brazilian journal of microbiology48(2017)489–492
h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Medical
Microbiology
Detection
of
vancomycin-resistant
enterococci
(VRE)
in
stool
specimens
submitted
for
Clostridium
difficile
toxin
testing
Sevim
Özsoy,
Arzu ˙Ilki
∗MarmaraUniversity,MedicalFaculty,DepartmentofMedicalMicrobiology,Istanbul,Turkey
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received14March2016 Accepted4December2016 Availableonline17March2017 AssociateEditor:AgnesFigueiredo
Keywords: Clostridiumdifficile
Enterococci
Vancomycinresistance(VRE) Colonization
a
b
s
t
r
a
c
t
TheaimofthisstudywastodeterminetheassociationbetweenClostridiumdifficile(C. diffi-cile)andvancomycin-resistantEnterococcus(VRE)andefficacyofscreeningstoolssubmitted forC.difficiletoxinassayforprevalenceofVRE.BetweenApril2012andFebruary2014,158 stoolsamplessubmittedforC.difficiletoxintotheMarmaraUniversityMicrobiology Labo-ratory,wereincludedinthestudy.Stoolsampleswereanalyzedbyenzymeimmunoassay test;VIDAS(bioMerieux,France)forToxinA&B.SampleswereinoculatedonchromIDVRE (bioMerieux,France)andincubated24hat37◦C.ManueltestsandAPI20STREP(bioMerieux, France)testwereusedtoidentifytheEnterococcispecies.Afterthespeciesidentification, vancomycinandteicoplaninMIC’swereperformedbyEtestandmolecularresistancegenes forvanAvsvanBweredetectedbypolymerasechainreaction(PCR).Ofthe158stool sam-ples,88weretoxinpositive.TheprevalenceofVREwas17%(n:19)intoxinpositiveshowever, 11.4%intoxinnegatives(n:70).AllVREisolateswereidentifiedasEnterococcusfaecium.These resultswereevaluatedaccordingtoFischer’sexactchi-squaretestandpvaluebetweenVRE colonizationandC.difficiletoxinpositivitywasdetected0.047(p<0.05).PPVandNPVwere 79%and47%respectively.Inourstudy,thepresenceofVREinC.difficiletoxinpositivesis statisticallysignificantcomparedwithtoxinnegatives(p<0.05).ScreeningforVREisboth additionalcostandworkloadforthelaboratories.ThereforeVREscreeningamongC.difficile
toxinpositivesamples,willbecosteffectivefordeterminationofhighriskpatientsinthe hospitalsespeciallyfordevelopingcountries.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Colonizationandinfectionwithvancomycin-resistant Entero-coccusfaecium(VRE) isanincreasingly commonproblem in
∗ Correspondingauthorat:MarmaraUniversity,MedicalFaculty,DepartmentofMedicalMicrobiology,Bas¸ıbüyük,MaltepeBas¸ıbüyüksok.
No.9/1,34854,Maltepe,Istanbul,Turkey. E-mail:[email protected](A. ˙Ilki).
hospitalsofmanycountriesworldwideanditsspreadis gen-erallyassociatedwithpoorhospitalhygienepractice.
Risk factors for VRE colonization include host char-acteristics (immunosuppression, neutropenia, and renal insufficiency),hospitalfactors(admissiontoanintensivecare
http://dx.doi.org/10.1016/j.bjm.2016.12.012
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
490
brazilian journal of microbiology48(2017)489–492unit (ICU) or oncologyward, proximityto a VRE-colonized patient,andextendedlengthofstay),andantimicrobialuse.1–3
VREcolonizationincreasesthepatientriskofdeveloping infections,suchasbloodstreaminfections.Rapidandaccurate identificationofVREiscrucialinthemanagementand treat-mentofbothcolonizedandinfectedpatientsandtoprevent the nosocomialspread ofthis resistant bacteria. Therefore screeningforVRE fromrectal swabsisaroutineprocedure inmosthospitals.However,routinescreeningforVRE repre-sentsafinancialburdenforhospitals,mainlyinthedeveloping countries. Therefore, it is important to select appropriate patientsforthisscreeningespeciallyinthehighriskwards.4
Clostridiumdifficileisanaerobicbacteriathatproduce mul-tipletoxinsincludingAandBtoxins(A&Btoxins)andcause diarrheaandinflammationininfectedpatients.VREandC. dif-ficilearebothnosocomialpathogensandthushavesimilarrisk factorsincludingantibiotictreatmentandhospitalization.5
Inthisstudyweaimedtoassesstheprevalenceof gastroin-testinalcolonizationbyVREinstoolsamplessubmittedforC. difficiletoxintestingandtoassessthecostandadvantagesof thislaboratory-basedsurveillance.
Materials
and
methods
Samples
StoolssubmittedforC.difficiletoxintestingbetweenApril2012 andFebruary 2014,attheMarmaraUniversityMicrobiology Laboratory,wereincludedinthestudy.
C.difficiletoxindetection
Stoolsampleswereanalyzedusinganenzyme-linked fluores-cent(ELFA)assay,VIDAS® C.difficileToxinA&B(BioMérieux, France). Stoolswere mixedwith 200Ldistilled waterand centrifugedat12,000×g.Supernatant(300L)wasloadedin tothetestwell.After75min,testresultswereevaluatedas <0.13-negative,≥0.13-<0.37-intermedate,≥0.37positive.
Enterococcusspp.identification
Samples were inoculated on chromID VRE (BioMérieux, France) and incubated at 37◦C in normal atmosphere and examinedforgrowthafter24–48h.Purplecolonieson chro-mIDVREwere presumptivelyidentifiedasVRE.AfterGram staining,positivecocciwerethensubculturedtosheepblood agarandincubatedat37◦Cinnormalatmosphere,and exam-inedafter24h.InadditiontocolonymorphologyandGram staining,catalasereaction,growthin6.5%NaCland pyrroli-donylaminopeptidaseactivitywereusedfortheidentification atgenuslevel.6Additionally,API20STREP(BioMérieux,France)
testwasusedtoidentifytheenterococciatspecieslevels.
Vancomycinresistancedetection
After species identification, vancomycin and teicoplanin minimal inhibitoryconcentrations (MICs) were determined for enterococci using Etest method (Biomérieux, France). MICs were interpreted using the following breakpoints
accordingtoCLSIstandards:Vancomycin≤4g/mL (suscep-tible), 8–16g/mL(intermediate), ≥32g/mL(resistant); and teicoplanin≤8g/mL(susceptible),8–16g/mL(intermediate), ≥32g/mL(resistant).7
DetectionofthegenesvanAandvanB
ThesegenesweredetectedbymultiplexPCR-basedtest.Two orthreecoloniesofVREweresuspendedindistilledwaterand centrifugedat10,000×g.Thesuspensionwasthenboiledat 100◦Cfor10min.Then2.5LsupernatantwasaddedtoPCR Master Mix (Promega),primers(vanA, vanB)tomakea final volume of25L. Primers for van;vanA1, vanA2 primers(5 GGGAAAACGACAATTGC 3 and5 GTACAATGCGGCCGTTA 3, 732bp) and forvanBvanB1, vanB2primers (5 ATGGGAAGC-CGATAGTC3 and5 GATTTCGTTCCTCGACC3,635bp)were used.8 PCRamplificationwasperformed byinitial
denatur-ation94◦Cfor2min,denaturationat94◦Cfor1min,annealing at54◦Cfor1min,extensionat72◦Cfor1min(30cycles).PCR productswereloadedin1.5%agarosegelandstainedby ethid-iumbromideandvisualizedunderultraviolettransiluminator.
Statisticalmethods
AllresultswereevaluatedusingSPSS17.0versionand ana-lyzedaccordingtoFischer’sExactChi-Squaretestandp<0.05 wasacceptedassignificant.
Results
BetweenAugust2012andFebruary2014,158stoolspecimens submittedforC.difficiletoxinAassayweretestedforVREin theMicrobiologyLaboratoryofMarmaraUniversityHospital.
C.difficileinfectionisthemostimportantcauseofnosocomial diarrhea,andtheuseofantibioticshavebeenimplicatedasa majorriskfactorforthiscondition.Inourstudy,113(71.5%) ofthe158patientswerehospitalizedandtreatedwith antibi-otics.C.difficiletoxinwasdetectedin88(55.7%)specimens.
Ofthe88toxinpositivesamples,27(30.6%)werepositivefor enterococciwhereasofthe70negativesamples,8(11.4%)were positiveforthesebacteria.Atotalof35Enterococcusspp.were isolated inthespecimens.thirty-twooftheseisolateswere identifiedasE.faecium(91.4%)andthreeasE.faecalis(8.6%).
Of the 35 isolates 19 (54.2%) were vancomycin and teicoplaninresistant.AlltheresistantisolateswereE.faecium
and they carriedvan Agene. vanB gene wasnotdetected (Fig.1).
ThevancomycinMICsfortheenterococcalisolateswere ≥256whereasteicoplaninMICswere32–256g/mL.EtestMIC resultsshowed100%agreementwithvanAPCRdata.VREwas isolated from 15 (17%)of88 C.difficile toxin positive speci-mens,comparedwith4(5.7%)of70C.difficiletoxinnegative specimens(Table1).
Positive predictive value (PPV) and negative predictive value(NPV)were79%and47%respectively.Theseresultswere evaluatedaccordingtoFischer’sexactchi-squareandpvalue betweenVREcolonizationandC.difficiletoxinpositivitywas foundtobe0.047(p<0.05). Patientswhosestoolspecimens were positiveforC. difficiletoxin Awere significantlymore
brazilian journal of microbiology48(2017)489–492
491
M P 1 2 3 4 5 6 7 8 9 10
vanA positive isolates
732 bp
P: Positive control M: 100 bp DNA marker 11 12 13 14 15 16 17 18 19
Fig.1–PolymerasechainreactionamplificationofvanAgenesinthevancomycinresistantenterococciisolates.
Table1–C.difficiletoxinpositivityandVREcolonization.
Toxin VREpositive VREnegative Total
n(%) n(%) n(%)
Positive 15(17) 73(83) 88(100)
Negative 4(5.7) 66(94.3) 70(100)
Total 19(12.1) 139(87.9) 158(100)
likelythanthosewhosespecimenswerenegativetohaveVRE detected.
Discussion
From the early 1970s Enterococcus spp. became one of the most common pathogens in nosocomial infections. Thesebacteriaarecommonlyresistanttomostantibiotics.1–3
Because the increased VRE incidence, rapid identification methods are getting importance, specially for inpatients. Chromogenicmediumisoneoftheserapidmethodsbothfor identificationandspeciesisolation.ChromIDVRE(bioMerieux, France)mediumcontaining8g/mLvancomycin isa selec-tivefortheidentificationofVRE.Sensitivityandspecificityof themediumishigh.9,10 Inthestudy ofLedeboeretal.,120
stoolsampleswereinoculatedonchromIDVREandthe sensi-tivityofidentificationratesofE.faeciumandE.faecaliswere 85.4%and 90.0% respectivelywhereas specificity was100% inbothspecies.11Malignity,chronickidneyfailure,
neutrope-nia,transplantation chemotherapyare risk factors forVRE infection.Similar riskfactorsare alsoimportantforC. diffi-cile infections.Gerdinget al. first detectedtherelationship betweenVREdetectionandC.difficiletoxinpositives.12
Gar-buttetal.studiedthepresenceofC.difficiletoxinin215patient samplesanddetectedVREin41(19%)ofthese.13Inanother
study,Shayet al.detected10(21.7%)C. difficileinfection in the46VREpatientwithbloodstreaminfection(BSI).14Jordens
etal.foundthisratioas11%whereasRaffertyetal.detected 16.5%.15,16Leberetal.evaluated50stoolsamplesanddetected
VREcolonizationin3.2%ofthe specimenssubmittedforC. difficiletoxinassay.17
Inour study,VRE was detectedin 17%of the C.difficile
toxinpositivesamples.Ourresultswereverysimilartothose obtainedbyGarbuttetal.andRaffertyetal.13,16Similarly,
Fuji-tanietal.detectedVREcolonizationin88ofthe158C.difficile
toxin positivepatients.18 VRE colonizationwas detectedas
5.7%intoxinnegatives.PresenceofVREintoxinpositivesare statisticallysignificantcomparingtotoxinnegatives(p<0.05).
AllVREisolateshadvanAgene,nonevanBgenewasdetected.
vanBgeneisarareresistancegeneforourcountry.
VidasC.difficiletoxinassayisarapidandrelativelysimple test;howeverithaslowsensitivity.Thesensitivity,specificity andpositiveandNPVusingRT-PCRkitwere100%,98.3%,84.6% and100%,respectively,whileusingVidasC.difficiletoxinassay were 63.6%,100%,100%and 96.6%,respectively.19However,
RT-PCRisexpensiveandneedsexperiencedstaff.
During ourstudy period, VRE isolationratio were 12.9% (n:486)in3772rectalswabsinourlaboratory.C.difficiletoxin positivitywas3.8%,howeverVREpositivityinC.difficiletoxin positivesampleswere17%.Therefore,thesepatientsshould beconsideredatgreatrisktobecolonizedbyVRE. Gastroin-testinalsystemisoneofthemostimportantsourceofVRE. Todetectandisolatecolonizatedpatients,isrecommendedto preventtransmissionofVRE.However,routinescreeningfor VREcauses staffworkloadandincreasescostforboth hos-pitalsandlaboratories.VREscreeningamongC.difficiletoxin positivesamplescanbecosteffectiveandefficientstrategy especiallyfordevelopingcountries.Inastudy,fromour coun-try,theestimatedcostsforscreeningVREperiodicallywere $19,074 annually.20 ScreeningVRE from stoolsamples
sub-mitted for C.difficile toxin assay eliminates the need fora separatespecimencollection,whichmaybeasourceofstool contaminations.21 Itisalsolessinvasiveprocedure
compar-ingtorectalswabs.Indevelopingcountries,likeourcountry, hospitalsshoulddeterminetheirhighriskclinicsforVREto limittheirscreeningpriority.Appropriatechoiceofhighrisk patientslikeC.difficiletoxinpositives,willleadthescreening tobemorecosteffective.WefinallysuggestthatamongtheC. difficiletoxinnegativepatients,thoseatincreasedriskforVRE colonization,suchaspreviouslybeingcolonizedorinfected withVRE,beingtransferredfromhospitalwithVREoutbreak or high VRE colonization or infection rates on admission, shouldalsobetestedforVRE.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
ThisstudywassupportedbyagrantfromMarmaraUniversity ScientificResearchCommission,withagrantnumberof SAG-C-YLP-130213-0025.
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s
1. ZaasAK,SongX,TuckerP,PeriTM.Riskfactorsfor
developmentofvancomycinresistantenterococcal
bloodstreaminfectioninpatientswithcancerwhoare
colonizedwithvancomycin-resistantenterococci.ClinInfect
Dis.2002;35(10):1139–1146.
2. FrenchGL.Enterococciandvancomycinresistance.ClinInfect
Dis.1998;27:75–83.
3. FisherK,PhillipsC.Theecology,epidemiologyandvirulence
ofEnterococcus.Microbiology.2009;155:1749–1757.
4. HumphreysH.Controllingthespreadofvancomycin
resistantenterococci.Isactivescreeningworthwide?JHosp
Infect.2014;88(December(4)):191–198[Review].
5. SafdarN,MakiDG.Thecommonalityofriskfactorsfor
nosocomialcolonizationandinfectionwithantimicrobial
resistantStaphylococcusaureus,Enterococcus,Gramnegative
bacilli,Clostridiumdifficile,andCandida.AnnInternMed.
2002;136:834–844.
6. KonemanEW,AllenSD,JandaWM,ScreckenbergerPC,Winn
WC.Thegrampositivecoccipart2:Streptococciand streptococcus-likebacteria.Diag.Microbiol.10thed.
Philadelphia:J.B.LippincottCompany;2005:431–466.
7. ClinicalandLaboratoryStandardsInstitute:Performance StandardsforAntimicrobialSusceptibilityTesting;Twentieth InformationalSupplement.Wayne,PA:CLSIDocument
M100-S20;2010.
8. Dutka-Malen,EversS,CourvalinP.Detectionofglycopeptide
resistancegenotypesandidentificationtothespecieslevel
ofclinicallyrelevantenterococcibyPCR.JClinMicrobiol.
1995;33:24–27.
9. D’AgataEM,GautamS,GreenWK,TangYW.Highrateof
false-negativeresultsoftherectalswabculturemethodin
detectionofgastrointestinalcolonizationwith
vancomycin-resistantenterococci.ClinInfectDis.
2002;34(2):167–172.
10.AndersonNW,BuchanBW,YoungCL,etal.Multicenter
clinicalevaluationofVRESelectagarforidentificationof
vancomycin-resistantEnterococcusfaecalisandEnterococcus
faecium.JClinMicrobiol.2013;51(August(8)):2758–2760.
11.LedeboerNA,DasK,EvelandM,etal.Anewchromogenic
agarmedium,chromIDVRE,toscreenfor
vancomycin-resistantEnterococcusfaeciumandEnterococcus
faecalis.DiagnMicrobiolInfectDis.2007;59(4):477–479.
12.GerdingDN.Istherearelationshipbetween
vancomycin-resistantenterococcalinfectionandClostridium
difficileinfection?ClinInfectDis.1997;25(suppl2):S206–S210.
13.GarbuttJM,LittenbergB,EvanoffBA,SahmD,MundyLM.
Entericcarriageofvancomycin-resistantEnterococcusfaecium
inpatientstestedforClostridiumdifficile.InfectControlHosp
Epidemiol.1999;20:664–670.
14.ShayDK,MaloneySA,MontecalvoM,etal.Epidemiologyand
mortalityriskofvancomycinresistantenterococcal
bloodstreaminfections.JInfectDis.1995;172:993–1000.
15.JordensJZ,BatesJ,GriffithsDT.Faecalcarriageand
nosocomialspreadofvancomycin-resistantEnterococcus
faecium.JAntimicrobChemother.1994;34:515–528.
16.RaffertyME,McCormickMI,BoppLH,etal.
Vancomycin-resistantenterococciinstoolspecimens
submittedforClostridiumdifficilecytotoxinassay.Infect
ControlHospEpidemiol.1997;18:342–344.
17.LeberAL,HindlerJF,KatoEO,BrucknerDA,PeguesDA.
Laboratorybasedsurveillanceforvancomicin-resistant
enterococci:utilityofscreeningstoolspecimenssubmitted
forClostridiumdifficiletoxinassay.InfectControlHosp Epidemiol.2001;22:160–164.
18.FujitaniS,GeorgeWL,MorganMA,NicholsS,MurthyAR.
Implicationsforvancomycin-resistantEnterococcus
colonizationassociatedwithClostridiumdifficileinfections.
AmJInfectControl.2011;39(April(3)):188–193.
19.KimH,JeongSH,KimM,LeeY,LeeK.Detectionof
ClostridiumdifficiletoxinA/Bgenesbymultiplexreal-time
PCRforthediagnosisofC.difficileinfection.JMedMicrobiol.
2012;61:274–277.
20.Ulu-KilicA,ÖzhanE,AltunD,Perc¸inD,Günes¸T,AlpE.Isit worthscreeningforvancomycin-resistantEnterococcus faeciumcolonization?Financialburdenofscreeningina developingcountry.AmJInfectControl.2016;(January),
http://dx.doi.org/10.1016/j.ajic.2015.11.008.
pii:S0196-6553(15)01160-8.
21.LawrenceSJ,PuzniakLA,ShadelBN,GillespieKN,KollefMH,
MundyLM.Clostridiumdifficileintheintensivecareunit:
epidemiology,costs,andcolonizationpressure.InfectControl