In vitro interaction of micafungin with conventional and new antifungals against clinical isolates of Trichosporon, Sporobolomyces and Rhodotorula

Carolina Serena1, Marçal Mariné1, F. Javier Pastor1, Nicole Nolard2 and Josep Guarro1,*

1 Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain; 2 Scientific Institute of Public Health, Brussels, Belgium


* Corresponding author. Tel: +34-977-759359; Fax: +34-977-759322; Email: josep.guarro{at}urv.net

Received 2 March 2005; returned 11 March 2005; revised 21 March 2005; accepted 21 March 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The infections caused by basidiomycetous yeasts are often difficult to resolve. Combined therapy might be useful in those severe cases where a monotherapy was ineffective. The aim of this study was to evaluate the in vitro activity of combinations of micafungin with amphotericin B or fluconazole, itraconazole, voriconazole and ravuconazole against isolates of Trichosporon, Rhodotorula and Sporobolomyces.

Methods: Twenty-seven clinical isolates were tested, i.e. 10 of Trichosporon asahii, two of Trichosporon mucoides, five of Sporobolomyces salmonicolor and 10 of Rhodotorula glutinis. Drug interactions were assessed by the chequerboard technique using the NCCLS microdilution method (M27-A2). The fractional inhibitory concentration index (FICI) was used to classify drug interactions. Results were interpreted as follows: synergy (FICI ≤0.5), no interaction (FICI >0.5 and ≤4.0), or antagonism (FICI >4.0).

Results: Micafungin combined with amphotericin B showed the highest percentage of synergic interactions (78%) followed by micafungin/ravuconazole and micafungin/itraconazole (48% for each), and micafungin/fluconazole and micafungin/voriconazole (34% for each). Antagonism was not observed in any case.

Conclusions: Some of the combinations tested, especially micafungin/amphotericin B, have potential for the treatment of basidiomycetous yeast infections.

Keywords: basidiomycetous yeasts , antifungal susceptibility , combined therapy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The frequency of life-threatening fungal infections caused by Trichosporon species and other basidiomycetous yeasts has increased in recent years.14 The antifungal agents approved for the treatment of invasive yeast infections are limited. In general, the most common treatment of yeast infections is based on the use of amphotericin B and fluconazole. However, these drugs have repeatedly failed against infections caused by Trichosporon species, Rhodotorula glutinis and Sporobolomyces salmonicolor.3,5 Such a limitation, associated with amphotericin B toxicity, makes interesting the evaluation of the combination of new and conventional drugs to obtain better clinical results and to reduce the antifungal doses. Echinocandins are a new class of antifungal agents with novel targets that can constitute new alternatives that need testing, especially in combined therapies.6 Few studies have been done on the in vitro interaction of antifungal drugs against the above mentioned fungi.7 We have recently demonstrated that the combination of micafungin with amphotericin B is effective for the treatment of murine disseminated infection by two strains of Trichosporon asahii.8 This is very interesting because the infection caused by this species commonly does not respond to the recommended treatment with amphotericin B or fluconazole. However, since drug–drug interaction is frequently strain specific, and in that study only two isolates were tested, it is important to prove if this combination can be selected to suit all members of this species.

The aim of this study, therefore, was to determine if the combination micafungin/amphotericin B is active against more strains of T. asahii (n = 10) and thus to confirm the effectiveness of this combination, and also to determine if it is active against other pathogenic yeasts. In addition, we have evaluated the combination of micafungin with some other traditional and new azoles.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A total of 27 clinical isolates were tested (10 T. asahii, 2 Trichosporon mucoides, 10 R. glutinis and 5 S. salmonicolor) provided by the BCCM/IHEM Biomedical Fungi/Yeast collection, Centraalbureau voor Schimmelcultures (CBS) and Faculty of Medicine of Reus (FMR). The isolates were stored lyophilized and were subcultured on Sabouraud dextrose agar (SDA) for the study. The cultures were incubated at 35°C. Although a reference method for determining the in vitro activity of antifungal agents against these species does not exist, we have followed the guidelines of the NCCLS (M27-A2).9

Antifungal agents were obtained as pure powders. Amphotericin B (USP, Rockville, MD, USA), voriconazole (Pfizer Inc., Madrid, Spain), itraconazole (Janssen Pharmaceutica, Beerse, Belgium) and ravuconazole (Bristol-Myers Squibb Company, New Brunswick, NJ, USA) were diluted in dimethyl sulphoxide (Panreac Química S.A., Barcelona, Spain). Micafungin (Fujisawa Pharmaceutical Co. Ltd, Osaka, Japan) and fluconazole (Pfizer Inc., Madrid, Spain) were diluted in sterile distilled water. The MIC was defined as the lowest drug concentration that produced complete absence of growth. Drug interactions were assessed by a chequerboard microdilution method that also included the determination of the MIC of each drug alone in the same plate by using the parameters outlined in the NCCLS document M27-A2.9 Antifungal agents were placed in the rows or in the columns of the trays to perform all possible combinations, with concentrations from 4 to 0.06 mg/L for amphotericin B, 8 to 0.12 mg/L for itraconazole, voriconazole and ravuconazole, 64 to 1 mg/L for fluconazole and 32 to 0.06 mg/L for micafungin. Microplates were read at 48 h for T. asahii and T. mucoides and at 72 h for R. glutinis and S. salmonicolor. Candida krusei ATCC 6258 and C. parapsilosis ATCC 22019 were included as quality controls.

The fractional inhibitory concentration index (FICI) was used to classify drug interaction. The FICI is the sum of the FIC of each of the drugs, which in turn is defined as the MIC of each drug when used in combination divided by the MIC of the drug when used alone. Interaction was considered synergic if the FIC index was ≤0.5, non-existent if it was >0.5 and ≤4, and antagonistic if it was >4.10


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Table 1 shows the in vitro interactions obtained with the five antifungal drug combinations tested against the clinical isolates of the four species. Of the 135 combinations evaluated, 65 were synergic, and the rest showed no interaction. No antagonistic interactions were produced in any case. Micafungin combined with amphotericin B showed the highest percentage of synergic interactions (78%) when all the isolates were studied together, followed by micafungin/ravuconazole and micafungin/itraconazole (48%) and micafungin/fluconazole and micafungin/voriconazole (34%).


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Table 1. Interactions of MFG with AMB and various azoles against Trichosporon asahii, Trichosporon mucoides, Rhodotorula glutinis and Sporobolomyces salmonicolor

 
Against T. asahii, as has been reported previously, when we tested the antifungals alone only the triazoles showed in vitro activity.11 However, here the combination micafungin/amphotericin B demonstrated synergic interactions against all the strains tested, with MIC values for one or the two combined antifungal drugs within the achievable plasma levels.6,12 The other combinations showed a lower percentage of synergic interactions (20–40%) against the isolates of this species. The response to the antifungals of the two strains of T. mucoides was very different. Practically all the drugs showed high MICs against one strain and low MICs against the other. But also for this species, the combination micafungin/amphotericin B demonstrated synergic interactions against the two strains tested. The combinations micafungin/fluconazole and micafungin/itraconazole only showed synergy against one strain. The other antifungal combinations did not show synergy against either of the two strains of T. mucoides tested.

In our study, the amphotericin B MICs against R. glutinis were relatively low (0.25–0.5 mg/L), which agrees with the favourable clinical outcomes observed in infections by Rhodotorula spp. treated with this drug.1,4 However, due to the high toxicity of this drug, other alternatives have to be explored. In our study, micafungin and azoles, with the exception of ravuconazole, were generally inactive against this species. Diekema et al.13 obtained similar results. They tested the activity of eight antifungal agents alone against Rhodotorula species and only amphotericin B and ravuconazole showed low MIC values against this fungi. For this reason, it is an interesting result that in our study all the antifungal combinations showed a high percentage of synergic interactions (60–80%) with the exception of micafungin/fluconazole that only showed 20% of synergic interactions. For S. salmonicolor, the amphotericin B MICs were very variable with a 6-fold difference between the lowest and the highest values (0.25–16 mg/L). But, in general, these values were high, similar to those of fluconazole and micafungin. Against this species, all the drug–drug interactions were strain dependent. The best combination was micafungin/itraconazole with 60% synergic interactions. The other combinations only showed 20–40% synergic interactions. Although these percentages are not as high as for the other species tested, some of these combined therapies could be helpful when amphotericin B fails.

One of the most significant results of this study has been the good activity showed by the combination micafungin/amphotericin B against T. asahii, which was synergic against the 10 strains tested. This confirms the in vivo effectiveness demonstrated by this combination against two strains of this fungus.8 T. asahii is the most frequent species of those tested in this study and causes severe human infections. The fact proved here that the susceptibility of this species to this combination is general and probably not strain dependent emphasizes the potential of this combined therapy for trichosporonosis treatment. Further in vivo studies are warranted to elucidate the potential usefulness of these combination therapies against the species tested in this study.


    Acknowledgements
 
This work was supported by a grant from Fondo de Investigaciones Sanitarias from the Ministerio de Sanidad y Consumo of Spain (PI 020114).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Anatoliotaki M, Mantadakis E, Galanakis E et al. Rhodotorula species fungemia: a threat to the immunocompromised host. Clin Lab 2003; 49: 49–55.[Medline]

2 . Groll AH, Walsh TJ. Uncommon opportunistic fungi: new nosocomial threats. Clin Microbiol Infect 2001; 7: 8–24.[CrossRef]

3 . Wildfeuer A, Seidl HP, Paule I et al. In vitro evaluation of voriconazole against clinical isolates of yeasts, moulds and dermatophytes in comparison with itraconazole, ketoconazole, amphotericin B and griseofulvin. Mycoses 1998; 41: 309–19.[ISI][Medline]

4 . Zaas AK, Boyce M, Schell W et al. Risk of fungemia due to Rhodotorula and antifungal susceptibility testing of Rhodotorula isolates. J Clin Microbiol 2003; 41: 5233–5.[Abstract/Free Full Text]

5 . Ebright JR, Fairfax MR, Vazquez JA. Trichosporon asahii, a non-Candida yeast that caused fatal septic shock in a patient without cancer or neutropenia. Clin Infect Dis 2001; 33: 28–30.[CrossRef]

6 . Denning DW. Echinocandin antifungal drugs. Lancet 2003; 362: 1142–51.[CrossRef][ISI][Medline]

7 . Ryder NS. Activity of terbinafine against serious fungal pathogens. Mycoses 1999; 42: 115–9.[ISI][Medline]

8 . Serena C, Pastor FJ, Gilgado F et al. Efficacy of micafungin in combination with other drugs in a murine model of disseminated trichosporonosis. Antimicrob Agents Chemother 2005; 49: 497–502.[Abstract/Free Full Text]

9 . National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Second Edition: Approved Standard M27-A2. NCCLS, Wayne, PA, USA, 2002.

10 . Mukherjee PK, Sheehan DJ, Hitchcock CA et al. Combination treatment of invasive fungal infections. Clin Microbiol Rev 2005; 18: 163–94.[Abstract/Free Full Text]

11 . Serena C, Pastor FJ, Ortoneda M et al. In vitro susceptibilities of uncommon basidiomycetous yeasts. Antimicrob Agents Chemother 2003; 48: 2724–6.[CrossRef][ISI]

12 . Bekersky I, Fielding RM, Dressler DE et al. Pharmacokinetics, excretion, and mass balance of liposomal amphotericin B (AmBisome) and amphotericin B deoxycholate in humans. Antimicrob Agents Chemother 2002; 46: 828–33.[Abstract/Free Full Text]

13 . Diekema DJ, Petroelje B, Messer SA et al. Activities of available and investigational antifungal agents against Rhodotorula species. J Clin Microbiol 2005; 43: 476–8.[Abstract/Free Full Text]





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