The purpose of the study was to evaluate the interlaboratory agreement of broth dilution susceptibility methods for five species of conidium-forming (size range, 2 to 7 m) filamentous fungi. The methods used included both macro-and microdilution methods that were adaptations of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (m27-P). The MICs of amphotericin B, fluconazole, itraconazole, miconazole, and ketoconazole were determined in six centers by both macro-and microdilution tests for 25 isolates of Aspergillus flavus, Aspergillus fumigatus, Pseudallescheria boydii, Rhizopus arrhizus, and Sporothrix schenckii. All isolates produced clearly detectable growth within 1 to 4 days at 35؇C in the RPMI 1640 medium. Colony counts of 0.4 ؋ 10 6 to 3.3 ؋ 10 6 CFU/ml (mean, 1.4 ؋ 10 6 CFU/ml) were demonstrated in 90% of the 148 inoculum preparations. Overall, good intralaboratory agreement was demonstrated with amphotericin B, fluconazole, and ketoconazole MICs (90 to 97%). The agreement was lower with itraconazole MICs (59 to 79% median). Interlaboratory reproducibility demonstrated similar results: 90 to 100% agreement with amphotericin B, fluconazole, miconazole, and ketoconazole MICs and 59 to 91% with itraconazole MICs. Among the species tested, the MICs for S. schenckii showed the highest variability. The results of the study imply that it may be possible to develop a reference method for antifungal susceptibility testing of filamentous fungi.Despite the lower volume of serious infections caused by filamentous fungi compared with that of serious infections caused by yeasts, the performance of antifungal susceptibility testing for these opportunistic pathogens is important in the clinical laboratory (8). Progress has been made in developing guidelines for the antifungal susceptibility testing of yeasts to deal with the standardization of different testing parameters such as inoculum preparation, medium composition and pH, length of incubation, and endpoint criteria (3,5,6,(9)(10)(11)(12). The standardization of these antifungal susceptibility testing steps has led to increased interlaboratory reproducibility which has opened the possibility of developing standards by adopting and tailoring these steps for antifungal susceptibility tests for the filamentous fungi. The first priority of the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests was to develop a standard for the preparation of inoculum suspensions. Among four procedures evaluated, a spectrophotometric method was recommended as the procedure that gives the least variable results for the preparation of inoculum suspensions of yeast cells (11). This recommendation has been substantiated by ensuing collaborative studies of the subcommittee and other studies (2,3,5,6,12).The spectrophotometric method has been evaluated further in a single laboratory for the preparation of conidial suspensions of selected medically important filamentous fungi (1). In t...

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...

The activities of fluconazole and voriconazole against isolates of Candida spp. (n ‫؍‬ 400) were tested by the E-test, disk diffusion, and the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 broth microdilution-based reference methods. More than 96% of isolates found to be susceptible to fluconazole by the reference method were identified as susceptible by the agar-based methods. Lesser degrees of correlation with the reference method were seen for isolates identified as resistant by the agar-based methods. Interpretive categories are not available for voriconazole, but results qualitatively similar to those for fluconazole were seen. The agar-based E-test and disk diffusion methods are reliable alternatives to the NCCLS M27-A2 reference microdilution method for isolates that test susceptible to fluconazole.The development of standardized antifungal susceptibility testing methods has been the subject of numerous studies during the last decade. Reference methods for yeasts (the National Committee for Clinical Laboratory Standards [NCCLS] M27-A2 method) and molds (the NCCLS M38-A method) are now available (16). Agar-based susceptibility testing methods have been a focus of interest for many researchers and include the classical disk diffusion (DD) methods and the E-test (ET) method (3, 6-10, 13, 14, 16-18). Those tests are very attractive due to their simplicity, reproducibility, and lack of requirements for specialized equipment (11,16). Recent studies have documented comparable results between those methods and the results of standard reference broth microdilution (MD) susceptibility testing (7,11,13).In this study, we compared the NCCLS M27-A2 MD method with the ET and DD methods for determination of the susceptibilities of 400 Candida species isolates to fluconazole and voriconazole. The ET and DD methods are well studied for fluconazole (3,(6)(7)(8)(9)(10)(11)14), and this work extends their usage to include voriconazole. MATERIALS AND METHODSIsolates. Four hundred bloodstream isolates of Candida species were randomly selected for testing. These included 205 isolates of Candida albicans, 56 isolates of C. tropicalis, 39 isolates of C. glabrata, 66 isolates of C. parapsilosis, 24 isolates of C. krusei, and 10 isolates of other species. The isolates were identified with the API 20C AUX system (Biomerieux Vitek, Hazelwood, Mo.) and were subsequently stored in sterile distilled water at room temperature until susceptibility tests were performed. Each isolate was subcultured at least twice on Sabouraud dextrose agar and incubated at 35°C prior to testing to ensure purity and optimal growth.Inoculum suspensions. Yeast inoculum suspensions were prepared as described for the NCCLS M27-A2 method (12). The turbidity was measured with a spectrophotometer at 530 nm and was adjusted to match a 0.5 McFarland density standard, resulting in a concentration of 1 ϫ 10 6 to 5 ϫ 10 6 yeast cells/ml. This inoculum was used directly for inoculation of agar plates (see below) or was diluted as needed for the MD ...

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 in vitro activities of amphotericin B, itraconazole, fluconazole, voriconazole, posaconazole, and ravuconazole against 39 isolates of Trichosporon spp. were determined by the NCCLS M27-A microdilution method. The azoles tested appeared to be more potent than amphotericin B. Low minimal fungicidal concentration/MIC ratios were observed for voriconazole, posaconazole, and ravuconazole, suggesting fungicidal activity.

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.

(1-->3)-beta-d-glucan is a well known cell wall constituent of fungal isolates that can be detected by assays in vivo and in vitro. Previous studies have shown that different fungal isolates may show different levels of reactivity with an assay for beta glucan. In this study we evaluated the in vitro reactivity of 127 clinical fungal isolates belonging to 40 different genera, with the Glucatell assay. The majority of the fungal isolates released high levels of beta glucan. Beta glucan test reactivity appears to be species-specific and this may reflect the beta glucan content of the organism.

Non-culture-based diagnostic strategies are needed for diagnosing invasive candidiasis (IC). We evaluated serial serum (133)-␤-D-glucan (BG) levels in patients in the surgical trauma intensive care unit (SICU) patients with clinical evidence of IC. Serum samples from patients admitted to the SICU for a minimum of 3 days were collected twice weekly and analyzed for BG by using a Fungitell kit with a positive cutoff of >80 pg/ml. Diagnosis of IC was done using a set of predefined and validated clinical practice-based criteria. A total of 57 patients consented to participate and were enrolled. The median ICU stay was 16 days (range, 3 to 51). A total of 14 of 57 (25%) false positives were observed in the first sample (ICU day 3) and, overall, 73% of the day 3 samples had higher BG levels than subsequent samples. On the date of clinical diagnosis of IC, the sensitivity of a positive BG for identifying invasive candidiasis was 87%, with a 73% specificity. In patients with evidence of IC, the median BG value was significantly higher than those without evidence of IC (171 versus 48 pg/ml, P ‫؍‬ 0.02), respectively. In the three patients with proven IC, BG was detected 4 to 8 days prior to diagnosis. BG serum detection may be a useful tool to aid in the early diagnosis of IC in SICU patients, particularly after day 3 and in patients with at least two positive samples drawn several days apart. Elevated BG levels within the first 3 days need to be further characterized.