Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin
Open accessAbstract
The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera , Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera , Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus , Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 μg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-μg disks, voriconazole 1- and 10-μg disks, two sources of caspofungin 5-μg disks [BBL and Oxoid], and posaconazole 5-μg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R , 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results, the optimal testing conditions for mold disk diffusion testing were (i) plain MH agar; (ii) incubation times of 16 to 24 h (zygomycetes), 24 h ( Aspergillus fumigatus , A. flavus , and A. niger ), and 48 h (other species); and (iii) the posaconazole 5-μg disk, voriconazole 1-μg disk, itraconazole 10-μg disk (for all except zygomycetes), BBL caspofungin 5-μg disk, and amphotericin B 10-μg (zygomycetes only).
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The study design followed CLSI M23-A3 requirements for establishing QC ranges and CLSI M51-A guidelines for agar diffusion testing of molds, except that only one disk lot was available for each antifungal agent instead of the recommended two lots from different manufacturers.
“The design followed the requirements described in the CLSI M23-A3 document for establishing QC ranges (with only one exception, as noted below) (3) and CLSI M51-A guidelines for agar diffusion testing of molds (5, 9); only one disk lot was available for each of the four antifungal agents instead of the recommended two lots from different manufacturers.”
From Quality Control Guidelines for Amphotericin B, Itraconazole, Posaconazole, and Voriconazole Disk Diffusion Susceptibility Tests with Nonsupplemented Mueller-Hinton Agar (CLSI M51-A Document) for Nondermatophyte Filamentous Fungi (2011) · cites this paper as [9]
The introduction says reference guidelines were not available for mold disk diffusion testing, while Materials and Methods says the study protocol used CLSI M38-A broth microdilution conditions and evaluated disk diffusion parameters. The study tested five antifungal agents, not four, and used caspofungin disks from two manufacturers. Our copy does not describe following M23-A3 or M51-A or an exception to a two-lot requirement. (LLM, conf=0.90)
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Experimental — accuracy not yet measuredIn a prior study, the zone diameters for P. variotii were narrower at 24 h than at 48 h with itraconazole, posaconazole, and amphotericin B, but the ranges were wider at both incubation times when the voriconazole disk was tested.
“In a study that identified testing conditions for disk diffusion testing of nondermatophyte molds (9), the zone diameters were narrower for the P. variotii isolate at 24 h than at 48 h with itraconazole, posaconazole, and amphotericin B (7- to 12-mm versus 9- to 12-mm intervals), but the ranges were wider at both incubation times when the voriconazole disk was tested (13- to 14-mm intervals).”
From Quality Control Guidelines for Amphotericin B, Itraconazole, Posaconazole, and Voriconazole Disk Diffusion Susceptibility Tests with Nonsupplemented Mueller-Hinton Agar (CLSI M51-A Document) for Nondermatophyte Filamentous Fungi (2011) · cites this paper as [9]
Table 3 (Materials and Methods; page unavailable) reports P. variotii zone ranges at 24 and 48 h. The intervals widen for itraconazole and amphotericin B, but narrow for posaconazole (35–46 mm at 24 h versus 37–45 mm at 48 h), contrary to that part of the claim. The voriconazole 1-µg disk has comparatively wide intervals at both times (40–53 and 41–53 mm). (LLM, conf=0.90)
Experimental — accuracy not yet measuredData for C. krusei ATCC 6258 and P. variotii ATCC MYA-3630 were not collected under M23-A3 guidelines for the selection of QC isolates.
“data for these two isolates were not collected under M23-A3 guidelines for the selection of QC isolates (3, 9).”
From Quality Control Guidelines for Amphotericin B, Itraconazole, Posaconazole, and Voriconazole Disk Diffusion Susceptibility Tests with Nonsupplemented Mueller-Hinton Agar (CLSI M51-A Document) for Nondermatophyte Filamentous Fungi (2011) · cites this paper as [9]
Materials and Methods describes data collection for both QC isolates across five centers, and Results and Discussion reports their zone diameters. Neither section says whether the data were collected under M23-A3 guidelines for selecting QC isolates; searches for M23-A3 and QC-isolate selection found no passage that resolves this. (LLM, conf=0.72)
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Cites
Sabatelli, F., Patel, R., Mann, P. A., Mendrick, C. A., Norris, C. C., Hare, R., Loebenberg, D., Black, T. A., & McNicholas, P. M. (2006) · Antimicrobial Agents and Chemotherapy · doi:10.1128/aac.00163-06 · Cited by 491 · see citing works
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Research works printed but not linked: 1
- [12]12. Espinel-Ingroff, A., K. Boyle, and D. J. Sheehan. 2001. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 38: 293-304.
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- Authors
- Espinel-Ingroff, A., Arthington-Skaggs, B., Iqbal, N., Ellis, D., Pfaller, M. A., Messer, S., Rinaldi, M., Fothergill, A., Gibbs, D. L., & Wang, A.
- Affiliations
- Virginia Commonwealth University Medical Center, Richmond, Virginia
- Centers for Disease Control, Atlanta, Georgia
- Centers for Disease Control and Prevention (US)
- Women's and Children's Hospital (AU)
- University of Iowa (US)
- The University of Texas at San Antonio (US)
- Giles Scientific Inc., Santa Barbara, California
- Venue
- Journal of Clinical Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2007-06
- Volume
- 45
- Issue
- 6
- Pages
- 1811-1820
- ISSN
- 0095-1137 (ISSN-L)
- 1098-660X
- PMC
- PMC1933077
- OpenAlex
- W2133798991
- Cited by
- 182
- References
- 1
- Citations by year
- 2026: 13 · 2025: 17 · 2024: 14 · 2023: 18 · 2022: 10 · 2021: 7 · 2020: 6 · 2019: 8 · 2018: 3 · 2017: 3 · 2016: 6 · 2015: 10 · 2014: 5 · 2013: 6 · 2012: 7
- FWCI
- 8.18
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Clinical Microbiology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Adelaide Research & Scholarship (AR&S) (University of Adelaide) (submittedVersion)
- Funders
- Pfizer
- Topics
- Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Bacterial Identification and Susceptibility Testing
- Health Sciences › Medicine › Epidemiology › Fungal Infections and Studies
- Keywords
- antifungal susceptibility testing, filamentous fungi, voriconazole, posaconazole, itraconazole, amphotericin B, caspofungin, minimum effective concentration, inhibition zone diameter, Mueller-Hinton agar, incubation time, Zygomycetes, Aspergillus, Scedosporium, Paecilomyces lilacinus
- MeSH terms
- Caspofungin, Amphotericin B, Antifungal Agents, Culture Media, Fungi, Microbial Sensitivity Tests, Peptides, Cyclic, Pyrimidines, Triazoles, Reproducibility of Results, Itraconazole, Disk Diffusion Antimicrobial Tests, Echinocandins, Lipopeptides, Voriconazole
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