In Vitro Activities of Various Antifungal Drugs against Aspergillus terreus: Global Assessment Using the Methodology of the European Committee on Antimicrobial Susceptibility Testing

Abstract

This study presents in vitro susceptibility data for clinical (n = 48) and environmental (n = 31) isolates of Aspergillus terreus against nine antifungal agents. The methodology of the European Committee on Antimicrobial Susceptibility Testing was applied. Posaconazole and anidulafungin had the lowest and amphotericin B the highest MICs. No differences in susceptibility patterns were observed between environmental and clinical isolates.

Invasive fungal infections due to Aspergillus species have become a major cause of morbidity and mortality among immunocompromised patients (12, 19). Aspergillus fumigatus is most frequently isolated from clinical specimens, but other important species include Aspergillus flavus, Aspergillus niger, and Aspergillus terreus (15). A. terreus appears to have increased as a cause of opportunistic infections and is of serious concern because of in vivo and in vitro resistance to amphotericin B (AMB) (9, 11).

In this study we assessed the in vitro activities of nine antifungal drugs against clinical and environmental A. terreus isolates. DNA sequencing of the ribosomal internal transcribed spacer (ITS) was performed as well in order to confirm results from standard morphological identification.

The clinical strains were recovered from the Innsbruck University Hospital over a period of 10 years, from 1996 to 2006. A. terreus strains were obtained from 48 patients with hematological malignancies and invasive aspergillosis. Thirty-one environmental isolates were collected over a period of 4 years from Innsbruck, Tyrol, Austria. Isolates were stored either in water at room temperature or in 20% glycerol solution at −80°C and subcultured on Sabouraud agar at 30°C. Isolates were identified as A. terreus by means of macroscopic and microscopic characteristics (4, 16). Molecular methods applying the ITS1-5.8s-ITS2 ribosomal DNA region and the cytochrome b sequences by parsimony analysis (8) confirmed standard identification in our A. terreus strain collection. DNA segments were amplified with the primers ITS1 (5′-CCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and the primer rEME2 (5′-AAATAGCATAGAAAGGTAA-3′), E2 (5′-GGTATAGMTCTTAAWATAGC-3′), or E1m (5′-TGAGGTGCTACAGTTATTAC-3′) in a GeneAmp 9700 PCR system (Applied Biosystems) as described previously (17, 21).

For MIC testing, we used AMB (Sigma Aldrich Química, Madrid, Spain), itraconazole (ITC) (Janssen S.A., Madrid, Spain), voriconazole (VRC) (Pfizer S.A., Madrid, Spain), ravuconazole (RVC) (Bristol-Myers Squibb, Madrid, Spain), posaconazole (POS) (Schering-Plough Research Institute, Kenilworth, NJ), terbinafine (TBF) (Novartis, Basel, Switzerland), caspofungin (CPF) (Merck & Co., Inc., Rahway, NJ), micafungin (MCF) (Astellas Pharma Inc., Tokyo, Japan), and anidulafungin (ADF) (Pfizer S.A., Madrid, Spain). MICs were determined at least two times on different days and by following the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee on antifungal susceptibility testing (3, 10). A. fumigatus ATCC 204305 and A. flavus ATCC 204304 were used as quality control strains (2). The endpoint for AMB, azoles, and TBF was the antifungal concentration that produced a complete inhibition of visual growth at 48 h. For the echinocandins, the endpoint was defined as the minimum effective concentration (MEC). The in vitro data for 79 A. terreus isolates are shown in Table 1. The highest MICs were observed for AMB, followed by VRC, RVC, and CPF. The AMB MICs in our strain collection ranged between 0.5 and 8 μg/ml, and only a few isolates (15%) showed MICs of ≤1 μg/ml. This reflects the potential for primary polyene resistance in A. terreus, a fact which is well known and which is displayed by a worse outcome under AMB treatment (11). The introduction of new drugs for treatment of invasive aspergillosis improved the outcome of A. terreus infections (7, 11, 20), and VRC seems to be highly active against this species in the clinical setting. Yet among the azoles, our in vitro data showed VRC (MIC at which 90% of organisms were inhibited [MIC₉₀], 2 μg/ml) having the highest and POS (MIC₉₀, 0.12 μg/ml) and ITC (MIC₉₀, 0.5 μg/ml) the lowest MICs. These elevated VRC MICs disagree with reports from studies which employed the CLSI method (6, 18). It is known from earlier studies that EUCAST testing results in higher MICs than are found with the CLSI method (1). These findings need to be investigated in more detail. Similar elevated MICs were obtained for RVC; the lack of clinical data for this drug and A. terreus infections prevents conclusions from being drawn.

TABLE 1.

In vitro susceptibilities of clinical and environmental isolates of A. terreus to antifungal agents

Strain group (na)	Drug	MIC (μg/ml) of indicated agent for A. terreus strainsb
		Range	50%	90%	Mean
Clinical (48)	AMB	0.5-8	2	2	1.67
	ITC	0.06-0.5	0.125	0.5	0.21
	VRC	0.5-4	1	2	1.54
	RVC	0.5-4	1	2	1.42
	POS	0.03-0.5	0.12	0.12	0.11
	TBF	0.06-1	0.25	0.5	0.28
	CPF	0.03-4	1	2	1.34
	MCF	0.015-0.06	0.015	0.03	0.03
	ADF	0.015-0.06	0.015	0.03	0.02
Environmental (31)	AMB	0.5-8	2	2	1.77
	ITC	0.12-0.5	0.125	0.5	0.23
	VRC	0.5-4	1	2	1.62
	RVC	0.5-4	1	2	1.32
	POS	0.06-0.25	0.12	0.12	0.10
	TBF	0.12-0.5	0.25	0.5	0.29
	CPF	0.12-2	1	2	1.24
	MCF	0.015-0.06	0.015	0.03	0.03
	ADF	0.015-0.06	0.015	0.03	0.02

^an, no. of strains.

^bFor CPF, MCF, and ADF, values are MECs.

We were able to show clear differences in the in vitro activities for A. terreus within the class of lipopeptide agents. CPF showed higher MECs (MEC for 90% of organisms [MEC₉₀], 2 μg/ml) than ADF (MEC₉₀, 0.03 μg/ml) and MCF (MEC₉₀ 0.02 μg/ml) (P < 0.05). CPF MECs of <1 μg/ml were observed for only 13% of isolates tested. Since the modes of action and antifungal targets of CPF, ADF, and MCF are similar, these findings need further clarification. Odabasi et al. (13) compared the in vitro activities of several A. fumigatus and A. flavus strains against CPF, ADF, and MCF; such a tendency was not observed for these species. Espinel-Ingroff investigated 137 A. fumigatus strains and reported CPF geometric means of 0.75 μg/ml (5). One should note that the in vitro susceptibility method for the candins is still suboptimal and is influenced by several technical factors (14).

TBF was also active against A. terreus and might be placed as a reserve drug within the clinical setting. Comparison of MICs for the various drugs demonstrated that the susceptibility patterns among environmental and clinical isolates of A. terreus do not differ. Drugs displaying high MICs for clinical isolates also display high MICs for environmental isolates. Geometric mean MICs of AMB, VRC, RVC, and CPF were 1.77 μg/ml, 1.62 μg/ml, 1.32 μg/ml, and 1.24 μg/ml, respectively.

In conclusion, testing according to the EUCAST methodology revealed A. terreus to be highly susceptible to ADF, MCF, POS, and ITC. Our findings raise the question of which in vitro susceptibility testing method best predicts optimal treatment.