Comparison of Etest with Modified Broth Microdilution Method for Testing Susceptibility of Aspergillus spp. to Voriconazole

Abstract

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.

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 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 μg/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 ranging from 0.002 to 32 μg/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.

TABLE 1.

In vitro susceptibilities of 77 isolates of Aspergillus spp. to voriconazole as determined by the Etest and broth microdilution methods

Species (no. of isolates)	MIC (μg/ml)
	NCCLSa		E test
			24 h		48 h
	Range	50/90b	Range	50/90b	Range	50/90b
A. fumigatus (26)	0.03-0.12	0.06/0.12	0.06-0.5	0.12/0.25	0.06-0.5	0.25/0.5
A. flavus (21)	0.03-0.12	0.06/0.12	0.03-0.5	0.12-0.25	0.12-0.5	0.12/0.5
A. terreus (10)	0.03-0.25	0.12/0.25	0.06-0.12	0.12/0.25	0.12-0.25	0.25/0.25
A. niger (9)	0.03-0.12	0.06/-c	0.03-0.06	0.06/-	0.03-0.25	0.12/-
A. nidulellus (5)	0.03-0.5	0.12/-	0.03-0.25	0.12/-	0.03/-	0.12/-
A. glaucus (4)	0.03-0.12	-d/-	0.06-0.12	-/-	-/-	-/-
A. flavipes (2)	0.03-0.06	-/-	0.03-0.06	-/-	-/-	-/-
Total (77)	0.03-0.25	0.06/0.12	0.03-0.5	0.12/0.25	0.03-1	0.25/0.5

^aModified reference microdilution method MICs read at 48 h of incubation.

^b50/90, MIC_50/90.

^cMIC₉₀s were not calculated, since there were <10 isolates tested.

^dMIC₅₀s were not calculated since there were <5 isolates tested.

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).

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)	% Agreement
	24 h/48 ha	48 hb
A. fumigatus (26)	84.6	65
A. flavus (21)	95.2	100
A. terreus (10)	100	100
A. niger (9)	100	88
A. nidulellus (5)	100	100
A. glaucus (4)	100	50
A. falvipes (2)	100	100
Total (77)	93.5	84.4

^aEtest results read at 24 h and microdilution results read at 48 h.

^bBoth Etest and microdilution MICs read at 48 h.

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 organisms might be resistant to safely achievable levels of voriconazole (MICs, 0.25 to 0.5 μg/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.