In Vitro Activity of Anidulafungin against Selected Clinically Important Mold Isolates

Abstract

In this study, we evaluated the in vitro activity of anidulafungin against selected mold isolates. Anidulafungin showed promising activity against Bipolaris spicifera, Exophiala jeanselmei, Fonsecaea pedrosoi, Madurella mycetomatis, Penicillium marneffei, Phialophora verrucosa, Pseudallescheria boydii, Sporothrix schenckii, and Wangiella dermatitidis.

The incidence of invasive fungal infections has increased in the past two decades. Candida species and Aspergillus species are the most common etiologic agents causing invasive fungal infections. Although rare overall, Fusarium species are the second most common mold isolate after Aspergillus species (14, 15). Some other less common filamentous fungi, like Penicillium, Bipolaris, Pseudallescheria, and Scedosporium species, have also been emerging as causative agents of opportunistic infections in immunocompromised patients (7, 14, 24, 26, 31).

Amphotericin B has been historically accepted as the “gold standard” for the treatment of most fungal infections, although it is known to have poor outcomes in immunocompromised patients with severe mold infections (13). Alternative therapeutic agents, like new azoles and echinocandins, are under clinical evaluation (18). The relatively recent approvals of caspofungin for aspergillosis and voriconazole for aspergillosis, fusariosis, and scedosporidiasis have revolutionized the field of antifungal therapy (2, 4, 5, 8-11, 19, 29, 30).

Anidulafungin is an echinocandin with excellent in vivo and in vitro activities against Candida spp. and Aspergillus spp. (17, 20-23, 27, 28). The in vitro activity of anidulafungin against less common but clinically emerging filamentous fungi has been evaluated in a limited number of studies (6, 22, 25). We sought to evaluate its in vitro activity against selected mold isolates in comparison with the activities of voriconazole and amphotericin B.

A collection of 74 clinical mold isolates (Table 1) were tested. Isolates were obtained from the Department of Pathology, University of Texas, Medical Branch, Galveston. All isolates were stored at −80°C, and each isolate was subcultured on potato dextrose agar slants (Becton, Dickinson and Company, Sparks, Md.) at least twice to ensure purity and viability. The quality control strains were Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258.

TABLE 1.

Susceptibilities of 74 selected clinical mold isolates to anidulafungin, voriconazole, and amphotericin B

Isolate (n)	Time (h)	MIC-2 (μg/ml) ofanidulafungin		MIC (μg/ml)
				Amphotericin B		Voriconazole
		Range	Median	Range	Median	Range	Median
Absidia corymbifera (2)	24	2-8	NAa	2-4	NA	>16	NA
	48	8-16	NA	4-8	NA	>16	NA
Acremonium spp. (3)	48	1->16	>16	2->16	>16	0.25-1	1
	72	1->16	>16	2->16	>16	0.25-2	1
Alternaria alternata (2)	48	16	NA	8	NA	>16	NA
	72	>16	NA	16	NA	>16	NA
Basidiobolus ranarum (1)	24	16	NA	>16	NA	0.5	NA
	48	>16	NA	>16	NA	0.5	NA
Bipolaris spicifera (4)	48	0.5-4	2.5	1-2	1.5	2-16	2
	72	1-8	3	1-4	2	2-16	5
Cunninghamella bertholletiae (5)	24	0.25->16	>16	4-16	4	>16	>16
	48	0.25->16	>16	8-16	8	>16	>16
Exophiala jeanselmei (5)	48	0.125-2	0.125	2-8	2	0.5-2	0.5
	72	0.125-2	1	2-16	2	0.5-2	0.5
Fonsecaea pedrosoi (4)	48	0.5-2	1.5	1-4	1.5	0.06-0.25	0.25
	72	0.5-4	1.5	2-4	3	0.125-0.5	0.375
Madurella mycetomatis (3)	120	1	1	NDb	ND	4-8	4
Mucor sp. (3)	24	0.25-16	8	1	1	>16	>16
	48	>16	>16	2	2	>16	>16
Paecilomyces spp. (5)	48	0.03->16	8	8->16	>16	0.25-16	1
	72	0.03->16	8	16->16	>16	0.25-16	2
Penicillium mameffei (3)	48	0.5-2	2	0.5-8	8	0.03-0.06	0.03
	72	2	2	2-8	8	0.03-0.125	0.06
Phialophora verrucosa (5)	48	0.03-0.125	0.06	2-4	2	0.125-1	0.5
	72	0.03-0.25	0.125	2-4	2	0.125-1	1
Pseudallescheria boydii (5)	48	2-4	4	>16	>16	0.25-1	0.5
	72	2-8	4	>16	>16	0.5-1	0.5
Rhizomucor pusillus (2)	24	4-8	NA	1-2	NA	>16	NA
	48	8	NA	4	NA	>16	NA
Rhizopus spp. (2)	24	>16	NA	1-2	NA	8->16	NA
	48	>16	NA	1-2	NA	16->16	NA
Scedosporium apiospermum (5)	48	2->16	4	4->16	>16	0.5->16	0.5
	72	2->16	4	16->16	>16	0.5->16	1
Scedosporium prolificans (5)	48	8-16	8	>16	>16	>16	>16
	72	8->16	8	>16	>16	>16	>16
Scopulariopsis brevicaulis (1)	48	8	NA	>16	NA	>16	NA
	72	>16	NA	>16	NA	>16	NA
Sporothrix schenckii (3)	48	2	2	0.5-2	0.5	16->16	>16
	72	2-4	4	1-4	2	>16	>16
Wangiella dermatitidis (5)	48	0.5-2	2	2->16	4	0.5-2	0.5
	72	2-8	2	4->16	4	0.5-2	1
QC C. parapsilosis (22,019)c	48	2	NA	2	NA	0.06	NA
QC C. krusei (6,258)c	48	0.125	NA	2	NA	0.25	NA

^aNA, not applicable (n < 3).

^bND, not done due to the limited number of isolates.

^cMIC-2 ranges for quality control (QC) isolates tested with amphotericin B, voriconazole, and anidulafungin (1, 16).

Anidulafungin (Vicuron Pharmaceuticals, Inc., King of Prussia, Pa.), voriconazole (Pfizer Pharmaceutical Group, New York, N.Y.), and amphotericin B (Bristol-Myers Squibb, Wallingford, Conn.) were dissolved in 100% dimethyl sulfoxide (Fisher Chemicals, Fair Lawn, N.J.) and then were further diluted (1:50) in 2× RPMI 1640 medium (Sigma Chemical Company, St. Louis, Mo.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer according to the recommendations of National Committee for Clinical Laboratory Standards (NCCLS) approved standard M38-A (16) to yield two times the final strength required for the test. Amphotericin B was diluted 1:50 with 2× antibiotic medium 3 (AM3; BBL, Cockeysville, Md.) buffered with 0.1 M sodium phosphate to pH 7. Final drug concentrations were 0.03 to 16 μg/ml for all drugs. Serial dilutions of each drug were dispensed to microdilution plates and stored at −80°C until use.

In vitro testing was carried out following the NCCLS M38-A method. Briefly, each isolate was grown on potato dextrose agar slants at 35°C for a period of 7 days. The stock suspensions were prepared by covering the mature fungal colonies with 1 ml of sterile 0.85% saline and scraping gently. The turbidity of conidial spore suspensions was measured at 530 nm and adjusted to 80 to 82% transmittance, except for Pseudallescheria boydii and Rhizopus arrhizus, which were adjusted to 68 to 70% transmittance. The adjusted suspensions were diluted in distilled water 1:50 (except P. boydii, diluted 1:25) to obtain a final (2×) inoculum of 0.4 × 10⁴ CFU/ml. Microtiter trays were thawed, and 100 μl of fungal suspensions was added to each well. Microdilution plates were incubated at 35°C, and MICs were read visually at the optically clear (MIC with 100% inhibition of growth [MIC-0]) and prominent growth inhibition (MIC at which 50% of the isolates tested are inhibited [MIC-2]) endpoints after incubation for 48 and 72 h, or longer if necessary.

All isolates produced enough visible growth after 48 to 72 h, except for Madurella mycetomatis, which produced visible growth at about 120 h. Zygomycetes grew rapidly within 24 h. MIC-2 ranges and median MICs of anidulafungin and MIC ranges and median MICs of voriconazole and amphotericin B at 48 and 72 h are summarized in Table 1. For the zygomycetes, as recommended in NCCLS standard M38-A, 24- to 48-h MICs are presented because of their rapid growth. For the other isolates, when comparing 48- and 72-h MIC-2s, most of the values were similar or had a 1-dilution increase at 72 h.

Anidulafungin demonstrated potent to moderate activity against Bipolaris spicifera (MIC-2 range, 1 to 8 μg/ml), Exophiala jeanselmei (0.125 to 2 μg/ml), Fonsecaea pedrosoi (0.5 to 4 μg/ml), Madurella mycetomatis (1 μg/ml), Paecilomyces spp. (0.03 to >16 μg/ml), Penicillium marneffei (2 μg/ml), Phialophora verrucosa (0.03 to 0.25 μg/ml), Pseudallescheria boydii (2 to 8 μg/ml), Sporothrix schenckii (2 to 4 μg/ml), and Wangiella dermatitidis (2 to 8 μg/ml). It showed no activity against the zygomycetes. None of the three drugs showed in vitro activity against Absidia spp., Alternaria spp., Scedosporium prolificans, and Scopulariopsis brevicaulis. The MICs of amphotericin B were the lowest for most of the zygomycetes. Echinocandins prevent fungal growth by inhibiting β-glucan synthesis in the fungal cell wall. Therefore, we might expect echinocandins to have broad-spectrum antifungal activity because of the presence of β-glucan in cell wall structures of many different fungal species (Z. Odabasi, V. L. Paetznick, J. R. Rodriguez, E. Chen, M. R. McGinnis, and L. Ostrosky-Zeichner, 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-1021, 2003). However, echinocandins have thus far been used only against Aspergillus and Candida spp. Fungi having reduced β-glucan in their cell wall, like Cryptococcus and zygomycetes, are usually resistant to echinocandins; thus, the β-glucan content of the organism may be at least partially related to the suceptibility to these drugs (12, 22, 32). Caspofungin has shown activity against Exophiala jeanselmei, Fonsecaea pedrosoi, Paecilomycess variotii, and Scedosporium apiospermum but no activity against Rhizopus arrhizus, Paecilomyces lilacinus, and Scedosporium prolificans (3). This is similar to our findings with anidulafungin. In a previous study, anidulafungin showed variable activity against Bipolaris spp., Pseudallescheria boydii, and Sporothrix schenkii and some activity against three different Phialophora species (6). We found anidulafungin to be very active against five Phialophora verrucosa isolates. In another previous study, anidulafungin was also found to be active against one Acremonium spp., one Paecilomyces sp., and five Pseudallescheria boydii isolates (22), which is consistent with our findings.

In summary, against a limited number of isolates in our study, anidulafungin demonstrated promising in vitro activity for a variety of molds for which we have few alternative therapeutic options. Further investigation is warranted.