a Servicio de Microbiología Clínica, Hospital Universitario de Valme, Carretera de Cádiz s/n, E-41014 Sevilla; Spain b Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco, Bilbao, Spain
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In 1992, the National Committee for Clinical Laboratory Standards (NCCLS) proposed a standardized reference broth macrodilution method for antifungal susceptibility testing of yeast (M27-P). 11 A microdilution method following the NCCLS criteria has shown good correlation with these macrodilution methods and was incorporated in the NCCLS M27-T document. 12 The initially proposed version (M27-P) has been revised to the approved level (M-27A). 13 Irrespective of these advances the methods have some limitations, and further efforts are necessary for the development of simpler and more economical methods. 13,14
The Etest (AB Biodisk, Solna, Sweden) is a novel susceptibility testing method that involves placing a plastic strip carrying a continuous gradient of an antifungal agent on the surface of an inoculated agar plate. The Etest and the broth microdilution method, in accordance with the NCCLS recommendations, 13 were used to determine the MICs of fluconazole and of itraconazole for 402 yeast isolates representing five Candida spp. isolated from oropharyngeal infections in AIDS patients. The purpose of this study was to compare the results obtained by the Etest and broth microdilution methods.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A total of 402 yeast isolates recovered from oropharyngeal specimens from 145 AIDS patients attending the Valme University Hospital of Seville and the Department of Microbiology, País Vasco University, Bilbao, and seven quality control and reference isolates (Candida albicans ATCC 90028, C. albicans ATCC 64548, C. albicans ATCC 64540, Candida glabrata ATCC 2238, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, Candida parapsilosis ATCC 90018), 15,16 were studied. The clinical isolates consisted of 360 C. albicans, 17 C. tropicalis, nine C. krusei, nine C. glabrata and seven C. parapsilosis. Fungal identification was based on standard methods 17 as well as biochemical characterization with the YBC card in the Vitek system (API-bioMérieux, Montalieu Vercieux, France).
Antifungal agents
The Etest strips were provided by the manufacturer and had drug concentrations ranging from 0.016 to 256 mg/L for fluconazole and from 0.02 to 32 mg/L for itraconazole. The strips were stored at - 20°C until used. Reference grade powders of fluconazole (Pfizer Inc., Sandwich, UK) and itraconazole (Janssen Pharmaceutical, Beerse, Belgium) were used to prepare drug dilutions ranging from 0.125 to 64 mg/L for fluconazole and from 0.015 to 8 mg/L for itraconazole. A fluconazole stock solution of 6400 mg/L was prepared in 2% dimethylsulphoxide and an itraconazole stock solution of 800 mg/L in 100% dimethylsulphoxide. Ten-fold drug dilutions were diluted 1:5 with liquid RPMI 1640 medium described below to achieve the 2 x final concentrations.
Assay media
Liquid RPMI 1640 with L-glutamine and 2% glucose 18 without bicarbonate was buffered with morpholine- propane sulphonic acid (MOPS) to pH 7, and used in broth microdilution tests in sterile flat-bottomed microtitre plates (Nunc InterMed, LabClinics, Barcelona, Spain). Solidified RPMI 1640 with L-glutamine and 2% glucose but without bicarbonate was buffered with potassium phosphate to pH 7.0, solidified with 1.5% Bacto-agar (Difco, Detroit, MI, USA) and used for Etest MICs.
Susceptibility testing
The broth microdilution method was used as the reference method, as described by the NCCLS. 11,12,13 The inoculum was prepared from Sabouraud glucose agar subcultures incubated at 35°C for 24 h and the resulting suspension was adjusted spectrophotometrically to a density equivalent to a 0.5 MacFarland standard at 530 nm (1.5 x 10 6 cfu/mL). A working suspension was made by 1:1000 dilution of the suspension in RPMI medium and 100 µL of the diluted inoculum was added to each well. The final inoculum size was (0.5- 2.5) x 10 3 cfu/mL. The broth dilution tests were incubated at 35°C, and MICs were determined after 48 h incubation by observation of the presence or absence of visible growth. The endpoint for azole MICs was defined as the lowest concentration where a prominent decrease in turbidity was observed. 11,12,13
The Etest was performed according to the manufacturer's s instructions. The inoculum suspension was adjusted as described above. The solidified medium was inoculated by dipping a sterile cotton swab into the respective undiluted stock inoculum suspension and streaking evenly in three directions over the entire surface of a 150 mm diameter RPMI agar plate. The plate was permitted to dry for at least 15 min before the Etest strips with antifungal were placed on the medium surface. The incubation time was 48 h and the MIC was considered as the drug concentration at which the border of the elliptical inhibition zone intercepted the scale on the strip. As the Etest scale has a continuous gradient of concentrations instead of the two-fold dilutions that are tested by the broth dilution method, when the MIC determined by the Etest was intermediate it was raised to the next two-fold dilution level of the reference method for the sake of comparison. No information about the MIC determinations by the reference method was available during the reading of Etest results and vice versa.
Analysis of the results
The MICs at which 50% and 90% of the isolates tested were inhibited were determined for each azole. A comparison of azole MICs as determined by both methods was also performed. Essential agreement (EA) occurred when the MIC results by the Etest and reference methods were in exact agreement or were within two two-fold dilutions. The MICs were read by two different readers. All isolates showing discrepancies were repeated by both methods. We also studied the impact of MIC disagreements on the categories of susceptibility for the organisms tested by both methods, with the susceptibility breakpoints for the azoles published in the NCCLS M27-A document. 13,19,20
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The MICs determined by both methods for the 402 isolates tested demonstrated a broad range of susceptibility, as shown in Table I. It can be seen that the MIC 90 values of fluconazole by the Etest were one or two dilutions higher than the MICs determined by the reference method for all species studied. The details of the agreement between the MICs determined by the two methods are given in Table I. The overall EA was 74.5%, with agreement rates among five species of yeast tested covering a range from 66.6% (C. glabrata) to 88.2% (C. tropicalis). When we studied the impact of MIC disagreement between methods on the susceptibility interpretation for the yeasts tested, we found that 80% of C. albicans isolates were susceptible or resistant by both methods, whereas 14 isolates (3.8%) showed very major discrepancies (resistant by the reference method but susceptible by the Etest). Thirteen isolates (3.6%) showed major discrepancies (susceptible by the reference method but resistant by Etest) and 45 (12.5%) showed minor discrepancies. No species other than C. albicans showed very major discrepancies.
Comparison of itraconazole MICs
The MICs determined by the two methods for the 402 isolates tested covered a broad range, as
shown in Table II. The MIC
90 values were identical or ±1 dilution by the two methods, except for C. tropicalis (four dilutions higher by Etest). However, with this antifungal agent all the
species except C. parapsilosisshowed MIC
90 values 0.5 mg/L by both methods. The EA between the methods for
itraconazole is shown in Table II. The overall agreement was 61.7%,
covering a range from
42.9% (C. parapsilosis) to 88.8% (C. glabrata). The problems regarding
itraconazole MIC interpretation by Etest were fewer than for fluconazole. On the other hand, the
MIC disagreement between the methods produced a change in susceptibility interpretation with
45% of C. albicans tested, 27 (7.5%) isolates showing very major discrepancies, 20
(5.6%) showing major discrepancies and 129 (35.8%) showing minor discrepancies. No species
other than C. albicans showed very major discrepancies.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fluconazole MIC
90 values were higher (1- 2 dilutions) by the Etest than by broth microdilution for all
species studied. These data coincide with those obtained by other workers.
7,8,22 For itraconazole, the MIC
90 values by Etest were equal to those obtained by broth microdilution, and these
data also coincided with those obtained by Colombo et al.
7,8expnd-1
for most of the species studied. For C. albicans, MIC
90 values by the Etest were higher than those obtained by broth microdilution in the
studies by Columbo et al.,
7,8 and for C.
tropicalis our MIC90 values by Etest were four times higher than those by broth
microdilution. Our MIC90 results for both antifungal agents are in agreement with
those obtained by Runhke et al.
22 In spite of these discrepancies, all the species studied
except C. parapsilosis and C. tropicalis showed an MIC90 32
mg/L for fluconazole by both methods. With C. glabrata and C. krusei the
MIC90 was
64 mg/L, and 64 mg/L is the resistance breakpoint for fluconazole.
For itraconazole all the species except C. parapsilosis showed an MIC
90
0.5 mg/L by both methods.
The overall percentage agreement between the two methods, based on MICs within ±2 dilutions, was higher for fluconazole (74.5%) than for itraconazole (61.4%). These values were lower than those obtained by others, 7,8,23,24,25 who observed an EA >80% with both antifungal agents. On the other hand, Sewell et al. 9 obtained similar results to ours (EA <80%), whereas Ruhnke et al. 22 obtained lower percentage agreement than in our study for fluconazole and similar results for itraconazole. On studying the species separately, we observed that for fluconazole, C. tropicalis showed the highest percentage of agreement (88.2%), whereas for itraconazole, C. glabrata showed the highest percentage of agreement (88.8%).
The NCCLS document M27-A 13 includes susceptibility breakpoints for the azoles. When we analysed the impact of MIC disagreements between the two methods on susceptibility interpretation of the isolates tested, we found a high correlation for fluconazole. With 80% of isolates the MIC discrepancies did not produce a change in their susceptibility interpretation. For itraconazole the correlation was very poor (<60%) with a high percentage of isolates showing very major discrepancies. These discrepancies could be attributed to the difficulty in determining the endpoints for the azoles, because partial inhibition in an Etest ellipse was frequently observed. This difficulty in reading of the Etest plates for azole antifungals has been observed by other authors, 25,26,27,28 who noted in most cases an inhibition zone with diffuse edges and growth of microcolonies within the inhibition zone on RPMI agar. Sharper edges were observed on Casitone agar. C. albicans was the species that gave greatest problems in reading, but our data do not coincide with those published by Colombo et al., 7,8 who found greater problems with C. tropicalis. Pfaller et al. 26 found for C. krusei ATCC 6258 that MIC values by the Etest were higher than by the broth microdilution method, probably because the composition of the culture medium was different (a solidified medium with 2% glucose, which according to some workers 29 would allow the yeast to grow in the presence of fluconazole). As the MICs for the control isolates included in this study were within the established ranges, and our essential agreement for the isolates from clinical specimens was lower than found by other workers, 7,8,9,10 these discrepancies could exist because the isolates we studied were from HIV patients with oropharyngeal candidosis or candida colonization. As stated by Odds, 30 with antifungal susceptibility tests the published data show that the results are of scientifically proven worth only in a limited set of circumstances and that clinical correlation with MICs is weaker than ideal.
We can conclude that the Etest could be a useful alternative method for study of the susceptibility of yeasts to antifungal agents, because it is technically an easier method than broth dilution. However, because of the difficulty in reading the endpoint, further studies are necessary to determine the culture medium and incubation conditions which give clearer endpoints.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Dupont, B., Graybill, J. R., Armstrong, D., Laroche, R., Touze, J. E. & Wheat, L. J.(1992)).Fungal infections in AIDS patients.Journal of Medical and Veterinary Mycology 30, Suppl. 1, 19-28.[ISI][Medline]
3 . Vanden Bossche, H. (1997)).Mechanisms of antifungal resistance.Revista Iberoamericana de Micología 14, 44-9.
4 . Goodman, J. L., Winston, D. J., Greenfield, R. A., Chandrasekar, P. H., Fox, B., Kaizer, H. et al. (1992)).A controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation. New England Journal of Medicine 326, 845-51[Abstract]
5 . Redding, S., Smith, J., Farinacci, G., Rinaldi, M., Fothergill, A., Rhine-Chalberg, J. et al. (1994)).Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS: documentation by in vitro susceptibility testing and DNA subtype analysis.Clinical Infectious Diseases 18, 2402-42.
6 . Sanguinetti, A., Carmichael, J. K. & Campbell, K.(1993)). Fluconazole-resistant Candida albicans after long-term suppressive therapy.Archives of Internal Medicine 153, 1122-4.[Abstract]
7 . 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]
8 . Colombo, A. L., Barchiesi, F., McGough, D. A., Fothergill, A. W. & Rinaldi, M. G.(1995)).Evaluation of the Etest system versus a microtitre broth method for antifungal susceptibility testing of yeasts against fluconazole and itraconazole.Journal of Antimicrobial Chemotherapy 36, 93-100.[Abstract]
9 . 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]
10 . Espinel-Ingroff, A., Pfaller, M. A., 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]
11 . National Committee for Clinical Laboratory Standards.(1992)., Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Proposed Standard M27-P . NCCLS, Villanova, PA.
12 . National Committee for Clinical Laboratory Standards. (1995). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Tentative Standard M27-T . NCCLS, Villanova, PA.
13 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A . NCCLS, Villanova, PA.
14 . Cormican, M. G. & Pfaller, M. A.(1996). Standardization of antifungal susceptibility testing.Journal of Antimicrobial Chemotherapy 38, 561-78.[Abstract]
15 . Pfaller, M. A., Bale, M., Buschelman, B., Lancaster, M., Espinel-Ingroff, A., Rex, J. H. et al. (1995)).Quality control guidelines for National Committee for Clinical Laboratory Standards recommended broth macrodilution testing of amphotericin B, fluconazole and flucytosine. Journal of Clinical Microbiology 33, 1104-7.[Abstract]
16 . Rex, J. H., Pfaller, M. A., Lancaster, M., Odds, F. C., Bolmström, A. & Rinaldi, M. G.(1996)).Quality control guidelines for National Committee for Clinical Laboratory Standards-recommended broth macrodilution testing of ketoconazole and itraconazole.Journal of Clinical Microbiology 34, 816-17.[Abstract]
17 . Warren, N. G. & Hazen, K. C.(1995). Candida, Cryptococcus, and other yeasts of medical importance. In Manual of Clinical Microbiology, 6th edn (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F.C. & Yolken, R. H., Eds), pp. 723- 37. American Society for Microbiology, Washington, DC.
18 . Rodríguez-Tudela, J. L. & Martínez-Suárez, J.(1995)).Defining conditions for microbroth antifungal susceptibility tests: influence of RPMI and RPMI- 2% glucose on the selection of endpoint criteria.Journal of Antimicrobial Chemotherapy 35, 739-49.[Abstract]
19 . Ghannoum, M. A., Rex, J. H. & Galgiani, J. N.(1996)).Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome.Journal of Clinical Microbiology 34, 489-95.[Abstract]
20 . Rex, J. H., Pfaller, M. A., Galgiani, J. N., Bartlett, M. S., Espinel-Ingroff, A., Ghannoum, M. A. et al. (1997)).Development of interpretative breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro- in vivo correlation data for fluconazole, itraconazole, and Candida infections.Clinical Infectious Diseases 24, 235-47.[ISI][Medline]
21 . Brown, D. F. J. & Brown, L.(1991)).Evaluation of the Etest, a novel method of quantifying antimicrobial activity.Journal of Antimicrobial Chemotherapy 27, 185-90.[Abstract]
22 . Ruhnke, M., Schmidt-Westhausen, A., Engelmann, E. & Trautmann, M.(1996)).Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy.Journal of Clinical Microbiology 34, 208-11.[Abstract]
23 . Espinel-Ingroff, A.(1994)).Etest for antifungal susceptibility testing of yeasts.Diagnostic Microbiology and Infectious Disease 19,217 -20.[ISI][Medline]
24 . van Eldere, J., Joosten, L., Verhaeghe, A. & 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]
25 . Dannaoui, E., Colin, S., Pichot, J. & Piens, M. A.(1997)).Evaluation of the E test for fluconazole susceptibility testing of Candida albicans isolates from oropharyngeal candidiasis.European Journal of Clinical Microbiology and Infectious Diseases 16, 228-32[ISI][Medline]
26 . Pfaller, M. A., Messer, S. A., Bolmström, 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]
27 . Arikan, S., Gür, D. & Akova, M.(1997)).Comparison of Etest, microdilution and colorimetric dilution with reference broth macrodilution method for antifungal susceptibility testing of clinically significant Candida species isolated from immunocompromised patients.Mycoses 40, 291-6.[ISI][Medline]
28 . Torres-Rodríguez, J. M., Madrenys, N., Jiménez, J. & Saballs, P. (1997). Concentraciones mínimas inhibitorias de levaduras a cinco antifúngicos utilizando un micrométodo de dilución estandarizado y el Etest.Revista Iberoamericana de Micología 14,115 18.
29 . Espinel-Ingroff, A., Rodríguez-Tudela, J. L. & Martínez-Suárez, J. V.(1995)).Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution methods M27-P for in vitro testing fluconazole-resistant and -susceptible isolates of Candida albicans .Journal of Clinical Microbiology 33, 3154-8.[Abstract]
30 . Odds, F. C.(1997)).Personal opinion: can antifungal sensitivity tests predict clinical treatment outcomes. Revista Iberoamericana de Micología 14, 83-4.
Received 10 March 1998; returned 7 May 1998; revised 6 July 1998; accepted 4 January 1999