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Journal of Clinical Microbiology, November 2008, p. 3668-3671, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.01242-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Comparison between the Standardized Clinical and Laboratory Standards Institute M38-A2 Method and a 2,3-Bis(2-Methoxy-4-Nitro-5-[(Sulphenylamino)Carbonyl]-2H-Tetrazolium Hydroxide- Based Method for Testing Antifungal Susceptibility of Dermatophytes

Atef S. Shehata,^1,2 Pranab K. Mukherjee,² and Mahmoud A. Ghannoum^2*

Microbiology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt,¹ Center for Medical Mycology, Department of Dermatology, Case Medical Center and Case Western Reserve University, Cleveland, Ohio²

Received 1 July 2008/ Returned for modification 8 August 2008/ Accepted 21 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we determined the utility of a 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based assay for determining antifungal susceptibilities of dermatophytes to terbinafine, ciclopirox, and voriconazole in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. Forty-eight dermatophyte isolates, including Trichophyton rubrum (n = 15), Trichophyton mentagrophytes (n = 7), Trichophyton tonsurans (n = 11), and Epidermophyton floccosum (n = 13), and two quality control strains, were tested. In the XTT-based method, MICs were determined spectrophotometrically at 490 nm after addition of XTT and menadione. For the CLSI method, the MICs were determined visually. With T. rubrum, the XTT assay revealed MIC ranges of 0.004 to >64 µg/ml, 0.125 to 0.25 µg/ml, and 0.008 to 0.025 µg/ml for terbinafine, ciclopirox, and voriconazole, respectively. Similar MIC ranges were obtained against T. rubrum by using the CLSI method. Additionally, when tested with T. mentagrophytes, T. tonsurans, and E. floccosum isolates, the XTT and CLSI methods resulted in comparable MIC ranges. Both methods revealed similar lowest drug concentrations that inhibited 90% of the isolates for the majority of tested drug-dermatophyte combinations. The levels of agreement within 1 dilution between both methods were as follows: 100% with terbinafine, 97.8% with ciclopirox, and 89.1% with voriconazole. However, the agreement within 2 dilutions between these two methods was 100% for all tested drugs. Our results revealed that the XTT assay can be a useful tool for antifungal susceptibility testing of dermatophytes.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Dermatophytes are a group of related keratinophilic fungi that infect the superficial keratinized tissues (skin, hair, and nails) of humans and animals. They belong to three genera: Epidermophyton, Microsporum, and Trichophyton, each of which includes several recognized species (17). Infections caused by these fungi are among the most prevalent cutaneous infections globally, and the recent increase in the number of patients with immunocompromised states, such as AIDS, diabetes mellitus, cancer, and organ transplantation, has given these infections more prominence (4, 14, 16). Dermatophyte infections can be disfiguring, recurrent, and chronic (especially nail infections) and generally need long-term treatment with antifungal agents. Recurrent dermatophyte infections may result from poor compliance of the patient, infection with a new strain, or development of resistance to the antifungal agent used in treatment. Therefore, antifungal susceptibility testing of dermatophytes may be helpful for managing patients, especially since a number of new antifungal agents are now clinically available.

The standard broth microdilution M38-A2 method has been developed and adopted by the Clinical and Laboratory Standards Institute (CLSI) for testing of antifungal susceptibility of filamentous fungi, including the dermatophytes (6). Alternative colorimetry-based approaches for antifungal susceptibility testing have also been used to determine antifungal susceptibilities of yeasts (8), Aspergillus species (3, 7, 11-13), and zygomycetes (2, 5). One such colorimetric approach is based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-[(sulfenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT) by mitochondrial dehydrogenases and cell membrane ferric reductase of live organisms in the presence of an electron transfer agent (such as menadione) to form a colored formazan product (1, 9). Utility of the XTT method in antifungal susceptibility testing of dermatophytes has not been assessed. In this study, we evaluated whether the XTT method has utility in determining the antifungal susceptibilities of dermatophytes to terbinafine, ciclopirox, and voriconazole in comparison to the standard CLSI M38-A2 method.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Test isolates. We tested 46 clinical dermatophyte isolates: Trichophyton rubrum (n = 15), Trichophyton mentagrophytes (n = 7), Trichophyton tonsurans (n = 11), and Epidermophyton floccosum (n = 13). Two reference strains, T. mentagrophytes ATCC MYA 4439 and T. rubrum ATCC MYA 4438, were included as quality control isolates, as recommended by the CLSI (6). The CLSI-recommended MIC ranges for these control strains are 0.002 to 0.008 µg/ml for terbinafine, 0.5 to 2 µg/ml for ciclopirox, and 0.03 to 0.25 µg/ml for voriconazole for T. mentagrophytes ATCC MYA 4439 and 0.5 to 2 µg/ml for ciclopirox and 0.008 to 0.06 µg/ml for voriconazole for T. rubrum ATCC MYA 4438. Among the tested clinical isolates, four T. mentagrophytes isolates and six T. rubrum isolates were obtained from patients with dermatophyte infections in Egypt, while the rest of the isolates and the reference strains were obtained from the Center for Medical Mycology, Case Western Reserve University, Cleveland, OH.

Medium. Both CLSI and XTT assays were performed using RPMI 1640 medium with L-glutamine but without bicarbonate, buffered to pH 7.0 with 0.165 M 3-N-morpholinopropanesulfonic acid (Hardy Diagnostics, Santa Maria, CA).

Antifungal drugs. The antifungal susceptibilities of organisms were determined for three drugs: terbinafine (Novartis, East Hanover, NJ), ciclopirox (Sigma-Aldrich, St. Louis, MO), and voriconazole (Pfizer Inc., New York, NY). The drugs were prepared according to the guidelines of the CLSI M38-A2 method. First, these agents were obtained in powder form and dissolved in dimethylsulfoxide (DMSO) (Fisher Chemicals, Fair Lawn, NJ), and then they were serially diluted using RPMI 1640 to yield concentrations with double the desired final concentrations of 0.001 to 0.5 µg/ml for terbinafine and voriconazole and 0.06 to 32 µg/ml for ciclopirox. The antifungal agents were prepared in 96-well tissue culture plates (Becton Dickinson and Company, New Jersey). More terbinafine plates with higher final concentrations up to 64 µg/ml were prepared to retest isolates that had terbinafine MICs of >0.5 µg/ml. The prepared plates were stored at –80°C until use.

Inoculum. Dermatophyte isolates were subcultured on potato dextrose agar plates (Becton, Dickinson) at 30°C for 7 to 15 days. All of the tested strains sporulated well after this period. Stock inoculum suspensions were obtained from each strain by covering the fungal colonies with 10 ml of sterile saline and gently rubbing the colonies with the tip of a transfer pipette. The resulting conidial suspensions were transferred to sterile 15-ml centrifuge tubes. Collected conidia were filtered using sterile gauze, counted using a hemocytometer, and then adjusted to the desired density by adding RPMI 1640 medium to obtain a conidial suspension of 2 x 10³ to 6 x10³ CFU/ml. Plate counts were performed to verify the conidium concentrations by plating 0.01 ml of the adjusted conidial suspensions on Sabouraud dextrose agar to determine the viable number of conidia per milliliter. In these experiments, the plate counts ranged from 2 x 10³ to 4.5 x10³ CFU/ml.

CLSI M38-A2 method. One hundred microliters of the prepared cell suspension of each strain was added to each well of 96-well microtiter plates containing 100 µl of previously prepared antifungal drugs in RPMI 1640 to bring the drug dilutions and inoculums to the final desired test concentrations. After this step, the final concentration of DMSO in test wells became 1%. Growth and sterility controls were included for each isolate tested (growth control, RPMI medium with DMSO and organisms but with no drug added; sterility control, RPMI medium only, with no organisms or drug added). Each organism was tested in duplicate. The inoculated plates were incubated at 35°C for 4 to 7 days. The two quality control strains and all of the T. rubrum strains (15 strains) and T. mentagrophytes strains (7 strains) had sufficient growth after 4 days' incubation, while T. tonsurans (11 strains) grew sufficiently after 5 days and all 13 E. floccosum strains were incubated for up to 7 days. MICs were determined visually using an inverted reading mirror and were defined as the lowest drug concentration that caused 80% inhibition of the growth in comparison to the growth control.

XTT-based method. The susceptibility plates were prepared, inoculated, and incubated as with the CLSI method described above. Five hours before MIC determination, the plates were agitated to resuspend the fungal elements in the wells and 50-µl aliquots containing an XTT-menadione mixture (both from Sigma-Aldrich, St. Louis, MO) were added to each test well (to reach final concentrations of 200 µg/ml XTT and 25 µM menadione). The plates were then incubated in the dark for 5 h at 35°C. At the end of the incubation, the plates were centrifuged at 3,000 rpm for 5 min and the supernatant was transferred to wells of new plates using a multichannel pipetter. The optical density (OD) of the supernatant in each well was determined by measuring the absorbance at 490 nm using a microtiter plate reader (Bio-Rad, Hercules, CA). The absorbance values for antifungal agent-treated cells were compared with that of untreated cells (growth control) after subtraction of background OD (OD of the sterility control well that contained RPMI, XTT-menadione, and no organisms). The MIC for each drug/isolate combination was determined as the lowest drug concentration that resulted in an OD reading that was 20% of that of the drug-free growth control.

For both CLSI and XTT-based methods, the MIC range, MIC₅₀, and MIC₉₀ (the lowest drug concentrations that inhibited 50% and 90% of the isolates, respectively) were determined for each drug tested. The agreement between the two methods was expressed as a percentage agreement, which was determined by calculating the percentage of isolates that had XTT MICs which were either (i) the same as or within 1 dilution or (ii) within 2 dilutions of the standard CLSI-determined MICs.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, two different methods were used to determine the susceptibilities of dermatophytes to three antifungals; the first was the standard CLSI M38-A2 method and the second an XTT-based assay. MICs were determined for each tested drug against each of the tested isolates.

Antifungal susceptibility of quality control strains. Initially, the reference dermatophyte strains were used to determine whether the XTT-based assay could be used to determine antifungal susceptibilities of dermatophytes. The CLSI-determined MICs for these control strains were within the recommended ranges. The XTT MICs of the control strains were identical to the CLSI MICs for each of ciclopirox and voriconazole. Our study revealed that the CLSI and XTT-derived MICs for ciclopirox and voriconazole were 0.5 µg/ml and 0.125 µg/ml, respectively, for the reference T. mentagrophytes strain (ATCC MYA 4439) and 0.5 µg/ml and 0.015 µg/ml, respectively, for the T. rubrum reference strain (ATCC MYA 4438). However, when the drug was tested against the T. mentagrophytes reference isolate, the XTT-determined MIC of terbinafine was 1 dilution higher than the CLSI-determined MIC (0.015 µg/ml and 0.008 µg/ml, respectively). Using both methods, testing of terbinafine against the T. rubrum reference strain showed terbinafine resistance as well as agreement within 1 dilution, since the MICs were 4 µg/ml and 8 µg/ml for CLSI and XTT assays, respectively. These results provide confidence that the XTT-based method can be used to determine antifungal susceptibilities of the control isolates.

Antifungal susceptibilities of clinical isolates. Table 1 presents a comparison between the MIC ranges, MIC₅₀s, and MIC₉₀s that were obtained by both the CLSI and XTT methods for the clinical isolates with the three antifungals tested. This comparison revealed agreement between the two methods, since similar MIC ranges were obtained by both the CLSI and XTT assays for terbinafine when it was tested against the T. rubrum, T. mentagrophytes, and T. tonsurans strains (0.004 to >64 µg/ml, 0.008 to 0.06 µg/ml, and 0.015 to 0.5 µg/ml, respectively), for ciclopirox against the T. tonsurans and E. floccosum strains (0.06 to 0.5 µg/ml and 0.125 to 1 µg/ml, respectively), and for voriconazole against E. floccosum strains (0.015 to 0.03 µg/ml). This agreement was also apparent for T. rubrum strains, since both the CLSI and XTT MICs were similar for ciclopirox (0.125 to 0.5 µg/ml and 0.125 to 0.25 µg/ml, respectively) and for voriconazole (0.015 to 0.125 µg/ml and 0.008 to 0.25 µg/ml, respectively). Moreover, against both T. mentagrophytes and T. tonsurans, voriconazole had similar MIC ranges (0.001 to 0.125 µg/ml by CLSI and 0.002 to 0.125 µg/ml by XTT), and the MICs obtained with both methods differed by only 1 dilution. When T. mentagrophytes strains were tested against ciclopirox, the MICs were also comparable (0.06 to 0.25 µg/ml and 0.125 to 0.25 µg/ml for the CLSI and XTT methods, respectively). The same pattern of agreement was observed for E. floccosum with terbinafine, where XTT-based MICs were 1 dilution greater than those derived using the CLSI method (0.03 to 0.125 µg/ml and 0.015 to 0.06 µg/ml, respectively).

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TABLE 1. Comparison of MICs determined by CLSI M38-A2 method and XTT-based assay for clinical dermatophyte strains

Moreover, the XTT assay showed good agreement with the CLSI assay when tested using terbinafine-resistant strains (T. rubrum MRL 10220 and T. rubrum MRL 16575 were found to be resistant to terbinafine by both methods). For T. rubrum MRL 10220, terbinafine MICs agreed within 1 dilution (2 µg/ml and 4 µg/ml for CLSI and XTT, respectively), and for T. rubrum MRL 16575, terbinafine showed MICs of >64 µg/ml by both methods.

Comparison of MIC₅₀s and MIC₉₀s for the tested drugs against the dermatophyte isolates revealed a very close similarity between the two methods (Table 1). Both methods revealed almost equal MIC₉₀s for all tested drug-dermatophyte combinations. The only exception was susceptibility to terbinafine used against T. rubrum and E. floccosum isolates (agreement within 1 dilution) and to voriconazole used against T. tonsurans (agreement within 2 dilutions). As can be seen in Table 2, XTT-derived MICs for the majority of tested isolates and drugs were either identical or within 1 dilution of the CLSI-determined MICs, indicating agreement of the two methods. Complete agreement (100%) was noted within 2 dilutions for all tested isolate and antifungal agent combinations.

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TABLE 2. Agreement between MICs determined by XTT and CLSI M38-A2 methods (XTT MICs that were either identical to or within 1 twofold dilution of CLSI MICs)a

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The XTT-based method has been utilized in monitoring of metabolic activities of some fungi (2) and in testing of antifungal susceptibilities of yeast and filamentous fungi (2, 3, 7, 8, 11-13). To our knowledge, the current study is the first to test the utility of XTT for determining the antifungal susceptibilities of dermatophytes in comparison to the standardized CLSI M38-A2 method. Our results showed excellent agreement between the two methods, as shown by comparing the MIC ranges obtained by the two methods. This concordance between the CLSI and XTT methods was also apparent when the MIC₅₀s and MIC₉₀s for the tested drugs were compared (Table 1). MIC₅₀s were equal or within 1 dilution for all drug-dermatophyte combinations obtained. Additionally, MIC₉₀s were identical for all examined drugs against all tested isolates obtained using both techniques, with the exception of terbinafine used against E. floccosum and T. rubrum (where the difference was within 1 dilution) and voriconazole used against T. tonsurans (2-dilution difference). Similar agreements were reported in other studies comparing the XTT and CLSI methods for antifungal susceptibility testing of yeast and filamentous fungi (8, 12).

The agreement between the two methods (Table 2) was very strong and revealed that the overall percentages of XTT-determined MICs (equal or within 1 dilution) in comparison to CLSI MICs were as follows: 100% (46/46), 97.8% (45/46), and 89.1% (41/46) for terbinafine, ciclopirox, and voriconazole, respectively. For voriconazole, the agreement between the two methods was 100% with T. rubrum and E. floccosum, 80% with T. tonsurans, and 71.4% with T. mentagrophytes. On the other hand, the agreement within 2 dilutions between the two methods was 100% for the three tested drugs with all dermatophyte species and strains examined in this study.

In the current study, the XTT assay, similar to the CLSI microbroth method, was able to differentiate the antifungal agent-resistant dermatophyte isolates. In this regard, both methods gave high MICs when tested with terbinafine-resistant dermatophytes, and these MICs fell within 1 dilution for T. rubrum ATCC MYA 4438 and T. rubrum MRL 10220. This agreement was also apparent with testing of a third more resistant clinical isolate (T. rubrum MRL 16575), where both assays showed MICs of >64 µg/ml.

The use of XTT in antifungal susceptibility testing depends on a color change that is caused by reduction of this salt by mitochondrial dehydrogenase enzymes and/or a cell membrane ferric reductase enzyme of metabolically active cells (1, 9). Therefore, this XTT-based technique provides information about the metabolic activity and living status of the tested organisms. An additional advantage of this method is that the development of color is measured spectrophotometrically, facilitating more-quantitative determination of MICs, especially when the tested drugs have the problematic trailing endpoints as seen with some azole antifungal agents (10, 15).

The long incubation time after addition of XTT-menadione, which lasted for 5 h, can be a disadvantage. In some cases we found that the color change began to develop within 2 h after adding the XTT-menadione mixture, but that color was too weak to be measured in comparison to the color that developed after 5 h (data not shown). Additionally, we attempted to reduce the CLSI-recommended incubation time of 4 days and monitored XTT activity after incubating the test wells for 2 days. All isolates failed to give a color change with this shorter incubation time except for T. mentagrophytes, which gave a very faint color that was difficult to measure (data not shown). These findings may be explained by the slow growth rate of dermatophytes.

In conclusion, our data showed that the XTT-based method provides a colorimetric quantitative method for determining the antifungal susceptibilities of dermatophytes which is in agreement with the CLSI method.

 
This study was supported by funds through the Ministry of Higher Education, Egypt, and the Center for Medical Mycology, Case Western Reserve University, Cleveland, OH.

We thank Nancy Isham for her critical review of the manuscript.

 
* Corresponding author. Mailing address: Center for Medical Mycology, Department of Dermatology, Case Medical Center and Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106. Phone: (216) 844-8580. Fax: (216) 844-1076. E-mail: mahmoud.ghannoum{at}case.edu

Published ahead of print on 1 October 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Clinical Microbiology, November 2008, p. 3668-3671, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.01242-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shehata, A. S. Articles by Ghannoum, M. A. Search for Related Content PubMed PubMed Citation Articles by Shehata, A. S. Articles by Ghannoum, M. A.