JAC Advance Access originally published online on August 17, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):861-863; doi:10.1093/jac/dkl335
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Comparative study of disc diffusion and microdilution methods in susceptibility testing of micafungin against Candida species
1 Servicio de Microbiología Clínica, Hospital Universitario de Valme Seville 41014, Spain 2 Servicio de Bioestadística, Unidad Investigación, Hospital Universitario de Valme Seville, Spain
*Corresponding author. Tel: +34-955015480; Fax: +34-955015481; E-mail: estrella.martin.sspa{at}juntadeandalucia.es
Received 19 May 2006; returned 4 July 2006; revised 18 July 2006; accepted 19 July 2006
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Abstract |
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Objectives: To compare the Clinical Laboratory Standards Institute CLSI M44-A disc diffusion (DD) and M27-A2 broth microdilution (MD) methods for determining the susceptibility of Candida spp. to micafungin (FK463).
Patients and methods: A total of 355 clinical yeast isolates including 270 Candida albicans, 45 Candida glabrata, 24 Candida krusei, 11 Candida tropicalis and 5 Candida parapsilosis were studied. The MIC of micafungin was determined by following the CLSI M27-A2 guidelines (MD). Endpoints were defined as the lowest concentration of micafungin resulting in partial inhibition (IC50) of visual growth after 24/48 h of incubation at 35°C. Final concentrations were 0.008–4 mg/L of micafungin. DD testing was performed using a CLSI M44-A document with 2.5 µg micafungin discs. Zone diameter endpoints were read after 24/48 h of incubation at 35°C. Arbitrary breakpoints were 4 mg/L for MD and 15 mm for DD.
Results: The best correlation was observed when we read MD 48 h/DD 24 and 48 h (97%). When the reading was MD 24 h/DD 24 and 48 h the percentage of correlation was 95.2%.
Conclusions: The DD method performs well for testing the susceptibility of Candida spp. to micafungin. More studies involving more Candida strains with elevated MIC values are needed.
Keywords: susceptibility testing , antifungals , lipopeptides
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Introduction |
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The incidence of life-threatening mycoses has been increasing in individuals with compromised immune systems. Among the many species, Candida albicans is the most important microorganism implicated in fungal infection.1 Antifungal agents currently available for clinical use are inadequate against serious systemic fungal infections and they also have serious side effects. Therefore, there is an urgent need for the development of novel antifungal agents with superior therapeutic effect and high safety.2 Extensive use of fluconazole as a prophylaxis treatment in immunocompromised patients has selected Candida spp. that are resistant to this drug.3 For these reasons it is necessary to study new antifungal agents and their activities against these yeast isolates.
Micafungin (FK463) is a novel, semi-synthetic antifungal echinocandin-like lipopeptide that inhibits the synthesis of 1,3-ß-D-glucan, an essential polymeric polysaccharide in the cell wall of many pathogenic fungi. In vitro, micafungin demonstrated potent and broad-spectrum fungicidal activity against clinically relevant Candida spp. and potent inhibitory activity against Aspergillus spp.4
The CLSI M27-A2 document published in 2002 is considered a reference method for susceptibility testing of yeasts such as Candida spp.; however, it does not describe testing conditions for the echinocandins and there are no established breakpoints.5 Although the CLSI method for in vitro susceptibility testing is essential for standardization and to improve inter-laboratory reproducibility, it may not be the best or the most convenient method for all organisms or for routine use in clinical laboratories. Alternative methods such as the agar-based disc diffusion (DD) method are suitable because of their simplicity and low cost. In addition, these methods could improve the trailing effect that occurs with the broth microdilution (MD) method.6
In the present study, we investigated the applicability of the DD method for testing the susceptibility of Candida species to micafungin and compared these results with the MD method of the CLSI (CLSI document M27-A2) for Candida spp. strains isolated from the oral cavities of HIV-infected patients.
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Isolates
A total of 355 clinical yeast isolates including 270 C. albicans, 45 Candida glabrata, 24 Candida krusei, 11 Candida tropicalis and 5 Candida parapsilosis were studied. These isolates were recovered from the oral cavity of HIV-infected patients. Each strain represented a unique isolate from a patient attending the Valme University Hospital, Seville, Spain. Isolates were stored in Microbank cryogenic vials at –70°C.7 Identification of these strains was carried out by common mycological methods.
Susceptibility testing
MD method..
Stock solutions of standard antifungal powder of micafungin (FK463, Fujisawa Pharmaceutical Co., Japan) and inoculum suspension were prepared as outlined in CLSI document M27-A2.6 Final concentrations of micafungin were 0.008–4 mg/L. These trays were incubated at 35°C and MIC endpoints were determined after 24–48 h. The MICs of micafungin were read, by eye, as the lowest concentration that caused a significant diminution of growth compared with the control growth well. The endpoint used is directly related to the medium that we used. When using RPMI 1640, the endpoint is a prominent decrease in turbidity corresponding to 
50% inhibition in growth as determined spectrophotometrically.5 Use of 100% reduction in turbidity only comes into play when you are utilizing antibiotic medium 3 (M3). Some studies have demonstrated that the most reliable testing conditions for echinocandins and Candida are the use of RPMI broth and a prominent inhibition MIC endpoint.8
DD method.. The DD test was done using micafungin discs prepared in-house according to the CLSI M44-A guidelines.9 The disc concentration (2.5 µg/disc) was based on preliminary experiments in which blank discs were impregnated with 25 µL of suspension containing 100 mg/L caspofungin.8 Zone diameters (in mm) for the zone of complete inhibition were determined after 24 and 48 h of incubation at 35°C and compared with MICs determined by the CLSI M27-A2 MD method. Under these conditions, good growth was obtained for all the tested isolates.
An arbitrary breakpoint was established for both methods. Isolates with MICs 
4 mg/L and diameter inhibition zone (ZD) of <15 mm were considered to have high MIC values, and those isolates with MICs of <4 mg/L and diameter inhibition zone (ZD) 15 mm were considered to have low MIC values.
Quality control isolates including ATCC 22019 (C. parapsilosis), ATCC 6258 (C. krusei) and four type culture collection strains [C. albicans (ATCC 90028, ATCC 64550 and ATCC 64548) and C. tropicalis (ATCC 750)] were used in every batch tested.5,9
Statistical analysis
For the comparative evaluation of the DD and MD methods, arithmetic means (AMs) and MIC ranges were calculated for each genus-species combination. The percentage of agreement correlation was also calculated. The MIC-inhibition zone diameter datasets for micafungin were analysed by our modifications of the Metzler and DeHaan approach. For non-parametric correlation, Spearman's rho correlation coefficients were calculated. Statistical analysis was performed using SPSS software package, version 12 (SPSS, Chicago, IL, USA).
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Results and discussion |
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The results are expressed as MIC50s (MICs that inhibit 50% of the studied strains), MIC90s (MICs that inhibit 90% of the studied strains) and ranges for MD, and as AMs and ranges for DD (Table 1). Micafungin exhibited high antifungal activity against C. albicans, C. glabrata, C. krusei and C. tropicalis (MIC50
0.5 mg/L) by the MD method. However, C. parapsilosis showed high MICs (4 mg/L) compared with other Candida species as shown by others.5 Similar results were obtained at 24 and 48 h of incubation.
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For the DD method, Mueller–Hinton agar (Difco) supplemented with 2% glucose and methylene blue (0.5 mg/L) was used.9 Growth of all isolates was good at 24 h and the susceptibility results were similar after 24 and 48 h of incubation (Table 1). All species of Candida showed high ZD (AM
15 mm) except C. parapsilosis (AM < 15 mm) using the DD method (Table 1).
There was a good correlation between low MIC values and large inhibition zones for most organisms tested. This correlation was better for MD 48 h/DD 24 h than MD 24 h/DD 24 h (r = –0.43 and r = –0.39, respectively) (Figure 1a and b). However, the converse was not always true since we noted that a few isolates with elevated MIC after 48 h of incubation did not show small inhibition zones after 24 h. One possible explanation of this observation is a paradoxical regrowth.10 Figure 1(a) shows that DD is a good method for detecting isolates with low MICs, because all isolates with MICs <4 mg/L had 15 mm inhibition zones, except three isolates of C. glabrata. These three isolates of C. glabrata have inhibition zones of <15 mm and an MIC value of 1 mg/L. On the other hand, there were five isolates of C. albicans and three of C. parapsilosis for which the MIC was 4 mg/L but with zones 15 mm (36.3%). At 48 h MD detected 14 isolates with MICs 4 mg/L and <15 mm inhibition zones (63.6%) (Figure 1a). When 24 h DD was compared with 24 h MD (Figure 1b) there were 13 isolates with MIC values of 1 mg/L but small ZD (<15 mm) instead of high ZD because of the low MIC values observed. There were only four isolates with MIC values of 4 mg/L with ZD 15 mm (Figure 1b). MD at 24 h only detected six isolates with MIC values of 4 mg/L and only two of these were detected by DD (<15 mm). The best correlation (97%) was observed when we read MD 48 h/DD 24 h. There were discrepancies in both methods, but only in 11 cases at 48 h MD versus 24 h DD, instead of 17 cases at 24 h MD versus 24 h DD (97% and 95.2% correlation, respectively) (data not shown). Following the Metzler and Dehaan method, the percentage of false resistance (FR or major errors) and false susceptible (FS or very major errors) when we read 24 h MD/24 h DD was 33% and 5.5%, respectively. When the reading was at 48 h MD/24 h DD the results were FR = 36.3% and FS = 1.36%.
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Since the DD method is less time-consuming it could be a method of choice for a clinical laboratory. The ability of the DD method to determine the susceptibility of an individual isolate within 24 h appears to be its notable advantage.
In the present study, we used the DD assay for susceptibility testing of micafungin against Candida species and compared the results with the MD method. This is the first report to demonstrate a correlation between the in vitro susceptibility data from a broth MD technique and an agar-based method for micafungin in yeast. The regression statistics (Figure 1a) demonstrate a good correlation between 24 h zone sizes compared with the 48 h broth MD method. These data are in agreement with those reported for caspofungin.6 The correlation with the 24 h broth MD method was less significant (Figure 1b).
On the basis of our results, it is suggested that the DD test is a useful method for testing the activity of micafungin against Candida spp and it is possible to read it at 24 h. Additional studies with Candida strains with high micafungin MICs are necessary, as are multi-laboratory studies, to document the reproducibility of these methods and to establish quality control parameters. The clinical significance of these in vitro results should be determined by clinical trials.
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None to declare.
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Acknowledgements |
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We have not received funds for this article.
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References |
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1 Edmond MB, Wallace SE, McClish DK, et al. (1999) Nosocomial bloodstream infections in United States hospital: a three-year analysis. Clin Infect Dis 29:239–44.[Web of Science][Medline]
2 Rubio Calvo MC, Gil J, Ramírez de Ocáriz I, et al. (2003) Actividad in vitro de fluconazol, voriconazol y posaconazol frente a Candida spp. Rev Esp Quimioterap 16:227–32.
3 Cuenca-Estrella M, Mellado E, Diaz-Guerra TM, et al. (2000) Susceptibility of fluconazole-resistant clinical isolates of Candida spp to echinocandin LY303366, itraconazole and amphotericin. Antimicrob Agents Chemother 41:1835–6.
4
Denning DW. (1997) Echinocandins and pneumocandins a new antifungal class with a novel mode of action. J Antimicrob Chemother 40:611–4.
5 National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved standard M27-A2. NCCLS, Wayne, PA, USA, 2002.
6
Lozano-Chiu M, Page WN, Victor LP, et al. (1999) Disk diffusion method for determining susceptibilities of Candida spp. to MK-0991. J Clin Microbiol 37:1625–7.
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Espinel-Ingroff A, Montero D, Martin-Mazuelos E. (2004) Long-term preservation of fungal isolates in commercially prepared cryogenic microbank vials. J Clin Microbiol 42:1257–9.
8
Pfaller MA, Messer SA, Boyken L, et al. (2004) Further standardization of broth microdilution methodology for in vitro susceptibility testing of caspofungin against Candida species by use of an international collection of more than 3,000 clinical isolates. J Clin Microbiol 42:3117–19.
9 National Committee for Clinical Laboratory Standards. Method for Antifungal Disc Diffusion Susceptibility Testing of Yeasts: Propose Guideline M44-A. NCCLS, Wayne, PA, USA, 2004.
10
Stevens DA, Espiritu M, Parmar R. (2004) Paradoxical Effect of caspofungin: reduced activity against Candida albicans at high drug concentration. Antimicrob Agents Chemother 48:3407–11.
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