Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, 28220 Majadahonda (Madrid), Spain
Received 13 July 2004; returned 29 September 2004; revised 30 November 2004; accepted 15 December 2004
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Abstract |
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Methods: The review included studies using antifungal susceptibility testing reference procedures or commercial methods exhibiting high correlation rates with reference procedures (Etest and Sensititre YeastOne). The 131 organisms analysed were 77 Rhodotorula mucilaginosa, 45 Rhodotorula glutinis and nine Rhodotorula spp.
Results and conclusions: Fluconazole, itraconazole and voriconazole were inactive in vitro against the majority of isolates. Amphotericin B and flucytosine exhibited good activity, being reasonable alternatives for empirical treatment. Ravuconazole was more active in vitro than other azole agents and it could be considered as an extended-spectrum triazole and maybe as a therapeutic alternative in treating infections caused by Rhodotorula species.
Keywords: Rhodotorula , susceptibility of emerging yeasts , fluconazole resistance , ravuconazole
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Introduction |
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In order to give an insight into the management of this emerging infection, we have analysed the antifungal susceptibility profile of 29 clinical isolates of Rhodotorula spp. In addition, a review of literature on susceptibility results of these species is presented.
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Materials and methods |
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The clinical strains were subcultured on 4% malt extract/0.5% yeast extract agar (MEYA). After 10 days at 30°C, macroscopic and microscopic examinations were carried out. The isolates were identified by routine physiological tests:9 fermentation of and growth on carbon sources, growth on nitrogen sources, growth at various temperatures and ability to hydrolyse urea. Colonies of Rhodotorula spp. were rapid growing, smooth, glistening or dull, sometimes roughened, soft and mucous. They were cream to pink, coral red, orange or yellow in colour. Rhodotorula isolates did not ferment carbohydrates and produced urease enzyme.
The susceptibility testing strictly followed the recommendations proposed by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing for fermentative yeast (AFST-EUCAST, discussion document 7.1).10 These recommendations are based on the National Committee for Clinical Laboratory Standards (NCCLS) reference procedure described in document M27-A2,11 but include some modifications to allow for automation of the method and to permit the incubation period to be shortened from 48 to 24 h.12 Briefly, testing was carried out with RPMI 1640 medium supplemented with 2% glucose, and an inoculum size of 105 cfu/mL was used, as were flat-bottomed microdilution plates. Inoculum size was confirmed by plating on Sabouraud agar plates. MIC end points were determined spectrophotometrically at 530 nm after 24 and 48 h. In addition, in order to improve the growth of some organisms, a minor modification was included.13 That was, all microplates for testing were wrapped with film sealer to prevent the medium from evaporating, attached to an electrically driven wheel inside the incubator, agitated at 350 rpm and incubated at 30°C for 48 h. Candida parapsilopsis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control strains.
The antifungal agents used in the study were as follows: amphotericin B (SigmaAldrich Química S.A., Madrid, Spain), flucytosine (SigmaAldrich), fluconazole (Pfizer S.A., Madrid, Spain), itraconazole (Janssen Pharmaceutica, Madrid, Spain), voriconazole (Pfizer Ltd, Sandwich, UK) and ravuconazole (Bristol-Myers Squibb, Princeton, NJ, USA). They were obtained as standard powders and stock solutions were prepared in water (flucytosine and fluconazole) or 100% dimethyl sulphoxide (SigmaAldrich). Solutions were conserved at 70°C for each drug. The final concentrations tested ranged from 16 to 0.03 mg/L for amphotericin B, from 64 to 0.12 mg/L for flucytosine and fluconazole, and from 8 to 0.015 mg/L for itraconazole, voriconazole and ravuconazole. For amphotericin B, the MIC end points were defined as the lowest drug concentration exhibiting reduction in growth of 90% or more compared with that of the control growth. For flucytosine and azole drugs, the MIC end point was defined as 50% of inhibition.
In addition, we undertook a MEDLINE search using the keywords Rhodotorula, antifungal susceptibility testing and emerging yeasts pathogens, as well as text word searching. We included reports available on MEDLINE from 1992, the date of publication of the NCCLS reference procedure for susceptibility testing of yeasts.11 The review therefore included studies on Rhodotorula susceptibility testing carried out using reference methods of NCCLS and EUCAST. We also reviewed studies carried out using commercial methods that have exhibited high correlation rates with reference procedures in several comparative works, i.e. Etest and Sensititre YeastOne.14,15
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Results |
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Regarding R. glutinis, this species was somewhat more susceptible than strains of R. mucilaginosa, although isolates tested also had high MICs of fluconazole and itraconazole. Unfortunately, the small number of isolates studied did not allow us to carry out an adequate statistical analysis.
The MIC values for the two quality control strains were consistent within two or three two-fold dilutions. These values agreed with those in the discussion document 7.1 by EUCAST and the NCCLS document M27-A2.11,12
When searching MEDLINE, nine reports were found, including a total of 102 organisms divided into 52 R. mucilaginosa isolates, 41 clinical strains of R. glutinis, and nine of other species of Rhodotorula or identified as Rhodotorula spp.5,1623
Table 2 summarizes the MIC results of the nine reports reviewed. Regardless of method used for susceptibility testing, amphotericin B and flucytosine were the most active antifungal agents in vitro, with MICs 1 mg/L. As we found for 29 clinical isolates tested in our laboratory, fluconazole was inactive in vitro against 100 of 102 (98%) isolates reviewed. The other azole agents (voriconazole and itraconazole) largely exhibited MICs similar to those observed for the 29 isolates, with MIC values of or over 1 mg/L for a significant percentage of strains. However, the three studies that were carried out using Sensititre YeastOne as the method for susceptibility testing reported itraconazole MICs <1 mg/L for the great majority of strains tested.17,20,21
With regard to ravuconazole, it was only included in one of the studies reviewed. Serena et al. reported a good activity of this azole against 10 isolates of R. glutinis,19
with MIC values similar to those observed for the 29 isolates tested in this study.
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Discussion |
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Other azole compounds, with the exception of ravuconazole, also exhibited a poor activity against Rhodotorula isolates when susceptibility testing was done using NCCLS, EUCAST or Etest techniques. However, lower MIC values of azole agents were observed when Sensititre YeastOne was used.17,20,21 Studies done with this technique reported MICs of itraconazole and ketoconazole comparable to that of amphotericin B with MIC ranges of itraconazole and ketoconazole of 0.031.0 and 0.060.25 mg/L, respectively. The Sensititre YeastOne technique has proven its potential value for antifungal susceptibility testing of clinical strains of Candida species and other yeast-like organisms because of its high correlation with reference procedures.14 Manufacturers of the YeastOne panel recommend a reading time of 24 h and 48 or 72 h of incubation for Cryptococcus neoformans. Species of Rhodotorula usually also require a 48 or 72 h incubation period for MIC determination since MIC values after 24 h of incubation can be falsely underestimated because of poor growth of these species.24 Studies by Galan-Sanchez et al.21 and Garcia-Martos et al.17 state that panels were read at 24, 48 and 72 h, and MICs were determined when yeast growth of the positive control well was evident, i.e. a change in the colorimetric growth indicator from blue to red. If MICs reported are values after 48 or 72 h of incubation, the lack of agreement between the YeastOne panel and reference procedures should be noted for testing the susceptibility profile of these species.
In conclusion, taking into account our results in vitro and those published before, fluconazole, itraconazole and voriconazole are inactive in vitro against the majority of clinical isolates of Rhodotorula spp. Amphotericin B and flucytosine exhibit a good activity being reasonable alternatives for empirical treatment. Ravuconazole was more active in vitro than other azole agents and it could be considered as an extended-spectrum triazole and maybe as a therapeutic alternative in treating infections caused by Rhodotorula species. Finally, prompt identification of isolates causing deep mycosis is important in selecting the most appropriate antifungal therapy.
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References |
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