In Vitro Activity of the New Azole Isavuconazole (BAL4815) Compared with Six Other Antifungal Agents against 162 Cryptococcus neoformans Isolates from Cuba

Abstract

Cuban Cryptococcus isolates (n = 165) were tested in vitro against amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole, giving MIC₉₀ values of 0.25, 8, 4, 0.25, 0.125, 0.016, and 0.016 μg/ml, respectively. Isavuconazole and posaconazole seem to be potentially active drugs for treating cryptococcal infections.

Antifungals available for therapy of cryptococcosis are limited to amphotericin B, flucytosine, and fluconazole; however, the side effects associated with administration of amphotericin B and flucytosine may restrict their use (17, 25). A new generation of triazoles, including posaconazole, voriconazole, ravuconazole, and isavuconazole, has been developed. These agents possess potent broad-spectrum activity and favorable pharmacokinetic profiles (14, 18, 23). Isavuconazole is a water-soluble triazole that is suitable for oral and intravenous administration; its active moiety, BAL4815, is a potent inhibitor of ergosterol biosynthesis. In vitro, the active drug shows activity against all major opportunistic and pathogenic fungi (14, 20, 21, 23).

Previous studies have been done regarding the activity of the new azoles against a wide variety of fungi. This report summarizes the in vitro activities of isavuconazole and six other antifungal drugs on a large number of Cuban Cryptococcus isolates from clinical and environmental origins by a broth microdilution method in accordance with the CLSI (formerly NCCLS) M27-A2 guidelines (12). To our knowledge, this is the first report evaluating the susceptibility of this yeast to this new triazole and the largest study ever done with Cuban Cryptococcus strains.

(Part of this work was presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 27 to 30 September 2006 [10a].)

A total of 165 Cryptococcus strains were included: 117 were obtained from clinical samples, including one isolated from a cheetah imported from South Africa. Forty-five strains were isolated from pigeon guano in three different geographical locations of Cuba: Havana City (n = 8), Cienfuegos (n = 6), and Pinar del Río (n = 31). Species identification was initially performed by standard mycological methods (9), and samples were stored on sterile water at room temperature until the study was carried out. Before use, the identities of all isolates were confirmed with a commercial identification system (Auxacolor 2; Bio-Rad, Marnes-la-Coquette, France). Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 strains were used for quality control.

Standard antifungal powders of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole were used and were obtained from Sigma (The Netherlands), Valeant Pharmaceuticals (The Netherlands), Pfizer Central Research (United Kingdom), Janssen-Cilag (The Netherlands), Pfizer Central Research (United States), Schering Plough (United States), and Basilea Pharmaceutica (Switzerland), respectively. The stock solutions of the drugs were prepared in the appropriate solvent (12).

Broth microdilution testing was performed in accordance with the guidelines in CLSI document M27-A2 (12). The final concentrations of the antifungal agents were 0.016 to 8 μg/ml for amphotericin B, itraconazole, voriconazole, and posaconazole; 0.063 to 32 μg/ml for flucytosine and fluconazole; and 0.004 to 4.00 μg/ml for isavuconazole. Drug-free and yeast-free controls were included. The MICs at 48 and 72 h were read optically and spectrophotometrically at 420 nm after agitation. The MIC was defined as the lowest concentration of drug showing no growth for amphotericin B and a prominent reduction of growth (≥50%) for the other antifungals compared to the drug-free growth control.

All strains were identified as Cryptococcus neoformans var. grubii, except one obtained from an animal (Cryptococcus gattii) and two obtained from environmental samples from Pinar del Rio (Cryptococcus albidus and Cryptococcus albidisimilus). No differences between visual and spectrophotometric readings were observed, and the MICs for the quality control strains were all within the reference ranges (data not shown). Table 1 summarizes the in vitro susceptibilities of all the isolates according to the origin of the strain. In this study, most of the C. neoformans isolates showed quite uniform patterns of susceptibility to the antifungal agents tested. All of the strains were susceptible to amphotericin B, voriconazole, posaconazole, and isavuconazole.

TABLE 1.

In vitro susceptibilities of Cryptococcus isolates to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole according to origin and underlying disease of patients

Origin (no. of strains)	Antifungal agent	MIC (μg/ml)
		Range	50%	90%
Environmental (45)	Amphotericin B	0.016-0.5	0.25	0.5
	Flucytosine	0.5-64	4	8
	Fluconazole	0.25-64	4	8
	Itraconazole	0.016-0.5	0.063	0.25
	Voriconazole	0.016-0.25	0.063	0.25
	Posaconazole	0.016-0.125	0.31	0.63
	Isavuconazole	0.002-0.031	0.004	0.016
Clinical (117)	Amphotericin B	0.031-1	0.25	0.25
	Flucytosine	0.063-8	4	8
	Fluconazole	0.25-8	2	4
	Itraconazole	0.016-1	0.031	0.25
	Voriconazole	0.016-0.25	0.031	0.125
	Posaconazole	0.016-0.5	0.004	0.016
	Isavuconazole	0.002-0.063	0.004	0.016
AIDS patients (85)	Amphotericin B	0.031-1	0.25	0.25
	Flucytosine	0.125-8	4	8
	Fluconazole	0.25-8	2	2
	Itraconazole	0.016-0.5	0.031	0.125
	Voriconazole	0.016-0.25	0.031	0.125
	Posaconazole	0.016-0.125	0.031	0.125
	Isavuconazole	0.002-0.063	0.008	0.016
Non-AIDS patients	Amphotericin B	0.125-1	0.25	1
(32)	Flucytosine	0.063-8	2	4
	Fluconazole	0.25-8	2	8
	Itraconazole	0.016-1	0.031	0.063
	Voriconazole	0.016-0.25	0.063	0.063
	Posaconazole	0.016-0.5	0.063	0.125
	Isavuconazole	0.004-0.063	0.008	0.031
Total	Amphotericin B	0.016-1	0.25	0.25
	Flucytosine	0.063-64	4	8
	Fluconazole	0.25-64	2	4
	Itraconazole	0.016-1	0.031	0.25
	Voriconazole	0.016-0.25	0.031	0.125
	Posaconazole	0.002-0.125	0.004	0.016
	Isavuconazole	0.002-0.063	0.004	0.016

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

When all of the strains were considered together, the widest ranges and highest MICs were for flucytosine (0.063 to 64 μg/ml) and fluconazole (0.25 to 64 μg/ml). The lowest MICs were for isavuconazole, voriconazole, and posaconazole.

The environmental isolates seem to be less susceptible to fluconazole than the clinical ones.

Strains obtained from human immunodeficiency virus-negative patients showed lower MIC₉₀s, especially for amphotericin B and fluconazole, compared with those from AIDS patients.

Our results on the in vitro activities of the main antifungal drugs against C. neoformans are similar to those published previously (3, 7, 8, 16, 19, 24). Amphotericin B has long successfully been used to treat various yeast and mold infections. Unfortunately, its clinical use is hindered by intrinsic nephrotoxicity (5). There are no defined amphotericin B breakpoints by CLSI for C. neoformans. For this reason, breakpoints were used according to Nguyen and Yu (13) and Lozano et al. (11). They define an isolate as resistant with a MIC of ≥2 μg/ml, which was found to be associated with therapeutic failure. Although isolates from non-HIV patients showed higher amphotericin B MICs, the studied strains appear to be susceptible to this drug.

Flucytosine is a drug with a limited spectrum of action that includes Candida spp. and C. neoformans (5). The CLSI M27-A2 document recommends that isolates for which MICs are 32 μg/ml be regarded as resistant to this drug (12). By this definition, none of the studied clinical isolates was resistant; nevertheless, a justified fear of the emergence of resistance has led to its use in combination with amphotericin B in vitro and in patients with cryptococcosis (4, 7, 22, 25).

Among the azoles, fluconazole showed the lowest activity. In fact, previous reports have already demonstrated the low activity of this drug against C. neoformans isolates, even though it has proven to be more active in vivo (1). According to these authors, the good therapeutic results obtained are largely attributable to its high concentrations in cerebrospinal fluid. Although no resistance has been found in the present collection of isolates, we continue paying attention to the emerging resistance to this antifungal agent due to its widespread use as primary prophylaxis in AIDS patients.

Fortunately, the antifungal armamentarium has increased during the past two decades with the addition of several agents and, although the echinocandins seem to be inactive, other new triazoles are expected to be licensed shortly (5, 14). Voriconazole has in vitro activity against cryptococcal yeasts (15) including those that are resistant in vitro to fluconazole (13). Our results show that voriconazole was more potent than itraconazole against Cuban Cryptococcus isolates. However, these promising in vitro results should be complemented by clinical confirmation.

Posaconazole and isavuconazole belong to the latest generation of azole antifungal agents that are being investigated for their role in treating serious infections due to yeasts and molds. In addition to potent activity, they are well tolerated and offer a diminished toxicity profile compared with other currently marketed systemic antimycotics and their pharmacokinetics are characterized by slow elimination, low plasma clearance, and extensive tissue distribution (14, 26, 27).

Posaconazole is the broadest-spectrum azole licensed to date. It exhibits linear pharmacokinetics in volunteers (6) and has demonstrated safety and efficacy comparable to those of fluconazole in human and animal studies, although there is no intravenous formulation available (26). In agreement with our results with Cryptococcus, other in vitro studies have documented a potency and spectrum of activity against clinically important yeasts and Aspergillus spp. similar to those of itraconazole and superior to those of fluconazole. Consistent with these results, in vivo studies have demonstrated efficacy in treating infections due to Candida spp. and C. neoformans with this antifungal agent (2, 10).

The prodrug isavuconazole is a water-soluble triazole precursor that is suitable for oral and intravenous administration. Its active moiety, BAL4815, is a potent inhibitor of ergosterol biosynthesis (14, 20, 21). In vitro, the drug shows broad-spectrum activity against all major opportunistic and true pathogenic fungi (14, 23). In animals as well as in humans, the pharmacokinetics of this drug are characterized by slow elimination, low plasma clearance (approximately 10% of liver blood flow), and extensive tissue distribution (20, 21). To our knowledge, this is the first report evaluating the action against Cryptococcus spp. of isavuconazole, which seems to be the most effective antifungal drug in terms of in vitro activity. In conclusion, our results indicate that there has been no significant shift in the MICs of amphotericin B and fluconazole for Cuban C. neoformans (8), despite the widespread use of these agents in persons with AIDS. Given the high oral bioavailability and the well-tolerated nature of the new azoles, they might become an important addition to the armamentarium of antifungal agents: both voriconazole and posaconazole show strong antifungal activity against Cuban Cryptococcus strains in vitro, while isavuconazole showed even lower MICs. These promising results still need to be correlated with clinical outcome.