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Journal of Clinical Microbiology, September 2004, p. 4358-4360, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4358-4360.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Comparison of the Sensititre YeastOne Colorimetric Antifungal Panel with a Modified NCCLS M38-A Method To Determine the Activity of Voriconazole against Clinical Isolates of Aspergillus spp.

Carmen Castro,1 M. Carmen Serrano,1 Bernardo Flores,1 Ana Espinel-Ingroff,2 and Estrella Martín-Mazuelos1*

Servicio de Microbiología, Hospital Universitario de Valme, Seville, Spain,1 Division of Infectious Diseases, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia2

Received 10 February 2004/ Returned for modification 5 April 2004/ Accepted 27 May 2004


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ABSTRACT
 
The susceptibilities of 63 isolates of Aspergillus spp. to voriconazole were evaluated by a modified NCCLS M38-A method and the Sensititre YeastOne method. The overall agreement was 82.5%, ranging from 100% for Aspergillus niger and Aspergillus terreus to 62.5% for Aspergillus flavus. Discrepancies between the methods were due to higher Sensititre MICs. The Sensititre YeastOne method could have potential value for susceptibility testing of Aspergillus spp. to voriconazole.


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TEXT
 
Over the past decades, the incidence of invasive fungal infections has increased, especially those caused by Aspergillus species (18). The treatment of choice for infected patients remains amphotericin B, although alternative drugs with activities against Aspergillus species are becoming available for clinical use, including antifungal azoles and echinocandins (6, 18). Because the number of serious infections caused by Aspergillus spp. has increased, as has the resistance of Aspergillus spp. to established agents, determination of the in vitro susceptibilities of Aspergillus isolates to both established and investigational agents is necessary (6).

There is a great need for an easier and reproducible method for in vitro susceptibility testing of filamentous fungi as a guide to selecting and monitoring antifungal therapy. Alternatives to the NCCLS method are currently under investigation, including two commercial methods, E-test and Sensititre YeastOne, which have been evaluated for yeast and molds (1, 3, 5, 6, 9, 10, 11, 13-17).

Our study represents an evaluation of the suitability of Sensititre YeastOne for determining the susceptibilities of Aspergillus spp. isolates to voriconazole. This test is a commercial colorimetric panel that consists of a disposable tray which contains dried serial dilutions of five antifungal agents in individual wells. The wells also contain an oxidation-reduction indicator (Alamar Blue) to generate clear-cut endpoints based on a visually detectable color change. MICs obtained by this method were compared to those obtained by the modified reference broth microdilution method (14).

A total of 63 clinical Aspergillus isolates, comprising 24 Aspergillus fumigatus isolates, 16 Aspergillus flavus isolates, 9 Aspergillus terreus isolates, 8 Aspergillus niger isolates, 4 Aspergillus glaucus isolates, and 2 Aspergillus nidulans isolates, were included in this study. These isolates were recovered from clinical specimens of patients at the Valme University Hospital, Seville, Spain, and the Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University, Richmond.

Culture, identification, and preservation of strains were done by using routine mycological methods (4, 7). Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were used as quality control strains for susceptibility testing procedures, and Aspergillus fumigatus ATCC 204305 and Aspergillus flavus ATCC 204304 were used as reference strains.

The broth microdilution method was performed according to modified NCCLS standard M38-A guidelines using RPMI 1640 medium supplemented with 2% glucose (Sigma Chemical Co., St. Louis, Mo.), and the results were compared with Sensititre YeastOne results. Voriconazole in powder form was provided by Pfizer (Central Research Division, Sandwich, United Kingdom). The drug was dissolved in dimethyl sulfoxide (Sigma-Aldrich Química, S.A.) and diluted 1:100 in RPMI medium at a final concentration of 0.03 to 16 mg/liter.

Inoculum suspensions were prepared from day 3 to day 4 cultures grown on potato dextrose agar (Difco) at 30°C and adjusted spectrophotometrically (530 nm) to optical densities that ranged from 0.09 to 0.11. The final inoculum was between 0.5 x 104 and 5 x 104 CFU/ml as demonstrated by quantitative colony counts. Drug-free and cell-free controls were included. Readings were made after 48 h of incubation at 35°C. The MIC endpoint for voriconazole was the lowest drug concentration that prevents any discernible growth.

Sensititre YeastOne panels (Trek Diagnostic Systems, Ltd., East Grimstead, United Kingdom) containing serial twofold dilutions of the drug (0.008 to 16 mg/liter) were used. Inoculum suspensions were prepared in the same way as for the M38-A method, and the adjusted suspensions were diluted 1:100 in YeastOne RPMI medium (American BioOrganics, Buffalo, N.Y). The dried YeastOne panels were rehydrated, and 100 µl of the working suspension was dispensed into each well. The panels were covered with seal strips and incubated at 35°C.

Twenty-four hours of incubation was not sufficient for the complete conversion of Alamar Blue to its pink derivative, but a bright pink color was attained for the growth controls of all strains after 48 h of incubation. Colorimetric voriconazole MICs were interpreted as the lowest concentration of antifungal solution causing a change from pink (growth) to blue (no growth).

Data analysis. MIC ranges and the concentration of voriconazole that produced the total inhibition of growth of 50% and 90% of the isolates tested (MIC50 and MIC90, respectively) were obtained for each species. Discrepancies among MIC endpoints of no more than twofold dilutions were used to calculate the percentage of agreement between the methods.

Table 1 summarizes the susceptibilities of the 63 Aspergillus strains to voriconazole and the percentage of agreement between the results of the Sensititre and NCCLS microdilution methods after 48 h of incubation. The MICs for quality control strains and reference strains were within the published range for both methods (2, 12).


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  		TABLE 1. Susceptibilities of 63 strains of Aspergillus spp. to voriconazole and agreement between Sensititre YeastOne and reference microdilution method testing determined after 48 h of incubation by the NCCLS and Sensititre YeastOne methodsb

All the strains were inhibited with ≤0.5 mg of voriconazole/liter and ≤1 mg of voriconazole/liter by the M38-A and colorimetric methods, respectively. MICs obtained by Sensititre were usually higher than those obtained by the M38-A method. In general, Sensititre MIC50s and MIC90s were 1 twofold dilution higher than those of the reference method, as shown in detail in Table 1.

The overall agreement between colorimetric and M38-A methods was 82.5%; the highest agreement between the methods was obtained for A. niger and A. terreus isolates (100%) followed by A. fumigatus isolates (87.5%), and the lowest agreement was obtained for A. flavus isolates (62.5%). Martín-Mazuelos et al. (10), testing with itraconazole, also found the best agreement for A. niger isolates, followed by A. fumigatus and A. flavus isolates. Discrepancies (greater than ±2 dilutions) between the MICs determined by the Sensititre and NCCLS M38-A methods were demonstrated for nine isolates (14.28%) tested against voriconazole: three A. fumigatus isolates, three A. flavus isolates, two A. glaucus isolates, and one A. terreus isolate. These discrepancies were always due to MICs that were higher by Sensititre than by the NCCLS M38-A method.

In the reference microdilution method, all the strains were inhibited by ≤0.5 mg of voriconazole/liter; similar results were found by other authors (5, 6, 8, 15, 18). Practically no differences were observed among both microdilution methods; by the Sensititre method, all the strains were inhibited by 1 mg of drug/liter. This result is in concordance with previous studies using Sensititre with another azole antifungal agent, itraconazole (10, 14).

Overall, higher voriconazole MICs were obtained by the Sensititre method; similar results were obtained by Martín-Mazuelos et al. (10) and Sánchez Sousa et al. (14) with itraconazole. In contrast, Meletiadis et al. (11) found that itraconazole MICs generated by Sensititre YeastOne were lower than those generated by the NCCLS method.

We have used MICs obtained after 48 h of incubation to compare the two methods because by the reference method the growth was more obvious after 48 h for all species, and in the case of the Sensititre method, 24 h of incubation was not sufficient for the complete conversion of Alamar Blue to its pink derivate. For itraconazole, Meletiadis et al. (11) found that the level of agreement between the methods was greater after 48 h of incubation (84.6%) than that after 24 h of incubation (44.6%). Martín-Mazuelos et al. (10) also found the best agreement after 48 h of incubation for this antifungal agent (90.2%). In contrast, Sánchez Sousa et al. (14) found better agreement when colorimetric results after 24 h of incubation were compared with reference broth microdilution MICs after 48 h of incubation.

In conclusion, on the basis of data from this study, the colorimetric method has potential value for the performance of susceptibility testing of filamentous fungi, so Sensititre could be a good and reliable alternative for in vitro antifungal susceptibility testing. However, more studies are required to determine which methods show the best agreement with in vivo results.


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FOOTNOTES
 
* Corresponding author. Mailing address: H. U. Valme, Ctra. Cádiz s/n, Seville, Spain. Phone: 034-955015480. Fax: 034-955015481. E-mail: estrella.martin.sspa{at}juntadeandalucia.es. Back


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Journal of Clinical Microbiology, September 2004, p. 4358-4360, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4358-4360.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Espinel-Ingroff, A. (2006). Comparison of Three Commercial Assays and a Modified Disk Diffusion Assay with Two Broth Microdilution Reference Assays for Testing Zygomycetes, Aspergillus spp., Candida spp., and Cryptococcus neoformans with Posaconazole and Amphotericin B. J. Clin. Microbiol. 44: 3616-3622 [Abstract] [Full Text]  

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