Quality Control Guidelines for Amphotericin B, Itraconazole ...

JOURNAL OF CLINICAL MICROBIOLOGY, July 2011, p. 2568–2571 0095-1137/11/$12.00 doi:10.1128/JCM.00393-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 49, No. 7

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䌤 A. Espinel-Ingroff,1* E. Canton,2 A. Fothergill,3 M. Ghannoum,4 E. Johnson,5 R. N. Jones,6 L. Ostrosky-Zeichner,7 W. Schell,8 D. L. Gibbs,9 A. Wang,9 and J. Turnidge10,11 VCU Medical Center, Richmond, Virginia1; HUP La Fe, Research Center, Valencia, Spain2; University of Texas Health Science Center, San Antonio, Texas3; Case Western Reserve University, Cleveland, Ohio4; The HPA Centre for Infections, Kingsdown, Bristol, United Kingdom5; JMI Laboratories, North Liberty, Iowa6; University of Texas-Houston Medical School, Houston, Texas7; Duke University Medical Center, Durham, North Carolina8; Giles Scientific, Inc., Santa Barbara, California9; Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia10; and University of Adelaide, Adelaide, South Australia 5005, Australia11 Received 23 February 2011/Returned for modification 11 April 2011/Accepted 22 April 2011

Although Clinical and Laboratory Standards Institute (CLSI) disk diffusion assay standard conditions are available for susceptibility testing of filamentous fungi (molds) to antifungal agents, quality control (QC) disk diffusion zone diameter ranges have not been established. This multicenter study documented the reproducibility of tests for one isolate each of five molds (Paecilomyces variotii ATCC MYA-3630, Aspergillus fumigatus ATCC MYA-3626, A. flavus ATCC MYA-3631, A. terreus ATCC MYA-3633, and Fusarium verticillioides [moniliforme] ATCC MYA-3629) and Candida krusei ATCC 6258 by the CLSI disk diffusion method (M51-A document). The zone diameter ranges for selected QC isolates were as follows: P. variotii ATCC MYA-3630, amphotericin B (15 to 24 mm), itraconazole (20 to 31 mm), and posaconazole (33 to 43 mm); A. fumigatus ATCC MYA-3626, amphotericin B (18 to 25 mm), itraconazole (11 to 21 mm), posaconazole (28 to 35 mm), and voriconazole (25 to 33 mm); and C. krusei, amphotericin B (18 to 27 mm), itraconazole (18 to 26 mm), posaconazole (28 to 38 mm), and voriconazole (29 to 39 mm). Due to low testing reproducibility, zone diameter ranges were not proposed for the other three molds. The Clinical and Laboratory Standards Institute (CLSI) has developed a reference method for disk diffusion antifungal susceptibility testing of nondermatophyte filamentous fungi (molds; CLSI documents M51-A and M51-S1) (5, 6). These documents describe guidelines for testing the susceptibility of opportunistic molds to triazoles, amphotericin B, and caspofungin. However, the M51 documents do not describe quality control (QC) parameters for any agent-isolate combination (5, 6). For QC purposes, two reference isolates (Candida krusei ATCC 6258 and Paecilomyces variotii ATCC MYA-3630) have been listed (6); data for these two isolates were not collected under M23-A3 guidelines for the selection of QC isolates (3, 9). The purpose of the present multicenter study (eight centers) was to select QC isolates among five molds and one yeast strain and to establish zone diameter limits (in millimeters) for disk diffusion tests of amphotericin B, itraconazole, posaconazole, and voriconazole.

MATERIALS AND METHODS Study design. 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. The inter- and intralaboratory reproducibility of 10 replicate tests performed for each drug and strain combination were determined by the CLSI M51-A disk diffusion method in each laboratory on 10 different days and between three separate batches of nonsupplemented Mueller-Hinton (MH) agars. The same coded set of five molds and the C. krusei ATCC 6258 isolate were sent to each laboratory. Each isolate was tested in each laboratory with amphotericin B, itraconazole, posaconazole and voriconazole following a standard protocol (3, 5) that included the description of the reading conditions to be evaluated at both 24 (Aspergillus fumigatus, A. flavus, P. variotii, and C. krusei) and 48 h (A. terreus and Fusarium verticillioides) (3, 5, 9). Isolates. The set of isolates included A. flavus ATCC MYA 3631, A. fumigatus ATCC MYA-3626, A. terreus ATCC MYA-3633, Fusarium verticillioides (moniliforme) ATCC MYA-3629, P. variotii ATCC MYA-3630, and C. krusei ATCC 6258. Antifungal susceptibility testing. The five molds and the C. krusei isolate were tested by the M51-A disk diffusion method in each center with the three lots of nonsupplemented MH agar (lots 09314, 09315, and 09316B; Hardy Diagnostics, Santa Maria, CA) on 10 different working days (3). A total of 240 zone diameters were determined for each isolate-drug combination. Briefly, mold stock inoculum suspensions were prepared from 7-day cultures grown on potato dextrose agar (Remel, Lenexa, KS) and adjusted spectrophotometrically to optical densities that ranged from 0.09 to 0.11 (2, 5, 9). The inoculum suspension of the QC yeast isolate was prepared from 24 h cultures grown on Sabouraud dextrose agar (Hardy Diagnostics) and adjusted by the spectrophotometer to the density of a 0.5 McFarland standard. The entire surface of each 150-mm nonsupplemented MA agar plate was

* Corresponding author. Mailing address: VCU Medical Center, 3804 Dover Rd., Richmond, VA 23221. Phone: (804) 358-5895. Fax: (804) 828-3097. E-mail: [email protected]. 䌤 Published ahead of print on 4 May 2011. 2568

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TABLE 1. Results of replicate tests in eight laboratories using 10-␮g amphotericin B disks on MH agara

TABLE 2. Results of replicate tests in eight laboratories using 10-␮g itraconazole disks on MH agara

No. of times each zone diam was recordedb Zone diam (mm)

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

P. variotii ATCC MYA-3630

2 10 11 18 42 37 关 28 33 35 10 10 3 1

C. krusei ATCC 6258

No. of times each zone diam was recordedb

A. fumigatus ATCC MYA-3626

Zone diam (mm)

No zone 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

1

兴

1 1 6 38 30 关 63 48 22 18 5 2

兴

8 18 45 49 关 69 23 11 10 4 2

2569

兴

4 2

a

The MH agar was not supplemented. Horizontal lines designate the proposed QC limits for each strain, and the brackets indicate the overall mean zone diameter for each data set. The QC zone diameter limits are based on 10 replicates of one disk on each of three lots of MH agar in eight laboratories per strain. The QC limits proposed (percent included) for each strain were as follows: P. variotii ATCC MYA-3630, 15 to 24 mm (97.5%); C. krusei ATCC 6258, 18 to 27 mm (97.1%); and A. fumigatus ATCC MYA-3626, 18 to 25 mm (97.1%). The percent values indicate the number of zone diameters that fell within the proposed QC limits, as calculated for the data from all eight laboratories. b

inoculated with the undiluted inoculum suspension. Disks of amphotericin B and itraconazole (10 ␮g of lots DB11 and JG4, respectively; Abtek Biologicals, Liverpool, United Kingdom), posaconazole (5 ␮g of lot 91115641; BD Diagnostics, Sparks, MD) and voriconazole (1 ␮g of lot 9070731; BD Diagnostics) were placed onto the surface of each lot of inoculated MH agar plate no closer than 32 mm from center to center when amphotericin B and itraconazole were tested and no closer than 55 mm from center to center when posaconazole and voriconazole were tested (5). The plates were incubated for 24 to 48 h at 35°C. Colony counts were performed in each center for at least one of the inoculum suspensions prepared for each isolate on the 10 testing days; for the five molds and the yeast isolate, ⬎99% of the inocula were within the target ranges (0.4 ⫻ 106 to 5 ⫻ 106 CFU/ml [molds] and 1 ⫻ 106 to 5 ⫻ 106 CFU/ml[C. krusei]) or similar reproducibility as in previous collaborative studies (92 to 95%) (1, 2, 7, 8). Zone diameters were determined by visual examination to the nearest whole millimeter at the point at which there was 80 to 100% growth inhibition; slight trailing growth around the inhibition zone edges and hyphal elements extending into the zone were ignored when the triazoles were tested but not when amphotericin B was tested (5, 9). Data analysis. From the data gathered, the mean, median, standard deviation, and Gavan et al. statistics were calculated (3, 10); potential outlier results by individual laboratory participants were also identified (11). The initial proposed zone diameter QC ranges were established by calculating the 95% confidence interval around the mean, rounded down and up to nearest millimeter for the lower and upper limit, respectively (11). For initial QC ranges with intervals ⬎ 11 mm, the initial range was then adjusted toward the mean (ⱖ95% of the observed zone diameter data points fell within the adjusted limits); initial ranges that suggested excessive interlaboratory variation were not proposed for that combination of QC strain and drug.

RESULTS AND DISCUSSION The performance of antimicrobial disk diffusion susceptibility testing is monitored by the introduction of at least one QC strain for which zone diameter ranges (in millimeters) of acceptable reproducibility have been established (3). To establish


6 3 14 22 18 43 关 36 24 29 8 25 3 4 3 2

兴

C. krusei ATCC 6258

1 1 16 31 60 关 45 40 27 8 8 1 1 1

兴


1 3 2 21 27 42 关 47 41 21 15 11 7 2

兴

a

The MH agar was not supplemented. Horizontal lines designate the proposed QC limits for each strain, and the brackets indicate the overall mean zone diameter for each data set. The QC zone diameter limits are based on 10 replicates of one disk on each of three lots of MH agar in eight laboratories per strain. The QC limits proposed (percent included) for each strain were as follows: P. variotii ATCC MYA-3630, 20 to 31 mm (96.3%); C. krusei ATCC 6258, 18 to 26 mm (98.3%), and A. fumigatus ATCC MYA-3626, 11 to 21 mm (98.8%). The percent values indicate the number of zone diameters that fell within the proposed QC limits, as calculated for the data from all eight laboratories. b

such QC limits, zone diameters from at least seven independent laboratories from distinct institutions should be included in the final analysis; we were able to include data from the eight laboratories for each of the proposed QC ranges depicted in Tables 1 to 4 except for the combination of posaconazole and P. variotii (3, 10, 11). Our proposed QC zone diameter limits were determined with 96.3 to 100% of the 240 (or 210) observed zone diameter values from the eight (or seven) laboratories. Only one disk of each drug was available at the time of the study, and this continues to be the case at this time. Although there is no agreed standard for a maximum allowable QC range, we chose 12 mm as the maximum allowable range for each set of drug-isolate combination limit. The established ranges for QC zone diameter limits of disk diffusion tests for Candida spp. are ⱕ12 mm (CLSI M44-S3) (4). We found no significant differences between the three lots of MH agar or consistent discrepancies between the laboratories included. Therefore, results were pooled for the analysis, and the chosen QC ranges fell within a ⱕ12-mm interval (3). Tables 1 to 4 specify the zone diameter distributions obtained by the M51-A method with the three triazoles and amphotericin B for three of the six isolates tested (5); the tables also present the proposed QC diameter ranges for these three agent-isolate combinations and provide the corresponding percentages of

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TABLE 3. Results of replicate tests in eight laboratories using 5-␮g posaconazole disks on MH agara

TABLE 4. Results of replicate tests in eight laboratories using 1-␮g voriconazole disks on MH agara

No. of times each zone diam was recordedb Zone diam (mm)

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50


1 1 6 5 20 38 33 关 27 25 24 18 10 2

兴

C. krusei ATCC 6258

3 8 38 27 39 关 28 38 32 19 6 1 1

兴

No. of times each zone diam was recordedb


2 1 4 8 30 56 关 66 48 15 8 2

Zone diam (mm)

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

兴

a

The MH agar was not supplemented. Horizontal lines designate the proposed QC limits for each strain, and the brackets indicate the overall mean zone diameter for each data set. The QC zone diameter limits are based on 10 replicates of one disk on each of three lots of MH agar in eight laboratories per strain, except for the P. variotii isolate (data from one laboratory was dropped). The QC limits proposed (percent included) for each strain were as follows: P. variotii ATCC MYA-3630, 33 to 43 mm (99%); C. krusei ATCC 6258, 28 to 38 mm (99.6%); and A. fumigatus ATCC MYA-3626, 28 to 35 mm (97.9%). The percent values indicate the number of zone diameters that fell within the proposed QC limits, as calculated for the data from all eight laboratories. b

agreement and mean zone diameters. QC zone diameter ranges were proposed with all four antifungal agents evaluated only for the C. krusei ATCC 6258 and A. fumigatus ATCC MYA-3626 strains. However, QC zone diameter ranges are also proposed for P. variotii ATCC MYA-3630 for three of the four agents. 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). Similar results were obtained in the present study with this isolate (17 mm, initial estimated QC interval). Visual inspection of the raw data suggested that one participating laboratory may have been an outlier (11). If data from this laboratory are excluded from the analysis, the calculated interval drops to an unacceptable 13 mm for P. variotii. Therefore, we propose QC zone diameter ranges for P. variotii ATCC MYA-3630 with the other three agents (amphotericin B, itraconazole, and posaconazole; Tables 1 to 4) but not for voriconazole. However, we proposed QC zone diameter ranges

C. krusei ATCC 6258


1

1 5 16 28 35 29 关 34 43 27 12 4 3 3

兴

1 15 3 15 48 关 54 56 29 12 4 2

兴

a

The MH agar was not supplemented. Horizontal lines designate the proposed QC limits for each strain, and the brackets indicate the overall mean zone diameter for each data set. The QC zone diameter limits are based on 10 replicates of one disk on each of three lots of MH agar in eight laboratories per strain. The QC limits proposed (percent included) for each strain were as follows: C. krusei ATCC 6258, 29 to 39 mm (98.3%), and A. fumigatus ATCC MYA-3626, 25-33 mm (98.3%). The percent values indicate the number of zone diameters that fell within the proposed QC limits, as calculated for the data from all eight laboratories. b

for the A. fumigatus ATCC MYA-3626 strain with the four antifungal agents. No QC ranges were recommended for the other three strains evaluated (F. verticillioides ATCC MYA-3629, A. flavus ATCC MYA-3631 and A. terreus ATCC MYA-3633), because some results did not meet the M23-A3 document requirements for QC strain selection or the zone range interval was ⬎12 mm. However, the results were also acceptable with both posaconazole and voriconazole disks and the A. flavus (zone diameter ranges, 27 to 37 mm and 25 to 36 mm; interlaboratory agreement, 99.2 and 97.5%, respectively) and A. terreus (zone diameter ranges, 33 to 41 mm and 23 to 33 mm; interlaboratory agreement, 97.5 and 96.3%, respectively, data not shown in the tables) strains. Our data for the F. verticillioides isolate and posaconazole also provided acceptable reproducibility (zone diameter range, 21 to 32 mm; interlaboratory agreement, 100%). Therefore, we proposed the use of these three isolates and zone diameter ranges as reference strains. In conclusion, this is the first study that has proposed QC and reference zone diameter limits (in millimeters) for the three strains selected as QC isolates (A. fumigatus ATCC MYA-3626, P. variotii ATCC MYA-3630, and C. krusei ATCC 6258) for testing clinically relevant antifungal agents (amphotericin B, itraconazole, posaconazole, and voriconazole) against nondermatophyte molds. These QC ranges would assist in monitoring the performance of in vitro antifungal disk diffusion susceptibility testing by the CLSI M51-A and alternative methods.

VOL. 49, 2011

QC GUIDELINES FOR SUSCEPTIBILITY TESTS FOR MOLDS ACKNOWLEDGMENTS

This study was partially supported by a grant from Pfizer. We thank Schering and BD Diagnostics for donating posaconazole and voriconazole disks, respectively, and Hardy Diagnostics for donating the MH agar plates.

6.

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