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Journal of Clinical Microbiology, May 1998, p. 1450-1452, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Comparison of a 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenylamino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) Colorimetric Method with the Standardized National Committee for Clinical Laboratory Standards Method of Testing Clinical Yeast Isolates for Susceptibility to Antifungal Agents

S. P. Hawser,1,* H. Norris,2 C. J. Jessup,2 and M. A. Ghannoum2

Hoechst Marion Roussel, F-93235 Romainville, France,1 and Mycology Reference Laboratory, Center for Medical Mycology, University Hospitals of Cleveland, and Department of Dermatology, Case Western Reserve University, Cleveland, Ohio 44106-50282

Received 4 December 1997/Returned for modification 5 January 1998/Accepted 31 January 1998

    ABSTRACT
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MICs for clinical Candida and Cryptococcus isolates were determined by a method incorporating the colorimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), and the results were compared with MICs obtained by the National Committee for Clinical Laboratory Standards approved standard method (M27-A). One hundred percent of all isolates demonstrated agreement within 2 dilutions between the MICs of amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine obtained by the two methods. These data suggest that an XTT-based method could provide a useful means for the determination of antifungal susceptibility of yeasts.

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The National Committee for Clinical Laboratory Standards (NCCLS) has recently developed a standardized broth-based method (M27-A) of testing pathogenic yeasts for susceptibility to antifungal agents (4). Although this method demonstrates good intra- and interlaboratory agreement, MICs of fungistatic agents such as fluconazole and itraconazole are often difficult to determine because of trailing endpoints caused by the partial inhibition of fungal growth. In attempts to overcome this shortcoming, investigators have developed a number of alternative testing methods, including modifications of the NCCLS method (8) or reading the MIC endpoints in a different manner, such as by spectrophotometry (7) and colorimetry (5, 6, 11). Colorimetric methods are attractive, since they have the potential to generate clear-cut endpoints, based on a visually detectable color change. A colorimetric method utilizing the oxidation-reduction colorimetric indicator Alamar blue, for example, has been shown to generate clear-cut endpoints for fluconazole and itraconazole for yeast clinical isolates and to yield MICs that correlate well with those determined by the NCCLS standard method (5, 6, 11). An alternative colorimetric method utilizes the tetrazolium salt 2,3-bis(2-methoxy-4- nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazo- lium hydroxide (XTT). XTT is a yellow salt that is reduced by mitochondrial dehydrogenases of metabolically active yeast cells to a dark blue XTT formazan product (2, 10). This color change can be assessed visually, similar to the Alamar blue method. In order to evaluate the potential utility of an XTT colorimetric method, we determined the MICs of five antifungal agents for clinical yeast isolates and compared them to those obtained by the standard NCCLS microtiter method (M27-A).

Clinical isolates of Candida spp. and Cryptococcus neoformans were obtained from the Center for Medical Mycology, Cleveland, Ohio, and included Candida albicans (n = 29), C. tropicalis (n = 17), C. lusitaniae (n = 11), C. guilliermondii (n = 6), C. glabrata (n = 12), C. parapsilosis (n = 18), C. krusei (n = 5), and C. neoformans (n = 20). C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were tested concurrently as quality control (QC) isolates. Fungi were maintained on Sabouraud dextrose agar. Prior to susceptibility testing, the isolates were placed on fresh agar plates and grown for 24 h at 35°C.

The susceptibility of each isolate to amphotericin B (Sigma Chemical Co., Saint Louis, Mo.), fluconazole (Pfizer Pharmaceuticals Group, New York, N.Y.), itraconazole and ketoconazole (Janssen Pharmaceuticals, Beerse, Belgium), and flucytosine (Sigma) was assessed in two ways. (i) Susceptibility testing was performed with 96-well round-bottom tissue culture plates as described in the M27-A procedure for the susceptibility testing of yeasts. The MIC was read visually as described in the NCCLS M27-A document (4). (ii) The second assay of susceptibility testing was a modification of the previously described XTT colorimetric method (2). Briefly, the susceptibility plates were prepared as for the NCCLS method. Three hours prior to the endpoint reading, the plates were agitated, and 50 µl of an XTT-phenazine methosulfate (PMS) mix (final concentrations of 200 µg/ml for XTT and 19.13 µg/ml for PMS [both purchased from Sigma]) was added to all wells. The plates were incubated for 3 h at 35°C to allow for color development. The MIC in the XTT assay was determined visually as the lowest concentration of antifungal agent at which no color change occurred.

The XTT MICs of the NCCLS QC strains correlated strongly with those obtained by the M27-A method (Table 1) and were confirmed in duplicate and by retesting of these organisms up to five times on different days (Tables 1 and 2). Furthermore, the ranges of MICs obtained by the XTT and NCCLS methods were almost identical for all Candida spp. and C. neoformans (Table 2).

                              
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  		TABLE 1.   Comparison of NCCLS- and XTT-based MIC data for antifungal susceptibility testing of QC strains of Candida

                              
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  		TABLE 2.   Comparison of NCCLS- and XTT-based MIC data for antifungal susceptibility testing of clinical isolates of Candida and C. neoformans

In order to more closely analyze the agreement between the XTT and NCCLS readings, agreements were expressed as percentage agreement in terms of (i) having the same MIC or a MIC within 1 dilution or (ii) having a MIC within 2 dilutions. Overall, there was an excellent agreement between the two MIC readings. For example, for C. albicans isolates, the levels of agreement were 97% for amphotericin B, 94% for itraconazole, 100% for flucytosine, 94% for fluconazole, and 97% for ketoconazole, as determined for those isolates for which the XTT-based MIC was either the same as or within 1 dilution of the NCCLS MIC (Table 3). Furthermore, the remainder of the XTT MICs fell within 1 dilution of the NCCLS MICs (Table 3).

                              
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  		TABLE 3.   Agreement between XTT- and NCCLS-derived MICs

Analysis of the agreement between the XTT and NCCLS MICs for the non-C. albicans species indicated that 100% of the C. glabrata, C. tropicalis, and C. guilliermondii isolates exhibited XTT MICs that were equal to or within 1 dilution of the NCCLS MIC (Table 3). Agreement was also excellent for C. krusei (80 to 100%). However, agreement was more variable for C. parapsilosis and C. lusitaniae isolates, 72 to 89% and 45 to 91% of which exhibited agreement within 1 dilution, with the remainder of the isolates exhibiting agreement within 2 dilutions (Table 3). With regard to C. neoformans, all of the XTT MICs fell within 1 dilution, suggesting 100% agreement between the two methods.

Tetrazolium salts have previously been used to determine the metabolic rates of higher eukaryotic cells and to assess the effects of cytotoxic agents (3), to provide a nondestructive and continuous spectrophotometric measurement of cell respiration (9), and to describe differences between the susceptibilities of adherent and nonadherent Candida cells (2). The M27-A MIC (4) is a useful means for the determination of MICs, although some difficulty may still be encountered in determining the MICs of certain agents, for example, azoles. Therefore, in the search for a sensitive method that assists the reading of MICs, we decided to develop the XTT assay as a colorimetric visual method. Our data show that the advantage of the XTT MIC is that one reads a colored endpoint, making reading of MICs easier. In contrast, the disadvantage of the XTT-based method is that it requires the additional step in order to add the XTT-PMS mix to the microtiter plate wells.

In this study, we have shown that agreement between the XTT and NCCLS MICs was excellent for the majority of the Candida isolates tested. For example, all of the XTT MICs fell within 2 dilutions compared to the NCCLS MICs, thus representing 100% agreement between the two methods. In addition, comparison of the MIC ranges further demonstrated the strong correlation between the two assay procedures. The only case of modest variation between these MIC determinations was observed for certain strains of C. parapsilosis and C. lusitaniae. The small variation among these isolates was not unexpected, given that previous studies with the Alamar blue reagent also showed some variation in comparison to the NCCLS reading (11). The XTT MIC was also useful for determining the susceptibility of C. neoformans. Excellent agreement between the values obtained by the two methods was observed.

In conclusion, the XTT-based method could prove to be a useful alternative to the NCCLS method. Taken together, alternative means of reading the MICs for yeast isolates such as the XTT assay, Alamar blue (5, 6), and the E-test (1) would provide a choice to the clinical microbiology laboratory when performing antifungal susceptibility testing of yeasts. Work comparing the XTT and NCCLS methods to determine the suitability of using an XTT-based method for the evaluation of the antifungal susceptibilities of filamentous fungi is currently under way.

    FOOTNOTES

* Corresponding author. Mailing address: Hoechst Marion Roussel, 102 Route de Noisy, F-93235 Romainville, France. Phone: 33-1-4991-3097. Fax: 33-1-4991-3116.

    REFERENCES
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1. Colombo, A. L., F. Barchiesi, D. A. McGough, A. W. Fothergill, and M. G. Rinaldi. 1995. Evaluation of the E-test versus a microtitre broth method for antifungal susceptibility testing of yeasts against fluconazole and itraconazole. J. Antimicrob. Chemother. 36:93-100[Abstract/Free Full Text].
2. Hawser, S. 1996. Comparisons of the susceptibilities of planktonic and adherent Candida albicans to antifungal agents: a modified XTT tetrazolium assay using synchronized C. albicans cells. J. Med. Vet. Mycol. 34:149-152[Medline].
3. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63[Medline].
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10. Tellier, R., M. Krajden, G. A. Grigoriew, and I. Campbell. 1992. Innovative endpoint determination system for antifungal susceptibility testing of yeasts. Antimicrob. Agents Chemother. 36:1619-1625[Abstract/Free Full Text].
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Journal of Clinical Microbiology, May 1998, p. 1450-1452, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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