Comparison of microbiological diagnosis of urinary tract infection in young children by routine

13 Pages·2017·0.84 MB·English

RESEARCHARTICLE
Comparison of microbiological diagnosis of
urinary tract infection in young children by
routine health service laboratories and a
research laboratory: Diagnostic cohort study
KateBirnie1,AlastairD.Hay2,MandyWootton3,RobinHowe3,AlasdairMacGowan4,
PennyWhiting1,5,MichaelLawton1,BrendanDelaney6,HarrietDowning2,JanDudley7,
WilliamHollingworth1,CatherineLisles8,PaulLittle9,KathrynO’Brien10,Timothy
a1111111111
Pickles8,KateRumsby9,EmmaThomas-Jones8,JudithVanderVoort11,Cherry-
a1111111111
AnnWaldron8,KimHarman9,KerenzaHood8,ChristopherC.Butler10,12,JonathanA.C.
a1111111111
Sterne1*
a1111111111
a1111111111 1 SchoolofSocialandCommunityMedicine,UniversityofBristol,Bristol,UnitedKingdom,2 Centrefor
AcademicPrimaryCare,NIHRSchoolofPrimaryCareResearch,SchoolofSocialandCommunityMedicine,
UniversityofBristol,Bristol,UnitedKingdom,3 SpecialistAntimicrobialChemotherapyUnit,PublicHealth
WalesMicrobiologyCardiff,UniversityHospitalWales,Cardiff,UnitedKingdom,4 NorthBristolNHSTrust,
Bristol,UnitedKingdom,5 KleijnenSystematicReviewsLtd,York,UnitedKingdom,6 NIHRBiomedical
ResearchCentreatGuy’sandStThomas’NHSFoundationTrustandKing’sCollegeLondon,Departmentof
OPENACCESS PrimaryCareandPublicHealthSciences,London,UnitedKingdom,7 BristolRoyalHospitalforChildren,
UniversityHospitalsBristol,NHSFoundationTrust,Bristol,UnitedKingdom,8 SouthEastWalesTrialsUnit
Citation:BirnieK,HayAD,WoottonM,HoweR,
(SEWTU),InstituteforTranslation,Innovation,MethodologyandEngagement,SchoolofMedicine,Cardiff
MacGowanA,WhitingP,etal.(2017)Comparison
University,Cardiff,UnitedKingdom,9 PrimaryCareandPopulationSciencesDivision,Universityof
ofmicrobiologicaldiagnosisofurinarytract Southampton,Southampton,UnitedKingdom,10 CochraneInstituteofPrimaryCare&PublicHealth,School
infectioninyoungchildrenbyroutinehealth ofMedicine,CardiffUniversity,Cardiff,UnitedKingdom,11 DepartmentofPaediatricsandChildHealth,
servicelaboratoriesandaresearchlaboratory: UniversityHospitalofWales,Cardiff,UnitedKingdom,12 NuffieldDepartmentofPrimaryCareHealth
Diagnosticcohortstudy.PLoSONE12(2): Sciences,UniversityofOxford,Oxford,UnitedKingdom
e0171113.doi:10.1371/journal.pone.0171113
*[email protected]
Editor:MartinChalumeau,UniversiteParis
Descartes,FRANCE
Received:July19,2016 Abstract
Accepted:January16,2017
Published:February15,2017
Objectives
Copyright:©2017Birnieetal.Thisisanopen
accessarticledistributedunderthetermsofthe
Tocomparethevalidityofdiagnosisofurinarytractinfection(UTI)throughurineculture
CreativeCommonsAttributionLicense,which
betweensamplesprocessedinroutinehealthservicelaboratoriesandthoseprocessedin
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal aresearchlaboratory.
authorandsourcearecredited.
DataAvailabilityStatement:Weareunableto
Populationandmethods
sharesupportingdataduetoethical
considerations.TheUniversityofBristolResearch Weconductedaprospectivediagnosticcohortstudyin4808acutelyillchildrenaged<5
DataServicehasexaminedthestudyconsentform
yearsattendingUKprimaryhealthcare.UTI,definedaspure/predominantgrowth(cid:21)105
andparentinformationsheetapprovedbyour
institutionalethicscommittee,andhasadvisedthat CFU/mLofauropathogen(thereferencestandard),wasdiagnosedatroutinehealthservice
wecannotshareanydatafromthisstudy, laboratoriesandacentralresearchlaboratorybycultureofurinesamples.Wecalculated
includingaggregategroupdata.Thisisbecausethe
areasunderthereceiver-operatorcurve(AUC)forUTIpredictedbypre-specifiedsymp-
consentformincludesconsentforsharingdata
toms,signsanddipsticktestresults(the“indextest”),separatelyaccordingtowhethersam-
withregulatoryauthorities,butnoothersharingis
permitted.Interestedresearchersmaysenddata pleswereobtainedbycleancatchornappy(diaper)pads.
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 1/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
requestsandqueriesto:SouthWestCentralBristol Results
ResearchEthicsCommittee+44(0)2071048028
251(5.2%)and88(1.8%)childrenwereclassifiedasUTIpositivebyhealthserviceand
[email protected].
UniversityofBristolResearchDataService+44(0) researchlaboratoriesrespectively.Agreementbetweenlaboratorieswasmoderate(kappa=
[email protected]. 0.36;95%confidenceinterval[CI]0.29,0.43),andbetterforcleancatch(0.54;0.45,0.63)
Funding:ThisworkwassupportedbytheNational thannappypadsamples(0.20;0.12,0.28).Incleancatchsamples,theAUCwaslowerfor
InstituteforHealthResearch(NIHR)asaHealth healthservicelaboratories(AUC=0.75;95%CI0.69,0.80)thantheresearchlaboratory
TechnologyAssessmentProgramme(project
(0.86;0.79,0.92).ValuesofAUCwerelowerinnappypadsamples(0.65[0.61,0.70]and
number08/66/01).TheDUTYstudywaspublished
infullasanNIHRHealthTechnologyAssessment 0.79[0.70,0.88]forhealthserviceandresearchlaboratorypositivity,respectively)thanclean
(HTA)monograph.Theviewsandopinions catchsamples.
expressedthereinarethoseoftheauthorsanddo
notnecessarilyreflectthoseoftheHTA
programme,NIHR,NHSortheDepartmentof Conclusions
Health.JonathanSternewassupportedbyNIHR
TheagreementofmicrobiologicaldiagnosisofUTIcomparingroutinehealthservicelabora-
SeniorInvestigatorAwardNF-SI-0611-10168.AH
isfundedbyNIHRResearchProfessorship(NIHR- torieswitharesearchlaboratorywasmoderateforcleancatchsamplesandpoorfornappy
RP-02-12-012).KBwassupportedbyaMedical padsamplesandreliabilityislowerfornappypadthanforcleancatchsamples.Positive
ResearchCouncilUKfellowship(RD1826).PW
resultsfromtheresearchlaboratoryappearmorelikelytoreflectrealUTIsthanthosefrom
wasemployedbyKleijnenSystematicReviewsLtd.
routinehealthservicelaboratories,manyofwhich(particularlyfromnappypadsamples)
Thefundersprovidedsupportintheformof
salariesforauthors[KB,AH,MW,RH,AM,PW, couldbeduetocontamination.Healthservicelaboratoriesshouldconsideradoptingproce-
ML,BD,HD,JD,WH,CL,PL,KOB,TP,KR,ETJ, duresusedintheresearchlaboratoryforpaediatricurinesamples.Primarycareclinicians
JV,CAW,KiH,KH,CB,JS],butdidnothaveany
shouldtrytoobtaincleancatchsamples,eveninveryyoungchildren.
additionalroleinthestudydesign,datacollection
andanalysis,decisiontopublish,orpreparationof
themanuscript.Thespecificrolesoftheseauthors
arearticulatedinthe‘authorcontributions’section.
Competinginterests:NoneforallexceptforP.
LittlewhoisamemberoftheNIHRJournals Introduction
LibraryBoardandhasprovidedconsultancywork
toBayerPharmaceuticals.Thisdoesnotalterour Urinarytractinfection(UTI)affects6%ofacutelyunwellchildrenpresentingtoUKgeneral
adherencetoPLOSONEpoliciesonsharingdata practice.[1]Timelydiagnosisandtreatmentmayalleviateshort-termsymptomsandcould
andmaterials. potentiallypreventlong-termadverseconsequencessuchasrenalscarring,impairedrenal
growth,recurrentpyelonephritis,impairedglomerularfiltration,hypertension,endstagerenal
disease,andpre-eclampsia.[2–4]Howeverestablishingadiagnosisinpre-orearly-schoolaged
childrenischallenging;manyarepre-verbalandcollectionofuncontaminatedurinesamples
isdifficult.[5]UKNationalInstituteforHealthandClinicalExcellence(NICE)guidelinessay
thata“cleancatch”sampleistherecommendedmethodforurinecollection,buturinecollec-
tionpadsareadvisedifthisisnotpossible.[6]TheAmericanAcademyofPediatricspractice
clinicalguidelinesrecommendthaturineiscollectedbycatheterizationorsuprapubicaspira-
tioninyoungchildren[7],butthesecollectionmethodsareinvasiveandmaybeunacceptable
toparents,andsoareuncommoninUKprimarycare.
Laboratorydiagnosisisbasedoncolonycountsfollowingculture.UTIistypicallycaused
byasingleorganismpresentinhighconcentration,usually(cid:21)105colony-formingunits(CFU)
permL.[8]Laboratoryguidelinesdifferregardingtheextentofgrowthrequiredtoconfirm
UTI.[9,10]NICEguidelinesdonotprovideadefinitivethreshold.[6]NationalHealthService
(NHS)laboratoriesaretheroutinehealthservicelaboratoriesintheUKandtheyfollowthe
UKStandardsforMicrobiologicalInvestigation[10]forexaminationofurine,butapplication
variesbetweenlaboratories.
TheaimofthisstudywastocomparethevalidityofdiagnosisofUTIthroughurineculture
betweensamplesprocessedinroutinehealthservicelaboratoriesandthoseprocessedina
researchlaboratory,usingdatafromadiagnosticcohortstudyamongunselectedchildrenaged
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 2/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
<5yearspresentingtoprimarycareinEnglandandWaleswithacuteillnesses.Becausethereis
noindependentreference(“gold-standard”)testfordiagnosisofUTI,wecouldnotdirectly
assessthediagnosticaccuracyofcultureresults.Wethereforeevaluatedthevalidityofdiagnosis
byexaminingassociationsofpre-specifiedparent-reportedsymptoms,clinician-reportedsigns,
andurinedipsticktestresultswithurineculturepositivityinthetwotypesoflaboratory.
Populationandmethods
TheDiagnosisofUrinaryTractinfectioninYoungchildren(DUTY)studywasamulticentre,
prospective,diagnosticcohortstudy.Themethodsofrecruitmentaredescribedindetailinthe
studyprotocol.[11]Childrenwereeligibleiftheywereaged<5years,presentedtoprimary
carewithanyacuteillnessepisodeof<28daysdurationandhadconstitutionalorurinary
symptomsassociatedwiththeiracuteillness.Childrenwereexcludediftheywerenotconstitu-
tionallyunwell,hadaneurogenicorsurgicallyreconstructedbladder,usedaurinarycatheter,
presentedwithtrauma,orhadtakenantibioticswithinthepastweek.Werecruitedpartici-
pantsfrom233primarycaresites(225GeneralPractitioner[GP]practices,fourWalk-inCen-
tresandfourpaediatricEmergencyDepartments)acrossEnglandandWalesbetweenApril
2010andApril2012.Clinicianswereaskedtorecruitconsecutiveeligiblechildren.Following
writteninformedparentalconsent,datawerecollectedonpersonaldetails,medicalhistory,
presentingsymptomsandresultsoftheclinicalexaminationincludingaclinician-reported
globalimpressionofillnessseverity(score0–10).Multi-centreethicalapprovalwasgranted
forthisstudybytheSouthWestSouthmeadResearchEthicsCommittee,Ref#09/H0102/64.
Urinesampleswereobtainedbycleancatchwherepossible,forchildrenwhoweretoilet
trainedorforwhomtheparent/guardianwashappytoattemptsuchcollection.‘Newcastle
NappyPads’wereusedforchildrenstillusingnappies(diapers)whoseparent/guardiandid
notthinkcleancatchwouldbesuccessful.TheResearchNurse,wearingdisposablegloves,
removedthepadandsqueezedtheurineintoasterilebowl.Samplesweredipsticktestedfor
blood,protein,glucose,ketones,nitrite,leukocyteesterase,pHandspecificgravityusingSie-
mens/BayerMultistix8SG.
Urinesamplesweresplitintotwofractionswiththepriorityfractionsenttotheroutine
healthservicelaboratoryusuallyusedbytherecruitingsite.Thesamplesweresenttothelocal
routineheathservicelaboratoryusingthesite’snormalmethodoftransport.Allsampleswere
senttothelaboratoryassoonaspossibleaftercollection,butwererefrigeratediftransportto
thelaboratorywasdelayedformorethanfourhours.Ifsufficienturinewasavailable,asecond
fractionwassenttotheSpecialistAntimicrobialChemotherapyUnit,PublicHealthWales
MicrobiologyLaboratory(“ResearchLaboratory”;RL).Samplesweresenttoroutineheathser-
vicelaboratoriesusingsterileurinecontainers,andtheresearchlaboratoryviaRoyalMailSafe-
boxesTMinaurineMonovettecontainingboricacid.HealthServicelaboratoriesusedlocal
StandardOperatingProcedures(SOPs)andreportedbacterialgrowth(<103;103-<105;or
(cid:21)105CFU/mL),purity(pure/predominant;mixedgrowth2species;mixedgrowth>2spe-
cies),speciationforuptotwospecies,andmicroscopyforwhiteandredcells.Alllocalhealth
servicelaboratorieswere‘ClinicalPracticeAccredited’andNHSlaboratorySOPswereusedto
processDUTYurinesamples.AlllocalhealthservicelaboratorySOPswerebasedonthePub-
licHealthEnglandguidelinefortheinvestigationofurine.[10]Asummaryoftheseprocesses
isgiveninS1Table.Intheresearchlaboratory,automatedmicroscopywasperformedusing
theIRISIQ200Sprint(InstrumentationLaboratories)then50μLculturedontochromogenic
agarandColumbiabloodagarusingaspiralplater(DonWhitley,UK)(S2Table).Absolute
colonycounts(range101−1010CFU/mL)wererecordedforallorganismspresent,andspecies
identification(usingaPhoenixautomatedID/ASTsystem[BDdiagnostics]plusconventional
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 3/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
methods)fororganismspresentat(cid:21)103CFU/mL.Sensitivitiestofirstlineantimicrobialswere
recordedforpure/predominantculturesandthepresenceofantimicrobialsubstancesinvesti-
gatedbyinhibitionofgrowthofBacillussubtilis(NCTC10400).
Analyseswererestrictedtosampleswithresultsfrombothhealthserviceandresearchlabo-
ratories.UropathogensweredefinedasmembersoftheEnterobacteriaceaefamily.Therefer-
encestandardwasUTI,definedaccordingtoUKguidelines[6]aspure/predominantgrowth
(cid:21)105CFU/mLofauropathogen.Forhealthservicelaboratories,sampleswithpure/predomi-
nantgrowthofauropathogenat(cid:21)105CFU/mLwereconsideredUTIpositive.Fortheresearch
laboratory,sampleswithgrowthof(cid:21)105CFU/mLofasingleuropathogen(“puregrowth”)or
growthof(cid:21)105CFU/mLofauropathogenwith(cid:21)3log differencebetweengrowththisand
10
thenextspecies(“predominantgrowth”)wereconsideredUTIpositive.Agreementwas
assessedusingkappastatistics.Becausewefoundthatagreementbetweenhealthserviceand
researchlaboratorieswasbetterforsamplescollectedthroughcleancatchthanforthosecol-
lectedusingnappypads,mostanalyseswerestratifiedbyurinecollectionmethod.Analyses
wereadditionallystratifiedbyurinecollectionmethodandage(0to<2,2to<3and3to<5
years).FurtherdetailsofstudymethodsareprovidedinS1Text.
Apriori,weselectedthe“indextest”forthisstudytobeasmallnumberofsymptoms,signs
anddipsticktestresultsreportedintheliteraturetobeclearlyrelatedtoUTI[12]:urinarysymp-
toms(pain/cryingwhenpassingurine,passingurinemoreoften,changesinurineappearance);
temperature(cid:21)39˚C,andnitriteorleukocytepositiveresultsfromurinedipsticktests.Wedecided
apriori(basedonlyoninspectionofsymptomfrequencies)todichotomisesymptomresponse
categoriesas“no,slightornotknown”and“moderateorsevere”.Observersassessingthe“index
test”differedfromandwereblindtothereferencestandard(andviceversa).
Weusedmultivariablelogisticregression,includingtheselectedsymptoms,signsanddip-
sticktestresults,toquantifyassociationswithUTIpositivity.Fromthelogisticregression
equation,weplottedReceiverOperatingCharacteristic(ROC)curvesandusedtheareaunder
theROCcurve(AUC)with95%confidenceinterval(CI)toquantifydiagnosticaccuracy.The
maximumvalueoftheAUCis1(perfectprediction)whileavalueof0.5correspondstono
associationwithanypredictor.WeestimatedAUCs:(1)stratifyingbyage(<3and(cid:21)3years),
(2)allowingfor“notknown”categoriesforvariablesforwhichtheseoccurredsufficientlyfre-
quently,(3)stratifyingbywhethersampleswerecollectedatthesurgeryorathome,(4)strati-
fyingbytimebetweentakingurinesampleandlaboratorysamplereceipt(<24hoursand(cid:21)24
hours),(5)stratifyingroutineheathservicelaboratoryresultsaccordingtoextentofpure/pre-
dominantgrowth((cid:21)105,(cid:21)103-<105CFU/mL),(6)stratifyingresearchlaboratoryresults
accordingtoextentofpure/predominantgrowth((cid:21)107,(cid:21)106-<107,(cid:21)105-<106,(cid:21)104-<105,
(cid:21)103-<104CFU/mL),(7)stratifyingaccordingtowhetherwhitebloodcellcountwas<30or
(cid:21)30/mm3and(8)stratifyingresearchlaboratoryresultsaccordingtowhethergrowthwas
pureorpredominant.AnalyseswerecarriedoutusingStataTMversion12.
Results
Of7163childrenrecruitedtothestudy,4828hadresultsfrombothlaboratoriesand4808had
informationavailableoncandidatepredictors(S1Fig).Thechildrenwhowereincludedinthis
studywereolder(meanage29months)comparedtochildrenwhowererecruitedtoDUTY,
butwerenotincludedinthisstudy(meanage21months).Therewerenogenderdifferences
(49.0%vs49.6%male,forthoseincludedandnotincludedinourstudy,respectively),but
therewasasmalldifferenceinethnicity(83.3%whiteinourstudyvs80.3%whiteinthosewho
wererecruitedbutnotincluded).Mostchildrenwhowereincludedinthestudy(4543,94.5%)
wererecruitedfromGPsurgeries(Table1).Therewereapproximatelyequalnumbersofboys
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 4/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
Table1. Characteristicsofchildrenandurinesamplescollectedviacleancatchornappypads,forthe4808childrenwithbotharoutinehealthser-
vicelaboratoryandresearchlaboratoryresult.
Variable Category Age<3years Age3–5years
Gender Male 1439(49.9%) 919(47.8%)
Female 1445(50.1%) 1005(52.2%)
Age(years) 0to<1 1016(35.2%) 0
1to<2 942(32.7%) 0
2to<3 926(32.1%) 0
3to<4 0 1099(57.1%)
4to<5 0 825(42.9%)
Ethnicity White 2429(84.2%) 1575(81.9%)
Non-white 431(14.9%) 328(17.1%)
Notknown 24(0.8%) 21(1.1%)
Recruitmentsite GPsurgery 2716(94.2%) 1827(95.0%)
Emergencydepartment 128(4.4%) 66(3.4%)
Walkincentre 40(1.4%) 31(1.6%)
Samplemethod Cleancatch 758(26.3%) 1861(96.7%)
Nappypad 2126(73.7%) 63(3.3%)
Locationofsamplecollection Surgery 1470(51.0%) 1477(76.8%)
Home 1414(49.0%) 447(23.2%)
Timebetweenclinicalexamandtakingurinesample <24hours 2683(93.0%) 1853(96.3%)
(cid:21)24hours 201(7.0%) 71(3.7%)
Timebetweentakingurinesampleandlaboratorysamplereceipt Healthservicelaboratory<24hours 2045(70.9%) 1432(74.4%)
Healthservicelaboratory(cid:21)24hours 839(29.1%) 492(25.6%)
Researchlaboratory<24hours 816(28.3%) 608(31.6%)
Researchlaboratory(cid:21)24hours 2068(71.7%) 1316(68.4%)
Pain/cryingwhenpassingurine Noorslightproblem 1812(62.8%) 1734(90.1%)
Moderateorsevereproblem 92(3.2%) 125(6.5%)
Notknown 980(34.0%) 65(3.4%)
Passingurinemoreoften Noorslightproblem 1618(56.1%) 1604(83.4%)
Moderateorsevereproblem 228(7.9%) 256(13.3%)
Notknown 1038(40.0%) 64(3.3%)
Changesinurineappearance Noorslightproblem 2206(76.5%) 1539(80.0%)
Moderateorsevereproblem 297(10.3%) 226(11.8%)
Notknown 381(13.2%) 159(8.3%)
Dayorbedwettingwhenpreviouslydry Noorslightproblem 364(12.6%) 1551(80.6%)
Moderateorsevereproblem 45(1.6%) 164(8.5%)
Wearsnappiesdayandnight 2377(82.4%) 70(3.6%)
Notknown 98(3.4%) 139(7.2%)
HistoryofUTI No 2699(93.6%) 1708(88.8%)
Yes 81(2.8%) 140(7.3%)
Notknown 104(3.6%) 76(4.0%)
Temperature <39˚C 2780(96.4%) 1843(95.8%)
(cid:21)39˚C 104(3.6%) 81(4.2%)
Urinedipstick Negative 2511(87.1%) 1881(97.8%)
nitrite Positive 373(12.9%) 43(2.2%)
(Continued)
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 5/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
Table1. (Continued)
Variable Category Age<3years Age3–5years
Urinedipstickleukocytes Negative/trace 2423(84.0%) 1715(89.1%)
Positive 461(16.0%) 209(10.8%)
Routinehealthservicelaboratoryresult Negative 2695(93.5%) 1862(96.8%)
Positive 189(6.6%) 62(3.2%)
Researchlaboratoryresult Negative 2833(98.2%) 1887(98.1%)
Positive 51(1.8%) 37(1.9%)
doi:10.1371/journal.pone.0171113.t001
andgirls.Atotalof2884children(60%)wereaged<3yearsand140children(2.9%)wereaged
<3months.Urinesampleswerecollectedusingcleancatchfor758(26.3%)of2884children
aged<3yearsand1861(96.7%)of1924childrenaged3–5years.Amongchildrenaged<3
years,sampleswereobtainedinthesurgeryin1470(51.0%)childrenaged<3yearsand1477
(76.8%)aged3–5years.94%ofsampleswereprovidedwithin24hoursofclinicalexaminations.
Healthservicelaboratorytransportsystemswerefasterthantheresearchlaboratorywith72.3%
vs.29.6%samplesarrivinginthelaboratorywithin24hours.Parentsreportedthefollowing
symptomsintheirchildrenasamoderateorsevereproblem:painorcryingwhenpassingurine
217(4.5%),passingurinemoreoften484(10.1%),dayorbedwettingwhenpreviouslydry209
(4.3%)andchangeinurineappearance523(10.9%).AhistoryofUTIwasreportedin221
(4.6%)children,140ofwhomwereaged(cid:21)3years.185(3.8%)childrenhadatemperature
(cid:21)39˚C,andfeveratanytimeduringtheillnesswasamoderate/severeproblemin2581(53.7%)
participants.Bothnitrite(12.9%comparedwith2.2%)andleukocyte(16.0%comparedwith
10.8%)urinedipstickpositivityweremorecommoninchildrenaged<3than(cid:21)3years.Weare
notawareofanyadverseeventsresultingfromthemeasurementof“index”orreferencetests.
Atotalof251(5.2%)and88(1.8%)sampleswereclassifiedUTIpositivebyhealthservice
andresearchlaboratories,respectively.Thecausativeorganismdistributionsweresimilar
betweenlaboratories;inthehealthservicelaboratorythecausativeorganismswere:E.coli
71.7%,other/unknowncoliforms22.3%andProteusspp.6.0%;intheresearchlaboratory:E.
coli84.1%,othercoliform(Klebsiellaspp.,Enterobacterspp.,Serratiaspp.,Citrobacterspp.,
Morganellaspp.)10.2%,Proteusspp.5.7%.Routinehealthservicelaboratorypositivitywas
morecommoninchildrenaged<3years(6.6%)thaninthoseaged(cid:21)3years(3.2%).Bycon-
trast,ratesofresearchlaboratorypositivityweresimilarintheseagegroups(1.8%and1.9%,
respectively).Only64(1.3%)sampleswerepositiveinbothlaboratories.In187(3.9%),the
healthservicelaboratoryresultwaspositivebutresearchlaboratoryresultnegativewhilein24
(0.5%)theresearchlaboratoryresultwaspositivebuthealthservicelaboratoryresultnegative
(Table2).Incleancatchsamples,104(4.0%)and59(2.3%)sampleswereclassifiedUTIposi-
tivebyhealthserviceandresearchlaboratories,respectively.Innappypadsamples,147(6.7%)
wereclassifiedasUTIpositiveinhealthservicelaboratories,and29(1.3%)sampleswereclassi-
fiedUTIpositivebytheresearchlaboratory.Thedistributionofclinicianglobalillnessseverity
scaleforroutineheathserviceandresearchlaboratoryUTIpositiveisshowninS2Fig.The
mostcommonclinicaldiagnosesinthechildrenwhowerenotUTIpositiveinthehealthser-
viceandresearchlaboratories,respectively,were‘upperrespiratorytractinfection’(31.0%and
31.3%),‘viralillness’(16.6%and17.7%)andotitismedia(10.0%and9.8%).
Overallagreementbetweenthehealthserviceandresearchlaboratorieswasmoderate
(kappa=0.36;95%CI0.29,0.43;Table2).Agreementwasbetterforcleancatchsamples(0.54;
0.45,0.63)thanfornappypads(0.20;0.12,0.28).Forchildrenaged(cid:21)3years,toofewnappy
padsampleswereavailabletoallowassessmentofreliability.Forcleancatchsamples,reliability
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 6/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
Table2. Extentofagreementbetweenhealthservicelaboratory(HSL)andresearchlaboratory(RL)results.
Agegroupandsamplecollectionmethod N HSL-ve,RL-ve HSL-ve,RL+ve HSL+ve,RL-ve HSL+ve,RL+ve Kappa 95%CI
Bothcollectionmethods 4808 4533 24 187 64 0.36 (0.29,0.43)
Cleancatch 2619 2501 14 59 45 0.54 (0.45,0.63)
Nappypad 2189 2032 10 128 19 0.20 (0.12,0.28)
(cid:21)3years 1924 1852 10 35 27 0.53 (0.41,0.65)
Cleancatch 1861 1792 10 32 27 0.55 (0.43,0.67)
Nappypad 63 60 0 3 0 N/A N/A
<3years 2884 2681 14 152 37 0.29 (0.21,0.36)
Cleancatch 758 709 4 27 18 0.52 (0.37,0.67)
Nappypad 2126 1972 10 125 19 0.20 (0.12,0.28)
<2years 1958 1809 7 121 21 0.23 (0.15,0.31)
Cleancatch 173 155 0 12 6 0.47 (0.23,0.72)
Nappypad 1785 1654 7 109 15 0.19 (0.10,0.27)
(cid:21)2and<3years 926 872 7 31 16 0.44 (0.29,0.59)
Cleancatch 585 554 4 15 12 0.54 (0.36,0.72)
Nappypad 341 318 3 16 4 0.27 (0.05,0.50)
-ve:negative,+ve:positive,HSL:healthservicelaboratory,RL:researchlaboratory.N/A:cannotcomputekappastatisticbecausenosampleswere
classifiedaspositivebytheresearchlaboratory.
doi:10.1371/journal.pone.0171113.t002
wassimilarinchildrenaged(cid:21)3years(0.55;0.43,0.67)and<3years(0.52;0.37,0.67),which
wasbetterthanfornappypadsamplesinchildrenaged<3years(0.20;0.12,0.28).Similarpat-
ternswereseenwhencomparisonswerefurtherstratifiedintoagegroups<2and(cid:21)2to<3
years,suggestingthatlowerreliabilitywasattributabletothenappypadsamplingratherthan
child’sage.Agreementbetweenthehealthserviceandresearchlaboratorieswaslowwhen
bothleukocyteandnitritedipsticktestresultswerenegative(kappa0.26[95%CI0.12,0.40]
forcleancatchsamplesand0.05[-0.02,0.11]fornappypadsamples,S3Table).
Therewaslittleevidencethatpassingurinemoreoftenortemperature(cid:21)39˚Cwereassoci-
atedwithUTIpositivity(Table3andS4Table).Associationsofpainorcryingwhenpassing
urine,anddipsticknitriteandleukocytepositivity,weremarkedlystrongerincleancatchthan
nappypadsamplesandwithresearchlaboratorythanhealthservicelaboratorypositivity.
Associationswithchangeinurineappearancedidnotdiffermarkedlybetweenhealthservice
andresearchlaboratories.Forbothcleancatchandnappypadsamples,valuesoftheAUC
werelowerforhealthservicethanresearchlaboratories(Table3andFig1).Forcleancatch
samplestheAUCwas0.75(95%CI0.69,0.80)forhealthservicelaboratorypositivityand0.86
(0.79,0.92)forresearchlaboratorypositivity.ValuesoftheAUCweremarkedlylowerin
nappypadsamples:0.65(0.61,0.70)forhealthservicelaboratorypositivityand0.79(0.70,
0.88)forresearchlaboratorypositivity.
Forcleancatchsamples,thevaluesoftheAUCweresimilarforchildrenaged<3and(cid:21)3
years,forbothhealthservicelaboratoryandresearchlaboratorypositivity(S5Table).Forthe
researchlaboratory,butnothealthservicelaboratories,AUCvalueswerehigherforsamples
collectedinsurgerycomparedwiththosecollectedathome.AUCvaluesweresimilarinsam-
plesreceivedbybothlaboratorieswithin24hourscomparedtosamplesreceivedafter24
hours,exceptfornappypadsamplessenttotheresearchlaboratory.Forbothhealthservice
andresearchlaboratoriestheAUCincreasedwithincreasingthresholdofpure/predominant
growthcount.Forresearchlaboratorypositivity,valuesoftheAUCweremarkedlylowerfor
pure/predominantgrowth<105CFU/mL.ValuesoftheAUCweremarkedlyhigherin
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 7/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
Table3. Resultsfrommultivariablelogisticregressionmodelsexaminingassociationsofsymptoms,signsandurinedipsticktestswithseparate
routinehealthserviceandresearchlaboratoryresults.
Cleancatch Nappypad
OR(95%CI) p OR(95%CI) p
Healthservicelaboratories
Pain/cryingpassingurine 2.9(1.6,5.1) <0.001 1.1(0.4,3.1) 0.838
Passingurinemoreoften 0.6(0.3,1.1) 0.073 0.7(0.3,1.5) 0.370
Changeinurineappearance 3.0(1.8,4.9) <0.001 2.1(1.3,3.5) 0.005
Temperature(cid:21)39˚C 1.7(0.8,3.8) 0.157 0.7(0.2,2.2) 0.526
Dipstick:nitrite+ve 7.6(4.1,14.1) <0.001 2.0(1.4,2.9) 0.001
Dipstick:leukocyte+ve 3.1(1.9,5.1) <0.001 3.1(2.1,4.4) <0.001
Nobservations(N+ve) 2619(104) 2189(147)
AUC(95%CI) 0.75(0.69,0.80) 0.65(0.61,0.70)
Researchlaboratory
Pain/cryingpassingurine 6.0(3.0,11.8) <0.001 1.4(0.3,7.0) 0.716
Passingurinemoreoften 0.8(0.4,1.7) 0.543 1.2(0.3,4.4) 0.839
Changeinurineappearance 3.1(1.6,6.1) 0.001 3.1(1.2,7.9) 0.019
Temperature(cid:21)39˚C 1.7(0.6,5.1) 0.333 1.1(0.1,8.8) 0.930
Dipstick:nitrite+ve 11.2(5.4,23.1) <0.001 5.2(2.4,11.3) <0.001
Dipstick:leukocyte+ve 5.3(2.8,10.0) <0.001 4.1(1.9,8.9) <0.001
Nobservations(N+ve) 2619(59) 2189(29)
AUC(95%CI) 0.86(0.79,0.92) 0.79(0.70,0.88)
OR:oddsratio,+ve:positive,N:number.
doi:10.1371/journal.pone.0171113.t003
sampleswithwhitebloodcellcount(cid:21)30/mm3,exceptforresearchlaboratorypositivityin
nappypadsamples.TherewaslittleevidencethatvaluesoftheAUCwerehigherforresearch
laboratorypositivitywithpure,comparedwithpredominant,growth.
Discussion
Basedonalarge,unselectedcohortofchildrenpresentingwithacuteillnesstoprimarycarein
EnglandandWales,reliabilityofmicrobiologicaldiagnosisofUTIwasworseusingnappypad
thancleancatchsamples.Theprevalenceofmicrobiologicalpositivitywasmuchhigherfor
healthservicelaboratoriesthantheresearchlaboratory,particularlyfornappypadsamples.
Associationsofmicrobiologicalpositivitywithpre-specifiedsymptoms,signsandurinedip-
sticktestresultswerelowerforhealthservicelaboratoriesthantheresearchlaboratory,andfor
nappypadthancleancatchsamples.ThereliabilityofmicrobiologicaldiagnosisofUTIthus
appearsbetterfortheresearchlaboratorythanforhealthservicelaboratories:theseresultssug-
gestthatmanyofthepositiveresultsreportedbyhealthservicelaboratories,particularlythose
fromnappypadsamples,couldbeduetocontamination.Discriminationimprovedwith
increasingbacteriuriaconcentrationandpyuria.
Toourknowledge,thisisthelargestandmostgeneralizableprimarycare-basedstudycom-
paringthediagnosticperformanceofhealthservicelaboratorieswitharesearchlaboratory,
andusingnappypadandcleancatchcollectionmethods.However,ourstudyhaslimitations,
includingthepotentialimpactofattrition;33%ofchildrenwhowererecruitedtotheDUTY
studywerenotincludedinthisanalysis.Childrenwhowerenotincludedwereyounger,
highlightingthedifficultiesofobtainingurinesamplesfromtheyoungerchildren.Thenum-
berofUTIpositivesampleswasrelativelysmall,especiallyforcleancatchsamplesinyounger
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 8/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
Clean catch Nappy pad
Routine health service laboratories
1
1

5 5
0. 0.

0 0
y 1 0.5 0 1 0.5 0
vit
siti
n
e
S
Research laboratory
1
1

5 5
0. 0.

0 0
1 0.5 0 1 0.5 0
Specificity
Fig1.ROCcurvesfrommultivariablelogisticregressionmodelsexaminingassociationsof
symptoms,signs,andurinedipsticktestswithurineculturepositivityinroutinehealthservice
laboratoriesandtheresearchlaboratory.
doi:10.1371/journal.pone.0171113.g001
childrenandfortheresearchlaboratory.Weminimisedasymptomaticbacteriuriabyonly
recruitingchildrenwithconstitutionalorurinarysymptomsassociatedwiththeiracuteillness,
suchthatallchildrenfoundtohavesignificantbacteriuriawithauropathogenicorganism
wouldbeconsideredtohaveaUTI.Weminimisedselectionbias,aswherepossiblewe
recruitedconsecutivechildren;andweaskedsitestokeepascreeninglogofpatientswhowere
approachedbutdidnottakepartinthestudyandthereasonsforthis.Observersassessingthe
“indextests”differedfromandwereblindtothereferencestandard(andviceversa),thusmin-
imisingreviewerbias.Adisadvantageofourstudydesignisthatwedonotknowwhichsam-
plesweresenttohealthservicelaboratoriesincontainerswithboricacidorwhichoneswere
refrigeratedpriortotransport,sowereunabletoperformexploratoryanalysesofhowthese
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 9/13
ComparisonoflaboratoryperformanceinthediagnosisofUTIinyoungchildren
factorsmayhaveinfluencedcultureresults.Neitherwereweabletoexploretheimpactofdif-
ferencesbetweenroutinehealthservicelaboratoryproceduresandprocesses.Timetolabora-
toryreceipt(within24hoursorgreaterthan24hours)didnotappeartoinfluenceresults.We
werenotabletoobtainasufficientvolumeofurinetosendalargeenoughfractiontothe
researchlaboratoryforallchildrenwhosubmittedaurinesample,asweprioritisedtheroutine
healthservicelaboratoryfractioninordertoensurethatcliniciansweresentlaboratoryresults
forclinicalpurposes.
Thereisnotuniversalagreementonthevalueofdipsticktestingingeneral,[6]andspecifi-
callyleukocyturia,inthediagnosisofUTIinchildren.Furthermore,recommendedbacteriuria
thresholdsdifferbetweenlaboratoryguidelines.Europeanpaediatricguidelinessuggesta
thresholdof(cid:21)104CFU/mLifsymptomsarepresentand(cid:21)105CFU/mLifnosymptomsare
presentformid-streamspecimens,andlowerthresholdsforspecimenscollectedbybladder
catheterisationorsuprapubicaspiration.[9]TheUKStandardsforMicrobiologicalInvestiga-
tionsdonotgivespecificpaediatricguidancebutsuggestathresholdofasingleorganism
(cid:21)1x104CFU/mLindicatesUTI,thoughleukocyturiaisnotrequiredandotherthresholdsare
discussed.[10]USguidancerequiresbothleukocyturiaplusathresholdof(cid:21)5x104CFU/mL.
[7]Partoftheexplanationisthatleukocyturiahasbeenidentifiedinchildrenwithfeverbutno
UTI.[13]SincestudyurinesampleswereprocessedbyUKlaboratories,wewereobligedtouse
theUKdefinition,whichdoesnotincludeleukocyturia.Oneadvantageofthiswasthatit
allowedanassessmentofthestrengthofassociationbetweenleukocyturiaandroutinehealth
service/researchlaboratoryconfirmedUTI,whichwouldnothavebeenstatisticallyvalidhad
leukocyturiabeenincorporatedintolaboratories’definitions.Bothleukocyteandnitritedip-
stickpositivitywereassociatedwithmicrobiologicallyconfirmedUTIinbothroutinehealth
serviceandresearchlaboratories,andagreementbetweenhealthserviceandresearchlabora-
toryresultswaspoorwhenneitherdipstickresultwaspositive,whichmaybebecauseUTIcul-
turepositivityismorelikelytobeduetocontaminationwhendipstickresultsarenegative.
Thus,ourresultssupporttheusefulnessofdipsticksasanearpatienttestinchildrenwithsus-
pectedUTI.
Microbiologicalexaminationofurinerequiresquantificationofbacteriaanddifferentiation
ofmixedfrompuregrowths.Thepourplatemethodhasprovedtoolabour-intensivegiventhe
largenumberofsamplessubmittedtoroutinemicrobiologylaboratoriesintheUK:in2012
663,355samples(12,689fromchildrenaged<5years)weresubmittedinWalesalone,equat-
ingtosome12.1msamplesannually(250000fromchildrenaged<5years)inEnglandand
Wales.Theneedforrapidthroughputledtodevelopmentofmethodsusingcalibratedloops,
filterpaperstrips,ormultipointmethodstodeliverastandardinoculum.[14–16]Allwerevali-
datedagainstviablecountsperformedbypourplatesorthemethodofMilesandMisra.[17]
TheStandardsforMicrobiologicalInvestigationfollowedbymostUKlaboratoriesprovide
optionsforurinecultureusingthesemethodstoinoculateCLEDorChromogenicagar:diffi-
cultiesindefiningmixedgrowthsandachievingaccuratebacterialcountsmaybeduetosmall
volumesofurineinoculatedontosmallareasofagar.[14,18]Spiralplating,whichwasusedby
theresearchlaboratoryandinvolvesamuchlargerinoculum(50μL)overanentireagarplate,
quantifiesbacterialcountsmoreaccuratelyandallowsdifferentiationofmixedcultures.[19]
Furtherimprovementsmightbeachievedthroughbettertransportprocedures.
OurresultssuggestthatthediagnosticperformanceofroutineUKroutinehealthservice
laboratorytestingmaybesub-optimal,andcouldleadtoovertreatmentandunnecessary
investigations.Inadults,resultsfromurinemicrobiologycanbeinterpretedlightofthe
patient’spresentation.Howeverinyoungchildrenthedifficultiesinobtaininguncontami-
natedsamples,togetherwiththenon-specificnatureofthepresentingsymptoms,meanthere
isgreaterrelianceonthelaboratoryresult.Moredetailedroutinemicrobiologicalexamination
PLOSONE|DOI:10.1371/journal.pone.0171113 February15,2017 10/13