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RESEARCHOpenAccessAsetofnovelmultiplexTaqmanreal-timePCRsforthedetectionofdiarrhoeagenicEscherichiacolianditsuseindeterminingtheprevalenceofEPECandEAECinauniversityhospitalChristophHardegen1,SabineMessler1,BirgitHenrich1,KlausPfeffer1,JensWürthner2,ColinRMacKenzie1*AbstractBackground:AccuratemeasurementoftheincidenceofdiarrhoeagenicE.
coliinpatientswithdiarrhoeaishinderedbythecurrentmethodsofdetectionandvariesfromcountrytocountry.
InordertoimprovethediagnosisofdiarrhoeagenicE.
coli(DEC),wedevelopedasetofmultiplexTaqManreal-timePCRsdesignedtodetecttherespectivepathogensfromanovernightstoolculture.
Methods:OvertheperiodJan.
2006toDec.
2006allstoolspecimens(n=1981)receivedwereinvestigatedforEPECandEAEC.
Results:Ofthese,371specimenshadnogrowthofEnterobacteriaceae.
Oftheremaining1610specimens144(8,9%)werepositiveforEPECand78(4,8%)positiveforEAEC.
AmongtheEPECpositivestoolspecimens28(19,4%)werereceivedfromthetropicaldiseasesunit,49(34%)fromthepaediatricdept.
and67(46,5%)fromtheremainderofthewards.
TheEAECweredistributedasfollows:39(50%)-tropicaldiseases,19(24,4%)-paediatricsand20(25,6%)otherwards.
ProportionatelymoreEAECandEPECwerefoundinchildrenlessthan3yearsofagethanotheragegroups.
Inonly22,2%ofthedetectedEPECand23%ofEAECwastheinvestigationrequestedbyhospitalstaff.
Conclusions:Thisis,toourknowledge,thefirststudyusingamultiplexTaqManPCRforthesuccessfuldetectionofdiarrhoeagenicE.
coli.
Inconclusion,duetothehighprevalenceofDECdetected,investigationofEPECandEAECshouldberecommendedasaroutinediagnostictestforpatientswithinfectiousdiarrhoea.
BackgroundInfectiousdiarrhoeaisacommoncomplaintamongpatientsseekingmedicaladviceand,despiteprogressindiagnosisandtreatment,remainsoneoftheleadingcausesofmorbidityandmortalityworldwide[1,2].
Thespectrumofpathogensresponsibleforsuchinfec-tionsvarieswithageandgeographicallocation.
Aviralpathogenesisisprobablythemaincauseofdiarrhoeainindustrialisedcountries[1,3],howeversystematicsurveyshaveshownthatdiarrhoeagenicE.
coli(DEC)areacommoncauseofdiarrhoeainbothdevelopinganddevelopedcountries.
Thesepathogens,especiallyEAEC,mayroutinelybeunderestimatedasacauseofdiarrhoeaduetounder-representationofrequestsanddifficultyrecognisingthesepathogensinthelaboratory[4,5].
TodatesixcategoriesofDEChavebeendefinedonthebasisofspecificvirulenceproperties[6,7].
Entero-toxigenicE.
coli(ETEC)causediarrhoeaviasecretionofheatstable(ST)and/orheatlabile(LT)enterotoxin.
EnteroinvasiveE.
coli(EIEC)strainsarecloselyrelatedtoShigellaspp.
andtheresponsiblegenesarecarriedonthepINVplasmid.
Shiga-toxinproducingorenterohae-morrhagicE.
coli(STEC/EHEC)causebothanon-bloodydiarrhoeaaswellasahaemorrhagiccolitiswhichmaytriggerhaemolyticuraemicsyndrome(HUS).
ThevirulencepropertiesdefiningEHECaretheShigatoxins*Correspondence:colin.
mackenzie@uni-duesseldorf.
deContributedequally1InstituteofMedicalMicrobiologyandHospitalHygiene,Heinrich-Heine-University,Universittsstrae1,40225Düsseldorf,GermanyHardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/52010Hardegenetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
1and2(Stx1,Stx2)and,whenpresent,thepathogeni-cityislandLEE("locusofenterocyteeffacement").
Enter-opathogenicE.
coli(EPEC)showlocalisedadherencetothesmallintestineandcauseahistopathological"attach-ingandeffacing(A/E)lesion"mediatedthroughviru-lencefactorsencodedwithintheLEE.
StrainsadditionallycontainingtheEPECadherencefactor(EAF)plasmidhavebeentermed"typical"EPEC,whereasstrainsthatlacktheEAFplasmidarereferredtoas"atypical"EPEC.
EnteroaggregativeE.
coli(EAECorEAggEC)wasfirstdescribedin1987[8]andhasbeenassociatedwithacutediarrhoeainchildren,HIVpositiveindividuals,andasacauseoftravellers'diar-rhoea[9].
Moreoverithasbeenlinkedtopersistentdiarrhoeaandevengrowthretardationinchildren[10].
Numerousfindingssuggestthatthereisaninflamma-toryprocessunderlyingtheinfection[11,12].
ThePCR-targetusedinthisreportisasequenceontheplasmidpCVD432onwhichthegenescodingforthefimbrialstructure(aggregativeadherencefimbriaI)andfurthervirulencefactorsarelocated[13].
ThesixthcategoryisthediffuselyadherentE.
coli(DAEC)inwhichthebac-teria,unlikeEAECdonotadheretothehostcellsinmicrocoloniesbutratherdiffuselyoverthecellsurface.
NoPCRhasbeendevelopedyettodetectDAECstrains.
SinceDECcannotbediagnosedadequatelybycultureandbiochemicalcriteriaalone,identificationofthesestrainsisdifficult.
ManyPCRtechniquesdetectingvar-iousgenescodingforvirulencetraitsofthedifferentcategoriesofDEChavebeenreported[14-18].
WehavedevelopedthreenewmultiplexTaqManPCRassays,designedtoberuninparallel,todetectETEC,EIEC,EHEC,EPECandEAECsimultaneously.
InadditiontomanystudiesfromdevelopingcountriesrecentreportsunderlinetheimportanceofEPECandEAECindevelopedcountries[5,19-21].
InGermanyverylimiteddataareavailable.
ThereforetheaimofthisstudywastoevaluatethelocalimportanceofEPECandEAECinaGermanUniversityhospitalinrelationtotherequestingclinicaldepartmentandagegroup,usingthenewlydevelopedreal-timePCRasadiagnostictool.
MethodsStudypopulationFromJanuary2006toDecember2006atotalof1981faecalsamplesreceivedforSalmonella,ShigellaandCampylobacterspp.
culturewereincludedinthescreen-ingforEPECandEAEC.
Ofthese371specimenshadnogrowthofEnterobacteriaceaehence1610samplesweretestedforEPECandEAECbyuseofthePCRmethoddescribedbelow.
StoolsampleswereobtainedfrommostdepartmentsoftheUniversityHospitalofDüsseldorf,Germany.
Thepatientagerangedfromafewdaysto98years.
DNApreparationAllfaecalsamplesreceivedwereplatedonMacConkeyAgarandincubatedfor18hat37°C.
Abacterialsus-pensionfromallcolonieswasmadebyrinsingtheentireplatewith2mlofsterilesaline.
Thebacterialsuspen-sionwasdilutedtoMacFarlandStandard1,0(approx.
3*108CFU/ml)usingdistilledwater.
Thissuspensionwasheatedat95°Cfor15minutes.
2,5μl(correspond-ingtoapproximately7,5*105CFU)wereapplieddirectlytothemultiplexPCR.
Theremainderofthesamplewasstoredat-20°C.
TaqManPCRThreedifferentmultiplexTaqManPCRsweredesignedandwereallrunusingthesametimeandtemperaturesettingsonaBioradiCyclersystem.
Atwostepamplifi-cationprofilewasusedasfollows:95°Cfor10min.
fol-lowedby45cyclesof15s.
at95°Cand60s.
at60°Cforannealingandelongation.
ThePCRcontained10pmolofeachprimer,2pmolofthetargetprobes,5pmoloftheinternalinhibitioncontrolprobe(allfromEurogen-tec,Cologne,Germany),100copiesofinhibitioncontrolplasmid,2,5μlsample,12,5μlofNoROXPCRMaster-mix(Qiagen,Hilden,Germany)andwasdilutedwithdistilledwatertoafinalvolumeof25μl.
TwotargetsequenceswereusedforEPECamplifica-tion:a107bpsequenceoftheenteropathogenicE.
coliadherencefactor(EAF)plasmid[22]anda189bplongfragmentoftheintimingene(eae)withinthelocusofenterocyteeffacementbaseduponanalignmentofvar-iouspublishedintiminsubtypesequences(designedwithsupportofTIBMOLBIOL,Berlin,Germany).
ThetargetsequenceforEAECwasa152bpsequenceofthepCVD432plasmid[23].
ThesecondmultiplexPCRwasdesignedtodetectETECandEIEC.
TargetsforETECwerea107bpfragmentoftheSTanda113bpsequenceoftheLTgene.
ForEIECa107bpsequenceofthevirulenceplasmidessentialforinvasiveness,pINVwastargeted.
ThethirdPCRdetectsEHECbytargetinga87bpfragmentoftheshiga-toxin1(stx1)anda82bpsequenceoftheshiga-toxin2(stx2)gene.
AllprobeswerelabelledwithfluorophoresandquenchersaslistedinTable1.
IsolatesofsequencedpathogensservedaspositivecontrolswhileanonpathogenicE.
colistrain(ATCC25922)wasusedasnegativecontrol.
SincethesequencesofpINVandstx1arealsopresentinshigella,inallspecimenspositiveforeitheroftheseaninfectionduetoshigellawasexcludedbyculture.
PCR-baseddetectionmethods,especiallyfromfaecalsamplesarepronetoinhibitionofamplification[24].
Asaninternalcontrol(IC)specimenswerespikedwithsynthesisednucleotidesequencesusinganunrelatedprobesequencefromtheretrotransposonNinjafromDrosophilasimulans(AB110070)inthepCRII-TOPOVector(Invitrogen,Karlsruhe,Germany)flankedbytheHardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page2of7respectiveprimersusedinthePCR[25].
Aserialdilu-tionoftargetedtemplateinaMacFarlandstandard1,0apathogenicE.
colisuspensionrevealedreliablefluores-cencesignalstoameannumberof1,5bacterialgen-omesper2,5μlforalltargets.
Nocrossreactivitywithvariousbacteria(Streptococcus,Staphylococcus,Entero-coccus,Pseudomonas,Klebsiella,Proteus,Citrobacter,Salmonella,Yersinia,Campylobacter,Aeromonascaviae,ClostridiumdifficileandEnterobacterspp.
)otherthantheknownpositivitywithShigelladysenteriaeusingtheEHECPCRwasdetected.
ResultsAllstoolsampleswereobtainedfromtheUniversityHospitalofDüsseldorf.
Altogether144(8,9%)specimenswerepositiveforEPECand78(4,8%)positiveforEAEC,including17samplesthatwerepositiveforbothpatho-gens.
AnexampleofanamplificationplotofadoubleinfectionisshowninFig.
1.
FromthesamplespositiveforEPEConly7(4,9%)werepositiveforbotheaeandEAF(typicalEPEC),makingatypicalEPECthepredomi-nantpathotype.
FivesampleswerepositiveforEAFonly.
Alleaepositivespecimenswereadditionallyexam-inedforthepresenceofstx1andstx2andwereallnega-tive.
IncomparisontotheseresultsSalmonellaspp.
wasfoundin38(2,4%),Campylobacterspp.
in39(2,4%)andShigellaspp.
inonly1(0,06%).
Thedataweredividedintothreegroupsdependingontherequestingdepartment:thedepartmentfortropicaldiseases,paediatricsandalastgroupofalltheTable1PrimersandprobesformultiplexTaqManPCRTargetPrimerorprobenameOligonucleotidesequence(5'3')Ampliconsize/Accessionnr.
Multiplex1:(EPEC/EAEC)EAFEP-1forEP-2revEP-SprobeGTTCTTGGCGAACAGGCTTGTCTTAAGCCAGCTACCATCCACCCCy5-AGTACTGACGTGCAGGTCGCCTGTTCG-BHQ-3107bpX76137eaeEAE-SforEAE-B1revEAE-B2revEAE-TMprobeACTGGACTTCTTATTRCCGTTCTATGCTAAGCGGGTATTGTTACCAGACCTAAACGGGTATTATCACCAGAROX-AATCCTGATCAATGAAGACGTTATAGCCCA-BBQ189bp*Z11541/AB040740pCVD432EA-1forEA-2revEA-SprobeAGGTTTGATATTGATGTCCTTGAGGATCAGCTAATAATGTATAGAAATCCGCTGTTFAM-CATGTTCCTGAGAGTGCAATCCCAGACATTAC-TAMRA152bpX81423Multiplex2:(ETEC/EIEC)STgeneST-1forST-2revST-SprobeCTGGTTTTGATTCAAATGTTCGTGTCCTGAGGGAAAGGTGAAAAAGACROX-TTGATTTCTTCATATTACCTCCGGACATGGCA-BHQ-2107bpM34916LTgeneLT-1forLT-2revLT-SprobeAGCGGCGCAACATTTCAGTTGGTCTCGGTCAGATATGTGATTCFAM-TCGAAGTCCCGGGCAGTCAACATATAGA-TAMRA113bpS60731ipaHEi-1forEi-2revEi-SprobeGAACTCAAATCTTGCACCATTCACGTCCGTCCGAGAACAATTAAGCy5-ATCCCCGACACCGTTTGTGAGTTTCACT-BHQ-3107bpAY206439Multiplex3:(EHEC)stx1slt1-1forslt1-2revslt1-SprobeCTTCCATCTGCCGGACACATAATTAATACTGAATTGTCATCATCATGCATROX-AAGGAAACTCATCAGATGCCATTCTGGCA-BHQ-287bpZ36899stx2slt2-1forslt2-2revslt2-SprobeGACGTGGACCTCACTCTGAACTGTCCCCACTCTGACACCATCCFAM-TACTCCGGAAGCACATTGCTGATTCGC-TAMRA82bpL11079InternalControlDrosophilasimulans+ICprobeHEX-ATGCCTCTTCACATTGCTCCACCTTTCCT-BHQ1AB110070+Thebold,italiclabelledprimersalsodetecttheinternalcontrolplasmidintherespectivePCR*AlignmentofvariousintiminsubtypesequencesHardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page3of7remainingdepartments.
Thedepartmentfortropicaldis-easesgroupconsistedof150samplesonlybuthadarelativelyhighpositivityratewith28EPECpositiveand39EAECpositivesamples.
18,7%ofallspecimenstestedwithinthisgroupwerepositiveforEPECand26%forEAEC.
Mostpatientsseeninthisdepartmenthaveatra-velhistoryand,withtheexceptionofonly4patients,allwerebetween18and65yearsofage.
Fromthepaedia-tricwards458specimensweretestedand49EPECposi-tiveand19EAECpositivesamplesweredetectedshowinganprevalenceof10,7%forEPECand4,1%forEAEC.
Fromtheremaining1002samplesfromallotherdepartments67(6,7%)weretestedpositiveforEPECand20(2%)positiveforEAEC.
Table2showstheresults.
SincemostreportsonDECshowdifferentincidencesdependingonagewedividedthesamplesaccordingtoagegroupsasseeninFig.
2.
ApeakprevalenceforEPECwasfound(16,3%)intheagegroupbetweenoneandtwoyears.
Intheagegroupsofpatientsbelowoneyearofageandbetweentwoandfiveyearsthepreva-lencevariedfrom9,1%to10,2%,whichisslightlyhigherthaninthegroupsbetweenfiveand18years(7,6%)andthegroupabove65yearsofage(4,9%).
Theprevalenceinthegroupbetween18and65years(10,2%)wasunex-pectedlyhigh,butofthe74positivesamples26weresentbythetropicaldiseasesdepartment,indicatingahistoryoftravel.
Withoneexception(onepositivespeci-menintheagegroupbelowoneyear),allspecimensfromthedepartmentoftropicaldiseaseswerefrompatientsover18yearsofage.
ForEAECaclearpeakwasseenintheagegroupbetweentwoandthreeyearswithanprevalenceof14,5%.
Belowtwoyearsofageandbetweenthreeandfiveyearstheprevalencerangedfrom1,3%upto3,3%.
Noneofthesesampleswerefromthetropicaldiseasesdepartment.
Intheagegroupsbetweenfiveand18and18and65asmallerpeakwasfoundwithprevalenceof4,6%and7%respectively.
1,7%ofEAECpositivespecimenswerefrompatientsover65.
Thepeakinthegroupbetween18and65yearswasmostlyduetosamplesfromthedepartmentfortropicaldiseases(36outof51).
Intheagegroupfiveto18years,onespecimenandinthegroupover65years,twospecimenswerereceivedfromthisdepartment.
InourhospitalallstoolspecimensfromchildrenageduptooneyeararetestedforEPECroutinely,howeveritwasnotedthatrequestsforDECareveryrarelymadeforpatientsabovethisage.
ThereforewelookedatthenumberofDECcasesfoundbyroutineinvestigationsincomparisontothetotalnumberofcasesfoundbysys-tematicscreeningofallspecimensreceived.
Table2showsthefindingsforEPECandEAEC.
15EPECposi-tivesamplesfromchildrenbelowoneyearofageweredetectedbyroutinescreeningandofthepositiveresultsforEPECorEAECinthegroupofsamplesfromchil-drenbetweenoneandfiveyearsofagenonewasdetectedbyroutinerequest.
Intheagegroupbetween5and18yearsofageonlyone(EPECpositive)specimenwasactuallyrequestedbythepaediatricdepartmentand1(EAECpositive)specimenfromthedepartmentfortropicaldiseases.
Between18and65yearsarelativelylargenumberofpositiveresultswasfoundinscreeningFigure1AmplificationplotofadoubleinfectionwithatypicalEPECandEAEC.
Table2NumberofstoolsamplestestedaccordingtodepartmentandpositivityrateforEPECandEAECDepartmenttestedEPEC(%)EAEC(%)tropicaldiseases15028(18,7)39(26,0)paediatrics45849(10,7)19(4,1)others100267(6,7)20(2,0)Hardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page4of7allreceivedstoolspecimens,butonly16(21,6%)EPECpositiveand15(29,4%)EAECpositivespecimensweredetectedduetoaspecificrequestfromtherespectivedepartment.
Furthermore,exceptforthreepositiveEPECspecimensandonepositiveEAECspecimen,allofthespecimensweresentbythedepartmentfortropi-caldiseases.
Inthegroupabove65yearsinonlytwoofthespecimensthatwerepositiveforEAECwastheinvestigationroutinelyrequested,againbythedepart-mentfortropicaldiseases.
DiscussionDetectingDECreliesgreatlyonPCRtechniquesandmanyreportsareavailableonproceduresusedtoiden-tifyallcategoriesofDEC[13-18,26].
Mostofthesetech-niqueshowever,arebasedonstandardPCR.
Wehavedevelopedanovelmultiplexreal-timePCRwhich,withthreereactionsrunparalleltooneanother,identifiesthe5majorcategoriesofDECsimultaneously.
ToourknowledgethisisthefirstreporteduseofaTaqManPCR,whichprovidesbothaconvenientdiagnostictoolandavoidstheriskofcrosscontaminationduetopostamplificationhandling.
Byrinsingallthecoloniesfromtheagarplatewithsalineratherthanpickingrepresentativecoloniesashasbeendescribedintheliterature,wewereabletoincreasethesensitivityofthePCR.
Thismethodisquick,simpleandinexpensive.
Thereal-timePCRwasabletodetect1,5genomeequivalentsperassay.
AsreportedbytheRobertKochInstitute,Berlin,Ger-many,ofallreportableintestinalinfectionsin2006inGermany,DEC,exceptingEHEC,wasthefifthlargestgroupofpathogensafterNorovirus,Rotavirus,Salmo-nellaandCampylobacterspp.
Inourstudyreportedhere,theprevalenceofEPEC(8,9%)andEAEC(4,8%)exceededthoseforsalmonellaandcampylobacter,each2,4%.
ItispossiblethattheincidenceofEPECandEAECthroughoutGermanyisfarhigherthanthatreported.
ThisdiscrepancymayverywellbeduetothelowlevelofroutinerequestsforDECasweobservedinthestudyperiodinthishospital.
UnfortunatelynodatacanbeprovidedheretocomparetheincidenceofDECtothatofviralpathogenssinceinthisstudystoolspeci-menswerenotsystematicallytestedforviralpathogens.
However,inthepaediatricpopulation(theagegroupunder18yearsinthisstudy),manyspecimenswereadditionallytestedforvirusesandoutof40EPECposi-tivesamplesadditionallytestedforviruses,16wereadditionallypositiveforrotavirus,norovirusoradeno-virus.
4specimenspositiveforEPECwereadditionallypositiveforSalmonellaspp.
(1)orCampylobacterspp.
(3).
ThisleavesEPECasthesoleidentifiedpathogenin50%ofallcases.
EAECwastheonlypathogendetectedin45%ofthesamplesinthisagegroup.
Inninecasesthespecimenwaspositiveforrotavirus,norovirusoradenovirusaswell,whereasinthreesamplesCampylo-bacterspp.
wasfoundasaco-infectionandonespeci-menwastestedpositiveforSalmonellaspp.
EventhoughthisstudyprovidesnosystematicdataonviraltestingwemustassumethatviralinfectionsoutnumberthecasesofdiarrhoeaduetoDECinchildren.
Itispos-siblethatmorestoolsamplesweresubmittedforviraldetectiononlyandthustheprevalenceforEPECandEAECfoundinthisstudymaystillbeanunderestima-tioninthisagegroup.
AstrongassociationbetweenEPECanddiarrhoeainchildrenhasbeenreported[27],yetwealsofoundarelativelyhighprevalenceforEPECinolderpatients.
AlargeproportionofEPEC(andEAEC)infectionsmaywellbeassociatedwithtravel,sinceahighnumberofpositivespecimenswithinthegroupbetween18and65yearswereobtainedfromthedepartmentfortropicaldiseases.
ConcerningtheassociationofatypicalEPECwithdiarrhoeafindingsarecontradictory[27-29],butitcontinuestobethemostprevalentpathotypeofEPECfoundinindustrialisedcountries[27,29,30],whichwealsonotedinourstudy.
AsubsetoftheheterogeneousgroupofatypicalEPEChasbeenfoundinpatientswithbloodydiarrhoea,(insomecasesleadingtoHUS).
TheyresembledEHECaccordingtotheirserotype,virulenceprofileandmultilocussequencetypes,leadingtotheassumptionthatthesestrainsmightbeEHECthathaveatsomestagelosttheshigatoxingeneduringinfection[31].
UnusualinthisstudyisthedetectionofEAFwith-outtheeaegeneasmostofteneaeisfoundalone(atypi-calEPEC)orwithEAF.
ItisquestionablewhetherstrainspositiveforEAFbutnotforeaeshouldbeclassi-fiedasEPECandwhethertheyactuallyarepathogenic.
FurtherinvestigationisrequiredastowhetherthisisduetoasequencevariationwithintheintimingeneFigure2ThepercentofspecimenspositiveforEPEC(whitesquare)andEAEC(blacksquare)inagegroupscomparedtoallspecimensreceivedfrompatientsintherespectiveagegroup.
Hardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page5of7resultinginafailureofamplificationorwhetherperhapsotherbacterialstrainshaveacquiredtheEAFplasmid.
OnelimitationofthisstudyistheinabilityofthemethodtodistinguishbetweenthosepatientswithadoubleinfectionwithtwoormoreDECandthosepatientswhoareinfectedwithasinglebacteriumcarry-ingmorethanonevirulencegene.
Becauseweuseapoolofbacteriafromanovernightculture,itisnotpos-sibletotracetheresultbacktoasinglebacterialclone.
Inthisstudywefound17specimenswhichwereposi-tiveforbothEPECandEAEC.
Innocasewasthedis-tinctionbetweenasinglebacteriumcarryingbothfactorsoradoubleinfectionclinicallyrelevant.
Never-theless,itwouldbeinterestingtodeterminethefre-quencyofmultiplegenecarriagebyasinglecloneandwearecurrentlyprospectivelyattemptingtotracebacktothegene-carryingclone(s).
ConclusionsInourstudywefindtheprevalenceforEPECandEAECtobeunexpectedlyhighinDüsseldorf,andweconcludethatallpatientswithdiarrhoealdiseaseshouldberouti-nelytestedforthesepathogens,especiallychildrenbelowtheageoffive,returningtravellersandinthosespecimensinwhichnootherpathogencanbeidentified.
WealsodemonstratetheuseofanovelmultiplexPCRforthedetectionofdiarrhoeagenicE.
colifromstoolspecimens.
Authordetails1InstituteofMedicalMicrobiologyandHospitalHygiene,Heinrich-Heine-University,Universittsstrae1,40225Düsseldorf,Germany.
2TranslationalPharmacologyDiscoveryMedicine,GlaxoSmithKline,GunnelsWoodRoad,StevenageSG12NY,UK.
Authors'contributionsCHandSMperformedallthemolecularstudies,collectedthedataandwrotethemanuscript,JWandKPdesignedthestudyandmolecularprobesandprimers,BHandCMcarriedoutthedesignandcoordinationofthestudyandtookpartinthewritingofthemanuscriptAllauthorshavereadandapprovedthefinalmanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
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FrankeJ,FrankeS,SchmidtH,SchwarzkopfA,WielerLH,BaljerG,BeutinL,KarchH:NucleotidesequenceanalysisofenteropathogenicEscherichiacoli(EPEC)adherencefactorprobeanddevelopmentofPCRforrapiddetectionofEPECharboringvirulenceplasmids.
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doi:10.
1186/1476-0711-9-5Citethisarticleas:Hardegenetal.
:AsetofnovelmultiplexTaqmanreal-timePCRsforthedetectionofdiarrhoeagenicEscherichiacolianditsuseindeterminingtheprevalenceofEPECandEAECinauniversityhospital.
AnnalsofClinicalMicrobiologyandAntimicrobials20109:5.
PublishwithBioMedCentralandeveryscientistcanreadyourworkfreeofcharge"BioMedCentralwillbethemostsignificantdevelopmentfordisseminatingtheresultsofbiomedicalresearchinourlifetime.
"SirPaulNurse,CancerResearchUKYourresearchpaperswillbe:availablefreeofchargetotheentirebiomedicalcommunitypeerreviewedandpublishedimmediatelyuponacceptancecitedinPubMedandarchivedonPubMedCentralyours—youkeepthecopyrightSubmityourmanuscripthere:http://www.
biomedcentral.
com/info/publishing_adv.
aspBioMedcentralHardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page7of7
coliinpatientswithdiarrhoeaishinderedbythecurrentmethodsofdetectionandvariesfromcountrytocountry.
InordertoimprovethediagnosisofdiarrhoeagenicE.
coli(DEC),wedevelopedasetofmultiplexTaqManreal-timePCRsdesignedtodetecttherespectivepathogensfromanovernightstoolculture.
Methods:OvertheperiodJan.
2006toDec.
2006allstoolspecimens(n=1981)receivedwereinvestigatedforEPECandEAEC.
Results:Ofthese,371specimenshadnogrowthofEnterobacteriaceae.
Oftheremaining1610specimens144(8,9%)werepositiveforEPECand78(4,8%)positiveforEAEC.
AmongtheEPECpositivestoolspecimens28(19,4%)werereceivedfromthetropicaldiseasesunit,49(34%)fromthepaediatricdept.
and67(46,5%)fromtheremainderofthewards.
TheEAECweredistributedasfollows:39(50%)-tropicaldiseases,19(24,4%)-paediatricsand20(25,6%)otherwards.
ProportionatelymoreEAECandEPECwerefoundinchildrenlessthan3yearsofagethanotheragegroups.
Inonly22,2%ofthedetectedEPECand23%ofEAECwastheinvestigationrequestedbyhospitalstaff.
Conclusions:Thisis,toourknowledge,thefirststudyusingamultiplexTaqManPCRforthesuccessfuldetectionofdiarrhoeagenicE.
coli.
Inconclusion,duetothehighprevalenceofDECdetected,investigationofEPECandEAECshouldberecommendedasaroutinediagnostictestforpatientswithinfectiousdiarrhoea.
BackgroundInfectiousdiarrhoeaisacommoncomplaintamongpatientsseekingmedicaladviceand,despiteprogressindiagnosisandtreatment,remainsoneoftheleadingcausesofmorbidityandmortalityworldwide[1,2].
Thespectrumofpathogensresponsibleforsuchinfec-tionsvarieswithageandgeographicallocation.
Aviralpathogenesisisprobablythemaincauseofdiarrhoeainindustrialisedcountries[1,3],howeversystematicsurveyshaveshownthatdiarrhoeagenicE.
coli(DEC)areacommoncauseofdiarrhoeainbothdevelopinganddevelopedcountries.
Thesepathogens,especiallyEAEC,mayroutinelybeunderestimatedasacauseofdiarrhoeaduetounder-representationofrequestsanddifficultyrecognisingthesepathogensinthelaboratory[4,5].
TodatesixcategoriesofDEChavebeendefinedonthebasisofspecificvirulenceproperties[6,7].
Entero-toxigenicE.
coli(ETEC)causediarrhoeaviasecretionofheatstable(ST)and/orheatlabile(LT)enterotoxin.
EnteroinvasiveE.
coli(EIEC)strainsarecloselyrelatedtoShigellaspp.
andtheresponsiblegenesarecarriedonthepINVplasmid.
Shiga-toxinproducingorenterohae-morrhagicE.
coli(STEC/EHEC)causebothanon-bloodydiarrhoeaaswellasahaemorrhagiccolitiswhichmaytriggerhaemolyticuraemicsyndrome(HUS).
ThevirulencepropertiesdefiningEHECaretheShigatoxins*Correspondence:colin.
mackenzie@uni-duesseldorf.
deContributedequally1InstituteofMedicalMicrobiologyandHospitalHygiene,Heinrich-Heine-University,Universittsstrae1,40225Düsseldorf,GermanyHardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/52010Hardegenetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
1and2(Stx1,Stx2)and,whenpresent,thepathogeni-cityislandLEE("locusofenterocyteeffacement").
Enter-opathogenicE.
coli(EPEC)showlocalisedadherencetothesmallintestineandcauseahistopathological"attach-ingandeffacing(A/E)lesion"mediatedthroughviru-lencefactorsencodedwithintheLEE.
StrainsadditionallycontainingtheEPECadherencefactor(EAF)plasmidhavebeentermed"typical"EPEC,whereasstrainsthatlacktheEAFplasmidarereferredtoas"atypical"EPEC.
EnteroaggregativeE.
coli(EAECorEAggEC)wasfirstdescribedin1987[8]andhasbeenassociatedwithacutediarrhoeainchildren,HIVpositiveindividuals,andasacauseoftravellers'diar-rhoea[9].
Moreoverithasbeenlinkedtopersistentdiarrhoeaandevengrowthretardationinchildren[10].
Numerousfindingssuggestthatthereisaninflamma-toryprocessunderlyingtheinfection[11,12].
ThePCR-targetusedinthisreportisasequenceontheplasmidpCVD432onwhichthegenescodingforthefimbrialstructure(aggregativeadherencefimbriaI)andfurthervirulencefactorsarelocated[13].
ThesixthcategoryisthediffuselyadherentE.
coli(DAEC)inwhichthebac-teria,unlikeEAECdonotadheretothehostcellsinmicrocoloniesbutratherdiffuselyoverthecellsurface.
NoPCRhasbeendevelopedyettodetectDAECstrains.
SinceDECcannotbediagnosedadequatelybycultureandbiochemicalcriteriaalone,identificationofthesestrainsisdifficult.
ManyPCRtechniquesdetectingvar-iousgenescodingforvirulencetraitsofthedifferentcategoriesofDEChavebeenreported[14-18].
WehavedevelopedthreenewmultiplexTaqManPCRassays,designedtoberuninparallel,todetectETEC,EIEC,EHEC,EPECandEAECsimultaneously.
InadditiontomanystudiesfromdevelopingcountriesrecentreportsunderlinetheimportanceofEPECandEAECindevelopedcountries[5,19-21].
InGermanyverylimiteddataareavailable.
ThereforetheaimofthisstudywastoevaluatethelocalimportanceofEPECandEAECinaGermanUniversityhospitalinrelationtotherequestingclinicaldepartmentandagegroup,usingthenewlydevelopedreal-timePCRasadiagnostictool.
MethodsStudypopulationFromJanuary2006toDecember2006atotalof1981faecalsamplesreceivedforSalmonella,ShigellaandCampylobacterspp.
culturewereincludedinthescreen-ingforEPECandEAEC.
Ofthese371specimenshadnogrowthofEnterobacteriaceaehence1610samplesweretestedforEPECandEAECbyuseofthePCRmethoddescribedbelow.
StoolsampleswereobtainedfrommostdepartmentsoftheUniversityHospitalofDüsseldorf,Germany.
Thepatientagerangedfromafewdaysto98years.
DNApreparationAllfaecalsamplesreceivedwereplatedonMacConkeyAgarandincubatedfor18hat37°C.
Abacterialsus-pensionfromallcolonieswasmadebyrinsingtheentireplatewith2mlofsterilesaline.
Thebacterialsuspen-sionwasdilutedtoMacFarlandStandard1,0(approx.
3*108CFU/ml)usingdistilledwater.
Thissuspensionwasheatedat95°Cfor15minutes.
2,5μl(correspond-ingtoapproximately7,5*105CFU)wereapplieddirectlytothemultiplexPCR.
Theremainderofthesamplewasstoredat-20°C.
TaqManPCRThreedifferentmultiplexTaqManPCRsweredesignedandwereallrunusingthesametimeandtemperaturesettingsonaBioradiCyclersystem.
Atwostepamplifi-cationprofilewasusedasfollows:95°Cfor10min.
fol-lowedby45cyclesof15s.
at95°Cand60s.
at60°Cforannealingandelongation.
ThePCRcontained10pmolofeachprimer,2pmolofthetargetprobes,5pmoloftheinternalinhibitioncontrolprobe(allfromEurogen-tec,Cologne,Germany),100copiesofinhibitioncontrolplasmid,2,5μlsample,12,5μlofNoROXPCRMaster-mix(Qiagen,Hilden,Germany)andwasdilutedwithdistilledwatertoafinalvolumeof25μl.
TwotargetsequenceswereusedforEPECamplifica-tion:a107bpsequenceoftheenteropathogenicE.
coliadherencefactor(EAF)plasmid[22]anda189bplongfragmentoftheintimingene(eae)withinthelocusofenterocyteeffacementbaseduponanalignmentofvar-iouspublishedintiminsubtypesequences(designedwithsupportofTIBMOLBIOL,Berlin,Germany).
ThetargetsequenceforEAECwasa152bpsequenceofthepCVD432plasmid[23].
ThesecondmultiplexPCRwasdesignedtodetectETECandEIEC.
TargetsforETECwerea107bpfragmentoftheSTanda113bpsequenceoftheLTgene.
ForEIECa107bpsequenceofthevirulenceplasmidessentialforinvasiveness,pINVwastargeted.
ThethirdPCRdetectsEHECbytargetinga87bpfragmentoftheshiga-toxin1(stx1)anda82bpsequenceoftheshiga-toxin2(stx2)gene.
AllprobeswerelabelledwithfluorophoresandquenchersaslistedinTable1.
IsolatesofsequencedpathogensservedaspositivecontrolswhileanonpathogenicE.
colistrain(ATCC25922)wasusedasnegativecontrol.
SincethesequencesofpINVandstx1arealsopresentinshigella,inallspecimenspositiveforeitheroftheseaninfectionduetoshigellawasexcludedbyculture.
PCR-baseddetectionmethods,especiallyfromfaecalsamplesarepronetoinhibitionofamplification[24].
Asaninternalcontrol(IC)specimenswerespikedwithsynthesisednucleotidesequencesusinganunrelatedprobesequencefromtheretrotransposonNinjafromDrosophilasimulans(AB110070)inthepCRII-TOPOVector(Invitrogen,Karlsruhe,Germany)flankedbytheHardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page2of7respectiveprimersusedinthePCR[25].
Aserialdilu-tionoftargetedtemplateinaMacFarlandstandard1,0apathogenicE.
colisuspensionrevealedreliablefluores-cencesignalstoameannumberof1,5bacterialgen-omesper2,5μlforalltargets.
Nocrossreactivitywithvariousbacteria(Streptococcus,Staphylococcus,Entero-coccus,Pseudomonas,Klebsiella,Proteus,Citrobacter,Salmonella,Yersinia,Campylobacter,Aeromonascaviae,ClostridiumdifficileandEnterobacterspp.
)otherthantheknownpositivitywithShigelladysenteriaeusingtheEHECPCRwasdetected.
ResultsAllstoolsampleswereobtainedfromtheUniversityHospitalofDüsseldorf.
Altogether144(8,9%)specimenswerepositiveforEPECand78(4,8%)positiveforEAEC,including17samplesthatwerepositiveforbothpatho-gens.
AnexampleofanamplificationplotofadoubleinfectionisshowninFig.
1.
FromthesamplespositiveforEPEConly7(4,9%)werepositiveforbotheaeandEAF(typicalEPEC),makingatypicalEPECthepredomi-nantpathotype.
FivesampleswerepositiveforEAFonly.
Alleaepositivespecimenswereadditionallyexam-inedforthepresenceofstx1andstx2andwereallnega-tive.
IncomparisontotheseresultsSalmonellaspp.
wasfoundin38(2,4%),Campylobacterspp.
in39(2,4%)andShigellaspp.
inonly1(0,06%).
Thedataweredividedintothreegroupsdependingontherequestingdepartment:thedepartmentfortropicaldiseases,paediatricsandalastgroupofalltheTable1PrimersandprobesformultiplexTaqManPCRTargetPrimerorprobenameOligonucleotidesequence(5'3')Ampliconsize/Accessionnr.
Multiplex1:(EPEC/EAEC)EAFEP-1forEP-2revEP-SprobeGTTCTTGGCGAACAGGCTTGTCTTAAGCCAGCTACCATCCACCCCy5-AGTACTGACGTGCAGGTCGCCTGTTCG-BHQ-3107bpX76137eaeEAE-SforEAE-B1revEAE-B2revEAE-TMprobeACTGGACTTCTTATTRCCGTTCTATGCTAAGCGGGTATTGTTACCAGACCTAAACGGGTATTATCACCAGAROX-AATCCTGATCAATGAAGACGTTATAGCCCA-BBQ189bp*Z11541/AB040740pCVD432EA-1forEA-2revEA-SprobeAGGTTTGATATTGATGTCCTTGAGGATCAGCTAATAATGTATAGAAATCCGCTGTTFAM-CATGTTCCTGAGAGTGCAATCCCAGACATTAC-TAMRA152bpX81423Multiplex2:(ETEC/EIEC)STgeneST-1forST-2revST-SprobeCTGGTTTTGATTCAAATGTTCGTGTCCTGAGGGAAAGGTGAAAAAGACROX-TTGATTTCTTCATATTACCTCCGGACATGGCA-BHQ-2107bpM34916LTgeneLT-1forLT-2revLT-SprobeAGCGGCGCAACATTTCAGTTGGTCTCGGTCAGATATGTGATTCFAM-TCGAAGTCCCGGGCAGTCAACATATAGA-TAMRA113bpS60731ipaHEi-1forEi-2revEi-SprobeGAACTCAAATCTTGCACCATTCACGTCCGTCCGAGAACAATTAAGCy5-ATCCCCGACACCGTTTGTGAGTTTCACT-BHQ-3107bpAY206439Multiplex3:(EHEC)stx1slt1-1forslt1-2revslt1-SprobeCTTCCATCTGCCGGACACATAATTAATACTGAATTGTCATCATCATGCATROX-AAGGAAACTCATCAGATGCCATTCTGGCA-BHQ-287bpZ36899stx2slt2-1forslt2-2revslt2-SprobeGACGTGGACCTCACTCTGAACTGTCCCCACTCTGACACCATCCFAM-TACTCCGGAAGCACATTGCTGATTCGC-TAMRA82bpL11079InternalControlDrosophilasimulans+ICprobeHEX-ATGCCTCTTCACATTGCTCCACCTTTCCT-BHQ1AB110070+Thebold,italiclabelledprimersalsodetecttheinternalcontrolplasmidintherespectivePCR*AlignmentofvariousintiminsubtypesequencesHardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page3of7remainingdepartments.
Thedepartmentfortropicaldis-easesgroupconsistedof150samplesonlybuthadarelativelyhighpositivityratewith28EPECpositiveand39EAECpositivesamples.
18,7%ofallspecimenstestedwithinthisgroupwerepositiveforEPECand26%forEAEC.
Mostpatientsseeninthisdepartmenthaveatra-velhistoryand,withtheexceptionofonly4patients,allwerebetween18and65yearsofage.
Fromthepaedia-tricwards458specimensweretestedand49EPECposi-tiveand19EAECpositivesamplesweredetectedshowinganprevalenceof10,7%forEPECand4,1%forEAEC.
Fromtheremaining1002samplesfromallotherdepartments67(6,7%)weretestedpositiveforEPECand20(2%)positiveforEAEC.
Table2showstheresults.
SincemostreportsonDECshowdifferentincidencesdependingonagewedividedthesamplesaccordingtoagegroupsasseeninFig.
2.
ApeakprevalenceforEPECwasfound(16,3%)intheagegroupbetweenoneandtwoyears.
Intheagegroupsofpatientsbelowoneyearofageandbetweentwoandfiveyearsthepreva-lencevariedfrom9,1%to10,2%,whichisslightlyhigherthaninthegroupsbetweenfiveand18years(7,6%)andthegroupabove65yearsofage(4,9%).
Theprevalenceinthegroupbetween18and65years(10,2%)wasunex-pectedlyhigh,butofthe74positivesamples26weresentbythetropicaldiseasesdepartment,indicatingahistoryoftravel.
Withoneexception(onepositivespeci-menintheagegroupbelowoneyear),allspecimensfromthedepartmentoftropicaldiseaseswerefrompatientsover18yearsofage.
ForEAECaclearpeakwasseenintheagegroupbetweentwoandthreeyearswithanprevalenceof14,5%.
Belowtwoyearsofageandbetweenthreeandfiveyearstheprevalencerangedfrom1,3%upto3,3%.
Noneofthesesampleswerefromthetropicaldiseasesdepartment.
Intheagegroupsbetweenfiveand18and18and65asmallerpeakwasfoundwithprevalenceof4,6%and7%respectively.
1,7%ofEAECpositivespecimenswerefrompatientsover65.
Thepeakinthegroupbetween18and65yearswasmostlyduetosamplesfromthedepartmentfortropicaldiseases(36outof51).
Intheagegroupfiveto18years,onespecimenandinthegroupover65years,twospecimenswerereceivedfromthisdepartment.
InourhospitalallstoolspecimensfromchildrenageduptooneyeararetestedforEPECroutinely,howeveritwasnotedthatrequestsforDECareveryrarelymadeforpatientsabovethisage.
ThereforewelookedatthenumberofDECcasesfoundbyroutineinvestigationsincomparisontothetotalnumberofcasesfoundbysys-tematicscreeningofallspecimensreceived.
Table2showsthefindingsforEPECandEAEC.
15EPECposi-tivesamplesfromchildrenbelowoneyearofageweredetectedbyroutinescreeningandofthepositiveresultsforEPECorEAECinthegroupofsamplesfromchil-drenbetweenoneandfiveyearsofagenonewasdetectedbyroutinerequest.
Intheagegroupbetween5and18yearsofageonlyone(EPECpositive)specimenwasactuallyrequestedbythepaediatricdepartmentand1(EAECpositive)specimenfromthedepartmentfortropicaldiseases.
Between18and65yearsarelativelylargenumberofpositiveresultswasfoundinscreeningFigure1AmplificationplotofadoubleinfectionwithatypicalEPECandEAEC.
Table2NumberofstoolsamplestestedaccordingtodepartmentandpositivityrateforEPECandEAECDepartmenttestedEPEC(%)EAEC(%)tropicaldiseases15028(18,7)39(26,0)paediatrics45849(10,7)19(4,1)others100267(6,7)20(2,0)Hardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page4of7allreceivedstoolspecimens,butonly16(21,6%)EPECpositiveand15(29,4%)EAECpositivespecimensweredetectedduetoaspecificrequestfromtherespectivedepartment.
Furthermore,exceptforthreepositiveEPECspecimensandonepositiveEAECspecimen,allofthespecimensweresentbythedepartmentfortropi-caldiseases.
Inthegroupabove65yearsinonlytwoofthespecimensthatwerepositiveforEAECwastheinvestigationroutinelyrequested,againbythedepart-mentfortropicaldiseases.
DiscussionDetectingDECreliesgreatlyonPCRtechniquesandmanyreportsareavailableonproceduresusedtoiden-tifyallcategoriesofDEC[13-18,26].
Mostofthesetech-niqueshowever,arebasedonstandardPCR.
Wehavedevelopedanovelmultiplexreal-timePCRwhich,withthreereactionsrunparalleltooneanother,identifiesthe5majorcategoriesofDECsimultaneously.
ToourknowledgethisisthefirstreporteduseofaTaqManPCR,whichprovidesbothaconvenientdiagnostictoolandavoidstheriskofcrosscontaminationduetopostamplificationhandling.
Byrinsingallthecoloniesfromtheagarplatewithsalineratherthanpickingrepresentativecoloniesashasbeendescribedintheliterature,wewereabletoincreasethesensitivityofthePCR.
Thismethodisquick,simpleandinexpensive.
Thereal-timePCRwasabletodetect1,5genomeequivalentsperassay.
AsreportedbytheRobertKochInstitute,Berlin,Ger-many,ofallreportableintestinalinfectionsin2006inGermany,DEC,exceptingEHEC,wasthefifthlargestgroupofpathogensafterNorovirus,Rotavirus,Salmo-nellaandCampylobacterspp.
Inourstudyreportedhere,theprevalenceofEPEC(8,9%)andEAEC(4,8%)exceededthoseforsalmonellaandcampylobacter,each2,4%.
ItispossiblethattheincidenceofEPECandEAECthroughoutGermanyisfarhigherthanthatreported.
ThisdiscrepancymayverywellbeduetothelowlevelofroutinerequestsforDECasweobservedinthestudyperiodinthishospital.
UnfortunatelynodatacanbeprovidedheretocomparetheincidenceofDECtothatofviralpathogenssinceinthisstudystoolspeci-menswerenotsystematicallytestedforviralpathogens.
However,inthepaediatricpopulation(theagegroupunder18yearsinthisstudy),manyspecimenswereadditionallytestedforvirusesandoutof40EPECposi-tivesamplesadditionallytestedforviruses,16wereadditionallypositiveforrotavirus,norovirusoradeno-virus.
4specimenspositiveforEPECwereadditionallypositiveforSalmonellaspp.
(1)orCampylobacterspp.
(3).
ThisleavesEPECasthesoleidentifiedpathogenin50%ofallcases.
EAECwastheonlypathogendetectedin45%ofthesamplesinthisagegroup.
Inninecasesthespecimenwaspositiveforrotavirus,norovirusoradenovirusaswell,whereasinthreesamplesCampylo-bacterspp.
wasfoundasaco-infectionandonespeci-menwastestedpositiveforSalmonellaspp.
EventhoughthisstudyprovidesnosystematicdataonviraltestingwemustassumethatviralinfectionsoutnumberthecasesofdiarrhoeaduetoDECinchildren.
Itispos-siblethatmorestoolsamplesweresubmittedforviraldetectiononlyandthustheprevalenceforEPECandEAECfoundinthisstudymaystillbeanunderestima-tioninthisagegroup.
AstrongassociationbetweenEPECanddiarrhoeainchildrenhasbeenreported[27],yetwealsofoundarelativelyhighprevalenceforEPECinolderpatients.
AlargeproportionofEPEC(andEAEC)infectionsmaywellbeassociatedwithtravel,sinceahighnumberofpositivespecimenswithinthegroupbetween18and65yearswereobtainedfromthedepartmentfortropicaldiseases.
ConcerningtheassociationofatypicalEPECwithdiarrhoeafindingsarecontradictory[27-29],butitcontinuestobethemostprevalentpathotypeofEPECfoundinindustrialisedcountries[27,29,30],whichwealsonotedinourstudy.
AsubsetoftheheterogeneousgroupofatypicalEPEChasbeenfoundinpatientswithbloodydiarrhoea,(insomecasesleadingtoHUS).
TheyresembledEHECaccordingtotheirserotype,virulenceprofileandmultilocussequencetypes,leadingtotheassumptionthatthesestrainsmightbeEHECthathaveatsomestagelosttheshigatoxingeneduringinfection[31].
UnusualinthisstudyisthedetectionofEAFwith-outtheeaegeneasmostofteneaeisfoundalone(atypi-calEPEC)orwithEAF.
ItisquestionablewhetherstrainspositiveforEAFbutnotforeaeshouldbeclassi-fiedasEPECandwhethertheyactuallyarepathogenic.
FurtherinvestigationisrequiredastowhetherthisisduetoasequencevariationwithintheintimingeneFigure2ThepercentofspecimenspositiveforEPEC(whitesquare)andEAEC(blacksquare)inagegroupscomparedtoallspecimensreceivedfrompatientsintherespectiveagegroup.
Hardegenetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:5http://www.
ann-clinmicrob.
com/content/9/1/5Page5of7resultinginafailureofamplificationorwhetherperhapsotherbacterialstrainshaveacquiredtheEAFplasmid.
OnelimitationofthisstudyistheinabilityofthemethodtodistinguishbetweenthosepatientswithadoubleinfectionwithtwoormoreDECandthosepatientswhoareinfectedwithasinglebacteriumcarry-ingmorethanonevirulencegene.
Becauseweuseapoolofbacteriafromanovernightculture,itisnotpos-sibletotracetheresultbacktoasinglebacterialclone.
Inthisstudywefound17specimenswhichwereposi-tiveforbothEPECandEAEC.
Innocasewasthedis-tinctionbetweenasinglebacteriumcarryingbothfactorsoradoubleinfectionclinicallyrelevant.
Never-theless,itwouldbeinterestingtodeterminethefre-quencyofmultiplegenecarriagebyasinglecloneandwearecurrentlyprospectivelyattemptingtotracebacktothegene-carryingclone(s).
ConclusionsInourstudywefindtheprevalenceforEPECandEAECtobeunexpectedlyhighinDüsseldorf,andweconcludethatallpatientswithdiarrhoealdiseaseshouldberouti-nelytestedforthesepathogens,especiallychildrenbelowtheageoffive,returningtravellersandinthosespecimensinwhichnootherpathogencanbeidentified.
WealsodemonstratetheuseofanovelmultiplexPCRforthedetectionofdiarrhoeagenicE.
colifromstoolspecimens.
Authordetails1InstituteofMedicalMicrobiologyandHospitalHygiene,Heinrich-Heine-University,Universittsstrae1,40225Düsseldorf,Germany.
2TranslationalPharmacologyDiscoveryMedicine,GlaxoSmithKline,GunnelsWoodRoad,StevenageSG12NY,UK.
Authors'contributionsCHandSMperformedallthemolecularstudies,collectedthedataandwrotethemanuscript,JWandKPdesignedthestudyandmolecularprobesandprimers,BHandCMcarriedoutthedesignandcoordinationofthestudyandtookpartinthewritingofthemanuscriptAllauthorshavereadandapprovedthefinalmanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Received:4November2009Accepted:22January2010Published:22January2010References1.
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