Performance of “RESIST-3 O.K.N. K-SeT” immunochromatographic assay for the detection of OXA-48 like, KPC, and NDM carbapenemases in Klebsiella pneumoniae in Turkey

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Clinical

Microbiology

Performance

of

“RESIST-3

O.K.N.

K-SeT”

immunochromatographic

assay

for

the

detection

of

OXA-48

like,

KPC,

and

NDM

carbapenemases

in

Klebsiella

pneumoniae

in

Turkey

Pınar

Sa ˘gıro ˘glu

_,

_Ufuk

_Hasdemir

_,

_Güls¸en

_Altınkanat

_Gelmez

_,

_Burak

_Aksu

_,

Onur

Karatuna

_,

_Güner

_Söyletir

a_{MarmaraUniversitySchoolofMedicine,DepartmentofMedicalMicrobiology,Istanbul,Turkey} b_{PrivateMelikgaziHospital,MedicalMicrobiology,Kayseri,Turkey}

c_{AcibademUniversitySchoolofMedicine,DepartmentofMedicalMicrobiology,Istanbul,Turkey}

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Articlehistory:

Received26April2017 Accepted5February2018 Availableonline1March2018 AssociateEditor:ElizabethMarques

Keywords: Klebsiellapneumoniae Immunochromatographictest NDM KPC OXA-48

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Inthisstudy,theperformanceofthe“RESIST-3O.K.N.K-SeT”(CorisBioConcept,Gembloux, Belgium)immunochromatographicassaywasevaluatedin132Klebsiellapneumoniae com-prising102carbapenemresistantand30carbapenemsusceptibleisolates.Genotypically knownisolatesofGramnegativebacteria(n=22)includingvariousspecieswerealsotested bytheassayascontrols.Theisolatestestedbytheimmunochromatographicassayandalso wererunPCRforblaKPC,blaIMP,blaVIM,blaNDM,andblaOXA-48.TheratesofblaNDM,blaOXA-48,

andblaKPCincarbapenemresistantisolateswerefoundat52.9%,39.2%,and2.0%,

respec-tively.BothblaNDMandblaOXA-48werefoundinsix(5.9%)isolates.Theresultsoftheassay

showed100%concordancewiththoseobtainedbyPCRin132K.pneumoniae.Theagreement betweenthetwomethodswasfoundtobeidenticalattheisolatelevel.Theassayalso correctlydetectedallgenotypicallyknownisolatesofEscherichiacoli,Serratiamarcescens, Cit-robacterfreundii,Enterobactercloacae,K.pneumoniaecarryingblaKPC,blaNDM,and/orblaOXA-48.

Ontheotherhand,theassaydidnotexhibitanycross-reactionincontrolisolatesharboring

blaIMPandblaVIM.WeconcludethattheRESIST-3O.K.N.K-SeTisareliable,rapid,anduser

friendlytestandwerecommenditforroutinediagnosticlaboratories.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Multi-drugresistantKlebsiellapneumoniaeisolatesareofgreat concernworldwide.1_{Althoughcarbapenemsarewidelyused}

∗ _{Correspondingauthor.}

E-mail:drpinarsa@gmail.com(P.Sa ˘gıro ˘glu).

antibioticsinthetreatmentofinfectionscausedbymulti-drug resistant K. pneumoniae, nosocomial infections due to car-bapenemresistantK.pneumoniae(CRKp)havealsoincreased dramatically in the last decade.2 _{The mainmechanism of}

carbapenemresistanceinK.pneumoniaehasbeenassociated with the production of carbapenem hydrolyzing enzymes. Various types of class A, B, and D type carbapenemases have been identified in K. pneumoniae. KPCs are the most

https://doi.org/10.1016/j.bjm.2018.02.002

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

commonlyseenclassAcarbapenemasesandendemicinthe USA,Colombia,Brasil,Argentina,Italy,Poland,China,Taiwan, Israel and Greece.1–3 _{Among class Bcarbapenemases, VIM}

andIMPhavespreadinmanycountriessincetheearly2000s andareendemicinSouthernEuropeandAsia-Pacificregion.2

AnotherclassBcarbapenemase,NDM,hasalsospreadrapidly andbecomeaglobalthreatsince2008.4_{InclassD,OXA-48type}

carbapenemaseshavebecomeprevalentamongK.pneumoniae

andothermembersofEnterobacteriaceaeinTurkey,Morocco, Tunisia,Libya,Algeria,Egypt,andIndia.1–3,28,29

The rapid and accurate laboratory diagnosis of carbapenemase-producing isolates is critical to enable the timely application of infection control measures. Standard susceptibilitytesting cannotspecify themechanismof car-bapenem resistance. An ideal diagnostic method should be rapid and able to detect all types of threatening car-bapenemases sensitively and specifically. Currently, there are various approaches to phenotypic tests based on dif-ferent principles. These include inhibitor based approach (combination disk test, double disk synergy test, gradient diffusionstrips),detectionbasedoncarbapenemhydrolysis (cloverleaf method, colorimetric assays, Carba NP, Blue-CARBA,carbapeneminhibitionmethod,starch-iodineassay, spectrometry, electrochemicalassay).5–10 _{Although someof}

thesemethodsexhibithighperformanceindiagnosisofKPC, IMP,VIMandNDMproducers;noneofthemishighlysensitive and specific for detecting OXA-48 producers.5–11 _{This may}

lead to challenges in the diagnosis of OXA-48 producers especiallyinregionswherethistypeofcarbapenemasesare prevalent. Moreover mostof the methods described above aretime-consuming.Recentstudieshaverevealedpromising results with immunochromatographic assays for easy and rapiddetectionofspecificcarbapenamases.6,12–19

In this study we aimedto evaluatethe performanceof anewlymarketedimmunochromatographicassay,“RESIST-3 O.K.N.K-SeT”(CorisBioConcept,Gembloux,Belgium) target-ing OXA-48 like, KPC,and NDMtypecarbapenemases in a collectionofcarbapenemresistantK.pneumoniae.

Materials

and

methods

Studyisolates

Atotalof132non-duplicatedclinicalK.pneumoniaeisolates collectedintwouniversityhospitalsbetweenJuly2014and December2016were included.102out of132K. pneumoniae

were resistantto ertapeneminroutinetesting byan auto-mated antimicrobial susceptibility test system (VITEK® 2, bioMérieux,Marcyl’Etoile,France).Therestingisolateswere susceptibletocarbapenemsinroutinetesting(Table1).

ReferenceandPCRcontrolstrains

Toassessthespecificityofthenewimmunochromatographic assay, acollection (n=22)of Gramnegative bacteriawhich have been previously characterized by PCR and/or DNA sequencingforthepresenceofblaKPC,blaIMP,blaVIM,blaNDM,

andblaOXA-48 geneswere alsostudiedwithRESIST-3O.K.N.

K-SeT (Table1). Escherichia coliATCC 25922 were used asa referencestraininantimicrobialsusceptibilitytesting. Bacterialidentification

Identification ofthe isolateswas confirmed byMALDI-TOF mass spectrometry(VITEK® MS,bioMérieux, Marcyl’Etoile, France).

Antimicrobialsusceptibilitytesting

Standard disk diffusiontest was performed on all isolates to confirm the ertapenem susceptibility results obtained with the VITEK® 2system.20 _{In addition, ertapenem MICs}

weredeterminedbythegradientdiffusionstriptest(Etest®, bioMérieux,Marcyl’Etoile,France)intheisolates.The Euro-pean Committee on Antimicrobial Susceptibility Testing (EUCAST v 6.0) standards used as interpretive criteria for antimicrobialsusceptibilitytesting.

Polymerasechainreaction

Carbapenemaseencodinggenes,blaKPC,blaIMP,blaVIM,blaNDM,

and blaOXA-48, were investigated by in house single PCR

methodtoconfirmphenotypictestresultsin132K.pneumoniae

isolates.21,24–26

Immunochromatographicassay

All isolates(132K. pneumoniae and 22 genotypically known Gram negativebacteria)weretested bythe RESIST-3O.K.N. K-SeTaccordingtothemanufacturer’sinstructions.Briefly,a singlecolonyonColumbiaagar+5%sheepblood(bioMérieux, Marcy l’Etoile, France) was suspended in 10 drops of lysis buffer.Threedropsofthesuspensionwerethenaddedonto theteststrip.Theresultswerereadwiththenakedeyewithin 15minutesatroomtemperature(Fig.1).

Results

Atotalof132K.pneumoniaeisolates(102CRKpand30CSKp) wereevaluatedforthepresenceofOXA-48like,KPC,andNDM carbapenemasesbytheRESIST-3O.K.NK-SeT immunochto-motographicassay.TheresultsoftheRESIST-3O.K.N.K-SeT assaywerecomparedthoseobtainedbyPCRtargetingthese genes(Table1).ErtapenemMICsinCRKpisolatesrangedfrom 1to>32␮g/mL(MIC50:32␮g/mL,MIC90:>32␮g/mL)bythe

gra-dient diffusionstriptest. Rates ofblaNDM, blaOXA-48, blaKPC,

genes inCRKp isolateswerefound tobe52.9%,39.2%,and 2.0%,respectively,byPCR.CombinedpositivityforbothblaNDM

and blaOXA-48 genes was found in six (5.9%) isolates. The

carbapenemase genes blaIMP and blaVIM were notdetected

in any of 132 study isolates. The results of the RESIST-3 O.K.N. K-SeT assay showed 100% concordance with those obtainedbyPCR.Theagreementbetweenthetwomethods was foundtobeidenticalattheisolatelevel.TheRESIST-3 O.K.N.K-SeTassaydidnotexhibitanycross-reactionin geno-typically known bacteria (n=8) harboringblaIMP and blaVIM

Table1–TheresultsofRESIST-3O.K.N.K-SeTimmunochromatographicassayin132clinicalKlebsiellapneumoniaeand

22controlisolatescomparedtoPCR.

PCRresults

n Gene(s) RESISTO.K.N.results Source

Studyisolates

CRa_{K.pneumoniae 40 bla}

OXA-48 OXA-48positive Thisstudy

54 blaNDM NDMpositive Thisstudy

6 blaOXA-48+blaNDM OXA-48+NDMpositive Thisstudy

2 blaKPC KPCpositive Thisstudy

CSb_{K.pneumoniae 30 ND}c _{Negative Thisstudy}

Genotypicallyknowncontrolisolates

Enterobactercloacae 4 blaIMP Negative Ourlaboratory

E.cloacae 1 blaNDM NDMpositive Ourlaboratory

Escherichiacoli 2 blaNDM NDMpositive Ourlaboratory

Serratiamarcescens 2 blaOXA-48 OXA-48positive Ourlaboratory

Citrobacterfreundii 1 blaNDM NDM Ourlaboratory

Klebsiellaoxytoca 1 blaIMP Negative Ourlaboratory

K.pneumoniae 2 blaIMP Negative d

K.pneumoniae4936 1 blaOXA-48 OXA-48positive Ref.No.21

K.pneumoniae0555 1 blaKPC KPCpositive Ref.No.22

K.pneumoniae05-506 1 blaNDM NDMpositive Ref.No.4

Pseudomonasaeruginosa 1 blaVIM Negative Ref.No.23

E.coli 5 NDc _{Negative Ourlaboratory}

a _{Carbapenemresistant.} b _{Carbapenemsusceptible.} c _Notdetected.

d _{Oneisolatefromourlaboratory,oneisolatefromRef.No.23.}

1 2 3 4 5 6 7 8 9 10 11

Fig.1–EvaluationofthetestresultsintheRESIST-3O.K.N.K-SeTassay.1;KPCproducingK.pneumoniae,2;OXA-48

producingK.pneumoniae,3;NDMproducingK.pneumoniae,4;OXA-48andNDMco-producingK.pneumoniae,5;Carbapenem susceptibleK.pneumoniaeasnegativecontrol,6;IMPproducingE.cloacae,7;NDMproducingE.cloacae,8;OXA-48producing S.marcescens,9;NDMproducingE.coli,10;NDMproducingC.freundii,11;CarbapenemsusceptibleE.coliasnegativecontrol.

showedagreementingenotypicallyknown(n=9) Enterobacte-riaceaeisolates(Escherichiacoli,Serratiamarcescens,Citrobacter freundii, Enterobacter cloacae, K. pneumoniae) carrying blaKPC,

blaNDM,and/orblaOXA-48genes(Table1).Falsepositiveresult

wasnotobservedinfivecarbapenemsusceptibleE.coliisolates whichwereknownnegativeforblaKPC,blaIMP,blaVIM,blaNDM,

andblaOXA-48.

Discussion

Infections due to carbapenemase producing K. pneumoniae

pose great concerns since they are associated with high morbidityand mortality.1–3 _{Rapid and specific diagnosis of}

carbapenemaseproducersplaysacrucialroleinpreventing the spread ofCRKp among hospitalizedpatients. Although PCR isrecommended as ‘gold standard’in carbapenemase detection,5,18,27_{intermsoflaboratoryuse,ithasseveral}

unfa-vorablepropertiesthatlimitits usefulnessinmanyroutine diagnosticlaboratories.5,27_{Thesepropertiesincludedifficulty}

in application, and the need for specific equipment and trainedpersonnel.5,8,9_{Moreover,PCRcannotrevealthe}

expres-sionstateofagene.Theavailabilityofareliable,rapid,anda simplephenotypicassaytodetectcarbapenemaseswouldbe ofgreatbenefit.Furthermore,suchatestshouldbeableto identifymostoftheprevalentcarbapenemaseswith epidemi-ologicalsignificance.Variousphenotypicmethodsdeveloped sofarareconsideredasreliableassaysespeciallyindetecting

classAandBcarbapenemasesbymanyauthors.5–11_However

thesemethodsremained unsatisfactoryinthediagnosis of OXA-48producers.Afewimmunochromatographicassaysin themarkethavebeenshownasreliableandfastfordetecting OXA-48and/orKPCproducers,separately.12–17_{Unfortunately,}

thesetests are notable toidentifythe mostcommon car-bapenemases,simultaneously.Inthisstudy,weevaluatedthe performanceofarecentlyintroducedRESIST-3O.K.N.K-SeT immunochromatographicassay.Itisthefirstassayinthe mar-kettargetingthedetectionofKPC,NDM,andOXA-48together. InourCRKpcollection,RESIST-3O.K.N.K-SeTresults exhib-itedexcellentconcordancewithPCR,yielding100%sensitivity forthetestedisolates(Table1).Theassayalsodetectedfive NDM,threeOXA-48andoneKPCproducerscorrectlyin geno-typicallyknowncollectionofE.cloacae,E.coli,S.marcescens, C.freundii,K.pneumoniae(Table1).Consideringthespecificity ofthe test, all carbapenem susceptibleisolates ofK. pneu-moniae (n=30) and E. coli (n=5) and the isolates harboring

blaIMP,blaVIMgeneswerefoundtobenegativebytheassay.

Theseresultsindicatethehighspecificity(100%)ofRESIST-3 O.K.N.K-SeT.TheassayalsodetectedcoproductionofNDM andOXA-48inclinicalisolatesharboringblaNDMandblaOXA-48

genes. Recent studies have remarked the emergence ofK. pneumoniaeand otherEnterobacteriaceaeproducing both car-bapenemasesfromTurkeyandsomeothercountries.31–40_For

that,ourresultwasfoundtobesignificantinterms ofthe reliabilityofRESIST-3O.K.N.K-SeTindetectingNDMand OXA-48co-producers.WehavealimitednumberofKPCproducers inourcollection.Therefore,itisnoteasytoreacha conclu-sionabout the reliabilityof theRESIST-3 O.K.N. K-SeT test inKPC detection. Asstated inmanyreports KPCis notan endemiccarbapenemaseinourcountry.1–3,28–30_Tworecently

publishedreportshaveshownthattheRESIST-3O.K.N.K-SeT exhibitedthehighperformanceinthedetectionofKPC pro-ducersaswellastheproducersofNDMandOXA-48.18,19 _As

concludedbytheauthorsofthesetwostudieswealsosuggest thattheassayhasalimitationintermsofdetectingother car-bapenemasessuchasIMPandVIMproducers.Wesuggestthat thereisstillaneedfordifferentassaysindetectionthe iso-latesproducingcarbapenemasesotherthanKPC,NDM,and OXA-48.

Besides the evaluation ofRESIST-3 O.K.N. K-SeT perfor-mance,ourwork hasalsorevealed someoutputs regarding theprevalentcarbapenemasesinourCRKpcollection. Accord-ingly, the most common carbapenemase has been NDM (52.9%), followedby OXA-48 (39.2%).These two carbapene-maseshavebeenfoundtogetherin5.9%ofCRKpisolates.We considertheseresultstobeveryremarkableintermsof revea-lingthepredominanceofNDMinourCRKpcollection.Almost all studies publishedfrom Turkeyso far, haveemphasized OXA-48prevalenceinthecountry.21,28,31,33_Althoughthe

pres-enceofvariousreportsfromTurkeynotifyingtheemergence ofNDMinthecountry,thisisthefirstreporthighlightingthe veryhighrateofNDMinanextendedcollectionofCRKpinour region.31–35_{MoreoverthedetectionofCRKpisolatesharboring}

NDMtogetherwithOXA-48wasconsideredasapremonitory outcomeofthestudy becauseitshows thespread ofCRKp NDMandOXA-48co-producers.31–40_{Ontheotherhand,this}

reporthasbeenthethirdonefromTurkeypresentingthe isola-tionofKPCproducingK.pneumoniae,sofar.41,42_Kuskucuetal.

alsoisolatedKPCenzymesintwoE.coliisolatesinTurkey.43_In

manyothercountries,includingTurkey’sneighboring coun-tryGreece,thepredominantenzymetypeisKPC.Theabsence ofanyregionalspreadorinstitutionaloutbreaksduetoKPC inTurkeymaybeassociatedwiththealreadyhighburdenof otherresistancedeterminantsamongK.pneumoniaeisolates, andthegeneticfailureofthesporadicKPCcasestotransfer theirplasmidscarryingtheblaKPCgene.

Finally,weconcludethattheRESIST-3O.K.N.K-SeTassay isareliable, rapid,user-friendlytestand werecommendit forroutinediagnosticlaboratoriesespeciallyinregionswhere OXA-48andNDMproducersareprevalent.

Conflicts

of

interest

Theauthorsdeclarethattherearenoconflictsofinterest.

Acknowledgments

WethankCigdemKayacanforgrantingtheOXA-48,IMP-1and VIM-4positivestrains.

Thisstudywassupportedbyinternalfundingandpartly supportedbyin-kindgrantfromZemedMedikalTekstilveDıs Tic.Ltd.Sti.,Istanbul,Turkey.

ThisstudywasapprovedbytheMarmaraUniversity Clini-calResearchEthicsCommittee(DecisionNo:09.2017.224).

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