Departments of Medical Microbiology, 1 University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen; 2 Canisius-Wilhelmina Hospital, PO Box 9015, 6500 GS, Nijmegen, The Netherlands
Received 5 November 2001; returned 4 June 2002; revised 1 July 2002; accepted 27 September 2002
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Abstract |
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Introduction |
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Intravenous amphotericin B is the treatment of choice for zygomycosis. Nevertheless the use of amphotericin B is limited by its severe side effects, and new drugs that may have a role in the management of these infections are needed. Despite antifungal therapy, mortality remains high, particularly in disseminated zygomycosis.1
Although it is generally assumed that there is no indication for the use of azole drugs in treating zygomycosis,2,3 susceptibility studies in vitro and in vivo are very scarce. Moreover, it has been shown that azole compounds alone4 or in combination5 had beneficial effects in animal models of Rhizopus infection. Recently, the efficacy of itraconazole in the treatment of Absidia infection in mice has been evaluated.6,7
Terbinafine is another sterol biosynthesis inhibitor, primarily used for superficial mycoses, but its current applications are being extended. Although low MICs of terbinafine for some zygomycete strains have been reported8 the potential of this drug for zygomycosis is largely unknown.
Although there is no standardized method for susceptibility testing of filamentous fungi, an adaptation of the technique used for yeasts has shown good intra- and interlaboratory reproducibility9,10 and has been proposed as a standard method for conidium-forming fungi.11 For zygomycetes, data generated in vitro with NCCLS-based techniques are scarce and a low number of strains have been tested.8,1219
One of the problems in the determination of MICs is that visual reading is time-consuming and subjective. Spectrophotometric MIC endpoint determination has shown good agreement with standardized visual reading for Candida2024 and Cryptococcus.25 More recently, good agreement between visual and spectrophotometric reading has been reported for susceptibility testing of filamentous fungi.2628
The aim of this study was (i) to compare the activity of conventional and new antifungals against strains belonging to different genera of zygomycetes, and (ii) to evaluate a spectrophotometric method of MIC endpoint determination.
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Materials and methods |
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A total of 36 zygomycete isolates, mostly of clinical origin, belonging to different genera of the order Mucorales were tested. These comprised 15 Rhizopus spp. (eight Rhizopus oryzae and seven Rhizopus microsporus), six Mucor spp. (three Mucor hiemalis, one Mucor circinelloides, one Mucor racemosus and one Mucor rouxii), 10 Absidia corymbifera, three Rhizomucor spp. (two Rhizomucor pusillus and one Rhizomucor miehei), one Cunninghamella bertholletiae and one Apophysomyces elegans.29 Two reference strains, Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were included as quality controls.
Medium
RPMI 1640 medium with L-glutamine but without sodium bicarbonate (Gibco-BRL, Life Technologies, Woerden, The Netherlands) buffered to pH 7.0 with 0.165 M MOPS (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used as test medium.
Inoculum
Isolates were grown on Sabouraud dextrose agar for 7 days at 30°C and stock spore suspensions were prepared by washing the surface of the slants with 2 mL of sterile saline containing 0.05% Tween 80. For A. elegans, sporulation was obtained by culturing the mycelium in sterile distilled water supplemented with 0.1% yeast extract for 10 days at 37°C. Spore suspensions were counted with a haemocytometer and then diluted into RPMI to a concentration of 2 x 104 spores/mL (twice final concentration). Inoculum sizes were checked by quantitative colony counts on Sabouraud dextrose agar.
Antifungal susceptibility testing
MICs were determined by a microdilution technique following the NCCLS guidelines11 with slight modifications.
The drugs that were tested included itraconazole (Janssen Pharmaceutica, Beerse, Belgium), posaconazole [(SCH56592), Schering-Plough Research Institute, Kenilworth, NJ, USA], voriconazole (Pfizer Central Research, Sandwich, UK), terbinafine (Novartis Pharma, Basel, Switzerland), 5-fluorocytosine (ICN Pharmaceuticals, Zoetermeer, The Netherlands) and amphotericin B (Bristol-Myers Squibb, Woerden, The Netherlands). Drugs were dissolved in dimethylsulphoxide, except for 5-fluorocytosine, which was dissolved in water. The drug dilutions were prepared as twice the strength of the final concentration by following the additive two-fold drug dilution NCCLS scheme.11 The final concentrations of the antifungal agents were 0.01516 mg/L for itraconazole, posaconazole and amphotericin B, 0.03 32 mg/L for terbinafine and voriconazole, and 0.25256 mg/L for 5-fluorocytosine.
Incubation and MIC determination
On the day of the test, each well of the microtitre plates containing 100 µL of the diluted drug concentration was inoculated with 100 µL of the inoculum suspension. Microtitre plates were incubated at 37°C and MICs were determined visually and spectrophotometrically after 16, 24 and 48 h incubation. MIC determination was performed in duplicate with similar results.
Visual MIC determination. Microtitre plates were read visually with the aid of a concave mirror, and the growth in each well was compared with that of the growth control. Each well was then given a numerical score according to the NCCLS guidelines: 4, no reduction in growth; 3, growth reduction of 25%; 2, growth reduction of 50%; 1, growth reduction of 75% or more; and 0, absence of growth (optically clear). Visual reading was always performed before spectrophotometric reading. MIC endpoints were defined as the lowest drug concentration that had a score of 0 for amphotericin B and a score of 2 for the other drugs.
Spectrophotometric MIC determination. Spectrophotometric readings were performed with an automated microplate reader spectrophotometer (Rosys Anthos ht3; Anthos Labtec Instruments GmbH, Salzburg, Austria) at 405 nm. MIC endpoints were defined as the lowest drug concentration that led to an inhibition of 95% or more for amphotericin B and to an inhibition of 50% or more for the other drugs.
Data analysis
MICs for 50% (MIC50s) and 90% (MIC90s) for the isolates tested were determined for genera for which 5 and
10 isolates were available, respectively. For calculation, the high off-scale MICs were converted to the next highest concentration and the low off-scale MICs were left unchanged. The difference in the distributions of MICs was determined by the KruskalWallis test or the Friedman test, as appropriate. Discrepancies among MIC endpoints of no more than one dilution step were used to calculate the percentage agreement between visual and spectrophotometric readings. The correlation between the two methods was also assessed by the Spearman correlation coefficient. Statistical analyses were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). Statistical significance was defined as P
0.05.
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Results |
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The MICs of the different drugs for the two NCCLS quality control isolates C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were within the expected range.11,30
Table 1 summarizes the in vitro activity of the six antifungal agents tested against the 36 strains, as determined by visual reading after 24 h of incubation. Overall, amphotericin B and posaconazole were the most active drugs. Amphotericin B was active against most of the strains. However, two strains (one C. bertholletiae and one A. elegans) showed high amphotericin B MICs of 2 mg/L. All the strains were highly resistant to 5-fluorocytosine (MIC > 256 mg/L). Voriconazole was significantly less active than amphotericin B, itraconazole, posaconazole and terbinafine (P < 0.001). For all the strains, the MIC of voriconazole was 2 mg/L and the overall MIC90 was 32 mg/L. A wide range of itraconazole and terbinafine MICs was obtained for Rhizopus spp. and Mucor spp. For the other genera (Absidia spp., Rhizomucor spp., C. bertholletiae and A. elegans), MICs of itraconazole and terbinafine were
1 mg/L. Some strains were very susceptible to these two drugs with MICs as low as 0.03 mg/L.
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Influence of incubation time
The influence of incubation time on MIC values was investigated by reading the microplates after 16, 24 and 48 h of incubation. For 94% of the strains sufficient growth to determine MICs was obtained after 16 h incubation (data not shown). A significant increase in the MICs of all the drugs was noted between 16 and 24 h incubation (P < 0.05) and between 24 and 48 h incubation (P < 0.01 to P < 0.001), with the exception of amphotericin B MICs, which were not statistically different at 16 and 24 h. Variation of geometric mean MICs of the different antifungal drugs with incubation time is shown in Figure 1. An increase in the MIC values, between 24 and 48 h incubation, of 2 dilution steps was noted in 56% of the strains for itraconazole and in 32% of the strains for terbinafine (data not shown). For these two antifungals, a dramatic increase in the MIC of six dilution steps was noted at 48 h for some strains.
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Of the 2160 expected MICs (1080 visual/spectrophotometric pairs) for the 36 isolates tested, 1764 (882 pairs) were available for analysis. Absence of detectable growth after 16 and/or 24 h incubation and absence of spectrophotometric reading precluded MIC determination in 198 pairs. Very good agreement of 9196% was observed for amphotericin B and posaconazole (Spearman coefficient of correlation of 0.810.98). For itraconazole, voriconazole and terbinafine, agreements ranged from 71% to 85% (coefficient of correlation of 0.680.94). For all the drugs, agreement was not dependent on the incubation time. The number of dilution differences between the two methods of endpoint reading is shown in Table 2. Of the 122 MIC pairs with different visual and spectrophotometric MIC results, the spectrophotometric method yielded lower MICs than the visual method in 92% of the cases and higher MICs in only 8% of the cases.
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Discussion |
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All the strains were highly resistant to 5-fluorocytosine, which is in accordance with previous reports.16,18,32 Amphotericin B was active against most of the strains. Previous susceptibility studies in vitro have also demonstrated good activity of this antifungal, and experimental studies have shown that amphotericin B was active in vivo in animals infected with Rhizopus46,31 or Absidia.6,7
Azole drugs are considered ineffective against zygomycetes.2,3 Nevertheless, some studies have shown that azole compounds may be active in animal models of zygomycosis.4,5 Moreover, recent results in vitro suggest that some zygomycete strains (particularly Absidia strains) are inhibited by relatively low concentrations of itraconazole.15,19,3234 In this study, we tested three different azoles: itraconazole, voriconazole and posaconazole. High voriconazole MICs of 2 mg/L were found for all the strains, and the overall MIC50 was 16 mg/L. These results are consistent with previous studies.13,15,17 For itraconazole, a wide range of MICs, from 0.03 to 32 mg/L, was found. Absidia and Rhizomucor, in particular, exhibited low itraconazole MICs. Recently, MICs of itraconazole ranging from 0.25 to 1 mg/L have been reported for 10 strains of A. corymbifera.15,19 Moreover, in a murine model of Absidia infection, itraconazole therapy was shown to increase the rate of survival of infected animals.6,7 Itraconazole MICs ranged from 0.25 to 32 mg/L for Rhizopus spp. and there was no correlation between MIC values and species (i.e. MIC distribution was no different for R. microsporus and R. oryzae). Itraconazole MICs ranging from 0.25 to >32 mg/L have been reported for Rhizopus spp. in previous studies using NCCLS-based techniques.13,1518
Posaconazole is a new azole antifungal agent with broad-spectrum activity.3 There have been few studies that examine the in vitro activity of posaconazole against zygomycetes. In vitro data are available only for Rhizopus spp.,3,12,16 and MICs ranged from 0.5 to 2 mg/L. In the present study, posaconazole showed good activity in vitro with an overall MIC50 of 0.25 mg/L. Since the activity of posaconazole has not been yet studied in vivo in the treatment of zygomycosis, the activity of this antifungal should be validated in animal models.
Primarily designed for superficial mycoses, terbinafine is a sterol biosynthesis inhibitor that may be effective for the treatment of systemic fungal infections such as aspergillosis35 or pseudallescheriasis.36 In the present study, a wide range of MICs was obtained for terbinafine. The drug was very active against all the Absidia strains and against some Rhizopus and Mucor isolates. Terbinafine has been tested in vitro against only a small number of isolates of Mucor and Rhizopus and found to have poor activity. Nevertheless, low terbinafine MICs have been reported for some strains.8 We found a sharp difference in susceptibility to terbinafine between R. oryzae and R. microsporus, the latter being susceptible to the drug.
The results in vitro obtained in this study demonstrate that the zygomycetes consist of a heterogeneous group with variable antifungal susceptibility. Therefore, no general conclusions about the antifungal susceptibilities of all zygomycetes can be drawn from the results obtained from one genus or one species.
Zygomycetes are fast-growing fungi, and the NCCLS recommendation for Rhizopus is to read MICs after 24 h incubation.11 Recently, an analysis of growth characteristics of different filamentous fungi has demonstrated that the end of the log phase of growth for R. microsporus was already reached after 16 h incubation in RPMI.37 For this reason, we have determined and compared MICs after 16, 24 and 48 h of incubation. For most of the strains used in the present study, sufficient growth to determine MICs was obtained after 16 h incubation, although some strains required 24 h incubation. A significant increase in the MICs was noted between 24 and 48 h incubation. It is not known whether MICs obtained after 24 h are more clinically relevant than those obtained after 48 h incubation. For this reason, studies of correlation between MICs determined in vitro after 24 and 48 h incubation and activity of the drugs in vivo are warranted.
Visual determination of MICs is time-consuming and subjective. Determination of MICs spectrophotometrically has shown good agreement with standardized visual reading for Candida albicans,21,22 other Candida species20,23,24 and Cryptococcus neoformans.25 Nevertheless, for filamentous fungi, only a few studies have compared spectrophotometric and visual methods for reading MICs2628 and, to our knowledge, no study has been carried out with zygomycetes.
In this study we found good agreement between the two methods of reading for all the antifungal drugs. Therefore it can be concluded that spectrophotometric reading is a valuable alternative to visual reading for MIC determination. When discrepancies between visual and spectrophotometric methods were noted, MICs determined spectrophotometrically were lower than those read visually. These differences could reflect the difficulty in determining 50% inhibition of growth visually.
In summary, our results suggest the following. (i) Some of the conventional and investigational antifungals, besides amphotericin B, have in vitro activity against zygomycetes. Therefore, further studies in vitro and in vivo are warranted. (ii) Within zygomycetes, there are differences between genera and between species in terms of their antifungal susceptibilities. (iii) Incubation time is an important variable for MIC determination in zygomycetes and the more relevant incubation time remains to be determined. (iv) A spectrophotometric procedure for MIC determination is a valuable alternative to visual reading.
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Acknowledgements |
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The Eurofung Network consists of the following participants: Emmanuel Roilides, co-ordinator, and Nicos Maglaveras, Aristotle University, Thessaloniki, Greece; Tore Abrahamsen and Peter Gaustad, Rikshospitalet National Hospital, Oslo, Norway; David W. Denning, University of Manchester, Manchester, UK; Paul E. Verweij and Jacques F. G. M. Meis, University of Nijmegen, Nijmegen, The Netherlands; Juan L. Rodriguez-Tudela, Instituto de Salud Carlos III, Madrid, Spain; George Petrikkos, Athens University, Athens, Greece.
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Footnotes |
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Members of The Eurofung Network are listed in the Acknowledgements.
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References |
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