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Mycology

Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin

A. Espinel-Ingroff, B. Arthington-Skaggs, N. Iqbal, D. Ellis, M. A. Pfaller, S. Messer, M. Rinaldi, A. Fothergill, D. L. Gibbs, A. Wang
A. Espinel-Ingroff
1Virginia Commonwealth University Medical Center, Richmond, Virginia
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  • For correspondence: avingrof@vcu.edu
B. Arthington-Skaggs
2Centers for Disease Control, Atlanta, Georgia
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N. Iqbal
2Centers for Disease Control, Atlanta, Georgia
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D. Ellis
3Women's and Children's Hospital, North Adelaide, South Australia, Australia
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M. A. Pfaller
4University of Iowa, Iowa City, Iowa
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S. Messer
4University of Iowa, Iowa City, Iowa
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M. Rinaldi
5University of Texas, San Antonio, Texas
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A. Fothergill
5University of Texas, San Antonio, Texas
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D. L. Gibbs
6Giles Scientific Inc., Santa Barbara, California
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A. Wang
6Giles Scientific Inc., Santa Barbara, California
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DOI: 10.1128/JCM.00134-07
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  • FIG. 1.
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    FIG. 1.

    Interference of MGM agar with voriconazole disks for A. terreus (A) and with caspofungin disks for P. lilacinus (B), where low MICs corresponded to very small or no inhibition zone diameters. These conflicting results were not observed on MH agar.

  • FIG. 2.
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    FIG. 2.

    Correlation of broth microdilution MICs and posaconazole 5-μg disk diffusion zone diameters on MH agar for 483 mold isolates in three laboratories. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 h and 48 h). ZD, zone diameter.

  • FIG. 3.
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    FIG. 3.

    Correlation of broth microdilution MICs and itraconazole 10-μg disk diffusion zone diameters on MH agar for 483 mold isolates in three laboratories. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 h and 48 h). ZD, zone diameter.

  • FIG. 4.
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    FIG. 4.

    Correlation of broth microdilution MICs and voriconazole 1-μg disk diffusion zone diameters on MH agar for 483 mold isolates in three laboratories. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 h and 48 h). ZD, zone diameter.

  • FIG. 5.
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    FIG. 5.

    Correlation of broth microdilution MECs and caspofungin 5-μg disk (BBL) diffusion zone diameters on MH agar for 483 mold isolates in three laboratories. MECs were determined at the recommended times (24 to 48 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 h and 48 h). ZD, zone diameter.

  • FIG. 6.
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    FIG. 6.

    Correlation of broth microdilution MICs and amphotericin B 10-μg disk diffusion zone diameters on MH agar for 483 mold isolates in three laboratories. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 h and 48 h). ZD, zone diameter.

Tables

  • Figures
  • TABLE 1.

    Species and numbers of mold isolates tested at three of five centers

    SpeciesNo. of isolates
    Absidia corymbifera 15
    Alternaria sp.23
    Bipolaris spicifera 18
    Aspergillus fumigatus 56
    A. flavus 38
    A. niger 38
    A. terreus 40
    A. nidulans 31
    Fusarium solani 29
    F. moniliforme 28
    F. oxysporum 20
    Mucor circinelloides 17
    Mucor ramosissimus 5
    Rhizopus arrhizus 37
    Rhizopus microsporus var. rhizopodiformis10
    Paecilomyces lilacinus 24
    Scedosporium apiospermum 28
    S. prolificans 26
        Total483a
    • ↵ a Another set of 72 isolates was tested at another two centers to obtain reproducibility data.

  • TABLE 2.

    Reproducibility of inhibition zone diameters, in millimeters, obtained by the disk diffusion method with five antifungal agents for 72 mold isolates at two of the five centersa

    Antifungal agent and mediumb% within 2-SD zone at:
    24 h48 h72 h
    Itra
        MH969188
        MGM878282
    Posa
        MH979592
        MGM969694
    Vori-1
        MH979493
        MGM979590
    Vori-10
        MH929392
        MGM929392
    Cas-BBL
        MH1009695
        MGM969487
    Cas-Oxoid
        MH1009797
        MGM10010097
    Amb
        MH959595
        MGM1009591
    • ↵ a Percentages of replicate zone diameters within 2 standard deviations of the mean; each of the 72 isolates was tested on 3 different days in two of the five laboratories with each antifungal agent. Inhibition zone diameters, in millimeters, on both MH and MGM agar were read at 16 to 24, 48, and 72 h.

    • ↵ b Itra, itraconazole 10-μg disk; Posa, posaconazole 5-μg disk; Cas-BBL and Cas-Oxoid, caspofungin 5-μg disks from two sources; Vori-1 and Vori-10, voriconazole 1-μg and 10-μg disks, respectively; Amb, amphotericin B 10-μg disk.

  • TABLE 3.

    Inhibition zone diameters obtained on MH agar by the disk diffusion method for two QC isolatesa

    QC isolateb and antifungal agentZone range in millimeters (% agreement) at:
    24 h48 h72 h
    Paecilomyces variotii ATCC 3630
        Itra25-31 (99)23-31 (94)24-31 (97)
        Posa35-46 (100)37-45 (97)37-44 (97)
        Vori-140-53 (94)41-53 (94)41-53 (100)
        Vori-1056-64 (97)60-71 (86)61-73 (89)
        Cas-BBL23-29 (100)23-30 (97)24-30 (97)
        Cas-Oxoid23-34 (100)23-34 (97)22-33 (89)
        Amb13-22 (94)9-19 (83)10-19 (89)
    Candida krusei ATCC 6258
        Itra21-30 (100)20-29 (100)20-28 (100)
        Posa31-43 (97)30-41 (97)30-41 (97)
        Vori-129-38 (100)25-35 (100)24-36 (97)
        Vori-1047-59 (92)46-60 (97)53-61 (92)
        Cas-BBL14-24 (100)18-27 (97)17-25 (97)
        Cas-Oxoid21-29 (100)21-30 (100)21-32 (100)
        Amb19-27 (100)15-25 (97)13-25 (97)
    • ↵ a Tests were performed 5 to 16 times for each isolate in each of the five laboratories for each disk and incubation time on MH agar.

    • ↵ b Itra, itraconazole 10-μg disk; Posa, posaconazole 5-μg disk; Cas-BBL and Cas-Oxoid, caspofungin 5-μg disks from two sources; Vori-1 and Vori-10, voriconazole 1-μg and 10-μg disks, respectively; Amb, amphotericin B 10-μg disk.

  • TABLE 4.

    Correlation between inhibition zone diameters (in millimeters) obtained by the disk diffusion method and reference M38-A MICs (amphotericin B and triazoles) and MECs (caspofungin) for 483 mold isolates at three of the five centersa

    Antifungal agent and incubation time (h)bCorrelation coefficient (R value)c for:
    MHMGM
    Itra
        240.79ND
        480.750.58
        720.740.67
    Posa
        240.77ND
        480.710.65
        720.710.71
    Vori-1
        240.85ND
        480.830.87
        720.820.53
    Vori-10
        240.83ND
        480.800.85
        720.830.58
    Cas-BBL
        240.92ND
        480.880.96
        720.900.87
    Cas-Oxoid
        240.93ND
        480.890.88
        720.900.87
    Amb
        240.68ND
        480.430.32
        720.310.54
    • ↵ a MICs were read at 24 h (zygomycetes), 72 h (Scedosporium sp.), and 48 h (other species) as recommended in the CLSI M38-A document. Caspofungin MECs were read at 24 h (all species except for Scedosporium sp., which were read at 48 h). Each MIC or MEC was plotted against inhibition zone diameters in millimeters obtained on both MH and MGM agars at each of the three incubation times; results from three centers were combined for linear regression analysis.

    • ↵ b Itra, itraconazole 10-μg disk; Posa, posaconazole 5-μg disk; Cas-BBL and Cas-Oxoid, caspofungin 5-μg disks from two sources; Vori-1 and Vori-10, voriconazole 1-μg and 10-μg disks, respectively; Amb, amphotericin B 10-μg disk.

    • ↵ c ND, not determined. The 48-h results for MGM medium encompassed 24- and 48-h zones of inhibition; 71% of the 24-h results were reported as no growth.

  • TABLE 5.

    Agreement of inhibition zone diameters (disk diffusion method) with MICs or MECs (M38-A method) according to assigned susceptible, intermediate, and resistant categories for 483 mold isolates with five antifungal agents as obtained in three laboratories

    Antifungal agent, method, and mediuma% of MICs by interpretive categoryb% Error% Agreementc
    SIRMinorMajorVery major
    Posaconazole
        BMD78913
        Disk, 5 μg834138.10.20.292
    Itraconazole
        BMD541135
        Disk, 10 μg5284012.82.51.883
    Voriconazole
        BMD59932
        Disk, 1 μg561439.74.60.286
    Caspofungin
        BMD51346
        Disk, 5 μg, BBL501493.310.296
    Amphotericin B
        BMD492427
        Disk, 10 μg58123023.12.71.773
    • ↵ a BMD, MICs and MECs, in micrograms per milliliter, were determined at the recommended incubation times (24 to 48 h for caspofungin and 24 to 72 h for the other agents) by the CLSI M38-A broth microdilution method. Disk inhibition zone diameters, in millimeters, were determined with the optimal medium (MH agar) and incubation times (16 to 24 h and 48 h) by the disk diffusion method.

    • ↵ b Percentages of MICs or MECs and inhibition zone diameters, in millimeters, that were within the assigned posaconazole, itraconazole, voriconazole, and caspofungin categories (S [susceptible], MIC or MEC of ≤1 μg/ml, ≥17 mm; I [intermediate], MIC or MEC of 2 μg/ml, 14 to 16 mm; R [resistant], MIC or MEC of ≥4 μg/ml, ≤13 mm) and amphotericin B MIC and zone diameter categories (susceptible, MIC of ≤1 μg/ml, >15 mm; intermediate, MIC of 2 μg/ml, 13 to 14 mm; resistant, MIC of ≥4 μg/ml, 12 mm).

    • ↵ c Percent categorical agreement between inhibition zone diameters, in millimeters, and MIC or MEC results.

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Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin
A. Espinel-Ingroff, B. Arthington-Skaggs, N. Iqbal, D. Ellis, M. A. Pfaller, S. Messer, M. Rinaldi, A. Fothergill, D. L. Gibbs, A. Wang
Journal of Clinical Microbiology Jun 2007, 45 (6) 1811-1820; DOI: 10.1128/JCM.00134-07

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Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin
A. Espinel-Ingroff, B. Arthington-Skaggs, N. Iqbal, D. Ellis, M. A. Pfaller, S. Messer, M. Rinaldi, A. Fothergill, D. L. Gibbs, A. Wang
Journal of Clinical Microbiology Jun 2007, 45 (6) 1811-1820; DOI: 10.1128/JCM.00134-07
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    • ABSTRACT
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KEYWORDS

antifungal agents
Disk Diffusion Antimicrobial Tests
fungi

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