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Home > Chemial News > Pharma News > Rezafungin In Vitro Activity against Contemporary Nordic Clinical Candida Isolates and Candida auris Determined by the EUCAST Reference Method

Rezafungin In Vitro Activity against Contemporary Nordic Clinical Candida Isolates and Candida auris Determined by the EUCAST Reference Method

Marie Helleberg 2021-07-22

INTRODUCTION


Echinocandins are the recommended first-line treatment for candidemia and invasive candidiasis. The currently available echinocandins—anidulafungin, caspofungin, and micafungin—are dosed intravenously once daily. Echinocandins target the enzyme complex 1,3-β-d-glucan synthase, which is comprised of two subunits: a regulatory GTP-binding protein, Rho1, and a catalytic component, Fks, which is encoded by three highly homologous genes, FKS1, FKS2, and FKS3. Alterations in the hot spots of Fks1 and Fks2 (Candida glabrata only) result in reduced sensitivity to echinocandins and elevated MIC values across various Candida species (1).


Rezafungin is a novel echinocandin that is currently in phase 3 development. It has activity against Aspergillus and Candida species, including C. auris, an emerging pathogen with high rates of drug resistance that spreads readily in health care facilities and can cause severe nosocomial infections (2).


Rezafungin is a structural analogue of anidulafungin with a choline moiety at the C-5 ornithine position, which results in increased stability (3). The safety and pharmacokinetic (PK) profile, with a long half-life of approximately 130 h, allow for once-weekly dosing (4). The epidemiological cutoff values and clinical breakpoints for rezafungin against common Candida species have not yet been established due to interlaboratory variation in MIC values for the Candida species with the highest susceptibility to echinocandins (such as C. albicans and C. tropicalis) (5).


The objectives of this study were (i) to examine the in vitro activity of rezafungin against a large sample of contemporary clinical isolates of Candida and other yeasts from Nordic countries and clinical isolates of C. auris from India, (ii) to determine the pattern of rezafungin resistance mutations in contemporary clinical non-wild-type Candida isolates, and (iii) to examine how fks mutations affect susceptibility to rezafungin and the degree of cross-resistance between rezafungin and other echinocandins.

 

RESULTS


Rezafungin against quality control strains.


C. albicans CNM-CL F8555, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 were tested 19, 81, and 108 times, respectively, during the study period. The MIC results for C. albicans CNM-CL F8555 were as follows: modal MIC, 0.03 mg/liter; MIC50, 0.03 mg/liter; range, 0.03 to 0.06 mg/liter. The MICs against C. parapsilosis ATCC 22019 were as follows: modal MIC, 2 mg/liter; MIC50, 2 mg/liter; range, 0.5 to 2 mg/liter. The MICs against C. krusei ATCC 6258 were as follows: modal MIC, 0.125 mg/liter; MIC50, 0.125 mg/liter; range, 0.03 to 0.125 mg/liter. Thus, for all three quality control (QC) strains, the range spanned ≤3 2-fold dilutions.


Rezafungin in vitro activity against Nordic clinical yeast isolates.


Rezafungin MIC distributions for the most common Candida species are shown in Table 1. Rezafungin had in vitro activity, with MIC90s of <0.5 mg/liter for all the most common Candia species except C. parapsilosis, where the MIC range was 1 to 4 mg/liter. The modal MICs for C. albicans and C. glabrata were 0.06 and 0.125 mg/liter, respectively. The statistical wild-type upper limits (WT-UL) (99%) were 0.25 mg/liter for most Candida species except C. albicans (0.125 mg/liter) and C. parapsilosis (4 mg/liter). The visual WT-UL was within one dilution of the statistical WT-UL for all species.

 

Rezafungin in vitro activity against Candida auris.


The rezafungin modal MIC for Indian clinical C. auris was 0.25 mg/liter (Table 1), which is two and one 2-fold dilutions higher than for C. albicans and C. glabrata, respectively. The rezafungin visual WT-UL and statistical WT-UL (97.5% and 99%) were 0.5 mg/liter.

The in vitro activity of rezafungin was similar to those of anidulafungin and micafungin (modal MIC, one 2-fold dilution higher). Rezafungin was more active than amphotericin B and fluconazole on a milligram-per-liter basis. Voriconazole and isavuconazole displayed bimodal and trimodal distributions, making comparisons with these drugs complex (Table 3).

 

Resistance mutations in non-wild-type strains.


We identified 18 (14.8%) rezafungin non-wild-type strains among the Indian C. auris isolates and 26 (2.1%) among the most common Nordic Candida strains: C. albicans (n = 11; 1.9%), C. glabrata (n = 10; 3.3%), C. tropicalis (n = 2; 2.7%), C. dubliniensis (n = 2; 2.9%), and C. krusei (n = 1; 1.2%) (Table 1). Alterations in Fks1 and/or Fks2 hot spots (or within ±3 amino acids) were found in all 26 Nordic isolates and in 8/18 C. auris non-wild-type isolates. The Fks1/Fks2 hot spot mutations, as well as the MICs of rezafungin and comparators, are summarized in Table 4. Rezafungin MICs for isolates with fks hot spot mutations were 0.25 to 2 mg/liter for the Nordic isolates but 8 to 16 mg/liter for C. auris isolates. The increase in rezafungin MICs (number of 2-fold dilutions) conferred by substitution of phenylalanine for serine in Fks1 or Fks2 hot spots was 2 to 4 for C. glabrata and C. krusei but 5 to 6 for C. auris. The increase in MIC conferred by the S639F mutation in C. auris was even more marked for anidulafungin and micafungin (>8 2-fold dilutions).

 

Rezafungin in vitro activity against Nordic clinical isolates.


Rezafungin had species-specific in vitro activity similar to that of anidulafungin and micafungin and was more active than fluconazole on a milligram-per-liter basis against the most common Candida spp., except C. parapsilosis.

 

The proportion of Candida spp. with rezafungin MICs above the WT-UL was low (<3%) and, for C. albicans isolates, significantly lower than for micafungin. Although the proportion of C. albicans isolates with micafungin MICs greater than the ECOFF was higher than the proportion with rezafungin MICs greater than the WT-UL, the data did not indicate that the C. albicans isolates were less susceptible or had higher rates of resistance to micafungin than to rezafungin (Table 5). This difference is likely explained by technical issues, as the mode of the micafungin MIC distribution in our study was in general one 2-fold dilution higher than that in the data set that formed the basis for the EUCAST ECOFF setting, which confers a risk of misclassification of some wild-type isolates as resistant. The echinocandins are highly potent compounds with a tendency to adhere to plastic. Microdilution testing of echinocandins has previously been associated with variation, as previously reported for caspofungin, anidulafungin, and rezafungin, and the data found here suggest that may also be the case occasionally for micafungin, a challenge that needs attention (6,–8).

 

Rezafungin in vitro activity against Candida auris.

Rezafungin had in vitro activity against clinical C. auris isolates; however, approximately 15% of the isolates were classified as non-wild type using a statistical 97.5% endpoint, and 8 (6.6%) harbored Fks1 hot spot alterations and had high MICs of 8 to 16 mg/liter. These mutant strains were also resistant to other echinocandins and fluconazole but susceptible to amphotericin B. Our results are in accordance with a study by Berkow and Lockhart showing in vitro activity of rezafungin against C. auris isolates but echinocandin cross-resistance of those harboring the S639P alteration at the same codon, using the CLSI methodology (12). However, that study did not include comparisons with azoles or amphotericin B.

In a previous study based on a neutropenic mouse model, the estimated MIC ceiling to achieve 1-log-kill target exposures against C. auris was 1 to 2 mg/liter, and for the stasis target it was 2 to 4 mg/liter (13). In the present study, a total of 14.8% of the tested C. auris isolates had an MIC of ≥1 mg/liter, and 9% had an MICof ≥2 mg/liter. Thus, a significant proportion of the clinical C. auris isolates had MICs that were at or above the above-mentioned estimated MIC ceilings. The MICs for anidulafungin and micafungin against these isolates were even higher, suggesting that liposomal amphotericin B may be a more appropriate choice for empirical treatment of severe C. auris infections until results of susceptibility testing are available in regions where this fks genotype is prevalent.

 

Resistance mutations in non-wild-type strains.

Among the Nordic Candida strains, only 26 non-wild-type isolates with mutations in fks target genes were detected. Locke et al. found that there was a low potential for resistance development for rezafungin, similar to other echinocandins, and that rezafungin-selected strains had MICs that were lower than or equal to those for the majority of strains generated under selection with anidulafungin and caspofungin (14). We found cross-resistance between rezafungin and the comparator echinocandins. Generally, the relative increase in rezafungin MICs conferred by fks mutations was comparable to or slightly less than those for anidulafungin and micafungin. In addition, it was codon dependent, with the most pronounced MIC elevations for alterations involving S645 in C. albicans and C. dubliniensis and the corresponding codon in the other Candida species (S663 in C. glabrata, S654 in C. tropicalis, and S659 in C. krusei). Of note, a differential impact on susceptibility was observed for the Fks1 hot spot S639F substitution in C. auris. Although this mutation caused significant elevation in MICs for rezafungin, in agreement with a study by Berkow and Lockhart showing a significant MIC elevation of isolates with an S639P alteration, the MIC elevation was more than three and four 2-fold-dilutions greater for the two other echinocandins. These findings suggest that the MIC elevation depends on the codon involved, the species in question, and the substitution, as previously suggested for other Candida spp. (15, 16).

In conclusion, rezafungin displayed broad in vitro activity against Candida spp., including C. auris. Adoption of center-specific WT-UL allowed a reliable categorization of isolates as wild type or non-wild type, which was supported by molecular analysis, an approach that may be helpful until official ECOFFs and clinical breakpoints are established. Few Nordic strains, but a notable proportion of the Indian C. auris isolates, had elevated MICs due to a mutation in the fks1 target gene that conferred echinocandin cross-resistance. fks1 mutations raised rezafungin MICs slightly less than the anidulafungin and micafungin MICs in most Candida spp., but notably less in C. auris.

 

MATERIALS AND METHODS

Isolates.

A total of 1,293 clinical yeast isolates (19 Candida and 13 other yeast species) received at the Danish mycology reference laboratory in 2017-2018 were included in the study. Of these 1,293 isolates, 1,226 were from Denmark (841 blood isolates and 385 isolates from tissue, pus, or swabs) and 67 were isolates referred from other Nordic countries due to clinical suspicion of antifungal resistance (Norway, 32; Sweden, 32; Greenland, 1; and Faroe Islands, 2). The species distribution (number of isolates) was as follows: C. albicans (569), C. dubliniensis (68), C. glabrata (328), C. krusei (82), C. lusitaniae (20), C. parapsilosis (61), C. tropicalis (73), other Candida spp. (41), and other yeast species (36). In addition, 122 clinical isolates of C. auris were collected from individual patients in six tertiary-care hospitals in India from 2010 to 2015. The isolates were mainly obtained from blood from patients with candidemia (n = 100) and samples from patients with other invasive Candida infections (n = 22), which included tissue, pleural fluid, and pus.

 

Species identification.

Identification methods, including the use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (Bruker, Bremen, Germany), were performed as previously described (15), with the addition of DNA sequencing as described below when needed.

 

Susceptibility testing.

Rezafungin (Cidara Therapeutics, San Diego, CA, USA) pure substance was stored in aliquots at −70 to −80°C, and stock solutions were prepared in dimethyl sulfoxide (DMSO) (5,000 mg/liter; Sigma-Aldrich, Brøndby, Denmark). EUCAST MICs were determined following E.Def 7.3.1 methodology (17). The final drug concentration range studied was 0.004 to 4 mg/liter, except for C. auris (range, 0.004 to 256 mg/liter). The following comparator compounds were also investigated (final concentration range and source of compound in parentheses): anidulafungin (0.004 to 4 mg/liter; Pfizer A/S, Ballerup, Denmark), micafungin (0.004 to 4 mg/liter; Astellas Pharma Inc., Tokyo, Japan), amphotericin B (0.004 to 4 mg/liter; Sigma-Aldrich), fluconazole (0.03 to 32 mg/liter for Nordic isolates and 0.5 to 256 mg/liter for C. auris; Sigma-Aldrich), and voriconazole (0.004 to 4 mg/liter; Pfizer A/S, Ballerup, Denmark). Cell culture-treated microtiter plates (Nunc MicroWell 96-well microplates; catalog no. 167008; Thermo Fisher Scientific) were used throughout. The microtiter plates were prepared with 2-fold drug dilutions in double-concentration medium according to the EUCAST methodology and frozen at −80°C prior to use. The EUCAST QC strains C. albicans CNM-CL F8555, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 were tested in parallel.

 

Molecular identification and fks gene sequence analysis.

Sequencing of internal transcribed spacer regions ITS1 and ITS2 was performed, and DNA sequence analysis of echinocandin target hot spots in fks1, and for C. glabrata also in fks2, was performed for non-wild-type isolates as previously described (18). The sequences obtained were compared to relevant reference sequences.

 

Data management.

MIC ranges and modal MIC (the most common MIC), MIC50, and MIC90 values were calculated. EUCAST ECOFFs and EUCAST clinical breakpoints for fungi (version 9.0) were adopted for wild-type and susceptibility classification where available. The WT-UL, defined as the upper MIC value where the wild-type distribution ends, were determined for rezafungin following principles for setting EUCAST ECOFFs. However, as the values reported here are not formally accepted EUCAST rezafungin ECOFFs, we used the term “WT-UL” to avoid confusion. The conventional method for determining the ECOFF is a visual inspection of histograms of the MICs for single species (the eyeball method) (19). Additionally, WT-UL were determined statistically using 97.5% and 99% endpoints and the EUCAST ECOFFinder program (19). A “non-wild-type” isolate was defined as an isolate with an MIC above the statistical WT-UL (97.5%).

 

Yeast

n

No. with MIC (mg/liter)a:

 


 

MIC range (mg/liter)

MIC50 (mg/liter)

MIC90 (mg/liter)

WT-ULb (mg/liter)

 


 

0.016

0.03

0.06

0.125

0.25

0.5

1

2

4

≥8

Visual

97.5%

99%

Nordic isolates

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 C. albicans

569

28

221

282

27

8

2

1

 

 

 

0.016 to 1

0.06

0.06

0.125

0.125

0.125

 C. glabrata

328

 

1

87

214

16

5

1

4

 

 

0.03 to 2

0.125

0.125

0.25

0.25

0.25

 C. krusei

82

 

2

26

47

6

 

1

 

 

 

0.03 to 1

0.125

0.125

0.25

0.25

0.25

 C. tropicalis

73

 

2

19

43

7

 

1

1

 

 

0.03 to 2

0.125

0.25

0.25

0.25

0.25

 C. dubliniensis

68

 

8

28

29

1

 

1

1

 

 

0.03 to 2

0.06

0.125

0.25

0.25

0.25

 C. parapsilosis

61

 

 

 

 

 

 

8

40

13

 

1 to 4

2

4

4

4

4

 C. lusitaniae

20

 

 

 

10

8

2

 

 

 

 

0.125 to 0.4

0,125

0,25

0.5

0.25

0.25

 S. cerevisiae

15

 

 

 

6

8

1

 

 

 

 

0.125 to 0.5

0.25

0.25

0.5

0.5

0.5

    Other Candida

55

 

2

16

7

1

11

11

6

 

1

0.03 to >4

0.5

2

ND

ND

ND

    Other yeast

22

 

2

2

1

 

 

1

 

2

14

0.03 to >4

>4

>4

ND

ND

ND

Indian isolates

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    C. auris

122

 

 

3

22

63

16

7

3

 

8

0.06 to 16

0.25

1

0.5

0.5

0.5

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