Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Antimicrobial Agents and Chemotherapy
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Susceptibility

Time of Incubation for Antifungal Susceptibility Testing of Aspergillus fumigatus: Can MIC Values Be Obtained at 24 Hours?

Juan Luis Rodriguez-Tudela, Laura Alcazar-Fuoli, Ana Alastruey-Izquierdo, Araceli Monzon, Emilia Mellado, Manuel Cuenca-Estrella
Juan Luis Rodriguez-Tudela
Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: jlrtudela@isciii.es
Laura Alcazar-Fuoli
Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ana Alastruey-Izquierdo
Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Araceli Monzon
Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Emilia Mellado
Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Manuel Cuenca-Estrella
Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AAC.00933-07
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

A collection of Aspergillus fumigatus isolates was used to check if MICs can be read at 24 h. At 24 h, the geometric mean MIC of itraconazole for resistant isolates was determined to be 5.11 mg/liter, but the MIC was read as 16 mg/liter at 48 h. At 24 h, MICs for 51.5% of resistant strains were determined to be ≤2 mg/liter. MICs must be obtained at 48 h.

The incidence of invasive mold infections has increased in the last decades. The mortality due to these infections is very high, and outcomes are determined mainly by the risk factors of the patients. Although the correlation between the results of antifungal susceptibility testing and the outcomes for patients is far from perfect, several reports have described poorer responses to therapy by those infections caused by isolates for which MICs are high. Lass-Flörl et al. (11) reported favorable outcomes for 20% of patients with invasive disease caused by Aspergillus terreus who were treated with amphotericin B, in comparison with 47% of amphotericin B-treated patients with disease caused by other Aspergillus spp. The amphotericin B MICs for A. terreus have been shown to be higher than those for A. fumigatus (11, 20), and therefore, the poorer responses of patients were attributed to higher amphotericin B MICs. In addition, resistant strains of A. fumigatus have recently been reported (2-5, 8, 13, 14, 21).

Antifungal susceptibility testing has been standardized for molds (16). The optimal susceptibility testing conditions for the detection of azole-resistant strains were defined by Espinel-Ingroff et al. (6). The recommendation was to estimate the end point as the complete inhibition of visual fungal growth at 48 h. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) started the standardization of the antifungal susceptibility testing of molds, and now the standard is pending final approval (10). In previous work (7), we observed that the time of incubation significantly affected the final MIC readings and that sometimes a major increase (two to six dilutions) occurred between readings at 24 and 48 h. Most A. fumigatus strains are capable of growth in RPMI 1640 within 24 h. Thus, there is a chance to obtain end points at 24 h and deliver the results to the clinicians at an earlier time.

The aim of this study was to check if the MICs of antifungal drugs for A. fumigatus can be read at 24 h instead of at 48 h. To achieve this, we have compared a large collection of clinical isolates of A. fumigatus for which MICs of azole drugs vary from low to high.

Microorganisms.

One hundred fifty itraconazole-susceptible A. fumigatus strains and 33 A. fumigatus strains resistant to itraconazole were included. The mechanisms of resistance of these strains have been described previously (5, 12-15).

Antifungal susceptibility testing.

Microdilution testing was performed by following the EUCAST methodology (10). This standard is similar to approved standard M38-A published by the Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards) (16), with the following modifications: (i) RPMI 1640 is supplemented with glucose to reach a 2% concentration, (ii) the inoculum size is between 2 × 105 and 5 × 105 CFU/ml, and (iii) inocula are prepared by counting spores in a hematocytometer (1, 18, 19). A. fumigatus ATCC 204305 and A. flavus ATCC 204304 were used as quality control strains (16).

The antifungal agents used were amphotericin B (range, 0.03 to 16 mg/liter; Sigma Aldrich Química), itraconazole (range, 0.015 to 8 mg/liter; Janssen S.A.), voriconazole (range, 0.015 to 8 mg/liter; Pfizer S.A.), ravuconazole (range, 0.015 to 8 mg/liter; Bristol-Myers Squibb), and posaconazole (range, 0.015 to 8 mg/liter; Schering-Plough Research Institute).

End points were recorded at 24 and 48 h and defined as the antifungal concentration that produced a complete inhibition of visual growth. MICs for resistant strains were determined at least two times on different days (range of repetitions, 2 to 11).

Statistical calculations.

Statistical analysis was done with the Statistical Package for the Social Sciences (version 15.0; SPSS S.L). Both on-scale and off-scale results were included in the analysis. The off-scale MICs were converted to the next concentration up or down.

Table 1 shows the geometric mean (GM) MICs and the ranges obtained at 24 and 48 h for susceptible and itraconazole-resistant isolates. For all itraconazole-resistant isolates, the GM MIC of itraconazole was 16 mg/liter at 48 h. However, at 24 h the GM MIC was 5.11 mg/liter, and itraconazole MICs for some strains were as low as 0.25 mg/liter. This result was not isolate dependent but rather due to the poor reproducibility of MICs read at 24 h for itraconazole-resistant strains. As the testing of all isolates was repeated at least two times, the analyses of the MICs obtained at 24 h showed that MICs for 17 (51.5%) of 33 itraconazole-resistant strains were read as ≤2 mg/liter at least once. For susceptible strains, the 24-h GM was 0.16 mg/liter and the 48-h GM was 0.33 mg/liter (Table 1). For voriconazole, ravuconazole, and posaconazole, the differences were less striking, and GMs showed one twofold-dilution difference between 24- and 48-h readings (Table 1) for all strains tested. As exceptions, there was one isolate not growing at 24 h and MICs of posaconazole for two further isolates were shown to be 2 mg/liter at 24 h and 16 mg/liter at 48 h.

View this table:
  • View inline
  • View popup
TABLE 1.

GM MICs and ranges of MICs (mg/liter) obtained at 24 and 48 h for azole drug-susceptible and -resistant A. fumigatus isolates

In this study, the MICs for a large collection of susceptible and itraconazole-resistant strains of A. fumigatus have been analyzed and the results obtained at 24 and 48 h have been compared. Several articles have associated poor outcomes for patients with infections caused by isolates or species for which MICs are high (9, 11, 17, 20). Therefore, the quick detection of resistant isolates can influence the outcome for the patient, allowing a prompt change of the antifungal treatment. The growth of most clinical isolates of A. fumigatus is apparently adequate at 24 h in antifungal susceptibility testing microplates with RPMI 1640-2% glucose. Furthermore, we have used an inoculum size of 2 × 105 to 5 × 105 CFU/ml, 10-fold larger than the one recommended by CLSI document M38-A. This inoculum provided better growth of A. fumigatus than smaller inocula, making the visual estimation of the end point straightforward. Our results do no support reading end points at 24 h because MICs for 51.5% of the itraconazole-resistant strains were determined to be ≤2 mg/liter at least once at that time point. As itraconazole MICs for some isolates were as high as 16 mg/liter at 24 h, one may consider the possibility of accepting the results for these isolates at 24 h. However, we detected such high MICs of itraconazole for two isolates at 24 h but posaconazole MICs of 2 mg/liter at 24 h and 16 mg/liter at 48 h.

In summary, the detection of resistant strains of A. fumigatus by means of EUCAST-standardized antifungal susceptibility testing for molds requires 48 h of incubation. Too many false-negative results for resistant strains were detected at 24 h, making MIC reading at this time point unreliable.

ACKNOWLEDGMENTS

This work was funded in part by grants MPY1175/06 and PI05/32 from the Instituto de Salud Carlos III and SAF2005-06541 from the Ministerio de Educacion y Ciencia and by the Spanish Network for Research in Infectious Diseases (REIPI RD06/0008). L. Alcazar-Fuoli holds a postdoctoral contract from the EU-STREP project (LSHM-CT-2005-518199). A. Alastruey-Izquierdo holds a predoctoral fellowship from Fondo de Investigaciones Sanitarias (grant FI05/00856).

FOOTNOTES

    • Received 19 July 2007.
    • Returned for modification 6 September 2007.
    • Accepted 2 October 2007.
  • Copyright © 2007 American Society for Microbiology

REFERENCES

  1. 1.↵
    Aberkane, A., M. Cuenca-Estrella, A. Gomez-Lopez, E. Petrikkou, E. Mellado, A. Monzon, and J. L. Rodriguez-Tudela. 2002. Comparative evaluation of two different methods of inoculum preparation for antifungal susceptibility testing of filamentous fungi. J. Antimicrob. Chemother.50:719-722.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Chen, J., H. Li, R. Li, D. Bu, and Z. Wan. 2005. Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma. J. Antimicrob. Chemother.55:31-37.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.
    Dannaoui, E., E. Borel, M. F. Monier, M. A. Piens, S. Picot, and F. Persat. 2001. Acquired itraconazole resistance in Aspergillus fumigatus. J. Antimicrob. Chemother.47:333-340.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.
    Dannaoui, E., D. Garcia-Hermoso, J. M. Naccache, I. Meneau, D. Sanglard, C. Bouges-Michel, D. Valeyre, and O. Lortholary. 2006. Use of voriconazole in a patient with aspergilloma caused by an itraconazole-resistant strain of Aspergillus fumigatus. J. Med. Microbiol.55:1457-1459.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    Diaz-Guerra, T. M., E. Mellado, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2003. A point mutation in the 14α-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother.47:1120-1124.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Espinel-Ingroff, A., M. Bartlett, V. Chaturvedi, M. Ghannoum, K. C. Hazen, M. A. Pfaller, M. Rinaldi, and T. J. Walsh. 2001. Optimal susceptibility testing conditions for detection of azole resistance in Aspergillus spp.: NCCLS collaborative evaluation. Antimicrob. Agents Chemother.45:1828-1835.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Gomez-Lopez, A., A. Aberkane, E. Petrikkou, E. Mellado, J. L. Rodriguez-Tudela, and M. Cuenca-Estrella. 2005. Analysis of the influence of Tween concentration, inoculum size, assay medium, and reading time on susceptibility testing of Aspergillus spp. J. Clin. Microbiol.43:1251-1255.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    Howard, S. J., I. Webster, C. B. Moore, R. E. Gardiner, S. Park, D. S. Perlin, and D. W. Denning. 2006. Multi-azole resistance in Aspergillus fumigatus. Int. J. Antimicrob. Agents28:450-453.
    OpenUrlCrossRefPubMed
  9. 9.↵
    Kontoyiannis, D. P., G. P. Bodey, H. Hanna, R. Hachem, M. Boktour, E. Girgaway, M. Mardani, and I. I. Raad. 2004. Outcome determinants of fusariosis in a tertiary care cancer center: the impact of neutrophil recovery. Leuk. Lymphoma45:139-141.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    Lass-Florl, C., M. Cuenca-Estrella, D. W. Denning, and J. L. Rodriguez-Tudela. 2006. Antifungal susceptibility testing in Aspergillus spp. according to EUCAST methodology. Med. Mycol.44:S319-S325.
    OpenUrlCrossRef
  11. 11.↵
    Lass-Florl, C., K. Griff, A. Mayr, A. Petzer, G. Gastl, H. Bonatti, M. Freund, G. Kropshofer, M. P. Dierich, and D. Nachbaur. 2005. Epidemiology and outcome of infections due to Aspergillus terreus: 10-year single centre experience. Br. J. Haematol.131:201-207.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    Mellado, E., T. M. Diaz-Guerra, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2001. Identification of two different 14-α sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species. J. Clin. Microbiol.39:2431-2438.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    Mellado, E., G. Garcia-Effron, L. Alcazar-Fuoli, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2004. Substitutions at methionine 220 in the 14α-sterol demethylase (Cyp51A) of Aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. Antimicrob. Agents Chemother.48:2747-2750.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    Mellado, E., G. Garcia-Effron, L. Alcazar-Fuoli, W. J. G. Melchers, P. E. Verweij, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2007. A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob. Agents Chemother.51:1897-1904.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Mellado, E., G. Garcia-Effron, M. J. Buitrago, L. Alcazar-Fuoli, M. Cuenca-Estrella, and J. L. Rodriguez-Tudela. 2005. Targeted gene disruption of the 14-α sterol demethylase (cyp51A) in Aspergillus fumigatus and its role in azole drug susceptibility. Antimicrob. Agents Chemother.49:2536-2538.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. National Committee for Clinical Laboratory Standards, Wayne, PA.
  17. 17.↵
    Perfect, J. R., K. A. Marr, T. J. Walsh, R. N. Greenberg, B. Dupont, J. Torre-Cisneros, G. Just-Nubling, H. T. Schlamm, I. Lutsar, A. Espinel-Ingroff, and E. Johnson. 2003. Voriconazole treatment for less-common, emerging, or refractory fungal infections. Clin. Infect. Dis.36:1122-1131.
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    Petrikkou, E., J. L. Rodriguez-Tudela, M. Cuenca-Estrella, A. Gomez, A. Molleja, and E. Mellado. 2001. Inoculum standardization for antifungal susceptibility testing of filamentous fungi pathogenic for humans. J. Clin. Microbiol.39:1345-1347.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Rodriguez-Tudela, J. L., E. Chryssanthou, E. Petrikkou, J. Mosquera, D. W. Denning, and M. Cuenca-Estrella. 2003. Interlaboratory evaluation of hematocytometer method of inoculum preparation for testing antifungal susceptibilities of filamentous fungi. J. Clin. Microbiol.41:5236-5237.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Steinbach, W. J., J. R. Perfect, W. A. Schell, T. J. Walsh, and D. K. Benjamin, Jr. 2004. In vitro analyses, animal models, and 60 clinical cases of invasive Aspergillus terreus infection. Antimicrob. Agents Chemother.48:3217-3225.
    OpenUrlFREE Full Text
  21. 21.↵
    Trama, J. P., E. Mordechai, and M. E. Adelson. 2005. Detection of Aspergillus fumigatus and a mutation that confers reduced susceptibility to itraconazole and posaconazole by real-time PCR and pyrosequencing. J. Clin. Microbiol.43:906-908.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Time of Incubation for Antifungal Susceptibility Testing of Aspergillus fumigatus: Can MIC Values Be Obtained at 24 Hours?
Juan Luis Rodriguez-Tudela, Laura Alcazar-Fuoli, Ana Alastruey-Izquierdo, Araceli Monzon, Emilia Mellado, Manuel Cuenca-Estrella
Antimicrobial Agents and Chemotherapy Nov 2007, 51 (12) 4502-4504; DOI: 10.1128/AAC.00933-07

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Antimicrobial Agents and Chemotherapy article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Time of Incubation for Antifungal Susceptibility Testing of Aspergillus fumigatus: Can MIC Values Be Obtained at 24 Hours?
(Your Name) has forwarded a page to you from Antimicrobial Agents and Chemotherapy
(Your Name) thought you would be interested in this article in Antimicrobial Agents and Chemotherapy.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Time of Incubation for Antifungal Susceptibility Testing of Aspergillus fumigatus: Can MIC Values Be Obtained at 24 Hours?
Juan Luis Rodriguez-Tudela, Laura Alcazar-Fuoli, Ana Alastruey-Izquierdo, Araceli Monzon, Emilia Mellado, Manuel Cuenca-Estrella
Antimicrobial Agents and Chemotherapy Nov 2007, 51 (12) 4502-4504; DOI: 10.1128/AAC.00933-07
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • Microorganisms.
    • Antifungal susceptibility testing.
    • Statistical calculations.
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

antifungal agents
Aspergillus fumigatus
microbial sensitivity tests

Related Articles

Cited By...

About

  • About AAC
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • AAC Podcast
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AACJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0066-4804; Online ISSN: 1098-6596