brazilian journal of microbiology48(2017)782–784
h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Clinical
Microbiology
Evaluation
of
a
selective
chromogenic
medium
for
detecting
vancomycin-resistant
enterococci
Renata
Oliveira
Soares
∗,
Adriana
Medianeira
Rossato,
Gustavo
Enck
Sambrano,
Neidimar
Cezar
Corrêa
Tolfo,
Juliana
Caierão,
Thiago
Galvão
da
Silva
Paim,
Pedro
Alves
d’Azevedo
FederalUniversityofHealthSciencesofPortoAlegre,PortoAlegre,RS,Brazil
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Articlehistory:
Received7June2016 Accepted1March2017 Availableonline8June2017 AssociateEditor:AgnesFigueiredo
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Rapididentificationofvancomycin-resistantenterococci(VRE)canassistinchoosingthe appropriatetreatmentandpreventingVREspread.TheperformanceofchromIDTMVREagar wasevaluatedusing184clinicalisolatesofEnterococcusspp.andreferencestrains.Thetest hadasensitivityof95.52%butalowspecificityof30%.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Vancomycin-resistantenterococci(VRE)areamongthemajor agentsofhealthcare-associatedinfectionsandareconsidered apublichealthproblem.RapidVREidentificationcanassist inchoosingthe appropriatetreatment and preventingVRE spread.1,2 Theaimofthis studywastoevaluatethe
perfor-manceofaselectivechromogenicmediumforthedetection anddifferentiationofvancomycin-susceptibleand-resistant
EnterococcusfaeciumandEnterococcusfaecalis.
Vancomycin-susceptible enterococci (VSE) isolated from casesofinfection (n=50) andVRE clinical isolates(n=134), includingthosecollectedfrom surveillancerectalswab cul-tures (n=62) and from cases of infection (n=72), were evaluated(Table1).Thefollowingreferencestrainswerealso included:VREstrain(E.faecium,n=1)andVSEstrains(E.hirae, n=1;E.gallinarum,n=2;E.faecium,n=1;andE.faecalis,n=4).
Allisolateswerepreviouslyidentifiedbyphenotypic meth-ods(hydrolysisofesculininthepresenceofbile,production ofpyrrolidonylarylamidase,growthinbrothcontaining6.5%
∗ Correspondingauthor.
E-mail:[email protected](R.O.Soares).
NaCl,andnegativecatalasetestevidentiatedbytheabsence ofeffervescence).3Polymerasechainreactionwasalsousedto
confirmthepresenceofthegenusEnterococcusanddistinguish thespeciesaccordingtomethodspreviouslydescribedbyKe et al.5 andKaryamaet al.4 Allisolateswere obtainedfrom
theculturecollectionoftheGram-positiveCocciLaboratory –UFCSPAandstoredinskimmilk(DifcoTM)at−20◦C. Van-comycinminimuminhibitoryconcentrationwasdetermined bybroth microdilutionaccording toCLSIguidelines (2015)6
and byEtest® accordingtothemanufacturer’sinstructions. ThechromIDTMVRE(bioMérieux,BrazilS/A)assayswere per-formedintwosteps.First,theisolatesthatwerepreviously storedinskimmilkweregrowninbile-esculinagartoconfirm thepresenceofenterococciandcheckculturepurity.Second, puresamplesweregrownintrypticasesoyagarfor24h, fol-lowedbysingle-colonygrowthinchromIDTMVREagarat37◦C. After24h,plateswithanygrowthwereconsideredVRE posi-tive.Anegativeresultwasdefinedasa48-hincubationperiod
http://dx.doi.org/10.1016/j.bjm.2017.03.005
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
brazilian journal of microbiology48(2017)782–784
783
Table1–Clinicalisolatesusedtoevaluatedthe performanceofthechromIDTMVRE.
Species Susceptibilityto vancomycina Origin S R Infection Surveillance culture E.faecalis 43 47 47 43 E.faecium 7 87 75 19 Total 50 134 122 62 a S,susceptible;R,resistant.
TP = Test positive, TN = Test negative
VRE
0 20 40 60 80 100
VSE
TP
TN
Enterococcal isolates
(%)
Fig.1–Schematicrepresentationoftheresultsobtained usingchromIDTMVRE.
withoutanybacterialgrowth.Accordingtothemanufacturer’s instructions,chromIDTMVREagarallowstheidentificationof speciesbasedonthedetectionofenzymeactivity.Therefore,
E.faeciumwasstainedpurpleandE.faecaliswasstained blue-green.
OftheE.faecalisVREtested(n=47),41werestained blue-green,5werestainedgray,and1isolatedidnotgrow.AllE. faeciumVREtested(n=87)werestained purple.AmongVSE isolates(n=50),15 didnotgrow(10 E.faecalis and5E. fae-cium)and 35 (33 E. faecalisand 2E. faecium) showed some growthattheedgesoftheplatesinthecorrespondingcolor ofeachspecies,whichmaysuggestfalse-positiveresults.All VSE-referencestrainstested(n=8)didnotshowany visible growthinthechromogenicmedium.Fig.1showsaschematic representationoftheresultsobtainedwithchromIDTM VRE agar.
ThechromIDTM VREagarhadasensitivityof87.23%and 100%fordetectingE.faecalisVREandE.faeciumVRE, respec-tively, and acombined sensitivityand specificityof95.52% and30.00%,respectively,fordetectingVRE.Nodifferencewas observedinthespecificityandsensitivityat24and48h.The positivepredictivevalue(correspondingtothepercentageof VREthattestedpositiveinchromIDTMVRE)was78.53%(95% confidenceintervals;C.I.=72.22–84.83%)andthenegative pre-dictive value was71.43% (95% C.I.=52.10–90.75%)(Table2). Regarding sensitivity,similar resultshavebeenobtainedin previous studies evaluatingthe performanceofchromIDTM
VRE.1,7–14Inrelationtothespecificity,previousstudieshave
obtainedvalueshigherthan95%,1,7,8,11incontrasttothelow
specificityobservedinthisstudy.
Colonieswithnon-discriminatorystaining(gray,dark,or colorless) havebeen reportedinsomestudies.10,15 Wealso
observedVREcolonieswithagrayishshade,whichmaylead tofalse-negativeresults.
AmongallVREisolates,100%oftheE.faeciumgrewwithin 24h.Thesamewasobservedinpreviousstudies.11,14
Accord-ingtoGrabschetal.,824-hidentificationofVREallowsearlier confirmationofcolonizationbythesestrains,facilitates infec-tioncontrol,andhelpstoavoidthespreadofmicroorganisms. However,oneE.faecalisVREisolatedidnotgrowevenafter 48h,whichmaysuggestthatsomestrainscanexhibita differ-entbehaviorand/orrequiremoretimetogrowinthemedium. Delmasetal.1comparedgrowthbeforeandafteran
enrich-mentstepinbile-esculinagarsupplementedwithvancomycin in order to select only VRE strains. The enrichment step improvedtheperformanceofchromIDTMVREat24hof incu-bation. Other studies have shown that strains incubated overnightinanenrichmentbrothcontainingvancomycinas afirst stepfollowedbytheuse ofchromIDTM VREresulted inimprovedspecificityorsensitivity.1,10,11,14,16However,this
methodisonlyuseful forfecal specimensdue tothelarge number ofdifferentmicroorganismsthatcanbepresentin thesesamples.
MoststudiesevaluatingchromIDTMVREperformancehave usedonlyfecalspecimens(stoolsamplesand rectalswabs) or only resistant strains. In our study, we evaluated well-characterizedvancomycin-resistantand-susceptibleisolates inordertoobservethepossibleoccurrenceoffalse-positive results,becauseincorrectlyprescribedantibioticshavea nega-tiveclinicalimpact.Despitethedatapresentedhere,apossible limitationofthisstudyisthatfecalsampleswerenotincluded,
Table2–EvaluationofchromIDTMVREindetectingtruepositivevancomycin-resistantenterococci.
ChromIDTMVREagar Goldstandard(VRE)a Goldstandard(VSE)a Total
Testpositive 128 35 163
Testnegative 6 15 21
Total 134 50 184
Sensitivity 95.52%(95%C.I.=92.02–99.02%)
Specificity 30.00%(95%C.I.=17.29–42.70%)
Positivepredictivevalue 78.53%(95%C.I.=72.22–84.83%)
Negativepredictivevalue 71.43%(95%C.I.=52.10–90.75%)
Accuracy 77.72%(95%C.I.=71.70–83.73%)
784
brazilian journal of microbiology48(2017)782–784becausethedensityofmicroorganismsinaclinicalspecimen mayaffectthecorrectdiagnosis.
Inconclusion,thechromIDTMVREagarisarapidand use-fultoolforthe screeningand identification ofVRE,with a goodsensitivityofabout96.00%.However,becausespecificity (30.00%)waslimitedbyfalsepositiveVREmainly,we recom-mendfurtherVREidentificationbyconventionalteststoavoid misinterpretation.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
This study was supported in part by the Coordenac¸ão de Aperfeic¸oamento de Pessoalde Nível Superior and Univer-sidade Federal de Ciências da Saúde de Porto Alegre. We alsothankthebioMérieux, BrazilS/A forthe supplyofthe chromIDTMVREagarplates.
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