Objectives: Polymyxin resistance has been increasing in many regions, and appropriate determination ofpolymyxin susceptibility is now a major challenge worldwide. Many clinical laboratories rely on gradientdiffusion methods to assess polymyxin susceptibility, although broth microdilution (BMD) is the onlymethod currently recommended by the CLSI and EUCAST. The aim of this study was to assess theperformance of the polymyxin B (PMB) Etest in a setting with a high prevalence of KPC-producingKlebsiella pneumoniae (KPC-KP).Methods: A commercial Etest susceptibility testing method was evaluated and compared with thereference BMD method, considering isolates with a minimum inhibitory concentration (MIC) 2 mg/L forPMB as susceptible to this drug. A total of 310 clinical KPC-KP isolates were evaluated.Results: Susceptibility was significantly higher by Etest compared with BMD (82.6% vs. 75.8%). The MIC50,MIC90and modal MICs for PMB were 0.25, 32 and 0.25 mg/L (27.1%) by BMD and 0.5, 16 and 0.5 mg/L(49.7%) by Etest, respectively. Although categorical agreement was 90.0%, there was poor essentialagreement (50.6%). A high rate (34.7%) of very major errors (VMEs) and a relatively low rate (2.1%) ofmajor errors were found.Conclusion: The considerable number of resistant isolates in this study allowed an accurate estimation ofVME rates and, consequently, a more comprehensive assessment of susceptibility testing for polymyxins.Etest did not meet fully the acceptance criteria for US FDA requirements. These data do not support theuse of this commercial method for determining PMB MICs in carbapenem-resistant Enterobacterales populations

Aquino, Valério Rodrigues

Barth, Afonso Luis

Kremer, Thaysa Guglieri

Magagnin, Cibele Massotti

Nunes, Aline Gabrielle Alves

Oliveira, Vanessa Pimentel de

Wink, Priscila Lamb

Zavascki, Alexandre Prehn

Journal of Global Antimicrobial Resistance

English

Lume 5.8

Journal of Global Antimicrobial Resistance 22 (2020) 40–42Short CommunicationPerformance of polymyxin B Etest in a setting of high prevalence ofKPC-producing Klebsiella pneumoniaeAlexandre P. Zavasckia,b,c, Cibele Massotti Magagninc,d, Priscila Lamb Winkc,e,*,Vanessa Pimentel de Oliveirab, Aline Gabrielle Nunesc, Thaysa Guglieri Krummerf,Valério Rodrigues Aquinog, Afonso Luís Barthc,eaDepartment of Internal Medicine, Medical School, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazilb Infectious Diseases Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazilc Laboratório de Pesquisa em Resistência Bacteriana (LABRESIS), Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazild Laboratório Weinmann–Grupo Fleury, Porto Alegre, Brazile Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazilf School of Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazilg Laboratório de Microbiologia, Hospital de Clínicas de Porto Alegre, Porto Alegre, BrazilA R T I C L E I N F OArticle history:Received 13 December 2019Received in revised form 27 January 2020Accepted 10 February 2020Available online 20 February 2020Keywords:Broth microdilutionCarbapenem resistanceColistinGradient testKlebsiella pneumoniaePolymyxin BA B S T R A C TObjectives: Polymyxin resistance has been increasing in many regions, and appropriate determination ofpolymyxin susceptibility is now a major challenge worldwide. Many clinical laboratories rely on gradientdiffusion methods to assess polymyxin susceptibility, although broth microdilution (BMD) is the onlymethod currently recommended by the CLSI and EUCAST. The aim of this study was to assess theperformance of the polymyxin B (PMB) Etest in a setting with a high prevalence of KPC-producingKlebsiella pneumoniae (KPC-KP).Methods: A commercial Etest susceptibility testing method was evaluated and compared with thereference BMD method, considering isolates with a minimum inhibitory concentration (MIC) 2 mg/L forPMB as susceptible to this drug. A total of 310 clinical KPC-KP isolates were evaluated.Results: Susceptibility was significantly higher by Etest compared with BMD (82.6% vs. 75.8%). The MIC50,MIC90 and modal MICs for PMB were 0.25, 32 and 0.25 mg/L (27.1%) by BMD and 0.5, 16 and 0.5 mg/L(49.7%) by Etest, respectively. Although categorical agreement was 90.0%, there was poor essentialagreement (50.6%). A high rate (34.7%) of very major errors (VMEs) and a relatively low rate (2.1%) ofmajor errors were found.Conclusion: The considerable number of resistant isolates in this study allowed an accurate estimation ofVME rates and, consequently, a more comprehensive assessment of susceptibility testing for polymyxins.Etest did not meet fully the acceptance criteria for US FDA requirements. These data do not support theuse of this commercial method for determining PMB MICs in carbapenem-resistant Enterobacteralespopulations.© 2020 The Authors. Published by Elsevier Ltd on behalf of International Society for AntimicrobialChemotherapy. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Contents lists available at ScienceDirectJournal of Global Antimicrobial Resistancejournal home page : www.e l sev ier .com/ loca te / jgar1. IntroductionKlebsiella pneumoniae is among the most important causes ofnosocomial infections, and resistance to carbapenems mediatedby carbapenemase production, especially K. pneumoniae carbape-nemase (KPC), is highly prevalent in several countries [1].* Corresponding author. Present address: Laboratório de Pesquisa em ResistênciaBacteriana (LABRESIS), Hospital de Clínicas de Porto Alegre, 2350 Ramiro BarcelosSt., Porto Alegre, RS 90.035-903, Brazil.E-mail address: pwink@hcpa.edu.br (P.L. Wink).http://dx.doi.org/10.1016/j.jgar.2020.02.0062213-7165/© 2020 The Authors. Published by Elsevier Ltd on behalf of International SocietND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Mainstream therapy for KPC-producing K. pneumoniae (KPC-KP)has been the polymyxins, either polymyxin B (PMB) or colistin, incombination with a second antimicrobial agent [2]. Although newantimicrobial drugs showing promising activity against KPC-KPisolates have recently been launched, the polymyxins will remainthe cornerstone of therapy where these new drugs are notavailable as well as for non-KPC-producing carbapenem-resistantEnterobacterales [3].Resistance to polymyxins has been increasing in many regionsand has been associated with higher mortality in patients infectedby KP-KPC isolates [4]. For this reason, appropriate determinationof susceptibility to this class of drugs is of paramount importance.y for Antimicrobial Chemotherapy. This is an open access article under the CC BY-NC-A.P. Zavascki et al. / Journal of Global Antimicrobial Resistance 22 (2020) 40–42 41There is no standard for polymyxin disk diffusion testing, whichhas not shown acceptable performance, possibly due to poordiffusion of polymyxins in agar. The European Committee onAntimicrobial Susceptibility Testing (EUCAST) and the Clinical andLaboratory Standards Institute (CLSI) Polymyxin BreakpointWorking Group recommends that methods other than brothmicrodilution (BMD) should not be used for susceptibility testingof polymyxins [5]. Although this recommendation as well as otherpublished studies have been mostly based on colistin, it has beenassumed that same results would be found for PMB since colistinand PMB are highly similar molecules [6,7].Use of the reference BMD method for susceptibility testing maynot be practical in many diagnostic microbiology laboratories, thusEtest remains an attractive option in these facilities. The aim of thiswork was to assess the performance of PMB Etest in a setting with ahigh prevalence of KPC-KP.2. Materials and methods2.1. Bacterial isolatesFrom April 2017 to April 2018, K. pneumoniae isolated frompatients admitted to Hospital de Clínicas de Porto Alegre (PortoAlegre, Brazil) that were intermediate or resistant to meropenem orertapenem by disk diffusion according to the CLSI were evaluatedfor the presence of carbapenemase-encoding genes. The presenceof carbapenemase genes (blaNDM-1, blaKPC-2, blaVIM-type, blaGES-type,blaOXA-48-like and blaIMP-type) was evaluated by multiplex high-resolution melting real-time PCR [8]. Only one isolate per patient(the first positive for the presence of blaKPC-2) was included in thisanalysis. Bacterial identification was performed by matrix-assistedlaser desorption/ionisation time-of-flight mass spectrometry(MALDI-TOF/MS) (bioMérieux, Marcy-l’Étoile, France). All isolatesincluded in this study were recovered from the same medical centreand were stored in 16% glycerol at 80 C.2.2. Minimum inhibitory concentration (MIC) determinationMICs of PMB were determined by Etest and BMD. The PMB Etestwas performed on fresh clinical isolates using Mueller–Hinton agar(NewProv, Pinhais, PR, Brazil) according to the manufacturer’sprotocol. Etest MICs were rounded up to the nearest two-folddilution as read by BMD (i.e. MIC = 1.5 mg/L was considered 2 mg/L). Isolates were stored at 80 C and were subsequently tested byBMD.The BMD method was performed in duplicate using polystyreneplates with cation-adjusted Mueller–Hinton broth (BectonDickinson, Franklin Lakes, NJ, USA). The PMB solution was obtainedfrom Sigma-Aldrich (St Louis, MO, USA). PMB was tested attwo-fold dilutions over the concentration range 64–0.125 mg/L.Fig. 1. Comparison of minimum inhibitory concentrations (MICs) by the reference stapneumoniae clinical isolates (n = 310). Numbers represent the occurrences observed at ealimits between testing methods. Dashed lines indicate the resistant MIC breakpoints (4in bold.Supplementation with polysorbate-80 was not performed [5].Escherichia coli ATCC 25922 and Pseudomonas aeruginosaATCC 27853 were used as quality control strains. Isolates with anMIC of 2 mg/L for PMB were considered susceptible [5].2.3. Definitions and analysisRates of essential agreement (EA), categorical agreement (CA), verymajor error (VME) and major error (ME) were determined. EAwas defined as an MIC result within a two-fold dilution of the gold-standard(BMD)result.CAoccurredwhentheinterpretationoftheMICresults was the same (i.e. susceptible or resistant). VME was defined asfalse susceptibility and ME as false resistance by Etest.3. ResultsA total of 310 KPC-KP isolates were included in the study, amongwhich 75 (24.2%) and 54 (17.4%) were resistant to PMB by BMD andEtest, respectively. The MICs of PMB ranged from 0.125–64 mg/L byBMD and from 0.125–512 mg/L by Etest (Fig. 1). The MIC50 and MIC90values for PMB were 0.25 mg/L and 32 mg/L for BMD and 0.5 mg/L and16 mg/L for Etest, respectively. The modal MICs were 0.25 mg/L(84 isolates; 27.1%) and 0.5 mg/L (154 isolates; 49.7%). CA was 90.0%(279/310). EA was observed in 157 (50.6%) of tests. Of 75 resistantisolates by BMD, 26 were categorised as susceptible by Etest, resultingin a VME rate of 34.7% Of 235 susceptible isolates by BMD, 5 werecategorised as resistant by Etest, resulting in a ME rate of 2.1% (Fig. 1).4. DiscussionClinical laboratories often rely on gradient diffusion methods orautomated systems for susceptibility testing for polymyxins asthey are less laborious [8]. These methods have been shown tobe unreliable for colistin (polymyxin E) compared with the gold-standard method of BMD [9]. However, very few studies haveassessed gradient tests for PMB [10–13].In the present study, the antimicrobial activity of PMB wasevaluated against selected contemporary bacterial pathogensconsisting exclusively of KPC-KP (n = 310). Although CA was90.0%, the results presented unacceptably high and low rates ofVME and EA, respectively. Using the US Food and DrugAdministration (FDA) requirements for commercial antimicrobialsusceptibility testing systems (EA  90%, CA  90%, VME  1.5%, ME 3.0%) [13], Etest did not meet the acceptance criteria for EA andVME, presenting EA and VME values well below and above the FDArequirements, respectively.Noteworthy, eight VMEs occurred in isolates with an MIC of4 mg/L by BMD (Fig. 1). However, this did not impact the finding ofan unacceptably high VME rate since only two of these presentedan MIC of 2 mg/L by Etest, which would be in EA with thendard method broth microdilution (BMD) and Etest for KPC-producing Klebsiellach point. Grey boxes indicate essential agreement (EA) measurements  1 log2 MIC mg/L) according to CLSI-EUCAST criteria [5]. Very major errors (VMEs) are indicated42 A.P. Zavascki et al. / Journal of Global Antimicrobial Resistance 22 (2020) 40–42corresponding BMD result. In contrast, five isolates presenting anMIC of 32 mg/L by BMD demonstrated MIC  1.0 mg/L by Etest.Four other studies have compared Etest with BMD for PMB incarbapenem-resistant Enterobacterales [10–13]. Only Latet al. foundthat the PMB Etest overestimates resistance in comparison withBMD; indeed, their study evaluating 48 KPC-KP also reported a lowEA(19%) butonly 2%VME and, incontrasttothecurrentstudy, the MErate was 23% [10]. On the other hand, the other three studies areconsistent with the findings of the current study [11–13]. Chew et al.tested 76 carbapenem-resistant Enterobacterales, including only 2 K.pneumoniae isolates, and reported an EA of 48.7%, a VME rate of26.1% and ME rate of 1.9% [11], which were all similar to thoseobserved in the current study (50.6%, 34.7% and 2.1%, respectively).Another study tested 39 Enterobacterales isolates, including 15 K.pneumoniae, and reported an EA of 61%, a VME rate of 8% and a CA ofonly 88%; no ME was reported [12]. Finally, Kulengowski et al.assessed 70 isolates of carbapenem-resistant Enterobacterales,including 34 K. pneumoniae, and reported overall very poor results,with only 80% and 10% of CA and EA, respectively, and an astonishing88% of VME; no ME was reported [13]. Interesting, the vast majorityof PMB MICs by Etest in the sample of that study were 0.5 mg/L [13],which is similar with the modal MIC of 0.5 mg/L by Etest in thecurrent study. It is important to state that these previous reports hada limited sample size with a relatively small number of resistantisolates and this might have affected a more accurate estimation ofVME rates. Moreover, only the report of Chew et al. [11] mentionedthat polysorbate-80 (P-80) was not used as supplementation to theBMD methodology, but the other studies did not mention it. BMDwithout P-80 supplementation is currently recommended as thereference method [5] as P-80 in itself has some antibacterial activityand may act synergistically with polymyxins to spuriouslylower MICs, especially for organisms for which MICs are near thebreakpoints and/or epidemiologic cut-off values of 1–2 mg/L[14,15].In the present study, a high prevalence of PMB resistance with awide range of polymyxins MICs was found. The considerablenumber of resistant isolates allows an accurate estimation of VMErates and, consequently, a more comprehensive assessment ofsusceptibility testing for polymyxins.Some limitations of this study must be acknowledged. All isolateswere recovered from the same medical centre, thus clonalrelatedness cannot be ruled out. In addition, it should be mentionedthat Etest and BMD were performed at two different times: Etestwas tested initially and BMD was subsequently performed onisolates that had been stored in 16% glycerol at 80 C. Therefore,occasionally thaw–frozen cycles and passage bias might haveaffected some BMD MIC results. Finally, as in previous studies, onlyone brand of PMB was evaluated. Although we would not expect adistinct result, it may be worthwhile to compare different brands inadditional studies.In conclusion, in a high number of KPC-KP isolates with a broadMIC distribution, this study found an unacceptably high rate of VMEof Etest for PMB. The high level of false susceptibility presented byEtest forPMB has greatclinical impactsince itmayleadto inadequatetreatment and clinical failure. The current data do not support thecontinued use of Etest strips by clinical laboratories for determiningPMB MICs in carbapenem-resistant Enterobacterales.FundingThis work was supported by Instituto Nacional de Pesquisa emResistência Antimicrobiana (INPRA), Brazil [INCT/CNPq: 465718/2014-0and INCT/FAPERGS: 17/2551-0000514-7] and by Fundo de Incentivo àPesquisa e Eventos do Hospital de Clínicas de Porto Alegre.Competing interestsAPZ has received honoraria for speaking engagements andconsultancy from Merck, Cipla and Pfizer and a research grant fromPfizer. All other authors declare no competing interests.Ethical approvalThis study was approved by the Ethical Committee of Hospitalde Clínicas de Porto Alegre (Porto Alegre, Brazil) [16-0444].AcknowledgmentAPZ is a research fellow of the National Council for Scientificand Technological Development (CNPq), Ministry of Science andTechnology, Brazil.References[1] Bonomo RA, Burd EM, Conly J, Limbago BM, Poirel L, Segre JA, et al.Carbapenemase-producing organisms: a global scourge. Clin Infect Dis2018;66:1290–7, doi:http://dx.doi.org/10.1093/cid/cix893.[2] Tsuji BT, Pogue JM, Zavascki AP, Paul M, Daikos GL, Forrest A, et al. 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Colistin and polymyxin B minimalinhibitory concentrations determined by Etest found unreliable for Gram-negative bacilli. Ochsner J 2017;17:239–42.[13] Kulengowski B, Ribes JA, Burgess DS. Polymyxin B Etest1 compared with gold-standard broth microdilution in carbapenem-resistant Enterobacteriaceaeexhibiting a wide range of polymyxin B MICs. Clin Microbiol Infect2019;25:92–5, doi:http://dx.doi.org/10.1016/j.cmi.2018.04.008.[14] Albur M,NoelA, Bowker K, MacgowanA. Colistin susceptibility: timefora review.J Antimicrob Chemother 2014;69:1432–4, doi:http://dx.doi.org/10.1093/jac/dkt503.[15] Humphries RM. Susceptibility testing of the polymyxins: where are we now?Pharmacotherapy 2015;35:22–7, doi:http://dx.doi.org/10.1002/phar.1505.

Performance of polymyxin B Etest in a setting of high prevalence of KPC-producing Klebsiella pneumoniae

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Performance of polymyxin B Etest in a setting of high prevalence of KPC-producing Klebsiella pneumoniae

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