofthe6polymorphismsanalysed,genotypingsuccessratewas≥95%.

Theirminorallelefrequencieswereinlinewiththosereportedintheliterature

(SupplementaryTable3).Forbothgroups,sevencommonhaplotypeswerederived

fromthe6polymorphisms(SupplementaryFigure1),whichisconsistentwithother

previousstudiesinWhites(Table2)[9,34].

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PresenceofthemutantalleleforallindividualMBL2variantshadasignificantinfluence

onserumMBLconcentrationacrossallpatients,exceptfortheXalleleencodedby

rs7096206(P=0.30;SupplementaryTable3).AlltheassembledMBL2haplotypesalso

significantlyimpactedonserumconcentrations,exceptforhaplotypeLXPAwherethere

wasnodifferencecomparedwiththeoverallmedianvalue(P=0.34;Table2).Genotypic

andhaplotypicanalysesdemonstratedthatthepresenceofavariantalleleforanyofthe

threeexonicvariants(rs1800451,rs1800450andrs5030737)werethemajor

contributingfactorsforlowerMBLconcentrations(Table2andSupplementaryTable

3).

Patientswithhigh‐expressinggenotypeshadamedianserumMBLconcentrationof714

ng/ml,comparedwith190ng/mlwithintermediate‐expressinggenotypes,and32

ng/mlwithlow‐expressinggenotypes(P<0.001;Table3;Figure2A).Thecontributionof

theXallele,seeminglyinsignificantwhenevaluatedonanindividualbasis

(SupplementaryTable3),becameapparentwithagradualdecreasewhencompared

withtheequivalentgenotypescontainingtheYalleleintherankorder:XA/YA<YA/YA;

XA/XA<XA/YA,andXA/YO<YA/YO(Table3;Figure2B).

MBLdeficiencycut‐offpointsinrelationtohaplotypegroups

Intotal59(13%),93(21%)and258(58%)patientshadserumMBLconcentrations

below50,100and500ng/ml,respectively.Whenthesedatawerecomparedwiththe

“expressing”genotypegroups,78%(42/54)and68%(59/87)ofthosewith

concentrationsbelow50and100ng/ml,respectively,werelowexpressors,compared

to28%(66/236)ofthosewithaconcentrationlessthan500ng/ml(Supplementary

Table4).Thecorrespondingfiguresforhighexpressorswere4%(2/54),6%(5/87)and

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30%(70/236),respectively.Similarly,96%(52/54)and93%(81/87)ofthosewith

concentrationsbelow50and100ng/ml,respectively,carriedthedeficient(O)

haplotypes,comparedto65%(153/236)ofthosewithaconcentrationlessthan500

ng/ml(SupplementaryTable4).Basedontheresultsabove,onlythe50and100ng/ml

cut‐offsweretakenforwardforfurtheranalysis,whichisconsistentwithprevious

literature[30,31].

ComparisonofMBLlevelsversusCDIdiseaseoutcomes

SerumMBLconcentrationsareshowninSupplementaryTable5.Analysisusingboth

<50and<100ng/mlascut‐offpointstosignifydeficiencyidentifiednosignificant

differencesbetweenCDIcasesandAADcontrols(P=0.79andP=0.09,respectively)

(Table4).EvaluationoftheclinicaloutcomesinCDIcasesshowedasignificant

associationwithCDIrecurrence(P<0.01forboth;Table4)withoddsratiosof3.18and

2.61atthe<50and<100ng/mlcut‐offpoints,respectively.Noassociationwas

identifiedwithanyoftheotheroutcomesincludingprolongedsymptoms,30‐day

mortalityanddiseaseseverityatbaseline(Table4).Despitethestrongcorrelation

observedbetweengenotypes/haplotypesandserumMBLconcentrationsinthiscohort,

nosignificantassociationswereidentifiedbetweenhigh‐,intermediate‐andlow‐

expressinggenotypesandCDIdiseaseoutcomes(SupplementaryTable6).

TherewasaninversecorrelationbetweenMBLandCRPserumconcentrations

(Pearson’sCorrelationCoefficientR2=‐0.16,P=0.001;SupplementaryFigure2).No

significantcorrelationwasidentifiedwithwhitecellcount(R2=‐0.04,P=0.44).

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Discussion

StudiesevaluatingtheroleofMBLininfectiousandimmunediseaseshavefocusedon

eithergenotype,phenotype,oroccasionallyonbothparameters.Thelatterapproachis

preferredasitcanshowdiscordancebetweengenotypeandphenotype.Thisstudyis

oneofthelargerdisease‐relatedstudiesconcurrentlyinvestigatingboth

genotypic/haplotypicvariantsandserumconcentrationsinWhites(Supplementary

Table1)andisthefirsttodemonstrateanassociationbetweenserumMBL

concentrations,butnotgenotype,andrecurrenceofCDIwithin90daysusingtwo

distinctcut‐offvaluesforMBLdeficiency.

Themechanisticbasisoftheassociationisunclear.Withotherbacterialandviral

infections,MBListhoughttobecapableofbindingtothecellsurfacesofinvasive

pathogenstherebystimulatingadownstreamimmuneresponse.However,thisdoesnot

seemtobethecasewithC.difficilewherebindingofMBLhasbeenshowntobelow[24].

ThissuggeststhatMBLdeficiencydoesnotpersepredisposetoCDIandisconsistent

withtheobservedlackofdifferenceincirculatingconcentrationsofMBLbetweenCDI

casesandAADcontrols.MBLhasotherfunctionsincludingmodulationofinflammation

andclearanceofapoptoticcells[35].TheformermayberelevanttoCDI,whereMBL

maybeactingasamodulatorofthedisease.Consistentwiththis,clinicalmanifestations

ofMBLdeficiencyappeartobeofmorerelevanceeitherininfantswhentheimmune

systemisstillmaturingorinsusceptiblegroupswhenthereisanassociated

immunodeficiency[36],suchasinhospitalizedelderlypatientsorfollowingmajor

clinicalinterventions.However,thesearehypothesesthatneedfurtherinvestigation.

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AlthoughMBLconcentrationsremainrelativelyconstantinindividualsduetogenetic

determinants,MBLisknowntobearelativelymodestacutephasereactant[37].Thisis

insharpcontrasttootheracutephaseproteinssuchasCRPwhoseconcentrationscan

increasesharplyby10to1,000‐foldduringacuteinflammation[38].ElevatedCRP

concentrationshavepreviouslybeenshowntobeassociatedwithvariousCDIoutcomes

includingdiseaseseverityandrecurrence[25,39].Consistentwiththis,lowMBL

concentrationshavebeenassociatedwithanincreaseinthelevelofCRP[40],andwith

ourfindingsoftheassociationwithCDIrecurrenceandinversecorrelationwithCRP.In

keepingwiththeimmunomodulatoryeffectofMBL,itisknownthatlowconcentrations

leadtoincreasedsecretionofthepro‐inflammatorycytokinesinterleukin‐6,interleukin

1‐betaandTNFalpha[40,41],allofwhichhavealsobeenshowntobeelevatedin

responsetoCDI[42,43].

ThegeneticarchitectureoftheMBL2geneiscomplex(Figure1)withtheexistenceof

numerouscommonfunctionalpolymorphismsandhaplotypes(Figure1,Tables2and3,

andSupplementaryTable3).MBL2haplotypefrequenciesandthecorrespondingimpact

onserumMBLconcentrationswereinlinewiththosepreviouslyreported[9,13](Table

2).ThiswasalsoevidentafterstratificationofMBLhaplotypesbasedonpreviously

definedexpressiongenotypes[32,33]withcarriersoflow‐expressinggenotypes

showingmuchlowerserumMBLconcentrationsthanbothintermediate‐andhigh‐

expressinggenotypes(32ng/mlversus190and714ng/ml,respectively;Table3).

DespitethestrongassociationobservedbetweenMBL2genotypesandserumMBL

concentrations,andtheassociationbetweenMBLconcentrationsandCDIrecurrence,

therewasnoassociationbetweenMBLgenotypeandCDIoutcomes.Otherstudieshave

alsoidentifiedassociationswithproteinlevels,butnotwithgenotype(Supplementary

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Table1),highlightingtheneedtoevaluatebothMBLgenotypeandphenotypein

infectionandotherimmuneconditions.ThelackofassociationbetweenMBLgenotype

anddiseaseoutcomemaybeduetotheincompletegeneticpenetranceofMBLgenetic

variationonphenotype.Inthisstudy,only78%and68%ofthelow‐expressing

genotypesaccountedfordeficientserumlevelsusingthecut‐offvaluesof<50and<100

ng/ml,respectively(SupplementaryTable4).Geneticheterogeneityduetofunctionally

relatedgenessuchasL‐ficolin,MASP2,andsurfactantproteinsmayalsoplayarole,but

thisneedsfurtherinvestigation.

Ourstudysoughttoadheretoastringentmethodologythroughtheuseofarelatively

largecohortsizeandextensiveQC,butitisnotwithoutitslimitations.Althoughthereis

lesschanceofMBLconcentrationsbeingconfoundedbyinfection‐relatedeventswhen

comparedtootherresponsemarkers,oneofthecleardrawbacksofthisworkisthelack

oflongitudinalmeasurements,whichisnowbeingaddressedinanewprospective

study.TheeffectofproteinsfunctionallyrelatedtoMBL,andothermarkersof

inflammation,andtherelativerolestheyplayindiseasemodulationneedsfurther

investigation.PreviousstudieshaveusedvariousdefinitionsforMBLdeficiency,with

commonlyusedcut‐offsrangingfrom50[30]to500ng/ml[32].Itisthusdifficultto

compareresultsacrossdifferentstudygroupsgiventheheterogeneityofplatforms,

profileofcohortsandstandardsadoptedforthemeasurementofMBL.Discrepancies

betweenstudiescouldbeduetolowsamplesizes,poorassayperformanceand

differencesintechniquesadoptedbylaboratories.Wehavetriedtoovercomesomeof

theselimitationsbyevaluatinganumberofcut‐offlevelsbutthereisaneedfor

internationalconsensusandharmonisationinthisarea.

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Inconclusion,ourdatasuggestthatlowserumMBLconcentrationsmayactasa

predictorofCDIrecurrence.Furtherworkisneededtovalidatethesefindingsinan

independentcohortofpatientsandtoevaluatethemechanisticbasisofthisassociation.

Thisareaofresearchwouldalsobeadvancedthroughconsensusondefinitionsof

deficiency,standardisationofmethodsemployedformeasurementofserum

concentrations,andfurtherevaluationofthegenotype‐phenotyperelationships.

Funding

ThisworkwasfundedbytheNationalInstituteforHealthResearch(NIHR)Biomedical

ResearchCentreinmicrobialdiseasesinLiverpool,andbytheMedicalResearchCouncil

[MR/K000551/1].WewouldalsoliketothanktheNIHRforPhDstudentfundingfor

AndrewSwale[BRF‐2011‐028].MPisaNIHRSeniorInvestigator.

Conflictofinterests

Noneoftheauthorshadanyconflictinginterests.

Acknowledgements

Wethankpatientsfortakingpartinthestudy,andallcliniciansandotherhealthcare

professionalswhohelpedwiththerecruitment.

Contributions

AS,FMandMPwrotethepaper.FMandMPconductedthestudydesignandML

recruitedthepatients.ML,ASandFMcollectedclinical,admissionandfollow‐up

information.AS,FM,PRandTLperformedthelaboratorywork.RK,ASandFM

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performedthestatisticalanalysis.MP,NBandMBledtheclinicalandmicrobiological

aspectsofthestudy.Allauthorscriticallyreviewedthemanuscriptandapprovedthe

finalversionofthearticle,includingtheauthorshiplist.

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Table1–DemographicsofpatientswithClostridiumdifficileinfection(CDI)and

antibiotic‐associateddiarrhea(AAD)

Patient’scharacteristics CDICases(n=308)

AADControls(n=145)

Gender–Female,n(%) 177/308(57) 81/142(57)

Age–Meaninyears(SD) 70.1(16.4) 65.0(17.6)

BMI–Mean(SD) 24.6(6.8) 26.9(6.9)

Presenceofimmunosuppression–n(%) 52/307(17) 35/144(24)

Presenceofrenalcomorbidity–n(%) 157/307(51) 82/144(57)

Presenceofdiabetes–n(%) 58/307(19) 39/144(27)

CharlsonComorbidityscore–Median(IQR) 1.0(0.0‐2.0) 1.0(0.0‐2.0)

Timedelay(testing/recruitment)–Median(IQR) 3.0(2.0‐4.0) 2.0(2.0‐3.0)

ClinicalParameters

Durationofsymptoms≥10days–n(%) 175/290(60)a 32/134(24)

All‐causemortalitywithin30days–n(%) 26/305(9) 5/142(4)

All‐causemortalitywithin1yr–n(%) 95/271(35)b 25/141(18)

Diseaseseverityatbaseline–n(%) 127/308(41) ‐

Recurrencewithin90days–n(%) 83/220(38) ‐

%:percentage;AAD:Antibiotic‐associateddiarrhea;BMI:bodymassindex;CDI:Clostridiumdifficileinfection;IQR:Interquartilerange;n:number;SD:Standarddeviation;Differencesbetweencaseandcontrolgroupswerefoundtobestatisticallysignificant:P<0.0001a&P<0.001b,respectively;

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Table2–MBLserumconcentrationsacrossMBL2haplotypesinpatientswithClostridiumdifficileinfectionandantibiotic‐associateddiarrhea

HYPA LYPA LYQA LXPA HYPD LYPB LYQC

Presenceofhaplotype

n(%frequency) 213(29) 44(6) 143(19) 170(23) 55(7) 108(15) 11(1)

Median,ng/ml(Range)

612(17‐3,981)

587(0‐2,500)

529(0‐3,981)

428(0‐2,968)

157(0‐815)

73(0‐637)

48(0‐492)

Absenceofhaplotype

n(%frequency) 198(9) 367(17) 268(13) 241(11) 356(17) 303(14) 400(19)

Median:Absence,ng/ml(Range)

171(0–2,374)

388(0‐3,981)

324(0‐2,968)

377(0‐3,981)

484(0‐3,981)

568(0‐3,981)

420(0‐3,981)

P‐value* <0.001 0.04 <0.001 0.34 <0.001 <0.001 0.001

n:number;%freq.:Percentagefrequency;*P‐valueswerecalculatedusingaMann‐WhitneytestcomparingMBLserumconcentrationsagainstthepresence/absenceofeachindividualhaplotype

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Table3–MedianserumMBLconcentrationsacrosspreviouslydefinedexpressiongenotypegroups*MBLexpressiongroup Genotype n Median(ng/ml) Combinedmedian(ng/ml)

HighYA/YA 124 854

714XA/YA 113 561

IntermediateXA/XA 16 270

190YA/YO 91 175

LowXA/YO 41 32

32YO/YO 26 31

*ExpressiongroupsdefinedaccordingtoEisenetal.2008[32]

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Table4–AnalysisofClostridiumdifficileinfectiondiseaseoutcomesversusserumMBLconcentrationbasedondeficiencycut‐offsof50and100ng/ml

Case(n=308) Control(n=145) P‐value OR(95%CI)<50ng/ml

41(13%) 18(12%) 0.79a

1.09(0.58‐2.06)

<100ng/ml

70(23%)

23(16%)

0.09b

1.61(0.93‐2.79)

Death(n=26) Survival(n=276) P‐value OR(95%CI)<50ng/ml

3(12%) 37(13%) 0.78c

1.22(0.31‐4.82)

<100ng/ml

5(19%)

64(23%)

0.84c

0.88(0.27‐2.89)

≥10days(n=174)* <10days(n=113)* P‐value OR(95%CI)<50ng/ml

27(16%) 10(9%) 0.10d

1.89(0.88‐4.08)

<100ng/ml

42(24%)

22(20%)

0.35d

1.32(0.74‐2.35)

Recurrence(n=81)* Non‐recurrence(n=136)* P‐value OR(95%CI)<50ng/ml

18(22%) 13(10%) <0.01e

3.18(1.40‐7.24)

<100ng/ml

29(36%)

24(18%)

<0.01e

2.61(1.35‐5.04)

Severe(n=125) Non‐severe(n=180) P‐value OR(95%CI)<50ng/ml

16(13%) 25(14%) 0.78d

0.91(0.46‐1.79)

<100ng/ml

29(23%)

41(23%)

0.93d

1.02(0.60‐1.76)

n:number;OR:oddsratio;CI:confidenceinterval;

P‐values&ORswerecalculatedusingunivariatelogisticregressionandadjustedforthepresenceof

significantcovariates:aAge,BMI,timedelaybetweentestingpositiveandrecruitment&thepresenceof

diabetes;bAge,BMI,timedelaybetweentestingpositiveandrecruitment&thepresenceofdiabetesand

immunosuppressivetherapy;cAge,BMI,scoreonCharlsonComorbidityIndexanddiseaseseverityat

baseline;dNocovariateswerefoundtobesignificant&thereforeP‐valueremainsunadjusted;eAge;

* Data regarding duration of symptoms and disease recurrence was unavailable for 18 and 60 ofourcases,

respectively.Fordiseaserecurrence,afurther28patientshaddiedwithinthefollow‐upperiodpriorto

experiencinganyrecurrentsymptomsandthereforecouldnotbeincludedintheanalysis.SerumMBLlevel

wasunavailableforafurther3individualswhowerethereforeexcludedfromanalysisacrossalloutcomes;

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FIGURELEGENDS

Figure1–SchematicrepresentationofthemajorMBL2isoformandgenetic

polymorphisms.Polymorphismsresponsibleforthehaplotypesthatultimately

determineMBLexpressionlevelsareindicatedbytheredarrows.*Inthisstudy,

rs10556764(6bpdeletion)wasusedasaproxySNPforrs7095891

Figure2–MedianserumMBLconcentrationsinrelationto:(A)3‐tiergrouping

basedonproposedexpressionprofiles;and(B)individualgenotypicgroups

withinproposedexpressionprofiles.MedianserumMBLconcentrationswere

determinedacrosspreviouslydefinedexpressionprofiles:high(YA/YA&XA/YA),

intermediate(XA/XA&YA/YA)andlow(XA/YO&YO/YO).Medianlevelswerealso

determinedforthe6individualgenotypicgroupsacrossallexpressionprofiles.

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