Antifungal susceptibility patterns of yeast isolates causing bloodstream infections

Francesco Barchiesi1,*, Giuseppina Caggiano2, Monia Maracci1, Daniela Arzeni1, Giorgio Scalise1 and Maria Teresa Montagna2

1 Istituto di Malattie Infettive e Medicina Pubblica, Università degli Studi di Ancona, Azienda Ospedaliera Umberto I°, Via Conca 60020, Torrette di Ancona, Ancona; 2 Dipartimento di Medicina Interna e Medicina Pubblica, Sezione Igiene, Università degli Studi di Bari, Italy

Received 29 July 2002; returned 6 October 2002; revised 31 October 2002; accepted 4 November 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A broth microdilution method following the NCCLS recommendations was used for testing fluconazole, itraconazole, posaconazole, 5-fluorocytosine and amphotericin B against 83 yeast isolates causing bloodstream infections in 59 patients hospitalized between January 1998 and June 2001 at the University Hospital of Bari, Italy. Isolates belonged to four species of Candida and three other yeast genera. Of the isolates, 97%, 95% and 100% were susceptible to fluconazole, itraconazole and posaconazole, respectively. Similarly, 97% and 100% of the isolates were susceptible to 5-fluorocytosine and amphotericin B, respectively.

Keywords: antifungal susceptibility, yeast isolates


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobial resistance is an important concern with all major groups of pathogenic microorganisms, including fungi.1 The real extent of the resistance problem among fungi causing bloodstream infections is unknown.

In this study, we analysed the antifungal susceptibility profiles of yeast isolates causing bloodstream infections in patients hospitalized between January 1998 and June 2001 at the University Hospital of Bari, Italy. The antifungal activities of three triazoles (fluconazole, itraconazole and posaconazole), 5-fluorocytosine and amphotericin B were investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Sources of isolates

A total of 83 clinical yeast isolates were used in this study. The strains were isolated from the blood of 59 patients hospitalized in six departments of the University Hospital of Bari, Italy, from January 1998 to June 2001. The departments included: the Intensive Care Unit [number of patients (n) = 19], Oncology Paediatric Unit (n = 16), Surgery (n = 11), Neonatology (n = 8), Transplant Unit (n = 3) and Haematology (n = 2). Sixty of 83 isolates represented the first isolation strain (one patient had a mixed fungaemia). They included: 28 strains of Candida albicans, 23 strains of Candida parapsilosis, three strains of Candida tropicalis, two strains each of Candida glabrata and Trichosporon capitatum and one strain each of Saccharomyces cerevisiae and Hansenula anomala. There were 23 patients for whom fungaemia, sustained by the same yeast species of the first isolation, persisted despite antifungal therapy. Therefore, an additional 23 isolates (11 C. albicans, 10 C. parapsilosis, one C. tropicalis and one T. capitatum) were available for antifungal susceptibility testing. Intervals between the first and last isolation ranged from 6 to 27 days. Yeast isolates were identified at the species level by conventional morphological and biochemical methods, and were stored at room temperature. Before the initiation of the study, yeast isolates were subcultured on antimicrobial agent-free medium to ensure viability and purity. C. albicans ATCC 90029 and Candida krusei ATCC 6258 were used as quality controls and tested in each run of the experiments.2,3

Antifungal agents

Standard powders of fluconazole (Pfizer, Inc., New York, NY, USA), itraconazole (Janssen, Beerse, Belgium) and posaconazole (Schering-Plough Research Institute, Kenilworth, NJ, USA) were obtained from their respective manufacturers. 5-Fluorocytosine and amphotericin B were purchased from Sigma (Milan, Italy). Serial two-fold dilutions were prepared as recommended by the NCCLS.3 Final dilutions were made in RPMI 1640 medium (Sigma) buffered to pH 7.0 with 0.165 M MOPS buffer (Sigma). Following incubation at 35°C for 48 h, the MICs of triazoles and 5-fluorocytosine were determined as the lowest concentration at which a prominent decrease in turbidity relative to the growth control well was observed. Amphotericin B MIC was determined as the lowest concentration at which no growth was detectable.3

Definitions

Interpretative criteria for susceptibility to fluconazole, itraconazole and 5-fluorocytosine were those published by Rex et al.4 and by the NCCLS.3 According to that previously reported by Nguyen et al.5 and Pfaller et al.,1 we selected a breakpoint of >1.0 mg/L to define isolates as amphotericin B resistant. Owing to preliminary pharmacokinetic data, and according to that reported by Pfaller et al.,1 we selected a breakpoint of 1 mg/L to define isolates as posaconazole resistant.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antifungal susceptibilities of the two ATCC strains were within the ranges reported previously for all drugs.2 The MICs of the five antifungal agents against 60 yeast isolates representing the first isolation strains are shown in Table 1. 97% of the isolates were susceptible to fluconazole. Only two strains of T. capitatum showed an MIC of 32 mg/L. Similarly, 95% of the isolates were susceptible to itraconazole. There were three strains for which the itraconazole MIC was >=0.25 mg/L: two strains of C. glabrata (0.25 and 0.5 mg/L) and one strain of H. anomala (1.0 mg/L). 100% of the isolates were susceptible to posaconazole and amphotericin B. 98% of the isolates were susceptible to 5-fluorocytosine. There were 23 patients for whom fungaemia, sustained by the same yeast species of the first isolation, persisted despite antifungal therapy. Therefore, an additional 23 isolates were available for antifungal susceptibility testing. Twenty patients were receiving liposomal amphotericin B at 3 mg/kg/day, whereas three patients were receiving fluconazole at 400 mg/day. Antifungal susceptibility results of these isolates were identical or similar (within one dilution) to those reported for the first isolation strains against all drugs (data not shown).


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Table 1.  In vitro activities of five antifungal agents against 60 yeast isolates representing the first isolation strains in 59 patients considered in this study.a
 
Although this antifungal susceptibility survey has been conducted in a limited geographical area, the present data confirmed that the majority of yeast isolates causing bloodstream infections are susceptible in vitro to the antifungal agents most commonly used in clinical practice. Our results agree with those reported recently by Pfaller et al.1 Of note, all strains tested in our study were susceptible to the new investigational triazole, posaconazole. It must be noted that in our analysis there was a high prevalence of C. albicans and C. parapsilosis, and that other species of Candida, characterized by a low susceptibility profile to the triazoles, such as C. glabrata or C. krusei, accounted for few isolates, or they were absent. This fact underlines epidemiological differences in yeast species occurrence among countries. Importantly, we found that isolates causing episodes of breakthrough fungaemia in 23 patients retained the same susceptibility profile as those causing the first episode of fungaemia. The latter finding leads to the following inferences. First, it seems from these data that MIC determination has no evident clinical relevance, at least in this type of fungal infection. Since the majority of patients were infected with isolates that were susceptible in vitro to all antifungal agents, it is hard to understand why there were so many breakthrough infections. One can hypothesize that host factors were more important than susceptibility test results in determining clinical outcome in such patients. Secondly, the majority of patients with breakthrough infections were undergoing amphotericin B therapy. It has been reported that NCCLS methodology does not represent a suitable technique for detection of amphotericin B resistance.3 Therefore, even though a significant variation of amphotericin B MIC could have occurred between isolates taken before and during treatment, this procedure might not be sensitive enough to detect it.

In conclusion, we confirmed that up to 95% of yeast isolates causing bloodstream infections are susceptible in vitro to antifungal agents. Clearly, prospective MIC-directed clinical trials are warranted to further elucidate the potential predictive utility of antifungal susceptibility testing in this setting.


    Acknowledgements
 
This work was supported in part by a grant from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (cofin 2001) and by a grant from Istituto Superiore di Sanità (III AIDS project, contract no. 50C.29), Rome, Italy.


    Footnotes
 
* Corresponding author. Tel: +39-071-5963467; Fax: +39-071-5963468; E-mail: cmalinf{at}popcsi.unian.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Pfaller, M. A., Diekema, D. J., Jones, R. N., Sader, H. S., Fluit, A. C., Hollis, R. J. et al. (2001). International surveillance of bloodstream infections due to Candida species: frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole, voriconazole of isolates collected from 1997 through 1999 in the SENTRY antimicrobial surveillance program. Journal of Clinical Microbiology 39, 3254–9.[Abstract/Free Full Text]

2 . Barry, A. L., Pfaller, M. A., Brown, S. D., Espinel-Ingroff, A., Ghannoum, M. A., Knapp, C. et al. (2000). Quality control limits for broth microdilution susceptibility tests of 10 antifungal agents. Journal of Clinical Microbiology 38, 3457–9.[Abstract/Free Full Text]

3 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA, USA.

4 . Rex, J. H., Pfaller, M. A., Galgiani, J. N., Bartlett, M. S., Espinel-Ingroff, A., Ghannoum, M. A. et al. (1997). Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitroin vivo correlation data for fluconazole, itraconazole, and Candida infections. Clinical Infectious Diseases 24, 235–47.[ISI][Medline]

5 . Nguyen, M. H., Clancy, C. J., Yu, V. L., Yu, Y. C., Morris, A. J., Snydman, D. R. et al. (1998). Do in vitro susceptibility data predict the microbiological response to amphotericin B? Results of a prospective study of patients with Candida fungemia. Journal of Infectious Diseases 177, 425–30.[ISI][Medline]





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