JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1998, p. 153–156 0095-1137/98/$04.0010 Copyright © 1998, American Society for Microbiology
Vol. 36, No. 1
Interpretation of Trailing Endpoints in Antifungal Susceptibility Testing by the National Committee for Clinical Laboratory Standards Method SANJAY G. REVANKAR,1* WILLIAM R. KIRKPATRICK,1 ROBERT K. MCATEE,1 ANNETTE W. FOTHERGILL,1 SPENCER W. REDDING,1 MICHAEL G. RINALDI,2 1,2 AND THOMAS F. PATTERSON University of Texas Health Science Center at San Antonio1 and Audie Murphy Division, South Texas Veterans Health Care System,2 San Antonio, Texas Received 14 July 1997/Returned for modification 22 September 1997/Accepted 10 October 1997
Trailing endpoints remain a problem in antifungal susceptibility testing using the National Committee for Clinical Laboratory Standards (NCCLS) method. For isolates for which trailing endpoints are found, MICs of 64 mg/ml at 48 h are usually observed. In a study of human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis, we identified three patients with multiple serial isolates for which trailing endpoints were observed with fluconazole. At 24 h, MICs were generally 64 mg/ml, while the organism remained susceptible by agar dilution testing with fluconazole. Most episodes of oropharyngeal candidiasis with trailing-endpoint isolates responded to doses of fluconazole as low as 100 mg/day. Two patients had both susceptible and trailing-endpoint isolates by NCCLS broth macro- and microdilution testing; these isolates were found to be the same strain by pulsed-field gel electrophoresis using restriction fragment length polymorphisms. Another patient had two different strains, one for which trailing endpoints were observed and one which was susceptible at 48 h. Trailing endpoints may be seen with selected isolates of a strain or may be a characteristic finding for most or all isolates of a strain. In addition, with isolates for which trailing endpoints are observed, reading the endpoint for the NCCLS method at 24 h may be more appropriate. (This work was presented at the 97th General Meeting of the American Society for Microbiology, Miami Beach, Fla., 6 May 1997 [12a]).
Antifungal susceptibility testing of yeasts continues to develop into a clinically useful tool. The National Committee for Clinical Laboratory Standards (NCCLS) method has been widely used and standardized, with good in vitro-in vivo correlation in the setting of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-infected patients (3, 7, 11, 12). We have developed an agar dilution method that uses fluconazole to screen for resistant yeast isolates and that has excellent correlation with the NCCLS method (8, 9). Recently, interpretive breakpoints were proposed for antifungal testing of yeasts with fluconazole by the NCCLS method, with MICs of #8 mg/ml indicating that the organism is susceptible, MICs of 16 to 32 mg/ml indicating that it is susceptible but dose dependent, and MICs of $64 mg/ml indicating that it is resistant (13). Trailing endpoints remain a problem in antifungal testing of certain yeast isolates by the NCCLS method, in which 48-h MICs are several dilutions above the 24-h values. However, no standardized definition exists for the phenomenon of trailing. In addition, the interpretation and clinical significance of trailing endpoints have not been established. We examined three HIV-positive patients with recurrent oropharyngeal candidiasis from whom we repeatedly isolated strains of Candida albicans for which we found trailing endpoints by NCCLS testing, and we performed agar dilution susceptibility and restriction fragment length polymorphism (RFLP) analyses for each strain in addition to examining the clinical outcomes of the treatment.
MATERIALS AND METHODS Patients. Three HIV-infected patients were selected from a study of recurrent oropharyngeal candidiasis. All had multiple serial isolates for which trailing endpoints were found by antifungal susceptibility testing with fluconazole by the NCCLS method. Patients were treated with 200 mg of fluconazole on day 1, followed by 100 mg of fluconazole per day for 7 days or until complete clinical response which was defined as the resolution of symptoms and clearance of lesions, occurred. The daily dose of fluconazole was increased as needed to achieve clinical response. Clinical evaluation by the same provider occurred weekly during therapy. Clinical samples for culture were taken weekly during episodes of thrush and quarterly for surveillance cultures. Samples were obtained by culture swab of individual lesions and by patients swishing and spitting 10 ml of sterile normal saline that was then used for culture. Antifungal susceptibility testing. (i) Agar dilution. Two types of media were utilized with and without fluconazole: (i) RPMI 1640-based agar and (ii) CHROMagar Candida chromogenic media (CHROMagar Company, Paris, France) with specific color patterns for yeasts to improve detection of non-C. albicans species: C. albicans (green), Candida krusei (lavender), Candida tropicalis (blue), and Candida glabrata (purple) (8). Fluconazole was added to these media at 8 and 16 mg/ml. One hundred microliters of swish solution was plated onto these media and incubated at 30°C for 48 h before growth was assessed. The addition of fluconazole assisted in differentiating resistant from susceptible yeasts: growths on media with and without fluconazole were compared as described previously (8). (ii) NCCLS method. Fungal cultures were performed as described above, and three to five colonies from each sample were submitted for MIC determination to correlate with appearance on fluconazole-containing media as either susceptible or resistant. MIC testing with fluconazole by the NCCLS broth macro- and microdilution methods (7) was performed at the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio. (iii) Trailing endpoints. Isolates were considered to have trailing endpoints by NCCLS testing if the 24-h MIC was ,8 mg/ml (which usually indicates susceptibility) and the 48-h MIC was $64 mg/ml (which usually indicates resistance). RFLP analysis. (i) Yeast plug preparation. Yeast strains were isolated on Sabouraud dextrose agar and grown at 30°C for 48 h. Colonies were suspended
* Corresponding author. Mailing address: University of Texas Health Science Center at San Antonio, Department of Medicine/ Infectious Diseases, 7703 Floyd Curl Dr., San Antonio, TX 782847881. Phone: (210) 567-4823. Fax: (210) 567-3303. E-mail:
[email protected]. 153
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FIG. 1. Multiple trailing-endpoint isolates recovered from patient 1 over a 6-month period from three episodes of symptomatic oropharyngeal candidiasis. Results of RFLP analysis are shown, demonstrating the persistence of the same strain, though trailing endpoints were observed for some isolates upon susceptibility testing. The patient responded to 100 mg of fluconazole per day for each episode of oropharyngeal candidiasis, despite the presence of the trailing-endpoint isolates. Numbers below the gel are MICs, in micrograms per milliliter.
in 2 ml of 75 mM NaCl–25 mM EDTA to a turbidity of approximately 2.0 McFarland. The suspension was centrifuged at 230 3 g for 10 min, and the pellet was resuspended in 1 ml of 75 mM NaCl–25 mM EDTA. Yeast plugs were made by mixing together 1 ml of 1.5% low-melting-point agarose in 125 mM EDTA (pH 7.5), 75 ml of zymolase (2,000 U/ml; ICN Biomedicals, Inc., Aurora, Ohio), and 1 ml of the cell suspension at 37°C. This mixture was distributed into plug molds and refrigerated for 1 h at 4°C. Spheroplasts were made by placing plugs in 4 ml of 0.5 M EDTA (pH 9.0–7.5% b-mercaptoethanol. Plugs were incubated overnight at 37°C and then rinsed with 5 ml of 50 mM EDTA, pH 7.5. Four milliliters of ESP (100 ml of 0.5 M EDTA, 10.2 ml of 10% sarcosyl, 2.0 mg of proteinase K) solution was added to each tube and incubated at 50°C overnight. Plugs were refrigerated at 4°C for 1 h. (ii) RFLP digestion. One third of each plug was incubated in a microcentrifuge tube in 1 ml of TE-PMSF (10 mM Tris, 1 mM EDTA [pH 7.5], and 40 mg of phenylmethylsulfonyl fluoride per ml) for 1 h at 50°C. This step was repeated (with fresh TE-PMSF). The plugs were then transferred to new tubes and incubated in 1 ml of TE alone for 30 min at room temperature. Then plugs were transferred to new microcentrifuge tubes, to which was added 300 ml of buffer M (Boehringer-Mannheim, Indianapolis, Ind.), and incubated for 30 min at 4°C. The buffer was removed and replaced with 200 ml of fresh buffer M. Forty units of the restriction endonuclease SfiI (Boehringer-Mannheim) was added, and the tubes were incubated for 12 to 16 h at 50°C. The digested plugs were placed in 1 ml of TE in new tubes for 1 h at 37°C and then stored at 4°C. (iii) CHEF gel electrophoresis. A 1.0% agarose gel was made in 0.53 TBE (13 TBE is 0.089 M Tris, 0.089 M boric acid, and 0.0025 M EDTA) buffer (ICN Biomedicals, Inc.), and one third of a plug was loaded into each well. This was placed in a contour-clamped homogeneous electric field (CHEF) gel chamber (model DR III; Bio-Rad, Hercules, Calif.) with the following parameters: 5-s initial time and 35-s final time at 6 V/cm for 24 h. The gel was then stained with 1 mg of ethidium bromide per ml for 30 min and photographed. (iv) RFLP analysis. Strains were considered unrelated if there was a difference of two or more bands between them, highly related if there was a difference of only one band, and identical if no difference in bands was seen.
RESULTS Each patient’s isolates were tested for susceptibility by the NCCLS method and by agar dilution. RFLP analysis was also performed for each isolate. Clinical data regarding response to therapy with fluconazole is presented as well. Patient 1. Multiple isolates for which trailing endpoints were found were recovered from clinical samples from three episodes of oropharyngeal candidiasis over a 6-month period. Results from RFLP analysis of serial isolates are shown in Fig. 1, which shows results for isolates from the same strain, though trailing endpoints were found for some of these isolates by NCCLS susceptibility testing. Testing by agar dilution with fluconazole revealed all trailing-endpoint isolates to be suscep-
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FIG. 2. Multiple trailing-endpoint isolates recovered from patient 2 over a 15-month period from 12 episodes of symptomatic oropharyngeal candidiasis. Results of RFLP analysis of these isolates are shown, demonstrating the presence of highly related strains, except in the case of an isolate from episode 12. There was significant variability among MICs, with trailing endpoints observed for some isolates upon susceptibility testing. Broth microdilution NCCLS testing yielded trailing endpoints for more of the strains. The patient responded to 100 to 200 mg of fluconazole per day for each episode of oropharyngeal candidiasis, despite the presence of the trailing-endpoint isolates. Numbers below the gel are MICs, in micrograms per milliliter.
tible (MIC ,8 mg/ml). The patient had a complete response to 100 mg of fluconazole per day for each episode of oropharyngeal candidiasis, despite the presence of the trailing-endpoint isolates. Patient 2. Multiple isolates for which trailing endpoints were found were recovered from clinical samples from 12 episodes of oropharyngeal candidiasis over a 15-month period. Results from RFLP analysis of these serial isolates are shown in Fig. 2, which, except for an isolate from episode 12, presents data for isolates from closely related strains. There was significant variability among MICs, and trailing endpoints were found for some isolates by NCCLS susceptibility testing. When broth microdilution NCCLS testing was used, trailing endpoints were found for more of the strains. Testing by agar dilution with fluconazole revealed all trailing-endpoint isolates to be susceptible (MIC ,8 mg/ml). The patient responded to 100 to 200 mg of fluconazole per day for each episode of oropharyngeal candidiasis, despite the presence of the trailing-endpoint isolates. Patient 3. Multiple isolates for which trailing endpoints were found were recovered from clinical samples from three episodes of oropharyngeal candidiasis over a 3-month period. In addition, for two isolates, MICs were .64 mg/ml at both 24 and 48 h by NCCLS testing. Results from RFLP analysis of serial isolates are shown in Fig. 3, which presents data for isolates from different strains: one strain that was susceptible and another for which trailing endpoints or MICs of .64 mg/ml at both 24 and 48 h by NCCLS susceptibility testing were consistently observed. Testing by agar dilution with fluconazole revealed all trailing-endpoint isolates to be susceptible (MIC ,8 mg/ml), including the isolates for which 24- and 48-h MICs were .64 mg/ml. The patient responded to 100 mg of fluconazole per day for all episodes of oropharyngeal candidiasis, despite the presence of the trailing-endpoint isolates. DISCUSSION Trailing endpoints occasionally complicate the interpretation of antimicrobial susceptibility testing of both bacteria and fungi. Early studies in bacterial susceptibility testing made note
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TRAILING ENDPOINTS IN ANTIFUNGAL SUSCEPTIBILITY TESTS
FIG. 3. Multiple trailing-endpoint isolates recovered from patient 3 over a 3-month period from three episodes of symptomatic oropharyngeal candidiasis. Results of RFLP analysis of some of these isolates are shown, demonstrating the presence of different strains: one strain that was susceptible and another for which trailing endpoints by susceptibility testing were consistently observed. The patient responded to 100 mg of fluconazole per day for each episode of oropharyngeal candidiasis, despite the presence of the trailing-endpoint isolates. Numbers below the gel are MICs, in micrograms per milliliter.
of the phenomenon, especially in the testing of sulfonamide compounds (2). Both broth and agar methods have been associated with trailing endpoints (2, 4, 5). The effect was attributed to the bacteriostatic action of the sulfonamides, with several generations being required before significant growth inhibition occurs. In addition, these isolates were not felt to be clinically resistant, despite high MICs. In one study, in vivo testing in mice showed no difference between susceptible isolates and those for which trailing endpoints were observed (6). Antifungal susceptibility testing has made considerable progress in recent years. Specifically, the NCCLS methods (broth macro- and microdilution) have been shown to be reproducible and clinically useful, with good in vitro-in vivo correlation in the setting of oropharyngeal candidiasis in HIVinfected patients (3, 11, 12). Occasionally, NCCLS testing of yeasts with fluconazole produces trailing endpoints, usually at 48 h, with growth evident at all concentrations of the drug. This may be due to the fungistatic activity of the azoles, which allows several generations of growth before significant inhibition occurs. Some studies have examined methods to lessen the trailingendpoint effect. Agitation of the broth in the NCCLS method has been shown to reduce the trailing-endpoint effect, though the incubation time in a study by Anaissie et al. was 24 h instead of the standard 48 h (1, 10). In the present study, trailing endpoints were observed for most of the isolates at 48 h. We obtained from HIV-infected patients multiple serial yeast isolates for which trailing endpoints were found by antifungal susceptibility testing by the NCCLS broth macro- and/or microdilution method. RFLP analysis was performed for these strains by CHEF gel electrophoresis, and it revealed that trailing endpoints may be seen with selected isolates of a strain or may be found for most or all isolates of a strain. This suggests that trailing endpoints may be associated with a particular genotype. For some of the isolates, MICs of .64 mg/ml at both 24 and 48 h were observed. Despite being associated with other isolates for which trailing endpoints were found by NCCLS testing, these isolates cannot be immediately characterized as trail-
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ing-endpoint isolates, since there clearly exist isolates of C. albicans for which similar MICs are found and which are truly resistant to fluconazole, both microbiologically and clinically. However, by agar dilution testing, these isolates were also found to be susceptible, i.e., MICs were ,8 mg/ml. Testing methods other than those of the NCCLS may be necessary to properly characterize these isolates, especially in instances where other susceptible (MIC , 8 mg/ml) or trailing-endpoint isolates are recovered from the same culture. It would not be possible to label these isolates as trailing-endpoint isolates without using other testing methods (such as agar dilution), as our definition requires a difference between 24- and 48-h MICs by the NCCLS method. Based on our experience, it appears that the trailing endpoint does not indicate resistance. There is other evidence to suggest that trailing-endpoint isolates are actually susceptible, including results from alternative testing methods and in vivo results from animal studies (14). Agar dilution screening at 48 h consistently predicted that these trailing-endpoint isolates would be susceptible to fluconazole, i.e., that MICs would be ,8 mg/ml. As a result, agar dilution may be more reliable for determining antifungal susceptibilities of these isolates. The reason for the differences in results between the agar and broth methods when the same media are used is unclear. Most cultures taken from the three patients with oropharyngeal candidiasis described here contained both susceptible and trailing-endpoint isolates, though the presence of the latter did not usually imply clinical resistance, as most episodes responded to doses of fluconazole as low as 100 mg/day. The incidence of trailing endpoints is not known, but they appear to be an infrequent yet difficult problem in antifungal susceptibility testing. Whether all strains for which trailing endpoints are observed are equally pathogenic is difficult to assess, as they are usually seen together with susceptible strains. The fact that they are repeatedly isolated from oropharyngeal candidiasis patients suggests that they may play a role in the pathogenesis of this disease. In summary, yeast isolates for which trailing endpoints were observed at 48 h by NCCLS testing were found to be susceptible by agar dilution, and episodes of oropharyngeal candidiasis with these isolates had responses to therapy with fluconazole similar to those in episodes with only susceptible isolates. Given the above information, yeast isolates for which trailing endpoints are observed with fluconazole appear to be susceptible, and therefore the NCCLS method result obtained at 24 h may be more appropriate for these isolates. With isolates for which MICs of .64 mg/ml are observed at both 24 and 48 h, alternate testing methods, such as agar dilution, may be used to confirm results obtained by the standard NCCLS method, especially in instances where trailing endpoints and/or MICs indicating susceptibility (,8 mg/ml) are observed for other yeast isolates from the same culture. ACKNOWLEDGMENTS This work was supported by grants from the National Institute of Dental Research (1 RO1 DE11381-01), the National Institute of Health (MO1-RR-01346) for the Frederic C. Bartter General Clinical Research Center, and Pfizer Inc. Chromogenic media were provided by CHROMagar. REFERENCES 1. Anaissie, E. J., V. L. Paetznick, L. G. Ensign, A. Espinel-Ingroff, J. N. Galgiani, C. A. Hitchcock, M. LaRocco, T. Patterson, M. A. Pfaller, J. H. Rex, and M. G. Rinaldi. 1996. Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eight-center collaborative study. Antimicrob. Agents Chemother. 40: 2387–2391.
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