Determination of Isavuconazole Susceptibility of Aspergillus and Candida Species by the EUCAST Method

Abstract

Isavuconazole is a novel expanded-spectrum triazole, which has recently been approved by the FDA as an orphan drug to treat invasive aspergillosis and is currently being studied in phase III clinical trials for invasive candidiasis. The susceptibility of relatively few clinical isolates has been reported. In this study, the isavuconazole susceptibilities of 1,237 Aspergillus and 2,010 Candida geographically diverse clinical isolates were determined by EUCAST methodology at four European mycology laboratories, producing the largest multicenter data set thus far for this compound. In addition, a blinded collection of 30 cyp51A mutant Aspergillus fumigatus clinical isolates and 10 wild-type isolates was tested. From these two data sets, the following preliminary epidemiological cutoff (ECOFF) values were suggested: 2 mg/liter for Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus; 4 mg/liter for Aspergillus niger; 0.25 mg/liter for Aspergillus nidulans; and 0.03 mg/liter for Candida albicans, Candida parapsilosis, and Candida tropicalis. Unfortunately, ECOFFs could not be determined for Candida glabrata or Candida krusei due to an unexplained interlaboratory MIC variation. For the blinded collection of A. fumigatus isolates, all MICs were ≤2 mg/liter for wild-type isolates. Differential isavuconazole MICs were observed for triazole-resistant A. fumigatus isolates with different cyp51A alterations: TR₃₄/L98H mutants had elevated isavuconazole MICs, whereas isolates with G54 and M220 alterations had MICs in the wild-type range, suggesting that the efficacy of isavuconazole may not be affected by these alterations. This study will be an aid in interpreting isavuconazole MICs for clinical care and an important step in the future process of setting official clinical breakpoints.

INTRODUCTION

The triazole agents have revolutionized antifungal therapy and are now the most common drug class used to treat fungal infections. However, currently licensed azoles have their limitations; for example, fluconazole has no anti-Aspergillus activity, itraconazole demonstrates poor bioavailability, voriconazole has challenging pharmacokinetics, and posaconazole has saturable absorption in its current oral formulation (1–6). Isavuconazole is a novel expanded-spectrum triazole. It has recently been approved by the FDA as an orphan drug to treat invasive aspergillosis, and phase III clinical trials for invasive candidiasis are ongoing. It has broad-spectrum, potent in vitro activity and a favorable pharmacokinetic-pharmacodynamic profile, although its clinical utility remains uncertain pending the outcome of the clinical trials (7).

Isavuconazole action includes in vitro activity against Aspergillus and Candida species, although the susceptibility of few clinical isolates has been studied (7–13). Epidemiological cutoff (ECOFF) values can assist in identifying isolates with raised MICs and/or a greater risk of the presence of a mechanism of resistance. ECOFFs are a valuable addition to help guide clinicians until data are obtained to enable the setting of clinical breakpoints. The process of setting ECOFF values, however, requires data from multiple laboratories, which are not currently available for Candida (by either the CLSI or the European Committee on Antimicrobial Susceptibility Testing [EUCAST] method) or for Aspergillus by the EUCAST method.

The aim of this study was to establish comprehensive multicenter isavuconazole MIC distributions for each of the five most common Aspergillus and Candida species by using EUCAST methodology. Clinical isolates from four European laboratories with mycological expertise were included, and the obtained MIC distributions were used to determine the ECOFF values of isavuconazole for these common pathogenic fungi.

MATERIALS AND METHODS

Isolates.

The following clinical Aspergillus isolates (a total of 1,237 isolates) were investigated: Aspergillus flavus complex (n = 215), Aspergillus fumigatus sensu stricto (n = 401), Aspergillus nidulans complex (n = 206), Aspergillus niger complex (n = 209), and Aspergillus terreus complex (n = 206). Finally, a blinded collection of 30 molecularly characterized azole-resistant and 10 wild-type A. fumigatus clinical isolates was studied (all laboratories tested the same blinded panel). The mutant strains had alterations at the following common molecular Cyp51A azole resistance hot spots: G54 (n = 10), TR₃₄/L98H (n = 10), and M220 (n = 10). All Aspergillus isolates were identified by standard microbiological techniques (14), and A. fumigatus sensu stricto was confirmed by thermotolerance at 48°C. Therefore, the use of the term “complex” is acknowledged for Aspergillus species other than A. fumigatus in the absence of detailed molecular identification, although for simplicity, it is not used throughout the manuscript.

For Candida species, the following isolates were studied: Candida albicans (n = 430), Candida glabrata (n = 385), Candida krusei (n = 399), Candida parapsilosis complex (n = 398), and Candida tropicalis (n = 398), with approximately 100 isolates of each species from each of the four European centers. Species identification was done according to standard procedures, including colony morphology on chromogenic agar (CHROMagar Co., Paris, France), microscopic morphology, growth at 35°C and 43°C (14), rapid tests for the identification of Candida dubliniensis and C. glabrata (Bichro-Dubli and Glabrata RTT; Fumouze Diagnostics, Simoco, Denmark), assimilation profile by the use of a commercial system (ATB ID32C; bioMérieux), or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). C. parapsilosis was not consistently separated from its sibling species (C. metapsilosis and C. orthopsilosis). The total number of Candida MIC determinations was 2,010.

Susceptibility testing.

Isavuconazole (Astellas Pharma Inc., Tokyo, Japan) MICs were determined by using EUCAST methodology (15, 16). The drug concentration range studied was 0.015 to 8 mg/liter. Although no accepted MIC ranges have yet been established for control strains for either CLSI or EUCAST testing, C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were included as common control strains across the four laboratories, and MICs were read at 24 ± 2 h (Table 1). Control isolate results were accepted where reproducibility was within 4-fold limits. Micafungin MICs were determined simultaneously for all Candida clinical strains as an additional control measure.

Table 1.

MIC results for the two control isolates at four mycology reference centers

Control isolate	Laboratory	No. of times when the following MIC (mg/liter) was obtained
		0.015	0.03	0.06	0.12	0.25	0.5
C. krusei ATCC 6258	1		2	1	1
	2	2	3	3
	3		13	1	1
	4			4	5	1
C. parapsilosis ATCC 22019	1	2	2
	2	2
	3		38
	4		9	1

ECOFF setting.

The data from each of the four laboratories were compared, pooled, and analyzed collectively. ECOFFs are defined as the highest MIC value of the wild-type distribution. The ECOFF for each species and isavuconazole was determined by using a standard nonstatistical approach (the eyeball method) (http://www.srga.org/Eucastwt/eucastdefinitions.htm). Aspergillus isolates for which the isavuconazole MIC was elevated compared to that of the other isolates of that species, and where indications of azole resistance were found (mutation found in the target gene or an itraconazole MIC of >4 mg/liter), were excluded from the ECOFF analysis. Excluded isolates are indicated in parentheses in Table 2 and included A. fumigatus (no. 4), A. nidulans (no. 6), and A. terreus (no. 6) isolates.

Table 2.

Isavuconazole MICs for clinical isolates of Aspergillus spp. determined at four mycology reference centers^a

^aThe modal MIC (the MIC most frequently found) is underlined for each data set, and the main wild-type distributions are shaded in gray. The dotted line indicates the proposed ECOFF. The numbers in parentheses indicate the number of non-wild-type isolates with either elevated itraconazole MICs (>4 mg/liter) or confirmed molecular resistance mutations. This information was available for isolates from laboratories 1 to 3 but unfortunately not for isolates from laboratory 4.

RESULTS

Aspergillus.

Table 2 details the isavuconazole MIC values for Aspergillus spp. MICs were lower for A. nidulans and higher for A. niger than for the other species tested. Modal MICs (the most commonly occurring MIC value) for 4/5 Aspergillus species tended to sit 1- to 4-fold (1 to 2 2-fold dilutions) higher in the strain collection from laboratory 4 than in those from the other centers. This was particularly evident for A. terreus, where the mode and MIC₅₀ were as high as 4 mg/liter for the data set from laboratory 4 but were 0.25 to 1 mg/liter for the other three data sets. Hence, this data set was excluded for A. terreus ECOFF determinations. The following Aspergillus ECOFFs for isavuconazole were then suggested: 2 mg/liter for A. fumigatus, 2 mg/liter for A. terreus, 2 mg/liter for A. flavus, 4 mg/liter for A. niger, and 0.25 mg/liter for A. nidulans. For laboratories 1 to 3, data on itraconazole susceptibility and/or the cyp51A profile were available for isolates with isavuconazole MICs above these ECOFFs. As shown in Table 2, 4/7 A. fumigatus, 6/11 A. nidulans, and 6/6 A. terreus isolates were found to have elevated itraconazole MICs and/or target gene mutations.

Blinded mutant/wild-type A. fumigatus collection.

The MICs of the wild-type strains in the blinded A. fumigatus collection (Table 3) were all within the expected range (0.25 to 2 mg/liter) compared to the wild-type distributions presented in Table 2. The MICs for the cyp51A mutant isolates were overall comparable to those for the wild-type isolates for strains harboring mutations affecting the G54 codon (G54E, G54R, G54V, or G54W) or the M220K or M220T alteration. Thus, three of the laboratories found MICs of ≤2 mg/liter for all these isolates, and laboratory 4 found MICs of ≤2 mg/liter for 10/15 strains, suggesting that susceptibility was not affected by these mutations. MICs of >2 mg/liter were found in at least two laboratories and overall for 25% of the readings for isolates harboring the M220I and the M220V mutations, suggesting a slight reduction of susceptibility. Finally, 72.5% of the MIC readings were >2 mg/liter for the TR₃₄/L98H strains (with MICs of up to >8 mg/liter), suggesting that isavuconazole may show reduced efficacy against this genotype (Table 3).

Table 3.

Isavuconazole MICs for a blinded collection of A. fumigatus strains with or without common resistance mutations

The intralaboratory variation, defined as the proportion of MICs within 3 dilutions, was 100% for laboratories 1, 2, and 3, except for one of the two M220I strains, for which the isavuconazole MIC was high (8 or >8 mg/liter), as determined by laboratories 1 and 2 but not by laboratories 3 (MIC of 2 mg/liter) and 4 (MIC of 1 mg/liter). The intralaboratory variation was slightly greater at laboratory 4: 85% were within 3 dilutions, but 15% differed by 4 dilutions. Notably, the visually determined no-growth endpoints for some of these A. fumigatus strains were more difficult to determine visually than for wild-type isolates.

Candida.

The isavuconazole MIC values for the Candida isolates are shown in Table 4. Isavuconazole MICs were generally low for C. albicans, C. parapsilosis, and C. tropicalis (modal MIC, ≤0.015 mg/liter) and higher for C. glabrata and C. krusei. The MIC distributions differed significantly between centers, with MICs being consistently lower in laboratories 2 and 3. This was particularly evident for distributions of C. glabrata and C. krusei, where the mode differed 4- to 32-fold (2 to 5 2-fold dilutions), presenting bimodal distributions when combined. Therefore, we abstained from defining ECOFFs for C. glabrata and C. krusei. It was less noticeable for C. albicans, C. parapsilosis, and C. tropicalis, possibly in part due to the fact that the MICs were truncated at 0.015 mg/liter. Particularly for C. albicans and C. tropicalis, the MIC distribution had a tail to the right of the mode including a number of isolates with elevated MICs, suggesting that at least some of these were non-wild-type isolates. Therefore, based on these data, we propose tentative ECOFFs for C. albicans, C. parapsilosis, and C. tropicalis of 0.03 mg/liter. More work is required to confirm these figures (or potentially revise them if appropriate).

Table 4.

Isavuconazole MICs for clinical isolates of Candida spp. determined at four mycology laboratories^a

^aThe modal MIC is underlined for each data set, and the main wild-type distributions are shown in gray. The dotted line indicates the proposed ECOFF. ND, not determined.

DISCUSSION

This study describes the in vitro susceptibilities of the largest in vitro collection of commonly encountered human-pathogenic fungi studied to date against the new antifungal isavuconazole and suggests species-specific ECOFFs for the five most common Aspergillus species and three Candida species.

A. nidulans isolates were more susceptible in vitro to isavuconazole (lower MICs) than the other Aspergillus species tested, while A. niger appeared to be less isavuconazole susceptible. There are relatively few Aspergillus isavuconazole MIC data sets in the literature using the EUCAST method, none of which included A. nidulans isolates and one of which focused on A. flavus only (8, 11). Together, those studies reported MICs similar to the ones in the present study. Thus, Perkhofer and colleagues also found higher MICs for A. niger than for A. fumigatus (11). This may be less surprising, as itraconazole and voriconazole MICs are also higher for this species than for A. fumigatus (EUCAST ECOFFs of 4 mg/liter and 2 mg/liter for these compounds, respectively, compared to 1 mg/liter for both agents and A. fumigatus) (17, 18). Additional isavuconazole susceptibility studies which used the CLSI method were also analogous (10, 12, 13, 19, 20). The clinical implications of these differences in susceptibility within species remain to be elucidated. However, they indicate that species-specific ECOFFs are required for this antifungal, reinforcing the need for diagnostic laboratories to accurately identify isolates to the species level. Although ECOFFs do not predict clinical outcome per se, they are a useful tool for interpreting MICs by allowing improved discrimination between wild-type isolates and isolates with resistance mechanisms. In this respect, it was noticeable that the majority or all of the A. fumigatus, A. terreus, and A. nidulans isolates with isavuconazole MICs above the suggested ECOFF also displayed reduced susceptibility to itraconazole and/or were found to harbor cyp51A mutations.

When the suggested ECOFF was applied for the blinded collection of A. fumigatus isolates with and without various well-described cyp51A mutations, all wild-type isolates were correctly classified as such by all four laboratories. The greatest MIC elevation was observed for the TR₃₄/L98H mutants, which is in agreement with the reported pan-azole in vitro resistance to the currently licensed azoles (21). In contrast, the isavuconazole MICs of the majority of the G54 and some of the M220 mutants (M220K and M220T) were within the wild-type range. Isolates with G54 alterations tend to demonstrate itraconazole and posaconazole resistance while maintaining voriconazole susceptibility, whereas isolates with M220 mutations have variable cross-resistance patterns (21). This suggests that there is differential activity of isavuconazole in A. fumigatus depending on the underlying cyp51A mutation. However, in vivo model and clinical studies are needed to clarify how these mutations respond to isavuconazole in clinical practice.

One laboratory consistently obtained higher MICs and a less clear separation between wild-type and mutant Aspergillus isolates, and particularly, the MIC distribution for A. terreus was remarkably higher than that determined by the other laboratories and was therefore excluded from the ECOFF determination. The underlying reason for this systematic deviation is not understood. Excluding the entire data set from the study would lead to a more homogenous data set; however, this would also mask the possibility that isavuconazole MIC testing may be associated with some challenges with respect to reproducibility, which needs further attention. Therefore, we included all four data sets but gave the MIC distributions from this laboratory less weight (accepted ECOFFs that bisected the right side of the MIC distributions from this laboratory) in order to not let this single data set drive the ECOFFs too high to be sensitive for identifying potential mutant isolates.

For Candida, the data showed that isavuconazole has potent in vitro activity against wild-type C. albicans, C. parapsilosis, and C. tropicalis isolates, with an MIC of ≤0.03 mg/liter for 91.5 to 96.0% of such isolates. In comparison, the isavuconazole MICs against C. glabrata and C. krusei were higher, and indeed, these species are generally considered poor targets for the azole class of drugs. There is a single recently reported Candida EUCAST MIC data set for isavuconazole in the literature, and there are two MIC data sets determined by CLSI methodology (9, 12, 20). These data sets also found higher MICs for C. glabrata and C. krusei than for the other species and also showed that interlaboratory variation may be a challenge. For example, a bimodal distribution was found for C. albicans in one study (9), and the CLSI MIC₅₀ for this species varied 32-fold (4 dilutions) from 0.002 mg/liter to 0.03 mg/liter between the three studies, which is comparable to the variation observed for the untruncated MIC distributions in the present study. Theoretical explanations for such variability could include differences in isolate collections, azole exposure in the patient populations, potency of drug powder and stocks, and MIC testing constituents, all of which could have a significant effect on the MIC. At the CLSI antifungal susceptibility testing committee meeting in January 2013, a multicenter study was presented, reporting that isavuconazole CLSI MICs values for a strain collection tested repeatedly with different batches of medium also showed unexpected MIC variation for some Candida isolates but not for Aspergillus isolates (http://www.clsi.org/standards-development/microbiology/subcommittee-on-antifungal). In this study, a single batch of manufactured microtiter plates was used by all participating laboratories, thus eliminating possible variation due to differences in the potency of the pure substance or in the preparation of isavuconazole stock solutions and dilutions. Furthermore, EUCAST micafungin MICs were simultaneously tested alongside isavuconazole MICs on the same Candida isolates in our study (see the micafungin EUCAST rationale document at http://www.eucast.org/), and no difference between centers was observed, suggesting that constituent variation was an unlikely cause. The discrepancy between the performances of the reference methods for isavuconazole testing of Aspergillus and Candida is somewhat intriguing. However, it is not inconceivable that some fungus-drug combinations may be more susceptible to change than others. Indeed, it was suggested previously that low-MIC organisms may be more sensitive to minor variations than those with higher MICs, as in the case of C. albicans versus C. parapsilosis and caspofungin (22). Hence, further studies on improvement of the reproducibility of microbroth susceptibility testing of isavuconazole are needed. However, in the meantime, laboratories should be aware that at least isolates with an MIC above the proposed ECOFFs presented in this study have a high risk of being non-wild-type organisms.

In conclusion, this is the largest data set of isavuconazole MICs available to date using EUCAST methodology, from which ECOFFs can be proposed, and includes a non-wild-type Aspergillus strain collection for comparison. Using these data, we have proposed species-specific ECOFF values for isavuconazole by the EUCAST method for the most common Aspergillus species associated with disease in humans and for some Candida species. Further work is required to determine the ECOFFs for C. glabrata and C. krusei and to investigate sources of interlaboratory variation. The interlaboratory variation in MIC distributions was most remarkable for these species, the cause of which remains unclear currently and requires additional investigation. Furthermore, with the acquisition of the required clinical and pharmacokinetic-pharmacodynamic data, ECOFFs will facilitate the process of breakpoint setting.