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In vitro antifungal activity of membrane-active peptides against dermatophytes and yeasts using broth microdilution method
Journal of Dermatological Science (2004) 35, 64—67
LETTER TO THE EDITOR
In vitro antifungal activity of membrane-active peptides against dermatophytes and yeasts using broth microdilution method Sir, Host defense molecules composed of small peptides have been recently isolated from a variety of natural sources and some of them have been reported to have antibacterial or antifungal effects [1,2]. The fact that novel membrane-active peptides (MAPs)–—KKVVFKVKFK (KSL), KKVVFKVKFKK (MP), showed antifungal activity against Candida albicans [3,4] prompted us to evaluate in vitro antifungal activity of the MAPs against varieties of dermatophytes and yeasts species. We also compared minimal inhibitory concentrations (MICs) of these peptides with those of well-known antifungal agents, terbinafine (TBF) and fluconazole (FCZ). A total of 43 strains of dermatophytes (35) and yeasts (8) were tested. Among them, 27 strains were clinical isolates obtained from two university hospitals in Korea, and 16 strains from commercial sources. Dermatophytes included eight species and 35 strains and yeasts tested were five species and eight strains. All clinical isolates were identified by conventional methods. MP and KSL were synthesized by biochemical methods in protein laboratory (Peptron Inc., Daejeon, South Korea), and the mixture of MP and KSL was made in the ratio of one to one. TBF (Novartis, Basel, Switzerland) and FCZ (Choongwae Pharma Corporation, Seoul, South Korea) were obtained from their manufacturers. All agents were prepared as stock solutions of 1 mg/ml and frozen at 20 8C until use. We performed in vitro susceptibility test by broth microdilution method mainly following the NCCLS M27-A [5] except inoculum preparation. Serial two-fold dilutions were done in RPMI 1640 medium to twice the final concentrations required for testing. On the basis of MIC results from our preliminary study, test concentrations for MAPs and FCZ were set from 0.12 to 64.0 mg/ml, while TBF was tested at 0.0001—16.0 mg/ml. Standardized inoculum was prepared to the desired concentrations ranging from 1:0 103 to 5:0 103 CFU (colony forming unit) per milliliter by counting the microconidia with hemocytometer. The tests were
performed in sterile, round-bottomed, 96-well microplates (tissue culture testplate; Sewon Co., Seoul, South Korea). Column 1 was filled with 200 ml drug-free medium to serve as sterility control. Column 12 was filled with 100 ml diluted inoculum plus 100 ml drug-free medium for growth control. Columns 2—11 were filled with 100 ml of diluted inoculum plus 100 ml serially diluted antifungal agents. The microplate contents were incubated at 35 8C with prevention against desiccation. The MIC reading was done after 48 h of incubation for yeasts and 72 h of incubation for dermatophytes. The growth in each well was compared with that of growth control. The MICs for TBF was defined as the lowest drug concentration that caused approximately 75% inhibition of fungal growth while those for FCZ and MAPs were defined at 50% growth inhibition. In accordance with the NCCLS M27-A, Candida parapsilosis (ATCC 22019) was used as quality control (QC) strain, and tested with each assay. The MIC values obtained for this QC strain were within expected ranges (2.0—8.0 mg/ml for FCZ). Antifungal susceptibility against TBF and FCZ was evaluated twice, and was indicated by the range of MIC values for each species with the exception of in vitro susceptibility of yeasts strains for FCZ. The mean MICs of MP (3:89 2:93 mg/ml) and KSL (3:44 3:33 mg/ml) for dermatophytes were not significantly different. Trychophyton schoenleinii (IHEM 5232) showed rather high MICs for MP (16.67 mg/ml) and KSL (20.00 mg/ml). The combination of MP and KSL showed synergistic effects on inhibition of Trychophyton violaceum, M. canis, E. floccosum, and M. audoinii (Table 1). In yeasts, however, the mean MIC value of MP (5:50 2:78 mg/ml) was significantly lower than that of KSL (15:00 7:93 mg/ml) (Table 2). Combination of MP and KSL did not produce synergistic effects. We also evaluated in vitro susceptibility of fungi against TBF and FCZ (Tables 1 and 2). The MICs of TBF for dermatophytes and yeasts were less than 0.008 and 0.06 mg/ml, respectively, similar to (dermatophytes) or lower than (yeasts) the results of previous study. In the case of FCZ, all dermatophyte species had the MICs over 0.5 mg/ml, and MICs for
0923–1811/$30.00 ß 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.02.006
Letter to the Editor
Table 1
65
MIC (mg/ml) values of different antifungals for dermatophytes
Strains T. rubrum STR1 STR2 STR3 STR4 STR5 KR1 KR18 ATCC 28188 Mean T. mentagrophytes STM1 STM2 STM5 STM6 STM7 STM9 IHEM 584 IHEM 6354 IHEM 10208 Mean M. canis SMC1 SMC2 SMC3 SMC4 SMC5 SMC6 IHEM 2483 Mean E. Floccosum SEF1 KEF1 IHEM 2536 Mean T. verrucosum STV2 STV3 IHEM 5480 Mean T. violaceum IFM 41074 IFM 41075 IHEM 4887 Mean
MP
KSL
MP þ KSL
3.00 3.00 3.67 2.33 1.00 3.67 1.00 5.33
1.00 2.33 2.33 1.00 2.33 1.00 1.00 2.33
3.83 4.00 4.00 4.00 4.00 1.00 1.00 2.00
2.88 1.45
1.67 0.71
2.98 1.40
3.00 1.00 1.00 2.33 1.00 1.00 1.00 2.33 2.33
5.33 2.33 1.00 1.00 1.00 1.00 1.00 2.33 2.33
3.67 2.33 2.33 2.33 2.33 2.33 2.33 2.33 2.33
1.67 0.82
1.92 1.43
2.48 0.45
2.33 5.00 6.67 6.67 8.33 6.67 2.33
2.33 2.33 5.00 5.00 5.33 5.00 2.33
1 1 5 5 2.33 2.33 2.33
5.43 2.32
3.90 1.48
2.71 1.67
3.67 3.67 3.67
3.67 3.67 3.67
2.33 2.33 2.33
3.67 0.00
3.67 0.00
2.33 0.00
5.00 5.00 5.00
5.00 5.00 5.00
5.00 5.00 5.00
5.00 0.00
5.00 0.00
5.00 0.00
5.00 3.67 3.67
5.00 2.33 2.33
3.00 3.67 1.00
4.11 0.77
3.22 1.54
2.56 1.39
5.00
6.67
1.00
TBF
FCZ
<0.008
>1
<0.001
>1
<0.008
>1
<0.008
>0.5
<0.008
>0.5
<0.008
>1
<0.008
>0.5
M. audoinii IHEM 10159
66
Letter to the Editor
Table 1 (Continued ) Strains
MP
KSL
MP þ KSL
TBF
FCZ
16.67
20.00
20.00
<0.008
>1
T. schoenleinii IHEM 5232 Total 3.89 2.93
3.44 3.33
3.31 3.17
MIC values of MP and KSL are from triplicate experiments and indicated by mean standard deviation. The MIC values of TBF and FCZ were measured for only one standard strain of each species, and are represented by range. STR, STM, SMC, SEF, and STV: strains from Seoul National University Hospital. KR and KEF: strains from Kyungpook University. ATCC: American type culture collection, Manassas, USA; IHEM: Institute of Hygiene and Epidemiology Culture Collection, Bruxelles, Belgium; IFM: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba, Japan; TBF: terbinafine; and FCZ: fluconazole.
yeasts ranged from 2 to 16 mg/ml. FCZ was superior to MAPs only for C. albicans strains. Our study demonstrated that the new MAPs and their combination have antifungal effects. For dermatophytes, MP and KSL had similar MIC ranges but the combination of both MAPs showed some synergistic effects whereas in yeasts MP was more effective and the combination revealed no superior effects. In comparison to well-established antifun-
Table 2
gal agents such as TBF or FCZ, neither MAPs showed superiority over TBF in dermatophytes and yeasts. However, MP turned out to be more potent than FCZ against yeasts except for C. albicans. We followed M27-A [5] from the National Committee for Clinical Laboratory Standards (NCCLS). NCCLS provides reference manuals to overcome inter-laboratory variability and clinical relevance of in vitro data. Although it recently produced
MIC (mg/ml) values of different antifungals for yeasts
Strains
MP
KSL
C. Guilliermondii SY2
4
16
SY3 ATCC 750
4 2
Mean
MP þ KSL
TBF
FCZ
4
<0.016
16
8 16
4 8
<0.016
8 8
3
12
6
SY4 ATCC 90028
8 8
32 16
32 16
Mean
8
24
24
ATCC 22019 ATCC 90018
2 8
8 16
4 8
Mean
5
12
6
5.50 2.78
15.00 7.93
C. Tropicalis
8
C. Albicans <0.03
4 4 4
C. Parapsilosis <0.06
8 2 5
Total 10.50 9.55
The MIC values of TBF for yeasts were measured for only one standard strain of each species except C. guilliermondii. SY: strain from Seoul National University Hospital; ATCC: American type culture collection, Manassas, USA; TBF: terbinafine; and FCZ: fluconazole.
Letter to the Editor
new reference method (NCCLS M38-P) [6] for in vitro susceptibility testing of filamentous fungi, it still lacks some important features such as representative species and antifungal agents. Considering incompleteness of reference method for dermatophytes, our experiment for dermatophytes followed NCCLS M27-A as well. Interpretive breakpoints of resistance by the NCCLS exist in candida species only for FCZ, itrconazole, and flucytosine [7,8]. For FCZ, MIC under 8 mg/ml is regarded as susceptible, ranges from 16 to 32 mg/ml as susceptible dose-dependent, and that over 64 mg/ml as resistant. In our experiment, MICs of FCZ for C. guilliermondii fell into the susceptible-dose dependent category, but this strain could be inhibited by MP and MP þ KSL (Table 2). Despite our data, it should be further evaluated whether MAPs could inhibit the growth of fungal strains resistant to known antifungal agents. In conclusion, MP and KSL clearly showed antifungal activity against dermatophytes and yeasts. However, they should go through toxicity, pharmacokinetics, and pharmacodynamics experiment before they could be considered as clinically useful antifungal agents.
Acknowledgements We thank Novartis and Choongwae Pharma Corporation for their kind donation of terbinafine and fluconazole. This work was supported by Korean Dermatological Foundation 2001 (Yang).
References [1] Barra D, Simmanco M. Amphibian skin: a promising resource for antimicrobial peptides. Trends Biotechnol 1995;13: 205—9. [2] Berkowitz BA, Bevins CL, Zasloff MA. Magainins: a new family of membrane-active peptides. Biochem Pharmacol 1990;39: 625—9. [3] Hong SY, Oh JE, Kwon MY, Choi MJ, Lee JH, Lee BL, et al. Identification and characterization of novel antimicrobial decapeptides generated by combinatorial chemistry. Antimicrob Agents Chemother 1998;42:2534—41.
67
[4] Hong SY, Oh JE, Lee KH. In vitro antifungal activity and cytotoxicity of a novel membrane-active peptides. Antimicrob Agents Chemother 1999;43:1704—7. [5] National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. Document M27A. Wayne, PA: National Committee for Clinical Laboratory Standards, 1997. [6] National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard. Document M38-P. Wayne, PA: National Committee for Clinical Laboratory Standards, 1998. [7] Espinel-Ingroff A, White T, Pfaller MA. Antifungal agents and susceptibility test. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH., editors. Manual of Clinical Microbiology, 7th ed. Washington DC: ASM Press; 1999. p. 1640—52. [8] Kontoyiannis DP, Lewis RE. Antifungal drug resistance of pathogenic fungi. Lancet 2002;359:1135—44.
Suk-Jin Choi Dong-Youn Lee Joo-Heung Lee Eil-Soo Lee Department of Dermatology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu Seoul 135-710, South Korea Jeong Aee Kim Department of Dermatology, Seoul National University College of Medicine, Seoul, South Korea Jung-Hyun Sung Samsung Biomedical Research Institute Seoul, Republic of Korea Jun-Mo Yang * Department of Dermatology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu Seoul 135-710, South Korea Samsung Biomedical Research Institute Seoul, Republic of Korea * Corresponding author. Tel.: þ82-2-3410-3541 fax: þ82-2-3410-3869 E-mail address: [email protected] (J.-M. Yang) 20 October 2003
LETTER TO THE EDITOR
In vitro antifungal activity of membrane-active peptides against dermatophytes and yeasts using broth microdilution method Sir, Host defense molecules composed of small peptides have been recently isolated from a variety of natural sources and some of them have been reported to have antibacterial or antifungal effects [1,2]. The fact that novel membrane-active peptides (MAPs)–—KKVVFKVKFK (KSL), KKVVFKVKFKK (MP), showed antifungal activity against Candida albicans [3,4] prompted us to evaluate in vitro antifungal activity of the MAPs against varieties of dermatophytes and yeasts species. We also compared minimal inhibitory concentrations (MICs) of these peptides with those of well-known antifungal agents, terbinafine (TBF) and fluconazole (FCZ). A total of 43 strains of dermatophytes (35) and yeasts (8) were tested. Among them, 27 strains were clinical isolates obtained from two university hospitals in Korea, and 16 strains from commercial sources. Dermatophytes included eight species and 35 strains and yeasts tested were five species and eight strains. All clinical isolates were identified by conventional methods. MP and KSL were synthesized by biochemical methods in protein laboratory (Peptron Inc., Daejeon, South Korea), and the mixture of MP and KSL was made in the ratio of one to one. TBF (Novartis, Basel, Switzerland) and FCZ (Choongwae Pharma Corporation, Seoul, South Korea) were obtained from their manufacturers. All agents were prepared as stock solutions of 1 mg/ml and frozen at 20 8C until use. We performed in vitro susceptibility test by broth microdilution method mainly following the NCCLS M27-A [5] except inoculum preparation. Serial two-fold dilutions were done in RPMI 1640 medium to twice the final concentrations required for testing. On the basis of MIC results from our preliminary study, test concentrations for MAPs and FCZ were set from 0.12 to 64.0 mg/ml, while TBF was tested at 0.0001—16.0 mg/ml. Standardized inoculum was prepared to the desired concentrations ranging from 1:0 103 to 5:0 103 CFU (colony forming unit) per milliliter by counting the microconidia with hemocytometer. The tests were
performed in sterile, round-bottomed, 96-well microplates (tissue culture testplate; Sewon Co., Seoul, South Korea). Column 1 was filled with 200 ml drug-free medium to serve as sterility control. Column 12 was filled with 100 ml diluted inoculum plus 100 ml drug-free medium for growth control. Columns 2—11 were filled with 100 ml of diluted inoculum plus 100 ml serially diluted antifungal agents. The microplate contents were incubated at 35 8C with prevention against desiccation. The MIC reading was done after 48 h of incubation for yeasts and 72 h of incubation for dermatophytes. The growth in each well was compared with that of growth control. The MICs for TBF was defined as the lowest drug concentration that caused approximately 75% inhibition of fungal growth while those for FCZ and MAPs were defined at 50% growth inhibition. In accordance with the NCCLS M27-A, Candida parapsilosis (ATCC 22019) was used as quality control (QC) strain, and tested with each assay. The MIC values obtained for this QC strain were within expected ranges (2.0—8.0 mg/ml for FCZ). Antifungal susceptibility against TBF and FCZ was evaluated twice, and was indicated by the range of MIC values for each species with the exception of in vitro susceptibility of yeasts strains for FCZ. The mean MICs of MP (3:89 2:93 mg/ml) and KSL (3:44 3:33 mg/ml) for dermatophytes were not significantly different. Trychophyton schoenleinii (IHEM 5232) showed rather high MICs for MP (16.67 mg/ml) and KSL (20.00 mg/ml). The combination of MP and KSL showed synergistic effects on inhibition of Trychophyton violaceum, M. canis, E. floccosum, and M. audoinii (Table 1). In yeasts, however, the mean MIC value of MP (5:50 2:78 mg/ml) was significantly lower than that of KSL (15:00 7:93 mg/ml) (Table 2). Combination of MP and KSL did not produce synergistic effects. We also evaluated in vitro susceptibility of fungi against TBF and FCZ (Tables 1 and 2). The MICs of TBF for dermatophytes and yeasts were less than 0.008 and 0.06 mg/ml, respectively, similar to (dermatophytes) or lower than (yeasts) the results of previous study. In the case of FCZ, all dermatophyte species had the MICs over 0.5 mg/ml, and MICs for
0923–1811/$30.00 ß 2004 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.02.006
Letter to the Editor
Table 1
65
MIC (mg/ml) values of different antifungals for dermatophytes
Strains T. rubrum STR1 STR2 STR3 STR4 STR5 KR1 KR18 ATCC 28188 Mean T. mentagrophytes STM1 STM2 STM5 STM6 STM7 STM9 IHEM 584 IHEM 6354 IHEM 10208 Mean M. canis SMC1 SMC2 SMC3 SMC4 SMC5 SMC6 IHEM 2483 Mean E. Floccosum SEF1 KEF1 IHEM 2536 Mean T. verrucosum STV2 STV3 IHEM 5480 Mean T. violaceum IFM 41074 IFM 41075 IHEM 4887 Mean
MP
KSL
MP þ KSL
3.00 3.00 3.67 2.33 1.00 3.67 1.00 5.33
1.00 2.33 2.33 1.00 2.33 1.00 1.00 2.33
3.83 4.00 4.00 4.00 4.00 1.00 1.00 2.00
2.88 1.45
1.67 0.71
2.98 1.40
3.00 1.00 1.00 2.33 1.00 1.00 1.00 2.33 2.33
5.33 2.33 1.00 1.00 1.00 1.00 1.00 2.33 2.33
3.67 2.33 2.33 2.33 2.33 2.33 2.33 2.33 2.33
1.67 0.82
1.92 1.43
2.48 0.45
2.33 5.00 6.67 6.67 8.33 6.67 2.33
2.33 2.33 5.00 5.00 5.33 5.00 2.33
1 1 5 5 2.33 2.33 2.33
5.43 2.32
3.90 1.48
2.71 1.67
3.67 3.67 3.67
3.67 3.67 3.67
2.33 2.33 2.33
3.67 0.00
3.67 0.00
2.33 0.00
5.00 5.00 5.00
5.00 5.00 5.00
5.00 5.00 5.00
5.00 0.00
5.00 0.00
5.00 0.00
5.00 3.67 3.67
5.00 2.33 2.33
3.00 3.67 1.00
4.11 0.77
3.22 1.54
2.56 1.39
5.00
6.67
1.00
TBF
FCZ
<0.008
>1
<0.001
>1
<0.008
>1
<0.008
>0.5
<0.008
>0.5
<0.008
>1
<0.008
>0.5
M. audoinii IHEM 10159
66
Letter to the Editor
Table 1 (Continued ) Strains
MP
KSL
MP þ KSL
TBF
FCZ
16.67
20.00
20.00
<0.008
>1
T. schoenleinii IHEM 5232 Total 3.89 2.93
3.44 3.33
3.31 3.17
MIC values of MP and KSL are from triplicate experiments and indicated by mean standard deviation. The MIC values of TBF and FCZ were measured for only one standard strain of each species, and are represented by range. STR, STM, SMC, SEF, and STV: strains from Seoul National University Hospital. KR and KEF: strains from Kyungpook University. ATCC: American type culture collection, Manassas, USA; IHEM: Institute of Hygiene and Epidemiology Culture Collection, Bruxelles, Belgium; IFM: Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba, Japan; TBF: terbinafine; and FCZ: fluconazole.
yeasts ranged from 2 to 16 mg/ml. FCZ was superior to MAPs only for C. albicans strains. Our study demonstrated that the new MAPs and their combination have antifungal effects. For dermatophytes, MP and KSL had similar MIC ranges but the combination of both MAPs showed some synergistic effects whereas in yeasts MP was more effective and the combination revealed no superior effects. In comparison to well-established antifun-
Table 2
gal agents such as TBF or FCZ, neither MAPs showed superiority over TBF in dermatophytes and yeasts. However, MP turned out to be more potent than FCZ against yeasts except for C. albicans. We followed M27-A [5] from the National Committee for Clinical Laboratory Standards (NCCLS). NCCLS provides reference manuals to overcome inter-laboratory variability and clinical relevance of in vitro data. Although it recently produced
MIC (mg/ml) values of different antifungals for yeasts
Strains
MP
KSL
C. Guilliermondii SY2
4
16
SY3 ATCC 750
4 2
Mean
MP þ KSL
TBF
FCZ
4
<0.016
16
8 16
4 8
<0.016
8 8
3
12
6
SY4 ATCC 90028
8 8
32 16
32 16
Mean
8
24
24
ATCC 22019 ATCC 90018
2 8
8 16
4 8
Mean
5
12
6
5.50 2.78
15.00 7.93
C. Tropicalis
8
C. Albicans <0.03
4 4 4
C. Parapsilosis <0.06
8 2 5
Total 10.50 9.55
The MIC values of TBF for yeasts were measured for only one standard strain of each species except C. guilliermondii. SY: strain from Seoul National University Hospital; ATCC: American type culture collection, Manassas, USA; TBF: terbinafine; and FCZ: fluconazole.
Letter to the Editor
new reference method (NCCLS M38-P) [6] for in vitro susceptibility testing of filamentous fungi, it still lacks some important features such as representative species and antifungal agents. Considering incompleteness of reference method for dermatophytes, our experiment for dermatophytes followed NCCLS M27-A as well. Interpretive breakpoints of resistance by the NCCLS exist in candida species only for FCZ, itrconazole, and flucytosine [7,8]. For FCZ, MIC under 8 mg/ml is regarded as susceptible, ranges from 16 to 32 mg/ml as susceptible dose-dependent, and that over 64 mg/ml as resistant. In our experiment, MICs of FCZ for C. guilliermondii fell into the susceptible-dose dependent category, but this strain could be inhibited by MP and MP þ KSL (Table 2). Despite our data, it should be further evaluated whether MAPs could inhibit the growth of fungal strains resistant to known antifungal agents. In conclusion, MP and KSL clearly showed antifungal activity against dermatophytes and yeasts. However, they should go through toxicity, pharmacokinetics, and pharmacodynamics experiment before they could be considered as clinically useful antifungal agents.
Acknowledgements We thank Novartis and Choongwae Pharma Corporation for their kind donation of terbinafine and fluconazole. This work was supported by Korean Dermatological Foundation 2001 (Yang).
References [1] Barra D, Simmanco M. Amphibian skin: a promising resource for antimicrobial peptides. Trends Biotechnol 1995;13: 205—9. [2] Berkowitz BA, Bevins CL, Zasloff MA. Magainins: a new family of membrane-active peptides. Biochem Pharmacol 1990;39: 625—9. [3] Hong SY, Oh JE, Kwon MY, Choi MJ, Lee JH, Lee BL, et al. Identification and characterization of novel antimicrobial decapeptides generated by combinatorial chemistry. Antimicrob Agents Chemother 1998;42:2534—41.
67
[4] Hong SY, Oh JE, Lee KH. In vitro antifungal activity and cytotoxicity of a novel membrane-active peptides. Antimicrob Agents Chemother 1999;43:1704—7. [5] National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. Document M27A. Wayne, PA: National Committee for Clinical Laboratory Standards, 1997. [6] National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard. Document M38-P. Wayne, PA: National Committee for Clinical Laboratory Standards, 1998. [7] Espinel-Ingroff A, White T, Pfaller MA. Antifungal agents and susceptibility test. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH., editors. Manual of Clinical Microbiology, 7th ed. Washington DC: ASM Press; 1999. p. 1640—52. [8] Kontoyiannis DP, Lewis RE. Antifungal drug resistance of pathogenic fungi. Lancet 2002;359:1135—44.
Suk-Jin Choi Dong-Youn Lee Joo-Heung Lee Eil-Soo Lee Department of Dermatology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu Seoul 135-710, South Korea Jeong Aee Kim Department of Dermatology, Seoul National University College of Medicine, Seoul, South Korea Jung-Hyun Sung Samsung Biomedical Research Institute Seoul, Republic of Korea Jun-Mo Yang * Department of Dermatology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu Seoul 135-710, South Korea Samsung Biomedical Research Institute Seoul, Republic of Korea * Corresponding author. Tel.: þ82-2-3410-3541 fax: þ82-2-3410-3869 E-mail address: [email protected] (J.-M. Yang) 20 October 2003