1 Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen; 2 Faculty of Health Sciences, Medical and Health Science Center, University of Debrecen; 3 Department of Microbiology and Biotechnology, Faculty of Food Science, Corvinus University of Budapest, Budapest, Hungary
Keywords: C. inconspicua , Etest , Fungitest , broth microdilution , NCCLS
Sir,
During the past 4 years, isolation of Candida inconspicua from clinical samples has increased in our laboratory.1 Fluconazole MIC values for C. inconspicua isolates are relatively high, as determined by standard broth microdilution (BMD), Etest or Fungitest,24 and according to the standard BMD method they fall mainly into the susceptible-dose dependent (S-DD) or resistant (R) categories, with few susceptible (S) isolates.13
Since fluconazole is the most widely used drug in the clinical setting, and in the case of rare Candida species susceptibility test results may precede definitive identification (24 or 48 h versus 48 or 72 h), the method used for susceptibility testing should be suitable to correctly determine the fluconazole susceptibility not only of the four most frequent species, but also of relatively frequently isolated rare species.
The aim of our study was to evaluate Etest, Fungitest and modified BMD methods as alternatives for determination of fluconazole susceptibility of our C. inconspicua clinical isolates and to compare their agreement with the standard method.
The isolates studied included 57 C. inconspicua strains isolated from 48 patients during a 3 year period (20012003). Twenty-two of the 42 hospitalized patients were immunocompromised and 15 were hospitalized in seven different intensive care units. Eleven patients received fluconazole previously. The majority of specimens were from the upper and lower respiratory tract (16 and 24, respectively), but wound, blood and genital isolates were also obtained. Inpatients were treated at 10 separate clinics at different times.
Identification of the isolates was carried out as described earlier.1 The reference BMD method was carried out according to the guidelines of the NCCLS document M27-A2.5 The pure fluconazole powder (Pfizer) was dissolved in sterile distilled water, and the final concentration range was 0.25128 mg/L. Yeast suspensions were prepared in RPMI 1640 medium and were adjusted to a final concentration of 103 cells/mL (standard inoculum size), or in the modified BMD method 104 cells/mL (large inoculum size). Drug-free purity controls and growth controls were included in each plate. The plates were incubated at 35°C and read visually after 24 and 48 h. Fluconazole MIC was defined as the lowest concentration that produced a prominent decrease in turbidity compared with that of the drug-free growth control.5 Quality control strains of Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were included in each test.
Etest (AB Biodisk, Solna, Sweden) and Fungitest (Bio-Rad SDP, formerly Sanofi Diagnostics Pasteur, France) methods were carried out according to the manufacturers' instructions. Etest MICs were recorded at 24 and 48 h. Fungitest was read after 48 h. Isolates were classified as S, S-DD or R according to the NCCLS guidelines.5
Fluconazole MICs obtained with the modified BMD tests and Etest read at 24 and 48 h were compared with the reference BMD MICs read at 48 h. Discrepancies between MICs of no more than ±1 dilution were used to calculate the percentage of agreement. Categorical agreement was defined when the applied method gave the same category result compared with the reference BMD category read at 48 h.
Results are summarized in Table 1. In vitro susceptibility test results demonstrated relatively high fluconazole MICs for C. inconspicua clinical isolates by all applied methods. Compared with the standard method, overall agreement of MICs obtained with the studied methods was excellent with the exception of the Etest read at 48 h. After 24 h of incubation, categorical agreement of the normal and large inoculum BMD method was very good, but out of five R strains three and two, respectively, were misdiagnosed as S-DD. Using a large inoculum BMD method read at 48 h and Etest read at 24 and 48 h, poor categorical agreements were observed, though all R isolates were diagnosed correctly. The Fungitest gave good categorical agreement compared with the standard method, but four of five R isolates were misdiagnosed as S-DD.
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Correct identification is a major step to adequate drug choice, mainly in the case of rare Candida species as we demonstrated in our previous study of C. inconspicua.1 However, if a clinician urgently needs fluconazole susceptibility results, before the correct identification becomes available, Etest read at 24 h safely detects the decreased fluconazole susceptibility of C. inconspicua. In a proven life-threatening C. inconspicua infection, the empirical fluconazole therapy should, of course, be switched to amphotericin B.6 Though the efficacy of caspofungin and voriconazole has not been tested in a systematic study, they seem to be reasonable alternatives.
References
1
.
Majoros, L., Kardos, G., Belák, Á. et al. (2003). Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures. Journal of Clinical Microbiology 41, 52503.
2
.
Barchiesi, F., Tortorano, A. M., Di Francesco, F. M. et al. (1999). In vitro activity of five antifungal agents against uncommon clinical isolates of Candida species. Journal of Antimicrobial Chemotherapy 43, 2959.
3
.
Pfaller, M. A., Diekema, D. J., Messer, S. A. et al. (2003). In vitro activities of voriconazole, posaconazole, and four licensed systemic antifungal agents against Candida species infrequently isolated from blood. Journal of Clinical Microbiology 41, 7883.
4 . Swoboda-Kopec, E., Kawecki, D., Wroblewska, M. et al. (2003). Epidemiology and susceptibility to antifungal agents of fungi isolated from clinical specimens from patients hospitalized in the Department of General and Liver Surgery of the Medical University of Warsaw. Transplantation Proceedings 35, 2298303.[CrossRef][ISI][Medline]
5 . National Committee for Clinical Laboratory Standards. (2002). Reference Method for Broth Dilution Testing of YeastsSecond Edition: Approved Standard M27-A2. NCCLS, Villanova, PA, USA.
6 . Baily, G. G., Moore, C. B., Essayag, S. M. et al. (1997). Candida inconspicua, a fluconazole-resistant pathogen in patients infected with human immunodeficiency virus. Clinical Infectious Diseases 25, 1613.[ISI][Medline]