In Vitro Activities of Voriconazole, Posaconazole, and Four Licensed Systemic Antifungal Agents against Candida Species Infrequently Isolated from Blood

Abstract

We determined the in vitro susceptibilities of 314 strains of Candida spp., representing 13 species rarely isolated from blood, to posaconazole and voriconazole as well as four licensed systemic antifungal agents (amphotericin B, flucytosine, fluconazole, and itraconazole). The organisms included 153 isolates of C. krusei, 67 isolates of C. lusitaniae, 48 isolates of C. guilliermondii, 10 isolates of C. famata, 10 isolates of C. kefyr, 6 isolates of C. pelliculosa, 5 isolates of C. rugosa, 4 isolates of C. lipolytica, 3 isolates of C. dubliniensis, 3 isolates of C. inconspicua, 2 isolates of C. sake, and 1 isolate each of C. lambica, C. norvegensis, and C. zeylanoides. MIC determinations were made by the National Committee for Clinical Laboratory Standards reference broth microdilution method and Etest (amphotericin B). Resistance to both amphotericin B and fluconazole was observed in strains of C. krusei, C. lusitaniae, C. guilliermondii, C. inconspicua, and C. sake. Resistance to amphotericin B, but not to fluconazole, was also observed among isolates of C. kefyr and C. rugosa. Posaconazole and voriconazole were active (MIC, ≤1 μg/ml) against 94 to 100% of these isolates. In contrast to the more common species of Candida causing bloodstream infection, these rare species appear to be less susceptible to the currently licensed systemic antifungal agents, with the exception of voriconazole. Continued surveillance will be necessary to detect the emergence of these species as more prevalent, resistant pathogens. The new triazoles appear to offer acceptable coverage of uncommon Candida sp. bloodstream infections.

The importance of Candida spp. as etiologic agents of bloodstream infection (BSI) in hospitalized patients is well established (9, 12, 17). Approximately 95% of Candida BSI are due to four species: C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis (32-34). The excellent activities of new and established systemic agents against these four species have been documented extensively (32, 33, 35), whereas very little is known regarding the susceptibility profiles of the less frequently isolated Candida species (10, 11, 15).

The number of fungal species causing invasive mycoses appears to be increasing steadily (15, 37). Recent individual reports and reviews address the issue of “new” and “emerging” fungal pathogens and the associated problems with identification and treatment of infection caused by these organisms (5, 6, 10, 15, 28, 29, 36, 40). Among the Candida spp. reported to cause BSI, more than 17 different species have been identified (15). Aside from C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis noted above, the 13 remaining species account for less than 5% of all Candida BSI. These species include C. krusei, C. lusitaniae, C. guilliermondii, C. rugosa, and C. dubliniensis, among others, all of which have been observed to occur in nosocomial clusters and/or to exhibit innate or acquired resistance to one or more established antifungal agents (7, 8, 14, 29, 45, 50). Given that these less common species may emerge as important pathogens in the future, it is prudent to describe the activities of both new and established antifungal agents as potential therapeutic options for infections due to these species. In the present study, we report the in vitro activities, determined by National Committee for Clinical Laboratory Standards (NCCLS) reference methods (22), of two new extended-spectrum triazoles, posaconazole and voriconazole, and of four additional licensed systemic agents against 13 of the less common species of Candida isolated from blood cultures of patients hospitalized in North America, Latin America, Europe, and the Asia-Pacific region.

MATERIALS AND METHODS

Organisms.

A total of 314 clinical isolates of Candida spp. obtained from 93 medical centers worldwide were tested. The collection included the following numbers of isolates: 153 isolates of C. krusei, 67 isolates of C. lusitaniae, 48 isolates of C. guilliermondii, 10 isolates of C. famata, 10 isolates of C. kefyr, 6 isolates of C. pelliculosa, 5 isolates of C. rugosa, 4 isolates of C. lipolytica, 3 isolates of C. dubliniensis, 3 isolates of C. inconspicua, 2 isolates of C. sake, and 1 isolate each of C. lambica, C. norvegensis, and C. zeylanoides (Table 1). were incident isolates obtained from blood cultures of 314 different patients with candidemia. Isolates were identified using Vitek and API yeast identification systems (bioMerieux, Inc., Hazelwood, Mo.) and supplemented by conventional methods as needed (15, 48). Isolates were stored as water suspensions until used. Prior to testing, each isolate was passaged at least twice on potato dextrose agar (Remel, Lenexa, Kans.) to ensure purity and viability.

TABLE 1.

Antifungal susceptibilities of rare Candida bloodstream isolates

Candida species	No. of isolates	Antifungal agentb	Cumulative % inhibited at the following MIC (μg/ml)a:
			0.06	0.12	0.25	0.5	1	2	4	8	16	32	64	128
C. krusei	153	Amphotericin B	0	0	0	1	7	34	86	99	100
		Flucytosine	1	1	2	3	3	4	5c	13	75	99	99	100
		Fluconazole		0	0	0	0	0	1	5d	18	59	98	100
		Itraconazole	0	1c	18	51	95	100
		Posaconazole	0	2	35	95	99	100
		Voriconazole	1	11	52	93	99	99	100
C. lusitaniae	67	Amphotericin B	2	23	51	88	98	98	98	100
		Flucytosine	70	87	90	91	94	94	94c	96	97	97	97	100
		Fluconazole		2	30	64	81	91	93	96d	97	99	100
		Itraconazole	3	46e	90	97	99	100
		Posaconazole	82	93	94	100
		Voriconazole	94	94	97	99	99	100
C. guilliermondii	48	Amphotericin B	3	26	63	95	97	97	97	97	97	100
		Flucytosine	27	65	88	96	96	98	100c
		Fluconazole		0	0	2	9	32	64	85d	94	94	94	96
		Itraconazole	2	10e	35	52	94	94	96	98
		Posaconazole	18	24	62	91	96	98	98	100
		Voriconazole	50	67	79	94	94	98	98	98
C. famata	10	Amphotericin B	20	30	50	70	100
		Flucytosine	20	80	90	90	90	90	90c	90	90	90	90	100
		Fluconazole		0	0	10	20	20	40	60d	100
		Itraconazole	0	10e	40	50	90	100
		Posaconazole	20	30	60	90	100
		Voriconazole	40	40	80	100
C. kefyr	10	Amphotericin B	0	0	0	0	40	60	80	90	100
		Flucytosine	30	80	80	80	100	100	100c
		Fluconazole		10	60	100	100	100	100	100d
		Itraconazole	20	50e	100
		Posaconazole	30	90	100
		Voriconazole	100
C. pelliculosa	6	Amphotericin B	0	0	17	50	100
		Flucytosine	50	50	50	50	50	50	50c	50	50	83	100
		Fluconazole		0	0	0	0	0	67	100d
		Itraconazole	0	0e	50	50	100
		Posaconazole	0	0	0	50	100
		Voriconazole	0	50	100
C. rugosa	5	Amphotericin B	0	0	20	40	60	60	100
		Flucytosine	20	20	40	60	80	80	80c	80	100
		Fluconazole		0	0	0	0	20	80	100d
		Itraconazole	40	60e	100
		Posaconazole	40	80	100
		Voriconazole	80	80	100
C. lipolytica	4	Amphotericin B	0	0	0	0	100
		Flucytosine	0	0	0	0	0	25	25c	50	75	75	100
		Fluconazole		0	0	0	0	0	75	75d	75	75	100
		Itraconazole	0	0e	0	0	75	75	75	100
		Posaconazole	0	0	0	25	75	75	100
		Voriconazole	25	75	75	75	100
C. dubliniensis	3	Amphotericin B	33	67	100
		Flucytosine	100	100	100	100	100	100	100c
		Fluconazole		0	100	100	100	100	100	100d
		Itraconazole	33	100e
		Posaconazole	100
		Voriconazole	100
C. inconspicua	3	Amphotericin B	0	0	0	0	0	33	67	67	100
		Flucytosine	0	0	0	0	33	33	33c	33	67	67	100
		Fluconazole		0	0	0	0	0	33	33d	33	33	67	100
		Itraconazole	0	0e	0	67	67	67	67	67				/PICK>
		Posaconazole	0	0	0	67	67	67	67	67
		Voriconazole	0	0	33	67	67	67	100
C. sake	2	Amphotericin B	0	0	0	50	50	50	100
		Flucytosine	50	100	100	100	100	100c
		Fluconazole		0	0	0	0	0	50	50d	50	50	50	50
		Itraconazole	0	0e	0	50	50	50	50	50
		Posaconazole	0	0	0	50	50	50	50	50
		Voriconazole	0	50	50	50	50	50	50	50
C. lambica	1	Amphotericin B	0	0	0	0	100
		Flucytosine	0	0	0	0	0	0	0c	0	0	100
		Fluconazole		0	0	0	0	0	0	0d	0	0	100
		Itraconazole	0	0e	100
		Posaconazole	0	0	100
		Voriconazole	0	0	0	100
C. norvegensis	1	Amphotericin B	0	0	0	0	100
		Flucytosine	0	0	0	0	0	0	0c	100
		Fluconazole	0	0	0	0	0	0	0	0d	100
		Itraconazole	0	0e	100
		Posaconazole	0	100
		Voriconazole	0	100
C. zeylanoides	1	Amphotericin B	0	0	100
		Flucytosine	100
		Fluconazole		100
		Itraconazole	100
		Posaconazole	100
		Voriconazole	100

^aMICs were determined by the NCCLS microdilution method (22).

^bAmphotericin B MICs were determined by Etest.

^cPercentage of isolates susceptible to flucytosine at the NCCLS breakpoint of ≤4 μg/ml.

^dPercentage of isolates susceptible to fluconazole at the NCCLS breakpoint of ≤8 μg/ml.

^ePercentage of isolates susceptible to itraconazole at the NCCLS breakpoint of ≤0.12 μg/ml.

Antifungal agents.

Standard antifungal powders of posaconazole (Sch 56592; Schering-Plough), voriconazole (Pfizer), fluconazole (Pfizer), itraconazole (Janssen), and flucytosine (Sigma) were obtained from their respective manufacturers. Stock solutions were prepared in dimethyl sulfoxide (voriconazole), polyethylene glycol (posaconazole and itraconazole), or water (fluconazole and flucytosine). Serial twofold dilutions were prepared exactly as outlined in NCCLS document M27-A (22). Final dilutions were made in RPMI 1640 medium (Sigma) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma). The final concentration of solvent did not exceed 1% in any well. Aliquots (100 μl) of each antifungal agent at a twofold final concentration were dispensed into the wells of plastic microdilution trays by using a Quick Spense II System (Dynatech Laboratories). The trays were sealed and frozen at −70°C until they were used.

Antifungal susceptibility studies.

Broth microdilution testing was performed in accordance with the guidelines in NCCLS document M27-A (22). The inoculum suspension was prepared by the spectrophotometric method of inoculum preparation and with a final inoculum of (1.5 ± 1.0) × 10³ cells/ml. A 100-μl yeast inoculum was added to each well of the microdilution trays. The final concentrations of the antifungal agents were 0.007 to 8 μg/ml for itraconazole, posaconazole, and voriconazole, 0.06 to 128 μg/ml for flucytosine, and 0.12 to 128 μg/ml for fluconazole. The trays were incubated at 35°C, and MIC endpoints were read after 48 h of incubation. Drug-free and yeast-free controls were included. The susceptibilities of Candida isolates to amphotericin B were determined by using Etest (AB BIODISK, Solna, Sweden) and by using RPMI 1640 agar with 2% glucose (Remel), as described previously (30).

Following incubation, the MICs of fluconazole, itraconazole, posaconazole, voriconazole, and flucytosine were read as the lowest concentration at which a prominent decrease (approximately 50%) in turbidity relative to the turbidity of the growth control was observed (22). Amphotericin B MICs determined by Etest were read after 48 h of incubation at 35°C and were determined to be at 100% inhibition of growth where the border of the elliptical inhibition zone intercepted the scale of the strip edge (28, 30, 47). Quality control was ensured by testing the NCCLS-recommended strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (4, 22).

The interpretive criteria for susceptibility to fluconazole, itraconazole, and flucytosine were those published by Rex et al. (38) and the NCCLS (22). The new triazoles, posaconazole and voriconazole, have not been assigned interpretive breakpoints. For purposes of comparison, and because pharmacokinetic data indicate that achievable levels for these agents in serum may range from 2 to 6 μg/ml, with sustained levels exceeding 1 μg/ml for the entire dosing interval (25, 27, 41, 42, 46), we have employed a susceptible breakpoint of ≤1 μg/ml for both agents. Interpretive criteria have not yet been defined for amphotericin B; however, for purposes of comparison, we have determined the percentage of isolates inhibited by ≤1 μg of amphotericin B per ml to be susceptible in this study.

RESULTS AND DISCUSSION

Table 1 summarizes the antifungal susceptibilities of 314 strains of rare Candida species isolated from blood cultures. These isolates were obtained during the course of Candida BSI surveillance studies and represent approximately 5% of the more than 6,000 Candida BSI isolates tested at the University of Iowa with NCCLS reference methods. All were isolated from blood cultures obtained from patients with signs and symptoms consistent with candidemia. Aside from this, we have no further evidence that these organisms were actually causing infection (15).

Most of the species listed in Table 1 have been described previously as causing serious infections in humans with severe underlying disease and often with vascular and/or peritoneal catheters (2, 5-8, 13-15, 18, 21, 23, 24, 28, 29, 43, 45, 50). Several species, such as C. krusei (13, 23, 50), C. lusitaniae (2, 14, 23, 28, 29), C. rugosa (8, 44), C. dubliniensis (45), C. inconspicua (5), C. guilliermondii (7), and C. norvegensis (2, 24), have been reported to express resistance to one or more antifungal agents and have occurred in nosocomial clusters, either in association with the selective pressure of an antifungal agent or related to intravascular catheters and breaks in infection control precautions (15). Thus, it is evident that these rare species may be considered opportunistic pathogens and that they may pose resistance problems for the currently licensed antifungal agents.

C. krusei is the most frequently encountered of these rare Candida species (13, 23, 31). C. krusei is best known for its propensity to emerge in settings where fluconazole is used for Candida prophylaxis (1, 23, 49, 50). The data in Table 1 clearly demonstrate the decreased susceptibility of C. krusei to fluconazole and also to both amphotericin B (7% of isolates were susceptible at ≤1 μg/ml) and flucytosine (5% were susceptible at ≤4 μg/ml). This pattern of decreased susceptibility to three licensed systemic antifungal agents has certainly been noted previously (11, 39) and is the basis for continued concern for this species as a possible emerging pathogen. Given this resistance profile, it is important to note that C. krusei is quite susceptible to both posaconazole and voriconazole (99% of isolates were susceptible at concentrations of ≤1 μg/ml).

C. lusitaniae is a species that is frequently mentioned in the literature as being capable of developing resistance to amphotericin B during the course of treatment (2, 14, 23, 26, 28, 29). Although it is clear that this species can develop secondary resistance to amphotericin B (14, 26), the frequency with which this resistance may be seen clinically is not known. Among the 67 BSI isolates of C. lusitaniae tested for amphotericin B resistance by Etest (Table 1), 98% were susceptible at concentrations of ≤1 μg/ml, and only one appeared highly resistant (MIC, 8 μg/ml). This level of susceptibility to amphotericin B is comparable to that reported for C. albicans (95% of isolates are susceptible) (35). Etest has been shown to be both sensitive and specific for detecting amphotericin B resistance among isolates of C. lusitaniae and other species of Candida (28, 29, 47). Based on these data, it appears that amphotericin B resistance is rare among incident BSI isolates of C. lusitaniae; however, if amphotericin B is used to treat infections due to this species, the patients should be monitored closely in order to detect the emergence of secondary resistance (39). In contrast to the results of previous studies (2a, 26, 51), C. lusitaniae appears to be quite susceptible to flucytosine (94% of isolates were susceptible, 3% were resistant). All four of the triazoles tested were very active against this species (96 to 100% of isolates were susceptible).

C. guilliermondii has been reported to be resistant to amphotericin B (7) and also to fluconazole (10, 11). Aside from a single report from Johns Hopkins (Baltimore, Md.) of in vitro amphotericin B resistance and clinical failure of amphotericin B therapy (7), resistance to the polyenes has not been widely appreciated for this species. Only 1 of 48 BSI isolates of C. guilliermondii tested by Etest appeared to be resistant to amphotericin B (MIC, 32 μg/ml) (Table 1). Likewise, 100% of these isolates were susceptible to flucytosine at the NCCLS breakpoint concentration of ≤4 μg/ml (Table 1). C. guilliermondii does appear to be less susceptible to fluconazole than the more common BSI isolates of Candida (85% of C. guilliermondii isolates were susceptible versus 97 to 99% of C. albicans, C. parapsilosis, and C. tropicalis isolates) (10, 11, 35). Although 94 to 96% of C. guilliermondii isolates were susceptible to posaconazole and voriconazole (MIC, ≤1 μg/ml), one isolate was resistant to both of these agents as well as fluconazole and itraconazole. A similar degree of cross-resistance among the azoles was reported for C. guilliermondii by Espinel-Ingroff et al. (11).

C. famata has been described as an agent of catheter-related fungemia (15, 43). This species appears to be susceptible to amphotericin B but is capable of expressing high-level resistance to flucytosine and fluconazole (10) (Table 1). Only 6 of the 10 isolates of C. famata tested herein were fully susceptible to fluconazole, but all were susceptible to posaconazole and voriconazole at concentrations of ≤1 μg/ml. Espinel-Ingroff (10) has also reported resistance to fluconazole among isolates of C. famata.

Disseminated infection due to C. kefyr (formerly C. pseudotropicalis) has been observed in a cancer patient (21), but otherwise there is little published information regarding this species. The susceptibility of C. kefyr to amphotericin B appears to be quite low (4 of 10 isolates were susceptible at ≤1 μg/ml); however, it is uniformly susceptible to flucytosine, fluconazole, itraconazole, posaconazole, and voriconazole (Table 1). These findings are consistent with those of other investigators (3, 10, 19). McGinnis and Rinaldi (20) and Espinel-Ingroff et al. (11) have also reported amphotericin B resistance among clinical isolates of C. kefyr.

Fewer than 10 isolates were available for each of the remaining nine species of Candida (Table 1). Among these species, decreased susceptibility to fluconazole has been reported for C. lipolytica (2a, 10, 11), C. dubliniensis (45), C. inconspicua (2a, 5), and C. lambica (10) and decreased susceptibility to amphotericin B, nystatin, and fluconazole has been reported for C. rugosa (8, 10, 11, 15). Although few in number, we have observed one or more isolates of these species with decreased susceptibility (MIC, >8 μg/ml) to fluconazole (C. lipolytica, C. inconspicua, C. sake, C. lambica, and C. norvegensis) and/or amphotericin B (MIC, >1 μg/ml; C. rugosa, C. inconspicua, and C. sake) (Table 1). In contrast, all isolates of C. pelliculosa, C. rugosa, C. lambica, C. norvegensis, and C. zeylanoides were susceptible to posaconazole and voriconazole at ≤1 μg/ml. Isolates of C. lipolytica, C. inconspicua, and C. sake showed decreased susceptibilities to the newer triazoles (MIC, >1 μg/ml) as well as to fluconazole and itraconazole (Table 1). The small number of each of these species tested in this study does not allow one to make firm conclusions regarding their susceptibility to any of the antifungal agents. Continued observation and testing of rare species when they occur, by NCCLS standardized methods, will ultimately provide clinically useful information.

Whether or not any of these 13 species may be considered to be emerging pathogens is difficult to say (15). Clearly, they are rare, and only by tracking BSI isolates of Candida species over time in the context of global surveillance programs can one eventually make such a determination (34). In any event, they may pose difficult problems for individual patients, and thus reporting of their in vitro susceptibility profiles may be helpful. The numbers of isolates of several of these species in the present study are small due to their infrequent isolation from blood cultures; however, by testing these isolates with a standardized method and reporting the percentages of isolates that are susceptible and resistant and the MICs in a continuous fashion rather than the MICs at which 50 and 90% of the isolates are inhibited, we hope to make the data more useful and available for ready comparison with that generated by other studies by the NCCLS method (16). The results of this study indicate that, compared with the more common species of Candida causing BSI, these rare Candida species appear to be less susceptible to the older licensed systemic antifungal agents. Thus, although less common, they pose a potential threat for immunocompromised patients in the future. Fortunately, the new extended-spectrum triazoles are very active against most of these species. It is notable, however, that isolates of some species (e.g., C. guilliermondii, C. lipolytica, and C. inconspicua) demonstrated cross-resistance to all of the azoles tested. By analogy with C. albicans and C. glabrata, these findings suggest that these rare species may possess multidrug efflux pumps and/or the ability to alter the target enzyme (14 α-demethylase) with resulting high-level resistance to all azoles (20, 35, 42, 48a). Continued surveillance will enable us to detect the true emergence and impact of the less common species of Candida.