JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2003, p. 5270–5272 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.11.5270–5272.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 41, No. 11
Comparison of Etest with Modified Broth Microdilution Method for Testing Susceptibility of Aspergillus spp. to Voriconazole M. C. Serrano,1 D. Morilla,1 A. Valverde,1 M. Cha´vez,1 A. Espinel-Ingroff,2 R. Claro,1 M. Ramírez,1 and E. Martín Mazuelos1* Servicio de Microbiología Clínica, Hospital Universitario de Valme, Seville 41014, Spain,1 and Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 239802 Received 22 January 2003/Returned for modification 30 March 2003/Accepted 21 July 2003
We compared the Etest with a broth microdilution method, performed according to a modified National Committee for Clinical Laboratory Standards guideline (M38-A), for determining the in vitro susceptibility of 77 isolates of Aspergillus spp. (26 A. fumigatus, 21 A. flavus, 10 A. terreus, 9 A. niger, 5 A. nidulellus, 4 A. glaucus, and 2 A. flavipes isolates). Overall, there was 92.2% agreement between both methods when Etest MICs were read at 24 h and 83.1% agreement when both methods were read at 48 h. When Etest MICs were read at 24 h, the agreement was >90% for all species tested except for A. fumigatus (84.6%). When Etest MICs were read at 48 h, the agreement ranged from 50 to 100%. The poorest agreement was seen with A. glaucus (50%) and A. fumigatus (65%). Where a discrepancy was observed between Etest and the reference method, the Etest MIC was generally higher. The Etest appears to be a suitable alternative procedure for testing the susceptibility of Aspergillus spp. to voriconazole. at the Valme University Hospital, Seville, Spain, and the Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University, Richmond. The isolates were stored as a conidial suspension in sterile distilled water with 25% glycerol at ⫺80°C until the study was done. Two quality control isolates (QC), Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019, were included as controls. The MICs for QC were in the established reference MIC ranges described by Barry et al. (4). The broth microdilution method was performed according to modified NCCLS guidelines to compare the MICs of voriconazole with the Etest results, since this agar-based method was performed in RPMI supplemented with 2% glucose (RPG). RPMI 1640 medium (Sigma Chemicals Co., St. Louis, Mo.) supplemented with 2% glucose, buffered with 0.165 M 3-(N-morpholino) propanesulfonic acid (MOPS) (Sigma Chemicals Co., St. Louis, Mo.), and adjusted to pH 7.0 was used throughout the study. The agar version of RPG was obtained by using RPMI 1640 medium (Sigma Chemicals) solidified with 1.5% Bacto agar (Difco Laboratories, Detroit, Mich.). Susceptibility testing was carried out in two different ways as follows. The isolates were subcultured for 3 to 4 days on potato dextrose agar (PDA) (Difco) at 30°C. The microdilution adaptation of the NCCLS M38-A method (16) was performed with twofold dilutions of voriconazole at concentrations ranging from 0.03 to 16 g/ml. The MIC was defined as the lowest drug concentration that showed prominent reduction in growth (approximately 50% or more) compared with growth in the drug-free well. The agar-based Etest was performed in accordance with the manufacturer⬘s instructions. The plates were inoculated with conidial suspensions prepared in sterile saline and adjusted to a concentration of 106 CFU/ml (68 to 82% transmittance at 530 nm) by swabbing the RPG agar in three directions. After excess moisture was absorbed into the agar and the surface was completely dry, Etest strips (voriconazole concentrations rang-
The incidence of invasive aspergillosis has increased considerably in the past few decades. During this period, the number of immunocompromised patients has markedly increased. Many factors have contributed to this increase and include the use of new and more aggressive therapies to treat solid tumors, myelomas, lymphomas, and leukemia; the chronic use of corticosteroids; the increasing number of patients who undergo organ transplant; and, finally, the spread of AIDS (8, 9). These infections are associated with significant morbidity and mortality despite therapy with amphotericin B, which remains the drug of choice in severely immunosuppressed patients (17, 19). Limitations associated with amphotericin B include its high toxicity and the in vitro tolerance or resistance of Aspergillus species, particularly A. terreus (24), so newer antifungal therapies with improved efficacy and reduced toxicity are needed to improve the treatment of invasive aspergillosis (19, 20, 23). Among these, voriconazole is a monotriazolic antifungal agent that has a wide spectrum of activity against yeasts (2–5, 14, 18, 27) and filamentous fungi (1, 7, 12, 13, 21, 22, 25). This azole has been tested extensively in broth-based procedures but has not been widely evaluated by agar-based testing methods (3, 11, 14, 15, 20). In the present study, we have studied Etest for testing the susceptibilities of 77 clinical isolates of Aspergillus spp. to voriconazole and compared these results with those obtained by a modified National Committee for Clinical Laboratory Standards (NCCLS) reference broth microdilution method (document M38-A) (16). There are previous studies that examined the Etest susceptibility of yeast species to voriconazole with excellent results (6). The 77 clinical isolates included 26 A. fumigatus, 21 A. flavus, 10 A. terreus, 9 A. niger, 5 A. nidulellus, 4 A. glaucus, and 2 A. flavipes strains, which were recovered from clinical specimens * Corresponding author. Mailing address: H.U. Valme, Ctra. Ca´diz s/n, Seville 41014, Spain. Phone: 034-955015480. Fax: 034-955015481. E-mail:
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TABLE 1. In vitro susceptibilities of 77 isolates of Aspergillus spp. to voriconazole as determined by the Etest and broth microdilution methods MIC (g/ml)
A. fumigatus (26) A. flavus (21) A. terreus (10) A. niger (9) A. nidulellus (5) A. glaucus (4) A. flavipes (2) Total (77)
E test
NCCLSa
Species (no. of isolates)
24 h
48 h
Range
50/90b
Range
50/90b
Range
50/90b
0.03–0.12 0.03–0.12 0.03–0.25 0.03–0.12 0.03–0.5 0.03–0.12 0.03–0.06 0.03–0.25
0.06/0.12 0.06/0.12 0.12/0.25 0.06/-c 0.12/-d/-/0.06/0.12
0.06–0.5 0.03–0.5 0.06–0.12 0.03–0.06 0.03–0.25 0.06–0.12 0.03–0.06 0.03–0.5
0.12/0.25 0.12–0.25 0.12/0.25 0.06/0.12/-/-/0.12/0.25
0.06–0.5 0.12–0.5 0.12–0.25 0.03–0.25 0.03/-/-/0.03–1
0.25/0.5 0.12/0.5 0.25/0.25 0.12/0.12/-/-/0.25/0.5
a
Modified reference microdilution method MICs read at 48 h of incubation. 50/90, MIC50/90. MIC90s were not calculated, since there were ⬍10 isolates tested. d MIC50s were not calculated since there were ⬍5 isolates tested. b c
ing from 0.002 to 32 g/ml) were applied to the RPG agar. Etest MICs were determined after 24 and 48 h of incubation at 35°C. The Etest MIC was read as the drug concentration at which the border of the elliptical inhibition zone intersected the scale on the antifungal test strip. Etest MIC endpoints were raised to the nearest twofold dilution value that matched the NCCLS concentration ranges to facilitate MIC comparisons by both methods. Discrepancies between MICs of no more than 2 dilutions were used to calculate the percentages of agreement. MIC ranges and MICs at which 50 and 90% of the isolates tested are inhibited (MIC50 and MIC90, respectively) were determined. Table 1 summarizes the in vitro susceptibilities of 77 Aspergillus isolates to voriconazole as determined at 48 h by the modified broth microdilution method and the corresponding 24- and 48-h Etest results. Insufficient growth at 24 h of incubation was observed to determine the endpoints by the microdilution method. Voriconazole showed similar activities for all Aspergillus spp., with MIC50 and MIC90 of 0.06 and 0.12 mg/liter, respectively. These values were similar to those obtained by the 24-h Etest MIC50 and MIC90 (0.12 and 0.25 mg/liter, respectively) and 48-h Etest MIC50 and MIC90 (0.25 and 0.5 mg/liter, respectively). The MIC90 obtained by Etest was 1 or 2 dilutions higher than that by microdilution. We found slightly lower voriconazole MICs for the different Aspergillus species tested than those reported by other authors (10, 13, 21, 25), although similar results have been reported for A. terreus by Sutton et al. (24). Table 2 summarizes the percentages of 24- and 48-h MIC Etest and broth dilution results that were within 2 dilutions. The agreement between the 24-h Etest and the 48-h microdilution MICs ranged from 84.6% for A. fumigatus to 100% for all species tested. However, the agreement between the 48-h Etest and 48-h microdilution was lower (50 to 100%). These data indicate that the Etest may be read earlier (at 24 h) than the broth microdilution method for the most common species of Aspergillus spp. encountered clinically. For A. flavus, voriconazole Etest MICs had a better correlation with reference values after 48 h (100%) than after 24 h (95.2%). Similar
results have been found when the Etest itraconazole results for Aspergillus spp. have been compared with broth dilution results (26). The latter authors also demonstrated that resistance to itraconazole among A. fumigatus and other molds can be detected by the Etest method. Pfaller et al. (19) found that for Aspergillus spp., the agreement between the itraconazole Etest MICs read at 24 h and the reference microdilution MICs read at 48 h was 100% with RPMI and Casitone media. When both tests were read at 48 h, the overall percentages of agreement were lower: 96% with RPMI and 80% with Casitone. In contrast to these results, Espinel-Ingroff found that the agreement between methods for voriconazole was independent of the Etest incubation time (93 to 100%) for four of five species tested at both incubation times and reading the MICs at the lowest drug dilution that showed 100% inhibition of growth (11). In general, the discrepancies between Etest and broth dilution results were due to higher Etest MICs; similar discrepancies have been documented with itraconazole and Aspergillus spp. (19, 26). Espinel-Ingroff found that the voriconazole Etest MICs were lower than NCCLS results (11). These higher MICs by Etest were not sufficiently high to indicate that these organ-
TABLE 2. Percentage of agreement of Etest MICs of voriconazole read at 24 and 48 h with modified reference microdilution MICs of voriconazole read at 48 h Species (no. of isolates)
A. fumigatus (26) A. flavus (21) A. terreus (10) A. niger (9) A. nidulellus (5) A. glaucus (4) A. falvipes (2) Total (77) a b
% Agreement 24 h/48 h
a
84.6 95.2 100 100 100 100 100 93.5
Etest results read at 24 h and microdilution results read at 48 h. Both Etest and microdilution MICs read at 48 h.
48 hb
65 100 100 88 100 50 100 84.4
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isms might be resistant to safely achievable levels of voriconazole (MICs, 0.25 to 0.5 g/ml). We have provided important documentation of the ability of Etest to generate voriconazole MIC data that are comparable to those obtained by the NCCLS broth microdilution method for Aspergillus spp. using RPMI supplemented with 2% glucose medium. The clinical value of these in vitro results should be determined in clinical trials. REFERENCES 1. Abraham, O. C., E. K. Manavathu, J. L. Cutright, and P. H. Chandrasekar. 1999. In vitro susceptibilities of Aspergillus species to voriconazole, itraconazole, and amphotericin B. Diagn. Microbiol. Infect. Dis. 33:7–11. 2. Arikan, S., M. Lozano-Chiu, V. Paetznick, S. Nangia, and J. H. Rex. 1999. Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against isolates of Aspergillus and Fusarium species. J. Clin. Microbiol. 37:3946–3951. 3. Barry, A. L., and S. D. Brown. 1996. In vitro studies of two triazole antifungal agents (voriconazole [UK-109,496] and fluconazole) against Candida species. Antimicrob. Agents Chemother. 40:1948–1949. 4. Barry A. L., M. A. Pfaller, S. D. Brown, A. Espinel-Ingroff, M. A. Ghannoum, C. Knapp, R. P. Rennie, J. H. Rex, and M. G. Rinaldi. 2000. Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J. Clin. Microbiol. 38:3457–3459. 5. Cha ´vez, M., S. Bernal, A. Valverde, M. J. Gutierrez, G. Quindos, and E. Martín Mazuelos. 1999. In-vitro activity of voriconazole (UK-109,496), LY303366 and other antifungal agents against oral Candida spp. isolates from HIV-infected patients. J. Antimicrob. Chemother. 44:697–700. 6. Chryssanthou, E., and M. Cuenca-Estrella. 2002. Comparison of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing proposed standard and the Etest with the NCCLS broth microdilution method for voriconazole and caspofungin susceptibility testing of yeast species. J. Clin. Microbiol. 40:3841–3844. 7. Cuenca-Estrella, M., J. L. Rodriguez-Tudela, E. Mellado, J. V. MartinezSuarez, and A. Monzon. 1998. Comparison of the in-vitro activity of voriconazole (UK-109,496), itraconazole and amphotericin B against clinical isolates of Aspergillus fumigatus. J. Antimicrob. Chemother. 42:531–533. 8. Denning, D. W. 1996. Therapeutic outcome in invasive aspergillosis. Clin. Infect. Dis. 23:608–615. 9. Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781–805. 10. Espinel-Ingroff, A. 1998. Comparison of in vitro activities of the new triazole SCH56592 and the echinocandins MK-0991 (L743,872) and LY303366 against opportunistic filamentous and dimorphic fungi and yeasts. J. Clin. Microbiol. 36:2950–2956. 11. Espinel-Ingroff, A., and A. Rezusta. 2002. Etest method for testing susceptibilites of Aspergillus spp. to new triazoles voriconazole and posaconazole and to established antifungal agents: comparison with NCCLS broth microdilution method. J. Clin. Microbiol. 40:2101–2107. 12. Johnson, E. M., A. Szekely, and D. W. Warnock. 1998. In-vitro activity of voriconazole, itraconazole and amphotericin B against filamentous fungi. J. Antimicrob. Chemother. 42:741–745. 13. Manavathu, E. K., J. L. Cutright, D. Loebenberg, and P. H. Chandrasekar. 2000. A comparative study of the in vitro susceptibilities of clinical and laboratory-selected resistant isolates of Aspergillus spp. to amphotericin B, itraconazole, voriconazole and posaconazole (SCH 56592). J. Antimicrob. Chemother. 42:229–234.
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