Comparison of EUCAST and CLSI broth microdilution methods for the susceptibility testing of 10 systemically active antifungal agents when tested against Candida spp.

Diagnostic Microbiology and Infectious Disease 79 (2014) 198–204

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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio

Mycology

Comparison of EUCAST and CLSI broth microdilution methods for the susceptibility testing of 10 Systemically active antifungal agents when tested against Candida spp. Michael A. Pfaller ⁎, Mariana Castanheira, Shawn A. Messer, Paul R. Rhomberg, Ronald N. Jones JMI Laboratories, North Liberty, Iowa

a r t i c l e

i n f o

Article history: Received 9 September 2013 Received in revised form 27 February 2014 Accepted 5 March 2014 Available online 17 March 2014 Keywords: EUCAST CLSI Candida Susceptibility testing

a b s t r a c t The antifungal broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was compared with Clinical and Laboratory Standards Institute (CLSI) BMD method M27-A3 for amphotericin B, flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole susceptibility testing of 357 isolates of Candida. The isolates were selected from global surveillance collections to represent both wild-type (WT) and non-WT MIC results for the azoles (12% of fluconazole and voriconazole results were non-WT) and the echinocandins (6% of anidulafungin and micafungin results were non-WT). The study collection included 114 isolates of Candida albicans, 73 of C. glabrata, 76 of C. parapsilosis, 60 of C. tropicalis, and 34 of C. krusei. The overall essential agreement (EA) between EUCAST and CLSI results ranged from 78.9% (posaconazole) to 99.6% (flucytosine). The categorical agreement (CA) between methods and species of Candida was assessed using previously determined CLSI epidemiological cutoff values. The overall CA between methods was 95.0% with 2.5% very major (VM) and major (M) discrepancies. The CA was N93% for all antifungal agents with the exception of caspofungin (84.6%), where 10% of the results were categorized as non-WT by the EUCAST method and WT by the CLSI method. Problem areas with low EA or CA include testing of amphotericin B, anidulafungin, and isavuconazole against C. glabrata, itraconazole, and posaconazole against most species, and caspofungin against C. parapsilosis, C. tropicalis, and C. krusei. We confirm high level EA and CA (N90%) between the 2 methods for testing fluconazole, voriconazole, and micafungin against all 5 species. The results indicate that the EUCAST and CLSI methods produce comparable results for testing the systemically active antifungal agents against the 5 most common species of Candida; however, there are several areas where additional steps toward harmonization are warranted. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The need for reproducible, clinically relevant antifungal susceptibility testing of Candida spp. has been prompted by the increasing number of infections, the expanding use of new and established antifungal agents, and the recognition of antifungal resistance as an important clinical problem (Arendrup et al., 2013; Cleveland et al., 2012; Kett et al., 2011; Ostrosky-Zeichner, 2013; Pakyz et al., 2011; Pfaller, 2012). The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antifungal Susceptibility Tests has standardized the broth microdilution (BMD) reference method for testing amphotericin B, flucytosine, the triazoles (including the investigational agent isavuconazole), and the echinocandins, against Candida spp. (CLSI, 2008b; 2012; Pfaller et al., 2012b, 2013b) and, most recently, has validated 24-h MIC readings for all agents (CLSI, 2012; Pfaller and Diekema, 2012; Pfaller et al., 2010a, 2011b, 2011c, 2012b, 2013b) and ⁎ Corresponding author. Tel.: +1-319-665-3370; fax: +1-319-665-3371. E-mail address: [email protected] (M.A. Pfaller). http://dx.doi.org/10.1016/j.diagmicrobio.2014.03.004 0732-8893/© 2014 Elsevier Inc. All rights reserved.

developed new species-specific clinical breakpoints (CBPs) (CLSI, 2012; Pfaller and Diekema, 2012; Pfaller et al., 2010a, 2011a, 2011d) and epidemiological cutoff values (ECVs) (Pfaller and Diekema, 2012; Pfaller et al., 2010b, 2010c, 2011b, 2012b) for these agents and several species of Candida. The new CBPs and ECVs replace the previously published non-species-specific CBPs for all of these agents (CLSI, 2008a), which were observed to lack sensitivity in discriminating wildtype (WT) strains of Candida (lack acquired or mutational resistance mechanisms) from non-WT strains (possess intrinsic or acquired resistance mutations) or where lack of clinical data precluded the establishment of CBPs (Pfaller, 2012; Pfaller and Diekema, 2012). In addition to the CLSI BMD method, the only other international standard method for antifungal susceptibility testing of yeasts is that of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Arendrup et al., 2012). The similarities and differences (minor) between the 2 BMD methods have been discussed previously (Alastruey-Izquierdo and Cuenca-Estrella, 2012; Espinel-Ingroff et al., 2005; Espinel-Ingroff et al., 2013a; Rodriguez-Tudela et al., 2007). The 2 methods have been harmonized so that there is a close agreement

M.A. Pfaller et al. / Diagnostic Microbiology and Infectious Disease 79 (2014) 198–204

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between MIC results when testing fluconazole and voriconazole against Candida to the extent that there are common CBPs for the 2 methods for some species of Candida (Arendrup et al., 2012; EspinelIngroff et al., 2013a; Pfaller and Diekema, 2012). Due to the international importance of these 2 methods in clinical testing and surveillance of antifungal resistance, there is a need to continue the process of harmonization for the testing of other new and established antifungal agents. In the present study, we examine the essential agreement (EA; MIC ± 2 log2 dilutions) between the 2 standardized methods for testing 10 antifungal agents (amphotericin B, flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole) against a collection of 357 clinical isolates of Candida selected to provide both WT and non-WT MIC phenotypes (using CLSI methods and ECVs) for most agents and species. We also provide an estimate of categorical agreement (CA; susceptibility results that fall within the same interpretive category) between the 2 methods by using the ECVs previously determined for each antifungal agent and species of Candida (Pfaller and Diekema, 2012) to categorize the isolates as WT (MIC, ≤ECV) or non-WT (MIC N ECV) as determined by each method.

amphotericin B) or in ≥50% (all other agents) inhibition of growth relative to that of the growth control.

2. Materials and methods

Table 1 ECVs for systemically active antifungal agents and Candida spp. determined by 24-h CLSI broth microdilution methods.a

2.1. Organisms A total of 357 clinical isolates of Candida spp. were selected from global surveillance collections (Pfaller et al., 2011f, 2013b, 2013c) to represent both WT and non-WT MIC results for the azoles (12.6% of fluconazole results were non-WT) and the echinocandins (6.4% of anidulafungin and micafungin results were non-WT). The study collection encompassed 5 species of Candida, including Candida albicans (114 isolates), C. glabrata (73 isolates), C. parapsilosis (76 isolates), C. tropicalis (60 isolates), and C. krusei (34 isolates). Species identification was established using Vitek (bioMerieux, Hazelwood, Missouri, USA), conventional reference methods (Howell and Hazen, 2011), and 28S and internal transcribed spacer (ITS) sequencing as described elsewhere (Pfaller et al., 2012c). The isolates were stored as water suspensions until used in the study. Prior to testing, each isolate was passaged at least twice onto potato dextrose agar (Remel) and CHROMagar Candida medium (Becton Dickinson and Company, Sparks, MD, USA) to ensure purity and viability. 2.2. Antifungal susceptibility testing All isolates were tested for in vitro susceptibility to amphotericin B, flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, isavuconazole, itraconazole, posaconazole and voriconazole using the CLSI (CLSI, 2012) and EUCAST(EUCAST, 2013) BMD methods. Reference powders of each agent were obtained from their respective manufacturers. Personnel performing the in vitro susceptibility studies were blinded to the results of the CLSI method compared to the EUCAST method. CLSI BMD testing was performed exactly as outlined in document M27-A3 (CLSI, 2008b) by using round-bottom trays and RPMI 1640 medium with 0.2% glucose, inocula of 0.5 × 103 to 2.5 × 103 cells/ml, and incubation at 35 °C. MIC values were determined visually after 24-h incubation as the lowest concentration of drug that caused complete inhibition (amphotericin B) or a significant diminution (≥50% inhibition; all other agents) of growth relative to that of the growth control. EUCAST BMD testing was performed exactly as outlined in document EDef 7.2 (Arendrup et al., 2012) by using flat-bottom trays and RPMI1640 medium with 2.0% glucose, inocula of 0.5 × 10 5 to 2.5 × 10 5 cells/ml, and incubation at 35 °C. MIC values were determined spectrophotometrically (at 490 nm), after 24-h incubation, as the lowest concentration of drug that resulted in complete (100%,

2.3. Quality control Quality control was performed as recommended in CLSI document M27-A3 (CLSI, 2008b) using C. krusei ATCC 6258 and C. parapsilosis ATCC 22019. 2.4. Analysis of results The MIC results for each triazole obtained with the EUCAST method were compared to those of the CLSI BMD method. High offscale BMD MIC results were converted to the next highest concentration and low off-scale MIC results were left unchanged. Discrepancies of more than 2 dilutions among MIC results were used to calculate the EA. The recently described CLSI ECVs for each agent and species (Table 1) were used to obtain CA percentages between the MIC values determined with the EUCAST method and those determined by the CLSI method. The ECVs were determined by the

Species

Antifungal agent

No. of isolates

ECV (μg/mL) WT

non-WT

C. albicans

Amphotericin B Flucytosine Anidulafungin Caspofungin Micafungin Fluconazole Itraconazole Posaconazole Voriconazole Amphotericin B Flucytosine Anidulafungin Caspofungin Micafungin Fluconazole Itraconazole Posaconazole Voriconazole Amphotericin B Flucytosine Anidulafungin Caspofungin Micafungin Fluconazole Itraconazole Posaconazole Voriconazole Amphotericin B Flucytosine Anidulafungin Caspofungin Micafungin Fluconazole Itraconazole Posaconazole Voriconazole Amphotericin B Flucytosine Anidulafungin Caspofungin Micafungin Fluconazole Itraconazole Posaconazole Voriconazole

9,252 8,007 4,283 4,283 4,283 8,059 14,716 8,619 8,619 3,117 3,387 1,236 1,236 1,236 2,240 5,769 2,415 2,415 3,107 3,165 1,238 1,238 1,238 2,117 4,894 2,278 2,279 2,062 2,046 996 996 996 1,771 3,624 1,895 1,895 577 499 270 270 270 473 809 508 507

≤2 ≤0.5 ≤0.12 ≤0.12 ≤0.03 ≤0.5 ≤0.12 ≤0.06 ≤0.03 ≤2 ≤0.5 ≤0.25 ≤0.12 ≤0.03 ≤32 ≤2 ≤2 ≤0.5 ≤2 ≤0.5 ≤4 ≤1 ≤4 ≤2 ≤0.5 ≤0.25 ≤0.12 ≤2 ≤0.5 ≤0.12 ≤0.12 ≤0.12 ≤2 ≤0.5 ≤0.12 ≤0.06 ≤2 ≤32 ≤0.12 ≤0.25 ≤0.12 ≤64 ≤1 ≤0.5 ≤0.5

N2 N0.5 N0.12 N0.12 N0.03 N0.5 N0.12 N0.06 N0.03 N2 N0.5 N0.25 N0.12 N0.03 N32 N2 N2 N0.5 N2 N0.5 N4 N1 N4 N2 N0.5 N0.25 N0.12 N2 N0.5 N0.12 N0.12 N0.12 N2 N0.5 N0.12 N0.06 N2 N32 N0.12 N0.25 N0.12 N64 N1 N0.5 N0.5

C. glabrata

C. parapsilosis

C. tropicalis

C. krusei

a

Data compiled from refs (Pfaller et al. 2010a, 2011b, 2011d, 2012b).

200


Table 2 In vitro susceptibilities of Candida spp. to 10 systemically active antifungal agents as determined by the CLSI and EUCAST broth microdilution methods. Antifungal agent

Species (no. tested)

Test method

MIC (μg/mL) Range

Mode

Amphotericin B

C. albicans (114)

CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST

0.12 -1 0.03 - 2 0.12 - 2 0.03–1 0.25–1 0.12–1 0.25–2 0.12–1 0.5–2 0.12–2 0.25–1 0.5–2 0.5–8 0.5–8 0.5 0.5 0.5–32 0.5–32 8–32 4–8 0.008–2 0.008–0.5 0.015–4 0.008–2 0.12–4 0.25–4 0.008–2 0.008–0.5 0.03–2 0.008–1 0.008–16 0.008–8 0.03–16 0.06–16 0.06–4 0.12–2 0.015–4 0.06–4 0.06–8 0.12–16 0.008–0.06 0.008–0.06 0.008–2 0.008–0.5 0.25–2 0.25–4 0.015–1 0.008–0.5 0.015–0.25 0.03–0.25 0.06 to N64 0.06 to N64 0.5 to N64 0.25 to N64 0.12 to N64 0.12 to N64 0.12 to N64 0.25 to N64 16 to N64 16 to N64 0.008–0.12 0.008–0.06 0.03–8 0.008–8 0.008–0.25 0.008–0.25 0.008–16 0.008–16 0.12–0.5 0.25–1 0.008–1 0.008–0.5

0.5 0.12 0.5 0.12 1 0.25 1 0.25 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 16 8 0.015 0.008 0.12 0.008 2 1 0.03 0.008 0.06 0.03 0.03 0.06 0.03 0.12 0.25 1 0.03 0.12 0.25 0.5 0.015 0.008 0.015 0.008 1 0.5 0.03 0.008 0.12 0.06 0.012 0.25 8 4 0.5 0.5 0.25 0.5 32 32 0.015 0.015 0.5 0.12 0.03 0.06 0.03 0.06 0.25 0.5 0.03 0.008

C. glabrata (73) C. parapsilosis (76) C. tropicalis (60) C. krusei (34) Flucytosine

C. albicans (89) C. glabrata (45) C. parapsilosis (48) C. tropicalis (31) C. krusei (17)

Anidulafungin


Caspofungin


Micafungin


Fluconazole


Isavuconazole


Itraconazole

C. albicans (114)

Table 2 (continued) Antifungal agent

EA (%)

Species (no. tested)

Test method

MIC (μg/mL)

C. glabrata (73)

CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST

0.06–8 0.008–8 0.008–1 0.015–0.25 0.015–8 0.008–8 0.12–2 0.06–0.5 0.008–1 0.008–0.5 0.06–4 0.008–8 0.015–0.5 0.008–0.12 0.015–8 0.008–8 0.12–2 0.03–0.5 0.008–2 0.008–2 0.008–4 0.008–8 0.008–1 0.008–1 0.008–16 0.015–8 0.06–1 0.12–1

83.3

C. parapsilosis (76)

75.3

C. tropicalis (60)

98.7

C. krusei (34)

93.3

Posaconazole

C. albicans (110) C. glabrata (65)

91.2


100.0 100.0

C. tropicalis (55)

100.0

C. krusei (29)

100.0 94.1

Voriconazole

C. albicans (114) C. glabrata (73)

82.5


45.2

C. tropicalis (60)

97.4

C. krusei (34)

Range

EA (%) Mode 1 0.25 0.12 0.03 0.06 0.03 0.25 0.12 0.03 0.008 1 0.25 0.06 0.015 0.03 0.03 0.25 0.06 0.008 0.008 0.25 0.12 0.015 0.015 0.03 0.03 0.25 0.5

83.6 71.1 88.3 85.3 87.3 75.4 65.8 89.1 69.0 99.1 97.3 100.0 93.3 100.0

93.3 91.2 95.6 94.5 98.7 91.7 97.1

method of Turnidge et al. (2006) and can be used as the most sensitive measure of the emergence of strains with reduced susceptibility to a given agent (Pfaller, 2012; Pfaller and Diekema, 2012). Very major (VM) discrepancies were identified when the CLSI BMD MIC was greater than the ECV for each agent and species, and when the EUCAST BMD MIC was less than or equal to the ECV. Major (M) discrepancies were identified when the isolates BMD MIC was greater than the ECV by the EUCAST method and less than or equal to the ECV by the CLSI method.

100.0

3. Results and discussion 100.0 100.0 94.4 100.0 99.1 95.9 98.7 100.0 100.0 100.0 69.0 100.0 89.3 100.0 81.6

Table 2 summarizes the in vitro susceptibilities of 357 isolates of Candida to the 10 systemically active antifungal agents as determined by the CLSI and EUCAST BMD methods. The MIC results were typical for each species and antifungal agent albeit enriched for non-WT strains for fluconazole and the echinocandins (Cleveland et al., 2012; Pfaller et al., 2012b, 2013b, 2013c). The EUCAST MIC results tended to be ≥1 doubling dilution lower than those determined by the CLSI method for most agents and species with the exception of caspofungin, where the EUCAST MIC results tended to be one or 2 doubling dilutions higher than the CLSI results. The overall EA between the EUCAST and CLSI methods ranged from a low of 78.9% (posaconazole) to 99.6% (flucytosine) (data not shown). Of the discrepancies noted between the EUCAST and CLSI BMD results, the MIC values generated by the CLSI method were higher than those obtained by the EUCAST method for 60% (fluconazole) to 100.0% (amphotericin B, flucytosine, anidulafungin, micafungin, itraconazole) of results for all agents with the exception of caspofungin, where 15 of 16 discrepancies (93.8%) were due to EUCAST MIC values that were higher than CLSI MIC results. The latter finding has been found in previous comparisons between these methods and speaks to methodological issues with testing of caspofungin by both reference methods (Arendrup et al., 2010; Arendrup et al., 2011; Espinel-Ingroff et al., 2013b). The largest number of discrepancies observed with the EUCAST and CLSI comparison occurred with C. glabrata tested against anidulafungin (40 discrepant results), with C. parapsilosis tested


201

Table 3 Categorical agreement between the results of the CLSI and EUCAST broth microdilution methods for 9 systemically active antifungal agents and Candida spp. using epidemiological cutoff values.a Species (no. tested)

C. albicans (114)

Antifungal agent (ECV [μg/mL])

Amphotericin B (2) b

Flucytosine (0.5) Anidulafungin (0.12) Caspofungin (0.12) Micafunginc (0.03) Fluconazole (0.5) Itraconazole (0.12) Posaconazoled (0.06) Voriconazole (0.03) C. glabrata (73)

Amphotericin B (2) Flucytosineb (0.5) Anidulafungin (0.25) Caspofungin (0.12) Micafunginc (0.03) Fluconazole (32) Itraconazole (2) d

Posaconazole (2) Voriconazole (0.5) C. parapsilosis (76)


Flucytosine (0.5) Anidulafungin (4) Caspofungin (1) Micafunginc (4) Fluconazole (2) Itraconazole (0.5) Posaconazoled (0.25) Voriconazole (0.12) C. tropicalis (60)

Amphotericin B (2) Flucytosineb (0.5) Anidulafungin (0.12) Caspofungin (0.12) Micafunginc (0.12) Fluconazole (2) Itraconazole (0.5) d

Posaconazole (0.12)

Test method

CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST

No. of isolates (%) with results

≤ECV

NECV

114 114 81 77 109 111 94 98 89 89 103 103 103 112 100 106 103 102 73 73 44 44 66 69 48 41 45 45 62 62 67 71 63 63 57 56 76 76 48 48 76 75 66 75 51 51 66 67 75 76 72 73 72 71 60 60 27 27 51 53 40 28 33 34 48 48 56 56 45 50

0 (0.0) 0 (0.0) 8 (9.0) 12 (13.5) 5 (4.4) 3 (2.6) 20 (17.5) 16 (14.0) 4 (4.3) 4 (4.3) 11 (9.6) 11 (9.6) 11 (9.6) 2 (1.8) 10 (9.1) 4 (3.6) 11 (9.6) 12 (10.5) 0 (0.0) 0 (0.0) 1 (2.2) 1 (2.2) 7 (9.6) 4 (5.5) 25 (34.2) 32 (43.8) 8 (15.1) 8 (15.1) 11 (15.1) 11 (15.1) 6 (8.2) 2 (2.7) 2 (3.1) 2 (3.1) 16 (21.9) 17 (23.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.3) 10 (13.2) 1 (1.3) 0 (0.0) 0 (0.0) 10 (13.2) 9 (11.8) 1 (1.3) 0 (0.0) 1 (1.4) 0 (0.0) 4 (5.3) 5 (6.6) 0 (0.0) 0 (0.0) 4 (12.9) 4 (12.9) 9 (15.0) 7 (11.7) 20 (33.3) 32 (53.3) 3 (8.3) 2 (5.6) 12 (20.0) 12 (20.0) 4 (6.7) 4 (6.7) 10 (18.2) 5 (9.1)

(100.0) (100.0) (91.0) (86.5) (95.6) (97.4) (82.5) (86.0) (95.7) (95.7) (90.4) (90.4) (90.4) (98.2) (90.9) (96.4) (90.4) (89.5) (100.0) (100.0) (97.8) (97.8) (90.4) (94.5) (65.8) (56.2) (84.9) (84.9) (84.9) (84.9) (91.8) (97.3) (96.9) (96.9) (78.1) (76.7) (100.0) (100.0) (100.0) (100.0) (100.0) (98.7) (86.8) (98.7) (100.0) (100.0) (86.8) (88.2) (98.7) (100.0) (98.6) (100.0) (94.7) (93.4) (100.0) (100.0) (87.1) (87.1) (85.0) (88.3) (66.7) (46.7) (91.7) (94.4) (80.0) (80.0) (93.3) (93.3) (81.8) (90.9)

CA (%)

% of isolates with discrepant results that were: VM

M

100.0

0.0

0.0

88.8

3.3

7.9

98.2

1.8

0.0

93.0

5.2

1.8

100.0

0.0

0.0

100.0

0.0

0.0

92.1

7.9

0.0

92.7

6.4

0.9

99.1

0.0

0.9

100.0

0.0

0.0

100.0

0.0

0.0

95.9

4.1

0.0

90.4

0.0

9.6

100.0

0.0

0.0

91.8

4.1

4.1

94.5

5.5

0.0

93.8

3.1

3.1

90.4

4.1

5.5

100.0

0.0

0.0

100.0

0.0

0.0

98.7

0.0

1.3

86.8

13.2

1.3

100.0

0.0

0.0

98.7

1.3

0.0

98.7

0.0

1.3

98.6

1.4

0.0

98.7

0.0

1.3

100.0

0.0

0.0

100.0

0.0

0.0

96.7

3.3

0.0

73.3

3.4

23.3

97.2

2.8

0.0

96.6

1.7

1.7

96.6

1.7

1.7

87.3

10.9

1.8

(continued on next page)

202


Table 3 (continued) Species (no. tested)

Antifungal agent (ECV [μg/mL])

Test method

Voriconazole (0.06)

CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST CLSI EUCAST

No. of isolates (%) with results

≤ECV

C. krusei (34)


Flucytosine (32) Anidulafungin (0.12) Caspofungin (0.25) c

Micafungin (0.12) Fluconazole (64) Itraconazole (1) Posaconazoled (0.5) Voriconazole (0.5)

48 (80.0) 39 (65.0) 34 (100.0) 34 (100.0) 17 (100.0) 17 (100.0) 32 (94.1) 32 (94.1) 24 (70.6) 13 (38.2) 21 (95.5) 21 (95.5) 33 (97.1) 33 (97.1) 33 (97.1) 34 (100.0) 27 (93.1) 29 (100.0) 33 (97.1) 31 (91.2)

CA (%)

NECV 12 (20.0) 21 (35.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (5.9) 2 (5.9) 10 (29.4) 21 (61.8) 1 (4.5) 1 (4.5) 1 (2.9) 1 (2.9) 1 (2.9) 0 (0.0) 2 (6.9) 0 (0.0) 1 (2.9) 3 (8.8)

% of isolates with discrepant results that were: VM

M

85.0

0.0

15.0

100.0

0.0

0.0

100.0

0.0

0.0

94.0

3.0

3.0

61.8

2.9

35.3

91.0

4.5

4.5

94.0

3.0

3.0

97.1

2.9

0.0

93.1

6.9

0.0

94.1

0.0

5.9

a CLSI, Clinical and Laboratory Standards Institute; EUCAST, European Committee on Antimicrobial Susceptibility Testing; ECV, epidemiological cutoff value; CA, categorical agreement; VM, very major discrepancy; M, major discrepancy. b Flucytosine was tested against 230 isolates (89 C. albicans, 45 C. glabrata, 48 C. parapsilosis, 31 C. tropicalis, 17 C. krusei). c Micafungin was tested against 255 isolates (93 C. albicans, 53 C. glabrata, 51 C. parapsilosis, 36 C. tropicalis, 22 C. krusei). d Posaconazole was tested against 332 isolates (110 C. albicans, 65 C. glabrata, 73 C. parapsilosis, 55 C. tropicalis, 29 C. krusei).

against posaconazole (25 discrepant results) and with C. albicans and C. parapsilosis tested against itraconazole (21 and 22 discrepant results, respectively). Regarding the individual Candida species, the EAs between the EUCAST and CLSI BMD results were N90% for all species tested against flucytosine, caspofungin, micafungin, fluconazole and voriconazole (Table 2). Whereas the EA was N90% for both amphotericin B and anidulafungin tested against C. parapsilosis, C. tropicalis, and C. krusei, the agreement between methods was poor for both of these agents when tested against C. albicans and C. glabrata. The reasons for these discrepancies is unclear but could be related to known differences in inoculum size and glucose content between the 2 methods. In contrast to our previous methods comparison with isavuconazole (Pfaller et al., 2013b), we observed poorer agreement between the 2 methods when testing C. glabrata and C. tropicalis, possibly due to a larger sample of isolates. Poor intermethod agreement was noted with all species tested against itraconazole and posaconazole. The poor solubility and lipophilic nature of these latter 2 antifungal agents may account for difficulties in methods comparison studies and has been cited by other authors (Chryssanthou, 2001; Espinel-Ingroff et al., 1999; Espinel-Ingroff et al., 2004; Pfaller et al., 2004). The ECVs for 9 of the agents (ECVs not yet available for isavuconazole) and the 5 Candida species are shown in Table 1. The ECVs were determined in previous studies using the CLSI BMD method (Pfaller et al., 2010a, 2010b, 2011a, 2011b, 2012b). The application of these ECVs allows both the assessment of the CA between methods and a means of discriminating WT strains from those with acquired resistance mutations (Arendrup et al., 2010; Pfaller et al., 2013c). The CA between the results obtained with the EUCAST method and those obtained with the CLSI method for each agent and Candida species are shown in Table 3. It should be noted that with the exception of amphotericin B, the collection of isolates included in this study contains a substantial number of non-WT strains for each antifungal agent-species comparison. Specifically, 12% of the MIC results for fluconazole and voriconazole were non-WT as were 6% of the results for anidulafungin and micafungin. The selection of non-WT strains for inclusion in this study was based strictly on the CLSI MIC phenotype and did not include an assessment of any specific resistance mechanism.

Overall the CA between the EUCAST and CLSI MIC results was 95.0% with 2.5% VM and M discrepancies (data not shown). The CA was N93% for all antifungal agents tested with the exception of caspofungin (84.6%) where 10% of the results were categorize as nonWT by the EUCAST method and WT by the CLSI method. This discrepancy may reflect the considerable intra-and interlaboratory variability in caspofungin MICs noted for both CLSI and EUCAST methods (Espinel-Ingroff et al., 2013b). The reason for this variability is currently under investigation. Regarding the individual Candida species, the CAs between the EUCAST and CLSI BMD MIC result were N90% for all organism-drug combinations with the exception of C. albicans and flucytosine (88.8%), C. parapsilosis and caspofungin (86.8%), C. tropicalis and caspofungin (73.3%), posaconazole (87.3%), and voriconazole (85.0%), and C. krusei and caspofungin (61.8%) (Table 3). The rate of VM discrepancies were b2.0% for 25 of the 36 organism-drug combinations and that of M discrepancies was b2.0% for 33 of the 36 combinations. The VM and M discrepancy rates may seem more elevated than normally encountered in part due to the use of ECVs to assess the CA where only 2 categories (WT and non-WT) were employed, as opposed to the use of clinical breakpoints where the susceptible and resistant categories are buffered by the application of intermediate or susceptible dose dependent categories (Pfaller et al., 2012a, 2013a). This provides the opportunity for minor error statistics rather than M or VM discrepancies for some comparisons. This study expands our earlier comparison evaluations of these 2 methods by including a robust collection of both WT and non-WT strains for most organism-drug combinations, as well as expanding the analysis to include older agents (amphotericin B, flucytosine and itraconazole) and the newer investigational triazole, isavuconazole. The results indicate that the EUCAST and CLSI methods produce comparable results for testing the systemically active antifungal agents against the 5 most common species of Candida. Problem areas identified include testing of amphotericin B, anidulafungin, and isavuconazole against C. glabrata, itraconazole and posaconazole against most species, and caspofungin against C. parapsilosis, C. tropicalis, and C. krusei. We confirm the excellent EA and CA between the 2 reference methods for testing fluconazole, voriconazole, and micafungin


(Pfaller et al., 2010c, 2011e). Despite this progress there are clearly some areas where additional steps toward harmonization are warranted. There are several limitations to this study: 1) none of the strains were characterized with respect to azole or echinocandin resistance mechanisms; 2) the lack of clinical breakpoints for several antifungal agents and species (CLSI and EUCAST) methods precludes a more standard intermethod comparison as does the lack of published ECVs for some organism-drug combinations with the EUCAST method; 3) the use of ECVs as categorical interpretive criteria may overestimate VM and M discords; 4) the study was limited to only the 5 most common species of Candida and 5) not all isolates were tested against all agents due to differences in antifungal drug availability at the various testing dates. Regarding the lack of isolates with documented resistance mechanisms, it should be noted that both methods have been documented to identify strains having azole and echinocandin resistance factors (Arendrup et al., 2010; Pfaller et al., 2010a, 2010c). Acknowledgments The global antifungal surveillance programs which served as the source of data used in the development of the manuscript were supported in part by Pfizer Inc. and by Astellas. We acknowledge the excellent technical assistance of S. Benning in the preparation of the manuscript. JMI Laboratories has received research and educational grants in 2011–2013 from Aires, American Proficiency Institute (API), Anacor, Astellas, AstraZeneca, Bayer, bioMerieux, Cempra, Cerexa, Contrafect, Cubist, Dipexium, Furiex, GlaxoSmithKline, Johnson & Johnson (J and J), LegoChem Biosciences, Meiji Seika Kaisha, Merck, Nabriva, Novartis, Pfizer, PPD Therapeutics, Premier Research Group, Rempex, Rib-X Pharmaceuticals, Seachaid, Shionogi, The Medicines, Theravance, and ThermoFisher Scientific. Some JMI employees are advisors/consultants for Astellas, Cubist, Pfizer, Cempra, Cerexa-Forest, J&J, and Theravance. In regards to speakers bureaus and stock options-none to declare. References Alastruey-Izquierdo A, Cuenca-Estrella M. EUCAST and CLSI: how to assess in vitro susceptibility and clinical resistance. Curr Fungal Infect Rep 2012;6:229–34. Arendrup MC, Garcia-Effron G, Lass-Florl C, Lopez AG, Rodriguez-Tudela JL, Cuenca-Estrella M, et al. Echinocandin susceptibility testing of Candida species: comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, disk diffusion, and agar dilution methods with RPMI and isosensitest media. Antimicrob Agents Chemother 2010;54:426–39. Arendrup MC, Rodriguez-Tudela JL, Park S, Garcia-Effron G, Delmas G, Cuenca-Estrella M, et al. Echinocandin susceptibility testing of Candida spp. Using EUCAST EDef 7.1 and CLSI M27-A3 standard procedures: analysis of the influence of bovine serum albumin supplementation, storage time, and drug lots. Antimicrob Agents Chemother 2011;55:1580–7. Arendrup MC, Cuenca-Estrella M, Lass-Florl C. EUCAST Technical Note on the EUCAST definitive document EDef 7.2: Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin Microbiol Infect 2012;18:E246–7. Arendrup MC, Dzajic E, Jensen RH, Johansen HK, Kjaeldgaard P, Knudsen JD, et al. Epidemiological changes with potential implication for antifungal prescription recommendations for fungaemia: data from a nationwide fungaemia surveillance programme. Clin Microbiol Infect 2013;19:E343–53. Chryssanthou E. Trends in antifungal susceptibility among Swedish Candida species bloodstream isolates from 1994 to 1998: comparison of the E-test and the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A reference method. J Clin Microbiol 2001;39:4181–3. Cleveland AA, Farley MM, Harrison LH, Stein B, Hollick R, Lockhart SR, et al. Changes in incidence and antifungal drug resistance in candidemia: results from populationbased laboratory surveillance in Atlanta and Baltimore, 2008-2011. Clin Infect Dis 2012;55:1352–61. Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts: 3rd informational supplement (M27-S3). Wayne, PA: CLSI; 2008a. Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts. 3rd ed., M27-A3. Wayne, PA: CLSI; 2008b. Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts: 4th informational supplement. Wayne, PA: CLSI; 2012. Espinel-Ingroff A, Pfaller M, Messer SA, Knapp CC, Killian S, Norris HA, et al. Multicenter comparison of the Sensititre YeastOne Colorimetric Antifungal Panel with the National Committee for Clinical Laboratory standards M27-A reference method for

203

testing clinical isolates of common and emerging Candida spp., Cryptococcus spp., and other yeasts and yeast-like organisms. J Clin Microbiol 1999;37:591–5. Espinel-Ingroff A, Pfaller M, Messer SA, Knapp CC, Holliday N, Killian SB. Multicenter comparison of the Sensititre YeastOne colorimetric antifungal panel with the NCCLS M27-A2 reference method for testing new antifungal agents against clinical isolates of Candida spp. J Clin Microbiol 2004;42:718–21. Espinel-Ingroff A, Barchiesi F, Cuenca-Estrella M, Pfaller MA, Rinaldi M, RodriguezTudela JL, et al. International and multicenter comparison of EUCAST and CLSI M27A2 broth microdilution methods for testing susceptibilities of Candida spp. to fluconazole, itraconazole, posaconazole, and voriconazole. J Clin Microbiol 2005; 43:3884–9. Espinel-Ingroff A, Cuenca-Estrella M, Canton E. EUCAST and CLSI: working together towards a harmonized method for antifungal susceptibility testing. Curr Fungal Infect Rep 2013a;7:59–67. Espinel-Ingroff A, Arendrup MC, Pfaller MA, Bonfietti LX, Bustamante B, Canton E, et al. Interlaboratory variability of caspofungin MICs for Candida spp. using CLSI and EUCAST methods: should the clinical laboratory be testing this agent? Antimicrob Agents Chemother 2013b;57:5836–42. EUCAST Breakpoint tables for interpretation of MICs and zone diameters. Version 3.0, January 2013. Available at http://www.eucast.org/clinical_breakpoints/. Accessed January 2, 2013. Howell SA, Hazen KC. Candida, Cryptococcus, and other yeasts of medical importance. In: Versalovic J, Carroll KC, Funke G, Jorgensen JH, Landry ML, Warnock DW, editors. Manual of clinical microbiology. 10th ed. Washington, DC: ASM Press; 2011. p. 1793–821. Kett DH, Azoulay E, Echeverria PM, Vincent JL. Candida bloodstream infections in intensive care units: analysis of the extended prevalence of infection in intensive care unit study. Crit Care Med 2011;39:665–70. Ostrosky-Zeichner L. Candida glabrata and FKS mutations: witnessing the emergence of the true multidrug-resistant Candida. Clin Infect Dis 2013;56:1733–4. Pakyz AL, Gurgle HE, Oinonen MJ. Antifungal use in hospitalized adults in U.S. academic health centers. Am J Health Syst Pharm 2011;68:415–8. Pfaller MA. Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment. Am J Med 2012;125:S3–13. Pfaller MA, Diekema DJ. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol 2012;50:2846–56. Pfaller MA, Espinel-Ingroff A, Jones RN. Clinical evaluation of the Sensititre YeastOne colorimetric antifungal plate for antifungal susceptibility testing of the new triazoles voriconazole, posaconazole, and ravuconazole. J Clin Microbiol 2004;42: 4577–80. Pfaller MA, Andes D, Diekema DJ, Espinel-Ingroff A, Sheehan D. Testing TCSfA. Wildtype MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods. Drug Resist Updat 2010a;13:180–95. Pfaller MA, Boyken L, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, et al. Wild-type MIC distributions and epidemiological cutoff values for the echinocandins and Candida spp. J Clin Microbiol 2010b;48:52–6. Pfaller MA, Castanheira M, Diekema DJ, Messer SA, Moet GJ, Jones RN. Comparison of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Etest methods with the CLSI broth microdilution method for echinocandin susceptibility testing of Candida species. J Clin Microbiol 2010c;48:1592–9. Pfaller MA, Andes D, Arendrup MC, Diekema DJ, Brown SD, Motyl M, et al. Clinical breakpoints for voriconazole and Candida spp. revisited: review of microbiologic, molecular, pharmacodynamic, and clinical data as they pertain to the development of species-specific interpretive criteria. Diagn Microbiol Infect Dis 2011a;70: 330–43. Pfaller MA, Boyken L, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, et al. Wild-type MIC distributions and epidemiological cutoff values for posaconazole and voriconazole and Candida spp. as determined by 24-hour CLSI broth microdilution. J Clin Microbiol 2011b;49:630–7. Pfaller MA, Boyken LB, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, et al. Validation of 24-hour posaconazole and voriconazole MIC readings versus the CLSI 48-hour broth microdilution reference method: application of epidemiological cutoff values to results from a global Candida antifungal surveillance program. J Clin Microbiol 2011c;49:1274–9. Pfaller MA, Diekema DJ, Andes D, Arendrup MC, Brown SD, Lockhart SR, et al. Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria. Drug Resist Updat 2011d;14:164–76. Pfaller MA, Espinel-Ingroff A, Boyken L, Hollis RJ, Kroeger J, Messer SA, et al. Comparison of the broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) with the 24-h CLSI BMD method for fluconazole, posaconzole, and voriconazole susceptibility testing of Candida species using epidemiological cutoff values. J Clin Microbiol 2011e;49:845–50. Pfaller MA, Moet GJ, Messer SA, Jones RN, Castanheira M. Geographic variations in species distribution and echinocandin and azole antifungal resistance rates among Candida bloodstream infection isolates: Report from the SENTRY Antimicrobial Surveillance Program (2008 to 2009). J Clin Microbiol 2011f;49:396–9. Pfaller MA, Chaturvedi V, Diekema DJ, Ghannoum MA, Holliday NM, Killian SB, et al. Comparison of the Sensititre YeastOne colorimetric antifungal panel with CLSI microdilution for antifungal susceptibility testing of the echinocandins against Candida spp., using new clinical breakpoints and epidemiological cutoff values. Diagn Microbiol Infect Dis 2012a;73:365–8. Pfaller MA, Espinel-Ingroff A, Castanheira M, Cuenca-Estrella M, Diekema DJ, Fothergill A, et al. Wild-type MIC distributions and epidemiological cutoff values for amphotericin B,

204


flucytosine, and itraconazole and Candida spp. as determined by CLSI broth microdilution. J Clin Microbiol 2012b;50:2040–6. Pfaller MA, Woosley LN, Messer SA, Jones RN, Castanheira M. Significance of molecular identification and antifungal susceptibility of clinically significant yeasts and moulds in a global antifungal surveillance program. Mycopathologia 2012c;174: 259–71. Pfaller MA, Diekema DJ, Procop GW, Rinaldi MG. Comparison of the Vitek2 yeast susceptibility system with CLSI microdilution for antifungal susceptibility testing of fluconazole and voriconazole against Candida spp., using new clinical breakpoints and epidemiological cutoff values. Diagn Microbiol Infect Dis 2013a;77:37–40. Pfaller MA, Messer SA, Rhomberg PR, Jones RN, Castanheira M. In vitro activity of isavuconazole and comparator antifungal agents tested against a global collection of opportunistic yeasts and molds. J Clin Microbiol 2013b;51:2608–16.

Pfaller MA, Messer SA, Woosley LN, Jones RN, Castanheira M. Echinocandin and triazole antifungal susceptibility profiles of opportunistic yeast and mould clinical isolates (2010-2011); Application of new CLSI clinical breakpoints and epidemiological cutoff values to characterize geographic and temporal trends of antifungal resistance. J Clin Microbiol 2013c;51:2571–81. Rodriguez-Tudela JL, Donnelly JP, Pfaller MA, Chryssantou E, Warn P, Denning DW, et al. Statistical analyses of correlation between fluconazole MICs for Candida spp. assessed by standard methods set forth by the European Committee on Antimicrobial Susceptibility Testing (E.Dis. 7.1) and CLSI (M27-A2). J Clin Microbiol 2007;45:109–11. Turnidge J, Kahlmeter G, Kronvall G. Statistical characterisation of bacterial wild-type MIC value distributions and the determination of epidemiological cut-off values. Clin Microbiol Infect 2006;12:418–25.

Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests.

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests.

Multicenter Comparison of the Etest and EUCAST Methods for Antifungal Susceptibility Testing of Candida Isolates to Micafungin.

Breakpoints for antifungal agents: an update from EUCAST focussing on echinocandins against Candida spp. and triazoles against Aspergillus spp.

Identification of Candida species and susceptibility testing with Sensititre YeastOne microdilution panel to 9 antifungal agents.

Comparing Etest and Broth Microdilution for Antifungal Susceptibility Testing of the Most-Relevant Pathogenic Molds.

Antifungal susceptibility testing of Candida spp. by relative growth measurement at single concentrations of antifungal agents.

Identification and antimicrobial susceptibility of 250 Bacteriodes fragilis subspecies tested by broth microdilution methods.

Comparison of Etests and Vitek 2 ® to broth microdilution for the susceptibility testing of Cryptococcus neoformans.

Collaborative investigation of broth microdilution and semisolid agar dilution for in vitro susceptibility testing of Candida albicans.

Comparison of M.I.C.E. and Etest with CLSI agar dilution for antimicrobial susceptibility testing against oxacillin-resistant Staphylococcus spp.

Activity of a long-acting echinocandin, CD101, determined using CLSI and EUCAST reference methods, against Candida and Aspergillus spp., including echinocandin- and azole-resistant isolates.

In-vitro Activity of 10 Antifungal Agents against 320 Dermatophyte Strains Using Microdilution Method in Tehran.

In Vitro Antifungal Susceptibility Testing of Candida Isolates with the EUCAST Methodology, a New Method for ECOFF Determination.

Recommendation for a Standardised Method of Broth Microdilution Susceptibility Testing for Porcine Bordetella bronchiseptica.

Use of Haemophilus test medium for broth microdilution antimicrobial susceptibility testing of Streptococcus pneumoniae.

Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI Broth Microdilution, and Etest Method.

Evaluation of agar dilution and broth microdilution methods to determine the disinfectant susceptibility.

Verification of an Automated, Digital Dispensing Platform for At-Will Broth Microdilution-Based Antimicrobial Susceptibility Testing.

Challenges, Issues and Warnings from CLSI and EUCAST Working Group on Polymyxin Susceptibility Testing.

Accuracy of the Thermo Fisher Scientific (Sensititre™) dry-form broth microdilution MIC product when testing ceftaroline.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically.

Assessment of caspofungin susceptibility of Candida glabrata by the Etest®, CLSI, and EUCAST methods, and detection of FKS1 and FKS2 mutations.

IDF standard for antimicrobial susceptibility testing of Lactobacilli.