JAC Advance Access originally published online on September 28, 2008
Journal of Antimicrobial Chemotherapy 2008 62(6):1277-1280; doi:10.1093/jac/dkn415
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Original research |
Rapid method for testing the susceptibility of Aspergillus fumigatus to amphotericin B, itraconazole, voriconazole and posaconazole by assessment of oxygen consumption
1 Department of Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal 2 IPATIMUP, Institute of Pathology and Molecular Immunology, University of Porto, Porto, Portugal 3 Virginia Commonwealth University, Medical Centre, Richmond, VA, USA
* Correspondence address. Department of Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal. Tel: +351-916035076; Fax: +351-225513603; E-mail: ricjparaujo{at}yahoo.com
Received 25 July 2008; returned 5 August 2008; revised 5 September 2008; accepted 9 September 2008
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Abstract |
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Objectives: Antifungal stress conditions affect fungal germination and growth. The assessment of oxygen consumption resulting from the challenge of Aspergillus fumigatus conidia with antifungal agents might be predictive of the susceptibility of this species to the agents evaluated.
Methods: The antifungal susceptibilities of A. fumigatus to amphotericin B, itraconazole, voriconazole and posaconazole were evaluated for 20 clinical strains by two methods: the rapid assessment of oxygen consumption and the CLSI M38-A2 microdilution method. For the determination of oxygen consumption, conidia were suspended in RPMI 1640 medium with two different concentrations of each antifungal drug (0.25 and 2 mg/L); the oxygen consumption was quantified in a biological oxygen monitor.
Results: A. fumigatus strains showed a wide spectrum of amphotericin B, itraconazole and voriconazole MICs (0.06 to >16 mg/L), but posaconazole MICs ranged from 0.06 to 1 mg/L. Distinct respiratory kinetics, which corresponded to the MIC results, were found. Strains with the highest itraconazole and voriconazole MICs grew faster, undoubtedly consuming the oxygen available in the liquid medium. The reproducibility of this new method was adequate (87%), as well as the agreement with the CLSI method (85%).
Conclusions: Although the potential of this new and rapid method (4–8 versus 48 h CLSI method) for evaluating the susceptibility of A. fumigatus to the antifungal agents has been demonstrated by these preliminary results, further collaborative studies with more isolates should better assess the value of this methodology for testing isolates in the clinical laboratory.
Keywords: Aspergillus spp. , conidial germination , hyphal growth , microdilution method , susceptibility testing
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Introduction |
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The CLSI has developed a method (M38-A2) for susceptibility testing of moulds.1 However, this methodology is time-consuming and cumbersome, and the MIC result for most moulds is obtained at 48 h. New and faster methods for mould antifungal susceptibility testing have been developed, such as the Etest and disc diffusion methods,2 fluorescent stains and colorimetric assays,3,4 as well as specific molecular markers.5 Aspergillus fumigatus remains the most clinically important mould, being frequently responsible for severe infections and high mortality rates. The objective of this first work was to develop an economic, rapid and practical method for antifungal susceptibility testing of A. fumigatus. The assessment of oxygen consumption following fungal germination and growth as a result of antifungal stress conditions might be predictive of the susceptibility pattern of fungal strains.
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Methods |
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Twenty clinical strains of A. fumigatus were evaluated. The determination of MICs of amphotericin B (Bristol-Myers SP, Dublin, Ireland), itraconazole (Janssen-Cilag, Saunderton, UK), voriconazole (Pfizer Inc., NY, USA) and posaconazole (Schering-Plough Farma, Cacém, Portugal) was performed by the CLSI M38-A2 broth microdilution method.1 The absence of visual growth defined the MIC of the four agents at 48 h. Quality control (QC) Candida parapsilosis ATCC 22 019 and Candida krusei ATCC 6258 isolates were included each time as controls; MICs for both QC isolates were within the recommended values.6
A. fumigatus conidia were kept frozen at –70°C in brain heart infusion (Difco, Detroit, MI, USA) with 5% glycerol until testing was performed. Briefly, conidial cells were harvested by flooding the colonies on the Sabouraud agar surface (incubation for 7 days at 35°C) with a PBS solution (Sigma-Aldrich, St Louis, MO, USA). The density of the resulting conidial suspension was evaluated optically by a Densimat photometer (BioMérieux sa, Marcy l’Étoile, France), as previously described.7 On the day of the test, 1 x 107 conidia/mL were suspended in RPMI 1640 medium (Sigma-Aldrich) supplemented with MOPS (Sigma-Aldrich) and 50 mg/L gentamicin (Sigma-Aldrich). The evaluation of oxygen consumption was performed using 1.5 mL volumes of the conidial suspension in the RPMI 1640 medium and two different concentrations of each of the four antifungal agents (0.25 and 2 mg/L). The tubes were incubated at 37°C for 12 h in a biological oxygen monitor (YSI Model 5300; YSI Inc., Yellow Springs, OH, USA). The 0.25 mg/L value corresponded to a susceptible value and the 2 mg/L indicated a decreased antifungal activity according to the recently proposed in vitro breakpoints (susceptible, MIC 
1 mg/L; intermediate, MIC 2 mg/L; and resistant, MIC 
4 mg/L) for the four agents evaluated.2 A drug-free RPMI medium tube was also included each time as a growth control. The assessment of oxygen consumption by A. fumigatus conidia was based upon the continuous monitoring and quantification of the oxygen available in the tested suspensions. All tests were performed in duplicate on different days.
Excel 2000 (Microsoft Corp., NY, USA) and SPSS 16.0 (SPSS Inc., IL, USA) applications were used for data analysis. The ANOVA test, using the Bonferroni correction, and Student’s t-test for paired samples were also used. Data were compared at a significance level of 0.05. A discrepancy was considered a ‘very major error’ whenever the strain was susceptible by oxygen consumption and resistant by the CLSI microdilution method (false susceptible result); a categorization of resistance by oxygen consumption with a corresponding categorization of susceptible by the broth microdilution method was considered a ‘major error’ (false resistant result). Cut-off values were based upon the resulting percentages of oxygen consumption when all susceptible or resistant strains were considered (Table 1).
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Results and discussion |
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A. fumigatus is usually highly susceptible to amphotericin B, itraconazole, voriconazole and posaconazole (MICs below 1 mg/L), thus resistant strains are rarely found.2,8 In this study, we included strains with a wide spectrum of MICs to three of the four agents evaluated, in order to evaluate the performance of our method in discriminating between susceptible from less-susceptible or resistant A. fumigatus strains. MICs of amphotericin B, itraconazole and voriconazole ranged from 0.06 to >16 mg/L, and those of posaconazole from 0.06 to 1 mg/L (Table 1).
The germination rate of A. fumigatus conidia is well known (4–6 h) after an incubation at 37°C in a rich medium such as the one (RPMI-1640) used in our study.9 However, the conidial germination rate can be reduced in the presence of an antifungal agent such as voriconazole, because the growth of the fungus is disturbed.10 Additionally, other cellular and metabolic mechanisms of conidial germination and hyphal growth have been shown to be affected in the presence of amphotericin B, itraconazole, voriconazole and posaconazole.3,4 The metabolic ability of Aspergillus species in the presence of fluorescent or colorimetric stains has already been described to be useful for the determination of antifungal susceptibility profiles.3,4 However, the differences in the respiratory capacity of susceptible versus resistant strains of A. fumigatus have not been previously evaluated as a way of determining antifungal susceptibility patterns.
In this preliminary study, distinct respiratory measurements were found among the strains, and overall these measurements corresponded to the different MIC categories. That is, strains with the highest itraconazole, voriconazole or amphotericin B MICs grew faster in presence of the drug when compared with the growth rate for strains for which low MICs were obtained. Because of that, the former strains consumed 49% test medium oxygen (P < 0.05) than the latter strains. For example, strains with voriconazole MICs of 0.125–1 mg/L consumed no oxygen (0%) in the presence of 2 mg/L voriconazole after 8 h of incubation, while the strain with a voriconazole MIC of 16 mg/L consumed 49% of oxygen (P < 0.05) (Figure 1 and Table 1). Similar curves were observed with itraconazole; although the set of isolates evaluated did not include isolates for which posaconazole MICs were above 1 mg/L (Table 1), the highest percentage of oxygen consumption among these isolates was 5% under the same testing conditions. In contrast, the distinction between isolates with amphotericin B MICs of 1 mg/L (0% to 8% oxygen consumption) and those with MICs of 2 mg/L (31% oxygen consumption) was observed in the presence of 0.25 mg/L amphotericin B after either 4 or 8 h of incubation (P < 0.05).
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Although both the correlation between the methods and the intra-laboratory reproducibility were excellent for the susceptible isolates by the conditions described above, the categorical agreement was 80% for itraconazole, 90% for voriconazole and 100% for amphotericin B. The method was not able to differentiate between the less-susceptible (six isolates with azole MICs of 2 mg/L) and susceptible (MICs
1 mg/L) strains to either itraconazole or voriconazole (Table 1).
In conclusion, our method of measuring the oxygen consumption of conidia was able to identify the few resistant A. fumigatus isolates to both voriconazole and itraconazole and those with decreased susceptibility to amphotericin B after 4–8 h of incubation. The oxygen consumption cut-off for susceptible strains to the four agents was 17% after 4–8 h in the presence of 0.25 or 2 mg/L of either drug (defined as the upper limit of oxygen consumption for susceptible strains), while the cut-off of oxygen consumption for azole resistance was 49% in the presence of 2 mg/L of drug (defined as the lower limit of oxygen consumption for azole-resistant strains). The cut-off percentage of oxygen consumption was 31% for the less-susceptible isolates to amphotericin B, but the best results were in the presence of 0.25 mg/L amphotericin B. This is an economic, rapid and practical method for the potential categorization of A. fumigatus isolates as either susceptible or less-susceptible to three of the four agents evaluated after 4–8 h. Further collaborative and multicentre studies with more resistant isolates are needed to better evaluate and validate the potential use of this method in the clinical laboratory. The clinical relevance of these in vitro preliminary results also needs to be determined.
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The laboratorial work was performed at the Department of Microbiology, Faculty of Medicine of Porto, Portugal, and it was financially supported by the department budget. R. A. had received a grant from Fundação para Ciência e Tecnologia (FCT; SFRH/BPD/26655/2006). IPATIMUP is partially supported by FCT and Programa Operacional Ciência e Inovação (POCI).
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We are grateful to Maria Luz Dias and Isabel Santos for their excellent technical assistance.
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References |
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1 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi—Second Edition: Approved Standard M38-A2 (2008) Wayne, PA, USA: CLSI.
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