braz j infect dis.2015;19(4):436–438
www .e l s e v i e r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Brief
communication
Evaluation
of
phenotypic
tests
to
detect
carbapenem-resistant
Enterobacteriaceae
in
colonized
patients
hospitalized
in
intensive
care
units
Leandro
Reus
Rodrigues
Perez
a,b,c,∗,
Diógenes
Rodrigues
c,
Cícero
Gomes
Dias
c,daProgramadePós-graduac¸ãoemCiênciasFarmacêuticas,UniversidadeFederaldoRioGrandedoSul,PortoAlegre,RS,Brazil
bLaboratóriodePesquisaemResistênciaBacteriana(LABRESIS),CentrodePesquisaExperimental(CPE),HospitaldeClínicasdePorto
Alegre,PortoAlegre,RS,Brazil
cHospitalMãedeDeus,PortoAlegre,RS,Brazil
dUniversidadeFederaldeCiênciasdaSaúdedePortoAlegre,PortoAlegre,RS,Brazil
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Articlehistory:
Received18November2014 Accepted12March2015 Availableonline18May2015
Keywords: Carbapenems Enterobacteriaceae KPC Surveillance
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Inthisstudy,weaimedtoevaluatetheperformanceofdifferentphenotypicteststodetect carbapenem-resistantEnterobacteriaceae.
Threedifferentphenotypicmethodswereevaluated:(1)combined-disktestofmeropenem plusphenylboronicacidorEDTAreadingafter24hand48h;(2)selective/chromogenicread after24h andafter48h;and(3) overnightselectiveenrichment brothcontaining10g ertapenemdiskfollowedbycultureonMacConkeyagar.Apositiveresultinatleastone ofthemethodswassubmittedtoPCRforblaNDM-1,blaOXA-48,blaKPC,blaSPM-1,blaIMP,andblaGES
detection.
Carbapenem-resistant Enterobacteriaceae was detected in 31 (30.4%) of 102 rec-tal swabs evaluated. All isolates showed to be KPC-2-producing organisms. Results showedexcellentagreementamongtheevaluated tests(positiveandnegative)(kappa= 0.88).
Itis importanttostate thatcombined-disktestwithphenylboronic acidis not suit-ableforbacterialidentification/isolation.Conversely,selective/chromogenicagarafter48h ofincubationshowed tobea usefultool, withthe advantageofpresumptivebacterial identification.
©2015ElsevierEditoraLtda.Allrightsreserved.
∗ Correspondingauthorat:UniversidadeFederaldoRioGrandedoSul,FaculdadedeFarmácia,2752IpirangaAvenue,90610-000Porto
Alegre,RS,Brazil.
E-mailaddress:leandro.reus@gmail.com(L.R.R.Perez). http://dx.doi.org/10.1016/j.bjid.2015.03.008
brazj infect dis.2015;19(4):436–438
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Nosocomial infections due to carbapenem-resistant Enterobacteriaceae(CRE)havebeenaworldwideproblemin thelast fewdecades.Falagaset al.1 haverecentlyreported that the number of deaths was significantly higher in patients with CRE infections in comparison to those with carbapenem-susceptibleEnterobacteriaceaeinfections.
Carbapenemase production has been considered as a majorchallenge formicrobiologicallaboratoriesdue tothe vast number ofgenes that encodes for productionof car-bapenemase,high abilitytodisseminate,and difficultiesin detecting.2–5
Theobjectiveofthisstudywastoevaluatethreedifferent typesofphenotypictestsforthedetectionofCREfromrectal swabscollectedfromhospitalizedpatients.
Aspartoftheroutinesurveillance,102rectalswabsforCRE screeningwereconsecutivelyobtainedfrompatients admit-tedto intensivecare unitsat HospitalMãe deDeus, Porto Alegre,fromJanuarythroughFebruary2014.
Each swab was initially plated on MacConkey agar (bioMérieux,Brazil)forgrowth,andcombined-disktest(CDT) wasappliedusing10gmeropenem (MER)disk(Oxoid,UK) alone,aMERdiskplus10lof40mg/mlphenylboronicacid (PBA)(Sigma–Aldrich,Germany)forKPCinhibition,andaMER disk plus 10 of0.1M EDTA(Sigma–Aldrich, Germany) for MBLinhibition.Bacterialgrowthwasscreenedat24h (CDT-24hwithPBAorEDTA)and48h(CDT-48hwithPBAandEDTA) ofincubation at37◦Cinambientair. Theresultsof inhibi-tionwereinterpretedaccordingtoapreviousreport.6Original swabs were inoculated on selective/chromogenic ChromID agar(bioMérieux,Brazil)forreadingafter24h(ChromID-24h) and48h(ChromID-48h)ofincubation.Suspectcolonieswere submitted toidentification and susceptibility testing. Also, each swab was subsequently suspendedin 5ml of tryptic soybrothtowhicha10gertapenem(ERT)diskwasadded (selectiveenrichmentbroth–SEBtest).Theinoculatedbroth wasincubatedovernightperiodat37◦Cforlaterplatingon MacConkeyagarcontaining10gertapenemand10gMER disks.7EnterobacterialcoloniesgrowingaroundMERandERT diskswerepickedup,subcultured, identifiedtothe species level,andsubjectedtosusceptibilitytestingbytheMicroScan automatedsystem(Siemens,USA).
Whenapositiveresultwasobtainedfromatleastoneof thetests,polymerasechainreaction(PCR)forthedetection ofblaNDM-1,blaOXA-48,blaKPC,blaSPM-1,blaIMP,andblaGESgenes wasapplied.8
StatisticalanalyseswerecarriedoutusingSPSSfor Win-dows,version13.0(SPSSInc.,Chicago,IL).Kappacoefficient
and95%confidenceintervals(CIs)weredeterminedforeach category(positiveandnegativeresults),inordertodetermine agreementamongthedistinctphenotypictests.9
CREwasdetectedin31 (30.4%)of102rectalswabs eval-uated.Forall,Klebsiellapneumoniaewasthesolespeciethat presentedapositiveresultinthephenotypictestsevaluated whileblaKPCwasthesolecarbapenemasegenedetected. Pos-itiveresultsweremoreoftenobservedinCDT-48hwithPBA (31observations),followedbyChromid-48,CDT-24withPBA, SEBandChromid-24(Table1).After48hofincubation,afalse positive result (none gene detected byPCR)was noted for twosamplesinCDTwithPBA.Also,onesamplewithno car-bapenemase gene grew on ChromID-48h. For all methods, weverifiedtheoccurrenceoffalse-negativeresults(negative resultbutpresenceofKPCgenedetected),mainlyin ChromID-24h(12cases)andSEBtest(6cases).
Some particular characteristics of each method should beevaluatedprioritsapplicationasasurveillancemethod. CDT withPBAshows tobean excellentand rapidmethod topredictthepresenceofKPC-producingCREand24h incu-bation wasenoughtoproduceareliableresult.Instudyby Pournaras et al.6 DCT with PBAwas able to detect and to differentiate KPC and/or MBL production, with the advan-tage ofobtaining resultswithinone day. Itisof note that this type oftestdoes notfavor the recoveryofthe isolate forlateranalyses,suchasspeciesidentification, antimicro-bialsusceptibilitytesting,ormoleculartyping.Vrionietal.3 found 95.1% accuracy with ChromID-24h. However, in our study,oneESBL-producingK.pneumoniaegrewon ChromID-48h, resulting in a false-positive detection compared with ChromID-24h.Despitesomeobserveddiscrepancies,all meth-odstestedshowedanalmostperfectagreement,asassessed by the kappa coefficient (kappa=0.82; 95% CI 0.75–0.88;
p<0.001).
ItshouldbepointedoutthattheSEBtestprotocolis rec-ommendedbytheCDC10andwithmodification(byusingERT as inhibitor substrate) bythe Brazilian Health Surveillance Agency–ANVISA.11Ithasbeenadoptedasstandardprotocol byBrazilianlaboratoriesinanefforttopreventCRE dissemi-nation.
Finally, ourresultsdemonstratethatdifferenttestshave similarperformancetodetectCREobtainedfromsurveillance rectal swabs.CDT withPBAproved tobeagood test,with the limitationofnotallowingforbacterialisolation. Useof aselective/chromogenicmedium,suchasChromID,may rep-resentausefultoolformicrobiologylabs,especiallyafter48h ofincubation.
Table1–NumberofpositiveandnegativeresultsforCREdetectionin102rectalswabsbyusingdifferentphenotypic methods.
Result CDT-24h CDT-48h ChromID-24h ChromID-48h SEBtest Kappa(95%CI)a
Positive 29 31 19 30 25 0.82(0.75–0.88)
Negative 73 71 83 72 77 0.82(0.75–0.88)
Total 102 102 102 102 102 0.82(0.75–0.88)
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braz j infect dis.2015;19(4):436–438Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
L.R.R.Perezisaresearchfellowofthe NationalCouncilfor ScientificandTechnologicalDevelopment(CNPq),Ministryof ScienceandTechnology,Brazil(165894/2013-0).
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