Comparison of Etest and a tablet diffusion test with the NCCLS broth microdilution method for fluconazole and amphotericin B susceptibility testing of Candida isolates

Maiken Arendrupa,*, Bettina Lundgrenb,c, Irene Møller Jensenc, Bo Sønder Hansena and Niels Frimodt-Møllera

a Departments of Clinical Microbiology and b Mycobacteriology, Statens Serum Institut, Artillerivej 5, DK-2300S, Copenhagen; c Department of Clinical Microbiology, University Hospital of Copenhagen, Herlev Hospital, Herlev, Denmark


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three methods were compared for the susceptibility testing of yeast isolates to fluconazole and amphotericin B: two fagar diffusion methods (Etest and a tablet diffusion test) and the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. Given as MIC50s (range), fluconazole endpoints were: for the 24 h broth microdilution test, 0.25 mg/L (0.06–32 mg/L); for the Etest, 0.38 mg/L (0.064–24 mg/L); and for the NCCLS broth microdilution test, 2 mg/L (0.06–>=64 mg/L). With breakpoints of <3 mg/L for susceptible and >16 mg/L for resistant, the Etest and the 24 h microdilution test classified the isolates in agreement with the classification obtained by the NCCLS method. Results obtained by Etest were in closer NCCLS method than those obtained with the tablet test. Amphotericin B endpoints were lower for the 24 h microdilution and Etests than MICs obtained by the NCCLS broth microdilution method. Reproducibility was high for all tests; however, disadvantages of both diffusion tests were microcolonies in the inhibition zone and dependence on stringent standardization of inoculum.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antifungal susceptibility testing is associated with difficulties related to a high dependency on standardized inoculum, media, pH, incubation time and temperature, and partial growth inhibition leads to difficulties in obtaining reproducible endpoints.14 The development of the National Committee for Clinical Laboratory Standards (NCCLS) reference method (M27-A)5 for broth dilution antifungal susceptibility testing of yeasts has been an important tool for standardizing susceptibility testing, and tentative breakpoints have been suggested for fluconazole, itraconazole and 5-fluorocytosine. However, many clinical microbio-logy laboratories find the dilution method labour intensive and therefore commercial diffusion tests are more attractive. Etest for antimycotic susceptibility testing has been investigated in several studies.14,6,7 In contrast, evaluations of the Rosco (Taastrup, Denmark) tablet diffusion test are few, do not include amphotericin B, and the relative performance of the two test methods has not been fully investigated.8,9 This study was undertaken to compare susceptibility testing methods with the two most widely used antifungal agents, amphotericin B and fluconazole, and by two commercial diffusion tests, Rosco Neo-Sensitabs and Etest. The NCCLS reference broth microdilution method with 48 h incubation was used as the ‘gold standard’ and special attention was paid to the influence of differences in incubation time of the three methods.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organisms

One hundred and thirteen clinical isolates from blood or spinal fluid were collected prospectively from 1994 to 1999. Twelve non-albicans isolates from the UK National Collection of Pathogenic Fungi (NCPF) (Bristol, UK) were included to provide a broader spectrum of isolates. The 125 isolates consisted of 92 Candida albicans, 13 Candida glabrata, nine Candida tropicalis, five Candida parapsilosis, two Candida krusei, two Candida kefyr, one Candida lusitaniae and one Candida guilliermondii. Isolates from the American Type Culture Collection (VA, USA) consisted of C. albicans ATCC 90028,5 C. krusei ATCC 6258,5 C. parapsilosis ATCC 22019,5 C. albicans ATCC 6454810 and C. albicans ATCC 64550,10 and were included as controls. The isolates were subcultured for 24 h on Sabouraud glucose agar before susceptibility testing.

Antifungal agents

A fluconazole (Pfizer, Ballerup, Denmark) stock dilution of 10 000 mg/L and an amphotericin B (Sigma–Aldrich, Vallensbæk Strand, Denmark) stock dilution of 5000 mg/L were prepared in dimethylsulphoxide.

Susceptibility testing

MICs and zone diameters were recorded after 24 and 48 h of incubation at 35°C, yielding a total of 1500 endpoints. To investigate the reproducibility of these results, 17 clinical isolates were tested twice and the five control strains were tested five to nine times.

Broth microdilution tests were performed according to NCCLS document M27-A.5 Microtitre plates were read spectrophotometrically at 490 nm, after mixing the wells by pipetting to resuspend yeast sediments. The MIC was defined as the lowest drug dilution resulting in 100% growth inhibition for amphotericin B and 80% growth inhibition for fluconazole. The following tentative breakpoints were applied: fluconazole susceptible (S), MIC <= 8 mg/L; susceptible dose-dependent (SDD), MIC >8–<64 mg/L; and resistant (R), MIC >= 64 mg/L.5

Etests were performed according to the manufacturer's instructions with a yeast suspension equivalent to 0.5 McFarland, RPMI agar plates (2% glucose, buffered to pH 7.0 with MOPS 34.6 g/L; Angus Buffers and Biochemicals, Niagara Falls, NY, USA) and Etest strips (AB Biodisk, Solna, Sweden).11 Endpoints were raised to the nearest two-fold dilution value in order to facilitate comparison with reference dilution MICs. NCCLS interpretative breakpoints were recommended by the manufacturer.11

The tablet test was performed according to the manufacturer's instructions with a yeast suspension equivalent to 0.5 McFarland, modified Shadomy agar plates (yeast nitrogen base containing glucose, asparagine and buffered with phosphate; Rosco) and Neo-Sensitabs tablets containing 15 µg of fluconazole or 10 µg of amphotericin B (Rosco).10 For C. krusei the inoculum was diluted 1:10 before inoculation of the plates. Endpoints were interpreted according to the manufacturer's instructions as follows. For fluconazole, S = zone diameters >=30 mm; intermediate susceptible (I) = zone diameters 23–29 mm; and R = zone diameters <=22 mm. For amphotericin B, S = zone diameters >=15 mm; I = zone diameters 10–14 mm; and R = zone diameters <=9 mm.10


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MIC determinations by microdilution method

Determination of fluconazole MICs by the NCCLS 48 h microdilution method divided clinical Candida isolates, other than C. glabrata, into two well-defined populations: one dominant population of susceptible isolates and a smaller population of resistant isolates [C. albicans (4), C. krusei (2), C. glabrata (1) and C. tropicalis (1)] (Table IGo). Resistant isolates with MICs <= 8 mg/L at 24 h incubation and also at 48 h, if applying a less restrictive endpoint criterion of 50% inhibition of growth as the breakpoint, are termed ‘trailing isolates’. Reading at 24 h resulted in significantly lower MIC values. The MIC50 (range) was 0.25 mg/L (0.06–32 mg/L) at 24 h (data not shown) in contrast to 2 mg/L (0.06–>=64 mg/L) at 48 h.


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Table I. Susceptibility to fluconazole as determined by the tablet diffusion and Etests, and comparison with MICs determined by the NCCLS broth microdilution method at 48 h incubation

 
The MIC50 (range) of amphotericin B by the NCCLS 48 h microdilution method was 1 mg/L (0.25–2 mg/L) (Table IIGo). Again, reading after 24 h of incubation resulted in lower MIC values, the MIC50 (range) being 0.25 mg/L (<0.03–1 mg/L) (data not shown).


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Table II. Susceptibility to amphotericin B determined by the tablet diffusion and Etests, and comparison with MICs determined by the NCCLS broth microdilution method at 48 h incubation
 
Comparison of diffusion tests with the reference NCCLS broth microdilution method

The tablet test classified 78 of 81 fluconazole S isolates as susceptible (96.3%) and two of eight R isolates as resistant (both C. krusei isolates). Three of five isolates classified as R by the tablet test were misclassified (Table IGo). Reading at 48 h changed the interpretation of susceptibility for 17 isolates (13.6%), 14 of which were non-albicans isolates. Twelve isolates were regarded as more resistant. In 12 cases (70.6%), the reclassification disagreed with the MIC determination by the NCCLS reference method (data not shown).

Etest interpreted with the NCCLS breakpoints identified all fluconazole S isolates as susceptible. In Table IGo, the S group are subdivided into two groups. As shown, all C. glabrata isolates had endpoints of >=3 mg/L, while this was only the case for two of 80 susceptible non-glabrata isolates (2.5%). Reading the Etest at 48 h reclassified eight of 125 isolates as more resistant. In seven cases, the reclassification was in agreement with the classification by the NCCLS reference method (data not shown). Non-albicans isolates that were reclassified as SDD or R had Etest endpoints >=3 mg/L at 24 h.

Isolates demonstrating the ‘trailing’ phenomenon were interpreted as S by both diffusion tests (Table IGo).

Determinations of amphotericin B susceptibility are presented in Table IIGo. Indicated as median (range), the zone diameters by the tablet test at 24 h were 22 mm (20–22 mm), 20 mm (18–25 mm), 20 mm (12–24 mm) and 18 mm (16–22 mm) for the isolates with MICs of 0.25, 0.5, 1 and 2 mg/L, respectively; thus a considerable overlap was seen.

A better correlation was observed between Etest endpoints and MICs determined by the reference method (Table IIGo). Etest readings at 48 h increased the endpoint for 10 of 125 isolates to >1 mg/L (range 1.5–64 mg/L).

Reproducibility of susceptibility testing

By the NCCLS broth microdilution method, there was 100% agreement within two log2 dilutions for clinical isolates and control strains at both incubation times and for both drugs. Susceptibility classification remained unchanged upon repeated testing, and MICs were within the established ranges for the control isolates.

The agreement of Etest endpoints with repeated testing of clinical isolates was 77.3% for amphotericin B at 24 h, and 100% for fluconazole; however, two SDD C. glabrata isolates would have been classified in a different susceptibility category on applying NCCLS tentative breakpoints. When control strains were tested repeatedly, the greatest agreement was seen for fluconazole at 24 h (93 versus 74% at 48 h), but at 48 h for amphotericin B (94 versus 90% at 24 h).

By the tablet test, the variation of zone diameter in relation to the mean zone diameter for clinical isolates tested twice was as follows (results given as mean variation in percentage of mean zone diameter): fluconazole at 24 and 48 h, 11.2 and 14%, respectively; and amphotericin B at 24 and 48 h, 13.3 and 12.2%, respectively. Repeated testing of control strains gave similar results (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The manufacturer of the Etest recommends 24 h incubation and breakpoints as suggested by the NCCLS. In this study, fluconazole and amphotericin B endpoints obtained by Etest were lower than the NCCLS MICs, this finding being in agreement with previous reports.7,12 Thus, for Etest determinations and for reading of the microdilution method at 24 h, application of interpretative breakpoints for fluconazole of <3 mg/L for S, 3–16 mg/L for SDD and >16 mg/L for R provide a more correct classification of fluconazole susceptibility according to the NCCLS reference method. Others have found Etest endpoints and those obtained by the reference method to be within the same range.1,13 This illustrates the necessity of including control strains and confirming the performance of diffusion tests by reference tests, since interlaboratory variations occur despite strict application of the manufacturer's guidelines.

For the tablet test, the best agreement with the NCCLS reference MICs was obtained at 24 h. Sandven et al.,8 who investigated the susceptibility to fluconazole by the tablet test at 48 h incubation, proposed alternative breakpoints of >=22 mm for susceptibility and <=15 mm for resistance. According to our study, this would have led to the misclassification of many C. glabrata isolates as susceptible. The readings of amphotericin B susceptibility at 24 and 48 h were similar. Although the zone diameter correlated with the MIC, considerable overlap was demonstrated for both drugs.

A high percentage of the C. albicans isolates were found to be fluconazole susceptible at 24 h incubation but resistant at 48 h using the broth microdilution test. The majority of these isolates demonstrated partial inhibition over a wide range of concentrations, and applying a less stringent endpoint criterion of 50% growth inhibition reclassified >85% of these isolates as susceptible. The reported number of trailing isolates varies from 3 to 40%, as described previously.9,13 One reason for this discrepancy is the use of visual versus spectrophotometric endpoint determination, the latter being more objective. Differences in test medium composition may also influence the number of trailing isolates.14

In conclusion, on the condition that isolates demonstrating the trailing phenomenon are susceptible in vivo, both Etest and broth microdilution susceptibility testing, with 24 h incubation and using the breakpoints for fluconazole suggested above, are suitable for antifungal susceptibility testing. Etest and broth microdilution were superior to the tablet diffusion test because of a better agreement with the susceptibility classification by the NCCLS reference method and a better discriminatory potential. Standardization of inoculum and differences in growth rates are less critical for the microdilution method owing to the automated endpoint reading and to the fact that the endpoint is relative to growth control. A disadvantage of diffusion tests is the difficulty associated with endpoint interpretation caused by microcolonies in the inhibition zone, leading to lower reproducibility when the test is performed by several technicians; for this reason we recommend the broth microdilution method.


    Acknowledgments
 
The authors thank Pfizer A/S (Ballerup, Denmark) for kindly providing the fluconazole powder for microdilution susceptibility testing.


    Notes
 
* Corresponding author. Tel: +45-44-88-38-79; Fax: +45-44-88-37-72; E-mail: mad{at}ssi.dk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Pfaller, M. A., Messer, S. A., Karlsson, A. & Bolmstrom, A. (1998). Evaluation of the Etest method for determining fluconazole susceptibilities of 402 clinical yeast isolates by using three different agar media. Journal of Clinical Microbiology 36, 2586–9.[Abstract/Free Full Text]

2 . Espinel-Ingroff, A., Pfaller, M., Erwin, M. E. & Jones, R. N. (1996). Interlaboratory evaluation of Etest method for testing antifungal susceptibilities of pathogenic yeasts to five antifungal agents by using Casitone agar and solidified RPMI 1640 medium with 2% glucose. Journal of Clinical Microbiology 34, 848–52.[Abstract]

3 . Wanger, A., Mills, K., Nelson, P. W. & Rex, J. H. (1995). Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates. Antimicrobial Agents and Chemotherapy 39, 2520–2.[Abstract]

4 . Espinel-Ingroff, A., Barchiesi, F., Hazen, K. C., Martinez-Suarez, J. V. & Scalise, G. (1998). Standardization of antifungal susceptibility testing and clinical relevance. Medical Mycology 36, Suppl. 1, 68–78.[ISI][Medline]

5 . National Commitee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.

6 . Pfaller, M. A., Messer, S. A., Bolmstrom, A., Odds, F. C. & Rex, J. H. (1996). Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates. Journal of Clinical Microbiology 34, 1691–3.[Abstract]

7 . van Eldere, J., Joosten, L., Verhaeghe, V. & Surmont, I. (1996). Fluconazole and amphotericin B antifungal susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution method compared with E-test and semiautomated broth microdilution test. Journal of Clinical Microbiology 34, 842–7.[Abstract]

8 . Sandven, P. (1999). Detection of fluconazole-resistant Candida strains by a disc diffusion screening test. Journal of Clinical Microbiology 37, 3856–9.[Abstract/Free Full Text]

9 . Canton, E., Peman, J., Carrillo-Munoz, A., Orero, A., Ubeda, P., Viudes A. et al. (1999). Fluconazole susceptibilities of bloodstream Candida sp. isolates as determined by National Committee for Clinical Laboratory Standards method M27-A and two other methods. Journal of Clinical Microbiology 37, 2197–200.[Abstract/Free Full Text]

10 . Rosco. (1998). Neo-Sensitabs User's Guide, 10th edition. Rosco, Taastrup, Denmark.

11 . AB Biodisk. (1997). Etest Technical Guide 4. AB Biodisk, Solna, Sweden.

12 . Sewell, D. L., Pfaller, M. A. & Barry, A. L. (1994). Comparison of broth macrodilution, broth microdilution, and Etest antifungal susceptibility tests for fluconazole. Journal of Clinical Microbiology 32, 2099–102.[Abstract]

13 . Colombo, A. L., Barchiesi, F., McGough, D. A. & Rinaldi, M. G. (1995). Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for azole antifungal susceptibility testing. Journal of Clinical Microbiology 33, 535–40.[Abstract]

14 . Marr, K. A., Rustad, T. R., Rex, J. H. & White, T. C. (1999). The trailing end point phenotype in antifungal susceptibility testing is pH dependent. Antimicrobial Agents and Chemotherapy 43, 1383–6.[Abstract/Free Full Text]

Received 31 March 2000; returned 9 August 2000; revised 27 October 2000; accepted 4 December 2000