Assessment of caspofungin susceptibility of Candida glabrata by the Etest®, CLSI, and EUCAST methods, and detection of FKS1 and FKS2 mutations

  • N. Bourgeois
  • C. Laurens
  • S. Bertout
  • Y. Balard
  • D. Krasteva
  • P. Rispail
  • L. Lachaud


Candida glabrata has emerged as a major pathogen in invasive candidiasis in recent years. Currently, guidelines for invasive candidiasis treatment recommend fluconazole or an echinocandin as the first-line therapy. Nevertheless, the resistance of Candida glabrata to echinocandin is an emerging problem and has been partly associated with mutations in the FKS1 and FKS2 genes. The Etest® is an appropriate method for determining antifungal susceptibility in emergency routine diagnosis. In this work, we evaluated the reliability of the Etest® in comparison with the two reference broth microdilution methods, Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST), to assess the caspofungin resistance of 193 isolates of Candida glabrata. The interpretation of minimum inhibitory concentration (MIC) values was also discussed according to different breakpoints. Moreover, FKS1 and FKS2 mutations were investigated for isolates with high MICs. Our results showed that the MIC50 value was similar to the MIC90 value for each method. The Etest® method showed the lowest MIC values, whereas EUCAST presented the highest. Categorical agreement between the Etest® and CLSI methods was 100 % and 36 % using the breakpoints proposed by Arendrup et al. (Antimicrob Agents Chemother 56(7):3965–3968, 2012) and Pfaller et al. (Int J Antimicrob Agents 38(1):65–69, 2011), respectively. Two isolates showed high MIC values with the three methods and both presented FKS2 mutations. A novel FKS2 mutation was also reported for one isolate. Future epidemiological studies should also evaluate the reliability of the Etest® to detect echinocandin resistance, as it remains a routine method.


Minimum Inhibitory Concentration Caspofungin Invasive Candidiasis Candida Glabrata EUCAST 
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The project was supported by investigator-initiated grants to L.L. from Merck. We also acknowledge the technical help of Valérie Fallot, Françoise Grino, Elsa Cazalet, and Myriam Leroyer.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Arendrup MC, Pfaller MA; Danish Fungaemia Study Group (2012) Caspofungin Etest susceptibility testing of Candida species: risk of misclassification of susceptible isolates of C. glabrata and C. krusei when adopting the revised CLSI caspofungin breakpoints. Antimicrob Agents Chemother 56(7):3965–3968PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Pfaller MA, Messer SA, Moet GJ, Jones RN, Castanheira M (2011) Candida bloodstream infections: comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008–2009). Int J Antimicrob Agents 38(1):65–69PubMedCrossRefGoogle Scholar
  3. 3.
    Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20(1):133–163PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr, Calandra TF, Edwards JE Jr et al (2009) Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 48(5):503–535PubMedCrossRefGoogle Scholar
  5. 5.
    Arendrup MC, Dzajic E, Jensen RH, Johansen HK, Kjaeldgaard P, Knudsen JD et al (2013) Epidemiological changes with potential implication for antifungal prescription recommendations for fungaemia: data from a nationwide fungaemia surveillance programme. Clin Microbiol Infect 19(8):E343–E353PubMedCrossRefGoogle Scholar
  6. 6.
    Cornely OA, Bassetti M, Calandra T, Garbino J, Kullberg BJ, Lortholary O et al; ESCMID Fungal Infection Study Group (2012) ESCMID* guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients. Clin Microbiol Infect 18(Suppl 7):19–37PubMedCrossRefGoogle Scholar
  7. 7.
    Lortholary O, Desnos-Ollivier M, Sitbon K, Fontanet A, Bretagne S, Dromer F et al (2011) Recent exposure to caspofungin or fluconazole influences the epidemiology of candidemia: a prospective multicenter study involving 2,441 patients. Antimicrob Agents Chemother 55(2):532–538PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Cota JM, Grabinski JL, Talbert RL, Burgess DS, Rogers PD, Edlind TD et al (2008) Increases in SLT2 expression and chitin content are associated with incomplete killing of Candida glabrata by caspofungin. Antimicrob Agents Chemother 52(3):1144–1146PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Beyda ND, Lewis RE, Garey KW (2012) Echinocandin resistance in Candida species: mechanisms of reduced susceptibility and therapeutic approaches. Ann Pharmacother 46(7–8):1086–1096PubMedCrossRefGoogle Scholar
  10. 10.
    Zimbeck AJ, Iqbal N, Ahlquist AM, Farley MM, Harrison LH, Chiller T et al (2010) FKS mutations and elevated echinocandin MIC values among Candida glabrata isolates from U.S. population-based surveillance. Antimicrob Agents Chemother 54(12):5042–5047PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Garcia-Effron G, Chua DJ, Tomada JR, DiPersio J, Perlin DS, Ghannoum M et al (2010) Novel FKS mutations associated with echinocandin resistance in Candida species. Antimicrob Agents Chemother 54(5):2225–2227PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Alexander BD, Johnson MD, Pfeiffer CD, Jiménez-Ortigosa C, Catania J, Booker R et al (2013) Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations. Clin Infect Dis 56:1724–1732PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST) (2008) EUCAST definitive document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts. Clin Microbiol Infect 14(4):398–405CrossRefGoogle Scholar
  14. 14.
    Durán-Valle MT, Gago S, Gómez-López A, Cuenca-Estrella M, Jiménez Díez-Canseco L, Gómez-Garcés JL et al (2012) Recurrent 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(6):3417–3419PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    EM Consulte (2004) Prise en charge des candidoses et aspergilloses invasives de l’adulte. Available online at:
  16. 16.
    Espinel-Ingroff A, Arendrup MC, Pfaller MA, Bonfietti LX, Bustamante B, Canton E et al (2013) Interlaboratory variability of Caspofungin MICs for Candida spp. Using CLSI and EUCAST methods: should the clinical laboratory be testing this agent? Antimicrob Agents Chemother 57(12):5836–5842PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Arendrup MC, Rodriguez-Tudela J-L, Park S, Garcia-Effron G, Delmas G, Cuenca-Estrella M et al (2011) Echinocandin susceptibility testing of Candida spp. Using EUCAST EDef 7.1 and CLSI M27-A3 standard procedures: analysis of the influence of bovine serum albumin supplementation, storage time, and drug lots. Antimicrob Agents Chemother 55(4):1580–1587PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • N. Bourgeois
    • 1
  • C. Laurens
    • 2
  • S. Bertout
    • 3
  • Y. Balard
    • 4
  • D. Krasteva
    • 3
  • P. Rispail
    • 4
  • L. Lachaud
    • 5
  1. 1.Laboratoire de Parasitologie-MycologieCHU de Nîmes et CHU de MontpellierMontpellierFrance
  2. 2.Laboratoire de BactériologieCHU de MontpellierMontpellierFrance
  3. 3.Laboratoire de Parasitologie-MycologieUniversité Montpellier 1/Faculté de PharmacieMontpellierFrance
  4. 4.Laboratoire de Parasitologie-MycologieCHU de Montpellier, Université Montpellier 1/Faculté de MédecineMontpellierFrance
  5. 5.Laboratoire de Parasitologie-MycologieCentre Hospitalier Universitaire CaremeauNîmes cedex 9France

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