Micafungin in combination with voriconazole in Aspergillus species: a pharmacodynamic approach for detection of combined antifungal activity in vitro

Russell E. Lewis1,2 and Dimitrios P. Kontoyiannis1,2,*

1 University of Houston College of Pharmacy, Houston, TX, USA; 2 Department of Infectious Diseases, Infection Control and Employee Health, Unit 402, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA


* Corresponding author. Tel: +1-713-792-6237; Fax: +1-713-745-6839; E-mail: dkontoyi{at}mdanderson.org

Received 28 April 2005; returned 1 June 2005; revised 11 August 2005; accepted 30 August 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Preclinical and clinical evidence indicate that echinocandin–triazole combinations provide enhanced killing versus triazoles alone against some Aspergillus isolates, however, in vitro test results designed to detect this combined effect are difficult to interpret.

Methods: We used a straightforward pharmacodynamic approach based on a microdilution format and a colorimetric analysis to harmonize growth end points.

Results: We detected a fourfold decrease in the EC90 of voriconazole when tested in combination with micafungin (4 mg/L) against isolates of Aspergillus fumigatus and Aspergillus terreus, but not against an isolate of Aspergillus flavus. Echinocandin-enhanced voriconazole activity was confirmed in A. fumigatus and A. terreus but not A. flavus by fluorescent morbidity staining and fluorescence microscopic analysis of damaged hyphae.

Conclusions: A microdilution-based pharmacodynamic method for testing antifungal combinations provides a less ambiguous description of the combined effects of antifungals against moulds and could be useful in reference laboratories that routinely evaluate the activity of antifungal combinations in vitro and in vivo.

Keywords: aspergillosis , echinocandins , triazoles


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Invasive aspergillosis (IA) has emerged as an important cause of death in heavily immunosuppressed patients. The poor outcome of this opportunistic mycosis following the use of antifungals given as monotherapy and the availability of safe and efficacious new antifungals such as the echinocandins1 and voriconazole2 have revitalized the interest in using antifungal combinations as a strategy to improve outcomes.3

Preclinical and clinical evidence have suggested a synergic interaction between echinocandins and triazoles against Aspergillus species.36 However, screening of clinical mould isolates for positive antifungal interactions in vitro is hampered by current methodological approaches such as the chequerboard dilution test, which is the most common method used to test antimicrobial combinations in research laboratories.7 Therefore, we sought to develop a more informative pharmacodynamic-based approach for the assessment of combined antifungal interactions in moulds. Using a straightforward microtitre plate assay with growth end points harmonized by a colorimetric indicator, we were able to compare the pharmacodynamics of the most active antifungal agent in an in vitro test system alone, and in combination with increasing concentrations of a second drug. We then analysed the inhibitory sigmoid dose–response curves for significant changes in the concentration–effect slope and in the effective concentration 90% (EC90) of the most lethal drug alone. This approach provided clear characterization of the pharmacodynamic interaction of two antifungals against Aspergillus species, and correlated with a second assay that described the degree of hyphal damage in Aspergillus species. We believe that this pharmacodynamic approach represents a straightforward and informative approach towards laboratory assessment of triazole–echinocandin combinations for Aspergillus species and would be useful in research laboratories that routinely perform similar assessment for antifungals against invasive moulds.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inoculum and drug preparation

We used three clinical isolates for testing (A. fumigatus 293, A. flavus 118, and A. terreus 0932) from the laboratory collection at the Mycology Research Laboratories at the University of Texas M.D. Anderson Cancer Center, Houston, TX, USA. Isolates were sub-cultured twice on potato dextrose agar slants (Remel, Lenexa, KS, USA) prior to testing. A standardized inoculum of conidia for each isolate was prepared by flooding 5- to 7-day-old cultures on potato dextrose agar slants with 5 mL of 0.85% sterile saline + 0.2% Tween 80 followed by 15 s of vortexing. The resultant suspension was then filtered twice through sterile syringes packed with glass wool to remove hyphal fragments. The conidial suspension was adjusted by haemocytometer to a concentration of 1–5 x 106 conidia/mL in sterile water. Drug stock solutions (1280 mg/L) were prepared for each antifungal in DMSO (voriconazole) or water (micafungin) from pure powders (voriconazole, Pfizer, Inc, Sandwich, UK; micafungin, Fujisawa Inc, Deerfield, IL, USA) and diluted in culture medium prior to testing.

Susceptibility testing and pharmacodynamic analysis

RPMI 1640 culture medium (Sigma, St Louis, MO, USA) buffered with 0.165 M MOPS (3-[N-morpholino] propanesulphonic acid) + 2% glucose, pH 7.0 served as the growth medium for all experiments. Stock solutions were then prepared in RPMI culture medium and serially diluted (32–0.0325 mg/L) for preparation of microtitre trays. The MIC of each antifungal was determined in triplicate using standardized methods (NCCLS M38-A).8 Briefly, conidial suspensions were diluted 1:50 in RPMI growth medium and dispensed (100 µL) into thawed microtitre trays containing serial twofold dilutions of antifungals. Trays were then incubated for 48 h at 37°C and voriconazole (VOR) MICs were read at 24 and 48 h as the lowest drug concentrations that showed absence of growth or complete growth inhibition (100% inhibition). For micafungin (MICA), the minimum effective concentration (MEC), defined as the minimal drug concentration producing short, stubby, hyper-acutely branching hyphae, was determined by microscopic examination of the microdilution plates.9 Candida parapsilosis ATCC 22019 served as a quality control isolate for each experimental run. XTT reduction assay was performed using the methods reported by Meletiadis et al.10 The association of formazan production versus fungal inoculum was determined by incubating standardized conidial suspensions of each isolate (105–102 conidia/mL) in RPMI growth medium for 24 h at 35°C as previously described (data not presented).10,11 Additional wells containing medium only (media controls) were included in each plate to correct for background absorbance and to verify growth medium sterility. After 22 h of incubation, trays were removed from the incubator and 50 µL of tetrazolium salt XTT (2,3-bis-(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide) solution (1 mg/mL) prepared in saline containing 125 µM menadione (Sigma) was added to each well and the tray was incubated for an additional 2 h. Trays were then removed from the incubator and absorbance of the XTT reduction product, formazan, was read at 492 nm for each well using a microplate spectrophotometer (Powerwave X Select, Biotech Instruments, Winooski, VT, USA). Absorbance readings were standardized in relation to unconverted XTT in the media control wells and plate absorbance (692 nm) to determine {Delta}OD 492 nm. All experiments were performed in triplicate.

Pharmacodynamic studies were performed by inoculating 100 µL of a 1:50 dilution of the standardized conidial suspension (1 x 106–2 x 106 conidia/mL) into wells of a flat-bottom 96-well microtitre tray containing serial twofold dilutions of voriconazole in RPMI growth medium (final concentration 0.0325–32 mg/L) with, or without micafungin (0, 0.25, 1 or 4 mg/L). Trays were then incubated for 24 h at 35°C and the absorbance of the XTT reduction product, formazan, was assayed as previously described. All experiments were performed in triplicate.

Fluorescence microscopy

Fluorescent viability staining and microscopy used a modification of methods reported by Bowman et al.12 Briefly, isolates were grown in RPMI medium for 18 h prior to the addition of voriconazole (final concentration 0.2 mg/L), micafungin (4 mg/L) or a combination of the antifungals (voriconazole 0.2 mg/L + micafungin 4 mg/L) to the culture medium. Concentrations of antifungals used for fluorescent staining were selected based on concentrations of antifungals that fell on the transitional portion of the dose–response curve in the pharmacodynamic studies. After incubation of hyphae for an additional 6 h, tubes were centrifuged at 10 000g for 5 min, culture media were carefully removed with a sterile pipette, and fungi were washed twice in MOPS pH 7 buffer solution. The morbidity dye, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC, Molecular Probes, Eugene, OR, USA) was then added from a 1 mg/mL stock in 100% ethanol to a final concentration of 2 mg/L and samples were incubated in the dark with shaking at room temperature for 1 h, washed twice again with MOPS pH 7 buffer, and resuspended for photomicrography.

Photomicrographs of hyphae were taken with an Olympus BX-51 microscope equipped with Nomarski optics and epifluorescence (FITC filter). All pictures were taken at x400 magnification using a computerized digital camera (Spot Diagnostics, Sterling Heights, MI, USA). Exposure times and light balance of micrographs were controlled through computer software (Image Pro Plus 4.1, Media Cybernetics, Silver Spring, MD, USA).

Hyphal damage was also confirmed by measuring total fluorescence with a fluorescein isothiocyanate (FITC) filter pair (excitation wavelength = 485 nm; emission wavelength = 538 nm) and 96-well Optiplates (Packard, Meriden, CT, USA) on a fluorescence microplate reader (Biotek FL600, Winooski, VT, USA). Samples of each stained hyphal suspension (400 µL) were added in triplicate to wells of the first Optiplate row and were serially diluted twofold to the last row of the plate in 200 µL of MOPS pH 7 buffer.

Data analysis

Median MIC values were calculated from experiments performed in triplicate. A four-parameter logistic model (Hill equation) using computer curve-fitting software (Prism 4, GraphPad Software, Inc, San Diego, CA, USA) was fitted to the experimental data performed in triplicate to derive EC50, EC90 and steepness of the inhibitory dose–response curve (Hill slope or coefficient). EC90 or the drug concentration resulting in 90% growth inhibition as assessed by XTT reduction was determined from fitted data using the equation: log EC50 = log EC90 – (1/Hill slope)*log [90/(100 – 90)]. Goodness of fit for each isolate/drug combination was assessed by R2 and the standard error of the EC50 value. If the standard error of the EC50 was ≥0.5 log, data were re-fitted using a fixed value for the top plateau of the regression curve based on formazan absorbance of the control wells. Significant changes in the antifungal activity of antifungal combinations were assessed by comparison of the inhibitory dose–response curves. Specifically, the mean Hill slope and EC90 plus 95% confidence intervals from experiments performed in triplicate of voriconazole alone versus each combination were compared to assess statistically significant changes in pharmacodynamic parameters of voriconazole activity. Statistical significance was set at a P value of <0.05.

Unpaired t-test was used to assess statistical differences in mean fluorescence between voriconazole 0.2 mg/L and the combination regimen (voriconazole 0.2 mg/L + micafungin 4 mg/L). Statistical significance was set at a P value of <0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antifungal susceptibility was determined in triplicate. The median voriconazole MIC for A. fumigatus, A. flavus and A. terreus was 0.25, 0.5 and 0.25 mg/L, respectively. The median micafungin MEC for A. fumigatus, A. flavus and A. terreus was 0.125, 0.125 and 0.5 mg/L, respectively. A linear relationship between XTT reduction to formazan, the colorimetrically assayed product, and starting inoculum was observed for isolates tested between 1 x 102 and 1 x 105 conidia/mL by XTT reduction assay (data not shown).10 Addition of voriconazole or micafungin to the culture decreased formazan in a concentration-dependent fashion.

Results of the pharmacodynamic analysis of voriconazole alone (0.013–32 mg/L) and in combination with micafungin (0.25, 1 and 4 mg/L) are presented in Figure 1. Overall, fit of the model to the data for each isolate/drug combination was considered adequate for assessing antifungal activity with R2 values ≥0.89 and standard error of the EC50 ≤0.5 log10. Voriconazole exhibited potent concentration-dependent antifungal activity against all three Aspergillus isolates with activity maximized as concentrations approached 1 mg/L. Voriconazole EC90s for A. fumigatus, A. flavus and A. terreus were 0.93, 0.68 and 0.86 mg/L, respectively and exceeded the NCCLS microdilution MIC for all three isolates. Similarly, the steepness of the voriconazole inhibitory dose–response curve was similar for all three isolates ranging between 0.96 and 1.47.



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Figure 1. Inhibitory dose–response curves of voriconazole (VOR) alone, or with increasing concentrations of micafungin (MICA). Mean absorbances of the formazan product ± standard deviations were plotted in relation to increasing voriconazole concentrations. A four-parameter logistic model (Hill equation) was then fitted to the data to determine the effective concentration 90% (EC90) and slope of the inhibitory dose–response curve (Hill slope) using the GraphPad Prism 4.0 software package. Mean best fit values and 95% CI were calculated by the software. Dotted lines represent the isolate VOR MICs as determined by NCCLS M38-A methodology.8

 
The addition of micafungin to voriconazole-containing microtitre wells changed the shape of the voriconazole inhibitory dose–response curve for A. fumigatus and A. terreus, but to a lesser degree for A. flavus (Figure 1). Consistently, this change in the dose–response curve was most pronounced at subinhibitory concentrations of voriconazole (Figure 1); where the activity of the azole was dramatically increased with the addition of micafungin. However, for purposes of assessing overall positive or negative interactions, the maximal or near-maximal activity of the antifungals (EC90) was assessed as this end point was considered to be more clinically relevant and more closely approaching achievable drug concentrations in vivo.

Micafungin decreased the median EC90 of voriconazole in a dose-dependent fashion for A. fumigatus and A. terreus. At the highest fixed concentration of micafungin (4 mg/L), the EC90 of voriconazole was reduced in A. fumigatus fourfold versus voriconazole alone (EC90 0.22 mg/L; 95% CI 0.10–0.27 versus EC90 0.93 mg/L; 95% CI 0.42–2.04), respectively, P < 0.05. Similarly, in A. terreus, the highest fixed concentration of micafungin (4 mg/L) reduced by nearly fourfold the EC90 of voriconazole compared with voriconazole alone (EC90 0.23 mg/L; 95% CI 0.11–0.56 versus EC90 0.86 mg/L; 95% CI 0.48–1.55), respectively. However, this reduction did not quite reach statistical significance (P > 0.05). For both A. fumigatus and A. terreus, a significant increase in the slope of the voriconazole dose–response curve was seen with the addition of micafungin 4 mg/L (Figure 1).

For A. flavus, micafungin exhibited minimal effects on the slope of the voriconazole dose–response curve or extent (EC90) of antifungal activity. At the highest fixed concentration of micafungin (4 mg/L), the EC90 of voriconazole was essentially unchanged versus voriconazole alone (EC90 0.68 mg/L; 95% CI 0.48–0.98 versus EC90 0.67 mg/L; 95% CI 0.38–1.19), respectively, P > 0.05.

The interaction between micafungin and voriconazole was further assessed by DiBAC staining. Control cells (non-drug exposed) demonstrated minimal diffuse or no fluorescence (Figure 2). Hyphal damage following exposure to micafungin (4 mg/L) was clearly evident for all three Aspergillus isolates and appeared to be concentrated at the hyphal tips. Voriconazole induced substantial hyphal damage that was appreciable in both apical and sub-apical hyphal compartments at a concentration of 0.2 mg/L (roughly correlating with mean EC50 for all three Aspergillus isolates). In agreement with the XTT reduction assay, greater fluorescence (more diffuse, and higher percentage of hyphal fragments) was seen in A. fumigatus and A. terreus with the combination of voriconazole 0.2 mg/L + micafungin 4 mg/L versus either drug alone. Analysis of the total A. fumigatus and A. terreus sample wells by the fluorescence microplate reader confirmed significantly higher fluorescence for the combination regimen versus voriconazole alone (P = 0.03, 0.05, respectively). An increase in fluorescence was not qualitatively evident with the combination for A. flavus, nor by total fluorescence analysis confirming the marginal to no improvement of voriconazole antifungal activity with the addition of micafungin to experimental samples (Figure 2).



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Figure 2. Detection of hyphal damage by fluorescence microscopy with the cellular morbidity dye DiBAC following exposure to voriconazole (VOR) 0.2 mg/L alone, or in combination with micafungin (MICA) 4 mg/L. Hyphae of each species were exposed to antifungals for 6 h after 18 h of pre-incubation in growth medium. Cells were then washed and stained with the fluorescent morbidity dye DiBAC. After additional wash steps, hyphae were examined by brightfield (grey boxes) and epifluorescence (dark boxes) microscopy at x400 using a fluorescein isothiocyanate (FITC) filter. Fluorescence in the dark boxes is indicative of early hyphal damage due to the antifungal(s).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite more than 20 years of published studies examining interactive effects of antifungal combinations for Candida, Cryptococcus and invasive moulds, in vitro methods for assessing and quantifying synergy or antagonism between antifungals remain poorly standardized.7 The simplest and most common approach for testing of antifungal combinations relies on the chequerboard dilution technique, which is an adaptation of conventional microdilution broth susceptibility testing into a two-dimensional array of serial concentrations of test compounds.7,13 Interpretation of the combined effect relies on determination of the MICs of each antifungal agent alone, then in combination with the second agent. The ratio of the MIC of each drug in combination versus alone is then summed into a calculation called the fractional inhibitory concentration index (FICI), which can be quantitatively related to qualitative concepts of combined drug effects such as synergy or antagonism.13

Although easy to set up and interpret, the chequerboard dilution method suffers from numerous methodological problems that may confound interpretation of antifungal combination testing for moulds. Chief among these problems for moulds is the fact that different classes of antifungal agents such as the echinocandins and triazole antifungals produce different patterns of growth inhibition, which confounds visual interpretation of the combined antifungal effects.3 Although these differences in growth pattern inhibition can be partially overcome through colorimetric viability dyes (e.g. MTT, XTT or Alamar Blue) that help harmonize indices of growth inhibition, synergic interactions identified by chequerboard dilution testing often predominate at antifungal concentrations well below the MIC and drug concentrations achieved in vivo.3 Considering that most antifungals used for the treatment of invasive moulds are given at high dosages, positive antifungal interactions identified at concentrations lower than those typically achieved in tissues with standard antifungal dosing or at concentrations of antifungal below the MIC would seem less relevant for translation to the clinic than interactions seen at maximal or near-maximal effects (i.e. EC90) of the most active individual agent alone.

Using this conceptual framework, we focused on the combined effect of micafungin plus voriconazole against three Aspergillus species by characterizing changes in the steepness of the dose–response curve and the EC90 of voriconazole when tested in combination with various clinically achievable concentrations of voriconazole. We have shown that for A. fumigatus and A. terreus, increasing clinically achievable concentrations of micafungin at or above the MEC resulted in a concentration-dependent increase in the steepness of the voriconazole concentration–effect curve and at maximal micafungin concentrations (4 mg/L) and significantly decreased the EC90 of voriconazole against these Aspergillus species (Figure 1). Conversely, the shape of the voriconazole sigmoid concentration–effect curve for A. flavus did not change significantly with increasing concentrations of micafungin, and the EC90 of the voriconazole + micafungin 4 mg/L combination was similar to voriconazole alone (0.67 versus 0.68 mg/L, respectively). The lack of a significant change of voriconazole EC90 for the A. flavus isolate was consistent with minimal concentration-dependent activity of micafungin alone against this isolate (Figure 1). Unlike A. fumigatus and A. terreus where micafungin alone reduced hyphal viability by ≥50% versus control at a concentration of 4 mg/L, hyphal viability was only reduced by roughly 25% in A. flavus.

Patterns of enhanced hyphal damage were confirmed with the voriconazole–micafungin combination for A. fumigatus and A. terreus, but were not observed for A. flavus by fluorescence microscopy and a microtitre plate assay using the fluorescent morbidity dye DiBAC. These data suggest that for some isolates, combinations of micafungin and voriconazole above the MIC can enhance the maximal killing effects of voriconazole against Aspergillus hyphae. Although standardized interpretations of pharmacodynamic parameters for antifungal combinations are not defined, this fourfold decrease in the EC90 of voriconazole for A. fumigatus and A. terreus isolates would be consistent with antifungal synergy, given the minimal antifungal activity of micafungin (i.e. high EC90). In comparison, the null effect of the combination for A. flavus isolate could be consistent with no interaction as defined by interpretive guidelines.7,13

In summary, we believe that this simple pharmacodynamic-focused approach to evaluate echinocandin–azole combinations for Aspergillus species at their near-maximal or maximal effect represents a promising and less ambiguous translational path for identifying antifungal combinations with enhanced killing activity that can be used to treat life-threatening invasive mould infections in patients. Although this approach does not provide the same depth of information on antifungal interactions as some other published methods based on zero-interaction theory (i.e. parametric surface-response models as reviewed by Meletiadis et al. or non-parametric models used to assess antibacterial combinations14), it can address the most clinically relevant question for triazole–echinocandin combinations for invasive aspergillosis, i.e. will the addition of an echinocandin to a triazole therapy enhance the activity of the triazole antifungal? Currently, additional studies are under way to assess the inter-laboratory reproducibility of this method and correlation with in vivo outcome in experimental models of invasive aspergillosis.


    Acknowledgements
 
We thank Nathaniel D. Albert for technical assistance. Supported in part by an educational grant from Fujisawa, Inc.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Maertens J, Raad I, Petrikkos G et al. Efficacy and safety of caspofungin for treatment of invasive aspergillosis in patients refractory to or intolerant of conventional antifungal therapy. Clin Infect Dis 2004; 39: 1563–7.[CrossRef][ISI][Medline]

2. Herbrecht R, Denning DW, Patterson TF et al. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. New Engl J Med 2002; 347: 408–15.[Abstract/Free Full Text]

3. Kontoyiannis D, Lewis R. Combination chemotherapy for invasive fungal infections: what laboratory and clinical studies tell us so far. Drug Resist Updat 2003; 6: 257–69.[CrossRef][ISI][Medline]

4. Kirkpatrick W, Perea S, Coco BJ et al. Efficacy of caspofungin alone and in combination with voriconazole in a Guinea pig model of invasive aspergillosis. Antimicrob Agents Chemother 2002; 46: 2564–8.[Abstract/Free Full Text]

5. Petraitis V, Petraitiene R, Sarafandi A et al. Combination therapy in treatment of experimental pulmonary aspergillosis: synergistic interaction between an antifungal triazole and an echinocandin. J Infect Dis 2003; 187: 1834–43.[CrossRef][ISI][Medline]

6. Marr K, Boeckh M, Carter R et al. Combination therapy for invasive aspergillosis. Clin Infect Dis 2004; 39: 797–802.[CrossRef][ISI][Medline]

7. Odds F. Synergy, antagonism and what the chequerboard puts between them. J Antimicrob Chemother 2003; 52: 1.[CrossRef][ISI][Medline]

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

9. Kurtz M, Heath I, Marrinan J et al. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase. Antimicrob Agents Chemother 1994; 38: 1480–9.[Abstract]

10. Meletiadis J, Mouton J, Meis J et al. Colorimetric assay for antifungal susceptibility testing of Aspergillus species. J Clin Microbiol 2001; 39: 3402–8.[Abstract/Free Full Text]

11. Lewis R, Wiederhold N, Klepser M. In vitro pharmacodynamics of amphotericin B, voriconazole, and itraconazole against Aspergillus, Fusarium, and Scedosporium spp. Antimicrob Agents Chemother 2005; 49: 945–51.[Abstract/Free Full Text]

12. Bowman J, Hicks P, Kurtz M et al. The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother 2002; 46: 3001–12.[Abstract/Free Full Text]

13. Eliopoulos G, Moellering R. Antimicrobial combinations. In: Lorian V, ed. Antibiotics and Laboratory Medicine, 4th edn. Baltimore, MD: Williams and Wilkins.

14. Meletiadis J, Meis J, Mouton J et al. Methodological issues related to antifungal drug interaction modeling for filamentous fungi. Rev Clin Microbiol 2002; 13: 101–17.





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