Comparison of a new colorimetric assay with the NCCLS broth microdilution method (M-27A) for antifungal drug MIC determination

Abstract

We evaluated a new microtiter assay for antifungal susceptibility testing based on a colorimetric reaction to monitor fungal substrate utilization. This new method (rapid susceptibility assay [RSA]) provides quantitative endpoint readings in less than 8 h compared with visual determination of MIC by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method, which requires a minimum of 48 h of incubation. In this study, we tested clinical isolates from each of the following species: Candida albicans (20 isolates), C. glabrata (20 isolates), C. krusei (19 isolates), C. tropicalis (19 isolates), and C. parapsilosis (28 isolates). RSA and NCCLS broth dilution methods were used to determine the MICs of amphotericin B, fluconazole, itraconazole, and 5-flucytosine for all 106 isolates. RPMI 1640 medium buffered with morpholinopropanesulfonic acid was used for both methods; however, glucose and inoculum concentrations in the RSA were modified. RSA MICs were determined as the lowest drug concentration that prevented glucose consumption by the organism after 6 h of incubation. MICs obtained from the RSA were compared with those obtained from the NCCLS M-27A method read at 24 and 48 h. MIC pairs were considered in agreement when the difference between the pairs was within 2 twofold dilutions. For the 106 isolates tested, amphotericin B and 5-flucytosine demonstrated the highest agreement in MICs between the two methods (100 and 98%, respectively), whereas fluconazole and itraconazole produced less favorable MIC agreement (63.2 and 61.3%, respectively). The azole MIC differences between the two methods were significantly reduced when lower inocula were used with a prolonged incubation time. This preliminary comparison suggests that this rapid procedure may be a reliable tool for the in vitro determination of MICs of amphotericin B and 5-flucytosine and warrants further evaluation.

FIG. 1

RSA susceptibility curve of AMB for an isolate of C. glabrata. Duplicate experiments with the same isolate are shown. The MIC for this organism was 2 μg/ml, as determined by RSA and the NCCLS method. Note the drug-saturation plateau above 2 μg/ml. The control well contained no drug and no yeast inoculum. An inoculum of an 0.5 McFarland standard was added to the wells containing 0 to 16 μg of drug per ml and incubated for 6 h before the complete color mix was added. OD values represent relative glucose concentrations.

FIG. 2

RSA susceptibility curve of 5-FC for an isolate of C. tropicalis (MIC = 2 μg/ml by NCCLS and RSA). Duplicate experiments with the same isolate are shown. See the legend to Fig. 1 for more information.

FIG. 3

(A) Representative RSA susceptibility curve of FLU for a C. glabrata isolate. The NCCLS MIC of FLU for this organism was ≤0.125 μg/ml. The RSA MIC falls in the arc between 0.25 and 1 μg/ml. Note the relatively small difference in OD between the wells with the highest drug concentration and the well without drug. (B) Plot of data for the same isolate as in panel A except that glucose control is included. Note the consumption of glucose even at high concentration of FLU (arrow). In the presence of the high-glucose control OD value, the curve appears flat and erroneously indicates resistance. Duplicate experiments with the same isolate are shown.

FIG. 4

(A) Representative RSA susceptibility curve of ITRA for a C. glabrata isolate. MICs obtained by RSA and NCCLS methods were both 0.5 μg/ml. (B) Plot of data for the same isolate as in panel A except that glucose control is included. Note the reduction in glucose level even at high concentrations of ITRA (arrow). In the presence of glucose control, the curve appears flat and erroneously indicates resistance. Duplicate experiments with the same isolate are shown.

FIG. 5

RSA curves of FLU for a C. albicans isolate (97-012) when inoculated with an undiluted 0.5-McFarland standard yeast suspension and incubated for 6 h (⧫) or with a 1/1,000-diluted inoculum and incubated for 19 h (■). The MICs for this organism are reported in Table 3.

FIG. 6

RSA curves of ITRA for a C. glabrata isolate (97-050). An undiluted 0.5-McFarland standard yeast suspension was used for inoculation and incubated for 6 h (⧫). The inoculum was diluted 1/1,000 and incubated for 19 h (■). The latter resulted in a MIC comparable to that obtained by the NCCLS method. MICs for this isolate are listed in Table 3.