a Department of Medicine, Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Box 041, Tufts University School of Medicine, 750 Washington Street, Boston, MA 02111, USA; b The Infectious Diseases Unit, Hospital Clinic of Barcelona, Barcelona, Spain
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Abstract |
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Introduction |
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We have developed a semi-solid agar antifungal susceptibility screening (SAAS) test that has performed well for laboratory yeast strains.5 The aim of this study was to determine whether preliminary screening of a yeast isolate from a clinical specimen using this test, sometimes even before species identification, could assist in real-time management of IFIs.
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Materials and methods |
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Patients were hospitalized at New England Medical Center over a 2 month period and diagnosed with IFI by blood cultures taken for evaluation of fever (seven patients), or by vitreal aspiration for evaluation of acute vision loss (one patient).
Definitions
Definitions used were: renal failure = estimated creatinine clearance <50 mL/min; neutropenia = absolute neutrophil count <500 cells/mm3 within 1 month of IFI; successful clinical outcome = resolution of symptoms (fever, eye pain and loss of visual acuity) on therapy; successful microbiological outcome = clearance of fungaemia on therapy without evidence of microbiological relapse.
Microbiology
The yeasts were subcultured onto corn meal and molybdate agar for rapid identification of Candida albicans by morphology and colour, respectively. Simultaneous subculture of the isolate onto a Sabouraud's dextrose agar plate was performed for SAAS testing. If necessary, final identification was performed with the Vitek and API 20C methods (bioMérieux, Marcy l'Étoile, France) in the clinical microbiology laboratory.
Antifungal susceptibility screening
The semi-solid heart infusion medium was prepared as described previously.5 In brief, heart infusion broth (Difco Laboratories, Detroit, MI, USA) was mixed with agar (0.5%) (Bacto Agar; Difco Laboratories) and pharmacy stock amphotericin B and fluconazole in concentrations reflecting achievable serum levels and/or the MIC of the drug. Concentrations of amphotericin B tested were 0.5 and 2 mg/L, and of fluconazole were 1, 8 and 40 mg/L. The drug mixtures and drug-free controls were stored in small lots in sterile, capped tubes. A just-turbid suspension (c. 0.5 McFarland standard) of bits of three to five single colonies from an overnight culture on Sabouraud's dextrose agar was prepared and a standard loopful was inoculated vertically into the semi-solid agar media containing drugs and drug-free control. The tubes were incubated at 35°C for 48 h. Susceptibility was visually assessed by complete inhibition of growth in amphotericin B-containing tubes and at least 75% reduction in growth in fluconazole tubes when compared with controls. Candida parapsilosis American Type Culture Collection (ATCC) 22019 was used as a quality control (QC) for each test by determining MICs within 1 log2 dilution of the NCCLS proposed QC range. No test results fell outside 1 log2 of the proposed range (14 mg/L).
The yeast isolates were each tested singly by the SAAS method after isolation in the clinical microbiology laboratory, usually within 8 days of isolation. Later, comparison of MICs obtained by two-fold dilutions of amphotericin B (0.0316 mg/L) and fluconazole (0.12564 mg/L, including 40 mg/L as in the SAAS test) was made between the NCCLS M27-A broth microdilution and SAAS methods.6
Antifungal treatment
All patients were treated in accordance with recommendations made by the infectious disease service and the primary team. When results of the SAAS test showed resistance to a drug, antifungal treatment was altered toward the active agent in accordance with the patient's clinical course.
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Results |
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Antifungal susceptibility results comparing the NCCLS broth microdilution and the SAAS methods are presented in Table 2. The SAAS test results compared favourably to those of the NCCLS reference method. In all instances when the SAAS test predicted fluconazole resistance (MIC > 40 mg/L), the NCCLS MIC result was
64 mg/L. Fluconazole MICs for the C. glabrata isolate were 32 and 40 mg/L by the NCCLS and SAAS test methods, respectively.
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Discussion |
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The accuracy of a new method of susceptibility testing may be assessed by comparing results with a standardized reference method. In this study, the MIC results achieved by the SAAS test compared favourably to those obtained by the NCCLS reference method. The high number of Candida spp. (five of six) resistant to fluconazole by both methods contrasts with recent reports of decreasing fluconazole resistance in blood isolates.1 Our results can-not be explained by prior fluconazole exposure in these patients and may reflect either the fact that most of the infections were non-albicans yeasts or the possibility of prior colonization with resistant organisms before the invasive illness. Nevertheless, all fluconazole-resistant organisms were accurately predicted by the screening test and later confirmed by the full SAAS and NCCLS MIC testing.
Ideally, susceptibility test results should correlate with clinical success of treatment with the active antifungal agent. Most experts agree that MICs achieved by the reference or other methods that predict antifungal resistance correlate best with clinical outcomes.9,10 To prove that susceptibility findings successfully influence patient management would require a much larger study of clinical failures successfully treated by guidance of screening susceptibility test results. In this small study of mostly Candida non-albicans infections in severely immunocompromised hosts, only one patient demonstrated clinical failure that was predicted by the screening test results. However, in the two patients with C. albicans infections, fluconazole would have been an optimal choice to avoid toxicities and increased costs, yet resistance was documented by both the screening test and NCCLS results. Early susceptibility screening may have contributed to their successful clinical and microbiological outcomes.
Our study is limited by the small number of patients managed in accordance with their clinical course and SAAS test results. Thus, generalization of the utility of the SAAS test to a larger population of patients with IFIs should be carried out with caution. Our patients' response to treatment may have simply reflected our use of amphotericin as the primary treatment. Additional limitations include that, while easy to perform, and reproducible in our hands, the SAAS test has not been validated in a multi-laboratory study and is subject to the inherent problems of subjective reading of results. Its use of a chemically undefined media and pharmacy stock antifungal agents, while rendering the procedure amenable to a busy clinical microbiology laboratory, precludes this test substituting for formal MIC determinations by the reference standard.
The simplicity of the SAAS test, the reproducibility of results demonstrated in laboratory yeast strains and the correlation with NCCLS MIC results shown here are exciting qualities of the test. For the eight patients studied here, we believe the test provided important early information that aided in their successful management. Further studies to assess the SAAS test's accuracy with currently available and new antifungal agents and its reproducibility in multiple laboratories are warranted.
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Acknowledgements |
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Notes |
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References |
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2 . Abi-Said, D., Anaissie, E., Uzun, O., Raad, I., Pinzcowski, H. & Vartivarian, S. (1997). The epidemiology of hematogenous candidiasis caused by different Candida species. Clinical Infectious Diseases 24, 11228.[ISI][Medline]
3 . Nguyen, M. H., Peacock, J. E., Jr, Morris, A. J., Tanner, D. C., Nguyen, M. L., Snydman, D. R. et al. (1996). The changing face of candidemia: Emergence of non-Candida albicans species and antifungal resistance. American Journal of Medicine 100, 61723.[ISI][Medline]
4 . Pfaller, M. A., Jones, R. N., Messer, S. A., Edmond, M. B., Wenzel, R. P. & the SCOPE Participant Group. (1998). National surveillance of nosocomial blood stream infection due to species of Candida other than Candida albicans: Frequency of occurrence and antifungal susceptibility in the SCOPE Program. Diagnostic Microbiology and Infectious Disease 30, 1219.[ISI][Medline]
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Provine, H. & Hadley, S. (2000). Preliminary evaluation of a semisolid agar antifungal susceptibility test for yeasts and molds. Journal of Clinical Microbiology 38, 53741.
6 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.
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Kirkpatrick, W. R., Turner, T. M., Fothergill, A. W., McCarthy, D. I., Redding, S. W., Rinaldi, M. G. et al. (1998). Fluconazole disk diffusion susceptibility testing of Candida species. Journal of Clinical Microbiology 36, 342932.
8 . Patterson, T. F., Revankar, S. G., Kirkpatrick, W. R., Dib, O., Fothergill, A. W., Redding, S. W. et al. (1996). Simple method for detecting fluconazole-resistant yeasts with chromogenic agar. Journal of Clinical Microbiology 34, 17947.[Abstract]
9 . Ghannoum, M. A., Rex, J. H. & Galgiani, J. N. (1996). Susceptibility testing of fungi: Current status of correlation of in vitro data with clinical outcome. Journal of Clinical Microbiology 34, 48995.[Abstract]
10 . Sheehan, D. F., Espinel-Ingroff, A., Moore, L. S. & Webb, C. D. (1993). Antifungal susceptibility testing of yeasts: A brief overview. Clinical Infectious Diseases 17, Suppl. 2, S494500.[ISI][Medline]
Received 18 April 2001; returned 6 October 2001; revised 12 November 2001; accepted 20 November 2001