In Vitro Susceptibilities of Candida and Cryptococcus neoformans Isolates from Blood Cultures of Neutropenic Patients

Abstract

Fluconazole-resistant Candida albicans and intrinsically fluconazole-resistant Candida species have been reported as bloodstream isolates. However, an association between the isolation of fluconazole-resistant Candida from the bloodstream and patient risk factors for fungemia has not been established. The purpose of this study was to determine the prevalence of fluconazole resistance in bloodstream isolates of Candida species and Cryptococcus neoformans collected from patients with neutropenia, one of the most important risk factors for fungemia. MICs of voriconazole, fluconazole, itraconazole, ketoconazole, amphotericin B, and flucytosine were determined by the National Committee for Clinical Laboratory Standards M27-A method (1997). Voriconazole, on a per-weight basis, was the most active azole tested. Fluconazole resistance (MIC ≥ 64 μg/ml) was not identified in any of the C. albicans (n = 513), Candida parapsilosis (n = 78), Candida tropicalis (n = 62), or C. neoformans (n = 38) isolates tested.

The incidence of systemic fungal infections has risen dramatically over the past 20 years (1, 7). This has been attributed principally to improved malignancy detection and increasingly immunosuppressive treatments, advances in organ transplantation, and a steady rise in the human immunodeficiency virus-positive population (1, 5). Other explanations have included the centralization of immunocompromised patients at tertiary-care institutions, improved blood culture techniques, and a greater appreciation of the pathogenicity of Candida species (1). Regardless, Candida species are the most common fungi isolated from the bloodstream and the major pathogens of invasive fungal infection (5). Azole resistance in systemic isolates of Candida albicans, although rare, has been reported and may be increasing (2, 3, 6). However, an association between the isolation of fluconazole-resistant Candida and underlying patient diagnoses or interventions has not been established. It is unclear from previous studies (2, 3, 6) if fluconazole-resistant Candida isolated from blood cultures represented breakthrough fungemia in AIDS patients with a history of azole therapy, infection of neutropenic patients following one or more febrile episodes treated with repeated courses of fluconazole, or de novo fluconazole resistance. Because many episodes of fungemia result from the ingress of a colonizing yeast into the systemic circulation during the transient, immunosuppressive effect of blood-cell-directed chemotherapy, this study was conducted to determine the prevalence of fluconazole resistance in bloodstream isolates of Candida species and Cryptococcus neoformans collected from neutropenic patients.

(This work was presented in part at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 24 to 27 September 1998.)

Candida species and C. neoformans isolates were acquired from stock blood cultures maintained by the Department of Clinical Microbiology, Health Sciences Centre, in Winnipeg, Canada, between 1986 and 1997. One isolate per patient infectious episode was chosen for susceptibility testing, and all isolates were from neutropenic (absolute neutrophil count ≤ 5 × 10² neutrophils/ml) patients. Isolates were identified to the species level by using colony morphology, germ tube formation, and API 20C AUX (bioMerieux, Hazelwood, Mo.) strips. The collection contained 513 C. albicans, 78 Candida parapsilosis, 66 Candida glabrata, 62 Candida tropicalis, and 38 C. neoformans isolates. Fewer than 10 isolates each of Candida krusei, Candida kefyr, Candida guilliermondii, and Candida lusitaniae were available and therefore were not included in the study.

Voriconazole and fluconazole were supplied by Pfizer Canada Inc. (Pointe-Claire/Dorval, Quebec, Canada), itraconazole and ketoconazole were supplied by Janssen/Ortho (North York, Ontario, Canada), amphotericin B was supplied by Bristol-Myers Squibb (Saint-Laurent, Quebec, Canada), and flucytosine (5FC) was supplied by Hoffmann-La Roche (Mississauga, Ontario, Canada). Stock solutions of voriconazole, fluconazole, itraconazole, ketoconazole, and amphotericin B were prepared in dimethyl sulfoxide, and 5FC stock solutions were prepared in water. The MIC doubling dilution ranges tested were 0.0078 to 8 μg/ml for voriconazole, itraconazole, ketoconazole, and amphotericin B and 0.0313 to 64 μg/ml for fluconazole and 5FC.

Candida species and C. neoformans isolates were subcultured onto Sabouraud agar prior to antifungal susceptibility testing. Antifungal MICs for Candida species and C. neoformans were determined in RPMI 1640 medium (with glutamine and without bicarbonate, pH 7.0) by the National Committee for Clinical Laboratory Standards M27-A microdilution reference method (4). C. albicans ATCC 90028, C. glabrata ATCC 90030, and C. neoformans ATCC 90112 were used as quality control organisms (1, 4). MICs were read after 48 h (Candida species) and 72 h (C. neoformans) of incubation at 35°C. MIC endpoints were determined for voriconazole as for other azoles as the lowest concentration of drug that inhibited fungal growth by 80% (4). Colony counts to confirm initial inocula were performed for each MIC determination.

The antifungal susceptibilities of the Candida species and C. neoformans isolates tested are presented in Table 1 as MIC ranges, MICs at which 50% of the isolates are inhibited (MIC₅₀s), and MIC₉₀s. Fluconazole-resistant (MIC ≥ 64 μg/ml) (4) isolates of C. albicans, C. parapsilosis, C. tropicalis, or C. neoformans were not detected (Table 1). As anticipated, fluconazole-susceptible dose-dependent isolates of C. glabrata were identified (Table 1). Significant changes in susceptibility (i.e., a fourfold or greater increase or decrease in MIC₉₀) to any of the six antifungal agents tested against C. albicans were not present between 1986 and 1997 (analysis of variance, P < 0.05) when isolates were grouped by year of isolation (data not shown). Due to the limited number of isolates, similar comparisons with individual non-C. albicans Candida species and C. neoformans were not calculated.

TABLE 1.

Activity of voriconazole, fluconazole, itraconazole, ketoconazole, amphotericin B, and 5FC against bloodstream isolates of Candida species and C. neoformans isolated from neutropenic patients at the Health Sciences Centre in Winnipeg, Canada, between 1986 and 1997

Yeast (no. of isolates)	Antifungal agent	MIC (μg/ml)a
		Range	50%	90%
Candida albicans (513)	Voriconazole	≤0.0078–0.5	0.0313	0.0625
	Fluconazole	≤0.0313–2	0.25	0.5
	Itraconazole	≤0.0078–0.5	0.0625	0.125
	Ketoconazole	0.0156–0.5	0.0625	0.125
	Amphotericin B	0.0625–1	1	1
	5FC	≤0.0313–64	0.125	1
Candida para-psilosis (78)	Voriconazole	≤0.0078–0.25	0.0313	0.125
	Fluconazole	0.25–2	0.5	1
	Itraconazole	0.0313–0.5	0.125	0.5
	Ketoconazole	0.0625–0.5	0.125	0.5
	Amphotericin B	0.5–1	1	1
	5FC	≤0.0313–0.5	0.125	0.25
Candida glabrata (66)	Voriconazole	0.125–2	1	2
	Fluconazole	1–32	8	16
	Itraconazole	0.0625–4	1	4
	Ketoconazole	0.25–4	1	2
	Amphotericin B	0.5–1	1	1
	5FC	≤0.0313–4	≤0.0313	4
Candida tropicalis (62)	Voriconazole	0.0156–1	0.125	0.25
	Fluconazole	0.25–16	1	2
	Itraconazole	0.0313–1	0.25	0.5
	Ketoconazole	0.0313–1	0.25	0.5
	Amphotericin B	0.5–1	1	1
	5FC	≤0.0313–>64	0.25	1
Cryptococcus neo-formans (38)	Voriconazole	0.0313–0.5	0.0625	0.25
	Fluconazole	0.125–8	2	8
	Itraconazole	≤0.0078–0.5	0.125	0.5
	Ketoconazole	≤0.0078–0.5	0.125	0.25
	Amphotericin B	0.5–1	1	1
	5FC	≤0.0313–8	2	8

^a50 and 90%, MIC₅₀ and MIC₉₀, respectively. 

Amphotericin B MICs were ≤1 μg/ml for all Candida species and C. neoformans isolates tested (Table 1). 5FC resistance (MIC ≥ 32 μg/ml) (4) was identified in three isolates of C. albicans and one isolate of C. tropicalis.

Voriconazole was the most active azole tested with twofold-higher activity than those of itraconazole and ketoconazole, and eightfold-higher activity than that of fluconazole, against C. albicans (Table 1). Against C. parapsilosis, voriconazole was fourfold more active than itraconazole and ketoconazole and eightfold more active than fluconazole (Table 1). Against C. glabrata, voriconazole’s activity was similar to that of ketoconazole, with twofold-higher activity than that of itraconazole and eightfold-higher activity than that of fluconazole (Table 1). Against C. tropicalis, voriconazole was twofold more active than itraconazole and ketoconazole and eightfold more active than fluconazole (Table 1). Against C. neoformans, voriconazole’s activity was similar to that ketoconazole, 2-fold better than that of itraconazole, and 16-fold better than that of fluconazole (Table 1). Voriconazole was more active than amphotericin B and 5FC against all Candida species except C. glabrata (Table 1). In general, isolates with decreased susceptibility to fluconazole (MIC ≥ 8 μg/ml), itraconazole (MIC ≥ 0.125 μg/ml), and ketoconazole (MIC ≥ 0.125 μg/ml) were less susceptible to voriconazole; however, voriconazole MICs were still lower than those of the other azoles.

This study demonstrated that fluconazole-resistant isolates of C. albicans and C. neoformans were not present in bloodstream isolates collected from neutropenic patients at the Health Sciences Centre, Winnipeg, Canada, between 1986 and 1997. Our data suggests that the transient neutropenia associated with blood-cell-directed chemotherapy is not uniquely associated with the isolation of intrinsically fluconazole-resistant Candida spp. (C. glabrata and C. krusei) or C. albicans with acquired fluconazole resistance. This observation is not surprising given knowledge that neutropenic patients at the Health Sciences Centre do not routinely receive fluconazole prophylaxis and receive short-term (1 to 2 weeks) fluconazole therapy only with the appearance of fever, as dictated by policy at our institution.

Another notable observation was that voriconazole, against the isolates tested, demonstrated modestly better activity than did itraconazole and significantly better activity than did fluconazole against Candida species, regardless of fluconazole susceptibility (Table 1). Voriconazole, a derivative of fluconazole, has previously demonstrated greater activity compared with itraconazole and a broader spectrum of activity and higher potency than fluconazole against Candida species, C. neoformans, Aspergillus species, and other fungi (2, 3, 6, 7). Our results confirm that voriconazole has promising antifungal activity against C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, and C. neoformans.

In conclusion, fluconazole resistance was not identified in the 513 isolates of C. albicans, 78 isolates of C. parapsilosis, 62 isolates of C. tropicalis, and 38 isolates of C. neoformans tested from blood cultures of neutropenic patients. As well, voriconazole, on a per-weight basis, was the most active antifungal agent tested.