In Vitro Activity of Isavuconazole against Opportunistic Fungal Pathogens from Two Mycology Reference Laboratories

Monitoring antifungal susceptibility patterns for new and established antifungal agents seems prudent given the increasing prevalence of uncommon species associated with higher antifungal resistance. We evaluated the activity of isavuconazole against 4,856 invasive yeasts and molds collected worldwide.

T he burden of invasive fungal infections (IFIs) for patients and health care systems is difficult to measure (1,2); however, it is well recognized that IFIs are associated with high morbidity and mortality rates and elevated health care costs. A higher prevalence of IFIs has been observed over the last 3 decades due to the increasing immunocompromised population, which includes individuals living with human immunodeficiency virus, transplant recipients, and cancer patients (1,(3)(4)(5)(6). Additionally, increases in the elderly population, neonates, and patients requiring invasive therapies also contribute to the higher IFI rates (4,7,8).
The systemically active antifungal armamentarium currently includes the polyenes, flucytosine, fluconazole, the extended-spectrum (mold-active) triazoles (isavuconazole, itraconazole, posaconazole, and voriconazole), and the echinocandins. Despite the fact that these agents cover the vast majority of opportunistic fungal pathogens and are increasingly employed in either a prophylactic or preemptive treatment strategy, breakthrough invasive fungal infections continue to be reported and increasingly involve yeasts and/or molds that are relatively uncommon and tend to exhibit decreased susceptibility to the available antifungal agents (27,29,31).
Isavuconazole, a mold-active triazole, may be administered orally or parenterally and offers advantages in terms of predictable pharmacokinetics and safety over the other mold-active triazoles, including itraconazole, posaconazole, and voriconazole (39)(40)(41)(42). Specifically, isavuconazonium sulfate (the prodrug formulation of isavuconazole) may be administered intravenously to patients with decreased renal function without the need for dose adjustment, due to the lack of cyclodextrin and minimal renal excretion (42).
Previous studies have documented activity of isavuconazole against common species of both Candida and Aspergillus (41,43). Isavuconazole is also active against many of the less common yeasts and molds, including members of the order Mucorales (44)(45)(46)(47), and has been approved by the U.S. Food and Drug Administration for the treatment of invasive aspergillosis and invasive mucormycosis (38-40, 42, 48, 49). Studies to assess the clinical activity of isavuconazole against Candida and uncommon yeasts and molds have been completed (42).
In the present study, we examined the in vitro activities of isavuconazole and comparator antifungal agents against 4,856 clinical fungal isolates (2,351 of Candida spp., 1,972 of Aspergillus spp., 97 of non-Candida yeasts, and 361 of non-Aspergillus molds, including 292 Mucorales isolates) collected in 2015 to 2016 from clinically significant infections as part of two fungal surveillance efforts: the global SENTRY Antimicrobial Surveillance Program (JMI Laboratories, North Liberty, IA [Candida spp., non-Candida yeasts, and rare molds]) and the Fungus Testing Laboratory (San Antonio, TX [Aspergillus spp. and Mucorales]). All isolates were tested using Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methods, species-specific clinical breakpoints (CBPs), and proposed epidemiological cutoff values (ECVs), where available, for each agent to detect emerging resistance among Candida spp., Aspergillus spp., and selected mucormycetes. Molecular and proteomic methods were used to confirm the identification of the less common species of Candida, non-Candida yeasts, and all filamentous fungi.

RESULTS
All fungal clinical isolates (species with 10 or more isolates) collected and tested in surveillance years 2015 and 2016 are presented in Table 1 Table 2). The cumulative frequencies of MIC distributions for isavuconazole are presented for Aspergillus species in Table 2.
Among the tested species of Aspergillus, the MIC values for isavuconazole ranged from 0.06 to Ն16 g/ml. The modal MIC for isavuconazole among all Aspergillus spp. was 0.5 g/ml, with a low modal MIC of 0.25 g/ml for A. nidulans and A. terreus SC and a high modal MIC of 4 g/ml for A. calidoustus and A. tubingensis. Isavuconazole ECVs have been defined for A. flavus, A. fumigatus, A. niger, and A. terreus (50). According to the species-specific ECVs, the vast majority of isolates represented wild-type (WT) strains of Aspergillus spp. (MIC Յ ECV; range, 83.2 to 100.0%) (Tables 2 and 3). The isavuconazole MIC values were elevated at Ն8 g/ml for 8 A. fumigatus isolates, which suggests resistance mediated by mutations in cyp51A.
The activity of isavuconazole against Mucorales isolates was generally lower than that seen with Aspergillus spp., with a MIC range of 0.25 to Ն16 g/ml (   (36) 0   (Table 3). There were 17 A. flavus isolates for which the isavuconazole MIC value was 2 g/ml, and if the ECV was increased from 1 g/ml to 2 g/ml, the percentage of WT would increase to 99.1%, comparable to that seen for the A. flavus SC, itraconazole (100.0% WT), and voriconazole (95.8% WT). Whereas the isavuconazole ECV for this species was determined using MIC values from 7 different laboratories (50), the reproducibility of the CLSI method for a single laboratory (Ϯone 2-fold dilution) should be kept in mind when evaluating such data. Given the potential for dose escalation with isavuconazole, it may be possible to  (Table 3) were comparable to that of itraconazole (2 g/ml) and voriconazole (2 g/ml) and higher than that of posaconazole (0.5 g/ml  (Table 3).

No. of isolates with MIC (g/ml) of
Activity of isavuconazole and comparators against Candida species isolates. The antifungal activities of isavuconazole, fluconazole, posaconazole, and voriconazole against 2,351 Candida isolates (10 species) as determined by CLSI BMD methods are shown in Table 5. Results are categorized using CLSI CBPs and/or ECVs, as appropriate. The majority of these isolates represented WT strains, as determined by the respective ECVs, and few (C. glabrata and C. parapsilosis) were resistant to triazoles, based on CBPs. Neither CBPs nor ECV values have been established for isavuconazole and Candida spp. Using species-specific breakpoints, 100.0% of C. albicans isolates were susceptible to fluconazole and voriconazole. Fluconazole and voriconazole were also active against C. parapsilosis (94.8 and 96.3% susceptible, respectively, at the CLSI CBP) and Candida tropicalis (97.9 and 97.9% susceptible, respectively, at the CLSI CBP). Voriconazole was also active against C. krusei (94.1% susceptible). Among the 10 species of Candida tested against posaconazole, 98.7% showed a WT phenotype based on the established ECVs (54). Only C. lusitaniae (76.5% WT) and C. guilliermondii (76.9% WT) exhibited greater than 3% strains non-WT to posaconazole ( Table 5).
The in vitro potency of isavuconazole against Candida spp. was most comparable to that of voriconazole. Based on MIC 90 values, isavuconazole was 2-to 16-fold more active than posaconazole against all species, although C. guilliermondii displayed much higher MIC 90 values for all agents (Table 5). C. guilliermondii is known to exhibit decreased susceptibility to fluconazole, posaconazole, and voriconazole (55)(56)(57), and this phenotype was apparent in isolates from the present study as well (23.1 to 53.8% non-WT [ Table 5]).

DISCUSSION
Several important observations can be made from this global survey. First, we have used molecular methods of species identification to further document the broad array of fungi implicated as causes of IFI in U.S. and non-U.S. medical centers. We have tested all fungi for susceptibility to isavuconazole and the other systemically active triazoles using reference CLSI BMD methods and have applied the most recent CBPs and ECVs to assess the relative activity of these important antifungal agents. In general, the more common species of Candida and Aspergillus remain susceptible to all the mold-active triazole antifungal agents. Resistance to multiple azoles is apparent in both C. glabrata and C. guilliermondii, and both species must be monitored closely for the emergence of multidrug resistance. Likewise, the azole-resistant non-fumigatus species of Aspergillus, such as A. calidoustus, A. lentulus, and A. tubingensis, along with emerging MDR strains of A. fumigatus, must be actively sought in clinical material and undergo accurate species identification as well as antifungal susceptibility testing to ensure optimal patient management (29,30,34,35). Whereas isavuconazole has been approved for the treatment of invasive mucormycosis (49), the available clinical and in vitro data to support this application have been limited to date (44)(45)(46)(47)(48)(49). In the present study, we have documented the variable activities of isavuconazole, itraconazole, and posaconazole across all of the Mucorales isolates tested and have confirmed the potentially useful activity of isavuconazole against select species of Rhizopus as determined by CLSI methods (44)(45)(46)(47). Given the modal MIC value of 1 g/ml for isavuconazole and species of Rhizopus, it is important to note that an analysis of real-world usage, along with an analysis of clinical trial samples, showed that drug concentrations of Ͼ1 g/ml are achieved with standard doses of isavuconazole (58).  In Vitro Activity of Isavuconazole Antimicrobial Agents and Chemotherapy Isavuconazole MIC distributions examined for Candida spp., Aspergillus spp., and the Mucorales from the most recent 2-year surveillance period (2015 to 2016) demonstrated little to no change in the distributions compared to reports from previous years (43,46,59,60), with activity comparable to those of itraconazole, posaconazole, and voriconazole. Isavuconazole and the other triazoles continue to be highly active against Aspergillus spp., but are less potent against the non-Aspergillus molds, including the Mucorales. The triazoles, including isavuconazole, appear to be more reliably active against the non-Candida yeasts than against rare molds, such as Fusarium spp.
In summary, the increasing application of molecular and proteomic methods of identification reveals a broad spectrum of opportunistic fungal pathogens. Isavuconazole exhibited excellent activity against most species of Candida and Aspergillus and is comparable to posaconazole and voriconazole against the less common yeasts and molds. Whereas most Candida and Aspergillus spp. remain susceptible to isavuconazole and the other triazoles, emergence of resistance during therapy, especially in patients with previous antifungal exposure, must be kept in mind. Given the extensive use of voriconazole in prevention and treatment of invasive aspergillosis, emergence of the Mucorales as breakthrough infections is a clear threat and underscores the importance of new agents, such as isavuconazole, in patients with invasive mucormycosis who are unable to tolerate amphotericin B therapy (42,49). Isolates were identified at participating institutions using methods routinely employed at the submitting laboratory, including the use of Vitek, MicroScan, API strips, and AuxaColor systems supplemented by conventional methods for yeast and mold identification (61)(62)(63). Isolates were submitted to JMI Laboratories (North Liberty, IA) or the Fungus Testing Laboratory (San Antonio, TX), where species identification was confirmed using morphological, biochemical, molecular, and proteomic methods (64)(65)(66). Yeast isolates were subcultured and screened using CHROMagar Candida (Becton, Dickinson, Sparks, MD) to ensure purity and to differentiate C. albicans/C. dubliniensis, C. tropicalis, and C. krusei. Additionally, biochemical tests, including Vitek 2 (bioMérieux, Hazelwood, MO), trehalose assimilation (for C. glabrata), or growth at 45°C (for C. albicans/C. dubliniensis), were used to identify common Candida species. Identity of isolates was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS [Bruker, Billerica, MA]). Isolates that were not identified by either phenotypic or proteomic methods, including all rare and sibling species, were identified using sequencebased methods as previously described (64).

MATERIALS AND METHODS
Identification of Aspergillus spp. and the Mucorales spp. was performed by combined morphology/phenotypic assessment and DNA sequence analysis. All rare and sibling species were identified by DNA sequencing. For morphological/phenotypic assessment, macroscopic and microscopic features were evaluated and temperature studies performed. For DNA sequence analysis, regions of the ␤-tubulin and calmodulin genes were amplified and sequenced. For Mucorales isolates, the internal transcribed spacer and D1/D2 regions were amplified and sequenced. Scedosporium spp. were also identified by amplifying and sequencing regions of the ␤-tubulin and calmodulin genes. Nucleotide sequences were examined using Lasergene software (DNAStar, Madison, WI) or Sequencher software (Gene Codes, Ann Arbor, MI) and then compared to database sequences using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Fusarium species isolates were analyzed for TEF sequence using the Fusarium-ID database through 2016 and the Fusarium multilocus sequence typing database (http://www.westerdijkinstitute.nl/fusarium/) (64). Results were considered acceptable if homology was Ͼ99.5% with other entries in the databases used for comparison. Sequences that were considerably different from the majority of entries for a species were considered outliers and were excluded from the analysis. The DNA sequence results were combined with the morphological/phenotypic assessment to assign a species identity to each isolate (67).