- Email: [email protected]
Evaluation of the in vitro activity of caspofungin against bloodstream isolates of Candida species from cancer patients: comparison of Etest and NCCLS reference methods
International Journal of Antimicrobial Agents 20 (2002) 468 /471 www.isochem.org
Evaluation of the in vitro activity of caspofungin against bloodstream isolates of Candida species from cancer patients: comparison of Etest and NCCLS reference methods M. Laverdiere *, C. Restieri, F. Habel Department of Microbiology-Infectious Diseases, Hoˆpital Maisonneuve-Rosemont, 5415 Boul. del’ Assomption, Montreal, Que., Canada H1T 2M4 Received 3 April 2002; accepted 21 May 2002
Abstract The in vitro activity of caspofungin (CAS) was compared with the activity of fluconazole, itraconazole and amphotericin B against 178 bloodstream Candida spp. from cancer patients. The activities were assessed using the reference NCCLS M-27A microdilution method and the Etest method. With both the NCCLS microdilution reference method and the Etest method, CAS was the most active agent (MIC90s 0.19 /0.5 mg/l) against Candida albicans , C. glabrata and C. tropicalis . CAS showed substantial activity against azole-resistant Candida. The percentages of agreement within 9/2 dilutions between the NCCLS reference microdilution method and Etest MICs ranged from 81 to 97%. CAS showed good in vitro activity against invasive azole-susceptible and azole-resistant Candida isolates. The CAS Etest MICs correlated well with the NCCLS reference MICs and may provide more choice for laboratories in assessing the activity of antifungal agents. The clinical correlation of these in vitro observations needs to be established. # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Caspofungin; Bloodstream isolates; Cancer patients; Etest
1. Introduction Candida bloodstream infections are important causes of morbidity and mortality in immunosuppressed cancer patients [1]. Although fluconazole (FCZ) and itraconazole (ITZ) have been shown to be effective against invasive candidiasis in compromised hosts, emerging resistance raises questions about their future clinical usefulness [2]. Among the newer antifungal agents, the glucan synthesis inhibitor caspofungin (CAS) has promising activity against pathogenic yeasts including most Candida species and may represent a promising agent against isolates with primary and secondary resistance to FCZ and ITZ [3,4]. We compared the in vitro activity of CAS with the activity of FCZ, ITZ and amphotericin B (AMB) against 178 bloodstream Candida isolates recovered from cancer patients, using the NCCLS M-
* Corresponding author. Tel.: /1-514-252-3817; fax: /1-514-2523898 E-mail address: [email protected] (M. Laverdiere).
27A microdilution reference method and the Etest method.
2. Materials and methods 2.1. Organisms A total of 178 bloodstream isolates of Candida spp. isolated from cancer patients between 1996 and 2000 and obtained from two Canadian centres (Hoˆpital Maisonneuve-Rosemont, Montre´al, Que´bec and The Health Sciences Center, Winnipeg, Manitoba) were selected for testing. 2.2. Antifungal agents Standard antifungal powders of (AMB), (BristolMyers-Squibb), ITZ (Janssen-Ortho), FCZ (Pfizer) and CAS (Merck Research Laboratories) were obtained from their respective manufacturers. Stock solutions
0924-8579/02/$ - see front matter # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 2 ) 0 0 2 4 0 - 6
M. Laverdiere et al. / International Journal of Antimicrobial Agents 20 (2002) 468 /471
were prepared in dimethyl sulfoxide (ITZ /AMB) or in water (FCZ /CAS). Serial twofold dilutions were made in RPMI 1640 medium (Gibco BRL) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) for all antibiotics except in antibiotic medium 3/2% glucose for AMB. The final concentration of the solvent did not exceed 1% in any of the wells. Etest strips (AB BIODISK Solna, Sweden) containing CAS were supplied by Merck Research Laboratories. 2.3. Susceptibility testing methods The antifungal susceptibility testing was performed by a broth microdilution reference method, according to the guidelines recommended by the NCCLS in document M27-A, and by the Etest agar-based method [5]. In the NCCLS method the final concentrations of the antifungal agents were 0.008 /8 mg/l for AMB, ITZ and CAS, and 0.25 /256 mg/l for FCZ. Drug-free and yeastfree controls were included. The trays were incubated in air at 35 8C and MIC endpoints were read after 48 h of incubation. Following incubation, the trays were visually examined and the growth in each well was
469
Fig. 1. Distribution of the isolates.
compared with that of the growth of the control (drug-free) well. The MICs of AMB and CAS were defined as the lowest concentration resulting in a complete inhibition of growth. The MICs of all the other compounds were defined as the lowest concentration that resulted in a prominent decrease in turbidity compared to that of growth-control wells using the turbidity numerical score proposed by the NCCLS M27A reference method. The Etest was performed according to the manufacturer’s recommendation using 90 mmdiameter plates containing RPMI 1640 medium supplemented with 2% glucose and MOPS. The MIC 50 and MIC 90 are the concentration of each antifungal agent
Table 1 Comparative in vitro activity of caspofungin against 178 Candida blood stream isolates from cancer patients Organism (no. of isolates)
Compound
MIC (mg/l) NCCLS method Range
MIC 50
All organisms (178)
CAS AMB FCZ ITZ
0.03 / 8 0.125 /2 0 0.25 / 256 0 0.008 / 8
0.125 0.25 1 0.125
Candida albicans (92)
CAS AMB FCZ ITZ
0.03 /0.5 0.125 /1 0 0.25 / 256 0 0.008 / 8
0.125 0.25 0.5 0.06
Candida glabrata (26)
CAS AMB FCZ ITZ
0.03 /4 0.25 /0.5 1 /128 0.06 / 8
0.06 0.5 8 0.5
Candida tropicalis (22)
CAS AMB FCZ ITZ
0.03 /0.25 0.125 /1 0.25 /256 0.06 / 8
0.06 0.25 1 0.25
Candida parapsilosis (20)
CAS AMB FCZ ITZ
0.06 /2 0.125 /1 0.25 /16 0.03 /0.5
1 0.25 0.5 0.06
FCZ resistant (12)
CAS AMB FCZ ITZ
0.03 /1 0.125 /2 64 / 256 0.125 / 8
ITZ resistant (21)
CAS AMB FCZ ITZ
0.03 /4 0.125 /2 0 0.25 / 256 1 / 8
0.03 1 128 1 0.25 0.5 32 2
MIC (mg/l) Etest method MIC 90 1 1 32 1
Range
MIC 50
MIC 90
0.032 / 32 0.047 / 32 0.064 / 256 0.006 / 32
0.19 0.38 0.38 0.125
0.032 /1 0.064 /1.5 0.064 / 256 0.006 / 32
0.125 0.25 0.25 0.064
0.25 0.5 1 0.25
0.125 /0.38 0.25 /1.5 0.5 / 256 0.047 /2
0.19 0.75 8 2
0.19 1 16 64
0.25 0.5 8 1
0.125 /0.75 0.25 /1 0.125 /8 0.032 /0.75
0.25 0.5 0.44 0.19
0.5 1 1 0.5
2 0.5 2 0.25
0.38 / 32 0.094 /1 0.125 /8 0.006 /0.75
1 0.5 0.3 0.03
2 0.75 0.75 0.064
0.25 0.5 4 0.25 0.5 1 16 2
0.5 1 512 16
0.094 /1 0.125 /4 0.125 / 256 0.016 / 32
1 1 512 16
0.125 /2 0.25 /2 0.125 /512 0.064 /64
1 1 16 2
0.25 0.625 16 1
1 1.5 512 64
0.19 0.75 8 2
0.5 1 512 64
M. Laverdiere et al. / International Journal of Antimicrobial Agents 20 (2002) 468 /471
470
Table 2 Distribution of differences between the MICs determined by Etest and reference NCCLS M27-A broth microdilution method Organism (no. of isolates) Compound No of isolates for which MICs determined by Etest differed from MICs determined by the NCCLS-A27 microdilution method
All organisms (178)
Candida albicans (92)
Candida glabrata (26)
Candida tropicalis (22)
Candida parapsilosis (22)
Candida kruseı¨
% Agreement within no of dilution (%)
E 4
3
2
1
0
1
2
3
E4
CAS FCZ ITZ AmphoB
9 9
2 13 6 2
6 23 19 7
28 42 31 39
32 49 31 55
46 28 31 46
38 4 33 26
20 3 9 2
CAS FCZ ITZ AmphoB
1 5 2
2 14 7 8
18 24 21 30
20 29 19 32
22 11 17 18
19 2 13 6
9
5 5
1 4
1 3 1
5 5 2 4
3 7 2 6
6 5 6 12
7 1 9 4
3 1 1
5
1 7 4 1
3 3 6 6
3 3 3 9
5
7
3
2 5
1
2 1 3 2
5 8 3 6
7 6 3 1
3
1 1 1 3
5 1 3 2
CAS FCZ ITZ AmphoB CAS FCZ ITZ AmphoB
3 1
CAS FCZ ITZ AmphoB
1 2
CAS FCZ ITZ AmphoB
3 1
1 1 1
3 4 1 3 7
1 1
necessary to inhibit 50 and 90% of the isolates, respectively. 2.4. Quality control C. parapsilosis ATCC 22019 and C. kruseı¨ ATCC 6258 were used for quality control. 2.5. Analysis of results: Etest MICs were compared with reference microdilution MICs. Since the Etest scale has a continuous gradient of concentrations, the MICs in-between twofold dilutions were raised to the next twofold level of the reference method for comparison. Off-scale MICs at the upper limit were converted to the next highest concentration, and off-scale results at the lower limit were left unchanged.
3. Results and discussion Forty-eight percent (48%) of the 178 isolates in our study were non-Candida albicans Candida (Fig. 1). The comparative in vitro activities of CAS, AMB, FCZ and ITZ are summarized in Table 1. With both the NCCLS
3
1 9 2 2 1
5
91
92
6 7 9 1
60 67 52 79
84 82 81 97
1 2 3
65 70 62 87
88 87 84 100
54 65 38 100
85 81 77 100
100 59 73 100
100 73 86 100
70 75 45 95
90 90 85 90
67 33 44 100
89 33 78 100
1 2
1 1 1
5 2
microdilution reference method and the Etest method, CAS showed substantial in vitro potency against C. albicans , C. glabrata and C. tropicalis but was less active against C. parapsilosis , activity against this species being less than that of AMB and ITZ. Lower potency of the echinocandin micafungin and LY303366 against C. parapsilosis and C. guilliermondi has also been previously observed by others [6,7]. Against FCZor ITZ-resistant Candida isolates, CAS retained activity comparable with that against azole-susceptible strains. The clinical relevance of such observations is unknown at the present since standardization of the susceptibility testing methods to assess the in vitro activity of the echinocandins have not been clearly defined and breakpoints are yet to be determined. In addition, clinical correlation between the in vitro and the in vivo responses has not yet been established. Table 2 summarizes the differences and the percentages of agreement between the Etest MICs and the NCCLS reference microdilution MICs. The percentage of agreement within 1 dilution ranges from 52% with ITZ to 60, 67 and 79% with CAS, FCZ and AMB, respectively. The agreement within two dilutions between the Etest and microdilution MICs were 81% (ITZ), 82% (FCZ), 84% (CAS) and 97% (AMB). Overall the Etest MICs correlated well with the NCCLS
M. Laverdiere et al. / International Journal of Antimicrobial Agents 20 (2002) 468 /471
reference method MICs. The increasing resistance rates of clinically significant yeasts and the different potencies of antifungal agents against the various Candida species, highlight the importance of antifungal MIC determinations. Several alternative methods, easier to perform than the recommended reference methods, have been suggested [8,9]. Similarly to Pfaller et al., our observations show that the Etest provides quantitative agar-based results and generates CAS MIC data that are comparable with those obtained by the NCCLS reference microdilution method [10]. In addition with CAS, trailing effects were observed with only 7% of our isolates compared with 41 and 38% with FCZ and ITZ, respectively. As a consequence, measurement of endpoints was considerably easier and more accurate with CAS than with azoles. In conclusion CAS was active against our azolesusceptible and azole-resistant bloodstream Candida from our cancer patients. The CAS Etest MICs correlated well with the reference MICs and may provide more choice for laboratories assessing the activity of antifungal agents. The clinical correlation of these in vitro observations needs to be established.
Acknowledgements We thank Dr Daryl Hoban for supplying the isolates from The Health Sciences Center in Winnipeg, Manitoba, Canada. This study was supported by a contribution from Merck Research Laboratories.
471
References [1] Uzun O, Anaissie EJ. Predictors of outcome in cancer patients with candidemia. Ann Oncol 2000;12:1517 /21. [2] Hong Nguyen M, Peacock JE, Morris AJ, et al. The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. Am J Med 1996;100:617 /23. [3] Bartizal KC, Gill CJ, Abruzzo GK, Flattery AM, Kong L, Scott PM. In vitro pre-clinical evaluation studies with the echinocandin antifungal MK-0991 (L-743,872). Antimicrob Agents Chemother 1997;41:2326 /32. [4] Marco F, Pfaller MA, Messer SA, Jones RN. Activity of MK0991 (L-743,872), a new echinocandin, compared to those of LY303,366 and four other antifungal agents tested against bloodstream isolates of Candida spp. Diagn Microbiol Infect Dis 1998;32:33 /7. [5] National Committee for Laboratory Standards. Reference method for broth dilution antifungal susceptibility of yeasts. Approved standards M27-A. National Committee for Laboratory Standards, Wayne, Pa; 1997. [6] Cuenca-Estrella M, Mellado E, Diaz-Guerra TM, Monzon A, Rodriguez-Tudela JL. Susceptibility of fluconazole-resistant clinical isolates of Candida spp. to echinocandin LY303366, itraconazole and amphotericin B. J Antimicrob Chemother 2000;46:475 /7. [7] Tawara S, Ikeda F, Maki K, et al. In vitro activities of a new lipopeptide antifungal agent, FK 463, against a variety of clinically important fungi. Antimicrob Agents Chemother 2000;44:57 /62. [8] Lozano-Chiu M, Nelson PW, Paetznick VL, Rex JH. Disk diffusion method determining susceptibilities of Candida spp to MK-0991. J Clin Microbiol 1999;37:1625 /7. [9] Chryssanthou E. Trends in antifungal susceptibility among Swedish Candida species bloodstream isolates from 1994 /1998: comparison of the Etest and the sensititre yeastOne colorimetric antifungal panel with the NCCLS M27-A reference method. J Clin Microbiol 2001;39:4181 /3. [10] Pfaller MA, Messer SA, Mills K, Bolmstrom A, Jones RN. Evaluation of Etest method for determining caspofungin (MK0991) susceptibilities of 726 clinical isolates of Candida species. J Clin Microbiol 2001;39:4387 /9.
Evaluation of the in vitro activity of caspofungin against bloodstream isolates of Candida species from cancer patients: comparison of Etest and NCCLS reference methods M. Laverdiere *, C. Restieri, F. Habel Department of Microbiology-Infectious Diseases, Hoˆpital Maisonneuve-Rosemont, 5415 Boul. del’ Assomption, Montreal, Que., Canada H1T 2M4 Received 3 April 2002; accepted 21 May 2002
Abstract The in vitro activity of caspofungin (CAS) was compared with the activity of fluconazole, itraconazole and amphotericin B against 178 bloodstream Candida spp. from cancer patients. The activities were assessed using the reference NCCLS M-27A microdilution method and the Etest method. With both the NCCLS microdilution reference method and the Etest method, CAS was the most active agent (MIC90s 0.19 /0.5 mg/l) against Candida albicans , C. glabrata and C. tropicalis . CAS showed substantial activity against azole-resistant Candida. The percentages of agreement within 9/2 dilutions between the NCCLS reference microdilution method and Etest MICs ranged from 81 to 97%. CAS showed good in vitro activity against invasive azole-susceptible and azole-resistant Candida isolates. The CAS Etest MICs correlated well with the NCCLS reference MICs and may provide more choice for laboratories in assessing the activity of antifungal agents. The clinical correlation of these in vitro observations needs to be established. # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Caspofungin; Bloodstream isolates; Cancer patients; Etest
1. Introduction Candida bloodstream infections are important causes of morbidity and mortality in immunosuppressed cancer patients [1]. Although fluconazole (FCZ) and itraconazole (ITZ) have been shown to be effective against invasive candidiasis in compromised hosts, emerging resistance raises questions about their future clinical usefulness [2]. Among the newer antifungal agents, the glucan synthesis inhibitor caspofungin (CAS) has promising activity against pathogenic yeasts including most Candida species and may represent a promising agent against isolates with primary and secondary resistance to FCZ and ITZ [3,4]. We compared the in vitro activity of CAS with the activity of FCZ, ITZ and amphotericin B (AMB) against 178 bloodstream Candida isolates recovered from cancer patients, using the NCCLS M-
* Corresponding author. Tel.: /1-514-252-3817; fax: /1-514-2523898 E-mail address: [email protected] (M. Laverdiere).
27A microdilution reference method and the Etest method.
2. Materials and methods 2.1. Organisms A total of 178 bloodstream isolates of Candida spp. isolated from cancer patients between 1996 and 2000 and obtained from two Canadian centres (Hoˆpital Maisonneuve-Rosemont, Montre´al, Que´bec and The Health Sciences Center, Winnipeg, Manitoba) were selected for testing. 2.2. Antifungal agents Standard antifungal powders of (AMB), (BristolMyers-Squibb), ITZ (Janssen-Ortho), FCZ (Pfizer) and CAS (Merck Research Laboratories) were obtained from their respective manufacturers. Stock solutions
0924-8579/02/$ - see front matter # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 2 ) 0 0 2 4 0 - 6
M. Laverdiere et al. / International Journal of Antimicrobial Agents 20 (2002) 468 /471
were prepared in dimethyl sulfoxide (ITZ /AMB) or in water (FCZ /CAS). Serial twofold dilutions were made in RPMI 1640 medium (Gibco BRL) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) for all antibiotics except in antibiotic medium 3/2% glucose for AMB. The final concentration of the solvent did not exceed 1% in any of the wells. Etest strips (AB BIODISK Solna, Sweden) containing CAS were supplied by Merck Research Laboratories. 2.3. Susceptibility testing methods The antifungal susceptibility testing was performed by a broth microdilution reference method, according to the guidelines recommended by the NCCLS in document M27-A, and by the Etest agar-based method [5]. In the NCCLS method the final concentrations of the antifungal agents were 0.008 /8 mg/l for AMB, ITZ and CAS, and 0.25 /256 mg/l for FCZ. Drug-free and yeastfree controls were included. The trays were incubated in air at 35 8C and MIC endpoints were read after 48 h of incubation. Following incubation, the trays were visually examined and the growth in each well was
469
Fig. 1. Distribution of the isolates.
compared with that of the growth of the control (drug-free) well. The MICs of AMB and CAS were defined as the lowest concentration resulting in a complete inhibition of growth. The MICs of all the other compounds were defined as the lowest concentration that resulted in a prominent decrease in turbidity compared to that of growth-control wells using the turbidity numerical score proposed by the NCCLS M27A reference method. The Etest was performed according to the manufacturer’s recommendation using 90 mmdiameter plates containing RPMI 1640 medium supplemented with 2% glucose and MOPS. The MIC 50 and MIC 90 are the concentration of each antifungal agent
Table 1 Comparative in vitro activity of caspofungin against 178 Candida blood stream isolates from cancer patients Organism (no. of isolates)
Compound
MIC (mg/l) NCCLS method Range
MIC 50
All organisms (178)
CAS AMB FCZ ITZ
0.03 / 8 0.125 /2 0 0.25 / 256 0 0.008 / 8
0.125 0.25 1 0.125
Candida albicans (92)
CAS AMB FCZ ITZ
0.03 /0.5 0.125 /1 0 0.25 / 256 0 0.008 / 8
0.125 0.25 0.5 0.06
Candida glabrata (26)
CAS AMB FCZ ITZ
0.03 /4 0.25 /0.5 1 /128 0.06 / 8
0.06 0.5 8 0.5
Candida tropicalis (22)
CAS AMB FCZ ITZ
0.03 /0.25 0.125 /1 0.25 /256 0.06 / 8
0.06 0.25 1 0.25
Candida parapsilosis (20)
CAS AMB FCZ ITZ
0.06 /2 0.125 /1 0.25 /16 0.03 /0.5
1 0.25 0.5 0.06
FCZ resistant (12)
CAS AMB FCZ ITZ
0.03 /1 0.125 /2 64 / 256 0.125 / 8
ITZ resistant (21)
CAS AMB FCZ ITZ
0.03 /4 0.125 /2 0 0.25 / 256 1 / 8
0.03 1 128 1 0.25 0.5 32 2
MIC (mg/l) Etest method MIC 90 1 1 32 1
Range
MIC 50
MIC 90
0.032 / 32 0.047 / 32 0.064 / 256 0.006 / 32
0.19 0.38 0.38 0.125
0.032 /1 0.064 /1.5 0.064 / 256 0.006 / 32
0.125 0.25 0.25 0.064
0.25 0.5 1 0.25
0.125 /0.38 0.25 /1.5 0.5 / 256 0.047 /2
0.19 0.75 8 2
0.19 1 16 64
0.25 0.5 8 1
0.125 /0.75 0.25 /1 0.125 /8 0.032 /0.75
0.25 0.5 0.44 0.19
0.5 1 1 0.5
2 0.5 2 0.25
0.38 / 32 0.094 /1 0.125 /8 0.006 /0.75
1 0.5 0.3 0.03
2 0.75 0.75 0.064
0.25 0.5 4 0.25 0.5 1 16 2
0.5 1 512 16
0.094 /1 0.125 /4 0.125 / 256 0.016 / 32
1 1 512 16
0.125 /2 0.25 /2 0.125 /512 0.064 /64
1 1 16 2
0.25 0.625 16 1
1 1.5 512 64
0.19 0.75 8 2
0.5 1 512 64
M. Laverdiere et al. / International Journal of Antimicrobial Agents 20 (2002) 468 /471
470
Table 2 Distribution of differences between the MICs determined by Etest and reference NCCLS M27-A broth microdilution method Organism (no. of isolates) Compound No of isolates for which MICs determined by Etest differed from MICs determined by the NCCLS-A27 microdilution method
All organisms (178)
Candida albicans (92)
Candida glabrata (26)
Candida tropicalis (22)
Candida parapsilosis (22)
Candida kruseı¨
% Agreement within no of dilution (%)
E 4
3
2
1
0
1
2
3
E4
CAS FCZ ITZ AmphoB
9 9
2 13 6 2
6 23 19 7
28 42 31 39
32 49 31 55
46 28 31 46
38 4 33 26
20 3 9 2
CAS FCZ ITZ AmphoB
1 5 2
2 14 7 8
18 24 21 30
20 29 19 32
22 11 17 18
19 2 13 6
9
5 5
1 4
1 3 1
5 5 2 4
3 7 2 6
6 5 6 12
7 1 9 4
3 1 1
5
1 7 4 1
3 3 6 6
3 3 3 9
5
7
3
2 5
1
2 1 3 2
5 8 3 6
7 6 3 1
3
1 1 1 3
5 1 3 2
CAS FCZ ITZ AmphoB CAS FCZ ITZ AmphoB
3 1
CAS FCZ ITZ AmphoB
1 2
CAS FCZ ITZ AmphoB
3 1
1 1 1
3 4 1 3 7
1 1
necessary to inhibit 50 and 90% of the isolates, respectively. 2.4. Quality control C. parapsilosis ATCC 22019 and C. kruseı¨ ATCC 6258 were used for quality control. 2.5. Analysis of results: Etest MICs were compared with reference microdilution MICs. Since the Etest scale has a continuous gradient of concentrations, the MICs in-between twofold dilutions were raised to the next twofold level of the reference method for comparison. Off-scale MICs at the upper limit were converted to the next highest concentration, and off-scale results at the lower limit were left unchanged.
3. Results and discussion Forty-eight percent (48%) of the 178 isolates in our study were non-Candida albicans Candida (Fig. 1). The comparative in vitro activities of CAS, AMB, FCZ and ITZ are summarized in Table 1. With both the NCCLS
3
1 9 2 2 1
5
91
92
6 7 9 1
60 67 52 79
84 82 81 97
1 2 3
65 70 62 87
88 87 84 100
54 65 38 100
85 81 77 100
100 59 73 100
100 73 86 100
70 75 45 95
90 90 85 90
67 33 44 100
89 33 78 100
1 2
1 1 1
5 2
microdilution reference method and the Etest method, CAS showed substantial in vitro potency against C. albicans , C. glabrata and C. tropicalis but was less active against C. parapsilosis , activity against this species being less than that of AMB and ITZ. Lower potency of the echinocandin micafungin and LY303366 against C. parapsilosis and C. guilliermondi has also been previously observed by others [6,7]. Against FCZor ITZ-resistant Candida isolates, CAS retained activity comparable with that against azole-susceptible strains. The clinical relevance of such observations is unknown at the present since standardization of the susceptibility testing methods to assess the in vitro activity of the echinocandins have not been clearly defined and breakpoints are yet to be determined. In addition, clinical correlation between the in vitro and the in vivo responses has not yet been established. Table 2 summarizes the differences and the percentages of agreement between the Etest MICs and the NCCLS reference microdilution MICs. The percentage of agreement within 1 dilution ranges from 52% with ITZ to 60, 67 and 79% with CAS, FCZ and AMB, respectively. The agreement within two dilutions between the Etest and microdilution MICs were 81% (ITZ), 82% (FCZ), 84% (CAS) and 97% (AMB). Overall the Etest MICs correlated well with the NCCLS
M. Laverdiere et al. / International Journal of Antimicrobial Agents 20 (2002) 468 /471
reference method MICs. The increasing resistance rates of clinically significant yeasts and the different potencies of antifungal agents against the various Candida species, highlight the importance of antifungal MIC determinations. Several alternative methods, easier to perform than the recommended reference methods, have been suggested [8,9]. Similarly to Pfaller et al., our observations show that the Etest provides quantitative agar-based results and generates CAS MIC data that are comparable with those obtained by the NCCLS reference microdilution method [10]. In addition with CAS, trailing effects were observed with only 7% of our isolates compared with 41 and 38% with FCZ and ITZ, respectively. As a consequence, measurement of endpoints was considerably easier and more accurate with CAS than with azoles. In conclusion CAS was active against our azolesusceptible and azole-resistant bloodstream Candida from our cancer patients. The CAS Etest MICs correlated well with the reference MICs and may provide more choice for laboratories assessing the activity of antifungal agents. The clinical correlation of these in vitro observations needs to be established.
Acknowledgements We thank Dr Daryl Hoban for supplying the isolates from The Health Sciences Center in Winnipeg, Manitoba, Canada. This study was supported by a contribution from Merck Research Laboratories.
471
References [1] Uzun O, Anaissie EJ. Predictors of outcome in cancer patients with candidemia. Ann Oncol 2000;12:1517 /21. [2] Hong Nguyen M, Peacock JE, Morris AJ, et al. The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. Am J Med 1996;100:617 /23. [3] Bartizal KC, Gill CJ, Abruzzo GK, Flattery AM, Kong L, Scott PM. In vitro pre-clinical evaluation studies with the echinocandin antifungal MK-0991 (L-743,872). Antimicrob Agents Chemother 1997;41:2326 /32. [4] Marco F, Pfaller MA, Messer SA, Jones RN. Activity of MK0991 (L-743,872), a new echinocandin, compared to those of LY303,366 and four other antifungal agents tested against bloodstream isolates of Candida spp. Diagn Microbiol Infect Dis 1998;32:33 /7. [5] National Committee for Laboratory Standards. Reference method for broth dilution antifungal susceptibility of yeasts. Approved standards M27-A. National Committee for Laboratory Standards, Wayne, Pa; 1997. [6] Cuenca-Estrella M, Mellado E, Diaz-Guerra TM, Monzon A, Rodriguez-Tudela JL. Susceptibility of fluconazole-resistant clinical isolates of Candida spp. to echinocandin LY303366, itraconazole and amphotericin B. J Antimicrob Chemother 2000;46:475 /7. [7] Tawara S, Ikeda F, Maki K, et al. In vitro activities of a new lipopeptide antifungal agent, FK 463, against a variety of clinically important fungi. Antimicrob Agents Chemother 2000;44:57 /62. [8] Lozano-Chiu M, Nelson PW, Paetznick VL, Rex JH. Disk diffusion method determining susceptibilities of Candida spp to MK-0991. J Clin Microbiol 1999;37:1625 /7. [9] Chryssanthou E. Trends in antifungal susceptibility among Swedish Candida species bloodstream isolates from 1994 /1998: comparison of the Etest and the sensititre yeastOne colorimetric antifungal panel with the NCCLS M27-A reference method. J Clin Microbiol 2001;39:4181 /3. [10] Pfaller MA, Messer SA, Mills K, Bolmstrom A, Jones RN. Evaluation of Etest method for determining caspofungin (MK0991) susceptibilities of 726 clinical isolates of Candida species. J Clin Microbiol 2001;39:4387 /9.