Isham, Nancy C. M(ASCP); Ghannoum, Mahmoud A. PhD
Center for Medical Mycology, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH.
This study was supposed by Pfizer, Inc, New York, NY.
Address correspondence and reprint requests to Mahmoud A. Ghannoum, PhD, Center for Medical Mycology, University Hospita;s of Cleveland, OH 44106. E-mail: firstname.lastname@example.org.
Caspofungin is generally regarded as fungicidal, and voriconazole is described as fungistatic against Candida species. In this study, we challenged these statements by testing the fungicidal activity of these 2 agents against 197 non-albicans Candida isolates. Minimum inhibitory concentration testing was performed by a microbroth dilution method according to the M27-A2 standard of the Clinical Laboratory Standards Institute, whereas minimum fungicidal concentration determinations were performed by subculturing the contents of each visibly clear well from the minimum inhibitory concentration panel. Fungicidal activity was defined as a 99.9% or greater reduction in the number of colony-forming unit per milliliter. Voriconazole demonstrated greater inhibitory activity than caspofungin against the non-albicans Candida tested and exhibited fungicidal activity against 5% of these strains. Interestingly, almost one third of the non-albicans yeast isolates tested versus caspofungin exhibited fungistatic rather than fungicidal activity. This investigation demonstrates the lack of universal fungicidal and fungistatic activity of caspofungin and voriconazole against non-albicans Candida strains.
With the increase in immunocompromised patient population because of human immunodeficiency virus/acquired immune deficiency syndrome, aggressive cancer therapies, and transplantation, the incidence of invasive fungal infections is on the rise. Candida species, almost half of which are non-albicans strains, are the fourth most common cause of bloodstream infection and are the leading cause of invasive fungal infection among hospitalized patients.1 Nosocomial bloodstream infections due to Candida species are associated with a 40% crude mortality rate in the United States, which is the highest rate relative to infectious bacterial species.2
Current antifungal agents for the treatment of invasive fungal infection often involve the use of voriconazole or caspofungin.3,4 In certain clinical settings such as difficult-to-manage meningitis, endocarditis, and osteomyelitis, the use of a fungicidal agent is preferred. Caspofungin is generally regarded as fungicidal, and voriconazole is described as fungistatic against Candida species.5,6 However, these statements are based on data derived using fungicidal testing methods that are not standardized and have a number of limitations, including the slower growth rate of fungi compared with bacteria and the morphological variation among fungal elements. In this study, we used a method that addresses another limitation, that of drug carryover.7 In testing a large panel of non-albicans Candida species, our data demonstrated variability of fungicidal and fungistatic activities of caspofungin and voriconazole against non-albicans Candida strains, which may have broad implications for the choice of antifungal therapy by clinicians.
Isolates tested were taken from the culture collection at the Center for Medical Mycology and included both reference and clinical strains. One hundred ninety-seven non-albicans yeast isolates were tested, including Candida glabrata (n = 43), Candida parapsilosis (n = 40), Candida tropicalis (n = 40), Candida krusei (n = 34), Candida guilliermondii (n = 10), Candida lipolytica (n = 10), Candida kefyr (n = 10), Candida lusitaniae (n = 7), and Candida dubliniensis (n = 3). Isolates were subcultured from frozen stock (−80°C) onto potato dextrose agar and passaged twice on this medium (24-hour incubation at 35°C) before testing.
Voriconazole and caspofungin powders were obtained from Pfizer, Inc (New York, NY) and Merck & Co, Inc (Whitehouse Station, NJ), respectively. Stock solutions for voriconazole and caspofungin were prepared in dimethylsulfoxide or sterile water, respectively, and stored at −80°C. Working dilutions were prepared in accordance with the M27-A2 standard of the Clinical Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) for the antifungal susceptibility testing of yeasts.8
Minimum inhibitory concentration (MIC) testing was performed according to the M27-A2 standard of the Clinical Laboratory Standards Institute,8 whereas minimum fungicidal concentration (MFC) determinations were carried out using the methods of Canton et al.7 In this MFC testing method, the criteria required to show cidality were made more stringent by increasing the inoculum size to 104 colony-forming units (CFU) per milliliter and by defining the MFC as the minimum concentration that resulted in a reduction of greater than 99.9% in CFU from the original inoculum, whereas fungistatic activity was defined as the minimum concentration that resulted in a reduction of less than 99.9%.
The total contents of each visibly clear well from the 96-well microtiter plate used to determine the MIC were subcultured by transferring 100 μL onto each of the 2 potato dextrose agar plates. To avoid antifungal carryover, the aliquots were allowed to soak into the center of the agar plate until dry and then were streaked across the entire plate with an inoculating loop to remove the cells from the drug (Fig. 1).
The MIC and MFC data are summarized in Tables 1 and 2. Our data showed that the antifungal activity of both voriconazole and caspofungin were strain specific because the MIC and MFC values for each drug varied depending on the strain tested. Overall, the MIC50 (defined as the lowest drug concentration to inhibit 50% of all isolates tested) of voriconazole was 3-fold lower than caspofungin, whereas the MIC90 (defined as the lowest drug concentration to inhibit 90% of all isolates tested) was 1-fold lower than caspofungin (Table 1).
As can be seen in Table 2, MFC data showed voriconazole to be cidal against 60% of C. lipolytica isolates, 14% of C. lusitaniae isolates, and 8% of C. parapsilosis isolates. On the other hand, MFC values of caspofungin demonstrated that this drug was fungistatic against 100% of C. guilliermondii isolates and 58% of C. parapsilosis isolates, with MFC values of greater than 16 μg/mL. Additionally, caspofungin exhibited fungistatic activity against 35% of C. tropicalis isolates, 21% of C. glabrata isolates, 12% of C. krusei isolates, and 14% of C. lusitaniae isolates.
In this study, we showed that voriconazole had more potent inhibitory activity than caspofungin against the non-albicans Candida tested, as measured by MIC. These data are in general agreement with previously published comparative studies of voriconazole and caspofungin.9,10 Moreover, voriconazole demonstrated fungicidal activity against different non-albicans species, as in the case of C. lipolytica where it was cidal against two thirds of the isolates tested. The overall cidality rate of voriconazole was 5%.
Importantly, caspofungin exhibited fungistatic and not fungicidal activity against almost one third of the non-albicans yeast isolates tested. These data demonstrate the lack of universal caspofungin cidality against non-albicans Candida and the selective cidality of voriconazole versus some isolates of C. lipolytica, C. lusitaniae, and C. parapsilosis. The finding that voriconazole is cidal against most C. lipolytica isolates tested may have important clinical implications, especially regarding the emergence of pediatric fungemia due to amphotericin B and fluconazole-resistant C. lipolytica strains.11,12
Previous cidality studies, each conducted under different study parameters, of caspofungin against non-albicans strains have demonstrated considerable variation in MFC values. For example, Bartizal, et al13 reported a caspofungin MFC90 range of 0.5 to 2.0 μg/mL against non-albicans strains using an inoculum of 0.5 × 103 to 2.5 × 105 CFU/mL, with MFC defined as the lowest concentration at which growth of fewer than 4 colonies occurred. Using this same inoculum size (0.5 × 103 to 2.5 × 105 CFU/mL) but defining the MFC as a greater than 99% reduction in growth, Romero et al14 reported a caspofungin MFC90 range of 2.0 to greater than 64 μg/mL against similar strains. Majoros et al15, using an inoculum size of 105 CFU/mL and an MFC definition of greater than 99.9% growth reduction, reported a caspofungin MFC90 of 0.5 μg/mL against Candida inconspicua. Clearly, standardization of the MFC assay will be essential for comparison of data from different researchers.
Data correlating MFC values with clinical outcome are currently lacking. No clinical trials have been conducted to compare the efficacy of a cidal versus a static drug in the treatment of patients with endocarditis, meningitis, or osteomyelitis (situations where a cidal drug is perceived advantageous). However, in a randomized double-blind study of caspofungin versus fluconazole for the treatment of esophageal candidiasis, the cidal drug caspofungin did not demonstrate a better clinical response than the static drug fluconazole (90% vs. 89%).16 Establishment of a standardized cidality method is necessary before in vitro-in vivo correlation can be adequately assessed.
It may be that performance of MFC assays on strains isolated from critically ill patients should be added to routine MIC testing to be able to identify a cidal agent for early treatment. This may be especially true for those with meningitis, osteomyelitis, or endocarditis infections involving critical organs and those that present drug penetration difficulties. Furthermore, combination studies to determine drug interactions should be conducted to demonstrate no antagonistic effects, which could have clinical significance in cases in which patients may already be receiving antifungal therapy. Above all, the universal perception of antifungal activity (cidal vs static) of any drug should be questioned, and efforts to standardize cidality testing should be encouraged.
The authors thank Pfizer, Inc for its generous support of this study.
1. Wenzel RP, Gennings C. Bloodstream infections due to Candida species in the intensive care unit: identifying especially high-risk patients to determine prevention strategies. Clin Infect Dis. 2005;41:S389-S393.
2. Zaoutis TE, Argon J, Chu J, et al. The epidemiology and attributable outcomes of candidemia in adults and children hospitalized in the United States: a propensity analysis. Clin Infect Dis. 2005;41:1232-1239.
3. Fluckiger U, Marchetti O, Bille J, et al. Treatment options of invasive fungal infections in adults. Swiss Med Wkly. 2006;136:447-463.
4. Nivoix Y, Zamfir A, Lutun P, et al. Combination of caspofungin and an azole or an amphotericin B formulation in invasive fungal infections. J Infect. 2006;52(1):67-74.
5. Kurtz M, Heath IB, Marrinan J, et al. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase. Antimicrob Agents Chemother. 1994;38:1480-1489.
6. Bopp LH, Baltch AL, Ritz WJ, et al. Antifungal effect of voriconazole on intracellular Candida glabrata, Candida krusei and Candida parapsilosis in human monocyte-derived macrophages. J Med Microbiol. 2006;55:865-870.
7. Canton E, Peman J, Viudes A, et al. Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species. Diagn Microbiol Infect Dis. 2003;45:203-206.
8. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard - Second Edition. CLSI document M27-A2 (ISBN 1-56238-469-4). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA; 2002.
9. Ghannoum M, D'Angelo M. Anidulafungin: a potent antifungal that targets Candida and Aspergillus. Infect Dis Clin Pract. 2005;13:1-14.
10. Espinel-Ingroff A, Pfaller M, Messer SA, et al. Multicenter Comparison of the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A2 Reference Method for Testing New Antifungal Agents against Clinical Isolates of Candida spp. J Clin Microbiol. 2004;42:718-721.
11. Shin JH, Kook DH, Hwang TJ, et al. Nosocomial cluster of Candida lipolytica fungemia in pediatric patients. Eur J Clin Microbiol Infect Dis. 2000;19:344-349.
12. Belet N, Ciftci E, Ince E, et al. Caspofungin treatment in two infants with persistent fungaemia due to Candida lipolytica. Scand J Infect Dis. 2006;38:559-562.
13. Bartizal K, Gill CJ, Abruzzo GK, et al. In vitro preclinical evaluation studies with the echinocandin antifungal MK-0991 (L-743,872). Antimicrob Agents Chemother. 1997;41:2326-2332.
14. Romero M, Canton E, Peman J, et al. Study of in vitro activity of caspofungin on non-Candida albicans yeast strains determined by two methods: M27-A2 and EUCAST. Rev Esp Quimioter. 2004;17:257-262.
15. Majoros L, Kardos G, Szabo B, et al. Caspofungin susceptibility testing of Candida inconspicua: correlation of different methods with the minimal fungicidal concentration. Antimicrob Agents Chemother. 2005;49:3486-3488.
16. Villanueva A, Gotuzzo E, Arathoon EG, et al. A randomized double-blind study of caspofungin versus fluconazole for the treatment of esophageal candidiasis. Am J Med. 2002;113:294-299.
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