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JAC Advance Access originally published online on July 10, 2007
Journal of Antimicrobial Chemotherapy 2007 60(3):658-661; doi:10.1093/jac/dkm247
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

In vitro susceptibilities to yeasts using the ATB® FUNGUS 2 method, compared with Sensititre Yeast One® and standard CLSI (NCCLS) M27-A2 methods

Josep M. Torres-Rodríguez* and Eidi Alvarado-Ramírez

Infectious Diseases and Mycology Research Unit (URMIM), IMIM/IMAS, Faculty of Medicine, Autonomous University of Barcelona, Spain

* Corresponding author. Tel: +34-933160400; Fax: +34-93160410; E-mail: jmtorres{at}imim.es

Received 20 March 2007; returned 14 May 2007; revised 7 June 2007; accepted 11 June 2007


   Abstract
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 Abstract
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Objectives: The microbroth ATB® FUNGUS 2 (ATBF2) method (bioMérieux, La Balme-les Grottes, France), designed for in vitro determination of the susceptibility of Candida spp. and Cryptococcus neoformans to antifungal agents, was evaluated with 100 yeasts and compared with Sensititre Yeast One® (SYO; Trek Diagnostic Systems, UK), considering CLSI M27-A2 as the reference method.

Methods: ATBF2 consists of ready-to-use strips including amphotericin B (AMB), 5-flucytosine, fluconazole and itraconazole for MIC determinations. Reproducibility of ATBF2 was determined. Two quality control strains and a panel of eight Candida isolates were tested five different times with the three methods. The essential agreements within ±2 log2 dilution between the ATBF2, SYO and M27-A2 methods were assessed. The yeast clinical isolates included were nine species of Candida (n = 80) and C. neoformans (n = 20).

Results: Inter- and intra-laboratory reproducibility, tested with the Candida panel, was ≥99%. MICs for the ATCC strains were within the expected ranges with the three methods. Visual and automated readings of ATBF2 presented good concordance, being lower with itraconazole. The overall essential agreements with the M27-A2 method were 94% and 99% for automated ATBF2 and visual ATBF2 readings, respectively. For SYO, the agreement was 91%. Percentages of agreements by drugs (automated ATBF2/visual ATBF2/SYO) were: 5-flucytosine, 97/100/90; AMB, 97/100/85; fluconazole, 93/97/95; and itraconazole, 89/98/95. Disagreement was higher between M27-A2 and SYO than between M27-A2 and ATBF2.

Conclusions: ATBF2 is an objective, reproducible and simple method for the accurate determination of MICs of the most common antifungal drugs in yeasts.

Keywords: antifungal susceptibilities , microdilution tests , Candida spp. , Cryptococcus neoformans


   Introduction
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Some alternative methods to the CLSI (formerly NCCLS) M27-A2 standard method for in vitro susceptibility testing have been commercialized and most of them have been evaluated.1–3 Ready-to-use tests should be reliable, reproducible and easy to perform for routine application in clinical laboratories.

ATB® FUNGUS 2 (ATBF2 bioMérieux, La Balme-les Grottes, France) is a modification of the former ATB FUNGUS and adds fluconazole and itraconazole to 5-flucytosine and amphotericin B, which were present in the prior strip, improving its clinical utility. Preliminary studies showed high reliability and reproducibility as well as good agreement with the CLSI standard method.4

The goal of this study was to assess MICs using the ATBF2 strip and the colorimetric microbroth method Sensititre Yeast One® (SYO; Trek Diagnostic Systems, UK) by testing 100 human clinical isolates of Candida spp. and Cryptococcus neoformans. MICs of both methods were referred to the microbroth dilution method described in the CLSI document.


   Materials and methods
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Yeast isolates

A total of 100 yeasts, isolated from clinical samples (oropharyngeal, vaginal, skin, nails, urine and CSF for C. neoformans), were tested: Candida albicans (n = 25), Candida glabrata (n = 15), Candida krusei (n = 10), Candida tropicalis (n = 6), Candida parapsilosis, Candida pelliculosa, Candida lipolytica and Candida guilliermondii (n = 5 each), Candida lusitaniae (n = 4) and C. neoformans (n = 20).

All isolates were identified using standardized morphological and biochemical methods (tube germination, chlamydospora production and API20C) (bioMérieux).

Another eight isolates of Candida spp. were used as a reproducibility panel, and two quality control strains C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were added. The reproducibility panel was tested five different times in two laboratories (URMIM, Barcelona, Spain and bioMérieux R&D).

Clinical isolates were stored at –20°C in skimmed milk and, when required, they were subcultured on Sabouraud glucose agar with chloramphenicol plates to ensure purity and optimal growth. When necessary, new biochemical tests were performed in order to confirm isolate identification.

MIC determination

Each isolate was tested in parallel using the two alternative commercial methods.

An ATBF2 strip is composed of 32 wells including a growth control and 4 antifungal drugs at different concentrations: 5-flucytosine from 0.5 to 64 mg/L (log2 dilutions), amphotericin B from 0.5 to 16 mg/L, fluconazole from 0.25 to 128 mg/L and itraconazole from 0.125 to 4 mg/L. The yeasts were suspended in sterile 0.85% NaCl to reach a turbidity equivalent to that of a 2 McFarland standard using a densitometer (DENSIMAT®, bioMérieux), and 20 µL of the suspension was added to the specific growth medium (ATBF2 medium). After homogenization, 135 µL was inoculated in each well. After incubation at 35°C, the strips were read either visually or automatically by the mini Api® instrument (bioMérieux) at 24–48 h for Candida species and at 48 h for C. neoformans isolates. The MICs were determined according to the following reading modes: no reduction or slight reduction in turbidity (scores 4 and 3) was considered as no inhibition and prominent decrease to no growth (scores 2 to 0) corresponded to inhibition for azoles and 5-flucytosine. Only optically clear was considered as susceptible to amphotericin B.

SYO tests several antifungal drugs, only the four in common with ATBF2 were considered here: 5-flucytosine, 0.03–64 mg/L; amphotericin B, 0.0008–16 mg/L; fluconazole, 0.125–256 mg/L; and itraconazole, 0.008–16 mg/L. Manufacturer's instructions were followed, 20 µL of a 0.5 McFarland standard yeast suspension was transferred into 11 mL of the RPMI broth and 100 µL was inoculated into each well of the microplate. The plates were visually read after 24–48 h of incubation at 35°C. The MIC was the lowest concentration of antifungal that produced a colour change from blue to red, corresponding to a significant inhibition of fungal growth.

The reference microdilution method was performed as recommended in the M27-A2 document.1 The following drugs and concentrations were tested: 5-flucytosine, 0.125–128 mg/L; amphotericin B, 0.03–32 mg/L; fluconazole, 0.125–128 mg/L; and itraconazole, 0.03–32 mg/L using RPMI 1640 medium in MOPS buffered to pH 7, and the yeast inocula were adjusted to a turbidity equivalent to that of a 0.5 McFarland standard at 530 nm wavelength using a spectrophotometer.

The incubation time was 24–48 h for the Candida spp. and 48 h for C. neoformans.

Reproducibility, essential agreement and category calculation

Reproducibility was the percentage agreement within ±1 log2 dilution. For each one of the four drugs and each reading mode, the essential agreements between ATBF2 and SYO with M27-A2 were assessed. Essential agreement was considered as the percentage agreement within ±2 log2 dilutions of the reference MIC. Categories were determined according to the M27-A2 document.


   Results
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MIC readings for the two quality control strains were within the limits reported in the CLSI M27-A2 document1 and those described in each one of the guides for the commercial methods.

The intra- and inter-laboratory reproducibility, corresponding to 320 MIC results obtained with the panel of eight isolates of Candida spp., was ≥99% for ATBF2.

MIC50 values for Candida spp. were within the susceptible range for all four antifungals, but for MIC90, only amphotericin B presented values ≤1 mg/L. MIC90s of fluconazole were within 32–64 mg/L, those of flucytosine within 8–16 mg/L and those of itraconazole within 1–4 mg/L.

All isolates of C. neoformans were within ≤0.016–0.5 mg/L for amphotericin B. MIC90 was within 2–16 mg/L for 5-flucytosine, 16–32 mg/L for fluconazole and 0.12–0.5 mg/L for itraconazole. In general, the highest MICs were obtained with the M27-A2 method.

No major difference was observed between the methods tested. MIC50 showed very good concordance between the standard method and ATBF2 (visual and automatic readings) and SYO. When MIC90 was considered, the concordance was good. Itraconazole presented the lowest concordance for Candida spp. The ranges of MICs observed with the three methods presented good consistency.

Distribution of categories for Candida spp. and C. neoformans showed the highest susceptibilities to amphotericin B and flucytosine and the lowest to itraconazole.

Essential agreement

Global agreement between ATBF2 and M27-A2 (Table 1) was 99% and 94%, respectively, for visual and automated readings. Global agreement between SYO and M27-A2 (Table 2) was 91%; amphotericin B gave the lowest essential agreement (85%).


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  		Table 1. Essential agreement (in per cent) between ATB® FUNGUS 2 and Sensititre Yeast One® (SYO), compared with the M27-A2 method in 100 isolates of yeasts (Candida spp. n = 80 and C. neoformans n = 20)

 


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  		Table 2. Essential agreement (in per cent) between two commercial methods in 80 isolates of Candida spp. and 20 isolates of C. neoformans

 
Considering each antifungal agent, the essential agreement of ATBF2 with the reference method for visual reading was 100% for amphotericin B and 5-flucytosine, 98% for itraconazole and 97% for fluconazole. Automated reading gave slightly lower results: 97% for amphotericin B and 5-flucytosine and 89% and 93% for itraconazole and fluconazole, respectively.

Category agreement

Categorizations in terms of susceptible, intermediate/susceptible dose-dependent and resistant were determined according to the breakpoints defined in the CLSI document.1

The level of resistance detection for Candida species and C. neoformans was equivalent for the three drugs. Four isolates of C. albicans presented major discrepancies between M27-A2 and the automated ATBF2 with itraconazole, but not with visual reading. Only one of these isolates presented major discordances with the SYO method.


   Discussion
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 Materials and methods
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Failure to respond to antifungal treatment may be due to microbiological resistance; for this reason, there is generally a need to have accurate methods for susceptibility testing. In vitro determination of resistance to antifungal agents is still far behind that of antibacterial agents. However, a standardized microbroth dilution method has been available from the CLSI and it is a useful tool yielding reproducible results and is valuable in the evaluation of new antifungal tests.1–3,5,6

Different ready-to-use commercial methods have been described and applied for antifungal testing of yeasts, but limited data on correlation with the M27-A2 methodology are available. Among the broth-based MIC systems, the ATBF2 method is an improvement of the former ATB® FUNGUS method7 including fluconazole and itraconazole, two of the most widely used antifungals in clinical practice, and the SYO colorimetric test is an adapted system of the microbroth CLSI method.

The present study demonstrates that ATBF2 presents a global essential agreement of >95% with visual reading. For automated reading, the highest agreement was observed for amphotericin B and 5-flucytosine and the lowest for fluconazole and itraconazole. The highest MICs obtained were due to the trailing phenomenon, particularly with C. tropicalis and some strains of C. albicans.

The lowest essential agreement between SYO and M27-A2 was found for amphotericin B (85%) and flucytosine (90%). Better agreements have been described by other authors in comparison with the M27-A method.8 From our experience, SYO seems to present a less discriminative performance for these antifungals. The use of other culture media such as Mueller–Hinton agar with glucose and methylene blue or the antibiotic medium 3 that gives a broader distribution of MICs could improve susceptibility testing, as has been described for amphotericin B.9

For SYO, turbidity is not considered but only the colour change. In this case, a trailing phenomenon can produce a slight change in colour, identical for all drug concentrations above the MIC.

In conclusion, the ATBF2 strip is a simple, objective, reproducible and accurate method for the determination of MICs for Candida spp. and C. neoformans against amphotericin B, 5-flucytosine, fluconazole and itraconazole. It can be considered a valuable alternative to the CLSI method and it is an appropriate method for in vitro resistance detection in yeasts for the four antifungals tested here.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
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 References
 
This study was supported by bioMérieux.


   Transparency declarations
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None to declare.


   Acknowledgements
 
We thank Genevieve Bossy and Laurence Nougier for their continuous support in data analysis and critical reading and we also thank Yolanda Morera for the technical help.


   References
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 Materials and methods
 Results
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1 National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Second Edition: Approved Standard M27-A2 (2002) Wayne, PA, USA: NCCLS.

2 Druetta A, Freydiere A, Guinet R, et al. Evaluation of five commercial antifungal susceptibility testing systems. Eur J Clin Microbiol Infect Dis (1993) 12:336–42.[CrossRef][Web of Science][Medline]

3 Lu JJ, Lee SY, Chiueh TS. In vitro antifungal susceptibility testing of Candida blood isolates and evaluation of the E-test method. J Microbiol Immunol Infect (2004) 37:335–42.[Medline]

4 Morera Y, Nougier L, Bossy G, et al. Comparison of the newly designed ATB Fungus 2 strip (bioMerieux) and NCCLS microdilution method for the in vitro antifungal susceptibility testing of yeasts. In Poster # 301 of the RICAI 2003 Congress, Paris.

5 Rex JH, Pfaller MA, Wash TJ, et al. Antifungal susceptibility testing: practical aspects and current challenges. Clin Microbiol Rev (2001) 14:643–58.[Abstract/Free Full Text]

6 López-Jodra O, Torres-Rodríguez JM, Méndez-Vasquez R, et al. In vitro susceptibility of Cryptococcus neoformans isolates to five antifungal drugs using a colorimetric system and the reference microbroth method. J Antimicrob Chemother (2000) 45:645–9.[Abstract/Free Full Text]

7 Quindós G, Salesa R, Carrillo-Muñoz A, et al. Multicenter evaluation of ATB Fungus. A standardized micromethod for yeast susceptibility testing. Chemotherapy (1994) 40:245–51.[Web of Science][Medline]

8 Davey KG, Szekely A, Johnson EM, et al. Comparison of a new commercial colorimetric microdilution method with a standard method for in-vitro susceptibility testing of Candida spp. and Cryptococcus. J Antimicrob Chemother (1998) 42:439–44.[Abstract/Free Full Text]

9 Park BJ, Arthington-Skaggs BA, Hajjeh RA, et al. Evaluation of amphotericin B interpretative breakpoints for Candida bloodstream isolates by correlation with therapeutic outcome. Antimicrob Agents Chemother (2006) 50:1287–92.[Abstract/Free Full Text]


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