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1 AAC Accepts, published online ahead of print on 26 August 2013 Antimicrob. Agents Chemother. doi: /aac Copyright 2013, American Society for Microbiology. All Rights Reserved. Comparison between the EUCAST procedure and the Etest for determination of the susceptibility of Candida spp. isolates to micafungin Laura Judith Marcos-Zambrano 1,2 Pilar Escribano 1,2,3 Cristina Rueda 5 Óscar Zaragoza 5 Emilio Bouza 1,2,3,4 Jesús Guinea 1,2,3,4 1 Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain. 2 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain. 3 CIBER Enfermedades Respiratorias-CIBERES (CD06/06/0058), Palma de Mallorca, Spain. 4 Medicine Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain. 5 Mycology Reference Laboratory, National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. Running title: Etest for micafungin susceptibility in Candida spp. Key words: Etest, microdilution, EUCAST EDef 7.2, Candida, antifungal susceptibility testing, micafungin, comparison Abstract word count: 75 Body text word count: 988 Correspondence: Jesús Guinea Servicio de Microbiología Clínica y Enfermedades Infecciosas Hospital General Universitario Gregorio Marañón C/Dr. Esquerdo, Madrid, Spain Phone: Fax: jguineaortega@yahoo.es 1 Downloaded from on September 27, 2018 by guest

2 18 ABSTRACT We compared the ability of the EUCAST EDef 7.2 and the Etest to detect the susceptibility to micafungin of 160 Candida and non-candida clinical isolates. Agreement was higher when Etest MICs were obtained after 24 h of incubation; essential agreement was 90%, and categorical agreement was >90%. False susceptibility was only seen for C. krusei (10%) and false resistance was observed in 6% of the isolates, ranging from 2.6% (C. glabrata) to 13% (C. albicans). Downloaded from on September 27, 2018 by guest 2

3 27 BODY TEXT The CLSI M27-A3 and EUCAST broth microdilution procedures are the gold standard for antifungal susceptibility testing and can detect micafunginresistant isolates (1-6). These procedures are comparable, and the results obtained correlated with clinical outcome (7-9). However, they are timeconsuming and difficult to implement in the clinical microbiology laboratories. The Etest (biomérieux) is a fast and cost-effective alternative, and agreement with CLSI M27-A3 is high when testing the susceptibility of Candida spp. to micafungin (10-12). Agreement between the Etest and the EUCAST procedure is also high for caspofungin (13, 14). We compared for the first time EUCAST and the Etest for detection of susceptibility to micafungin in a collection of Candida spp isolates. Organisms and identification. We studied 160 yeast strains obtained from patients with fungemia admitted to Gregorio Marañón hospital (2007 to 2013) (Table 1). The 17 control isolates (5 strains with fks hot spot (HS) mutations and 12 intrinsically echinocandin-resistant non-candida isolates) were included in the analysis (Tables 1, and 2). Antifungal susceptibility testing. The in vitro susceptibility was obtained by means of the EUCAST and the Etest methods. For EUCAST EDef 7.2., the plates were incubated for 24 h at 35ºC, and MIC values were determined spectrophotometrically at 530 nm (15, 16); a minimum threshold of optical density 0.3 was used for growth control wells to validate the reading of the plates. Non- Candida isolates were incubated with shaking at 30ºC. All MICs could be obtained 3

4 50 51 after 24h of incubation. C. krusei ATCC 6258 and C. parapsilosis ATCC were used as quality control strains Etest strips containing micafungin at concentrations ranging from to 32 µg/ml were used. The plates were incubated at 35ºC and read after 24 h and 48 h of incubation. The MIC value was defined as the lowest concentration of micafungin at which the zone of inhibition intersects the strip; microcolonies in the inhibition zone were not taken into account. Identification of fks mutations. HS1 and HS2 from the fks genes of the 5 Candida control isolates were amplified as previously described (17-19) (Table 2). The presence of fks mutations was also screened in the remaining isolates showing phenotypic resistance). Comparison between procedures. Off-scale results obtained using the Etest were transformed to the next higher dilution matching the scale used for EUCAST. Using the MICs obtained by EUCAST procedure after 24 h of incubation as the gold standard, the results obtained by Etest after 24 h and 48 h of incubation were studied. MIC discrepancies of no more than ±2 fold dilutions were used to calculate the essential agreement (EA) (20, 21). Categorical agreement (CA) was analyzed using the following species-specific clinical breakpoints: µg/ml (C. albicans), 2 µg/ml (C. parapsilosis), or µg/ml (C. glabrata) (22). For C. tropicalis and C. krusei we chose the following epidemiological cut-off values (ECOFFs) µg/ml, and 0.5 µg/ml, respectively (22) We considered discrepancies as very major errors or false susceptibility (VME) when the Etest classified a strain as susceptible and the EUCAST as 4

5 74 75 resistant and major errors or false resistance (ME) when a strain was classified as resistant by the Etest method and susceptible by EUCAST Table 1 shows the susceptibility of the 160 isolates to micafungin. The EA between both procedures was high (Table 3). After 48 h of incubation, agreement decreased for all species; for C. parapsilosis, growth was slow. The overall CA was high although differences were found between species (Table 4). No VMEs were seen for most of the species studied after 24 h of incubation, with the exception of C. krusei. The Etest correctly classified as resistant the 5 isolates with mutations in the fks genes and the 12 intrinsically micafungin-resistant non- Candida isolates. In previous studies, the EA between the Etest and EUCAST for caspofungin ranged from 87.7% (13) to 94% (14). Unfortunately, no CA was reported in either study. As with the results reported for caspofungin in previous studies, we found that the EA between the Etest and the EUCAST procedure for micafungin was very high after 24 h of incubation. However, 17% of the strains studied did not grow properly, and the Etest had to be read after 48 h of incubation. This delay could be a limitation of the Etest, particularly for C. parapsilosis isolates. The CA was also high, but the percentage of errors was species-dependent and lower when the Etest was read after 24 h. Of interest, VMEs was infrequent and was only seen with C. krusei (involving 10% of isolates), or with C. tropicalis 5

6 97 98 only after 48 h of incubation. The presence of VME of C. krusei to micafungin is a matter of concern owing to its intrinsic resistance to fluconazole The overall rate of MEs was 6%, ranging from 2.6% (C. glabrata) to 13% (C. albicans) after 24 h of incubation. The presence of MEs could necessitate the use of an alternative antifungal agent to the echinocandins. However, impact would be limited in the case of C. albicans, as most isolates are fluconazolesusceptible. The impact could also be negative for C. glabrata, as many clinicians prefer candins to fluconazole. We did not find mutations in the 15 isolates (C. albicans, n=10; C. glabrata, n=3; C. tropicalis, n=1; C. krusei, n=1) showing phenotypic resistance by EUCAST (n=1), Etest (n=11), or both (n=2). Our study is limited by the low number of micafungin-resistant and C. krusei isolates. Furthermore, all isolates sourced from a single hospital. Further studies should be carried out to prove whether higher micafungin MICs in the Etest is an intrinsic problem of C. krusei and C. tropicalis. However, we did include control isolates with well-characterized micafungin resistance mechanisms. To conclude, we found that the Etest was an effective procedure when screening for the presence of micafungin-resistant Candida spp. isolates if results are obtained after 24 h of incubation. Resistant strains, particularly of C. albicans, should be retested using a broth microdilution procedure in order to prove resistance to micafungin ACKNOWLEDGEMENTS 6

7 We would like to thank Thomas O Boyle for editing and proofreading the article. We would like to thank Elia Gómez de Pedrosa (Ramón y Cajal Hospital, Madrid), Alfredo Pérez Revilla (12 de Octubre Hospital, Madrid), Julio García Rodriguez (La Paz Hospital, Madrid), and Isabel Ruiz (Majadahonda Hospital, Madrid) for kindly providing us with 4 out of the fks mutant Candida isolates. This work was supported by grants from Fondo de Investigación Sanitaria (FIS) (grants number PI11/00167) and Astellas Pharma. P. Escribano is supported by a postdoctoral Sara Borrell contract (CD09/00230) from FIS. J. Guinea is supported by a Miguel Servet contract (MS09/00055) from FIS. L.J. Marcos-Zambrano is supported by a pre-doctoral grant (FI12/00265) from FIS. Cristina Rueda is supported by a postdoctoral Sara Borrell contract (CD11/00110) from FIS. This study does not present any conflicts of interest for its authors. 7

8 Tables and Figures TABLE 1. Activity of micafungin against the 160 isolates using the EUCAST EDef 7.2 procedure and the Etest read after 24 h and 48 h of incubation. MIC 90 µg/ml (range) Species (N) EUCAST Etest 24 h a Etest 48 h C. albicans ( ) ( ) ( ) C. parapsilosis 27 2 (0.25-2) 1 ( ) 2 ( ) C. glabrata ( ) ( ) ( ) C. tropicalis ( ) 1 ( ) 4 ( ) C. krusei 10 1 ( ) 0.25 ( ) 0.25 ( ) Candida spp. b 12 1 ( ) 1 ( ) 16 ( ) Other yeasts 12 8 ( ) 16 ( 16-16) 16 (1-16) Overall c ( ) 0.5 ( ) 4 ( ) a For Etest read at 24 h, 55.5% of C. parapsilosis, 5% of C. glabrata, 16.6% of Candida spp., and 66.6% of other yeasts did not grow and were excluded from the analysis. b Candida isolates belonging to species without proposed breakpoints or epidemiological cut-offs according to the EUCAST EDef 7.2 procedure. c Strains were identified after amplification and sequencing of the ITS1-5.8S-ITS2 region (23); the distribution of isolates was as follows: C. albicans, n=31; C. parapsilosis complex, n=27 (C. parapsilosis senso stricto, n=24; C. orthopsilosis, n=3); C. glabrata, n=40; C. krusei, n=10; C. tropicalis, n=28; Candida spp., n=12 (C. dubliniensis, n=5; C. guilliermondii, n=4; C. kefyr, n=1; C. lusitaniae, n=1; Pichia caribbica, n=1), and other yeasts [n=7 (Rhodotorula mucilaginosa), and n=5 (Trichosporon spp.)]. 8

9 TABLE 2. Antifungal susceptibility and mutations found in the fks genes of the 5 Candida isolates included as controls. All isolates were isolates from blood samples. Micafungin MIC (ug/ml) Species Mutation Region EUCAST Etest 24 h Etest 48 h Candida glabrata F649 HS1 fks Candida albicans F641S HS1 fks Candida tropicalis F641L HS1 fks Candida tropicalis R647G HS1 fks Candida tropicalis S645F HS1 fks Downloaded from on September 27, 2018 by guest 9

10 TABLE 3. Essential agreement. Percentage of strains in which the MIC differed ±1, ±2, and >±3 log dilutions over the reference method % ±1 % ±2 C. albicans 24H H H C. parapsilosis a 48H C. krusei 24H H C. tropicalis 24H H H C. glabrata a 48H H Candida spp. a, b 48H H Other yeast a 48H Overall 24H H a For Etest read at 24 h, 55.5% of C. parapsilosis, 5% of C. glabrata, 16.6% of Candida spp., and 66.6% of other yeasts did not grow and were excluded from the analysis. b Candida isolates belonging to species without proposed breakpoints according to the EUCAST EDef 7.2 procedure. 10

11 TABLE 4. Categorical agreement between methods using the breakpoints and epidemiological cut-offs (ECOFFs) established by the EUCAST EDef 7.2. The percentage of agreement and the percentage or errors are also shown. EUCAST Species C. albicans Etest 24 h a Etest 48 h S R %CA b VME b ME b S R %CA VME ME S R C. parapsilosis c S R C. glabrata S R C. krusei d S R C. tropicalis d S R Overall S R a For Etest read at 24 h, 55.5% of C. parapsilosis and 5% of C. glabrata did not grow at 24 h and were excluded from the analysis. b CA, categorical agreement; VME, very major error; ME, major error. c According to the EUCAST procedure, there is no Susceptible category for C. parapsilosis, and all strains with an MIC 2 µg/ml are considered Intermediate. d Owing to the lack of clinical breakpoints for these species, the categorical analysis was performed using the ECOFFs, and the strains were classified as wild-type or non wild-type instead of susceptible or resistant. 11

12 References 1. Pfaller MA, Diekema DJ Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to J Clin Microbiol 50: Perlin DS Resistance to echinocandin-class antifungal drugs. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy 10: Slater JL, Howard SJ, Sharp A, Goodwin J, Gregson LM, Alastruey- Izquierdo A, Arendrup MC, Warn PA, Perlin DS, Hope WW Disseminated Candidiasis caused by Candida albicans with amino acid substitutions in Fks1 at position Ser645 cannot be successfully treated with micafungin. Antimicrob Agents Chemother 55: García-Effrón G, Lee S, Park S, Cleary JD, Perlin DS Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-beta-D-glucan synthase: implication for the existing susceptibility breakpoint. Antimicrob Agents Chemother 53: Shields RK, Nguyen MH, Press EG, Kwa AL, Cheng S, Du C, Clancy CJ The presence of an FKS mutation rather than MIC is an independent risk factor for failure of echinocandin therapy among patients with invasive candidiasis due to Candida glabrata. Antimicrob Agents Chemother 56: Alexander BD, Johnson MD, Pfeiffer CD, Jiménez-Ortigosa C, Catania J, Booker R, Castanheira M, Messer SA, Perlin DS, Pfaller MA

13 Increasing Echinocandin Resistance in Candida glabrata: Clinical Failure Correlates With Presence of FKS Mutations and Elevated Minimum Inhibitory Concentrations. Clinical infectious diseases 56(12): Rodríguez-Tudela JL, Almirante B, Rodríguez-Pardo D, Laguna F, Donnelly JP, Mouton JW, Pahissa A, Cuenca-Estrella M Correlation of the MIC and dose/mic ratio of fluconazole to the therapeutic response of patients with mucosal candidiasis and candidemia. Antimicrob Agents Chemother 51: Rodríguez-Tudela JL, Martínez-Suárez JV, Dronda F, Laguna F, Chaves F, Valencia E Correlation of in-vitro susceptibility test results with clinical response: a study of azole therapy in AIDS patients. Journal of Antimicrobial Chemotherapy 35: Espinel-Ingroff A Clinical utility of in vitro antifungal susceptibility testing. Revista española de quimioterapia 13: Pfaller MA, Castanheira M, Diekema DJ, Messer SA, Moet GJ, Jones RN Comparison of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Etest methods with the CLSI broth microdilution method for echinocandin susceptibility testing of Candida species. J Clin Microbiol 48: Arendrup MC, García-Effrón G, Lass-Florl C, López AG, Rodríguez-Tudela JL, Cuenca-Estrella M, Perlin DS Echinocandin susceptibility testing of Candida species: comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, disk diffusion, and agar dilution methods with RPMI and isosensitest media. Antimicrob Agents Chemother 54:

14 Axner-Elings M, Botero-Kleiven S, Jensen RH, Arendrup MC Echinocandin susceptibility testing of Candida isolates collected during a 1- year period in Sweden. J Clin Microbiol 49: Dannaoui E, Paugam A, Develoux M, Chochillon C, Matheron J, Datry A, Bouges-Michel C, Bonnal C, Dromer F, Bretagne S Comparison of antifungal MICs for yeasts obtained using the EUCAST method in a reference laboratory and the Etest in nine different hospital laboratories. Clin Microbiol Infect 16: Chryssanthou E, Cuenca-Estrella M Comparison of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing proposed standard and the E-test with the NCCLS broth microdilution method for voriconazole and caspofungin susceptibility testing of yeast species. Journal of clinical microbiology 40: EUCAST definitive document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts. Clin Microbiol Infect 14: Arendrup MC, Cuenca-Estrella M, Lass-Florl C, Hope W EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin Microbiol Infect 18:E Durán-Valle MT, Gago S, Gómez-López A, Cuenca-Estrella M, Jiménez Díez-Canseco L, Gómez-Garces JL, Zaragoza O Recurrent 14

15 episodes of candidemia due to Candida glabrata with a mutation in hot spot 1 of the FKS2 gene developed after prolonged therapy with caspofungin. Antimicrob Agents Chemother 56: Desnos-Ollivier M, Bretagne S, Raoux D, Hoinard D, Dromer F, Dannaoui E Mutations in the fks1 gene in Candida albicans, C. tropicalis, and C. krusei correlate with elevated caspofungin MICs uncovered in AM3 medium using the method of the European Committee on Antibiotic Susceptibility Testing. Antimicrob Agents Chemother 52: García-Effrón G, Park S, Perlin DS Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints. Antimicrob Agents Chemother 53: Guinea J, Peláez T, Alcalá L, Bouza E Correlation between the E test and the CLSI M-38 A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus. Diagnostic microbiology and infectious disease 57: Guinea J, Peláez T, Alcalá L, Bouza E Comparison of Sensititre YeastOne with the NCCLS M38-A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus. Diagnostic microbiology and infectious disease 56:

16 European Committee on Antimicrobial Susceptibility Testing. Micafungin and Candida spp.: Rationale for the clinical breakpoints, version 1.0, White T, T. Bruns, S. Lee, and J. Taylor Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics., p. 322, PCR protocols: a guide to methods and applications. Academic Press, San Diego. Downloaded from on September 27, 2018 by guest 16

17 crossmark ERRATUM Erratum for Marcos-Zambrano et al., Comparison between the EUCAST Procedure and the Etest for Determination of the Susceptibility of Candida Species Isolates to Micafungin Laura Judith Marcos-Zambrano, a,b Pilar Escribano, a,b,c Cristina Rueda, e Óscar Zaragoza, e Emilio Bouza, a,b,c,d Jesús Guinea a,b,c,d Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain a ; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain b ; CIBER Enfermedades Respiratorias-CIBERES (CD06/06/0058), Palma de Mallorca, Spain c ; Medicine Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain d ; Mycology Reference Laboratory, National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain e Volume 57, no. 11, p , Page 5769: the second paragraph of the Acknowledgments section should read, This work was supported by grants from Fondo de Investigación Sanitaria (FIS; Plan Nacional de I D I ) (grant number PI11/00167), by the European Regional Development Fund (FEDER) A way of making Europe, and Astellas Pharma. P. Escribano is supported by a postdoctoral Sara Borrell contract (CD09/00230) from FIS. J. Guinea is supported by a Miguel Servet contract (CP09/00055) from FIS. L. J. Marcos-Zambrano is supported by a predoctoral grant (FI12/00265) from FIS. Cristina Rueda is supported by a postdoctoral Sara Borrell contract (CD11/00110) from FIS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Citation Marcos-Zambrano LJ, Escribano P, Rueda C, Zaragoza Ó, Bouza E, Guinea J Erratum for Marcos-Zambrano et al., Comparison between the EUCAST procedure and the Etest for determination of the susceptibility of Candida species isolates to micafungin. Antimicrob Agents Chemother 60:4429. doi: /aac Copyright 2016, American Society for Microbiology. All Rights Reserved. July 2016 Volume 60 Number 7 Antimicrobial Agents and Chemotherapy aac.asm.org 4429