Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
Keywords: minimal fungicidal concentration , Etest , Fungitest
Sir,
Candida inconspicua, similarly to Candida krusei, possesses primary decreased susceptibility to fluconazole.1 During the past few years, the frequency of isolation of C. inconspicua has increased in our laboratory.2 Optimal treatment of C. inconspicua infections is not established, but in the reported cases amphotericin B was generally found effective.1
The aim of our study was to determine the MICs and minimal fungicidal concentrations (MFCs) of amphotericin B and flucytosine against clinical isolates of C. inconspicua.
We isolated 57 C. inconspicua strains from 42 inpatients (in 10 different clinics) and six outpatients during a 3 year period (20012003). Only one isolate per patient was used in the study. Twenty-two inpatients were immunocompromised and 15 were hospitalized in seven intensive care units. The majority of specimens were upper and lower respiratory tract samples (16 and 24, respectively), but wound, blood and genital isolates were also obtained. No patients received amphotericin B or flucytosine before isolation of C. inconspicua. Strains were identified using rDNA RFLP.2
For susceptibility testing we used the standard broth microdilution (BMD) method3 and Fungitest. For screening amphotericin B resistance we used Etest. The reference BMD was performed as specified by NCCLS document M27-A2.3 For interpretation of flucytosine susceptibility we used the NCCLS criteria. For amphotericin B we regarded isolates with MICs of 1 mg/L as resistant.4
Etest (AB Biodisk) and Fungitest (Bio-Rad SDP) were performed according to the manufacturers' instructions. Etest MICs were recorded at 24 and 48 h. Fungitest was read after 48 h. The breakpoints used were 2 and 8 mg/L for amphotericin B and 2 and 32 mg/L for flucytosine.
The percentage of agreement was calculated using discrepancies of no more than ± one dilution. Categorical agreement was defined comparing the results to the reference BMD category read at 48 h.
MFC was determined as described by Cantón et al.5 using an 104 cfu/mL yeast inoculum, otherwise following the NCCLS guidelines. The starting cfu number was confirmed by serial 10-fold dilutions and plating 10 µL in duplicates onto Sabouraud agar plates. After 48 h the content of each well containing drug concentrations above the MIC was transferred onto drug-free Sabouraud agar plates. Plates were incubated at 35°C for 48 h. MFC was defined as the lowest drug concentration that killed 99.9% of the starting inoculum.
Results are summarized in Table 1. The majority of isolates were resistant (27/48, 56.3%) to amphotericin B. Overall agreement between the reference method and Etest read at 24 and 48 h was acceptable (76.9 and 87.5%, respectively), but categorical agreement was markedly poorer (41.7 and 45.8%, respectively). Using Fungitest all isolates proved to be susceptible (data not shown), thus all resistant strains were misdiagnosed as susceptible. This is due to higher breakpoints used by Fungitest (2 and 8 versus 1 mg/L).
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Flucytosine showed good in vitro activity against C. inconspicua clinical isolates (100% susceptible, Table 1). Fungitest results (data not shown) due to the lower susceptible breakpoint (2 mg/L) strongly underestimate the number of susceptible strains (14.4 versus 100%); the majority of isolates showed intermediate susceptibility (85.6%) to flucytosine. These inadequate breakpoints and that of amphotericin B corrupt the utility of Fungitest in amphotericin B and flucytosine susceptibility testing.
In contrast to the narrow MIC range (12 mg/L), MFC values of flucytosine for C. inconspicua varied between 464 mg/L (geometric mean 38.7 mg/L). Fifty percent of our isolates showed tolerance to flucytosine (MFC 32 x MIC); however, MFC90 (64 mg/L) is lower than the safely achievable serum concentration (<100 mg/L).6
Nguyen et al.4 postulated that MFCs of amphotericin B read at 48 h could be clinically more relevant than MICs at 48 h. In our study, we have found relatively low MFC values against C. inconspicua, similar to Cantón et al.5 against C. krusei. Our MFC results are higher than the resistant breakpoint of amphotericin B suggested by Nguyen et al.,4 thus further investigation is needed to determine the role of amphotericin B in the treatment of infections caused by C. inconspicua.
To our knowledge this study is the first to determine the MFC of flucytosine against any Candida species. As flucytosine proved to possess good fungistatic activity in our study as well as by Pfaller et al.,6 it has a place in antifungal therapy, particularly in the light of the findings of Pfaller et al.,6 i.e. all Candida species with the exception of C. krusei are susceptible to flucytosine.
References
1. Baily GG, Moore CB, Essayag SM et al. Candida inconspicua, a fluconazole-resistant pathogen in patients infected with human immunodeficiency virus. Clin Infect Dis 1997; 25: 1613.[ISI][Medline]
2.
Majoros L, Kardos G, Belák Á et al. Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures. J Clin Microbiol 2003; 41: 52503.
3. National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A2. NCCLS, Wayne, PA, USA, 2002.
4. Nguyen MH, Clancy CJ, Yu VL et al. Do the in vitro susceptibility data predict the microbiologic response to amphotericin B? Results of a prospective study of patients with Candida fungemia. J Infect Dis 1998; 177: 42530.[ISI][Medline]
5. Cantón E, Pemán J, Viudes A et al. Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species. Diagn Microbiol Infect Dis 2003; 45: 2036.[CrossRef][ISI][Medline]
6.
Pfaller MA, Messer SA, Boyken L et al. In vitro activities of 5-fluorocytosine against 8,803 clinical isolates of Candida spp.: global assessment of primary resistance using National Committee for Clinical Laboratory Standards susceptibility testing methods. Antimicrob Agents Chemother 2002; 46: 351821.
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