Geographic Variation in the Susceptibilities of Invasive Isolates of Candida glabrata to Seven Systemically Active Antifungal Agents: a Global Assessment from the ARTEMIS Antifungal Surveillance Program Conducted in 2001 and 2002

Abstract

We examined the susceptibilities to amphotericin B, flucytosine, fluconazole, posaconazole, ravuconazole, voriconazole, and caspofungin of 601 invasive isolates of Candida glabrata and grouped the isolates by geographic location: North America (331 isolates), Latin America (58 isolates), Europe (135 isolates), and Asia-Pacific (77 isolates). Caspofungin (MIC at which 90% of isolates tested are susceptible [MIC₉₀], 0.12 μg/ml; 100% of strains are susceptible [S] at a MIC of ≤1 μg/ml) and flucytosine (MIC₉₀, 0.12 μg/ml; 99.2% S) were the most active agents in all geographic regions. Fluconazole susceptibility was highest in the Asia-Pacific region (80.5% S, 3.9% resistant [R]) and lowest in North America (64% S, 10.3% R) and Latin America (62.1% S, 3.4% R). The extended-spectrum triazoles were most active in the Asia-Pacific region (90 to 96.1% S) and least active in North America (82.5 to 90.3% S). All 46 isolates that were resistant to fluconazole were susceptible to caspofungin (MIC₉₀, 0.06 μg/ml) and flucytosine (MIC₉₀, 0.12 μg/ml) and exhibited variable cross-resistance to posaconazole, ravuconazole, and voriconazole.

It is common practice in the literature to refer to species of Candida other than C. albicans as “non-albicans” Candida spp. and suggest that this group of organisms exhibits decreased susceptibility to fluconazole and other azoles (1, 8, 14-16, 39, 41). Although the MICs of fluconazole for species such as C. parapsilosis, C. tropicalis, and C. lusitaniae may be somewhat higher than those for C. albicans (MIC at which 90% of isolates tested are susceptible [MIC₉₀], 2 to 4 μg/ml versus 0.5 μg/ml, respectively), they remain overwhelmingly susceptible (MIC ≤ 8 μg/ml; 96 to 99% susceptible [S]) to fluconazole (22-25, 27, 28). Likewise, although C. krusei may be intrinsically resistant to fluconazole, it is very susceptible to the new extended-spectrum triazoles such as voriconazole and posaconazole (98 to 99% S at a MIC of ≤1 μg/ml) (22, 23, 25, 28). C. glabrata alone, among the non-albicans Candida spp., is truly less susceptible to fluconazole (MIC₉₀, 32 μg/ml; 57% S) and exhibits variable cross-resistance to the other triazoles (20, 22, 23, 25, 28). The extent of fluconazole resistance among bloodstream infection (BSI) isolates of C. glabrata has been shown to vary considerably throughout the United States (U.S.) (26). Although the frequency of C. glabrata as a cause of BSI has been shown to vary among different countries worldwide (5-7, 15, 22, 27), differences in the susceptibility of invasive (BSI, normally sterile sites, tissues, abscesses) isolates of C. glabrata to fluconazole and other systemically active agents among various geographic regions internationally have not been explored and compared directly.

In the present study we compare and contrast the in vitro activities of fluconazole and six systemically active antifungal agents, including voriconazole, posaconazole, ravuconazole, and caspofungin, against 601 recent clinically invasive isolates of C. glabrata. These isolates were obtained from 59 different study sites in 25 countries during the course of the ARTEMIS Global Antifungal Surveillance Program conducted in 2001 and 2002.

MATERIALS AND METHODS

Organisms.

A total of 3,997 clinical isolates of Candida spp. obtained from more than 60 medical centers in Europe, Latin America, North America, and the Asia-Pacific region were collected during the course of the ARTEMIS Global Antifungal Surveillance Program in 2001 and 2002. The isolates represented consecutive incident isolates from patients with candidemia or other invasive forms of candidiasis (isolates from sterile-site infections, tissue biopsy specimens, abscesses, and joint fluid) cared for at ARTEMIS participating hospitals. Of the 3,997 isolates, 15% (601 isolates) were C. glabrata, including 331 isolates (20% of all Candida spp. submitted from North America) from 21 study sites in North America, 58 isolates (8% of total submitted) from 8 sites in Latin America, 135 isolates (14% of total submitted) from 19 sites in Europe, and 77 isolates (11% of total submitted) from 11 sites in the Asia-Pacific region. A total of 25 countries were represented in the collection.

All isolates were identified by Vitek and API products (bioMérieux, St. Louis, Mo.), supplemented by conventional methods as required (11), and were stored as water suspensions until they were used. Prior to testing, each isolate was passaged on potato dextrose agar (Remel, Lenexa, Kans.) and CHROMagar (Hardy Laboratories, Santa Monica, Calif.) to ensure purity and viability.

Antifungal agents.

Standard antifungal powders of flucytosine (Sigma), fluconazole (Pfizer), voriconazole (Pfizer), posaconazole (Schering-Plough), ravuconazole (Bristol-Myers Squibb), and caspofungin (Merck) were obtained from their respective manufacturers. Stock solutions were prepared in water (caspofungin, fluconazole, and flucytosine), dimethyl sulfoxide (ravuconazole and voriconazole), or polyethyleneglycol (posaconazole). Serial twofold dilutions of each agent were prepared exactly as outlined in National Committee for Clinical Laboratory Standards (NCCLS) document M27-A2 (18). Final dilutions were made in RPMI 1640 medium (Sigma) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma). Aliquots (0.1 ml) of drug solutions at 2× final concentration were dispensed into the wells of plastic microdilution trays using a Quick Spense II System (Dynatech Laboratories, Chantilly, Va.). The trays were sealed and frozen at −70°C until they were used in the study.

Antifungal susceptibility studies.

Broth microdilution testing was performed in accordance with the guidelines in NCCLS document M27-A2 (18) by using the spectrophotometric method of inoculum preparation, an inoculum concentration of (1.5 ± 1.0) × 10³ cells/ml, and RPMI 1640 medium buffered to pH 7.0 with MOPS. Yeast inocula (0.1 ml) were added to each well of the microdilution trays. The trays were incubated in air at 35°C, and MIC endpoints were read after 24 h for caspofungin (28a) and after 48 h of incubation for all other drugs. Susceptibility of isolates to amphotericin B was determined using Etest (AB BIODISK, Solna, Sweden) and RPMI 1640 agar with 2% glucose as described previously (21).

Following incubation, the broth microdilution wells were read with the aid of a reading mirror; the growth in each well was compared with that of the growth control (drug-free) well. The MICs of fluconazole, flucytosine, posaconazole, ravuconazole, voriconazole, and caspofungin were defined as the lowest concentration that produced a prominent decrease in turbidity (approximately 50% reduction in growth) compared with that of the drug-free control (18; Pfaller et al., submitted for publication). 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 intersected the scale on the strip edge (21). Quality control was ensured by testing the NCCLS-recommended strains, C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (18).

Interpretive criteria for fluconazole (S, ≤8 μg/ml; S-dose dependent, 16 to 32 μg/ml; R, ≥64 μg/ml) and flucytosine (S, ≤4 μg/ml; intermediate, 8 to 16 μg/ml; R, ≥32 μg/ml) were those published by Rex et al. (31) and the NCCLS (18). Interpretive criteria have not yet been defined for amphotericin B; however, because the study of Nguyen et al. (19) suggested that amphotericin B MICs of >1 μg/ml may indicate clinically resistant isolates of Candida spp., we determined the percentage of isolates inhibited by drugs at concentrations of ≤1 μg/ml to be susceptible in this study. Likewise, the new triazoles, voriconazole, ravuconazole, and posaconazole, and the echinocandin caspofungin have not been assigned interpretive breakpoints. For purposes of comparison and because pharmacokinetic data indicate that achievable levels in serum for these agents may exceed 1 to 2 μg/ml throughout the dosing interval (2-4, 37), we have employed a susceptibility breakpoint of ≤1 μg/ml for all four agents.

RESULTS AND DISCUSSION

The overall susceptibility of the 601 isolates of C. glabrata to seven systemically active antifungal agents is shown in Table 1. Caspofungin (MIC₉₀, 0.12 μg/ml; 100% S at a MIC of ≤1 μg/ml) and flucytosine (MIC₉₀, 0.12 μg/ml; 99.2% S) were the most-active agents and fluconazole (MIC₉₀, 32 μg/ml; 66.2% S) was the least-active agent against these isolates. A total of 46 isolates (7.7%) exhibited high-level resistance (MIC, ≥64 μg/ml) to fluconazole.

TABLE 1.

In vitro susceptibilities of 601 clinical isolates of C. glabrata to seven antifungal agents^a

Antifungal agent	MIC (μg/ml)b		% Sc	No. of strains for which MIC (μg/ml) was:
	50	90		0.03	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64
Amphotericin B	1	2	75.2		2	11	22	152	265	137	9	2	1
Flucytosine	0.06	0.12	99.2		382	204	7	1	1	1		3			2
Fluconazole	8	32	66.2						3	56	161	178	138	19	46
Posaconazole	0.5	2	85.4		1	12	74	234	192	58	12	4	14
Ravuconazole	0.25	1	90.7	3	51	121	206	112	52	23	21	11	1
Voriconazole	0.25	1	92.8	7	80	199	188	59	25	18	21	4
Caspofungind	0.03	0.06	100	377	182	30	14	3

^aAll isolates tested by broth microdilution according to NCCLS M27-A2 guidelines (18) or by Etest (amphotericin B) as part of the Artemis Global Antifungal Surveillance Program conducted in 2001 and 2002.

^b50 and 90, MIC₅₀ and MIC₉₀, respectively.

^cPercentage of strains susceptible at MICs of ≤8 μg/ml (fluconazole), ≤4 μg/ml (flucytosine), or ≤1 μg/ml (all other agents).

^dCaspofungin MICs read as the lowest concentration at which prominent inhibition (∼50%) is observed at 24 h of incubation (Pfaller et al., submitted).

Among the new extended-spectrum triazoles, voriconazole was the most active (MIC₉₀, 1 μg/ml; 92.8% S at a MIC of ≤ 1 μg/ml) and posaconazole was least active (MIC₉₀, 2 μg/ml; 85.4% S at MIC ≤ 1 μg/ml). Amphotericin B MICs were ≤1 μg/ml for 75.2% of isolates, whereas MICs were greater than 2 μg/ml for 12 isolates (2% of total) as determined by the Etest method.

Fluconazole susceptibility was highest in the Asia-Pacific region (80.5%) and lowest in North America (64%) and Latin America (62.1%) (Table 2). Only 3.9% of isolates from the Asia-Pacific region were resistant to fluconazole, compared to 10.3% in North America. Likewise, the extended-spectrum triazoles were most active in the Asia-Pacific region (90 to 96.1% S) and least active in North America (82.5% to 90.3% S). Flucytosine and caspofungin were both very active against isolates from all regions, whereas amphotericin B was most active against isolates from North America (80.4% S) and least active against isolates from Latin America (63.8% S) and the Asia-Pacific region (64.9% S).

TABLE 2.

In vitro susceptibilities of 601 clinical isolates of C. glabrata to seven antifungal agents stratified by geographic location^a

Antifungal agent	Locationb	MIC (μg/ml)c		% Sd	No. of strains for which MIC (μg/ml) was:
		50	90		0.03	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64
Amphotericin B	N. Am.	1	2	80.4			9	15	98	144	62	2	1
	L. Am.	1	2	63.8				1	13	23	20		1
	Eur.	1	2	73.3		2	1	3	27	66	30	5		1
	A-Pac.	1	2	64.9			1	3	14	32	25	2
Flucytosine	N. Am.	0.06	0.12	99.4		220	104	2	1	1	1		1			1
	L. Am.	0.06	0.12	100		32	24	2
	Eur.	0.06	0.12	98.5		87	45	1					1			1
	A-Pac.	0.06	0.12	98.7		43	31	2					1
Fluconazole	N. Am.	8	64	64.0						3	33	84	92	74	11	34
	L. Am.	8	16	62.1							7	15	14	18	2	2
	Eur.	8	16	65.2							12	40	36	36	4	7
	A-Pac.	8	16	80.5							4	22	36	10	2	3
Posaconazole	N. Am.	0.5	2	82.5		1	7	44	129	92	34	11	2	11
	L. Am.	1	2	86.2			2	5	15	28	7			1
	Eur.	0.5	2	88.9			3	18	55	44	12	1	1	1
	A-Pac.	0.5	1	90.9				7	35	28	5		1	1
Ravuconazole	N. Am.	0.25	2	87.3		27	69	107	56	30	17	14	10	1
	L. Am.	0.25	1	94.8	1	4	11	18	15	6	2	1
	Eur.	0.25	1	95.6	2	16	34	37	27	13	2	4
	A-Pac.	0.25	1	93.5		4	7	44	14	3	2	2	1
Voriconazole	N. Am.	0.25	1	90.3	1	45	106	104	28	15	14	14	4
	L. Am.	0.25	0.5	96.6	2	7	17	18	8	4		2
	Eur.	0.12	0.5	95.6	2	20	52	36	15	4	3	3
	A-Pac.	0.25	0.5	96.1	2	8	24	30	8	2	1	2
Caspofungine	N. Am.	0.03	0.06	100	191	111	22	5	2
	L. Am.	0.03	0.06	100	41	16	1	5
	Eur.	0.03	0.06	100	98	29	5	2	1
	A-Pac.	0.03	0.06	100	47	26	2	2

^aAll isolates tested by broth microdilution according to NCCLS M27 A2 guidelines (18) or by Etest (amphotericin B).

^bAbbreviations for locations: N. Am., North America (21 sites, 331 isolates); L. Am., Latin America (8 sites, 58 isolates); Eur., Europe (19 sites, 135 isolates); A-Pac., Asia-Pacific (11 sites, 77 isolates).

^c50 and 90, MIC₅₀ and MIC₉₀, respectively.

^dPercentage of strains susceptible at MICs of ≤8 μg/ml (fluconazole), ≤4 μg/ml (flucytosine), or ≤1 μg/ml (all other agents).

^eCaspofungin MICs read as the lowest concentration at which prominent inhibition (∼50%) is observed at 24 h of incubation (Pfaller et al., submitted).

Among the 46 isolates of C. glabrata that were resistant to fluconazole, all were very susceptible to both caspofungin (MIC₉₀, 0.06 μg/ml; 100% S at a MIC of ≤0.25 μg/ml) and flucytosine (MIC₉₀, 0.12 μg/ml; 100% S at a MIC of ≤0.25 μg/ml). In contrast, considerable cross-resistance was observed among the extended-spectrum triazoles: voriconazole (MIC₉₀, 4 μg/ml; 13% S at a MIC of ≤1 μg/ml), posaconazole (MIC₉₀, 16 μg/ml; 4% S at a MIC of ≤1 μg/ml), and ravuconazole (MIC₉₀, 8 μg/ml; 8.7% S at a MIC of ≤1 μg/ml). All 46 of these isolates were resistant (MIC ≥ 1 μg/ml) to itraconazole as well as fluconazole (data not shown). Amphotericin B MICs were ≤1 μg/ml for 78.3% of these azole-resistant strains, and there were no isolates for which amphotericin B MICs exceeded 2 μg/ml.

There is no question that C. glabrata has emerged as an important, and potentially resistant, opportunistic fungal pathogen (9, 10, 26, 30, 35, 40). Trick et al. (40) have demonstrated that C. glabrata alone among Candida spp. has increased as a cause of BSI in U.S. intensive care units. Other studies have shown that in certain regions of the United States, C. glabrata both is a common cause of BSI and is also often resistant to fluconazole (26, 30, 35, 42).

The U.S. findings, however, may not be universal (15, 24, 27). Recent surveys from France (34), Italy (17, 38), Finland (29), Iceland (5), and Taiwan (6, 7) indicate that C. glabrata has not increased as a cause of BSI and other serious infections to the same extent as that seen in the United States, and furthermore, isolates from these countries may not be as resistant to fluconazole as isolates from the United States.

In the present study we have compared directly the activities of four major classes of antifungal agents (antimetabolites, azoles, echinocandins, and polyenes) against recent clinically invasive isolates of C. glabrata from four different geographic regions worldwide. We have confirmed that the azoles are less active against isolates of C. glabrata from North America than those from other regions and have shown that azoles have the greatest activity against isolates from the Asia-Pacific region. Furthermore, we have demonstrated that both a very old agent, flucytosine, and a very new agent, caspofungin, exhibit potent activity against virtually all isolates of C. glabrata irrespective of their geographic origin or azole susceptibility. Although one might expect that a novel, newly introduced agent such as caspofungin would be highly active, the excellent potency of flucytosine against C. glabrata is not widely appreciated (15, 36). Both of these agents may play an important role in optimizing therapy in regions where decreased susceptibility to azoles is common.

Although amphotericin B is generally recommended for primary therapy of serious infections due to C. glabrata (15, 32), it is slowly becoming apparent that this agent is not universally active against this species of Candida and that higher doses of amphotericin B (i.e., 1 mg/kg of body weight/day) may be required for optimal treatment (13, 14, 19, 32). The data presented in Tables 1 and 2 confirm these observations and also suggest that decreased susceptibility to amphotericin B may be more of an issue in some regions than others. Whereas amphotericin B MICs in excess of 2 μg/ml were observed in less than 1% of North America isolates, this phenotype was seen in 4.4% of European isolates (Table 2).

The new triazoles have been shown to have good activity against some, but not all, fluconazole-resistant Candida spp. (12, 20, 25, 33). Voriconazole MICs of 1 μg/ml or less have been reported for most fluconazole-resistant strains of C. albicans (25, 33) and for essentially all strains of C. krusei (12, 25). It has also been suggested that the new triazoles may be active against strains of C. glabrata that are resistant to fluconazole (12). Although it is true that C. glabrata isolates for which fluconazole MICs are 16 to 32 μg/ml (susceptible-dose dependent) appear susceptible to voriconazole and the other new triazoles (87 to 97% S at MIC ≤ 1 μg/ml), MICs are usually ≥4 μg/ml for those strains resistant to fluconazole (25, 28). This cross-resistance is readily apparent in the data presented in Table 3. The clinical significance of these in vitro data, however, has yet to be determined.

TABLE 3.

In vitro susceptibilities of 46 fluconazole- and itraconazole-resistant isolates of C. glabrata to six antifungal agents^a

Antifungal agent	No. of strains for which MIC (μg/ml) was:
	0.03	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64
Amphotericin B				1	16	19	10
Flucytosine		21	24	1
Posaconazole				1		1	18	8	4	14
Ravuconazole		1				3	10	21	10	1
Voriconazole		1				5	16	20	4
Caspofunginb	22	21	2	1

^aAll isolates tested by NCCLS M27-A2 broth microdilution methods (18) or by Etest (amphotericin B). Fluconazole (MIC ≥ 64 μg/ml) and itraconazole (MIC ≥1 μg/ml) resistance defined according to NCCLS M27-A2 guidelines (18).

^bCaspofungin MICs read as the lowest concentration at which prominent inhibition (∼50%) is observed at 24 h of incubation (Pfaller et al., submitted).

In summary we have provided a global comparison of the in vitro activities of seven systemically active antifungal agents, comprising four different classes, against clinically significant isolates of C. glabrata. This information documents the geographic variation in azole activity on a global scale and demonstrates the universal activity of flucytosine and caspofungin against this species. This information should be useful in optimizing therapy of infections due to C. glabrata and will also serve as a baseline for future studies regarding the emergence of antifungal resistance in this species.