JAC Advance Access originally published online on February 9, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):767-771; doi:10.1093/jac/dkl555
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In vitro susceptibility of Candida species to five antifungal agents in a German university hospital assessed by the reference broth microdilution method and Etest
Institute of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Germany
* Corresponding author. Tel: +49-621-383-2695; Fax: +49-621-383-3816; E-mail: ralf.fleck{at}imh.ma.uni-heidelberg.de
Received 1 September 2006; returned 14 October 2006; revised 23 November 2006; accepted 21 December 2006
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Abstract |
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Objectives: The aim of this study is to evaluate the susceptibilities of Candida spp. to the common antifungal agents in a German university hospital. Since quick results of in vitro testing are desirable, Etest and the CLSI broth microdilution (BMD) method (reference method) were compared, focusing on the validity of early readings.
Methods: A total of 512 Candida spp. isolates, including 174 from primarily sterile sites, were collected in the clinical routine. The yeasts were differentiated by CHROMagar and verified by API 20C AUX if necessary. In vitro susceptibilities to amphotericin B, flucytosine, fluconazole, voriconazole and caspofungin were determined using the BMD method described in the CLSI (formerly NCCLS) M27-A2 document and Etest. MICs were noted after 24 and 48 h of incubation.
Results: The most frequently isolated species was Candida albicans. Among the non-albicans species, Candida glabrata was the most prevalent, followed by Candida tropicalis, Candida parapsilosis and Candida krusei. MICs (mg/L) at which 90% of the strains were inhibited were 1 for amphotericin B, 32 for flucytosine, 8 for fluconazole, 0.25 for voriconazole and 1 for caspofungin. Susceptibility to fluconazole was 85.0% for C. glabrata and 5.3% for C. krusei, almost all other isolates were susceptible in over 90% except very rare species. The 48 h MIC values of Etest and BMD were in agreement (no more than 2 log2 dilutions) in 88.7% to 98.1% with categorical agreement rates of 91.6% to 98.2%, depending on the antifungal agent. Comparison of the 24 h MICs of both BMD and Etest with the 48 h MICs of the reference method showed categorical agreement in 94.9% to 99.2%. For caspofungin, however, a comparison of the categorical agreement was not possible due to the lack of interpretive breakpoints. The order of frequency and the resistance patterns of the isolates from primarily sterile sites and those of isolates from non-sterile sites did not differ.
Conclusions: No alarming resistances against the agents tested were found; however, owing to the relatively high frequency of C. glabrata with elevated fluconazole MICs, this species and, to a certain extent, C. krusei must be taken into consideration when choosing antifungal agents for calculated therapy. Etest is a reliable method for the susceptibility testing of Candida spp. and the 24 h readings of both Etest and BMD can serve as helpful preliminary results in most cases.
Keywords: caspofungin , voriconazole , fluconazole , flucytosine , amphotericin B
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Introduction |
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Candida spp. have been shown to be the fourth most common cause of nosocomial bloodstream infections1 and treatment failure due to drug resistance has been reported repeatedly.2 Furthermore, a shift from Candida albicans to non-albicans species has been noted, which may in part be explained by the overall higher fluconazole resistance rates among some of these species.3,4 The management of febrile patients at risk for fungal infections relies mainly on empirical therapy, which renders a good knowledge of the resistance situation indispensable. Mannheim University Hospital is a tertiary-level teaching hospital with 1400 beds, treating approximately 70 000 inpatients and 200 000 outpatients each year. Fluconazole is the most frequently used antifungal agent; however, especially in high-risk patients, newer agents are also in use. We carried out resistance testing by the CLSI (formerly NCCLS) reference broth microdilution (BMD) method M27-A25 and Etest. The aim of our study was 3-fold; first to get an overall idea of the resistance situation in this hospital that can be considered as being representative of hospitals of its size in Germany. Secondly, we evaluated the concordance of the 48 h MICs of the two testing methods. We thirdly assessed the validity of the 24 h MICs of both Etest and BMD in relation to the 48 h MICs of the reference method.
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Materials and methods |
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Study design
A total of 512 yeast isolates (including 174 from primarily sterile sites) recovered from 427 patients (some with various species) treated at Mannheim University Hospital were collected between 2004 and 2006. Primarily sterile sites include blood, intravasal catheter tips, abdominal cavity, CNS and pleural cavities. Owing to limited resources and in order to test a decent amount of non-albicans species, not all C. albicans from non-sterile sites were included in this study.
All fungal isolates were cultured on CHROMagar Candida (Becton Dickinson GmbH, Heidelberg, Germany) for identification and to exclude contaminants. If necessary, identification was verified using the API 20C AUX profile (BioMérieux Deutschland GmbH, Nürtingen, Germany).
Antifungal susceptibility testing was performed following both the reference BMD method (CLSI, document M27-A25) and Etest (AB Biodisk, Solna, Sweden). Wider discrepancies between the two methods as well as unexpectedly high or low MICs were verified by repeated testing and species verification with the API 20C AUX profile.
Reference BMD method. Standard antifungal powders of amphotericin B (Sigma Aldrich, product no. A4888), flucytosine (ICN Pharmaceuticals), fluconazole (Pfizer Pharmaceuticals Group), voriconazole (Pfizer Pharmaceuticals Group) and caspofungin (Merck & Co., Inc.) were obtained from the respective manufacturers and testing was performed following the CLSI5 guidelines. Quality control for each measurement was ensured by testing the five strains recommended by the CLSI with each batch of plates. Quality control limits were those established by Barry et al.6
Etest method. Etest strips (AB Biodisk) containing the antifungal agents mentioned earlier were obtained for comparative testing. Testing was performed on RPMI 1640 agar plates as recommended. Owing to limited availability of the Etest strips, only 157 isolates were tested against caspofungin.
Interpretation of results.
MICs for both methods were read after 24 and 48 h of incubation. The interpretive susceptibility criteria used for flucytosine, fluconazole and amphotericin B were those suggested by the CLSI.5 Voriconazole breakpoints were 
1 mg/L (susceptible) and 
4 mg/L (resistant).7 Owing to the lack of interpretive breakpoints for caspofungin, a categorical assignment was not possible.
Comparison of the two methods. The Etest MICs were rounded to the next higher concentration of BMD to simplify comparison and MIC values were considered in agreement when they were no more than 2 log2 dilutions apart. Minor errors were defined as susceptible or resistant by one method and susceptible-dose-dependent (S-DD) or intermediate by the other method. Major errors were defined as resistant according to Etest and susceptible according to BMD and very major errors were defined as susceptible according to Etest and resistant according to BMD. In order to assess the validity of the 24 h readings, Etest as well as BMD 24 h MICs were compared with the BMD 48 h MICs.
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Results and discussion |
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Isolates included 220 (43.0%) Candida albicans, 160 (31.3%) Candida glabrata, 60 (11.7%) Candida tropicalis, 29 (5.7%) Candida parapsilosis, 19 (3.7%) Candida krusei and 24 (4.6%) rare species with
5 isolates each (Candida lusitaniae, Candida famata, Candida kefyr, Candida sake, Candida inconspicua, Candida valida, Candida colliculosa, Candida utilis, Candida catenulata, Candida lipolytica, Candida membranaefaciens, Candida intermedia and Candida globosa). Antifungal susceptibilities are shown in Table 1. Species distribution among the 174 isolates from primarily sterile sites was 107 (61.5%) C. albicans, 42 (24.1%) C. glabrata, 9 (5.2%) C. tropicalis, 7 (4.0%) C. parapsilosis, 3 (1.7%) C. krusei and 6 (3.5%) rare species.
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Of all isolates, 96.3% were susceptible to amphotericin B with MICs of 1 mg/L or lower, according to BMD. Amphotericin B MICs of 2 mg/L were only found in C. glabrata (9 out of 160) and C. krusei (10 out of 19).
Resistance to flucytosine was identified in 12.3% of all isolates, the highest resistance rates were seen in C. krusei (84.2%) and C. tropicalis (58.3%).
Fluconazole resistance was seen in 2.5% (13 isolates), 90.4% were susceptible, leaving 7.1% categorized as S-DD. Five percent of all C. glabrata isolates were resistant, with another 10.0% being categorized as S-DD. This needs to be considered when C. glabrata is frequently isolated and fluconazole is a common choice for prevention and treatment of fungal infections. The highest fluconazole MICs were found among C. krusei with only 1 of the 19 isolates appearing susceptible and 3 being resistant; the remaining 15 isolates were categorized as S-DD.8 In contrast, all but one C. krusei isolate were categorized as resistant; with the Etest results. Since C. krusei is known to be intrinsically resistant to fluconazole, Etest seems to reflect the in vivo situation better than BMD.
There was no voriconazole resistance identified in our study, and MICs of 2.0 mg/L (S-DD) were only seen in five C. glabrata isolates.
The range of caspofungin MICs for all isolates was 0.031–4.0 mg/L; however, an MIC of 1.0 mg/L or lower was seen in 99.0% of all isolates. Higher MICs were only found in C. parapsilosis (3), C. globosa (1) and C. famata (1). This strongly indicates that caspofungin is highly effective against Candida spp.
The MIC50 and MIC90 values found for the 174 isolates from primarily sterile sites were the same as those found for the 338 isolates from other sites.
The Etest 48 h MICs were in agreement with the above results in 88.7% for amphotericin B, 97.9% for flucytosine, 97.5% for fluconazole, 96.3% for voriconazole and 98.1% for caspofungin. The categorical agreement rates of the respective MICs were somewhat different at 97.1% for amphotericin B, 97.1% for flucytosine, 91.6% for fluconazole and 98.2% for voriconazole.
Except for amphotericin B, Etest categorized more isolates as resistant than BMD. Fifteen isolates that were categorized as ‘likely resistant’ to amphotericin B by BMD were not detected by Etest (9 C. glabrata and 6 C. krusei). This seems to stand in contrast to previous observations,9 however, the higher numbers of C. glabrata and C. krusei isolates in this study might explain this discrepancy. In contrast, the MICs for these 15 isolates did not differ more than 2 log2 dilutions, except for one C. glabrata isolate. Nonetheless, it still remains unclear which of the two methods is better for the prediction of clinical outcome.10 With amphotericin B, we noted an MIC range of 0.25–2 mg/L with BMD and 0.031–2 mg/L with Etest, which led to some disagreement of MICs within the ‘susceptible’ group and explains the better categorical agreement as opposed to the agreement of the MICs (Table 2). In cases with borderline MICs of amphotericin B, an additional in vivo benefit with the use of lipid formulations of amphotericin B should be taken into consideration.
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MICs obtained with fluconazole were clustered around the breakpoints, which explains the slightly lower categorical agreement.
Nonetheless, these data strongly indicate that Etest is a reliable method for the susceptibility testing of yeasts.
The Etest 24 h MICs turned out to be equal or even superior to the Etest 48 h MICs with categorical agreement rates of 97.5% versus 97.1% for flucytosine, 94.9% versus 91.6% for fluconazole and 98.6% versus 98.2% for voriconazole and 96.3% versus 97.1% for amphotericin B. BMD 24 h MICs showed similar categorical agreement rates with the BMD 48 h MICs; for a comprehensive overview, refer to Table 2. Thus, especially in unambiguous cases with either low or high 24 h MICs, these values can very well serve as preliminary results for the clinician. The agreement rates of the MICs were also calculated and included in Table 2.
In conclusion, no alarming resistances among Candida spp. were found in our study; however, non-albicans species such as C. glabrata and C. krusei need to be considered in the clinical routine when choosing antifungal agents. With our results, we give further evidence that Etest is a reliable alternative to the reference method. Moreover, the 24 h readings of both Etest and BMD were found to be as reliable as the 48 h readings, thus allowing earlier optimization of antifungal therapy.
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Transparency declarations |
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R. F. has received funds for speaking at symposia organized on behalf of Pfizer. A. D. has received funds from Pfizer for the supervision of course participants and from MSD for photographic work. H. H. has received funds for speaking at symposia organized on behalf of Gilead, Valeant, MSD and Pfizer and is a member of the Gilead and MSD advisory boards.
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Acknowledgements |
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This study was supported by Gilead, Valeant, MSD and Pfizer.
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References |
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1 Pfaller MA, Jones RN, Doern GV, et al. (1998) International surveillance of bloodstream infections due to Candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the United States, Canada, and South America for the SENTRY Program. The SENTRY Participant Group. J Clin Microbiol 36:1886–9.
2 Kontoyiannis DP and Lewis RE. (2002) Antifungal drug resistance of pathogenic fungi. Lancet 359: pp. 1135–44.[CrossRef][ISI][Medline]
3 Wingard JR, Merz WG, Rinaldi MG, et al. (1991) Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. N Engl J Med 325:1274–7.[Abstract]
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Tortorano AM, Rigoni AL, Biraghi E, et al. (2003) The European Confederation of Medical Mycology (ECMM) survey of candidaemia in Italy: antifungal susceptibility patterns of 261 non-albicans Candida isolates from blood. J Antimicrob Chemother 52:679–82.
5 National Committee for Clinical Laboratory Standards. (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts–Second Edition: Approved Standard M27-A2(NCCLS, Wayne, PA, USA).
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Barry AL, Pfaller MA, Brown SD, et al. (2000) Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J Clin Microbiol 38:3457–9.
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Pfaller MA, Diekema DJ, Rex J, et al. (2006) Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints. J Clin Microbiol 44:819–26.
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Munoz P, Sanchez-Somolinos M, Alcala L, et al. (2005) Candida krusei fungaemia: antifungal susceptibility and clinical presentation of an uncommon entity during 15 years in a single general hospital. J Antimicrob Chemother 55:188–93.
9 Wanger A, Mills K, Nelson P, et al. (1995) Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates. Antimicrob Agents Chemother 39:2520–2.[Abstract]
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Park BJ, Arthington-Skaggs BA, Hajjeh RA, et al. (2006) Evaluation of amphotericin B interpretive breakpoints for Candida bloodstream isolates by correlation with therapeutic outcome. Antimicrob Agents Chemother 50:1287–92.
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