Fungicidal activities of commonly used disinfectants and antifungal pharmaceutical spray preparations against clinical strains of Aspergillus

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1 Ó Medical Mycology 2002, 40, Accepted 19 September 2001 Fungicidal activities of commonly used disinfectants and antifungal pharmaceutical spray preparations against clinical strains of Aspergillus and Candida species A. K. GUPTA*, I. AHMADy & R. C. SUMMERBELLz * Division of Dermatology, Department of Medicine, Sunnybrook and Women s College Health Sciences Center (Sunnybrook site), and the University of Toronto, Toronto, Ontario, Canada; ymedical Mycology, Laboratories Branch, Ontario Ministry of Health, Toronto, Ontario, Canada and Mediprobe Laboratories, Toronto, Ontario, Canada; zdepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada and Centraalbureau voor Schimmelcultures, Baarn, Netherlands The antifungal ef cacy of commercial chemical disinfectants and pharmaceutical antifungal agents against medically important moulds and yeast species was investigated. Chlorine, phenol, sodium dodecyl sulfate and quaternary ammonium salts were the chemical disinfectants, and bifonazole and terbina ne were the antifungal pharmaceutical products tested against clinical isolates of Aspergillus and Candida species. Fungal inocula were obtained from conidial preparations of two A. ochraceus strains and yeast cells of C. albicans, C. krusei and C. parapsilosis. The antifungal activities were evaluated either by determining the kill rate in a cell suspension media at different contact periods, or by examining the viability and growth on plates sprayed with the active ingredient. Chlorine (1%) was the only disinfectant with the ability to cause a rapid inactivation of all ve strains. Phenol (5%) was equally effective against Candida species; however, a number of A. ochraceus conidia were able to survive this treatment for up to 1 h. Benzalkonium chloride (0 5%) and cetrimide (0 5%) were also able to disinfect the three Candida species rapidly; however, these two quaternary ammonium compounds were relatively ineffective against A. ochraceus. In spray experiments, quaternary ammonium compounds had a fungicidal activity against Candida species and were fungistatic against A. ochraceus conidia. All ve fungal strains were able to resist 0 5% sodium dodecyl sulfate, present either in the suspension solution or on the sprayed plate. Of the two pharmaceutical antifungal products tested, bifonazole (1%) were essentially ineffective against all ve strains. Terbina ne (1%) had a fungicidal activity against A. ochraceus and C. parapsilosis. In suspension experiments, an exposure to 0 01% terbina ne required a contact period of 1 h for a complete inactivation of A. ochraceus conidia and an onset of fungicidal effect on C. parapsilosis yeast cells. Terbina ne was only moderately effective against C. albicans and was completely ineffective against C. krusei. Keywords antifungal drugs, Aspergillus ochraceus, Candida species, chemical disinfectants, fungicidal, fungistatic, nondermatophytes Correspondence: Aditya K. Gupta M.D., Ph.D. F.R.C.P.(C), 490 Wonderland Road South, Suite 6, London, Ontario, Canada N6K 1L6. Tel.: ; Fax: ; agupta@ execulink.com. Downloaded Ó 2002 from ISHAM, Medical Mycology, 40,

2 202 Gupta et al. Introduction Pathogenic strains of Candida and Aspergillus species are the primary cause of hospital-acquired fungal infections [1], and in recent years these yeast and mould infections have been recognized as serious threats to immunocompromised patients [2 4]. The distribution of these fungal pathogens appears to be widespread in our environment. In dermatological laboratory examinations, Candida and Aspergillus species are frequently isolated as super cial infections of skin and nails [5,6]. The shedding of dermatophytes and other fungal pathogens from infected individuals often leads to the contamination of recreation facilities in public arenas [7], and the eradication of this contamination from public facilities such as swimming pools has been found to be an arduous task [8]. These facts underscore the importance of nding adequate measures for eradicating fungal contaminants in our immediate environment. Several new disinfectants containing quaternary ammonium compounds as active agents have been introduced in the market in recent years. Many of these formulations are stated to have a broadly based antifungal activity; however, the evidence for their ef cacy against medically important fungi is not clear in the literature. The work of Ohta et al. [9] indicates a considerable variation in the ability of commercially available quaternary ammonium compounds to disinfect clinical strains belonging to Aspergillus and Candida species. In the US, all the Environmental Protection Agency (EPA)- registered quaternary-ammonium-compound-based antifungal formulations failed the disinfectant test against clinical strains of Aspergillus species [10]. These studies indicate some serious shortcomings in the ef cacy of prevalent antifungal commercial disinfectants, and there appears to be a need for new approaches to disinfecting pathogenic fungi in our environment. The work of Piérard et al. [11] with miconazole powder suggest that pharmaceutical products can be employed super cially as protective agents against infectious fungi present on external surfaces. Fortunately, a number of antifungal pharmaceutical products have become available in spray formulations in recent years. It is pertinent, therefore, to evaluate the ef cacy of antifungal drugs to disinfect fungal pathogens prevalent in our environment. Currently, the disinfection of various groups of medically important fungi is being investigated in our laboratory. Our work on the disinfection of dermatophytic species of Trichophyton has been presented previously [12]. The present paper deals with the disinfection of Aspergillus and Candida species. The antifungal activities of several commonly used chemical disinfectants and two recently introduced pharmaceutical antifungal sprays has been evaluated against standardized inocula of A. ochraceus, C. albicans, C. krusei and C. parapsilosis. Materials and methods Clinical isolates Freshly isolated strains of Aspergillus and Candida species were obtained from clinical material at Laboratories Branch of Ontario Ministry of Health. Table 1 lists two isolates of A. ochraceus and single isolates of C. albicans, C. krusei and C. parapsilosis employed in the present study. Cultures were maintained at 28 o C by monthly transfer on Sabouraud s agar medium (10 g l 1 of Bacto peptone 40 g l 1 of glucose 15 g l 1 of Difco agar). For experimental work, Candida yeast cells were obtained from 1-week-old fungal lawns grown on 15 ml of Sabouraud s agar in a 8 5-cm diameter petri dish. Conidia of A. ochraceus were obtained from 2 3- week-old fungal lawns grown on 80 ml of Sabouraud s agar in a 15-cm diameter petri dish. Preparation of fungal inocula The preparation of fungal inocula was carried out under axenic conditions. Conidial cultures of A. ochraceus were ooded with 20 ml of phosphate-buffered saline (PBS), ph 7 2, gently scraped with a glass rod and the resulting conidial suspension was gradually loaded with a Pasteur pipette on a 5-ml plastic column padded with a Table 1 Clinical isolates Species Vegetative morphology Designated OMH* number Inoculum cell density (cfu ml 1 ) A. ochraceus Filamentous FR A. ochraceus Filamentous SF C. albicans Yeast FR C. krusei Yeast FR C. parapsilosis Yeast FR *Ontario Ministry of Health laboratory specimen accession number.

3 Disinfectants and antifungals against Aspergillus and Candida 203 porous disk. Any hyphal debris on the top of the disk was cleared with a metal loop. The ltered conidial suspension was collected in a centrifuge tube and made up to 20 ml with PBS, ph 7 2. Candida cultures were scraped off gently with a heat-sterile scalpel and suspended in 20 ml of PBS, ph 7 2. These Candida preparations yielded homogenous turbid suspensions of yeast cells without any evidence of clumps or debris under microscopic examinations. Therefore, ltration was not considered necessary for these preparations. For cell density measurements, each preparation was serially diluted and at each dilution triplicate 100-m l frs were inoculated on 8 5-cm Sabouraud s agar plates. The inoculum was spread evenly on the plate surface with a metal loop. The cell density of the inoculum was estimated at the dilution yielding individual colonies per culture plate. The cell density values presented in Table 1 correspond to the 20-ml inoculum prepared for each strain. The disinfection studies were initiated within 2 h of inocula preparation. Preparation of disinfectant suspension solutions Stock solutions of 10% chlorine (sodium hypochlorite), 50% phenol, 5% benzalkonium chloride (BAC), 5% hexadecyltrimethyl ammonium bromide (cetrimide), 5% sodium dodecyl sulfate (SDS) were prepared in autoclaved distilled water. All reagents used were of analytical grade. Terbina ne stock was prepared by extracting a powdered 250-mg Sandoz Lamisil 1 tablet (Sandoz Pharmaceutical Corporation, East Hanover, NJ, USA) for 30 min in 2 5 ml of dimethyl sulfoxide, clarifying the extract by centrifugation at 1000g for 5 min and serially diluting the clari ed extract in 80% ethanol to obtain a 0 1% stock solution. All stock solutions were stored at room temperature. Disinfectant suspension solutions were prepared by adding 0 5 ml of stock solution to 4 ml of autoclaved PBS, ph 7 2. Negative controls were prepared by adding 0 5 ml of autoclaved water to 4 ml of PBS, ph 7 2. Measurement of kill rates The kill rate of each disinfectant solution was determined by examining the viability of fungal cells after 0-, 0 25-, 0 5-, 1 0- and 24-h periods of contact with the disinfectant solution. The treatment was initiated by adding 0 5 ml of quanti ed fungal inoculum to 4 5 ml of disinfectant solution and was terminated by centrifuging the suspension at 2000g for 5 min at room temperature and suspending the fungal pellet in 10 ml of fresh PBS, ph 7 2. To examine the presence of living cells in these suspensions, a tenfold dilution of A. ochraceus and a 1000-fold dilution of Candida suspensions were prepared, and triplicate 100-m l frs were inoculated on 10-ml Sabouraud s slants in 2 5 5cm screw-capped bottles. The inocula were spread evenly on agar slants with a metal loop and the slants were incubated horizontally at room temperature. The slants were examined regularly for the emergence of fungal colonies. For each contact time, the highest colony count obtained over a 7-day growth period was recorded as. Disinfectant spray formulations Disinfectant solutions of 5% phenol, 0 5% BAC, 0 5% cetrimide were prepared in autoclaved water from stock solutions and transferred to sterile 500-ml amber bottles tted with plastic spray assemblies. The spray bottle produced 0 6 ml of ne mist in each delivery. Commercially available brands of Lysol 1 domestic spray (Reckitt & Coleman Inc., Toronto, Ontario, Canada), Spray Nine 1 (Korkay System Canada Ltd., Gananoque, Ontario, Canada) and Brentdale germicidal spray (Brentdale Chemicals, Rexdale, Ontario, Canada), each containing a speci c formulation of quaternary ammonium compounds (Table 2), were obtained locally. Mycospor 1 spray containing 1% bifonazole (Bayer AG, Germany) and Lamisil 1 spray containing 1% terbina ne HCl (Novartis, Spartan, Kempton Park, South Africa) were obtained as physician samples. Table 2 Antifungal spray formulations Trade name Lysol 1 Spray Nine 1 Brentdale Mycospor 1 Lamisil 1 Active ingredients 0 1% n-alkyl (40% C 1 2, 50% C 14, 30% C 1 6 ) dimethylbenzylammonium chloride 0 15% n-alkyl (5% C 1 2, 60% C 14, 30% C 1 6, 5% C 18 ) dimethylbenzylammonium chloride 0 15% n-alkyl (68% C 12, 32% C 1 4 ) dimethylethylbenzylammoniumchloride 0 1% n-alkyl (5% C 12, 60% C 1 4, 30% C 1 6, 5% C 1 8 ) dimethylbenzylammonium chloride 0 1% n-alkyl (68% C 1 2, 32% C 14 ) dimethylethylbenzylammoniumchloride 1 6% Tetrasodium ethylenediamine tetraacetate 1% Bifonazole 1% Terbina ne hydrochloride Downloaded Ó 2002 from ISHAM, Medical Mycology, 40,

4 204 Gupta et al. Spray experiments For each spray treatment, triplicate 100-m l aliquots of quanti ed inoculum were evenly plated on 15 ml of Sabouraud s agar medium in 8 5-cm diameter petri dishes and left for 2 h at room temperature. Plates were treated with the given disinfectant by spraying a thin lm of about ml on the agar surface. This layer of spray on the agar plate covered with an unsealed lid evaporated completely within 3 days of incubation at room temperature. On day 3, a surface swab from of each plate was inoculated onto a 10-ml Sabouraud s slant in a cm bottle. Both sprayed plates and swab test slants were examined regularly, and the numbers of newly emerging colonies were recorded for a period of 14 days. Any spray formulation allowing more than 300 fungal colonies on the sprayed agar plate was categorized as noninhibitory. The spray was categorized as moderately inhibitory when the number of colonies on the plate ranged between 100 and 300. A fungistatic was recorded when a large area of the sprayed plate was essentially free of fungal growth, whereas the swab test from the colony-free plate surface was positive for fungal growth. The spray was categorized as fungicidal when the sprayed plate was essentially free of fungal growth and the swab test was negative for growth. Results Table 1 lists the two lamentous strains of A. ochraceus and the three yeast species of Candida grown from primary cultures of clinical isolates. Each isolate was transferred to fresh agar media every 4 weeks and used for disinfectant tests within 3 months of its isolation. Over this period, the subcultures of both Aspergillus strains and three Candida species maintained their normal colony appearance, thereby ruling out any apparent deterioration of these isolates on the growth media. In preliminary studies, a 24-h suspension of the fungal inoculum in PBS, ph 7 2, followed by a 5-min centrifugation at 2000g had no detectable effect on the viability of A. ochraceus conidia and Candida yeast cells (results not shown). The effect of each disinfectant on the ve fungal strains was evaluated either by determining the number of cells surviving after a given period of cell contact with the disinfectant in a suspension solution (Tables 3 and 4), or by counting the number of colonies able to grow on agar plates sprayed with the disinfectant (Tables 5 and 6). The presented in Tables 3 and 4 correspond to 100 m l of an appropriately diluted cell suspension that was inoculated on a 10-ml agar slant in a 16-ml tube. Under these conditions, the water controls for each of the ve fungal strains produced a dense growth with more than 100 overlapping colonies covering the entire agar slant. Any other treatment allowing normal growth with more than 100 colonies on the agar slant was, therefore, evaluated as noninhibitory. Similarly, in spray experiments (Tables 5 and 6), water controls produced a dense growth of more than 300 individual colonies on the 8 5-cm diameter agar plate, and therefore, any other spray treatment allowing the growth of more than 300 colonies on the plate was considered noninhibitory. In A. ochraceus, the inactivation of conidia occurred most rapidly in the presence of chlorine (Table 3). Both FR and SF-0643 were killed within 15 min of Table 3 Inactivation of A. ochraceus conidia in disinfectant suspension solutions Contact time Treatment Isolate no. 15 min 30 min 60 min 24 h Water FR > 100 > 100 > 100 > 100 SF > 100 > 100 > 100 > 100 1% Chlorine FR SF % Phenol FR SF % BAC FR > 100 > SF > 100 > % Cetrimide FR > 100 > SF > 100 > 100 > % SDS FR > 100 > 100 > 100 > 100 SF > 100 > 100 > 100 > % Terbina ne FR SF Values are averages of three agar slants () standard deviations.

5 Disinfectants and antifungals against Aspergillus and Candida 205 Table 4 Inactivation of C. albicans FR-00806, C. krusei FR and C. parapsilosis FR in disinfectant suspension solutions Contact time Treatment Species 15 min 30 min 60 min 24 h C. albicans > 100 > 100 > 100 > 100 Water C. krusei > 100 > 100 > 100 > 100 C. parapsilosis > 100 > 100 > 100 > 100 C. albicans % Chlorine C. krusei C. parapsilosis C. albicans % Phenol C. krusei C. parapsilosis C. albicans % BAC C. krusei C. parapsilosis C. albicans % Cetrimide C. krusei C. parapsilosis C. albicans > 100 > 100 > 100 > % SDS C. krusei > 100 > 100 > C. parapsilosis > 100 > 100 > 100 > 100 C. albicans > 100 > 100 > % Terbina ne C. krusei > 100 > 100 > 100 > 100 C. parapsilosis > 100 > 100 > Values are averages of three agar slants () standard deviations. contact with 1% chlorine in the suspension solution. With 5% phenol, a number of conidia were able to survive a contact of up to 60 min in the suspension media. However, a 24-h contact with 5% phenol in the suspension media and a 3-day incubation on phenol sprayed agar plates was found to be fungicidal against both strains of A. ochraceus (Tables 3 and 5). With 0 5% BAC or cetrimide in the suspension media, no inactivation of A. ochraceus was detectable for a period of 30 min, and even after 24 h of contact a large number of conidia were able to remain viable (Table 3). A 3-day incubation with BAC and cetrimide on sprayed plates caused a complete suppression of A. ochraceus conidia; however, the swab tests from these plates revealed that the effects of these two quaternary ammonium compounds were largely fungistatic in nature. The three Table 5 Inactivation of A. ochraceus by antifungal sprays Isolate no. FR SF Spray Water > 300 Noninhibitory > 300 Noninhibitory 5% Phenol 0 Fungicidal 0 Fungicidal 0 5 % BAC 0 Fungistatic 0 Fungistatic 0 5% Cetrimide 0 Fungistatic 3 2 Fungistatic 0 5% SDS > 300 Noninhibitory > 300 Noninhibitory Lysol 1 0 Fungistatic 4 1 Fungistatic Spray Nine 1 0 Fungistatic 0 Fungistatic Brentdale 4 2 Fungistatic 0 Fungistatic Mycospor 1 > 300 Noninhibitory > 300 Noninhibitory Lamisil 1 0 Fungicidal 0 Fungicidal Values are averages of three agar plates standard deviations. Downloaded Ó 2002 from ISHAM, Medical Mycology, 40,

6 206 Gupta et al. Table 6 Inactivation of C. albicans FR-00806, C. krusei FR and C. parapsilosis FR by antifungal sprays C. albicans C. Krusei C. parapsilosis Water > 300 Noninhibitory > 300 Noninhibitory > 300 Noninhibitory 5% Phenol 0 Fungicidal 0 Fungicidal 0 Fungicidal 0 5 % BAC 0 Fungicidal 0 Fungicidal 0 Fungicidal 0 5% Cetrimide 0 Fungicidal 0 Fungicidal 0 Fungicidal 0 5% SDS > 300 Noninhibitory > 300 Noninhibitory > 300 Noninhibitory Lysol 1 0 Fungicidal 0 Fungicidal 0 Fungicidal Spray Nine 1 0 Fungicidal 0 Fungicidal 0 Fungicidal Brentdale 0 Fungicidal 0 Fungicidal 0 Fungicidal Mycospor 1 > 300 Noninhibitory > 300 Noninhibitory > 300 Noninhibitory Lamisil Inhibitory > 300 Noninhibitory 0 Fungicidal Values are averages of three agar plates standard deviations. commercial spray formulations of quaternary ammonium compounds, Lysol 1, Spray Nine 1 and Brentdale, also produced a fungistatic effect against A. ochraceus conidia on agar plates. SDS was completely ineffective against A. ochraceus both in suspension media and on sprayed plates (Tables 3 and 5). Of the two pharmaceutical spray formulations used here, 1% bifonazole in Mycospor 1 was completely noninhibitory, whereas 1% terbina ne in Lamisil 1 spray was highly fungicidal against this mould (Table 5). Even with a lower concentration of 0 01% terbina ne in the suspension media, only a few conidia of A. ochraceus were able to survive the rst hour of contact with this drug (Table 3). Both 1% chlorine and 5% phenol were fungicidal within 15 min of contact with all three species of Candida (Table 4). A 15-min contact with 0 5% BAC was similarly lethal for C. krusei and C. parapsilosis cells, whereas a contact period of 60 min was required for a complete inactivation of C. albicans by this compound (Table 4). Similarly, with 0 5% cetrimide in the suspension media, C. krusei was killed within 15 min of contact whereas a few cells of C. albicans and C. parapsilosis survived for up to 60 min under these conditions (Table 4). The three Candida species were completely suppressed by BAC and cetrimide spray on the agar plate, and the swab tests showed the inhibition of Candida cells by the two quaternary ammonium compounds to be fungicidal (Table 6). Quaternary ammonium compounds in Lysol 1, Spray Nine 1 and Brentdale spray formulations were also fungicidal against the three Candida species. The presence of 0 5% SDS, either in the suspension media (Table 4) or on sprayed plates (Table 6), did not cause any inhibition in the three Candida species. Spraying agar plates with 1% bifonazole (Mycospor 1 ) was also noninhibitory to the Candida species (Table 6). The response of the three yeast species to the presence of terbina ne was, on the other hand, quite variable. A spray with 1% terbina ne had no effect on C. krusei, caused a moderate inhibition in C. albicans, and brought about a fungicidal inactivation in C. parapsilosis. With 0 01% terbina ne in the suspension media, a contact period of 24 h was required to cause a detectable inactivation in C. albicans and C. parapsilosis (Table 4). Discussion Disinfectants are de ned as chemical agents capable of removing infectious microbes other than bacterial spores [13]. A number of commonly used disinfectants are known to be relatively ineffective against fungi [14]. Moreover, not all fungal species are equally sensitive to a given product, and even different strains of the same fungal species may vary in resistance [14]. We have monitored the ability of antimicrobial agents both in suspension solutions and on sprayed culture plates. The relevance of these two measures of antifungal activities to the disinfection of various surfaces in our immediate environment and the adequacy of the methodology used is discussed elsewhere [12]. Chlorine was the only agent capable of killing within 15 min all ve fungal strains tested in the present study. Phenol was equally rapid in its fungicidal against the three Candida species; however, its against the two strains of A. ochraceus was considerably slower. Both chlorine and phenol are highly toxic and corrosive and their use has declined considerably in recent years [14]. In today s market, quaternary ammonium compounds have become the most common ingredients of disinfectant formulations. In our suspension experiments, a contact of up to 24 h with BAC and cetrimide caused only a partial inactivation of A. ochraceus

7 Disinfectants and antifungals against Aspergillus and Candida 207 conidia. A complete inactivation of A. ochraceus conidia by BAC, cetrimide and the three commercial formulations of quaternary ammonium compounds, Lysol 1, Spray Nine 1 and Brentdale was observed after a 3-day long exposure of these cells on sprayed plates. A high level of resistance in A. ochraceus strains to chemical disinfectants appears to be consistent with the data published on clinical isolates of A. fumigatus, A. versicolor, A. niger and A. terreus [9,10,16]. Quaternary ammonium compounds do not appear to be suf ciently potent against clinical strains of Aspergillus species. SDS, recently described [15] as an antimicrobial surfactant, was ineffective against the ve fungal strains tested in the present study. Terbina ne was notably fungicidal against both strains of A. ochraceus. No viable conidia of the two strains were found from agar plates sprayed with 1% terbina- ne. Even with a much lower concentration of 0 01% terbina ne used in the suspension media, only a few individual conidia remained viable after a 60-min contact, and were completely inactivated after a 24-h exposure to the drug. A concentration of 0 01% terbina ne was formulated for our contact experiments, because a similar level has been shown to separate terbina ne sensitive and resistant cells in Candida species [17]. The of terbina ne against Candida species was highly variable, with only C. parapsilosis showing a marked sensitivity to the presence of this drug. Spraying with 1% terbina ne resulted in a complete loss of viability in C. parapsilosis. However, the of terbina ne on C. parapsilosis appears to be slow, requiring a contact period of about 24 h with 0 01% terbina ne for the onset of fungicidal activity. It may be added here that for C. parapsilosis minimum inhibitory concentrations (MICs) values of terbina ne are reported to be in the range of m g ml 1 [17]. The presence of 0 01% (100 m g ml 1 ) terbina ne was, therefore, greatly in excess of that required to inhibit C. parapsilosis cells, so the observed delay in terbina ne against this yeast species cannot be attributed to the use of a suboptimal concentration of terbina ne in our suspension media. The apparent lag in terbina ne activity, therefore, appears to be an inherent phenomenon probably associated with the uptake rate and accumulation of the drug by C. parapsilosis. The pattern of resistance to terbina ne in Candida species C. krusei > C. albicans > C. parapsilosis observed in the present study agrees well with the data published by Ryder et al. [17]. Bifonazole was relatively ineffective in disinfecting the ve fungal strains tested in the present study. The observed tolerance of Candida to terbina ne and A. ochraceus to quaternary ammonium compounds indicate the limitations in applying any given class of antifungal agents as a common disinfectant against medically important fungi. The feasibility of formulating a combination of antimicrobial agents to target a broad group of fungal pathogens needs to be tested. These cocktails of antifungal agents could be a combination of both chemical disinfectants and antifungal drugs. Acknowledgements We thank Sal Albreish and his staff for a gift of Aspergillus and Candida cultures. References 1 Seeliger HP, Schroter G. Prevention of fungal infections in hospitalized patients. Immun infekt 1984; 143: Wildfeuer A, Seidl HP, Paul I, Haberreiter A. In vitro evaluation of voriconazole against clinical isolates of yeasts, moulds and dermatophytes in comparison with itraconazole, ketaconazole, amphotericin B and griseofulvin. Mycoses 1998; 41: Morrison VA, Haake RJ, Weisdorf DJ. A. 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