binafine, Caspofungin, Voriconazole, and Posaconazole against 30 Clinical Isolates ... Posaconazole, itraconazole and amphotericin B had lower MIC than the ...
Mycobiology 31(2): 95-98 (2003) Copyright © 2003 by The Korean Society of Mycology
In Vitro Antifungal Activities of Amphotericin B, Fluconazole, Itraconazole, Terbinafine, Caspofungin, Voriconazole, and Posaconazole against 30 Clinical Isolates of Cryptococcus neoformans var. neoformancs Young Ki Lee and Annette W. Fothergill
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Seoul Government Research Institute of Public Health & Environment 1 Fungus Testing Laboratory, Department of Pathology, The University of Texas Health Science Center at San Antonio (Received June 4, 2003) Aantifungal agents were tested against 30 clinical isolates of Cryptococcus neoformans var. neoformans using the NCCLS method (M27-A2). Posaconazole, itraconazole and amphotericin B had lower MIC than the remaining four antifungal agents. The MIC result for posaconazole was over 220-fold lower active than fluconazole. Fluconazole MICs for most isolates fell within the dose-dependant range. The overall MIC ranges and MIC50s were amphotericin B (0.03-0.25; 0.25), fluconazole (0.5-64; 16), itraconazole (0.015-1; 0.125), terbinafine (0.06->2; 1), caspofungin (8-32; 32), voriconazole (0.015-0.5; 0.25), and posaconazole (0.015-0.25; 0.06 µg/ml), respectively. In conclusion, the MIC50s of these drugs did not exhibit any sign of an upward shift with the exception of fluconazole and tendency cross-resistance between the seven drugs was not observed. We conclude that in vitro resistance to antifungal agents has not significantly changed despite the recent wide-spread use of triazoles for long-term treatment of Cryptococcal meningitis. KEYWORDS: Antifungal agents, Cryptococcal meningitis, Cryptococcus neoformans, MIC
In the 1980s with the increased incidence of AIDS, infections with C. neoformans was on the rise. However, during the 1990s due to the highly active antiretroviral therapy (HAART), incidence of Cryptococcosis was decreased. This remains one of the most serious mycoses that primarily affects HIV-infected patients and those receiving immunosuppressive treatment for cancer, organ transplantation, and other serious medical conditions (Brandt et al., 2001; Yildiran et al., 2000). The treatment of disease due to C. neoformans has improved dramatically over the last several decades because of establishement of antifungal agents; amphotericin B (1950s), flucytosine (1970s), fluconazole and itraconazole (1990s) (Saag et al., 2000; White et al., 1998). The antifungal drugs currently available for the treatment of invasive mycoses can be divided in 4 different classes on the basis of their mechanisms of action. alteration of membrane function (amphotericin B); inhibition of DNA or RNA synthesis (flucytosine); inhibition of ergosterol biosynthesis (azoles{fluconazole, itraconazole, voriconazole, posaconazole, and ravuconazole}, terbinafine); and inhibition of glucan syn thesis (echinocandins{caspofungin, micafungin, and anidulafungin}) (Perea and Patterson, 2002; Ryder et al., 1998). Current regimens for treatment of the disease remain focused on amphotericin B, with or without flucytosine, for induction treatment, while fluconazole remains the agent of choice for long-term maintenance treatment. For patients in whom fluconazole can not be used, itra-
conazole is an acceptable but less effective alternative (Sheehan et al., 1999; Yidliran et al., 2002). As described above, this gold standard method (combination therapy) has proven to be superior for treatment of Cryptococcal menginintis whether in HIV-negative or positive patients (Davey et al., 1998; Pappas et al., 2001). The azole antifungal agents in clinical use are classified as imidazoles (e.g., ketoconazole and miconazole, clotrimazole) or triazoles (e.g., itraconazole, fluconazole, voriconazole), respectively (Sheehan et al., 1999). The triazole antifungal agents are less toxic and better tolerated than amphotericin B alone or combined with flucytosine, so, have been frequently used in the treatment of cryptococcosis (Ana, 1998; Pfaller et al., 2001). In general, fungi can be intrinsically resistant to antifungal drugs (primary resistance) or can develop resistance in response to exposure to the drug during treatment (secondary resistance) (Perea and Patterson, 2002). Actually, the longterm use of fluconazole as maintenance therapy in persons with AIDS has generated concern that less susceptible strains may begin to emerge (Perfect et al., 1996). In a few studies, there have been some reports regarding the frequent development of secondary resistance during treatment (Brandt et al., 2001; Davey et al., 1998; Mondon et al., 1999). Recently, the National Committee for Clinical Laboratoty Standards (NCCLS) has established guidelines for standardized susceptibility testing of Yeasts with azoles, amphotericin B, and flucytosine in the form of the M27A2 broth dilution assay (NCCLS, 1997; Saag et al.,
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2000). This method is widely used in the evaluation of established antifungal agents because of its reproducible and convenient characteristics. To date, there have been few published reports of resistance to amphotericin B, itraconazole, terbinafine, caspofungin, voriconazole, or posaconazole and cross-resistant between these antifungal agents (Davey et al., 1998). The purpose of this investigation was designated to assess the in vitro antifungal activities and trends of crossresistance of 30 clinical isolates of Cryptococcus neoformans var. neoformans against seven drugs (amphoterin B, fluconazole, itraconazole, terbinafine, caspofungin, voriconazole, and posaconazole).
Materials and Methods Isolates. A total of 30 clinical yeast isolates were used in this study. Isolates were identified to the species level by conventional morphological and biochemical methods o and stored at 70 C on SABDEX agar slants Before testing, all isolates were subcultured on the sabouraud dextrose agar (Remel, Lenexa, KS) to ensure optimal growth characteristics. Antifungal agents. The antifungal agents tested were amphotericin B (Bristol-Meyers Squibb, USA), fluconazole, voriconazole (Pfizer, USA), itraconazole (Janssen Pharmaceutica, USA), posaconazole (Schering-Plough Research Institute, USA), caspofungin (Merk, USA), and terbinafine (Novartis, USA) and were provided as standard powders by the manufactures. Stock solutions were prepared in 50% of polyethylene glycol (itraconazole, terbinafine, voriconazole, and posaconazole) or distilled water (amphotericin B, fluconazole, caspofungin). Serial twofold dilutions were prepared as recommended in the NCCLS approved standard M27-A2. The final concentration of the solvent did not exceed 1% in any well. The drug dilutions were dispensed into 96-well microdilution plates that were then sealed and frozen at 70oC until needed. Antibiotic Medium 3 (Difco Laboratories, USA) was used for amphotericin B and caspofungin testing, while RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) (Angus buffers, Niagara Falls, NY,USA) was utilized for the remaining antifungal agents. Antifungal susceptibility testing. The reference microdilution method M27-A2 was used for in vitro antifungal testing. Final drug concentrations ranged from 16 to 0.03 µg/ml for amphotreicin B, from 64 to 0.125 µg/ml for fluconazole and caspofungin, from 8 to 0.015 µg/ml for itraconazole, voriconazole, and posaconazole, and from 2 to 0.004 µg/ml for terbinafine. A final inoculum of 3 0.5~2.5×10 cells was prepared using a spectrophotometer. Each microdilution well containing 100 µl of the 2×
drug concentrations was inoculated with 100 µl of the diluted (2×) inoculum suspension (final volume in each well was 200 µl). Growth and sterility control wells were included for each isolate tested. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control organisms and were included each time that a set of isolates was tested (Pfaller et al., 1995). After o inoculation, plates were incu- bated for 72 h at 35 C and readings were taken daily. Absorbance was determined spectrophotometrically at 630 nm after agitation of the plates. The MICs of fluconazole, itraconazole, voriconazole, posaconazole and terbinafine were defined as the lowest drug concentration exhibiting approximately 50% reduction of growth compared with the control well (Rex et al., 2001). The MICS of amphotericin B and caspofungin were defined as the lowest drug concentration giving 100% inhibition (optically clear).
Results Testing was done by the NCCLS broth microdilution method. The MIC ranges for amphotericin B, fluconazole, and itraconazole were within the expected ranges for the quality control strain, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258 (data not shown). Ranges for the other antifungal agents have not yet been established. Table 1. summarizes the in vitro antifungal activities of 30 clinical isolates of C. neoformans var. neoformans to seven drugs. The results are reported as MIC ranges and the MIC required to inhibit 50% of the isolates, respectively. A broad range of MICs were observed for both fluconazole and itraconazole. Fluconazole MICs ranged between 0.5 and 64 µg/ml, while itraconazole ranged between 0.015 and 1 µg/ml. The MIC50 results for caspofungin and posaTable 1. In vitro antifungal activities of amphoterincin B, fluconazole, itraconazole, terbinafine, caspofungin, voriconazole, and posaconazole against clinical isolates of Cryptococcus neoformans var. neoformans MIC (µg/ml)a Antifungal agent
48 h Range
Amphotericin Fluconazole Itraconazole Terbinafine Caspofungin Voriconazole Posaconazole a
B0.03~0.25 0.25~64 0.015~2 0.06~2 8~32 0.015~0.5 0.015~0.25
72 h MIC50 b
0.25 (0.144 ) 16 (11.8) 0.125 (0.066) 0.5 (0.483) 32 (18.4) 0.25 (0.162) 0.06 (0.046)
Range
MIC50
0.03~0.25 0.5~64 0.015~1 0.06~>2 8~32 0.015~0.5 0.015~0.25
0.25 (0.167) 16 (10.5) 0.125 (0.061) 1 (0.558) 32 (18.4) 0.25(0.182) 0.06 (0.046)
MICs were determined after 72 h of incubation for isolates of C. neoformans var. neoformans under the NCCLS conditions (NCCLS, 1997). b Mean MIC50.
In Vitro Antifungal Activities of Amphotericin B, etc. Against Cryptococcus neoformans
conazole at both 48 and 72 h remained the same, while results for the remaining antifungal agents were similar to within +1 dilution. According to the MIC50 values obtained at 72 h of incubation, posaconazole gave the lowest values (0.06 µg/ml), followed by itraconazole (0.125 µg/ml), amphotericin B (0.25 µg/ml), and voriconazole (0.25 µg/ml), respectively. The MIC50 results for posaconazole was over 220-fold lower active than fluconazole, while amphotericin B, displayed nearly identical activity to voriconazole.
Discussion To date, there are a few studies on the susceptibility of C. neoformans, however direct comparisons of MIC data between studies are complicated by variability in test methods (Saag et al., 2000). The NCCLS M27-A2 method includes details for testing C. neoformans but does not specify any interpretive breakpoints for amphotericin B, fluconazole, itraconazole, terbinafine, caspofungin, voriconazole, and posaconazole against C. neoformans, except for flucytosine (Kirkpatrick et al., 1998; Rex et al., 2001). Therefore, This study tested the in vitro antifungal activity of seven drugs using M27-A2, which is commonly used worldwide, and examined their resistance properties through comparing the results of other researches. Overall, According to the MIC50 values using the microdilution method, posaconazole gave the lowest vlaues (0.06 µg/ml), followed by itraconazole (0.125 µg/ml), amphotericin B (0.25 µg/ml), and voriconazole (0.25 µg/ml), respectively. These results on the in vitro suscepibilities of these antifungal agents against C. neoformans were similar to those published earlier (Pfaller et al., 2001). According to the result of a study by Brandt et al. (2001) on amphotericin B, fluconazole and itraconazole using the same test method, itraconazole appeared to be most active as its MIC range and MIC50 were 0.06~0.5 and 0.125 µg/ ml respectively, followed by amphotericin B (0.125~0.5, 0.25 µg/ml) and fluconazole (2~8, 4 µg/ml). There was no remarkable resistance property found. In addition, in a test by Pfaller et al., 2001, the MIC range and MIC50 of posaconazole, itraconazole and fluconazole were 0.015~1; 0.12, 0.03~1; 0.25, and 0.25->128; 8 µg/ml respectively, similar to the results of the previously mentioned. There are also many test reports showing similar results although the tests used different method (macrodilution method). According to the results of a study by Franzot and Hamdan, 1996, using antifungal agents against clinical and environmental isolates, the MIC range and MIC50 of itraconazole, amphotericin B and fluconazole were 0.03~ 0.25; 0.125, 0.25~1; 0.5, and 0.5~16; 4 µg, and in a test by Yildiran et al., 2002, posaconazole appeared to be most active (MIC range; MIC50, ≤0.015~≥8; ≤0.015), followed itraconazole (0.015~2; 0.015), voriconazole (≤0.125~
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32; ≤0.125), amphotericin B (0.125~0.5; 0.5) and fluconazole (≤0.125~≥64; 1 µg/ml). This is similar to the results of the present study, However, the MIC50 of voriconazole and amphotericin B in tests by Yildiran et al., 2002, and Ana, 1998, were opposite to those presented here. In a test by Ryder et al., 1998, (macrodilution method), the MIC range and MIC50 of terbinafine against C. neoformans were 0.06~0.125; 0.125 µg/ml respectively, which were fourfold different from the results of the present test. Caspofungin is not recommended for Cryptococcal meningitis (Roling et al., 2002) was included in this study simply because of the possibility of cross resistance. When comparing the result of the susceptibility of the seven antifungal agents used in this study with those of previous studies relating to C. neoformans, a dose-dependant response, as the MIC50 of fluconazole increased slightly (16 µg/ml), but the increase was not regarded as a remarkable enhancement of resistance. In conclusion, our results showed that the MIC ranges and MIC50s of these seven antifungal agents did not increase significantly compared to the other studies despite the recent widespread use of triazoles for long-term treatment of cryptococcal meningitis and evident possible cross-resistant between these drugs was not noted.
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