The antifungal susceptibilities of 232 pathogenic bloodstream Candida isolates collected during a recently completed trial comparing fluconazole (400 mg/day) with amphotericin B (0.5 mg/kg of body weight per day) as treatment for candidemia in the nonneutropenic patient were determined both by the National Committee for Clinical Laboratory Standards M27-P macrobroth methodology and by a less cumbersome broth microdilution methodology. For amphotericin B, M27-P yielded a very narrow range of MICs (0.125 to 1 g/ml) and there were no susceptibility differences among species. For fluconazole, a broad range of MICs were seen (0.125 to >64 g/ml), with characteristic MICs seen for each species in the rank order Candida albicans < C. parapsilosis Х Х C. lusitaniae < C. glabrata Х Х C. krusei Х Х C. lipolytica. The MIC distribution for C. tropicalis was bimodal and could not be ranked. Broth microdilution MICs were within one tube dilution of the M27-P MIC for Ն Ն90% of isolates with amphotericin B and for Ն Ն77% of isolates with fluconazole. For both methods, elevated MICs did not predict treatment failure. In the case of amphotericin B, the MIC range was too narrow to permit identification of resistant isolates. In the case of fluconazole, MICs for isolates associated with failure to clear the bloodstream consistently were equivalent to the median MIC for the given species. Successful courses of therapy were seen with four isolates from four patients despite MICs of Ն Ն32 g/ml. As MICs obtained by M27-P and similar methods correlate with responsiveness to fluconazole therapy in animal models and in AIDS patients with oropharyngeal candidiasis, the lack of correlation in this setting suggests that the MICs for these isolates are at or below the relevant fluconazole breakpoint for this dose of fluconazole and patient setting and that host factors such as failure to exchange intravenous catheters were more important than MIC in predicting outcome.Antifungal susceptibility testing remains in evolution. Recent work by the National Committee for Clinical Laboratory Standards has focused on the development of standard, reproducible methods for testing of yeast. The proposed method M27-P is a broth macrodilution method that has good interand intralaboratory reproducibility (5, 10). As MICs obtained by methods similar to M27-P have generally correlated well with outcome in various animal models of infection, it is anticipated that M27-P (or a method utilizing the principles of M27-P) will prove useful in prediction of the likelihood of response to a given antifungal agent (16). However, no breakpoints have yet been established for M27-P. During our recently completed trial of fluconazole versus amphotericin B for therapy of candidemia in nonneutropenic patients, we collected Candida bloodstream isolates from our patients (15). We now report the distribution of M27-P MICs seen in this collection of pathogenic Candida isolates, the correlation of these MICs with an easily performed broth microdilution variation of M27-P, and the ...

The National Committee for Clinical Laboratory Standards (NCCLS) proposed macrobroth reference method (M27P) for susceptibility testing of yeasts is technically difficult. We evaluated Etest, a simple agar-based MIC methodology, as a possible alternative. In studies of six yeast quality control strains, Etest yielded results identical to those obtained by the NCCLS reference method for both amphotericin B and fluconazole. In studies of 91 clinical Candida isolates, agreement ؎ 2 dilutions between the two methods was 95% for fluconazole with phosphate-buffered RPMI 1640 agar and 96 to 97% for amphotericin B with either MOPS (morpholinepropanesulfonic acid)-buffered RPMI 1640 agar or antibiotic medium 3 agar. While the two methods had excellent general agreement, testing of a collection of amphotericin B-resistant isolates demonstrated that, unlike the NCCLS reference method, Etest readily identified the resistant isolates and could do so with a defined medium. Etest is equivalent to the NCCLS proposed method for susceptibility testing of yeasts and superior in its ability to detect amphotericin B resistance.In vitro susceptibility testing of yeasts is fraught with numerous problems and affected by factors such as media, buffer, and inoculum density (11). A proposed standard (M27P) using RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) to pH 7.0 in a macrobroth format with incubation for 48 h at 35ЊC has been published by the National Committee for Clinical Laboratory Standards (NCCLS) (6). This method is time-consuming, expensive, and technically difficult to perform. Modifications of the NCCLS method using a microbroth format will simplify its implementation and have demonstrated results comparable to those for the proposed standard (5). Both of these methods are, however, particularly lacking in their ability to differentiate among amphotericin B-susceptible and -resistant isolates unless antibiotic medium 3 is substituted for RPMI 1640 (9, 11).Etest, a novel method utilizing a stable gradient of an antimicrobial agent, has been well documented as an accurate and simple method for bacterial susceptibility testing (12), and Etest has recently been shown to produce results comparable to those obtained with the NCCLS method for ketoconazole, itraconazole, and fluconazole (3, 4). We have extended this work by comparing the NCCLS method with Etest for testing of susceptibility of Candida isolates to both fluconazole and amphotericin B for a group of clinical isolates as well for a group of recently described quality control strains (7,8). In addition, having recently demonstrated that the NCCLS reference method has a limited ability to detect amphotericin Bresistant isolates unless antibiotic medium 3 is substituted for RPMI 1640 (9), we also studied the ability of Etest to identify these amphotericin B-resistant isolates. MATERIALS AND METHODSIsolates. Study strains included 91 yeast isolates from blood, a subset of those previously described by Rex et al. (9,10). A collection of previously desc...

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.

A multicenter study was conducted to expand the generation and analysis of data that supports the proposal of a reference method for the antifungal susceptibility testing of filamentous fungi. Broth microdilution MICs of amphotericin B and itraconazole were determined in 11 centers against 30 coded duplicate pairs of Aspergillus spp., Fusarium spp., Pseudallescheria boydii, and Rhizopus arrhizus. The effect of inoculum density (approximately 10 3 and 10 4 CFU/ml), incubation time (24, 48, and 72 h), and procedure of MIC determination (conventional and colorimetric [Alamar Blue] evaluation of growth inhibition) on intra-and interlaboratory agreement was analyzed. Based on intra-(97 to 100%) and interlaboratory (94 to 95%) agreement for both drugs, the overall optimal testing conditions identified were determination of colorimetric MICs after 48 to 72 h of incubation with an inoculum density of approximately 10 4 CFU/ml. These testing conditions are proposed as guidelines for a reference broth microdilution method.

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We have previously shown that use of antibiotic medium 3 in the microdilution variant of the M27-T test method permits detection of amphotericin B-resistant Candida isolates. As this medium is not standardized and our initial work used only a single lot of antibiotic medium 3, we studied the lot-to-lot variability of three commercial lots of antibiotic medium 3 obtained from two sources. The MICs obtained with the new lots were lower than those of with the original lot, but the new lots still consistently separated putatively resistant and putatively susceptible isolates and this permits proposal of possible breakpoints for this assay system.

We have developed an agar-based methodology for testing susceptibilities of Candida spp. to the new antifungal agent MK-0991, a glucan synthase inhibitor. Results obtained with this method correlated well with the results obtained by the National Committee for Clinical Laboratory Standards M27-A broth microdilution reference method. However, as noted with prior comparisons of broth- and agar-based systems, some isolates yielded inhibition zones which were not consistent with the MICs obtained for them. Understanding the implications of these differences will require testing in an in vivo system.