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Günter Rambach, Heidemarie Oberhauser, Cornelia Speth, Cornelia Lass-Flörl, Susceptibility of Candida species and various moulds to antimycotic drugs: use of epidemiological cutoff values according to EUCAST and CLSI in an 8-year survey, Medical Mycology, Volume 49, Issue 8, November 2011, Pages 856–863, https://doi.org/10.3109/13693786.2011.583943
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Abstract
A collection of 2,834 isolates of Candida spp. and 1,079 isolates of Aspergillus spp. and other moulds that were recovered between 2000 and 2007 in Tyrol, Austria, were examined for their susceptibility to antifungal drugs. The susceptibility of Candida spp. to amphotericin B (AMB), caspofungin (CPF), fluconazole (FLC), and voriconazole (VRC) were studied, while filamentous fungi were tested against AMB, CPF, VRC, itraconazole (ITC), and posaconazole (POS). As EUCAST and CLSI are currently revising their breakpoints for several antifungal agents, epidemiological cutoff values (ECVs) of these two guidelines were used to examine trends in susceptibility. For Candida spp. we noted increases in the percentage of non-wild type isolates which were resistant to CPF, FLC, and VRC. Most noticeably, we observed a change in: C. tropicalis (from 0.9–3.8%) and C. parapsilosis (from 4.0–6.0%) relative to CPF; C. parapsilosis (from 0.8–3.4%) and C. glabrata (from 11.0–20%) against FLC; and C. glabrata (from 3.0–12.0%) for VRC. Among the moulds, most Aspergillus spp. isolates were found to be susceptible to VRC, ITC, and POS, while AMB and POS were confirmed to be the most effective agents against zygomycetes. EUCAST and CLSI should continue their efforts to harmonize their methods of antimicrobial susceptibility testing (AST) and to define additional and shareable epidemiological cutoff values and clinical breakpoints.
Introduction
Invasive fungal infections (IFI) are associated with high rates of morbidity and mortality in at-risk patients. The frequency of IFI has been increasing over the last two decades, while there have been coincidental changes in the pattern of the etiologic species [1,2]. Although Candida albicans and Aspergillus fumigatus remain the predominant pathogens, other species of these two genera, as well as members of the zygomycetes, hyaline moulds and dematiaceous fungi have gained increasing importance [2–4]. There have been changes in risk factors and medical treatment [5], and several new antifungal agents have been approved. However, drug resistance mechanisms of pathogens to the various agents have evolved concurrently [6,7]. Rapid identification of the pathogenic fungi and antimicrobial susceptibility testing (AST) are vitally important to enable target-aimed treatment. Furthermore, continual surveillance of trends in susceptibility to antifungal agents is necessary to provide data for both empirical therapy recommendations and the assessment of putative interventional procedures.
In this study, we analyzed the in vitro susceptibility (S/R) rates of patient-derived Candida species and various moulds to commonly used antifungal drugs over an 8-year period. For Candida spp., the polyene amphotericin B (AMB), the echinocandin caspofungin (CPF) and the triazoles fluconazole (FLC) and voriconazole (VRC) were tested. The study period between 2000 and 2007 was subdivided into three periods to facilitate the detection of potentially emerging resistances. Filamentous fungi were cumulatively tested for their susceptibility to AMB, CPF, VRC, itraconazole (ITC), and posaconazole (POS). All susceptibility tests were performed by applying the Etest method to determine the minimal inhibitory concentrations (MICs) of the respective antifungal agents. This commercially available test was previously described as a simple and reliable method for AST that was comparable in performance to reference procedures [8,9], except for the determination of minimal effective concentrations (MEC) of moulds to echinocandins (see discussion). As clinical susceptibility breakpoints to several antifungal agents are currently being revised by the Clinical and Laboratory Standards Institute (CLSI), as well as by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), epidemiological cutoff values (ECVs) were applied to examine trends in susceptibility of the most important pathogenic Candida species and moulds to the antifungal agents.
Materials and methods
Isolates
Clinical samples were obtained from the University Clinics Innsbruck and other medical centres and practices in Tyrol, Austria. All were recovered from patient-derived samples collected between 2000 and 2007 and included various types of specimens such as tracheal lavage, respiratory tract tissue, blood and other normally sterile body fluids and tissue specimens from different deep sites. Candida isolates were obtained from blood and sterile tissue (50%) and superficial swabs (50%), whereas moulds were isolated from the respiratory tract (50%), biopsy (30%) and various other sites (20%).
The identification of Candida species was determined by application of the BBL CHROMagar™ Candida (BD, Schwechat, Austria) and the API ID 32C yeast identification system (bioMérieux, Vienna, Austria). Aspergillus spp. and the zygomycetes were identified according to their morphological characteristics and when needed, by use of ribosomal internal transcribed spacer (ITS) DNA sequencing.
In total, 2,834 isolates of Candida spp. and 1,079 isolates of Aspergillus spp. and other moulds (see tables for detailed numbers) were collected to determine their MICs against respective antifungals.
As the study period was subdivided into three intervals for Candida spp., the numbers of less frequently isolated species were too small to obtain convincing data, so they were excluded from the results. For these species, surveillance studies of larger scale are required. The small numbers of rare moulds and those Candida spp. that were not further identified were not included for the same reasons.
Antifungal susceptibility testing by Etest
Etest gradient strips of AMB, CPF, FLC, VRC, ITC and POS were obtained from the manufacturer AB BIODISK (Solna, Sweden). The Etest procedure was performed according to the manufacturer's instruction. Briefly, agar plates (RPMI 1640 with 2% glucose) were inoculated with sterile loops from stock suspensions which had been adjusted to a turbidity equivalent of 0.5 McFarland standard. Etest strips were placed onto the inoculated plates, and the plates incubated at 37°C. Determination of the MICs was made after 24 h incubation for yeasts and moulds and, if indicated, after 48 h. The MICs were read as the lowest drug concentration at which the border of the elliptical inhibition area intercepted the scale on the Etest strip.
Use of epidemiological cutoff values (ECVs) to examine trends in susceptibility to antifungal agents
CLSI and EUCAST are currently revising their susceptibility breakpoints (BPs) for several antifungal agents. Therefore, we did not use either the old BP or those that have not been approved as this might lead to misinterpretation of the results. For these reasons, we applied CLSI and EUCAST epidemiological cutoff values (ECVs) to examine probable trends in fungal susceptibility to various antifungals. These values have been established to distinguish wild-type (WT) populations of microorganisms from those that may have acquired resistance. ECVs for the various antifungals and the respective fungal species are obtained by considering the wild-type (WT) MIC distribution, the modal MIC for each distribution, and the inherent variability of the test [10].
In our results, the proportion of isolates with MICs lower than or equal to the respective ECVs were termed susceptible (S) WT isolates, while the others were considered to be resistant (R) non-WT isolates.
Both CLSI and EUCAST have published ECVs for Candida spp. against FLC (Table 3) and VRC (Table 4), whereas CLSI has defined ECVs for Candida spp. and the echinocandins (for CPF see Table 2). CLSI has also published ECVs for several Aspergillus spp. against VRC, ITC and POS, and EUCAST has done the same for A. fumigatus (Table 5). In cases of unavailable ECVs, we applied breakpoints that were used in several previous studies [2] (AMB; Zygomycetes in Table 5).
Results
Distribution of fungal species
C. albicans was the most frequently isolated species with a recovery rate of 67.8%, followed by C. glabrata (14.1%), C. tropicalis (6.8%), C. parapsilosis (3.8%) and C. krusei (3.6%). Other species, including those not elsewhere specified isolates, accounted for 3.7%. Aspergillus fumigatus was the most common mould species (66.5%), ahead of A. terreus (14%), A. flavus (6.7%) and A. niger (4.9%). All zygomycetes species (Lichtheimia spp., Mucor spp., Rhizopus spp. and Rhizomucor spp.) accounted for 4.4% of all mould isolates, with an additional 3.6% not further identified.
In vitro susceptibility of Candida spp.
Amphotericin B. Although non-lipid formulated AMB is now not routinely administered, this classical antifungal agent is still highly active against virtually all pathogenic species of Candida. Applying a breakpoint of ≥2 µg/ml, we found that up to 100% of the C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei isolates were susceptible and no noticeable changes in the drug's efficiency could be observed over the study period (Table 1). The same was observed for most of the other yeastlike fungal species (data not shown).
In vitro susceptibility to amphotericin B* | |||||||
2000–2002 | 2003–2005 | 2006–2007 | |||||
Species | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1922 | 98.9 | 1.1 | 99.9 | 0.1 | 100 | 0.0 |
C. glabrata | 401 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. tropicalis | 195 | 99.5 | 0.5 | 99.5 | 0.5 | 100 | 0.0 |
C. parapsilosis | 108 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. krusei | 102 | 98.2 | 1.8 | 100 | 0.0 | 98.2 | 1.8 |
In vitro susceptibility to amphotericin B* | |||||||
2000–2002 | 2003–2005 | 2006–2007 | |||||
Species | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1922 | 98.9 | 1.1 | 99.9 | 0.1 | 100 | 0.0 |
C. glabrata | 401 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. tropicalis | 195 | 99.5 | 0.5 | 99.5 | 0.5 | 100 | 0.0 |
C. parapsilosis | 108 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. krusei | 102 | 98.2 | 1.8 | 100 | 0.0 | 98.2 | 1.8 |
No ECVs available; applied resistance breakpoint: MIC ≥2 µg/ml.
In vitro susceptibility to amphotericin B* | |||||||
2000–2002 | 2003–2005 | 2006–2007 | |||||
Species | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1922 | 98.9 | 1.1 | 99.9 | 0.1 | 100 | 0.0 |
C. glabrata | 401 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. tropicalis | 195 | 99.5 | 0.5 | 99.5 | 0.5 | 100 | 0.0 |
C. parapsilosis | 108 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. krusei | 102 | 98.2 | 1.8 | 100 | 0.0 | 98.2 | 1.8 |
In vitro susceptibility to amphotericin B* | |||||||
2000–2002 | 2003–2005 | 2006–2007 | |||||
Species | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1922 | 98.9 | 1.1 | 99.9 | 0.1 | 100 | 0.0 |
C. glabrata | 401 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. tropicalis | 195 | 99.5 | 0.5 | 99.5 | 0.5 | 100 | 0.0 |
C. parapsilosis | 108 | 100 | 0.0 | 100 | 0.0 | 100 | 0.0 |
C. krusei | 102 | 98.2 | 1.8 | 100 | 0.0 | 98.2 | 1.8 |
No ECVs available; applied resistance breakpoint: MIC ≥2 µg/ml.
Caspofungin. While CPF was initially approved in 2001, its routine screening in Innsbruck did not begin before 2003. To date, CLSI has published ECVs for Candida spp. against CPF. Applying these cutoff values, we examined trends towards emerging resistance for the most important Candida spp. (Table 2). Over the study period, increases in the percentage of non-WT isolates (resistant) were observed for all tested Candida species, most noticeable C. albicans (from 0.9–2.8%), C. tropicalis (from 0.9–3.8%), and C. parapsilosis (from 4.0–6.0%) (Table 2).
In vitro susceptibility to caspofungin | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1522 | n.t | n.t | 99.1 | 0.9 | 97.2 | 2.8 |
C. glabrata | 0.12 | 201 | n.t | n.t | 99.9 | 0.1 | 99.0 | 1.0 |
C. tropicalis | 0.12 | 105 | n.t | n.t | 99.1 | 0.9 | 96.2 | 3.8 |
C. parapsilosis | 1 | 68 | n.t | n.t | 96.0 | 4.0 | 94.0 | 6.0 |
C. krusei | 0.25 | 52 | n.t | n.t | 99.1 | 0.9 | 98.0 | 2.0 |
In vitro susceptibility to caspofungin | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1522 | n.t | n.t | 99.1 | 0.9 | 97.2 | 2.8 |
C. glabrata | 0.12 | 201 | n.t | n.t | 99.9 | 0.1 | 99.0 | 1.0 |
C. tropicalis | 0.12 | 105 | n.t | n.t | 99.1 | 0.9 | 96.2 | 3.8 |
C. parapsilosis | 1 | 68 | n.t | n.t | 96.0 | 4.0 | 94.0 | 6.0 |
C. krusei | 0.25 | 52 | n.t | n.t | 99.1 | 0.9 | 98.0 | 2.0 |
n.t.: not tested.
In vitro susceptibility to caspofungin | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1522 | n.t | n.t | 99.1 | 0.9 | 97.2 | 2.8 |
C. glabrata | 0.12 | 201 | n.t | n.t | 99.9 | 0.1 | 99.0 | 1.0 |
C. tropicalis | 0.12 | 105 | n.t | n.t | 99.1 | 0.9 | 96.2 | 3.8 |
C. parapsilosis | 1 | 68 | n.t | n.t | 96.0 | 4.0 | 94.0 | 6.0 |
C. krusei | 0.25 | 52 | n.t | n.t | 99.1 | 0.9 | 98.0 | 2.0 |
In vitro susceptibility to caspofungin | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1522 | n.t | n.t | 99.1 | 0.9 | 97.2 | 2.8 |
C. glabrata | 0.12 | 201 | n.t | n.t | 99.9 | 0.1 | 99.0 | 1.0 |
C. tropicalis | 0.12 | 105 | n.t | n.t | 99.1 | 0.9 | 96.2 | 3.8 |
C. parapsilosis | 1 | 68 | n.t | n.t | 96.0 | 4.0 | 94.0 | 6.0 |
C. krusei | 0.25 | 52 | n.t | n.t | 99.1 | 0.9 | 98.0 | 2.0 |
n.t.: not tested.
Fluconazole. FLC is widely used as a first line and prophylactic drug and still is highly effective against C. albicans, C. tropicalis and C. parapsilosis (Table 3). However, through the use of ECVs we detected an increase in the percentage of non-susceptible isolates over the study period. These proportions rose from 1% (2000–2002) to 2.3% (2006–2007) for C. albicans, from 1.8% (2000–2002) to 3.1% (2006–2007) for C. tropicalis, and from 0.8% (2000–2002) to 3.4% for C. parapsilosis (2006–2007). A strong emergence of non-WT strains was found with C. glabrata, i.e., the proportion of isolates with a MIC higher than the ECV increased from 11% (2000–2002) to 16.5% (2003–2005) to 20.0% (2006–2007). C. krusei is well known for its high inherent resistance to fluconazole, which was also shown by our results.
(A) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 128 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
(B) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.5 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 64 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
(A) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 128 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
(B) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.5 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 64 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
(A) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 128 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
(B) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.5 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 64 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
(A) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 1 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 128 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
(B) | ||||||||
In vitro susceptibility to fluconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.5 | 1922 | 99.0 | 1 | 98.6 | 1.4 | 97.7 | 2.3 |
C. glabrata | 32 | 401 | 89.0 | 11.0 | 83.5 | 16.5 | 80.0 | 20.0 |
C. tropicalis | 2 | 195 | 98.2 | 1.8 | 98.3 | 1.7 | 96.9 | 3.1 |
C. parapsilosis | 2 | 108 | 99.2 | 0.8 | 98.7 | 1.3 | 96.6 | 3.4 |
C. krusei | 64 | 102 | 59.8 | 40.2 | 66.5 | 33.5 | 70.0 | 30.0 |
Comparison of EUCAST and CLSI ECVs did not result in any differences in the proportion of susceptible isolates (Table 3).
Voriconazole. Trends of emerging VRC resistance mechanisms were observed for all the most important pathogenic Candida species (Table 4). Applying EUCAST ECVs, this tendency was rather slight for C. parapsilosis (from 0.0–0.9%), while the percentage of non-WT isolates rose from 0.8% (2000–2002) to 2.5% (2006–2007) for C. albicans, and from 0.8–2.8% for C. tropicalis. Again, the most remarkable trend was detected with C. glabrata, which showed an increase in the proportion of non-WT isolates from 3.0% (2000–2002) to 7.0% (2003–2005) to 12.0% (2006–2007). The rate of emergence of non-susceptible C. krusei isolates is remarkably low in Tyrol, with an increase over the study period from only 1.2% (2000–2002) to 2.9% (2003–2005) to 4.4% (2006–2007).
(A) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1922 | 99.2 | 0.8 | 98.0 | 2.0 | 97.5 | 2.5 |
C. glabrata | 1 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.12 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 1 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
(B) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.03 | 1922 | 98.9 | 1.1 | 97.6 | 2.4 | 97.2 | 2.8 |
C. glabrata | 0.5 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.06 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 0.5 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
(A) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1922 | 99.2 | 0.8 | 98.0 | 2.0 | 97.5 | 2.5 |
C. glabrata | 1 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.12 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 1 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
(B) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.03 | 1922 | 98.9 | 1.1 | 97.6 | 2.4 | 97.2 | 2.8 |
C. glabrata | 0.5 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.06 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 0.5 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
(A) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1922 | 99.2 | 0.8 | 98.0 | 2.0 | 97.5 | 2.5 |
C. glabrata | 1 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.12 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 1 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
(B) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.03 | 1922 | 98.9 | 1.1 | 97.6 | 2.4 | 97.2 | 2.8 |
C. glabrata | 0.5 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.06 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 0.5 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
(A) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (EUCAST 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.12 | 1922 | 99.2 | 0.8 | 98.0 | 2.0 | 97.5 | 2.5 |
C. glabrata | 1 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.12 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 1 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
(B) | ||||||||
In vitro susceptibility to voriconazole | ||||||||
2000–2002 | 2003–2005 | 2006–2007 | ||||||
Species | ECV (CLSI 24 h; µg/ml) | No. of isolates | % S | % R | % S | % R | % S | % R |
C. albicans | 0.03 | 1922 | 98.9 | 1.1 | 97.6 | 2.4 | 97.2 | 2.8 |
C. glabrata | 0.5 | 401 | 97.0 | 3.0 | 93.0 | 7.0 | 88.0 | 12.0 |
C. tropicalis | 0.06 | 195 | 99.2 | 0.8 | 98.1 | 1.9 | 97.2 | 2.8 |
C. parapsilosis | 0.12 | 108 | 100 | 0.0 | 99.7 | 0.3 | 99.1 | 0.9 |
C. krusei | 0.5 | 102 | 98.8 | 1.2 | 97.1 | 2.9 | 95.6 | 4.4 |
Similar to FLC, almost no differences were detected by applying the ECVs according to EUCAST and CLSI, respectively (Table 4).
In vitro susceptibility of moulds
Aspergillus spp. A. fumigatus was by far the most frequently isolated Aspergillus species in Tyrol. Our results indicated high susceptibility to AMB, VRC, ITC, and POS. For the azoles, no differences were noticed in applying EUCAST and CLSI ECVs. For caspofungin, 20% of the isolates showed an in vitro MIC higher than the CLSI ECV (see discussion and Table 5).
Cumulative in vitro resistance (2000–2007) | |||||||
Species | No. of isolates | Amphotericin B*%R | Method (48 h) | Caspofungin %R (ECV µg/ml) | Voriconazole %R (ECV µg/ml) | Itraconazole %R (ECV µg/ml) | Posaconazole %R (ECV µg/ml) |
A. fumigatus | 717 | 0.3 | EUCAST CLSI | NA 20.0 (0.06) | 0.6 (1) 0.6 (1) | 0.3 (1) 0.3 (1) | 0.3 (0.25) 0.3 (0.5) |
A. terreus | 151 | 88.7 | EUCAST CLSI | NA 1.0 (0.06) | NA 0.7 (1) | NA 0.7 (1) | NA 0.0 (0.5) |
A. flavus | 72 | 2.8 | EUCAST CLSI | NA 13.0 (0.06) | NA 0.0 (1) | NA 1.4 (1) | NA 0.0 (0.5) |
A. niger | 53 | 0.0 | EUCAST CLSI | NA 8.0 (0.06) | NA 5.7 (2) | NA 12.0 (2) | NA 1.9 (0.5) |
Lichtheimia spp.** | 25 | 4.0 | n.t. | n.t. | 16.0 | 8.0 | |
Mucor spp.** | 9 | 44.4 | n.t. | n.t. | 44.4 | 44.4 | |
Rhizopus spp.** | 6 | 33.3 | n.t. | n.t. | 33.3 | 33.3 | |
Rhizomucor spp.** | 7 | 28.6 | n.t. | n.t. | 57.1 | 28.6 |
Cumulative in vitro resistance (2000–2007) | |||||||
Species | No. of isolates | Amphotericin B*%R | Method (48 h) | Caspofungin %R (ECV µg/ml) | Voriconazole %R (ECV µg/ml) | Itraconazole %R (ECV µg/ml) | Posaconazole %R (ECV µg/ml) |
A. fumigatus | 717 | 0.3 | EUCAST CLSI | NA 20.0 (0.06) | 0.6 (1) 0.6 (1) | 0.3 (1) 0.3 (1) | 0.3 (0.25) 0.3 (0.5) |
A. terreus | 151 | 88.7 | EUCAST CLSI | NA 1.0 (0.06) | NA 0.7 (1) | NA 0.7 (1) | NA 0.0 (0.5) |
A. flavus | 72 | 2.8 | EUCAST CLSI | NA 13.0 (0.06) | NA 0.0 (1) | NA 1.4 (1) | NA 0.0 (0.5) |
A. niger | 53 | 0.0 | EUCAST CLSI | NA 8.0 (0.06) | NA 5.7 (2) | NA 12.0 (2) | NA 1.9 (0.5) |
Lichtheimia spp.** | 25 | 4.0 | n.t. | n.t. | 16.0 | 8.0 | |
Mucor spp.** | 9 | 44.4 | n.t. | n.t. | 44.4 | 44.4 | |
Rhizopus spp.** | 6 | 33.3 | n.t. | n.t. | 33.3 | 33.3 | |
Rhizomucor spp.** | 7 | 28.6 | n.t. | n.t. | 57.1 | 28.6 |
NA: data not available; n.t.: not tested; *no ECVs available; applied breakpoint ≥2 µg/ml; **applied breakpoints (µg/ml): AMB ≥2, ITC >2, POS >2.
Cumulative in vitro resistance (2000–2007) | |||||||
Species | No. of isolates | Amphotericin B*%R | Method (48 h) | Caspofungin %R (ECV µg/ml) | Voriconazole %R (ECV µg/ml) | Itraconazole %R (ECV µg/ml) | Posaconazole %R (ECV µg/ml) |
A. fumigatus | 717 | 0.3 | EUCAST CLSI | NA 20.0 (0.06) | 0.6 (1) 0.6 (1) | 0.3 (1) 0.3 (1) | 0.3 (0.25) 0.3 (0.5) |
A. terreus | 151 | 88.7 | EUCAST CLSI | NA 1.0 (0.06) | NA 0.7 (1) | NA 0.7 (1) | NA 0.0 (0.5) |
A. flavus | 72 | 2.8 | EUCAST CLSI | NA 13.0 (0.06) | NA 0.0 (1) | NA 1.4 (1) | NA 0.0 (0.5) |
A. niger | 53 | 0.0 | EUCAST CLSI | NA 8.0 (0.06) | NA 5.7 (2) | NA 12.0 (2) | NA 1.9 (0.5) |
Lichtheimia spp.** | 25 | 4.0 | n.t. | n.t. | 16.0 | 8.0 | |
Mucor spp.** | 9 | 44.4 | n.t. | n.t. | 44.4 | 44.4 | |
Rhizopus spp.** | 6 | 33.3 | n.t. | n.t. | 33.3 | 33.3 | |
Rhizomucor spp.** | 7 | 28.6 | n.t. | n.t. | 57.1 | 28.6 |
Cumulative in vitro resistance (2000–2007) | |||||||
Species | No. of isolates | Amphotericin B*%R | Method (48 h) | Caspofungin %R (ECV µg/ml) | Voriconazole %R (ECV µg/ml) | Itraconazole %R (ECV µg/ml) | Posaconazole %R (ECV µg/ml) |
A. fumigatus | 717 | 0.3 | EUCAST CLSI | NA 20.0 (0.06) | 0.6 (1) 0.6 (1) | 0.3 (1) 0.3 (1) | 0.3 (0.25) 0.3 (0.5) |
A. terreus | 151 | 88.7 | EUCAST CLSI | NA 1.0 (0.06) | NA 0.7 (1) | NA 0.7 (1) | NA 0.0 (0.5) |
A. flavus | 72 | 2.8 | EUCAST CLSI | NA 13.0 (0.06) | NA 0.0 (1) | NA 1.4 (1) | NA 0.0 (0.5) |
A. niger | 53 | 0.0 | EUCAST CLSI | NA 8.0 (0.06) | NA 5.7 (2) | NA 12.0 (2) | NA 1.9 (0.5) |
Lichtheimia spp.** | 25 | 4.0 | n.t. | n.t. | 16.0 | 8.0 | |
Mucor spp.** | 9 | 44.4 | n.t. | n.t. | 44.4 | 44.4 | |
Rhizopus spp.** | 6 | 33.3 | n.t. | n.t. | 33.3 | 33.3 | |
Rhizomucor spp.** | 7 | 28.6 | n.t. | n.t. | 57.1 | 28.6 |
NA: data not available; n.t.: not tested; *no ECVs available; applied breakpoint ≥2 µg/ml; **applied breakpoints (µg/ml): AMB ≥2, ITC >2, POS >2.
A. terreus is an unusually frequent cause of infection in Tyrol. It is well known for its inherent resistance to AMB, which was as high as 88.7% of all isolates included in this study. All other tested antifungal agents showed excellent efficacy with rates of non-WT isolates ranging from 0.0% (POS) to 1.0% (CPF).
A. flavus was fairly susceptible to AMB, but highly susceptible to all tested azoles. In comparison, CPF seem to be ineffective against 13% of the isolates (see discussion). A. niger was found to be fully susceptible to AMB. Among the azoles, POS was most efficient (1.9% R), followed by VRC and ITC (5.7% and 12.0% R, respectively) (Table 5).
Zygomycetes. As echinocandins and VRC are known to be ineffective against zygomycetes, they were not tested. A high susceptibility rate to AMB was only noted with Lichtheimia (formerly called Absidia) spp. (4% R) among the clinical isolates collected in Tyrol, while the rates of non-susceptible isolates of Mucor spp., Rhizopus spp., and Rhizomucor spp. were 44.4%, 33.3%, and 28.6%, respectively. Similarly, Lichtheimia spp. were rather susceptible to ITC and POS (16.0% and 8.0% R, respectively), whereas high proportions of Mucor spp., Rhizopus spp., and Rhizomucor spp. were not inhibited by the azoles (Table 5). However, it must be remembered that these findings are based upon only a small number of isolates and must be confirmed in future studies.
Discussion
In this study, we examined the in vitro susceptibility of the most important pathogenic Candida species and moulds in Tyrol, Austria. The reasons for applying epidemiological cutoff values were twofold. First, the recommended AST methods differ between EUCAST and CLSI, and clinical breakpoints (CBPs) for several antifungal drugs are not available or being revised by EUCAST and CLSI. Consequently, it is difficult to employ CBPs in the present studies. In addition, CBPs do not appear to be suitable to distinguish wild-type isolates from those that possibly have acquired resistance and may even classify such isolates as being susceptible, as seen for the CLSI CBPs of Candida spp. to echinocandins [11,12]. Breakpoints for susceptibility testing should not divide wild-type distributions of important target species [13]. ECVs appear to be more reliable to detect putative trends in susceptibility to antifungal agents, as they are determined by statistical methods that are based on the respective WT MIC distributions and include inherent variability of AST methods [10,11,13,14]. In this study, we wanted to underline the benefits of previous agreements between EUCAST and CLSI, as well as the need to further harmonize AST standards and clinical breakpoints in the future.
The species distribution among the collected Candida isolates in Tyrol is quite average in comparison to a big global survey (197,619 isolates) [15], but the ratio of C. glabrata is considerably higher and that of C. parapsilosis lower.
Due to its high nephrotoxicity, non-lipid formulated amphotericin B (AMB) is mainly used today as a drug of last resort. Furthermore, ECVs for this antifungal agent have not been published. Nevertheless, applying a breakpoint of MIC ≥2 µg/ml, our results indicated that this agent is still highly effective against Candida species. Previous and current international studies have indicated that resistance of Candida spp. to AMB to be rare and mainly restricted to C. parapsilosis, C. krusei and C. lusitaniae [16–18]. Compared to these studies, C. parapsilosis and C. krusei recovered in Tyrol are highly susceptible (Table 1), while there were several resistant isolates among the small number of tested C. lusitaniae (data not shown).
Using CLSI ECVs, our results showed an increasing percentage of non-WT isolates with possibly acquired resistance to caspofungin with respect to the five most important Candida species (results, Table 2). While several previous studies could not find striking evidence for increasing resistance by using the CLSI clinical breakpoints [18–20], Pfaller et al. demonstrated that CLSI CBPs for echinocandins (>2 µg/ml for caspofungin, micafungin and anidulafungin) are clearly not sensitive enough to determine trends in resistance and may classify isolates with acquired resistance mutations as susceptible [11].
The use of the EUCAST and CLSI ECVs indicated a consistent increase of possibly resistant non-WT isolates (Tables 4 and 5) to fluconazole and voriconazole. Our results for fluconazole and C. glabrata matched those from global surveillance programs that applied CBPs and observed high rates of non-susceptible isolates of between 13% and 22.8% [15,21]. These rates varied between geographical regions, with the average percentage from 2001 to 2007 of 16.3% in the European Union remaining relatively constant [21]. Our results detected a slight, but constant increase of non-WT isolates of C. albicans, C. tropicalis and C. parapsilosis. As expected, the rates of non-susceptible isolates of C. krusei were high as this species is inherently resistant to FLC [22].
Similar to the findings relative to FLC, C. glabrata showed the most distinct tendency to emerging resistance to VRC. These results are in line with previous local and global antifungal surveillance studies that indicated high rates of non-susceptible isolates to azoles [21,23]. However, a general trend towards increased proportions of non-WT isolates was also noted with C. albicans, C. tropicalis and C. parapsilosis. In comparison to global studies, the rate of susceptible isolates of C. krusei to VRC in Tyrol is remarkably low [15].
EUCAST and CLSI have published epidemiological cutoff values for Candida species to FLC and VRC. As they are very similar, application of these ECVs to our collection of Candida isolates revealed almost identical data that appear particularly suitable to examine trends in susceptibility to these azoles. These ECVs can be considered a good example for future harmonized determinations of ECVs for other pathogenic fungi and antifungal agents, as well as for standard methods of antimicrobial susceptibility testing. In addition, CBPs should be adjusted in the light of these new insights to avoid the division of wild-type distributions [13].
Although fungal infections caused by Candida species are still more common that those by filamentous fungi, the latter, most noticeable Aspergillus species and zygomycetes, are increasing [4,24,25]. As expected, A. fumigatus was the most frequently recovered pathogenic Aspergillus species. The proportion of A. terreus (15% of Aspergillus isolates) is above average, which may be considered as a regional exception in Tyrol. This species is known as an emerging AMB-resistant opportunistic pathogen [26]. Interestingly, an exceptionally high incidence of invasive aspergillosis caused by this species was previously described for the University Hospital of Innsbruck, Tyrol, and the M. D. Anderson Cancer Center in Houston, Texas [24,27]. The reasons for this phenomenon in these geographically and climatically very different regions are still unclear.
Applying a breakpoint of >2 µg/ml, most isolates of A. fumigatus, A. flavus and A. niger were susceptible to amphotericin B. Today, lipid formulations of AMB serve as alternatives for the treatment of patients whose infections are caused by isolates that are resistant to other antifungal agents. The well-known inherent resistance of A. terreus to AMB was also confirmed by this study.
For caspofungin, the use of CLSI ECVs resulted in the recognition of extraordinarily high rates of Aspergillus isolates that were not susceptible to this antifungal (Table 5). This fact can be explained by the different AST methods employed to evaluate the susceptibility of moulds to echinocandins. While Etests were used in this study to measure MICs of agents, the determination of CLSI ECVs was based on the wild-type distribution of MECs [28]. In comparison, applying a breakpoint of MIC ≥2 µg/ml indicated full susceptibility of A. terreus and A. niger to CPF, as well as a high proportion of susceptible isolates of A. fumigatus and A. flavus (92.8% and 98.6%, respectively). A harmonization of methods for AST by EUCAST and CLSI, as well as the development of clinical breakpoints are still outstanding.
With the exception of A. niger, the tested triazole antifungal agents were shown to be highly effective against the most important Aspergillus species in Tyrol. Previous studies indicated growing evidence of an increasing frequency of azole resistance in A. fumigatus [29,30], but this could not be confirmed by our findings. EUCAST and CLSI have published similar ECVs for A. fumigatus to VRC, ITC, and POS, which resulted in identical rates of susceptible isolates. EUCAST has not yet published ECVs for A. terreus, A. flavus, and A. niger. Due to increasing relevance, several studies have recently examined the epidemiology, resistance and genetic diversity of A. terreus, while very little is known about the resistance mechanisms of A. flavus and A. niger [7,31].
Infections caused by zygomycetes are an emerging threat that is exacerbated by high resistance rates of these fungi to antifungal drugs. Previous studies showed echinocandins and VRC to be ineffective against zygomycetes. AMB and POS are currently considered the most potent weapons against zygomycetes [6,17,32–34].
Again, we would like to note that it is inevitable for EUCAST and CLSI to continue their efforts for the determination of shareable and universally valid epidemiological cutoff values and clinical breakpoints, as well as to harmonize methods for antimicrobial susceptibility testing in order to challenge the increasing threat by pathogenic fungi.
Declaration of interest: This study was carried out without any funding by commercial or non-commercial institutions. Cornelia Lass-Flörl received unrestricted grants from Pfizer, Gilead and Merck as well as speakers honoraria from Pfizer and Gilead. All other authors declare the absence of dual or conflicting interests.
References
This paper was first published online on Early Online on 27 May 2011.