We investigated the in vitro interaction of caspofungin and amphotericin B for clinical isolates of Aspergillus and Fusarium. Synergy tests were performed using the checkerboard method and following the NCCLS M38-P guidelines in Antibiotic Medium 3 broth supplemented to 2% glucose. Antagonism was not observed for any of the isolates tested. Caspofungin and amphotericin B were synergistic or synergistic to additive for at least half of the isolates.Echinocandins, amphophilic cyclic hexapeptides with an Nlinked acyl side chain, exhibit selective antifungal activity via inhibition of ␤-glucan synthesis. Although caspofungin has proven to be active in vivo against Aspergillus spp. (1, 2; J. Maertens, I. Raad, C. A. Sable, A. Ngai, and R. Berman, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1103Chemother., abstr. , 2000, it has limited in vitro activity when measured using a conventional MIC-0 (complete inhibition of growth) endpoint. Minimum effective concentration (MEC, in micrograms per milliliter) is a microscopic endpoint that may correlate better with the in vivo activity of the echinocandins. The MEC refers to the lowest concentration of the drug that results in the formation of aberrantly growing, unusual hyphal tips (7). We previously demonstrated that the MEC correlates well with the macroscopic MIC-2 (Ϸ50% reduction in turbidity, prominent decrease in growth visually) endpoint (3).While the distinctive mechanisms of action of caspofungin on Aspergillus hyphae make it a good candidate for use in combination with other antifungal agents (C. M. Douglas, J. C. Bowman, G. K. Abruzzo, A. M. Flattery, C. J. Gill, L. Kong, C. Leighton, J. G. Smith, V. B. Pikounis, K. Bartizal, M. B. Kurtz, and H. Rosen, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. J-1683, 2000), its behavior in combination has been little studied. In an effort to clarify whether enhanced activity against Aspergillus and Fusarium is achieved when caspofungin is combined with another antifungal agent acting via a different mechanism, we performed in vitro synergy studies for caspofungin combined with amphotericin B. The individual caspofungin and amphotericin B MICs (in micrograms per milliliter) were determined initially by using the NCCLS M38-P microdilution methodology (8) in Antibiotic Medium 3 and after 24 and 48 h of incubation. Antibiotic Medium 3 (BBL lot JD4ZSG; Becton Dickinson) was buffered by addition of 1 g of Na 2 HPO 4 and 1 g of NaH 2 PO 4 to each liter of medium (pH ϭ 7) and then supplemented to 20-g/liter glucose (AM3). It was previously shown that AM3 provided good growth and generated slightly lower amphotericin B MICs than did RPMI medium, particularly for some Aspergillus isolates (4). Consistent with data obtained with Candida and amphotericin B (9), this lowering effect might help to differentiate caspofungin-susceptible Aspergillus isolates from caspofungin-resistant ones, and we thus performed the susceptibility tests with AM3. Also, based on the observations that reading at earl...

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...

The trailing growth phenomenon seen when determining the susceptibilities of Candida isolates to the azole antifungal agents makes consistent endpoint determination difficult, and the M27-A method of the National Committee for Clinical Laboratory Standards addresses this problem by requiring an 80% reduction in growth after 48 h of incubation. For some isolates, however, minor variations of this endpoint criterion can produce up to 128-fold variations in the resulting MIC. To investigate the significance of this effect, isolates of Candida that exhibited various forms of trailing growth when tested against fluconazole were identified. The isolates were examined in a murine model of invasive candidiasis and were ranked by their relative response to fluconazole by using both improvement in survival and reduction in fungal burden in the kidney. The resulting rank order of in vivo response did not match the MICs obtained by using the M27-A criterion, and these MICs significantly overestimated the resistance of three of the six isolates tested. However, if the MIC was determined after 24 h of incubation and the endpoint required a less restrictive 50% reduction in growth, MICs which better matched the in vivo response pattern could be obtained. Minor variations in the M27-A endpoint criterion are thus required to optimize the in vitro-in vivo correlation for isolates that demonstrate significant trailing growth when tested against fluconazole.

In order to determine the current prevalence and incidence of fluconazole-resistant oropharyngeal candidiasis among human immunodeficiency virus (HIV)-infected patients, we conducted a prospective observational study of a consecutive series of HIV-infected patients. Of 128 enrolled patients, 70 patients completed four quarterly follow-up visits over a period of 1 year. Over this period, declining rates of carriage of Candida albicans (from 61% to 39%; P = .008) and of oropharyngeal candidiasis (from 30% to 4%; P < .001) were documented. Trends toward reduction in the frequency of fluconazole-resistant isolates (MIC, > or = 64 micrograms/mL) were also seen. During the survey period, the mean (median) number of antiretroviral agents used per patient rose from 0.5 (0) to 1.8 (2) (P < .001). Thus, rather than progression, we observed declining rates of oropharyngeal candidiasis, C. albicans carriage, and fluconazole-resistant C. albicans in a cohort of HIV-infected patients treated with increasingly effective antiretroviral therapy.

We compared the activities of amphotericin B, itraconazole, and voriconazole against clinical Aspergillus(n = 82) and Fusarium (n= 22) isolates by a microdilution method adopted from the National Committee for Clinical Laboratory Standards (NCCLS-M27A). RPMI 1640 (RPMI), RPMI 1640 supplemented to 2% glucose (RPMI-2), and antibiotic medium 3 supplemented to 2% glucose (AM3) were used as test media. MICs were determined after 24, 48, and 72 h. A narrow range of amphotericin B MICs was observed for Aspergillus isolates, with minor variations among species. MICs for Fusariumisolates were higher than those for Aspergillus isolates. MICs of itraconazole were prominently high for two previously defined itraconazole-resistant Aspergillus fumigatus isolates andFusarium solani. Voriconazole showed good in vitro activity against itraconazole-resistant isolates, but the MICs of voriconazole for F. solani were high. RPMI was the most efficient medium for detection of itraconazole-resistant isolates, followed by RPMI-2. While the significance remains unclear, AM3 lowered the MICs, particularly those of amphotericin B.