ABSTRACT
The activity of 7 antifungal agents against 3,557 invasive yeasts and molds collected in 29 countries worldwide in 2014 and 2015 was evaluated. Epidemiological cutoff values (ECVs) published in the Clinical and Laboratory Standards Institute (CLSI) M59 document were applied for species with no clinical breakpoints. Echinocandin susceptibility rates were 95.9% to 100.0% for the 5 most common Candida species, except for the rates for Candida parapsilosis to anidulafungin (88.7% susceptible, 100.0% wild type). Rates of fluconazole resistance ranged from 8.0% for Candida glabrata to 0.4% for Candida albicans. Seven Candida species displayed 100.0% wild-type amphotericin B MIC results, and Candida dubliniensis and Candida lusitaniae exhibited wild-type echinocandin MIC results. The highest fluconazole, voriconazole, and posaconazole MIC values for Cryptococcus neoformans var. grubii were 8 μg/ml, 0.12 μg/ml, and 0.25 μg/ml, respectively. Aspergillus fumigatus isolates were 100.0% wild type for caspofungin and amphotericin B, but 3 (0.8%) of these isolates were non-wild type to itraconazole (2 isolates) or voriconazole (1 isolate). Mutations in FKS hot spot (HS) regions were detected among 13/20 Candida isolates displaying echinocandin MICs greater than the ECV (16 of these 20 isolates were C. glabrata). Most isolates carrying mutations in FKS HS regions were resistant to 2 or more echinocandins. Five fluconazole-nonsusceptible C. albicans isolates were submitted to whole-genome sequencing analysis. Gain-of-function, Erg11 heterozygous, and Erg3 homozygous mutations were observed in 1 isolate each. One isolate displayed MDR1 promoter allele alterations associated with azole resistance. Elevated levels of expression of MDR1 or CDR2 were observed in 3 isolates and 1 isolate, respectively. Echinocandin and azole resistance is still uncommon among contemporary fungal isolates; however, mechanisms of resistance to antifungals were observed among Candida spp., showing that resistance can emerge and monitoring is warranted.
KEYWORDS: azoles, echinocandins, resistance
INTRODUCTION
The burden of invasive fungal infections (IFIs) for patients and health care systems is difficult to measure; 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 in the last few decades due to the increasing size of the population of immunocompromised individuals, which includes individuals living with human immunodeficiency virus, transplant recipients, and cancer patients (1, 2). Additionally, the increase in the numbers of individuals in the elderly, neonate, and patient populations requiring invasive therapies also contributes to the higher rates of IFIs.
Candida and Aspergillus species are among the most frequent causes of IFIs, and although isolates displaying resistance to clinically available antifungal agents are still uncommon, these isolates are increasingly reported worldwide (3, 4). For this reason, continuous monitoring of antifungal susceptibility patterns and mechanisms of resistance to clinically used antifungal agents is of increased importance. The SENTRY Antifungal Surveillance Program collects consecutive invasive fungal isolates from over 60 hospitals in North America, Europe, Latin America, and the Asia-Pacific region each calendar year and evaluates these isolates for susceptibility to the various antifungal agents clinically used to treat IFIs (5). In addition, the mechanisms of resistance to the echinocandins and azoles among isolates of Candida species displaying elevated MIC values for these agents are also investigated (6, 7).
Echinocandin resistance among Candida isolates is caused by alterations in the target of these antifungal agents, 1,3-β-d-glucan synthase, encoded by FKS (6). Mutations in 2 hot spot (HS) regions of FKS1 leading to amino acid substitutions are responsible for echinocandin resistance among the majority of Candida species; however, mutations in HS regions of FKS1 and FKS2 can lead to resistance among Candida glabrata isolates (8).
Azole resistance among Candida and Aspergillus isolates can be encoded by genes with target alterations and caused by the increased efflux of antifungal molecules (3, 4, 9). Target alterations in the ergosterol synthesis pathway, mainly in Erg11 (10) but also in Erg3 (11), have been associated with azole resistance development in Candida species. Additionally, alterations in various transcription factors that regulate the genes involved in ergosterol production and the regulation of efflux systems can also lead to azole resistance among clinical isolates (9). Azole resistance in Aspergillus spp. (largely Aspergillus fumigatus) involves a variety of mutations in CYP51 and its promoter (3, 4). As with Candida spp., efflux mechanisms may also contribute to azole resistance in A. fumigatus (2–4).
In this study, we evaluated the activity of 7 antifungal agents, tested using the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method, against 3,557 clinical fungal isolates collected during 2014 and 2015 in 29 countries. We applied species-specific breakpoints and the newly published epidemiological cutoff values (ECVs) listed in CLSI document M59 (12). Additionally, we used PCR methods and/or whole-genome sequencing analysis to investigate resistance mechanisms among Candida isolates displaying non-wild-type MIC values for echinocandins and among Candida albicans isolates displaying elevated fluconazole MIC values.
RESULTS
During 2014 and 2015, 3,557 fungal isolates were received and evaluated. These isolates consisted of the following: 2,809 (78.9%) Candida spp.; 152 (4.3%) yeasts belonging to other genera, including 89 (2.5%) Cryptococcus neoformans isolates; 511 (14.4%) Aspergillus isolates; and 85 (2.4%) other molds. Table 1 summarizes the results of the CLSI broth microdilution method for susceptibility to echinocandins, azoles, and amphotericin B for the species for which clinical breakpoints and/or ECVs have been established in CLSI documents (12, 13).
TABLE 1.
Activity of antifungal agents tested using the CLSI reference broth microdilution method against fungal organism groups according to clinical breakpoints and/or ECV interpretative criteriaa
| Organism (no. of isolates tested) and antifungal agent | MIC50 or MEC50 (μg/ml) | MIC90 or MEC90 (μg/ml) | % of isolates |
|||
|---|---|---|---|---|---|---|
| CLSI method |
ECV |
|||||
| S | R | WT | NWT | |||
| C. albicans (1,310) | ||||||
| Anidulafungin | 0.015 | 0.03 | 99.9 | 0.0 | 99.8 | 0.2 |
| Caspofungin | 0.015 | 0.03 | 99.8 | 0.2 | ||
| Micafungin | 0.015 | 0.03 | 99.8 | 0.2 | 99.8 | 0.2 |
| Fluconazole | ≤0.12 | 0.25 | 99.6 | 0.4 | ||
| Voriconazole | ≤0.008 | 0.015 | 99.9 | <0.1 | ||
| Posaconazole | 0.03 | 0.06 | ||||
| Amphotericin B | 1 | 1 | 100.0 | 0.0 | ||
| C. glabrata (514) | ||||||
| Anidulafungin | 0.06 | 0.12 | 95.9 | 2.5 | 97.5 | 2.5 |
| Caspofungin | 0.03 | 0.06 | 96.9 | 2.5 | ||
| Micafungin | 0.015 | 0.03 | 97.5 | 2.3 | 96.9 | 3.1 |
| Fluconazole | 4 | 16 | 92.0b | 8.0 | ||
| Voriconazole | 0.12 | 0.5 | ||||
| Posaconazole | 0.5 | 1 | ||||
| Amphotericin B | 1 | 1 | 100.0 | 0.0 | ||
| C. parapsilosis (417) | ||||||
| Anidulafungin | 2 | 4 | 88.7 | 0.0 | 100.0 | 0.0 |
| Caspofungin | 0.25 | 0.5 | 100.0 | 0.0 | ||
| Micafungin | 1 | 2 | 100.0 | 0.0 | 100.0 | 0.0 |
| Fluconazole | 0.5 | 2 | 95.7 | 3.8 | ||
| Voriconazole | 0.015 | 0.03 | 96.4 | 0.7 | ||
| Posaconazole | 0.06 | 0.12 | ||||
| Amphotericin B | 1 | 1 | 100.0 | 0.0 | ||
| C. tropicalis (264) | ||||||
| Anidulafungin | 0.015 | 0.03 | 100.0 | 0.0 | 100.0 | 0.0 |
| Caspofungin | 0.015 | 0.06 | 100.0 | 0.0 | ||
| Micafungin | 0.015 | 0.06 | 100.0 | 0.0 | 99.6 | 0.4 |
| Fluconazole | 0.25 | 0.5 | 96.2 | 2.7 | 96.2 | 3.8 |
| Voriconazole | 0.015 | 0.06 | 97.0 | 2.3 | ||
| Posaconazole | 0.03 | 0.12 | ||||
| Amphotericin B | 1 | 1 | 100.0 | 0.0 | ||
| C. krusei (93) | ||||||
| Anidulafungin | 0.06 | 0.12 | 100.0 | 0.0 | 100.0 | 0.0 |
| Caspofungin | 0.12 | 0.25 | 100.0 | 0.0 | ||
| Micafungin | 0.06 | 0.12 | 100.0 | 0.0 | 100.0 | 0.0 |
| Fluconazole | 32 | 64 | ||||
| Voriconazole | 0.25 | 0.5 | 100.0 | 0.0 | ||
| Posaconazole | 0.25 | 0.5 | ||||
| Amphotericin B | 1 | 2 | 100.0 | 0.0 | ||
| C. dubliniensis (58) | ||||||
| Anidulafungin | 0.03 | 0.12 | 100.0 | 0.0 | ||
| Caspofungin | 0.03 | 0.06 | ||||
| Micafungin | 0.03 | 0.06 | 100.0 | 0.0 | ||
| Fluconazole | ≤0.12 | 0.25 | ||||
| Voriconazole | ≤0.008 | 0.015 | ||||
| Posaconazole | 0.03 | 0.06 | ||||
| Amphotericin B | 0.5 | 1 | ||||
| C. lusitaniae (39) | ||||||
| Anidulafungin | 0.25 | 0.5 | 100.0 | 0.0 | ||
| Caspofungin | 0.12 | 0.25 | ||||
| Micafungin | 0.12 | 0.25 | 100.0 | 0.0 | ||
| Fluconazole | 0.5 | 1 | ||||
| Voriconazole | ≤0.008 | ≤0.008 | ||||
| Posaconazole | 0.06 | 0.12 | ||||
| Amphotericin B | 0.5 | 1 | ||||
| C. guilliermondii (14) | ||||||
| Anidulafungin | 2 | 4 | 92.9 | 0.0 | 100.0 | 0.0 |
| Caspofungin | 0.5 | 1 | 100.0 | 0.0 | ||
| Micafungin | 1 | 1 | 100.0 | 0.0 | 100.0 | 0.0 |
| Fluconazole | 2 | 64 | ||||
| Voriconazole | 0.06 | 1 | ||||
| Posaconazole | 0.5 | >8 | ||||
| Amphotericin B | 0.25 | 1 | ||||
| A. fumigatus (391) | ||||||
| Anidulafungin | ≤0.008 | 0.03 | ||||
| Caspofungin | 0.015 | 0.03 | 100.0 | 0.0 | ||
| Micafungin | ≤0.008 | 0.015 | ||||
| Itraconazole | 0.5 | 1 | 99.5 | 0.5 | ||
| Voriconazole | 0.5 | 0.5 | 99.7 | 0.3 | ||
| Posaconazole | 0.25 | 0.5 | ||||
| Amphotericin B | 1 | 2 | 100.0 | 0.0 | ||
| A. niger (15) | ||||||
| Anidulafungin | ≤0.008 | 0.03 | ||||
| Caspofungin | 0.015 | 0.03 | 100.0 | 0.0 | ||
| Micafungin | ≤0.008 | 0.015 | ||||
| Itraconazole | 0.5 | 1 | 100.0 | 0.0 | ||
| Voriconazole | 0.5 | 0.5 | 100.0 | 0.0 | ||
| Posaconazole | 0.25 | 0.5 | 100.0 | 0.0 | ||
| Amphotericin B | 1 | 2 | 100.0 | 0.0 | ||
| A. terreus (12) | ||||||
| Anidulafungin | ≤0.008 | 0.03 | ||||
| Caspofungin | 0.015 | 0.03 | 100.0 | 0.0 | ||
| Micafungin | ≤0.008 | 0.015 | ||||
| Itraconazole | 0.5 | 1 | 100.0 | 0.0 | ||
| Voriconazole | 0.5 | 0.5 | 100.0 | 0.0 | ||
| Posaconazole | 0.25 | 0.5 | 100.0 | 0.0 | ||
| Amphotericin B | 1 | 2 | 100.0 | 0.0 | ||
MEC50 and MEC90, minimum effective concentrations for 50% and 90% of isolates, respectively; CLSI, Clinical and Laboratory Standards Institute; S, susceptible; R, resistant; ECV, epidemiologic cutoff value; WT, wild-type; NWT, non-wild-type.
Susceptible dose dependent.
Anidulafungin, caspofungin, and micafungin displayed good activity against the 8 Candida species listed in Table 1. Echinocandin resistance was observed among 0.2% of the 1,310 C. albicans isolates tested. Two isolates belonging to this species displayed MIC values for resistance to caspofungin and micafungin, but 1 isolate displayed a susceptible result for anidulafungin (MIC, 0.25 μg/ml). C. glabrata isolates displayed the highest echinocandin resistance rates, which ranged from 2.3% (micafungin) to 2.5% (anidulafungin and caspofungin), and 14/514 (2.7%) of the isolates tested displayed resistance to at least 1 of the echinocandins tested. All Candida tropicalis and Candida krusei isolates tested were susceptible to the clinically available echinocandins. Anidulafungin inhibited 88.7% and 92.9% of the Candida parapsilosis and Candida guilliermondii isolates, respectively, at the current CLSI breakpoint. These isolates were susceptible/wild type to caspofungin and micafungin. Note that anidulafungin-intermediate C. parapsilosis and C. guilliermondii isolates (MIC, 4 μg/ml) were categorized as wild type when the ECVs recently published by CLSI were applied (12).
Resistance to fluconazole was observed among 0.4% of the C. albicans isolates, 3.8% of the C. parapsilosis isolates, and 2.7% of the C. tropicalis isolates tested. As expected, these resistance rates were higher than the rates of resistance to voriconazole, which were <0.1% for C. albicans, 0.7% for C. parapsilosis, and 2.3% for C. tropicalis. A total of 8.0% of the C. glabrata isolates displayed MIC values indicating resistance to fluconazole.
When the recently published ECVs for the Candida species-antifungal combinations that do not have clinical breakpoints were applied (12), all Candida lusitaniae and Candida dubliniensis isolates were considered wild type for anidulafungin and micafungin and all C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis isolates were wild type for amphotericin B (Table 1).
ECVs were also recently published for various Aspergillus species (12), and we applied these criteria for sensu stricto isolates of A. fumigatus, Aspergillus niger, Aspergillus terreus and for Aspergillus flavus species complex (SC) isolates (Table 1).
The echinocandins exhibited similar activities against A. fumigatus, and all isolates were considered wild type for caspofungin. ECVs for itraconazole and voriconazole identified 0.5% (2 isolates, 1 each from Italy and Thailand) and 0.3% (1 isolate from Germany) of the isolates, respectively, to be non-wild type. All A. fumigatus isolates were wild type for amphotericin B, and all A. niger, A. terreus, and A. flavus species complex isolates were considered wild type for caspofungin, itraconazole, posaconazole, voriconazole, and amphotericin B.
The distributions of the MICs of the echinocandins and triazoles for species for which there were ≥3 isolates of uncommon species of Candida and Aspergillus, the non-Candida yeasts, and non-Aspergillus molds are shown in Table 2. Given that these species are uncommon in most regions of the world and data concerning their in vitro susceptibility to most antifungals are lacking, we elected to display the results as the number of isolates at each MIC value so that these results may eventually be combined with similarly derived data to form a more robust understanding of the MIC profiles of these unusual species.
TABLE 2.
Activity of antifungal agents tested against uncommon yeast species with ≥3 isolates
| Organism/organism group (no. of isolates) | No. of isolates (cumulative %) at the following MIC (μg/ml): |
MIC50 (μg/ml) | MIC90 (μg/ml) | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.008 | 0.015 | 0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | >a | |||
| Candida haemulonii (4) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 2 (50.0) | 1 (75.0) | 1 (100.0) | 0.12 | |||||||||||||
| Caspofungin | 0 (0.0) | 1 (25.0) | 1 (50.0) | 2 (100.0) | 0.06 | |||||||||||||
| Micafungin | 0 (0.0) | 4 (100.0) | 0.12 | |||||||||||||||
| Fluconazole | 0 (0.0) | 1 (25.0) | 1 (50.0) | 2 (100.0) | 1 | |||||||||||||
| Voriconazole | 0 (0.0) | 2 (50.0) | 2 (100.0) | 0.015 | ||||||||||||||
| Posaconazole | 0 (0.0) | 2 (50.0) | 2 (100.0) | 0.06 | ||||||||||||||
| Amphotericin B | 0 (0.0) | 1 (25.0) | 3 (100.0) | 2 | ||||||||||||||
| Candida metapsilosis (8) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 4 (50.0) | 2 (75.0) | 1 (87.5) | 1 (100.0) | 0.25 | ||||||||||||
| Caspofungin | 0 (0.0) | 1 (12.5) | 5 (75.0) | 1 (87.5) | 1 (100.0) | 0.12 | ||||||||||||
| Micafungin | 0 (0.0) | 1 (12.5) | 3 (50.0) | 3 (87.5) | 1 (100.0) | 0.25 | ||||||||||||
| Fluconazole | 0 (0.0) | 1 (12.5) | 6 (87.5) | 1 (100.0) | 1 | |||||||||||||
| Voriconazole | 0 (0.0) | 3 (37.5) | 4 (87.5) | 0 (87.5) | 1 (100.0) | 0.03 | ||||||||||||
| Posaconazole | 0 (0.0) | 2 (25.0) | 5 (87.5) | 0 (87.5) | 1 (100.0) | 0.06 | ||||||||||||
| Amphotericin B | 0 (0.0) | 2 (25.0) | 6 (100.0) | 1 | ||||||||||||||
| Candida fabianii (3) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 3 (100.0) | 0.015 | |||||||||||||||
| Caspofungin | 0 (0.0) | 3 (100.0) | 0.015 | |||||||||||||||
| Micafungin | 0 (0.0) | 2 (66.7) | 1 (100.0) | 0.015 | ||||||||||||||
| Fluconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 4 | ||||||||||||||
| Voriconazole | 0 (0.0) | 1 (33.3) | 1 (66.7) | 1 (100.0) | 0.06 | |||||||||||||
| Posaconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 0.5 | ||||||||||||||
| Amphotericin B | 0 (0.0) | 3 (100.0) | 1 | |||||||||||||||
| Candida inconspicua (6) | ||||||||||||||||||
| Anidulafungin | 2 (33.3) | 2 (66.7) | 2 (100.0) | 0.015 | ||||||||||||||
| Caspofungin | 0 (0.0) | 2 (33.3) | 2 (66.7) | 1 (83.3) | 1 (100.0) | 0.03 | ||||||||||||
| Micafungin | 2 (33.3) | 0 (33.3) | 2 (66.7) | 1 (83.3) | 1 (100.0) | 0.03 | ||||||||||||
| Fluconazole | 0 (0.0) | 5 (83.3) | 1 (100.0) | 16 | ||||||||||||||
| Voriconazole | 0 (0.0) | 2 (33.3) | 4 (100.0) | 0.25 | ||||||||||||||
| Posaconazole | 0 (0.0) | 5 (83.3) | 1 (100.0) | 0.25 | ||||||||||||||
| Amphotericin B | 0 (0.0) | 1 (16.7) | 2 (50.0) | 3 (100.0) | 0.5 | |||||||||||||
| Candida kefyr (22) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 7 (31.8) | 7 (63.6) | 8 (100.0) | 0.06 | 0.12 | ||||||||||||
| Caspofungin | 11 (50.0) | 10 (95.5) | 1 (100.0) | ≤0.008 | 0.015 | |||||||||||||
| Micafungin | 0 (0.0) | 1 (4.5) | 11 (54.5) | 9 (95.5) | 1 (100.0) | 0.03 | 0.06 | |||||||||||
| Fluconazole | 6 (27.3) | 7 (59.1) | 8 (95.5) | 1 (100.0) | 0.25 | 0.5 | ||||||||||||
| Voriconazole | 15 (68.2) | 7 (100.0) | ≤0.008 | 0.015 | ||||||||||||||
| Posaconazole | 0 (0.0) | 4 (18.2) | 3 (31.8) | 6 (59.1) | 5 (81.8) | 4 (100.0) | 0.06 | 0.25 | ||||||||||
| Amphotericin B | 0 (0.0) | 3 (13.6) | 18 (95.5) | 1 (100.0) | 1 | 1 | ||||||||||||
| Candida lipolytica (5) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 4 (80.0) | 1 (100.0) | 0.25 | ||||||||||||||
| Caspofungin | 0 (0.0) | 2 (40.0) | 3 (100.0) | 0.25 | ||||||||||||||
| Micafungin | 0 (0.0) | 1 (20.0) | 3 (80.0) | 1 (100.0) | 0.5 | |||||||||||||
| Fluconazole | 0 (0.0) | 1 (20.0) | 2 (60.0) | 0 (60.0) | 0 (60.0) | 0 (60.0) | 2 (100.0) | 2 | ||||||||||
| Voriconazole | 0 (0.0) | 2 (40.0) | 1 (60.0) | 0 (60.0) | 0 (60.0) | 0 (60.0) | 0 (60.0) | 2 (100.0) | 0.03 | |||||||||
| Posaconazole | 0 (0.0) | 3 (60.0) | 0 (60.0) | 1 (80.0) | 1 (100.0) | 0.25 | ||||||||||||
| Amphotericin B | 0 (0.0) | 1 (20.0) | 4 (100.0) | 1 | ||||||||||||||
| Candida orthopsilosis (34) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 5 (14.7) | 9 (41.2) | 15 (85.3) | 5 (100.0) | 1 | 2 | |||||||||||
| Caspofungin | 0 (0.0) | 5 (14.7) | 15 (58.8) | 11 (91.2) | 3 (100.0) | 0.12 | 0.25 | |||||||||||
| Micafungin | 0 (0.0) | 3 (8.8) | 10 (38.2) | 17 (88.2) | 4 (100.0) | 0.5 | 1 | |||||||||||
| Fluconazole | 1 (2.9) | 3 (11.8) | 13 (50.0) | 9 (76.5) | 4 (88.2) | 1 (91.2) | 0 (91.2) | 1 (94.1) | 1 (97.1) | 0 (97.1) | 1 (100.0) | 0.5 | 4 | |||||
| Voriconazole | 6 (17.6) | 10 (47.1) | 9 (73.5) | 4 (85.3) | 2 (91.2) | 0 (91.2) | 2 (97.1) | 1 (100.0) | 0.03 | 0.12 | ||||||||
| Posaconazole | 0 (0.0) | 8 (23.5) | 8 (47.1) | 15 (91.2) | 2 (97.1) | 1 (100.0) | 0.12 | 0.12 | ||||||||||
| Amphotericin B | 0 (0.0) | 19 (55.9) | 15 (100.0) | 0.5 | 1 | |||||||||||||
| Candida pelliculosa (5) | ||||||||||||||||||
| Anidulafungin | 4 (80.0) | 1 (100.0) | ≤0.008 | |||||||||||||||
| Caspofungin | 3 (60.0) | 0 (60.0) | 2 (100.0) | ≤0.008 | ||||||||||||||
| Micafungin | 2 (40.0) | 2 (80.0) | 1 (100.0) | 0.015 | ||||||||||||||
| Fluconazole | 0 (0.0) | 3 (60.0) | 1 (80.0) | 1 (100.0) | 2 | |||||||||||||
| Voriconazole | 0 (0.0) | 2 (40.0) | 2 (80.0) | 1 (100.0) | 0.12 | |||||||||||||
| Posaconazole | 0 (0.0) | 1 (20.0) | 1 (40.0) | 2 (80.0) | 1 (100.0) | 0.5 | ||||||||||||
| Amphotericin B | 0 (0.0) | 1 (20.0) | 4 (100.0) | 0.5 | ||||||||||||||
| Cryptococcus neoformans (3) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 3 (100.0) | >8 | |||||||||||||||
| Caspofungin | 0 (0.0) | 3 (100.0) | >8 | |||||||||||||||
| Micafungin | 0 (0.0) | 3 (100.0) | >8 | |||||||||||||||
| Fluconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 4 | ||||||||||||||
| Voriconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 0.06 | ||||||||||||||
| Posaconazole | 0 (0.0) | 3 (100.0) | 0.25 | |||||||||||||||
| Amphotericin B | 0 (0.0) | 3 (100.0) | 0.5 | |||||||||||||||
| Cryptococcus neoformans var. grubii (78) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (1.3) | 0 (1.3) | 5 (7.7) | 72 (100.0) | >8 | >8 | |||||||||||
| Caspofungin | 0 (0.0) | 1 (1.3) | 8 (11.5) | 69 (100.0) | >8 | >8 | ||||||||||||
| Micafungin | 0 (0.0) | 1 (1.3) | 0 (1.3) | 2 (3.8) | 75 (100.0) | >8 | >8 | |||||||||||
| Fluconazole | 0 (0.0) | 1 (1.3) | 7 (10.3) | 34 (53.8) | 35 (98.7) | 1 (100.0) | 2 | 4 | ||||||||||
| Voriconazole | 0 (0.0) | 4 (5.1) | 37 (52.6) | 36 (98.7) | 1 (100.0) | 0.03 | 0.06 | |||||||||||
| Posaconazole | 0 (0.0) | 3 (3.8) | 13 (20.5) | 32 (61.5) | 30 (100.0) | 0.12 | 0.25 | |||||||||||
| Amphotericin B | 0 (0.0) | 1 (1.3) | 46 (60.3) | 31 (100.0) | 0.5 | 1 | ||||||||||||
| Cryptococcus neoformans var. neoformans (8) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (12.5) | 0 (12.5) | 2 (37.5) | 5 (100.0) | >8 | ||||||||||||
| Caspofungin | 0 (0.0) | 6 (75.0) | 2 (100.0) | 8 | ||||||||||||||
| Micafungin | 0 (0.0) | 1 (12.5) | 0 (12.5) | 0 (12.5) | 2 (37.5) | 5 (100.0) | >8 | |||||||||||
| Fluconazole | 0 (0.0) | 1 (12.5) | 4 (62.5) | 2 (87.5) | 1 (100.0) | 1 | ||||||||||||
| Voriconazole | 0 (0.0) | 2 (25.0) | 5 (87.5) | 1 (100.0) | 0.03 | |||||||||||||
| Posaconazole | 0 (0.0) | 3 (37.5) | 4 (87.5) | 1 (100.0) | 0.12 | |||||||||||||
| Amphotericin B | 0 (0.0) | 1 (12.5) | 7 (100.0) | 0.5 | ||||||||||||||
| Geotrichum clavatum (5) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (20.0) | 4 (100.0) | 4 | ||||||||||||||
| Caspofungin | 0 (0.0) | 2 (40.0) | 1 (60.0) | 2 (100.0) | 8 | |||||||||||||
| Micafungin | 0 (0.0) | 1 (20.0) | 0 (20.0) | 3 (80.0) | 1 (100.0) | 2 | ||||||||||||
| Fluconazole | 0 (0.0) | 1 (20.0) | 3 (80.0) | 1 (100.0) | 8 | |||||||||||||
| Voriconazole | 0 (0.0) | 4 (80.0) | 0 (80.0) | 1 (100.0) | 0.12 | |||||||||||||
| Posaconazole | 0 (0.0) | 2 (40.0) | 3 (100.0) | 0.5 | ||||||||||||||
| Amphotericin B | 0 (0.0) | 2 (40.0) | 3 (100.0) | 1 | ||||||||||||||
| Magnusiomyces capitatus (7) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (14.3) | 6 (100.0) | 4 | ||||||||||||||
| Caspofungin | 0 (0.0) | 1 (14.3) | 2 (42.9) | 4 (100.0) | >8 | |||||||||||||
| Micafungin | 0 (0.0) | 1 (14.3) | 0 (14.3) | 0 (14.3) | 2 (42.9) | 4 (100.0) | >8 | |||||||||||
| Fluconazole | 0 (0.0) | 1 (14.3) | 0 (14.3) | 0 (14.3) | 1 (28.6) | 5 (100.0) | 8 | |||||||||||
| Voriconazole | 0 (0.0) | 1 (14.3) | 0 (14.3) | 1 (28.6) | 4 (85.7) | 1 (100.0) | 0.25 | |||||||||||
| Posaconazole | 0 (0.0) | 1 (14.3) | 0 (14.3) | 1 (28.6) | 2 (57.1) | 3 (100.0) | 0.25 | |||||||||||
| Amphotericin B | 0 (0.0) | 7 (100.0) | 1 | |||||||||||||||
| Rhodotorula mucilaginosa (6) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 6 (100.0) | >8 | |||||||||||||||
| Caspofungin | 0 (0.0) | 2 (33.3) | 4 (100.0) | >8 | ||||||||||||||
| Micafungin | 0 (0.0) | 6 (100.0) | >8 | |||||||||||||||
| Fluconazole | 0 (0.0) | 1 (16.7) | 5 (100.0) | >128 | ||||||||||||||
| Voriconazole | 0 (0.0) | 1 (16.7) | 0 (16.7) | 3 (66.7) | 2 (100.0) | 4 | ||||||||||||
| Posaconazole | 0 (0.0) | 3 (50.0) | 2 (83.3) | 0 (83.3) | 0 (83.3) | 1 (100.0) | 1 | |||||||||||
| Amphotericin B | 0 (0.0) | 2 (33.3) | 4 (100.0) | 1 | ||||||||||||||
| Saccharomyces cerevisiae (19) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 4 (21.1) | 6 (52.6) | 7 (89.5) | 2 (100.0) | 0.12 | 0.5 | |||||||||||
| Caspofungin | 0 (0.0) | 2 (10.5) | 5 (36.8) | 6 (68.4) | 5 (94.7) | 1 (100.0) | 0.12 | 0.25 | ||||||||||
| Micafungin | 0 (0.0) | 3 (15.8) | 11 (73.7) | 3 (89.5) | 2 (100.0) | 0.12 | 0.5 | |||||||||||
| Fluconazole | 0 (0.0) | 2 (10.5) | 4 (31.6) | 4 (52.6) | 6 (84.2) | 3 (100.0) | 2 | 8 | ||||||||||
| Voriconazole | 0 (0.0) | 1 (5.3) | 7 (42.1) | 5 (68.4) | 5 (94.7) | 1 (100.0) | 0.06 | 0.12 | ||||||||||
| Posaconazole | 0 (0.0) | 3 (15.8) | 8 (57.9) | 5 (84.2) | 3 (100.0) | 0.25 | 1 | |||||||||||
| Amphotericin B | 0 (0.0) | 5 (26.3) | 14 (100.0) | 1 | 1 | |||||||||||||
| Trichosporon asahii (14) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 2 (14.3) | 12 (100.0) | >8 | >8 | |||||||||||||
| Caspofungin | 0 (0.0) | 2 (14.3) | 12 (100.0) | >8 | >8 | |||||||||||||
| Micafungin | 0 (0.0) | 14 (100.0) | >8 | >8 | ||||||||||||||
| Fluconazole | 0 (0.0) | 1 (7.1) | 0 (7.1) | 8 (64.3) | 1 (71.4) | 3 (92.9) | 0 (92.9) | 1 (100.0) | 4 | 16 | ||||||||
| Voriconazole | 0 (0.0) | 1 (7.1) | 5 (42.9) | 3 (64.3) | 4 (92.9) | 0 (92.9) | 1 (100.0) | 0.12 | 0.25 | |||||||||
| Posaconazole | 0 (0.0) | 1 (7.1) | 7 (57.1) | 4 (85.7) | 2 (100.0) | 0.25 | 1 | |||||||||||
| Amphotericin B | 0 (0.0) | 1 (7.1) | 13 (100.0) | 1 | 1 | |||||||||||||
| Trichosporon mycotoxinivorans (3) | ||||||||||||||||||
| Anidulafungin | 0 (0.0) | 3 (100.0) | >8 | |||||||||||||||
| Caspofungin | 0 (0.0) | 1 (33.3) | 2 (100.0) | >8 | ||||||||||||||
| Micafungin | 0 (0.0) | 1 (33.3) | 0 (33.3) | 2 (100.0) | >8 | |||||||||||||
| Fluconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 4 | ||||||||||||||
| Voriconazole | 0 (0.0) | 3 (100.0) | 0.12 | |||||||||||||||
| Posaconazole | 0 (0.0) | 2 (66.7) | 1 (100.0) | 0.25 | ||||||||||||||
| Amphotericin B | 0 (0.0) | 3 (100.0) | 1 | |||||||||||||||
> represents the dilution greater than the highest dilution tested.
Among the 100 isolates of the less common species of Candida encountered in 2014 and 2015, we identified 19 different species (8 with ≥3 isolates; Table 2). Notable observations included elevated echinocandin MICs (>0.5 μg/ml) among Candida orthopsilosis and Candida metapsilosis isolates (Table 2). The MIC values for the echinocandins against the very rare species of Candida were generally low (<0.5 μg/ml), except for Candida lipolytica (Table 2), Candida fermentati (1 isolate; anidulafungin MIC, 2 μg/ml; data not shown), and Candida quercitrusa (1 isolate; anidulafungin MIC, 2 μg/ml; data not shown).
Elevated fluconazole MIC values (MIC, >4 μg/ml) were observed for isolates of C. orthopsilosis, Candida inconspicua, C. lipolytica, and Candida pelliculosa (Table 2). Additional species for which fluconazole MIC results appeared to be elevated (MIC, >4 μg/ml) included single isolates of Candida auris (MIC, 64 μg/ml), Candida norvegensis (MIC, 16 μg/ml), Candida pararugosa (MIC, 32 μg/ml) and C. quercitrusa (MIC, 16 μg/ml; data not shown). The MIC values for both voriconazole and posaconazole were <1 μg/ml for each of these rare species except C. lipolytica (Table 2).
As expected, the echinocandins were inactive against many of the non-Candida yeasts (Table 2). Echinocandin MIC results of ≤0.5 μg/ml were observed with Saccharomyces cerevisiae. Echinocandin MIC values were >2 μg/ml for Cryptococcus neoformans, Candida gattii (2 isolates; data not shown), Geotrichum clavatum, Geotrichum silvicola (1 isolate, data not shown), Magnusiomyces capitatus, Rhodotorula mucilaginosa, Trichosporon asahii, and Trichosporon mycotoxinivorans (Table 2). Fluconazole MIC values were >4 μg/ml for G. clavatum, M. capitatus, R. mucilaginosa, S. cerevisiae, T. asahii, and T. mycotoxinivorans (Table 2). In contrast, voriconazole and posaconazole showed good activity (MIC, <1 μg/ml) against most of the non-Candida yeasts, with the exception being Rhodotorula mucilaginosa. Fluconazole MIC values were <8 μg/ml against all species of Cryptococcus, whereas the echinocandins were inactive (MIC, >8 μg/ml) against these organisms (Table 2).
The echinocandins were highly active against the uncommon species of Aspergillus (Table 3); only 2 isolates of Aspergillus nidulans SC exhibited a minimum effective concentration (MEC) of >0.06 μg/ml. MIC values for itraconazole, posaconazole, and voriconazole were all <1 μg/ml against isolates of the A. nidulans species complex, whereas 1 isolate each of Aspergillus lentulus, the Aspergillus ustus species complex, and Aspergillus versicolor showed elevated MIC results for all triazoles (data not shown).
TABLE 3.
Activity of antifungal agents tested against uncommon mold species with ≥3 isolates
| Organism/organism group (no. of isolates) | No. of isolates (cumulative %) at the following MIC/MEC (μg/ml): |
MIC50 (μg/ml) | MIC90 (μg/ml) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.008 | 0.015 | 0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | >a | |||
| Aspergillus nidulans SC (9) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 5 (55.6) | 2 (77.8) | 1 (88.9) | 1 (100.0) | 0.015 | ||||||||
| Caspofungin | 3 (33.3) | 1 (44.4) | 1 (55.6) | 0 (55.6) | 2 (77.8) | 0 (77.8) | 0 (77.8) | 0 (77.8) | 2 (100.0) | 0.03 | ||||
| Micafungin | 3 (33.3) | 2 (55.6) | 2 (77.8) | 2 (100.0) | 0.015 | |||||||||
| Itraconazole | 0 (0.0) | 1 (11.1) | 1 (22.2) | 7 (100.0) | 0.5 | |||||||||
| Voriconazole | 0 (0.0) | 1 (11.1) | 6 (77.8) | 2 (100.0) | 0.12 | |||||||||
| Posaconazole | 0 (0.0) | 1 (11.1) | 5 (66.7) | 3 (100.0) | 0.25 | |||||||||
| Amphotericin B | 0 (0.0) | 4 (44.4) | 5 (100.0) | 2 | ||||||||||
| Aspergillus nidulans (8) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 5 (62.5) | 2 (87.5) | 0 (87.5) | 1 (100.0) | 0.015 | ||||||||
| Caspofungin | 3 (37.5) | 1 (50.0) | 1 (62.5) | 0 (62.5) | 1 (75.0) | 0 (75.0) | 0 (75.0) | 0 (75.0) | 2 (100.0) | 0.015 | ||||
| Micafungin | 3 (37.5) | 1 (50.0) | 2 (75.0) | 2 (100.0) | 0.015 | |||||||||
| Itraconazole | 0 (0.0) | 1 (12.5) | 7 (100.0) | 0.5 | ||||||||||
| Voriconazole | 0 (0.0) | 6 (75.0) | 2 (100.0) | 0.12 | ||||||||||
| Posaconazole | 0 (0.0) | 5 (62.5) | 3 (100.0) | 0.25 | ||||||||||
| Amphotericin B | 0 (0.0) | 3 (37.5) | 5 (100.0) | 2 | ||||||||||
| Aspergillus versicolor (4) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 2 (50.0) | 1 (75.0) | 1 (100.0) | 0.015 | |||||||||
| Caspofungin | 0 (0.0) | 2 (50.0) | 1 (75.0) | 1 (100.0) | 0.015 | |||||||||
| Micafungin | 0 (0.0) | 3 (75.0) | 1 (100.0) | 0.03 | ||||||||||
| Itraconazole | 0 (0.0) | 3 (75.0) | 0 (75.0) | 1 (100.0) | 0.5 | |||||||||
| Voriconazole | 0 (0.0) | 2 (50.0) | 1 (75.0) | 0 (75.0) | 1 (100.0) | 0.25 | ||||||||
| Posaconazole | 0 (0.0) | 3 (75.0) | 1 (100.0) | 0.5 | ||||||||||
| Amphotericin B | 0 (0.0) | 4 (100.0) | 2 | |||||||||||
| Fusarium solani SC (15) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 15 (100.0) | >8 | >8 | ||||||||||
| Caspofungin | 0 (0.0) | 15 (100.0) | >8 | >8 | ||||||||||
| Micafungin | 0 (0.0) | 15 (100.0) | >8 | >8 | ||||||||||
| Itraconazole | 0 (0.0) | 15 (100.0) | >8 | >8 | ||||||||||
| Voriconazole | 0 (0.0) | 4 (26.7) | 10 (93.3) | 1 (100.0) | 8 | 8 | ||||||||
| Posaconazole | 0 (0.0) | 15 (100.0) | >8 | >8 | ||||||||||
| Amphotericin B | 0 (0.0) | 4 (26.7) | 10 (93.3) | 1 (100.0) | 2 | 2 | ||||||||
| Scedosporium apiospermum/Scedosporium boydii (11) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (9.1) | 0 (9.1) | 0 (9.1) | 0 (9.1) | 0 (9.1) | 1 (18.2) | 9 (100.0) | 4 | 4 | ||||
| Caspofungin | 0 (0.0) | 1 (9.1) | 0 (9.1) | 0 (9.1) | 0 (9.1) | 0 (9.1) | 3 (36.4) | 2 (54.5) | 1 (63.6) | 0 (63.6) | 4 (100.0) | 2 | >8 | |
| Micafungin | 0 (0.0) | 1 (9.1) | 0 (9.1) | 0 (9.1) | 6 (63.6) | 2 (81.8) | 0 (81.8) | 0 (81.8) | 0 (81.8) | 2 (100.0) | 0.5 | >8 | ||
| Itraconazole | 0 (0.0) | 1 (9.1) | 2 (27.3) | 4 (63.6) | 0 (63.6) | 4 (100.0) | 1 | 4 | ||||||
| Voriconazole | 0 (0.0) | 1 (9.1) | 0 (9.1) | 1 (18.2) | 2 (36.4) | 3 (63.6) | 4 (100.0) | 0.5 | 1 | |||||
| Posaconazole | 0 (0.0) | 1 (9.1) | 0 (9.1) | 4 (45.5) | 5 (90.9) | 1 (100.0) | 1 | 1 | ||||||
| Amphotericin B | 0 (0.0) | 1 (9.1) | 0 (9.1) | 2 (27.3) | 4 (63.6) | 4 (100.0) | 2 | >2 | ||||||
| Scedosporium aurantiacum (3) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (33.3) | 1 (66.7) | 1 (100.0) | 8 | |||||||||
| Caspofungin | 0 (0.0) | 3 (100.0) | >8 | |||||||||||
| Micafungin | 0 (0.0) | 3 (100.0) | >8 | |||||||||||
| Itraconazole | 0 (0.0) | 1 (33.3) | 0 (33.3) | 0 (33.3) | 0 (33.3) | 2 (100.0) | >8 | |||||||
| Voriconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 1 | ||||||||||
| Posaconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 2 | ||||||||||
| Amphotericin B | 0 (0.0) | 1 (33.3) | 2 (100.0) | >2 | ||||||||||
| Scedosporium (Lomentospora) prolificans (3) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 2 (66.7) | 0 (66.7) | 1 (100.0) | 4 | |||||||||
| Caspofungin | 0 (0.0) | 2 (66.7) | 1 (100.0) | 8 | ||||||||||
| Micafungin | 0 (0.0) | 1 (33.3) | 2 (100.0) | >8 | ||||||||||
| Itraconazole | 0 (0.0) | 3 (100.0) | >8 | |||||||||||
| Voriconazole | 0 (0.0) | 3 (100.0) | >8 | |||||||||||
| Posaconazole | 0 (0.0) | 3 (100.0) | >8 | |||||||||||
| Amphotericin B | 0 (0.0) | 3 (100.0) | >2 | |||||||||||
| Exophiala dermatitidis (4) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 1 (50.0) | 2 (100.0) | 8 | ||
| Caspofungin | 0 (0.0) | 1 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 1 (50.0) | 2 (100.0) | 8 | ||
| Micafungin | 0 (0.0) | 1 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 0 (25.0) | 3 (100.0) | >8 | |
| Itraconazole | 0 (0.0) | 1 (25.0) | 3 (100.0) | 0.5 | ||||||||||
| Voriconazole | 0 (0.0) | 3 (75.0) | 1 (100.0) | 0.12 | ||||||||||
| Posaconazole | 0 (0.0) | 3 (75.0) | 1 (100.0) | 0.25 | ||||||||||
| Amphotericin B | 0 (0.0) | 2 (50.0) | 1 (75.0) | 1 (100.0) | 0.5 | |||||||||
| Geosmithia (Rasamsonia) argillacea (3) | ||||||||||||||
| Anidulafungin | 2 (66.7) | 1 (100.0) | < = 0.008 | |||||||||||
| Caspofungin | 0 (0.0) | 3 (100.0) | 0.015 | |||||||||||
| Micafungin | 2 (66.7) | 1 (100.0) | < = 0.008 | |||||||||||
| Itraconazole | 0 (0.0) | 1 (33.3) | 0 (33.3) | 0 (33.3) | 0 (33.3) | 2 (100.0) | >8 | |||||||
| Voriconazole | 0 (0.0) | 3 (100.0) | >8 | |||||||||||
| Posaconazole | 0 (0.0) | 1 (33.3) | 2 (100.0) | 2 | ||||||||||
| Amphotericin B | 0 (0.0) | 2 (66.7) | 1 (100.0) | 1 | ||||||||||
| Rhizopus microsporus group (9) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 1 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 8 (100.0) | >8 | ||
| Caspofungin | 0 (0.0) | 1 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 8 (100.0) | >8 | ||
| Micafungin | 0 (0.0) | 1 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 8 (100.0) | >8 | |
| Itraconazole | 0 (0.0) | 2 (22.2) | 5 (77.8) | 1 (88.9) | 0 (88.9) | 0 (88.9) | 1 (100.0) | 1 | ||||||
| Voriconazole | 0 (0.0) | 1 (11.1) | 0 (11.1) | 0 (11.1) | 0 (11.1) | 7 (88.9) | 1 (100.0) | 8 | ||||||
| Posaconazole | 0 (0.0) | 1 (11.1) | 0 (11.1) | 1 (22.2) | 3 (55.6) | 2 (77.8) | 2 (100.0) | 0.5 | ||||||
| Amphotericin B | 0 (0.0) | 1 (11.1) | 6 (77.8) | 2 (100.0) | 1 | |||||||||
| Sarocladium kiliense (6) | ||||||||||||||
| Anidulafungin | 0 (0.0) | 2 (33.3) | 2 (66.7) | 2 (100.0) | 8 | |||||||||
| Caspofungin | 0 (0.0) | 2 (33.3) | 2 (66.7) | 0 (66.7) | 0 (66.7) | 0 (66.7) | 0 (66.7) | 2 (100.0) | 0.25 | |||||
| Micafungin | 0 (0.0) | 1 (16.7) | 1 (33.3) | 1 (50.0) | 0 (50.0) | 1 (66.7) | 0 (66.7) | 0 (66.7) | 2 (100.0) | 0.5 | ||||
| Itraconazole | 0 (0.0) | 1 (16.7) | 0 (16.7) | 0 (16.7) | 0 (16.7) | 0 (16.7) | 0 (16.7) | 5 (100.0) | >8 | |||||
| Voriconazole | 0 (0.0) | 6 (100.0) | 2 | |||||||||||
| Posaconazole | 0 (0.0) | 1 (16.7) | 0 (16.7) | 0 (16.7) | 0 (16.7) | 0 (16.7) | 0 (16.7) | 5 (100.0) | >8 | |||||
| Amphotericin B | 0 (0.0) | 1 (16.7) | 5 (100.0) | >2 | ||||||||||
> represents the dilution greater than the highest dilution tested.
Among the non-Aspergillus molds, only Rasamsonia argillacea consistently exhibited echinocandin MECs of ≤0.06 μg/ml (Table 3). Notably, MEC values of ≥8 μg/ml were observed for the majority of the Fusarium and Mucormycetes species. Similarly, the triazoles showed poor activity against many of these rare molds.
Posaconazole stands apart from voriconazole, in that it appears to be active against some clinical isolates of the mucoraceous molds both in vitro and in vivo (14, 15). Among the 13 isolates of the Rhizopus microsporus group (9 isolates) and Rhizopus oryzae species complex (4) tested in the present study, the voriconazole MICs were ≥8 μg/ml, whereas the MICs for posaconazole were ≤1 μg/ml for 11 isolates (84.6%; Table 3).
The typical antifungal susceptibility profile of Fusarium spp. is that of relative resistance to most antifungal agents (14). Among the small number of Fusarium isolates tested in the present study, MIC values were generally >4 μg/ml for both posaconazole and voriconazole against the 14 isolates of Fusarium solani and Fusarium spp. (Table 3). Although voriconazole and posaconazole exhibited only modest activity in vitro against isolates of Fusarium, both triazoles have successfully been used in some patients with amphotericin B-refractory fusariosis (16, 17).
Scedosporium apiospermum/Scedosporium boydii is generally considered resistant to amphotericin B, to which the clinical response is very poor (18), whereas both posaconazole and voriconazole have successfully been used for the treatment of central nervous system abscesses (19, 20). Posaconazole MIC results ranged from 0.12 to 2 μg/ml and voriconazole MIC results ranged from 0.03 to 1 μg/ml against the 11 Scedosporium apiospermum/S. boydii isolates tested in the present study (Table 3). Similar activities for these two agents against Scedosporium aurantiacum were observed, whereas Scedosporium (Lomentospora) prolificans exhibited MIC values of >8 μg/ml (Table 3).
Geographic differences in susceptibility patterns.
The isolates tested in this study were geographically distributed and were collected from Europe (52.4%), North America (25.5%), the Asia-Pacific region (13.8%), and Latin America (8.3%). We analyzed the susceptibility/wild-type patterns for the 5 main Candida species and A. fumigatus isolates stratified by continent (Fig. 1).
FIG 1.
Geographic differences in susceptibility and wild-type profiles for the main fungal species tested.
Echinocandin susceptibility rates were 99.2 to 99.6% for C. albicans isolates collected in North America, but all isolates from other regions were susceptible to these compounds. Similarly, echinocandin-resistant C. glabrata isolates were more common in North America, and the rates of susceptibility to these agents among isolates from this region ranged from 90.7% to 93.0%. Echinocandin susceptibility rates for C. glabrata were 98.1% to 99.6% in Europe and 98.3% to 100.0% in the Asia-Pacific region. All isolates from Latin America were susceptible to the echinocandins.
Differences in the rates of susceptibility to anidulafungin among the C. parapsilosis isolates from the 4 regions analyzed were observed. Isolates from the Asia-Pacific region displayed 82.2% susceptibility to anidulafungin, whereas these rates were 84.4% in Latin America, 89.6% in Europe, and 90.4% in North America.
Azole resistance rates differed slightly by geographic regions for C. albicans, C. parapsilosis, and C. tropicalis. Azole-resistant C. albicans isolates were detected in North America and Europe but not in the Asia-Pacific region and Latin America. Additionally, resistance to azoles among the C. parapsilosis isolates was not observed in the Asia-Pacific region, and no azole-resistant C. tropicalis isolates were noted in North America, although resistance to azoles was detected for these species in other regions. C. glabrata isolates displaying azole resistance were observed worldwide, and slightly higher rates were noted in North America and the Asia-Pacific region than in Europe and Latin America.
Among the A. fumigatus isolates, azole (itraconazole, posaconazole, and voriconazole) non-wild-type isolates were observed in the Asia-Pacific region and Europe but not in Latin America and North America. There were no azole non-wild-type isolates detected among the other species of Aspergillus.
Investigation of resistance mechanisms in Candida.
All 20 Candida isolates displaying echinocandin MIC values higher than the ECVs recently established by CLSI were screened for mutations in HS regions of the 1,3-β-d-glucan-encoding gene(s). The majority of echinocandin non-wild-type isolates (15/20, 75.0%) were from North America (Table 4). Three C. albicans isolates were tested, and only the 2 isolates displaying resistant MIC results for caspofungin and micafungin (1 μg/ml for both) exhibited mutations leading to an FKS1 HS1 alteration (S645P; Table 4). These 2 isolates displayed anidulafungin MIC values of 0.25 μg/ml and 0.5 μg/ml. The remaining isolate displayed a non-wild-type MIC for micafungin (0.06 μg/ml) and had zero FKS1 HS mutations.
TABLE 4.
Summary of FKS alterations detected among Candida isolates displaying echinocandin MIC values above the ECV
| Country or U.S. state | Organism | MIC (μg/ml) by CLSI methoda |
1,3-β-d-Glucan synthase mutationsb |
|||||
|---|---|---|---|---|---|---|---|---|
| Anidulafungin | Caspofungin | Micafungin | FKS1 HS1 | FKS1 HS2 | FKS2 HS1 | FKS2 HS2 | ||
| Italy | Candida albicans | 0.12 | 0.12 | 0.06 (>ECV) | WT | WT | NT | NT |
| New York | Candida albicans | 0.25 | 1 (R) | 1 (R) | S645P | WT | NT | NT |
| Indiana | Candida albicans | 0.5 | 1 (R) | 1 (R) | S645P | WT | NT | NT |
| New York | Candida glabrata | 0.25 | 0.5 (R) | 0.03 | WT | WT | P667T | NT |
| New York | Candida glabrata | 0.5 (R) | 0.25 | 0.25 (R) | WT | WT | S663P | WT |
| Slovenia | Candida glabrata | 0.5 (R) | 0.25 | 0.06 (>ECV) | WT | WT | WT | WT |
| Colorado | Candida glabrata | 0.25 | 0.12 | 0.06 (>ECV) | WT | WT | WT | WT |
| Indiana | Candida glabrata | 2 (R) | 2 (R) | 0.5 (R) | F625S | WT | F659Y | WT |
| Israel | Candida glabrata | 2 (R) | 2 (R) | 2 (R) | WT | WT | F659S | WT |
| California | Candida glabrata | 2 (R) | 8 (R) | 2 (R) | S629P | WT | WT | WT |
| California | Candida glabrata | 0.25 | 0.5 (R) | 0.25 (R) | WT | WT | F659S | WT |
| Australia | Candida glabrata | 0.06 | 0.5 (R) | 0.015 | WT | WT | WT | WT |
| New York | Candida glabrata | 2 (R) | 2 (R) | 2 (R) | WT | WT | S663P | WT |
| Canada | Candida glabrata | 4 (R) | >8 (R) | 4 (R) | S629P | WT | S663P | WT |
| Canada | Candida glabrata | 4 (R) | >8 (R) | 4 (R) | S629P | WT | S663P | WT |
| Georgia | Candida glabrata | 2 (R) | 2 (R) | 0.5 (R) | F625S | WT | WT | WT |
| California | Candida glabrata | 1 (R) | 1 (R) | 0.25 (R) | F625S | WT | WT | WT |
| Colorado | Candida glabrata | 0.5 (R) | 0.25 | 0.03 | WT | WT | WT | WT |
| Virginia | Candida glabrata | 0.12 | 0.06 | 0.12 (>ECV) | WT | WT | WT | WT |
| Turkey | Candida tropicalis | 0.06 | 0.06 | 0.12 (>ECV) | WT | WT | NT | NT |
Fifteen C. glabrata isolates displayed non-wild-type results for anidulafungin and micafungin MIC values according to the recently published ECVs (12). Among these isolates, 11 (73.3% of the isolates screened) harbored FKS HS alterations, including FKS2 HS1 S663P (4 isolates), FKS1 HS1 S629P (3 isolates), FKS1 HS1 F625S (3 isolates), or FKS2 HS1 F659S/Y (3 isolates). Three isolates carried double mutations that were either FKS1 HS1 S629P/FKS2 HS1 S663P or FKS1 HS1 F625S/FKS2 HS1 F659Y. The MICs for the FKS1 HS1 S629P/FKS2 HS1 S663P double mutant isolates recovered from Canada were highly elevated for caspofungin (>8 μg/ml) and were 4 μg/ml for anidulafungin and micafungin; however, the C. glabrata isolate from Indiana, USA, carrying FKS1 HS1 F625S/FKS2 HS1 F659Y exhibited modestly elevated MIC values at 2 μg/ml for anidulafungin and caspofungin and 0.5 μg/ml for micafungin, suggesting that these alterations might not have a cumulative effect. One additional mutation in FKS2 HS1, P667T, was observed in an isolate from New York State, and the MIC values for this isolate were low for anidulafungin (0.25 μg/ml) and micafungin (0.03 μg/ml) and the isolate was resistant only to caspofungin (MIC, 0.5 μg/ml). Four C. glabrata isolates carried zero mutations in FKS. These isolates displayed low MIC values overall, but 3 displayed MIC results indicating resistance to caspofungin and anidulafungin.
The micafungin MIC value for one C. tropicalis isolate was higher than the current ECV, but the isolate did not harbor mutations in FKS1 HSs.
Five C. albicans isolates displaying fluconazole-nonsusceptible MIC results (susceptible dose dependent or resistant; MIC, ≥4 μg/ml) were submitted to whole-genome sequencing analysis to detect alterations in genes associated with azole resistance. All isolates were from different geographic regions: Romania, France, United Kingdom, and two U.S. states (Washington and New York) (Table 5). The isolate from New York displayed resistance to fluconazole and other azoles, and the remaining 4 isolates were resistant to fluconazole only.
TABLE 5.
Results for 5 C. albicans isolates displaying fluconazole MIC values of ≥4 μg/ml
| Country or U.S. state | MLSTa | MIC (μg/ml)b |
Gain-of-function alterationsc |
Relative mRNA expression levele |
||||||
|---|---|---|---|---|---|---|---|---|---|---|
| FLU | VOR | Erg11 | Erg3 | MDR promoter allele sequenced | Erg11 | MDR1 | CDR1 | CDR2 | ||
| Romania | 3212 | 4 | 0.015 | A351V, A353T | 0.52 | 11.21 | 0.23 | 2.13 | ||
| France | 3213 | 8 | 0.015 | 0.83 | 86.87 | 0.48 | 5.33 | |||
| United Kingdom | 3216 | 8 | 0.06 | 1.15 | 200.64 | 2.55 | 0.92 | |||
| Washington | 3214 | 8 | 0.12 | 0.80 | 0.58 | 1.84 | 27.74 | |||
| New York | 3215 | 64 | 1 | G450E, G464S | A/A | 1.07 | 0.28 | 1.15 | 8.24 | |
MLDT, multilocus sequence type.
FLU, fluconazole; VOR, voriconazole.
Only gain-of-function mutations previously associated with azole resistance are listed. For a complete list of alterations in Erg11, Erg3, UPC2, MRR1, MDR1, CDR1, CDR2, and TAC1, see Table S2 in the supplemental material.
Homozygous alleles (A/A) in the MDR promoter confer higher levels of MDR1 expression and resistance to fluconazole.
mRNA expression levels were quantified relative to those of the 18S rRNA gene. Underlined data indicate increased expression levels.
Gain-of-function mutations in Erg11 and Erg3 that were previously detected in azole-resistant C. albicans isolates were observed in 1 isolate each. Erg11 substitutions G450E and G464S were homozygous, and Erg3 alterations A351V and A353 were heterozygous. The isolates displaying Erg11 homozygous mutations displayed higher fluconazole MIC results (MIC, 64 μg/ml) and voriconazole MIC results (MIC, 1 μg/ml) than the isolate carrying Erg3 heterozygous alterations (MIC results, 4 μg/ml and 0.015 μg/ml, respectively). One of these isolates (from New York State, USA) also exhibited mutations in the MDR promoter alleles described by Bruzual and Kumamoto (21) (Table 4).
A complete list of the alterations detected in Erg11, Erg3, UPC2, MDR1, MMR1, CRD1, CDR2, and TAC1 is provided in Table S2 in the supplemental material.
Expression assays using mRNA preparations showed that MDR1 levels were 11 to 200 times greater than the baseline level in C. albicans ATCC 90028 in 3 isolates displaying fluconazole-nonsusceptible MIC results but susceptible voriconazole MIC values (Table 5). One of these isolates also carried the A351V and A353T heterozygous Erg3 alterations. The level of CDR2 expression was elevated (27 times greater than the baseline level) in 1 isolate displaying a fluconazole MIC of 8 μg/ml and a voriconazole MIC of 0.12 μg/ml.
DISCUSSION
Despite the low antifungal resistance rates among Candida and Aspergillus isolates, continuous monitoring of antifungal susceptibility patterns and continuous determination of an understanding of mechanisms of resistance to antifungal agents seem prudent. Reports of breakthrough infections (22), the increasing prevalence of uncommon species refractory to clinically available antifungal agents (23, 24), and emerging resistance mechanisms (4, 25) highlight the importance of local and global surveillance.
Antifungal ECVs have been used to monitor the emergence of resistance for surveillance initiatives. These values are method-specific criteria that can segregate wild-type isolates from isolates that are likely to carry a resistance mechanism, designated non-wild type (26). ECVs for several organism-antifungal agent combinations have been published in the literature over the past few years and have been determined from the results from single or multiple laboratories and by the application of different criteria for the inclusion of isolates and/or the method of ECV determination. In this study, we applied the ECVs recently published by CLSI in the M59 document (12) that were established using the same rigorous criteria and the determination method described in the M57 document (27).
The CLSI-published ECVs used in this study are available for amphotericin B and 7 Candida species, anidulafungin and micafungin and C. dubliniensis and C. lusitaniae isolates, and 5 Aspergillus species and various antifungal agents. Additionally, we applied ECVs for Candida species that have CLSI clinical breakpoints listed in the M27-S4 document. Although ECVs should not be used in lieu of the clinical breakpoint, the categorization obtained by the use of ECVs would be acceptable to promote the early detection of emerging resistance for epidemiological purposes in a surveillance study.
The echinocandin resistance rates among the Candida species in the collection described here were low overall, but resistance among C. glabrata isolates was noted in 3 geographic regions, with resistant isolates predominantly being seen in North America. We used ECVs to select isolates for screening for alterations in the FKS HS region, and all isolates displaying anidulafungin and/or micafungin MIC values above the ECVs were tested. Most isolates carrying FKS HS mutations displayed resistance to 1 or more of the echinocandins. Isolates exhibiting MIC values above the ECV but below the resistance breakpoints did not carry alterations in the FKS HS region. This finding does not reduce the importance of the ECVs but illustrates that isolates with elevated MIC results but no known resistance mechanisms might be categorized as non-wild type. It also emphasizes that the species-specific clinical breakpoints are very good at detecting these isolates, as suggested in the literature (6, 28–31).
Additional organism-antifungal agent combinations are being evaluated by CLSI, and that information will expand the ability to provide interpretations and monitor the emergence of resistance for less common species.
C. albicans is the leading cause of candidemia worldwide, and this organism is usually responsible for one-third of the cases of candidemia in the United States and European countries (1–3, 5, 7, 32). Resistance to azoles and echinocandins has been described among isolates belonging to this species, and although we observed low rates of resistance to fluconazole among the C. albicans isolates surveyed, we decided to investigate the causes of resistance in 5 fluconazole-nonsusceptible (MIC, ≥4 μg/ml) isolates due to the importance of this pathogen.
Azole resistance in C. albicans is associated with alterations to various genes/alleles, promoter regions, and/or transcriptional regulators that might lead to resistance to fluconazole only or all azoles (9, 10). One isolate in this collection was resistant to all azoles and had homozygous mutations in Erg11 that have been recognized to cause azole resistance in other studies (10). The remaining isolates displayed elevated fluconazole MIC values due to the overexpression of MDR1, encoding fluconazole resistance only. The resistance mechanisms in 3 of these isolates were aligned with the MIC results, but 1 isolate overexpressing CDR2 was noted. Expression of CDR2 promotes resistance to all azoles, and although the voriconazole MIC value for this isolate (0.12 μg/ml) was considered susceptible, it is 2 dilution steps above the ECV of 0.3 μg/ml for this drug-organism combination, indicating that this is a non-wild-type strain (12).
We have included the antifungal susceptibility profiles of those species from the SENTRY program that have undergone sequence-based identification in order to provide MIC data not only for the relatively common species of Candida and A. fumigatus but also for those that may be less frequently encountered but still pose problems with the selection of the optimal therapy. In doing so, we have identified decreased susceptibility to both echinocandin and triazoles in several additional species of Candida, non-Candida yeasts, and non-Aspergillus molds. Whereas the emergence of azole resistance among A. fumigatus isolates has been reported by others (3, 4), we identified only 0.8% of A. fumigatus isolates (from 3 different countries) that warranted further investigation regarding acquired resistance mechanisms (because the MIC was greater than the ECV).
Recent studies using whole-genome methods for characterizing fungal organisms (16) and antifungal resistance (33) have taken different approaches. We performed whole-genome sequencing and analyzed polymorphisms in all genes previously associated with azole resistance in this species. These results elucidated the resistance mechanisms in 2 of 5 fluconazole-nonsusceptible C. albicans isolates tested. The remaining isolates displayed elevated levels of expression of efflux systems that could be detected only by mRNA expression assays, which are cumbersome, lack interlaboratory reproducibility, and are difficult to implement.
Both echinocandin resistance in Candida (2, 8) and azole resistance in A. fumigatus (3, 4) have a very good correlation with the presence of mutations in FKS and CYP51, respectively, raising the possibility that a molecular approach to detect resistance might be possible for clinical laboratories. A similar approach to the detection of azole resistance in Candida will be more difficult, and further studies are needed to evaluate azole resistance mechanisms in representative numbers of clinical isolates. Meanwhile, susceptibility testing is required to confirm resistance to these antifungal agents.
In summary, the activity of clinically available antifungal agents used to treat IFIs indicated that their activity is stable when the results were compared to the results of previous surveys (3, 5, 7), but resistant/non-wild-type isolates were noted regardless of geographic region. Tools for monitoring the emergence of resistance, such as ECVs and its mechanisms using whole-genome sequencing, are useful and should be applied.
MATERIALS AND METHODS
Organisms.
A total of 3,557 consecutive nonduplicate clinical isolates of fungi were collected from 66 hospitals located in 29 countries as part of a global surveillance initiative. Isolates were collected from patients with bloodstream infections (n = 1,930 strains), hospitalized patients with pneumonia (n = 806), patients with skin and skin structure infections (n = 95), patients with intra-abdominal infections (n = 35), patients with urinary tract infections (n = 33), and other/unknown specimens (n = 658). Isolates were submitted to JMI Laboratories (North Liberty, IA, USA), where identification was confirmed by morphological, biochemical, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and molecular methods as previously described (5, 34).
Susceptibility testing.
Yeasts were tested for in vitro susceptibility to anidulafungin, caspofungin, micafungin, fluconazole, posaconazole, voriconazole, and amphotericin B using the CLSI reference broth microdilution method (35). MIC results for all agents were read following 24 h of incubation when their activity against Candida spp. was tested, whereas MIC endpoints for the triazoles were read after 48 h of incubation when their activity against Cryptococcus species was tested. The CLSI reference broth microdilution method was performed to test the in vitro susceptibility of Aspergillus spp. and other molds to anidulafungin, caspofungin, micafungin, itraconazole, posaconazole, voriconazole, and amphotericin B (35). The triazole MIC and echinocandin minimal effective concentration (MEC) endpoints were determined as described in the CLSI guidelines (35).
Quality control was performed as recommended in CLSI documents M27-A3 (36) and M38-A2 (35) using C. krusei ATCC 6258 and C. parapsilosis ATCC 22019, and all results were within established ranges. While testing the molds, the quality control strains Aspergillus flavus ATCC 204304 and Aspergillus fumigatus MYA-3626 were also tested (35).
Screening for FKS HS region mutations.
Candida isolates with MIC values greater than the ECV for the echinocandin compounds were submitted to PCR and sequencing of the hot spot regions of the FKS genes encoding the 1,3-β-d-glucan synthase subunits. PCR amplification was carried out using previously described oligonucleotides for FKS1 and FKS2 HS regions 1 and 2 (6, 37).
Whole-genome sequencing analysis of fluconazole-nonsusceptible C. albicans isolates.
Five C. albicans isolates displaying fluconazole MIC values of ≥4 μg/ml were submitted to whole-genome sequencing. Total genomic DNA was used as input material for construction of a library prepared using the Nextera XT library construction protocol and index kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. Sequencing was performed on a MiSeq sequencer (Illumina). Reads were error corrected using the BayesHammer algorithm, and each sample was assembled using a reference-guided assembly in DNAStar SeqMan NGen (v.14.0) software (Madison, WI, USA). The sequences of Erg11, Erg3, UPC2, MDR1 (including the MDR1 promoter region), MRR1, and TAC1 were compared to the sequences of those genes from C. albicans ATCC 90028. The CDR1 and CDR2 sequences were compared to the sequences of those genes from C. albicans ATCC 10261.
Analysis of Erg11, CDR1, CDR2, and MDR1 expression by C. albicans.
The expression of Erg11, CDR1, CDR2, and MDR1 was determined by quantitative real-time PCR (qRT-PCR) using high-quality DNA-free RNA preparations. Total RNA was extracted from 2 × 107 mid-log-phase yeast cells grown in Sabouraud liquid medium (cell density at an optical density at 600 nm of 600, 0.25 to 0.3). Cells were harvested, suspended in 2 ml freshly prepared Y1 buffer containing 0.1% β-mercaptoethanol and 150 units of lyticase, and incubated for 10 to 30 min at 30°C with gentle shaking to generate spheroplasts. RNA was extracted from the spheroplasts using an RNeasy minikit (Qiagen, Hilden, Germany). Residual DNA was eliminated with RNase-free DNase (Promega, WI, USA). Quantification of mRNA and sample quality were assessed using an RNA 6000 Pico kit on an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Only preparations that had an RNA integrity number (RIN) of >6.5 and that showed no visual degradation were used for the experiments. Relative quantification of target genes was performed in triplicate by normalization to the level of an endogenous reference gene (the 18S rRNA gene) on a StepOne Plus instrument (Life Technologies, Carlsbad, CA, USA) using custom-designed or previously published (38) primers showing ≥97.0% efficiency. Transcription levels were considered significantly different if a 10-fold difference from the level of transcription in C. albicans ATCC 90028 was noted.
Supplementary Material
ACKNOWLEDGMENTS
The antifungal global surveillance program that served as the source of data for this article was supported in part by Pfizer Inc. and Astellas Pharma Global Development, Inc.
M. Castanheira, L. M. Deshpande, A. P. Davis, P. R. Rhomberg, and M. A. Pfaller are employees of or consultants for JMI Laboratories who were paid consultants to Pfizer Inc. in connection with the development of this article. JMI Laboratories contracted to perform services in 2016 for Achaogen; Actelion; Allecra Therapeutics; Allergan; AmpliPhi Biosciences; API; Astellas Pharma; AstraZeneca; Basilea Pharmaceutica; Bayer AG; BD; Biomodels; Cardeas Pharma Corp.; CEM-102 Pharma; Cempra; Cidara Therapeutics, Inc.; CorMedix; CSA Biotech; Cutanea Life Sciences, Inc.; Debiopharm Group; Dipexium Pharmaceuticals, Inc.; Duke; Entasis Therapeutics, Inc.; Fortress Biotech; Fox Chase Chemical Diversity Center, Inc.; Geom Therapeutics, Inc.; GSK; Laboratory Specialists, Inc.; Medpace; Melinta Therapeutics, Inc.; Merck & Co.; Micromyx; MicuRx Pharmaceuticals, Inc.; Motif Bio; N8 Medical, Inc.; Nabriva Therapeutics, Inc.; Nexcida Therapeutics, Inc.; Novartis; Paratek Pharmaceuticals, Inc.; Pfizer; Polyphor; Rempex; Scynexis; Shionogi; Spero Therapeutics; Symbal Therapeutics; Synlogic; TenNor Therapeutics; TGV Therapeutics; The Medicines Company; Theravance Biopharma; Thermo Fisher Scientific; VenatoRx Pharmaceuticals, Inc.; Wockhardt; Zavante Therapeutics, Inc. We have no speakers' bureaus or stock options to declare.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00906-17.
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