In vitro activity of terbinafine against medically important non-dermatophyte species of filamentous fungi

Guillermo Garcia-Effron, Alicia Gomez-Lopez, Emilia Mellado, Araceli Monzon, Juan L. Rodriguez-Tudela and Manuel Cuenca-Estrella*

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The activity in vitro of terbinafine against 442 clinical isolates of several species of filamentous fungi was analysed.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Terbinafine is an allylamine antifungal agent used in the treatment of dermatophytosis and other dermatomycoses with excellent results.1 Regarding deep mycoses, small clinical case series revealed the efficacy of the monotherapy with terbinafine in the treatment of refractory pulmonary aspergillosis of non-immunosuppressed patients,2 and other studies indicated that it could be effective in treating subcutaneous and some systemic mycoses as well.3 Moreover, combinations of azole agents and terbinafine are predicted to exhibit positive interaction since they act at different steps of the same pathway. In this sense, some reports have documented successful treatments of mycoses with terbinafine plus azole compounds, such as cases of deep infections due to Scedosporium prolificans.4

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fungi

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 (Sigma–Aldrich 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 1–5 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 Fisher’s exact test or by {chi}2 analysis. The significance of the differences in MICs was determined by the Student’s t-test (unpaired, unequal variance). P < 0.01 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The terbinafine MICs for the control organisms were consistent within three two-fold dilutions and ranged between 0.5–2.0 mg/L (mode 1.0 mg/L) for P. variotii ATCC 22319, and 1.0–4.0 mg/L (mode 2.0 mg/L) for A. fumigatus ATCC 9197.

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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Table 1. Susceptibility in vitro to terbinafine of species included in the study
 
First of all, terbinafine showed a potent activity in vitro against Aspergillus spp. with the exception of A. fumigatus. The GM of MICs of terbinafine for non-A. fumigatus species was 0.24 mg/L whereas the GM for A. fumigatus rose as high as 2.92 mg/L. This value was significantly higher than those observed for other Aspergillus species (P < 0.01 by the Student’s t-test).

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.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reports on susceptibility testing of terbinafine have increased since this antifungal compound has shown a high activity in vitro against a broad spectrum of pathogenic fungi. Our results indicate that MIC values varied according to species. A significant finding was that MICs for A. fumigatus were significantly higher than those for other Aspergillus species. Although breakpoints have not been established for interpreting susceptibility testing of terbinafine, high MICs (>=4 mg/L) were observed in 90 out of 142 (63.38%) isolates of A. fumigatus. This was described earlier by Moore et al.6 Their study also revealed that A. fumigatus appeared significantly less susceptible to terbinafine than other Aspergillus species. In contrast, another study has shown good activity of the allylamine against Aspergillus species, including A. fumigatus (MICs 0.03–1 mg/L).5 Conflicting results could largely be explained by differences in the methodologies used.5,6 In this sense, the inoculum size that was used in our study, 1–5 x 105 cfu/mL, is 10-fold higher than the inoculum recommended by the NCCLS M38-A protocol and a significant influence on MIC values could be expected. However, some reports have demonstrated that these inoculum sizes generate reproducible susceptibility data in vitro for Aspergillus spp. that can predict clinical outcome.10 In addition, higher inoculum size does not have a significant influence on MIC values of several antifungal agents (Table 2).


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Table 2. MIC ranges for control strains and amphotericin B, itraconazole and voriconazole
 
In contrast to A. fumigatus, non-A. fumigatus species seem to be susceptible in vitro to the allylamine. The prevalence of these species has grown over the past years,11 and terbinafine might represent a therapeutic option as monotherapy or in combination therapy for treating some selected cases. However, a small, randomized study that compared amphotericin B plus placebo with amphotericin B plus terbinafine (750 mg/day) in the treatment of invasive aspergillosis,12 showed a significantly higher mortality in the combination group. This study was not stratified per Aspergillus species, but the efficacy of terbinafine in treating aspergillosis has been understandably questioned.

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.


    Acknowledgements
 
This work was partially supported by research project 02/1196 from the Instituto de Salud Carlos III.


    Footnotes
 
* Corresponding author. Tel: +34-91-5097961; Fax: +34-91-5097966; E-mail: mcuenca-estrella{at}isciii.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Tosti, A., Piraccini, B. M. & Lorenzi, S. (2000). Onychomycosis caused by nondermatophytic molds: clinical features and response to treatment of 59 cases. Journal of the American Academy of Dermatology 42, 217–24.[CrossRef][ISI][Medline]

2 . Schiraldi, G. F., Cicero, S. L., Colombo, M. D. et al. (1996). Refractory pulmonary aspergillosis: compassionate trial with terbinafine. British Journal of Dermatology 134, Suppl. 46, 25–9.[ISI][Medline]

3 . Hay, R. J. (1999). Therapeutic potential of terbinafine in subcutaneous and systemic mycoses. British Journal of Dermatology 141, Suppl. 56, 36–40.[CrossRef][ISI][Medline]

4 . Howden, B. P., Slavin, M. A., Schwarer, A. P. et al. (2003). Successful control of disseminated Scedosporium prolificans infection with a combination of voriconazole and terbinafine. European Journal of Clinical Microbiology and Infectious Diseases 22, 111–3.[ISI][Medline]

5 . Jessup, C. J., Ryder, N. S. & Ghannoum, M. A. (2000). An evaluation of the in vitro activity of terbinafine. Medical Mycology 38, 155–9.[ISI][Medline]

6 . Moore, C. B., Walls, C. M. & Denning, D. W. (2001). In vitro activities of terbinafine against Aspergillus species in comparison with those of itraconazole and amphotericin B. Antimicrobial Agents and Chemotherapy 45, 1882–5.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (2002). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi: Approved Standard M38-A. NCCLS, Wayne, PA, USA.

8 . Cuenca-Estrella, M., Diaz-Guerra, T. M., Mellado, E. et al. (2001). Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida spp. Journal of Clinical Microbiology 39, 525–32.[Abstract/Free Full Text]

9 . Rodriguez-Tudela, J. L., Chryssanthou, E., Petrikkou, E. et al. (2003). Interlaboratory evaluation of hematocytometer method of inoculum preparation for testing antifungal susceptibilities of filamentous fungi. Journal of Clinical Microbiology 41, 5236–7.[Abstract/Free Full Text]

10 . Denning, D. W., Radford, S. A., Oakley, K. L. et al. (1997). Correlation between in-vitro susceptibility testing to itraconazole and in-vivo outcome of Aspergillus fumigatus infection. Journal of Antimicrobial Chemotherapy 40, 401–14.[Abstract]

11 . Gomez-Lopez, A., Garcia-Effron, G., Mellado, E. et al. (2003). In vitro activities of three licensed antifungal agents against Spanish clinical isolates of Aspergillus spp. Antimicrobial Agents and Chemotherapy 47, 3085–8.[Abstract/Free Full Text]

12 . Steinbach, W. J., Stevens, D. A. & Denning, D. W. (2003). Combination and sequential antifungal therapy for invasive aspergillosis: review of published in vitro and in vivo interactions and 6281 clinical cases from 1966 to 2001. Clinical Infectious Diseases 37, Suppl. 3, S188–224.[CrossRef][ISI][Medline]

13 . Clark, N. M. (1999). Paecilomyces lilacinus infection in a heart transplant recipient and successful treatment with terbinafine. Clinical Infectious Diseases 28, 1169–70.[ISI][Medline]

14 . Cuenca-Estrella, M., Gomez-Lopez, A., Mellado, E. et al. (2003). Scopulariopsis brevicaulis, a fungal pathogen resistant to broad-spectrum antifungal agents. Antimicrobial Agents and Chemotherapy 47, 2339–41.[Abstract/Free Full Text]





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