INTRODUCTION

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Fungal infections represent a significant cause of morbidity and mortality in immunocompromised patients. The majority of these infections are reported to be caused by Candida spp., which, at this moment, are the fourth most common group of isolates recovered from blood cultures in the United States (10). Rates of candidemia are also increasing substantially in Europe.

Although C. albicans continues to be the most common cause of invasive mycoses, in recent years non-C. albicans Candida species (e.g., C. parapsilosis, C. glabrata, C. tropicalis, or C. krusei) have become increasingly important causes of fungemia in hospitalized patients and account for more than 60% of Candida species isolated from blood in several institutions (5, 11).

Although amphotericin B has been the most effective antifungal agent against systemic infections, the introduction of new azole compounds (fluconazole [FZ], ketoconazole, itraconazole) with increased potency and safety has made long-term and low-toxicity treatments possible. However, the availability and increased use of these compounds may lead, in some cases, to the development of drug resistance. This situation requires the use of in vitro antifungal susceptibility tests when a deep-stated mycotic infection is present.

Since publication in 1995 and 1997 of standard guidelines (M27-A) for testing of the susceptibilities of yeasts to antifungal agents by both macro- and microdilution methods by the National Committee for Clinical Laboratory Standards (NCCLS) (12, 13), it is possible to study and compare the results of susceptibility tests reported in the literature.

We studied the susceptibilities to FZ of the Candida spp. isolated from blood in our hospital during 1995, 1996, and 1997 by the M27-A broth macrodilution method, a modified M27-A broth macrodilution method, and the agar diffusion method.

	MATERIALS AND METHODS

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Antifungal agent. A solution of 2 mg of FZ per ml in saline (FZ for intravenous infusion; Pfizer, Madrid, Spain) was used. Further dilutions were made in the assay medium. Neo-Sensitab tablets of FZ (15 µg) were provided by Rosco Diagnostica (Taastrup, Denmark) and were stored at room temperature.

Assay media. Two media were used: (i) standard RPMI 1640 medium and (ii) a modified (2% dextrose) standard RPMI 1640 medium (RPMI-G medium; GIBCO BRL, Izasa, S.A., Barcelona) (18).

Yeast isolates. A total of 143 yeast isolates were tested. They comprised 65 C. albicans, 58 C. parapsilosis, 6 C. krusei, 6 C. glabrata, and 8 C. tropicalis isolates. All isolates were recovered from cultures of blood from 143 patients (95% adults) with severe underlying illnesses or clinical conditions (leukemia, diabetes, human immunodeficiency virus infection, trauma, solid-organ transplantation, or surgery) hospitalized in the intensive care, hematology, burn, cardiac surgery, trauma, and oncology units in the Hospital La Fe, Valencia, Spain, from 1995 to 1997. Strains subsequently isolated from the same patient were excluded. Culture, identification, and preservation of strains were carried out by standard methods (1). C. albicans ATCC 64548, C. albicans ATCC 90028 (FZ-susceptible strains), and C. krusei ATCC 6258 (FZ-resistant strains) were included as controls with each batch of organisms tested.

Susceptibility testing. (i) Broth macrodilution method. The broth macrodilution method was performed as described in NCCLS document M27-A (13) with RPMI 1640 and RPMI-G media by the spectronic method for inoculum preparation. The final inoculum varied between 1 × 10³ and 5 × 10³ CFU/ml and was confirmed by plating 10 and 100 µl from the drug-free control tube onto Sabouraud dextrose agar (Difco). The final volume was 1 ml. The FZ concentrations tested ranged from 64 to 0.03 µg/ml. Drug-free and yeast-free controls were included. Readings were made visually after 48 h of incubation at 35°C in RPMI 1640 medium and after 24 h in RPMI-G medium, according to the criteria determined for the NCCLS proposed standard (1:5 dilution of the corresponding growth control) (12). The MIC was defined as that concentration of drug that produced an 80% reduction in turbidity compared with that of the drug-free control after 48 h of incubation in RPMI 1640 medium and after 24 h of incubation in RPMI-G medium. Discrepancies among MIC endpoints of no more than 2 dilutions were used to calculate the percent agreement. The MICs for all yeast strains that were inhibited by FZ concentrations of 1 µg/ml were redetermined. Isolates were classified as susceptible if the MIC was 8 µg/ml, susceptible, dose dependently, if the MIC was 16 to 32 µg/ml, and resistant if the MIC was 64 µg/ml (13).

(ii) Agar diffusion method. Disk diffusion tests were performed on modified Shadomy agar by following the manufacturer's recommendations (6). An FZ tablet (15 µg) was placed on the agar surface; subsequently, the diameters of the inhibition zones were measured with a metric vernier caliper after 24 h of incubation by following the criteria of the manufacturer both for the measurement of inhibition zones and for the interpretation of the diameters of the inhibition zones. For Candida spp. isolated from blood and sterile body fluids, strains with inhibition zones of 30 mm were considered susceptible, those with inhibition zones of <22 mm were considered resistant, and those with inhibition zones of 29 to 23 mm were considered intermediate.

	RESULTS

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The mode MICs of FZ for the reference strains were as follows: for C. albicans ATCC 90028 in RPMI 1640 and RPMI-G media, 0.25 and 0.12 µg/ml, respectively (ranges 0.12 to 0.5 µg/ml in both media); for C. albicans ATCC 64548, 0.25 and 0.25 µg/ml, respectively (range, 0.25 to 0.5 and 0.12 to 0.25 µg/ml, respectively); and for C. krusei ATCC 6258, 64 and 32 µg/ml, respectively (ranges, 64 and 16 to 32 µg/ml, respectively).

The susceptibilities of the yeast isolates to FZ obtained by the broth macrodilution method with the two media are presented in Table 1. As expected, all six isolates of C. krusei were resistant and susceptible, dose dependently, to FZ, with MICs of 32 µg/ml. The range of MICs for the Candida spp. isolated was wide for C. albicans and C. parapsilosis; C. albicans was the most susceptible organism tested. By the reference method, 1 µg of FZ per ml inhibited 93.85% of C. albicans isolates, 81.03% of C. parapsilosis isolates, 62.5% of C. tropicalis isolates, and 16.6% of C. glabrata isolates. For two strains (3.07%) of C. albicans and one strain (1.72%) of C. parapsilosis, FZ MICs were 16 µg/ml. The percent agreement between the MICs obtained in RPMI-G medium and those obtained by the reference broth macrodilution method ranged from 62.5 to 84.5% within ±1 log₂ dilution and 83.3 to 100% within ±2 log₂ dilutions. Except for C. krusei, for which agreement was 100% within ±2 log₂ dilution, C. parapsilosis was the species for which the best agreement was achieved for all criteria used (the same result for ±1 and ±2 log₂ dilutions). The major discrepancies were found for two strains of C. albicans: one was susceptible, dose dependently, in RPMI 1640 medium and the other was resistant in RPMI 1640 medium (MICs, 16 and >64 µg/ml, respectively), and both were susceptible in RPMI-G medium (MICs, 0.12 and 0.25 µg/ml, respectively). By the diffusion test, the inhibition zones for these strains were 23.2 and 22.3 mm, respectively. The MIC for the resistant strain was determined three times, and the same result was always obtained. However, when a less restrictive endpoint criterion (50% reduction of growth) was applied, the MICs were 4, 0.5, and 0.5 µg/ml in RPMI 1640 medium, but if the reading was taken at 24 h, the MIC was 0.12 µg/ml. In RPMI-G medium the MIC was 0.25 µg/ml all the three times. For the susceptible, dose dependently, strain, the MIC diminished to 0.06 µg/ml when the 50% reduction of growth criterion was applied.

The results of the agar diffusion test are presented in Table 2. A distribution of two well-defined populations can be observed: one dominant population with zone diameters of 23 to 29 mm for C. albicans and >30 mm for C. parapsilosis and a smaller population with zone diameters of <22 and <30 mm for C. albicans and between 23 and 29 mm for C. parapsilosis.

No correlation was found between MICs and inhibition zone diameters. In a comparison of the rank order of FZ susceptibilities according to MIC determined by the M27-A method with that according to inhibition zone diameters determined by the disk diffusion method, a major agreement for C. parapsilosis was found, in that for 37 strains that were susceptible according to their zone diameters, MICs were 4 µg/ml and for those that were resistant according to their zone diameters, MICs were between 0.5 and 8 µg/ml. Only 8 C. albicans strains were susceptible and 11 were resistant by the disk diffusion method. The only strain resistant by the agar dilution method was also resistant by the disk diffusion method.

	DISCUSSION

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FZ is a bistriazole antifungal agent that distributes widely in body tissues (9) and which has been shown to have few side effects coupled with good therapeutic activity. The increased use of this drug has given rise to the development of resistance among Candida spp.; as a result, it is important to monitor susceptibility. Since the NCCLS has a reference method and tentative breakpoints for three antifungal agents, results in the literature can be compared and any increase in the range of MICs can be detected. Performance of susceptibility tests by the NCCLS method on a large scale is difficult, but the agar diffusion test is easier for small laboratories. We have evaluated the ability of the agar diffusion method to separate strains that are susceptible and resistant to FZ with disks containing 15 µg of FZ. Previously, other investigators have evaluated a FZ diffusion test, but they used disks containing 25 µg (2, 15). Furthermore, the method described by Rodriguez-Tudela (18) was also evaluated but they used a microdilution method. That method, which is very similar to that of NCCLS, has the advantage of a shorter incubation time and simplified endpoint determination.

The MIC at which 90% of strains are inhibited (MIC₉₀) for C. albicans is 1 µg/ml, the MIC₉₀ for C. parapsilosis is 4 µg/ml, and the MIC₉₀ is for both C. glabrata and C. tropicalis is 64 µg/ml. According to the breakpoints stated by NCCLS, only one strain of C. albicans is resistant and another is susceptible, dose dependently; for C. parapsilosis only one strain is susceptible, dose dependently. These results are similar to those reported by other investigators who used the same method (3, 7, 20, 21).

The two dilution methods tested gave very similar results. Generally, MICs are 1 log₂ dilution lower, endpoints are easier to determine, and the trailing effect is minor in RPMI-G medium. C. albicans is the species that shows the greatest trailing effect. The overall agreement within 2 log₂ dilutions is 93.7%, which is lower than that reported by Espinel-Ingroff et al. (8), 97.7%, but they compared the M27-P macrodilution method with a microdilution method, using for the latter method RPMI-G medium as the culture medium, an inoculum size ranging from 1 × 10⁴ to 5 × 10⁴ CFU/ml, and C. albicans as the target yeast. The major discrepancy that we have found is for two strains of C. albicans: one susceptible, dose dependently, and the other resistant in RPMI 1640 medium (MICs, 16 and >64 µg/ml, respectively) and both susceptible in RPMI-G medium (MICs, 0.12 and 0.25 µg/ml, respectively). When a less restrictive endpoint criterion (50% reduction of growth) is applied for RPMI 1640 medium, the MICs decrease to 0.06 and 0.5 µg/ml, respectively, whereas for RPMI-G medium no changes are detected. This is a case in which the strain is susceptible at 24 h and resistant at 48 h, and Rex et al. (17) denominated a low/high strain.

The ability of the diffusion test to separate the strains susceptible and resistant to FZ is less. There is no correlation between zone size and MIC. Interestingly, inhibition zones are smaller for C. albicans, for which the geometric mean MIC was 0.29 µg/ml the mean inhibition zone was 25.7 mm, while for C. parapsilosis the geometric mean was 0.96 µg/ml and the mean inhibition zone was 31.52 mm. On the basis of the manufacturer's recommendations to interpret the inhibition zones and comparison of the MICs obtained by the reference method, only 13.8% of C. albicans strains and 65.5% of C. parapsilosis strains are well classified. Sandren et al. (19), using tablets of 15 µg, concluded that the diffusion test is an appropriate method for detection of FZ-resistant C. albicans strains. In our case, all strains susceptible by the diffusion test were also susceptible by the dilution method. The converse was not necessarily true. On the basis of our results we recommend, as Bille suggests (4), the determination of MICs of FZ for those strains resistant by the diffusion method.

When our results and those reported recently by other investigators (3, 5, 21, 22) are compared with those obtained by Price et al. (16) for Candida strains isolated over 5 years (1987 to 1992), it is interesting that the MIC range and MIC₉₀s have increased; for example, the MIC₉₀ for C. albicans has gone from 0.25 to 1 µg/ml.

The increasing use of FZ as antifungal therapy is resulting in a reduction in susceptibility and the appearance of resistant strains. For this reason, it would be convenient to carry out antifungal susceptibility studies in order to establish the in vitro activities of antifungal agents against local isolates and also to detect shifts toward resistance as early as possible.