Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain
Received 18 November 2003; returned 16 December 2003; revised 23 December 2003; accepted 8 March 2004
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Abstract |
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Methods: A broth microdilution test was carried out following the National Committee for Clinical Laboratory Standards reference method, with modifications described previously.
Results: The geometric mean (GM) of MICs of terbinafine for non-Aspergillus fumigatus species was 0.24 mg/L whereas the GM for A. fumigatus rose as far as 2.92 mg/L. Terbinafine showed a very strong activity in vitro against Penicillium spp., Paecilomyces spp., Trichoderma spp., Acremonium spp. and Arthrographis spp. with GMs <1 mg/L. However, some species such as Scedosporium spp., Fusarium spp., Scopulariopsis brevicaulis, and most of Mucorales exhibited high MICs of the allylamine with GMs 4 mg/L.
Conclusions: Overall, the GM of MICs of terbinafine was 1.57 mg/L, but significant differences in susceptibilities were seen between genera and species.
Keywords: Aspergillus spp., Spanish filamentous fungi, allylamines, antifungals
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Introduction |
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Terbinafine exhibits a broad spectrum of activity in vitro that includes yeasts, some species of moulds and dimorphic fungi.5,6 We have analysed the susceptibility in vitro to terbinafine of a large collection of strains of non-dermatophyte filamentous fungi in order to determine the activity of the allylamine against clinically relevant mould species, including those that are uncommonly isolated in clinical cases.
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Materials and methods |
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A collection of 442 filamentous fungi were included in the study. All strains were recovered from 81 Spanish hospitals through the years 2001 and 2002. The isolates were obtained from blood (7/442, 1.6%), respiratory tract specimens (260/442, 58.8%), skin samples (70/442, 15.8%), ophthalmic samples (16/442, 3.6%), ear specimens (33/442, 7.5%), tissue biopsy specimens (18/442, 4.1%), environmental samples (14/442, 3.2%) and other locations (24/442, 5.4%). Each isolate was obtained from a different patient. Aspergillus fumigatus ATCC 9197 and Paecilomyces variotii ATCC 22319 were included as control isolates in each set of experiments.
Antifungal susceptibility testing
Terbinafine (Novartis, Basle, Switzerland) was obtained as standard powder and stock solutions were prepared in 100% dimethyl sulphoxide (SigmaAldrich Química, Madrid, Spain). Assays were carried out in RPMI 1640 with L-glutamine buffered to pH 7 with 0.165 M MOPS and 10 M NaOH (Oxoid, Madrid, Spain), and supplemented with 18 g of glucose per litre.
A broth microdilution test was carried out following the National Committee for Clinical Laboratory Standards (NCCLS) reference method,7 with modifications described previously.8,9 The final concentrations of terbinafine ranged from 16 to 0.03 mg/L. Inoculum suspensions were prepared from fresh, mature (3- to 5-day-old) cultures in accordance with a methodology reported previously.9 The inoculum size was adjusted to a range of 1.0 x 106 to 5.0 x 106 spores/mL by microscopic enumeration with a cell-counting haemocytometer (Neubauer chamber; Merck, S.A., Madrid, Spain). All adjusted suspensions were quantified by plating on Sabouraud agar plates. The suspension was then diluted 1:10 with sterile distilled water to obtain a final working inoculum of 15 x 105 cfu/mL. The trays were inoculated with 0.100 mL into each well. The plates were incubated at 35°C for 48 h in a humid atmosphere. Visual readings were carried out with the help of a mirror. MICs were defined as the lowest concentration of the antifungal agent that completely inhibited the fungal growth.
Statistical analysis was carried out with the Statistical Package for the Social Sciences (SPSS, version 11.5.) (SPSS S.L., Madrid, Spain). Differences in proportions were determined by Fishers exact test or by 2 analysis. The significance of the differences in MICs was determined by the Students t-test (unpaired, unequal variance). P < 0.01 was considered significant.
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Results |
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With regard to clinical isolates tested, overall, terbinafine exhibited a good activity in vitro with a geometric mean (GM) of MICs of 1.57 mg/L. However, MIC values ranged between 0.03 and >16 mg/L, indicating that some isolates were resistant in vitro to the allylamine. Notably, the MIC90 (MIC causing inhibition of 90% of the isolates) was 16 mg/L. The in vitro activities of terbinafine classified per species of filamentous fungi are summarized in Table 1.
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In reference to other species of hyaline, non-phaeoid moulds, terbinafine showed a very strong activity in vitro against the majority of strains of Penicillium spp. (18/19, 94.73%), and Paecilomyces spp. strains, even against six Paecilomyces lilacinus isolates that were included in the study. Terbinafine had a good activity against Acremonium spp., Trichoderma spp. and Arthrographis spp. as well. However, some species such as Scedosporium apiospermum, Scopulariopsis brevicaulis, Chrysonilia sitophila and Cylindrocarpon spp. showed resistance in vitro to the allylamine. In reviewing terbinafine MICs for Fusarium spp., the antifungal compound had a very limited activity against the species most commonly isolated in clinical samples.
Terbinafine revealed a low activity in vitro against the species of phaeoid hyphomycetes included in this study. Isolates of Scedosporium prolificans were invariably resistant to the allylamine, and strains of Thermomyces and Scytalidium had terbinafine MICs of 4 mg/L. Other species of black fungi exhibited lower MICs, particularly a strain of Alternaria alternata whose MIC was 0.25 mg/L.
Turning to Mucorales, the GM for strains included in the study was 2.83 mg/L. For all the Rhizopus oryzae strains, the terbinafine MICs showed a constant value of 16 mg/L, and for the Rhizopus microsporus strains the values ranged between 1 and 16 mg/L. Isolates of Mucor circinelloides and Absidia corymbifera had average MIC values of terbinafine around 1.5 mg/L.
No significant differences were found when MIC values were compared per isolation site. No differences were observed in species distribution over the 2-year study period. Some associations were not analysed because of the small number of isolates.
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Discussion |
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Regarding other species, of particular note is the high activity reported for most of the isolates of Paecilomyces spp. and Penicillium spp., confirming previous studies.5 These organisms are common environmental moulds that are associated with soft tissue and deep infections in immunosuppressed patients. Paecilomyces is known to show high resistance in vitro against antifungal agents, particularly P. lilacinus. Notably, terbinafine monotherapy has been useful in treating infections due to this species.13
On the other hand, several species were resistant in vitro to terbinafine. Scedosporium spp. had very high MIC values. Fusarium spp. also showed high MICs, particularly F. solani isolates. Terbinafine has shown a limited efficacy in treating nail and skin infections due to Fusarium.1 This finding and the resistance in vitro to the allylamine should be taken into account in therapy of Fusarium infections. Strains of S. brevicaulis included in this study exhibited high MIC values of terbinafine as has been reported elsewhere.14 This species is one of the most common non-dermatophyte agents of onychomycosis and terbinafine is one of the standard treatments for this mycosis. These data could be taken into consideration for their potential clinical implications.
There is no standardized method available for testing terbinafine, since it is currently not included within reference methods for antifungal susceptibility testing, but our results merit more comprehensive clinical studies to determine the correlation between these data and the in vivo outcome.
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Acknowledgements |
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Footnotes |
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References |
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