Effect of media composition and in vitro activity of posaconazole, caspofungin and voriconazole against zygomycetes

Cristina Gil-Lamaignere1, Regina Hess1, Stefanie Salvenmoser1, Kathrin Heyn1, Reinhard Kappe2 and Frank-Michael C. Müller1,*

1 Department of Paediatrics III, Paediatric Pulmonology and Infectious Diseases, University of Heidelberg, Im Neuenheimer Feld 153, D-69120 Heidelberg, Germany; 2 Haema Institute of Laboratory Medicine at the Helios Medical Center, Erfurt, Germany


* Corresponding author. Tel: +49-6221-56-8356; Fax: +49-6221-56-33853; Email: frank-michael_mueller{at}med.uni-heidelberg.de

Received 23 November 2004; returned 12 January 2005; revised 5 March 2005; accepted 31 March 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The effect of different media and composition on the in vitro activity of posaconazole, caspofungin and voriconazole against 59 zygomycetes species was determined.

Methods: The media tested were RPMI 1640 medium with and without 2% glucose, antibiotic medium 3 (AM3) with and without 2% glucose, and high resolution (HR) medium.

Results: Posaconazole was significantly more active than caspofungin and voriconazole, both in RPMI 1640 medium with 2% glucose and in HR medium. Adding glucose improved the determination of end points, but had only minor influence on the MICs. MICs evaluated in AM3 were lower than in RPMI 1640 medium or HR medium.

Conclusions: The in vivo effect of posaconazole in zygomycosis needs further evaluation.

Keywords: rhizopus , mucor , absidia , rhizomucor , cunninghamella


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Zygomycosis is an aggressive and rapidly progressive infection occurring mostly in immunocompromised patients. Members of the genera Rhizopus, Mucor and Absidia are the most common causative agents. However, other zygomycetes, including species Rhizomucor, Cunninghamella, Apophysomyces and Saksenaea, have also been implicated in causing human diseases.1 Intravenous amphotericin B is the current treatment of choice for zygomycosis. However, the use of this drug is limited by its severe side effects. Despite antifungal therapy, mortality remains high, particularly in disseminated zygomycosis.2 It is generally assumed that there is no indication for the use of azole drugs in treating zygomycosis.1 However, there are few data from either in vitro susceptibility studies or in vivo animal models to support such an assumption. The results of in vitro susceptibility tests can be strongly influenced by the test medium. RPMI 1640 medium has been evaluated extensively for in vitro susceptibility testing of yeasts and moulds and has been shown to give reproducible results.2 Other media used include antibiotic medium 3 (AM3) and high resolution (HR) medium.3 The aims of this study were twofold; to evaluate the activity of three newer antifungal drugs, posaconazole, caspofungin and voriconazole against strains belonging to different genera of zygomycetes, and secondly to compare the three different media and assess the effect of adding glucose to RPMI 1640 medium and AM3.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Microplates containing drug dilutions were prepared in advance as large batches, frozen at –70°C and stored for a maximum of 1 month.

Test isolates

A total of 59 zygomycete isolates, either of clinical origin or from several National Reference Laboratories, belonging to different genera of the order Mucorales were tested. Some of the tested species are non-pathogenic for humans or have not been definitively shown to cause disease in humans. These comprised 28 Rhizopus spp. [18 Rhizopus arrhizus (former R. oryzae4 ), eight Rhizopus microsporus and two Rhizopus stolonifer], seven Mucor spp. (four Mucor circinelloides, two Mucor mucedo and one Mucor rouxii), 15 Absidia spp. (one Absidia coerulea, 11 Absidia corymbifera, one Absidia glauca, one Absidia pseudocylindrospora and one Absidia repens) and nine Cunninghamella spp. (three Cunninghamella bertholletiae, one Cunninghamella blakesleeana, two Cunninghamella echinulata and three Cunninghamella elegans). Two reference strains, Candida albicans ATCC #90028 and Candida parapsilosis ATCC #22019, were included as quality controls.

Media

Five different media were used for comparison: RPMI 1640 medium with L-glutamine (without sodium bicarbonate; Sigma–Aldrich, GmbH, Steinheim, Germany) buffered to pH 7.0 with 0.165 M MOPS (Sigma) was used as is (R–G) or supplemented (R+G) with 2% glucose (Applichem GmbH, Darmstadt, Germany). AM3 (lot 1000A2DKBF; Becton Dickinson and Company, MD, USA) was similarly supplemented (A+G) or not (A–G) with 2% glucose. Since batch-to-batch variability has been documented, only one lot was used for all the experiments. Finally, HR medium (Oxoid Ltd, Hampshire, UK) was buffered to pH 7.5 with 0.2 M phosphate buffer according to the manufacturer's instructions. All of the media were prepared and added to 96-well plates at twice their final concentration.

Inoculum

Isolates were grown on potato dextrose agar (Oxoid Ltd) plates for 7 days at 25°C and stock spore suspensions were prepared by washing the surface of the plates with 6 mL of phosphate-buffered saline (PBS; PAA Laboratories GmbH, Pasching, Austria). Spore suspensions were counted with a haemocytometer and then diluted in H2O to a concentration of 3 x 104 cfu/mL (twice final concentration).

Antifungal susceptibility testing

MICs were determined by a microdilution technique following the NCCLS guidelines5 with a modification of the incubation temperature: several preliminary tests (data not shown) have demonstrated that a temperature of 25°C was suitable for all pathogenic and non-pathogenic strains. The following drugs were tested: posaconazole (Schering-Plough Research Institute, Kenilworth, NJ, USA), caspofungin (Merck Sharp & Dohme Limited, Hertfordshire, UK) and voriconazole (Pfizer Central Research, Sandwich, UK). Drugs were dissolved in dimethyl sulphoxide (DMSO; SERVA Electrophoresis GmbH, Heidelberg, Germany), except for caspofungin, which was dissolved in water according to the manufacturer's instructions. The drug dilutions were prepared at twice the final concentration by following the additive 2-fold drug dilution NCCLS scheme.5 The final concentrations of the antifungal agents were 0.015–8 mg/L for posaconazole and voriconazole, and 0.03–16 mg/L for caspofungin.

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. The plates were incubated at 25°C and MICs were determined visually after 24 h of incubation. The growth in each well was compared with that of the growth control. For each drug, an inhibition of growth ≥90% was recorded as MIC1, and an inhibition of growth ≥50% was recorded as MIC2 (Table 1).8 MIC determination was performed in duplicate by two different people on separate days with similar results.


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Table 1. In vitro susceptibility of 59 zygomycete strains to posaconazole, caspofungin and voriconazole in RPMI 1640 medium with (R+G) or without glucose (R–G), AM3 with (A+G) or without glucose (A–G) and HR medium

 
Data analysis

The MICs for 50% (MIC50) and 90% (MIC90) of the isolates tested were determined for all the genera. Those MICs that were off-scale MICs at the upper-end were converted into the next highest concentration, those off-scale at the lower end were left unchanged. The difference in the distributions of MICs was determined by either the Friedman test or the Kruskal–Wallis test, as specified in the text. The MIC values obtained using different media were considered to agree when their difference was ≤2 two-fold dilutions. Statistical analyses were performed using GraphPad Instat version 3.05 for Windows (GraphPad Software, San Diego, CA, USA). Statistical significance was defined as P < 0.05.


    Results and discussion
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
MIC data

The MICs of the different drugs for the two NCCLS quality control isolates C. albicans ATCC #90028 and C. parapsilosis ATCC #22019 were within the expected range.5,6

Usually a complete inhibition (100%) is used as the end point for visual reading. We decided to use an inhibition of >90% (MIC1) as the end point for this study as 100% inhibition was not observed for a single strain, in all wells at all antifungal concentrations for the zygomycetes. For the MIC2, a growth inhibition >50% was chosen, because we were not capable of distinguishing between 50 and 75% inhibition by visual recording, since the growth of zygomycetes is much more disperse than Ascomycota.

Table 1 summarizes the in vitro activity of posaconazole, caspofungin and voriconazole in the different media. Overall, posaconazole was significantly more active than caspofungin and voriconazole, both in R+G and in HR media (P < 0.05 when comparing the MIC1 of posaconazole in HR medium with that of voriconazole; P < 0.001 for all other comparisons, as evaluated by the Friedman test). These data are in agreement with the findings of Sun et al.7 and Dannaoui et al.1 Dannaoui et al. compared the MICs obtained for six different drugs against 36 zygomycetes isolates; they reported that posaconazole had good overall activity. Their results and ours suggest that posaconazole might be active in vivo against this group of fungi. Dannaoui et al.8 and Sun et al.9 have demonstrated a good in vivo activity of posaconazole against disseminated zygomycosis in neutropenic and non-neutropenic mice.

Currently, the standard treatment of disseminated zygomycosis is still amphotericin B, even though the clinical response is poor, and morbidity and mortality are high. Azoles have not been used to manage these infections since the response rates are regarded as inadequate. Posaconazole has already been used to treat zygomycosis infection in patients.10

Agreement among the media

The five media supported growth of all isolates after 24 h of incubation. However, growth in RPMI 1640 and AM3 media supplemented with glucose was more robust than in the corresponding media lacking glucose. The presence or absence of glucose had little influence on the MIC values. For example, when comparing the MIC1 evaluated in RPMI 1640 medium with or without glucose, the percentage of agreement was higher than 75% for all genera except Mucor (agreement 71.4%). When this comparison was evaluated using the MIC2 end point, the agreement was 100% for all genera. Similarly, the agreement when comparing the MIC1 evaluated in AM3 with or without glucose was quite good (>85%), whereas the use of the MIC2 end point yielded 100% agreement for all genera. In addition, the presence of glucose promoted thicker fungal growth, providing a more dense control that made definition of end points easier. We therefore consider that glucose supplementation improves the performance of these media.

When comparing the data obtained in RPMI 1640 medium (±glucose) to that in HR medium, the agreement overall was good. The percentage agreements using the MIC2 and MIC1 end points were 100% and >70%, respectively (the one exception for MIC1 was between R–G medium and HR medium for Mucor spp., the agreement was 42.9%). The reasons for this difference need further investigation. In contrast, the agreement between AM3 (±glucose) and the other media was generally poor. Moreover, the average MICs obtained in AM3 were lower than those obtained in either HR medium or RPMI 1640 medium (Table 1). We observed that this was due to a difference among genera: the values obtained using AM3 for Absidia and Cunninghamella were significantly lower than those for Rhizopus and Mucor spp. (P < 0.05 as evaluated by the Kruskal–Wallis test). In contrast, there were no significant differences in the posaconazole MICs in either RPMI 1640 medium or HR medium.

AM3 was first proposed as a growth medium for MIC testing because it seemed to improve the detection of Candida strains resistant to amphotericin B.11 Against Ascomycota, we and others have previously reported differences in the MIC values of various drugs obtained using AM3 and other media. For instance, we observed that micafungin MICs against C. albicans and C. dubliniensis were lower when evaluated in AM3 than in HR medium.3 Similarly, the use of AM3 for caspofungin susceptibility testing led to a lowering of MIC end points when testing Aspergillus and Candida spp.12 However, Arikan et al.13 demonstrated that the effect of AM3 on the itraconazole and voriconazole MICs against Aspergillus spp. was variable and hard to predict; voriconazole MICs against Fusarium spp. were slightly lower in AM3. Against zygomycetes, Vitale et al.2 observed that the post-antifungal effect of amphotericin B was very similar in RPMI 1640 medium and in AM3. To our knowledge, this is the first time that the effect of several media including HR medium on MIC against zygomycetes has been evaluated. Based on our data, we believe that R+G is a good growth medium, since it yielded a good agreement with both R–G medium and HR medium. In addition, R+G medium provided the clearest end points.

In summary, our results indicate the following. (i) Posaconazole is active in vitro against zygomycetes at clinically relevant concentrations. Therefore, further in vivo studies are warranted. (ii) Within zygomycetes, there are differences between genera in terms of their antifungal susceptibilities. (iii) Growth medium is an important variable for MIC determination in zygomycetes, and the more convenient medium appears to be RPMI 1640 supplemented with 2% glucose. On the other hand, it is unknown if data obtained with AM3 are less relevant than those obtained with RPMI 1640 medium. Further in vivo studies need to address whether testing posaconazole is more reproducible with AM3 or with RPMI 1640 medium (or with HR medium).


    Acknowledgements
 
This work was partially supported by a research grant from Schering-Plough Research Institute, Kenilworth, NJ, USA.


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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