CHEMOTHERAPY SEPT Amphotericin B (AMPH-B), flucytosine (5- SDA PDA Procedure for disk method 1. Prepare agar plates containing two-fold dilution

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2 CHEMOTHERAPY SEPT Amphotericin B (AMPH-B), flucytosine (5- SDA PDA Procedure for disk method 1. Prepare agar plates containing two-fold dilutions of drugs and dry agar surface at room temperature for 1 h 2. Immerse a paper disk in an inoculum suspension and place it on the agar medium 3. Incubate at 30 t for 3 days 4. Measure the radius of growth zone of fungi (excluding the 8 mm disk) PYGA YNBDA 5. Prepare dose-response curves based on the radius at each drug concentration and estimate IC,D values of antifungals for filamentous fungi Fig. 1. Procedure for disk method. Details are described in Methods. Fig. 2. Effects of inoculum size, incubation temperature and agar medium on mycelium growth of Aspergillus fumigatus Readings were made daily for 2 to 6 days. The averages of triplicate measurements are plotted.

3 VOL.41 NO.9 糸状 菌 に対 す る抗 真菌 剤感受 性 試験 法 Table 1. Effects of inoculum perature and agar growth 4 days gillus niger size, incubation medium after tem- 様 に 求 め た on mycelium inoculation 943 画線 法 of Asper- 寒 天 プ レ ー トは デ ィス ク 法 と 同様 に 鯛 整 し,こ れ に FP conidia/mlの Zone radius (mm) 菌 液 を画 線 幣 抹 器 で 接種 した プ レ ー ト は30,3日 間 培 養 し,MICは 増殖が抑制 され て い る 薬 剤 の 最 小 濃 度 と し た II,結 果 デ ィス ク 法 の 操 作 手 順 糸 状 菌 の 分 生 子 を デ ィ ス ク を用 い て 寒 天 培 地 に接 種 す る と,放 射 状 に 菌 糸 が 発 育 す る の で,培 地 上の菌 の 増 殖 域 の 最 長 半 径 を ノ ギ ス で 測 定 し た 詳 細 は方 法 の 項 お よ びFig,1説 A.fumigetusの Inoculum size: 0 1 ~ 104, Averages of triplicate h) 1 ~ 108 conidia/ml. measurements are Aspergillus given. 明 文 に記 載 した 菌糸発 育 spp,の3株 に つ い て,4種 地 上 で のA.fumigatus8004の 度,お Fig. 3. Outgrowth sive response gillus flavus 106 fumigatus FP 1022 conidia/ml pattern to of Aspergillus amphotericin FP 1305 (right). incubated spp. (AMPH) (upper), Tests at were 30 Ž made for よ び 接 種 菌 量 の 影 響 を検 討 し た(Fig.2) on disk-inoculated and flucytosine. Aspergillus niger on SDA 3 days. 類 の寒天 培 菌 糸 発 育 に対 す る 温 FP medium plates Disks 1398 (left) with and its contained an and regresasper- Aspergillus inoculum of 1 x 使

4 CHEMOTHERAPY Fig. 4. Dose-response curves of amphotericin (AMPH), fluconazole (FLCZ), miconazole (MCZ) and flucytosine for Aspergillus fumi- atus FP Test was made on an inoculumg of 1 x 106 conidia/ml incubated at 30 Ž for 3 dyas. IC50 values were estimated as 50% inhibiti on points on the curves and are indicated

5 VOL.41 NO.9 Table 2. A comparison of various IC,. and MIC values for 3 strains of Aspergillus funsigatus (Đg/m1) Tests were made on PYG medium with an inoculum of 1 ~ 10' conidia/ml at 30t for 3 days. Disk; Disk method, A,20; turbidmetric method, wt; dry weight method, MIC; streak method. Fluconazole Fig. 5. A comparison of dose-response curves of fluconazole for Aspergillus fumigatus FP 1305 obtained by the disk method and traditional mycological methods. Details are described in the legend to Table 2. disk, disk; wt., dry weight; A620, turbidmetric method. 1) Galgiani J N: Antifungal susceptibility tests. Anti-

6 CHEMOTHERAPY microb. agents Chemother. 31: 1867 `4870, ) Pfaller M A, Rinaldi M G, Galgiani J N, Bartlett M S, Body B A, Espinel-Ingroff A, Fromtling R A, Hall G S, Hughes C E, Odds F C, Sugar A M: Collaborative investigation of variables in susceptibility testing of yeasts. Antimicrob. Agents Chemother. 34: , ) Shadomy S, Pfaller M A: Laboratory studies with antifungal agents: susceptibility tests and quantitation in body.fluids, In Balows A, Hausler W J, Herrmann K L Jr., Isengerg H D, Shadomy H J (ed), Manual of clinical microbiology, 5 th ed., pp.1173 `1183, American society for microbiolgy, 1991 (Washington, D. C.) 4) Boyle F T, Ryley J F, Wilson R G: In vitro-in vivo correlations with azole antifungals. In Fromtling R A (ed.), Recent trends in the discovery, development and evaluation of antifungal agents, pp. 31 `41, J. R. Prous science publishers, S. A., 1987 (Barcelona, Spain) 5) Troke P F, Marriott M S, Richardson K, Tarbit M H: The in vitro potency and in vivo activity of azoles, In St. Giorgiev V (ed.), Antifungal drugs, pp.284 `293. New York Academy of Sciences, 1988 (New York) 6) Galgiani J N, Stevens D A: Antimicrobial susceptibility testing of yeasts: a turbidimetric technique independent of inoculum size. Antimicrob. Agents Chemother. 10: 721 `726, ) Odds F C: Antifungal susceptibility testing of Candida spp. by relative growth measurement at single concentrations of antifungals agents. Antimicrob. Agents Chemother. 36: 1727 `4737, ) Pore R S: Antibiotic susceptibility testing of Candida albicans by flow cytometry. Curr. Microbial. 20: 323 `328, ) Tellier R, Krajden M, Grigoriew G A, Campbell I: Innovative endpoint determination system for antifungal susceptibility testing of yeasts. Antimicrob. Agents Chemother. 36: 1619 `1625, ) Yanagida T, Kogane F: Growth and cytochemical differentiation of mold colonies. J. Gen. Appl. Microbiol. 8: 201 `213, ) Bartnicki-Garcia S: Fundamental aspects of hyphal morphogenesis. In 23 rd symposium of the society for general microbilogy. University Press, 1973 (Cambridge) 12) Gooday G W, Trinci A P J: Wall structure and biosynthesis in fungi. Symposia of the society for general microbiology 30: 207 `251, ) Bardana E J Jr.: The clinical spectrum of aspergillosis-part 2: classification and description of saprophytic, allergic, and invasive variants of human disease. CRC Crit Rev Clin Lab Sci. 13: 85 14) Sch$nheyder H: Pathogenetic and serological aspects of pulmonary aspergillosis. Scand. J. Infect. Dis. Suppl. 51: 1 `62, ) Rhodes J C. Virulence factors in fungal pathogens. Microbiol. Sci. 5: 252 `254, ) Reichard U, BUttner S, Eiffert H, Staib F, RUchel R: Purification and characterisation of an extracellular serine proteinsase from Aspergillus fumigatus and its detection in tissue. J. Med. Microbiol. 33: 243 `251, ) Zhu W -S, Wojdyla K, Donlon K, Thomas P A, Eberle H I: Extracellular proteases of Aspergillus flavus fungal keratitis, proteases, and pathogenesis. Diagn. Microbiol. Infect. Dis. 13: 491 ` 497, ) Arruda L K, Platts-Mills T A E, Fox J W, Chapman M D: Aspergillus fumigatus allergen I, a major IgE-binding protein, is a member of the mitogillin family of cytotoxins. J. Exp. Med. 172: 1529 `1532, ) Arruda L K, Good M, Platts-Mills T A E, Chapman M D, Charlottesville V A: Asp f I, a major A. fumigatus allergen: homology to the cytotoxin mitogillinand measurements in spore, mycelial and culture filtrate extracts. J. allergy din. immunol. 87: 168, ) Wosten H A B, Moukha S M, Sietsma J H, Wessels J G H: Localization of growth and secretion of proteins in Aspergillus niger. J. Gen. Microbiol. 137: 2017 `2023, 1991

7 VOL.41 NO.9 Reproducible susceptibility testing of antifungals for filamentous fungi by an inhibitory effect on mycelium growth Katsuyuki Maki, Yoshiko Koyama, Shuichi Tawara and Toshiaki Kamimura New Drug Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Kashima, Yodogawa-ku, Osaka 532, Japan Sachiko Goto Department of Microbiology, Toho University School of Medicine A reproducible in vitro method for the determination of IC. values of antifungals for filamentous fungi is described. A paper disk in which conidium of the test fungi was immersed was placed on a susceptibility agar plate and then incubated under appropriate conditions. The fungal conidium developed radial mycelium around the disk. Fungal growth was monitored by a growth zone radius around the disk. The effects of inoculum size and temperature on the mycelium growth of Aspergillus fumigatus were examined using four media. During the test period, the daily radius extention was linear and was little affected by the inoculum size. The inhibiotory effects of four antifungals, amphotericin B, miconazole, fluconazole and flucytosine, against Aspergillus fumigatus were examined. Dose-response regression of the radius was observed, and the ICeo value was obtained. The inhibitory effect of fluconazole could be detected sensitively even at 10 ug/ml or less. These data were closer to those obtained by the turbidmetric method than those obtained by the dry weight method.

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