JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1996, p. 3208–3211 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 34, No. 12
Comparative Evaluation of Three Antifungal Susceptibility Test Methods for Candida albicans Isolates and Correlation with Response to Fluconazole Therapy MARKUS RUHNKE,1* ANDREA SCHMIDT-WESTHAUSEN,2 ELISABETH ENGELMANN,3 4 AND MATTHIAS TRAUTMANN Abteilung Innere Medizin und Poliklinik1 and Institut fu ¨r Medizinische Mikrobiologie,3 Virchow-Klinikum der Humboldt-Universita ¨t, and Zentrum fu ¨r Zahnmedizin, Universita ¨tsklinikum Charite´,2 13353 Berlin, and Abteilung Medizinische Mikrobiologie und Hygiene, Institut fu ¨r Mikrobiologie und Immunologie, Universita ¨t Ulm, 89075 Ulm,4 Germany Received 29 April 1996/Returned for modification 11 June 1996/Accepted 16 September 1996
In vitro susceptibilities were determined for 56 Candida albicans isolates obtained from the oral cavities of 41 patients with human immunodeficiency virus infection. The agents tested included fluconazole, itraconazole, ketoconazole, flucytosine, and amphotericin B. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P (M27-P micro), a broth microdilution technique using high-resolution medium (HR micro), and the Etest with solidified yeast-nitrogen base agar. The in vitro findings were correlated with in vivo response to fluconazole therapy for oropharyngeal candidiasis. For all C. albicans isolates from patients with oropharyngeal candidiasis not responding to fluconazole MICs were found to be >6.25 mg/ml by the M27-P micro method and >25 mg/ml by the HR micro method as well as the Etest. However, for several C. albicans isolates from patients who responded to fluconazole therapy MICs found to be above the suggested breakpoints of resistance. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B > fluconazole > flucytosine > ketoconazole > itraconazole. The appropriate rank order with best agreement between the M27-P micro method and the Etest was flucytosine > amphotericin B > fluconazole > ketoconazole > itraconazole. It could be concluded that a good correlation between in vitro resistance and clinical failure was found with all methods. However, the test methods used in this study did not necessarily predict clinical response to therapy with fluconazole. not responding to antifungal therapy with fluconazole were tested in parallel by two broth microdilution methods and the Etest. The MICs of amphotericin B (Sigma A2422), flucytosine (Sigma F-7129), fluconazole (Pfizer, Sandwich, United Kingdom), itraconazole (Janssen Biotech, Beerse, Belgium), and ketoconazole (Sigma K-1003) for Candida albicans isolates were determined by the NCCLS broth microdilution method according to the M27-P protocol (M27-P micro), the broth microdilution method developed by Troke and Pye, and the Etest (1, 9, 19). The Etest antifungal gradient strips were purchased from the manufacturer (Difco Laboratories, Augsburg, Germany) and stored at 2208C until used. Fifty-six C. albicans isolates from 41 patients were tested. All isolates were recovered from the oral cavities of patients with human immunodeficiency virus infection and recurrent OPC. For the purpose of the study, Candida isolates were selected either from fluconazole responders (before treatment) who were exposed to the drug for the first and/or second time (,14 days of therapy for OPC) or from nonresponders who were repeatedly exposed to fluconazole for at least 12 months. In some patients a second isolate was obtained from a different treatment episode. The OPC could be treated successfully (no more clinical signs and symptoms of pseudomembranous candidiasis) with fluconazole (100 mg/day for 5 to 7 days) in 27 of 41 patients (9 female and 18 male; CDC stage B2 to C3; median CD4 cell count, 42/ml [range, 1 to 300/ml]), but in 14 of 41 patients (3 female and 11 male; CDC stage C3; median CD4 cell count, 8/ml [range, 0 to 16/ml]) the OPC did not respond to fluconazole (300 to 400 mg/day for 7 days). Twenty-two C. al-
The predictive value of yeast antifungal susceptibility testing in correlation to clinical outcome still raises questions. The Subcommittee on Antifungal Susceptibility Tests of the National Committee for Clinical Laboratory Standards (NCCLS) proposed a broth macrodilution method for testing of yeasts (M27-P method) which appeared to give results similar to those of the broth microdilution method (7, 9, 11). Microdilution methods other than the RPMI 1640-based protocol M27-P have not been fully evaluated but may serve as alternatives to the NCCLS method (8). The broth macro- and microdilution method using a chemically defined medium (high-resolution antifungal assay medium [HR medium]) was developed primarily for testing of yeasts with fluconazole and showed encouraging results in a recent collaborative study (10, 18). In AIDS patients with oropharyngeal candidiasis (OPC) clinical response to treatment with fluconazole has been correlated with in vitro findings from individual methods (4, 12, 14). First studies with Candida strains comparing the NCCLS method and the ready-to use agar-based Etest produced comparable results (5, 6, 17). However, studies that compared different susceptibility test methods using clinical isolates are still limited (13). In this study, Candida isolates from human immunodeficiency virus-infected patients with OPC either responding or * Corresponding author. Mailing address: Virchow-Klinikum der Humboldt-Universita¨t, Department of Medicine, Division Hematology/Oncology, Augustenburger Platz 1, 13353 Berlin, Germany. Phone: 49-30-450-59268. Fax: 49-30-450-59910. Electronic mail address:
[email protected]. 3208
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bicans isolates from 14 patients with fluconazole-refractory OPC and 34 C. albicans isolates from 27 patients who responded to fluconazole were included in this study. The quality control organism included two reference isolates (A [C. albicans ATCC 90028] and B [Candida parapsilosis YO501]). MIC determinations were performed twice for each clinical isolate and each method to ensure reproducibility. Quality control isolates were tested five times by each method throughout the study. Tests were performed with the M27-P micro method, the HR micro method, and the Etest and were read at 24 and 48 h. For the final analysis, 48-h MIC readings were used with all methods, because the M27-P standard recommends reading at this time. Furthermore, for the HR micro method reading at 48 h has been shown to correlate with clinical outcome (16). The broth microdilution test (M27-P micro) was performed by following the guidelines in NCCLS document M27-P with use of the spectrophotometric method for inoculum preparation (9, 11). An inoculum of 0.5 3 103 to 2.5 3 103 cells per ml and RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma M1254) was used. Yeast inocula (100 ml) were added to each well of microdilution trays containing 100 ml of the antifungal drug solution (23 final concentration). The final concentrations ranged from 0.09 to 100 mg/ml for fluconazole and flucytosine and 0.02 to 25 mg/ml for ketoconazole, itraconazole, and amphotericin B. The trays were incubated in air at 358C. Drugfree and yeast-free controls were included. The MIC was defined as that concentration of the drug that completely inhibited growth (amphotericin B and flucytosine) or produced an 80% reduction of turbidity (fluconazole, itraconazole, and ketoconazole) compared with the drug-free control. The broth microdilution method (HR micro) was performed according to the method of Troke and Pye (19). An inoculum of 1 3 103 to 2 3 103 yeast cells per ml was mixed in synthetic high-resolution medium (HR medium; Oxoid CM845) which was prepared with 0.2 M phosphate buffer, pH 7.2. Yeast inocula (100 ml) were added to each well of microdilution trays containing 100 ml of twice the strength of the antifungal agent. Separate dilution series of all antifungal agents were made from the stock solution (1 mg/ml) by using the HR medium. The final concentrations ranged from 0.09 to 100 mg/ml for fluconazole and flucytosine and from 0.02 to 25 mg/ml for ketoconazole, itraconazole, and amphotericin B. The trays were incubated in air at 358C. Drug-free and yeast-free controls were included. The MIC endpoint was determined visually by recording the lowest concentration of the antifungal agent that prevented the appearance of visible growth. The Etest was performed by inoculation of 150-mm-diameter plates containing 60ml modified yeast-nitrogen base medium (YNB medium) mixed with agar with a sterile cotton swab using a yeast cell suspension adjusted spectrophotometrically to 0.5 McFarland standard turbidity. The moisture was allowed to dry for 15 min, and the antifungal strips were placed on the medium surface. Determination of MICs with the Etest was performed at the exact continuous concentration on the test strip, but the concentration was finally raised to the next twofold dilution level used with the microdilution methods for comparison. Etest strip concentrations ranged from 0.016 to 256 mg/ml for fluconazole and 0.002 to 32 mg/ml for amphotericin B, flucytosine, ketoconazole, and itraconazole. Discrepancies among MIC endpoints of no more than 2 dilutions were used to calculate the percent agreement. The MICs for the two control organisms were within a close range for all test methods (for A: fluconazole, 0.19 to 0.5
NOTES
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TABLE 1. Comparison of MICs of five antifungal agents determined by three antifungal susceptibility testing methods for 56 clinical isolates of C. albicans Antifungal agent
Fluconazole
Itraconazole
Ketoconazole
Amphotericin B
Flucytosine
MIC (mg/ml)b
Test methoda
Range
50%
90%
M27-P micro HR micro Etest M27-P micro HR micro Etest M27-P micro HR micro Etest M27-P micro HR micro Etest M27-P micro HR micro Etest
0.19–$100 0.09–$100 0.09–$256 0.09–$25 0.048–$25 0.012–$32 0.09–$25 0.02–$25 0.015–$32 0.048–$3.12 0.09–$3.12 0.008–$3.12 #0.09–12.5 0.09–25 #0.002–0.5
3.12 1.56 25 0.5 0.78 0.125 0.39 1.5 2 0.39 0.78 0.39 #0.09 0.19 #0.002
$100 $100 $256 25 25 $32 $25 $25 $32 0.78 1.5 0.78 0.39 0.78 0.09
a Following the principles of the NCCLS M27-P methodology broth microdilution testing (M27-P micro) was performed as previously described (9). The alternative methods were the Etest using YNB agar and a microdilution method (HR micro) using HR medium as described previously (1, 19). b 50% and 90%, MIC50 and MIC90, respectively.
mg/ml; itraconazole, 0.094 to 0.19 mg/ml; ketoconazole, 0.125 to 0.19 mg/ml; amphotericin B, 0.25 to 1.56 mg/ml; flucytosine, 0.047 to 0.78 mg/ml) (for B: fluconazole, 0.78 to 1.5 mg/ml; itraconazole, 0.064 to 0.31 mg/ml; ketoconazole, 0.125 to 0.78 mg/ml; amphotericin B, 0.78 to 1.0 mg/ml; flucytosine, 0.008 to 0.09 mg/ml). The range of MICs of all antifungal agents for C. albicans as well as the MICs at which 50% (MIC50) and 90% (MIC90) of isolates were inhibited are summarized in Table 1. In general, the MIC90 of the azoles fluconazole, itraconazole, and ketoconazole increased for at least 2 log2 dilutions for the C. albicans isolates compared with the MIC50. The Etest produced a MIC50 of fluconazole but not the other azoles of at least 3 log2 dilutions higher compared with that by the other methods. MIC testing with flucytosine gave comparable results among the three methods, but the Etest produced lower MICs than the microdilution methods. Amphotericin B MICs were clustered within a narrow range of 3 log2 of all Candida isolates and test methods. Agreement between the M27-P micro method and HR micro method was good for fluconazole (91%), flucytosine (83%), and amphotericin B (95%). In contrast, the agreement of the Etest results with those obtained with the M27-P micro method was 67% for fluconazole, 79% for flucytosine, and 78% for amphotericin B (Table 2). Agreement among the methods was lowest for ketoconazole and itraconazole. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B . fluconazole . flucytosine . ketoconazole . itraconazole, and that between the M27-P micro method and the Etest was flucytosine . amphotericin B . fluconazole . ketoconazole $ itraconazole. In vitro fluconazole resistance was defined as a MIC of $25 mg/ml by the three methods. For all C. albicans isolates (100%) from patients with OPC not responding to fluconazole MICs were found to be $25 mg/ml by the HR micro method and the Etest, but that was the MIC for only 86% by the M27-P micro method (for one isolate the fluconazole MIC was 6.25 mg/ml, and for two isolates MICs were 12.5 mg/ml). However, for 91%
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TABLE 2. Percent agreement of Etest and HR micro results with M27-P micro results for 56 C. albicans isolates at 48 h Antifungal agent
Fluconazole Itraconazole Ketoconazole Amphotericin B Flucytosine
Test method
% Agreementa
HR micro Etest HR micro Etest HR micro Etest HR micro Etest HR micro Etest
91 67 65 64 66 64 95 78 83 79
a Percentage of MICs that were equivalent to the M27-P micro method MICs within 2 dilutions.
of C. albicans isolates from patients responding to fluconazole therapy MICs were #12.5 mg/ml by the M27-P micro method, compared to 97% by the HR micro method and 85% by the Etest (Table 3). The Troke and Pye broth microdilution (HR micro) method produced an acceptable level of agreement with the NCCLS M27-P micro method for fluconazole, flucytosine, and amphotericin B. It was expected that, because HR medium was developed for in vitro testing of fluconazole, sharper fluconazole MIC endpoints would be determined with this medium. This assumption was true for most isolates. For the other azoles, ketoconazole and itraconazole, agreement between the M27-P micro method and the HR micro method and between the M27-P micro method and the Etest was low (65 to 66% and 64%, respectively). The Etest manufacturer offers five different solidified media for antifungal susceptibility testing of yeasts. In this study the solidified YNB medium was used for the Etest, because it has been proposed to be equally suited for testing all antifungal agents with one medium. However, using this medium failed to produce perfect agreement in the present study. Earlier studies comparing the M27-P micro method and the Etest (using RPMI 1640 agar) showed that MIC readings after 24 h showed better agreement than after 48 h (6, 17). Sewell et al. found 79% agreement after 48 h in contrast to 93% after 24 h in a study comparing the MICs of fluconazole for C. albicans using the NCCLS macrobroth dilution and the Etest (17). We found only a slightly better agreement with the Etest at 24 h (69% agreement at 24 h and 67% at 48 h for fluconazole [data not shown]). Partial inhibition of fungal growth in vitro often takes place over a range of all azole concentrations. It is recommended by
the NCCLS method and the Etest to determine the MIC endpoints at the concentration of the azole antifungal agents that reduces growth $80% compared with the growth control. In the present study excessive trailing at MIC endpoints was a problem only for some tests with the M27-P micro and the HR micro methods but frequently with the Etest. MIC endpoint determination was difficult and subjective with the Etest because of diffuse endpoints on the YNB agar with many clinical Candida isolates. All test methods produced high MICs for some C. albicans isolates from patients who responded to fluconazole therapy. The appropriate rank order of best correlation of in vivo and in vitro findings was HR micro . M27-P micro . Etest. However, a larger number of isolates need to be studied to make definite conclusions. Precise prediction of clinical failure by determining a high MIC is still controversial, because MIC breakpoints for antifungal agents are not yet defined. According to earlier suggestions we used a MIC breakpoint of $25 mg/ml for fluconazole to define in vitro resistance using the HR micro method (16, 18). Additionally, we used the clinical description of resistance defined as a failure to clear pseudomembranous oral candidiasis with daily dosages of 300 to 400 mg of fluconazole. Table 3 shows that for three C. albicans isolates from AIDS patients with OPC not responding to fluconazole, fluconazole MICs were ,25 mg/ml by the M27-P micro method. With the HR micro method as well as the Etest MICs for all C. albicans isolates from nonresponders were $25 mg/ml. Consequently, the breakpoint for the M27-P micro method may be at $12.5 mg/ml and should be $25 mg/ml for the other two methods. On the other hand, MICs of .12.5 mg/ml could be found with C. albicans isolates from patients who responded to fluconazole therapy for OPC (Table 3). Using these breakpoints, the lowest rate of false in vitro resistance to fluconazole was found with the HR micro method (one isolate). Higher rates of in vitro resistance to fluconazole in patients with a clinical response to fluconazole were found with the M27-P micro method (3 of 34 C. albicans isolates) and the Etest (5 of 34 C. albicans isolates). Barchiesi et al. defined in vitro fluconazole resistance as MICs for C. albicans of .8 mg/ml using the NCCLS broth macrodilution method (2). In contrast, after analysis of Candida bloodstream isolates from nonneutropenic patients with fungemia Rex et al. argued that the fluconazole breakpoint might be .64 mg/ml (15). However, the inverse correlation between log(MIC) and outcome as noted by Rex et al. was not seen in this study with isolates from the oral cavity. The MIC breakpoint for fluconazole resistance with the Etest was suggested by the manufacturer to be .8 mg/ml (3). In the
TABLE 3. Fluconazole MICs for 56 C. albicans isolates from 41 patients with human immunodeficiency virus infection and OPC either responding or not responding to oral fluconazole as defined by M27-P micro, HR micro, and Etest and mycologic response in patients No. of strains in: a
MIC (mg/ml)
#6.25 12.5 25 $50 Agreement with therapyc a b c
Nonrespondersb
Responders M27-P micro
HR micro
Etest
M27-P micro
HR micro
Etest
30 1 1 2 31 (91%)
33 0 0 1 33 (97%)
28 1 0 5 29 (85%)
1 2 7 13 19 (86%)
0 0 5 17 22 (100%)
0 0 0 22 22 (100%)
The total number of strains from responders was 34. The total number of strains from nonresponders was 22. For responders, a MIC of #12.5 mg/ml constituted agreement with therapy; for nonresponders, a MIC of $25 mg/ml constituted agreement with therapy.
VOL. 34, 1996
NOTES
present study the Etest MICs for fluconazole-resistant C. albicans isolates were as high as $256 mg/ml for 90% of the isolates. It may be even more difficult to establish the breakpoints for the azoles ketoconazole and itraconazole. In this study were included six C. albicans isolates from three patients who failed to respond to fluconazole but responded to itraconazole solution (400 mg/day). In the two microdilution methods itraconazole MICs were #3.12 mg/ml for the six C. albicans isolates from patients who responded to itraconazole. The Etest provided low MICs from clinical responders only for three of six C. albicans isolates (data not shown). Because agreement between all three test methods is low, information from these findings remains unclear. Barchiesi et al. found that itraconazole MICs for fluconazole-resistant C. albicans isolates were #2 mg/ml, which may serve as a breakpoint for in vitro susceptibility (2). Five C. albicans isolates from three patients who failed to respond to fluconazole therapy and who were treated with intravenous amphotericin B were included. The MICs were #1.56 mg/ml with all test methods, but all three patients failed to respond to intravenous amphotericin B (data not shown). However, further studies are needed for these antifungal agents to evaluate the prediction of response to therapy from MIC testing. Correlation of in vitro findings for ketoconazole and flucytosine with response to therapy could not be established in this study, because none of the patients had received treatment with these antifungal agents. It could be concluded that a good correlation between in vitro fluconazole resistance and clinical failure was found with all methods; however, the converse was not necessarily true. Good agreement between the M27-P micro method and the HR micro method was found when testing fluconazole, amphotericin B, and flucytosine. Discrepant results were obtained in some instances with MIC testing of fluconazole, since for several isolates from patients responding to this drug MICs were above the preliminary breakpoint for resistance. REFERENCES 1. AB Biodisk. 1994. Etest technical guide no. 4b. Antifungal susceptibility testing of yeasts. AB Biodisk, Solna, Sweden. 2. Barchiesi, F., A. L. Colombo, D. A. McGough, A. W. Fothergill, and M. G. Rinaldi. 1994. In vitro activity of itraconazole against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients infected with human immunodeficiency virus. Antimicrob. Agents Chemother. 38: 1530–1533. 3. Bolmstro¨m, A., A. Karlsson, A. Wiman, and K. Mills. 1995. Validation of Etest MICs for antifungal agents tested on different media as compared to broth microdilution. 7th European Congress of Clinical Microbiology and Infectious Diseases, Vienna, Austria, 26 to 30 March 1995. Poster 793. 4. Cameron, M. L., W. A. Schell, S. Bruch, J. A. Bartlett, H. A. Waskin, and J. R. Perfect. 1993. Correlation of in vitro fluconazole resistance of Candida isolates in relation to therapy and symptoms of individuals seropositive for human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 37: 2449–2453.
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