In Vitro Susceptibility Testing Methods for Caspofungin against Aspergillus and Fusarium Isolates

Abstract

We investigated the relevance of prominent reduction in turbidity macroscopically (MIC) and formation of aberrant hyphal tips microscopically (minimum effective concentration; MEC) in measuring the in vitro activity of caspofungin against Aspergillus and Fusarium. Caspofungin generated low MICs and MECs against Aspergillus, but not for Fusarium. While MICs increased inconsistently when the incubation time was prolonged, MEC appeared as a stable and potentially relevant endpoint in testing in vitro caspofungin activity.The echinocandins are a group of lipopeptide antifungal agents that contain a cyclic hexapeptide nucleus and act via inhibition of (1,3)-␤-D-glucan synthase. Novel echinocandins, including LY303366, L-733,560, and caspofungin (formerly referred to as MK-0991 and as L-743,872) (1, 4, 5, 9-12, 15, 17, 19, 21-24) are currently under investigation.A reproducible and clinically relevant method for susceptibility testing of echinocandins has not been fully established yet. One of the undetermined test parameters is the MIC endpoint to be used for measuring the in vitro activity. Echinocandins exhibit fungicidal or fungistatic activity against Candida spp. (8, 9). The MICs of echinocandins for Candida have been determined so far as either the least concentration of the drug that produces 100% inhibition of growth (21,22) or that producing 80% reduction in turbidity (9, 13).Testing the in vitro activity of echinocandins against Aspergillus spp. is more complicated. Echinocandins are active against Aspergillus both in vitro (5,10,20) and in vivo (1, 16). However, the assessment of in vitro activity requires distinct evaluation. Instead of a complete macroscopic growth inhibition, partial inhibition is seen in which the fungus microscopically produces short, stubby, and highly branched hyphae (6, 9, 16). Kurtz et al. (16) proposed that the drug concentration at which these morphological changes were first observed be called the minimum effective concentration (MEC). Nevertheless, neither the proper method to be used for the detection of the in vitro activity of caspofungin and other echinocandins against molds (5,10,20) nor the clinical significance of MEC has been fully defined.This study was designed to comparatively evaluate the two endpoints, MIC and MEC, in the determination of the in vitro activity of caspofungin against clinical Aspergillus and Fusarium isolates. The effect of incubation period and test media on both MIC and MEC was also investigated.(This work was presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, 26 to 29 September 1999, in San Francisco, Calif., as abstr. no. J-160.)The test organisms were comprised of 82 Aspergillus and 22 Fusarium strains. Two itraconazole-resistant A. fumigatus strains, kindly provided by D. W. Denning, were also included (7). Each isolate was tested in duplicate. One of the clinical isolates (strain no. 2-160; A. fumigatus) was included in each run for quality control.Caspofungin was provided by Merck Research La...

Activities of Caspofungin, Itraconazole, Posaconazole, Ravuconazole, Voriconazole, and Amphotericin B against 448 Recent Clinical Isolates of Filamentous Fungi

“…Caspofungin, using MEC as the in vitro susceptibility testing endpoint (1,15), also had excellent in vitro activity against all species of Aspergillus tested, inhibiting 90% of isolates at an MEC of 0.06 g/ml and Ͼ98% at an MEC of Յ1 g/ml. Caspofungin has been approved by the U.S. Food and Drug Administration for use in refractory cases of invasive asper- (2,13,14,26).…”

“…In particular, the MEC endpoint for echinocandins recognizes that they do not produce complete macroscopic growth inhibition of Aspergillus spp. but rather partial inhibition associated with the development of short, stubby, highly branched, and abnormal hyphae (1,15). Of particular note, the triazoles and caspofungin all had excellent in vitro activity against A. terreus (100% inhibited at an MIC or MEC of Յ1 g/ml), a species against which amphotericin B demonstrated poor in vitro activity (38% inhibited at an MIC of Յ1 g/ml) and against which amphotericin B has poor clinical efficacy (12).…”

“…The stock conidial suspension (10 6 spores/ml) was diluted to a final inoculum concentration of 0.4 ϫ 10 4 to 5 ϫ 10 4 CFU/ml and dispensed into the microdilution wells. The inoculated microdilution trays were incubated at 35°C and read at 48 h. The MIC endpoint for the azoles and amphotericin B was defined as the lowest concentration that produced complete inhibition of growth, whereas the minimum effective concentration (MEC) endpoint for caspofungin was defined according to published methods (1,15). Quality control.…”

Multilaboratory Evaluation of In Vitro Antifungal Susceptibility Testing of Dermatophytes for ME1111

“…One case in point is the susceptibility testing of echinocandins against Aspergillus species. It was found that the minimum effective concentration, defined as the lowest concentration with visible morphological changes in growth patterns from filamentous to granular signifying abnormal hyphal growth, correlates better with clinical outcome than with the traditional MIC endpoint for echinocandins against Aspergillus strains (3,12,13).…”

New Methods To Assess Susceptibilities of Aspergillus Isolates to Caspofungin

“…This is largely because in broth culture methods (National Committee for Clinical Laboratory Standards [NCCLS] standard M38-A) (12), CAS-exposed Aspergillus spp. demonstrate growth in every well, making end points of growth and MICs difficult to determine (1). Kurtz et al were the first to propose the term minimal effective concentration (MEC) to denote the lowest echinocandin concentration at which the fungi display microscopic morphological changes (11), and this measure is currently used by investigators to report CAS susceptibilities (1,17,18).…”