(2) VOL. 32, 1994. and after (repeated) antifungal treatment with FCZ. After shaking the sample, 10 and 100 ,lI of washout fluid was plated onto two separate Sabouraud dextrose agar agar plates containing 4% dextrose (Becton Dickinson 011584362). By using a two-step incubation procedure (20°C [room temperature] and 37°C over 48 h), C. albicans colonies were counted after 48 h and, for non-C. albicans yeasts, after 120 h as CFU per milliliter, and one colony was transferred onto rice agar for the detection of pseudomycelia and chlamydospores (diagnostic for C. albicans). Detection of germ tube formation as an additional diagnostic test for C. albicans was done in bovine serum after 4 h of incubation. All Candida isolates were identified on the basis of rice agar morphology and germ tube. testing: FCZ, ketoconazole, itraconazole, saperconazole (some isolates only), and 5-FC. FCZ was obtained from Pfizer/ Heinrich Mack Nachfolger (Illertissen, Germany), ketoconazole (K 1003) was purchased from Sigma Chemical Co. (Deisenhofen, Germany), itraconazole was obtained from Janssen Pharmaceutical (Neuss, Germany), saperconazole was obtained from R. W. Johnson Pharmaceutical Research Institute (Raritan, N.J.), and 5-FC (13321 CW) was purchased from Aldrich Chemie GmbH (Steinheim, Germany). FCZ and 5-FC powders were dissolved in sterile water, while ketoconazole, itraconazole, and saperconazole powders were dissolved with dimethyl sulfoxid (D-5879; Sigma). The stock solutions were. prepared immediately prior to susceptibility testing. Antifungal susceptibility testing. Two test methods were applied for antifungal susceptibility testing: a broth microdilution technique and an AD technique. The broth microdilution assay (MD1) was done with high-resolution (HR) antifungal assay medium (from Pfizer Central Research, Sandwich, United Kingdom) according to the instructions of the ongoing German collaborative study for standardized antifungal susceptibility testing of FCZ (protocol FCA-D-92-511 by Pfizer/ Heinrich Mack Nachfolger) for all C. albicans isolates but, additionally, in a slightly modified form, for the first 63 C. albicans isolates only (MD2), which was recommended earlier by Troke and Pye (42) (Pfizer Central Research). The MD1 assay was done by the method by Troke and Pye (42) (MD2), but with 0.2 M phosphate buffer instead of MOPS (3-Nmorpholinepropanesulfonic acid (no. M1254; Sigma). Briefly, for the MD1 test, the HR liquid medium for MIC (antifungal concentration) testing was prepared with 0.2 M phosphate puffer (pH 7.5) to reach a final pH of 7.2 and was mixed with a fresh Candida colony to give a density of 103 cells per ml. Separate dilution series of all antifungal agents were made from the stock dilution (400 ,ug/ml) by using the HR medium to give final concentrations from 400 to 0.02 ,ug/ml. Finally, in 24-well microtiter plates, 15 dilutions with different antifungal concentrations (each of 100 pul) were mixed with 900 pd of Candida suspension, the plates were incubated at 37°C, and MICs were determined at 24 and 48 h. The MIC end point was determined by visual inspection by recording the lowest concentration of the antifungal agents which prevented the appearance of visible growth when viewed with the naked eye. Small spots of growth (fewer than five single spots) were ignored. The MIC was the. concentration in the first well in. which there was no marked growth other than the small spots of growth. All values were assessed in relation to the values for the drug-free control. This method differed slightly from the. 2093. MD1 test established later in that we used 0.3 M MOPS buffer to adjust the HR medium to pH 7.0 with an inoculum of 103 cells per ml. The AD method was also performed on the first 63 C. albicans isolates only. The AD method was established by Gordon et al. (13) by using a semisolid medium. In the present study, dilution series of antifungal agents were made with sterile distilled water from the stock dilution (400 to 0.02 p,g/ml), resulting in 15 different concentrations. A total of 1 ml of each antifungal dilution was added to 9 ml of freshly prepared molten supplemented yeast nitrogen base (6.7 g of yeast nitrogen base [no. 392-15-9; Difco Laboratories], 10 g of D-glucose [no. 15.896-8; Aldrich Chemie], 1.5 g of L-asparagine [no. A4159; Sigma]) and 100 ml of distilled water as described in detail elsewhere (13). The various agar-antifungal agent mixtures were pipetted into 15 wells of 24-well microtiter plates, and the plates were stored at 4°C until use for susceptibility testing. For in vitro testing, a yeast suspension was prepared with sterile 0.9% NaCl. Finally, an inoculum of 2.0 x 102 cells per ml was added to each well, which was filled with semisolid agar. Incubation was done at 37°C for 48 h, with MICs read at 24 and 48 h. The MIC end point was obtained from the well with the lowest concentration of azole in which little or no growth was visually detected, with the control yeast culture used as the reference. A small, so-called ghost sediment sometimes seen in wells containing azoles at inhibitory concentrations for C. albicans was considered no growth. C. albicans ATCC 10231 (MIC, 0.313 to 0.625 ,ug of FCZ per ml) and C. albicans ATCC 76615 (MIC, 1.56 pug of FCZ per ml) were used as reference cultures for all tests. Both reference strains were kindly provided by A. Schmalreck Pfizer/Heinrich. Mack Nachfolger. Candida isolates were determined to be susceptible to FCZ if MICs were <6.25 pug/ml, intermediately susceptible if MICs were .6.25 to <25 pug/ml, and resistant if MICs were .25 pug/ml, according to previous recommendations (41, 42). Statistical analysis. The distribution of values among two groups (MD1 versus AD, MD1 versus MD2) was compared by the Wilcoxon rank sum test for paired data when it was assumed that the medium of the difference was zero or there was no difference between MIC results in the two groups. A P value of .0.05 was considered statistically significant. All P values are two-tailed. RESULTS Isolation of Candida species. Between October 1989 and August 1993, 65 subjects with symptomatic HIV type 1 infection were treated with FCZ at least once for OPC. Treatment for all episodes consisted of 100 mg of FCZ per day orally for 5 to 21 days, which led in all cases to a rapid clinical cure of the signs and symptoms of OPC unless resistance occurred. Of the 65 subjects, 38 patients had one or two episodes of OPC that were treated with FCZ (two samples per patient). A total of 27 patients had recurrent OPC with more than two recurrences of OPC (3 to 28 samples per patient; mean, 4.5 samples per patient). From 305 oral washouts obtained before and after therapy with FCZ, 302 Candida strains were isolated (201 C. albicans [66%], 50 Torulopsis [Candida] glabrata [16.5%], 35 Candida krusei [12%], 11 Candida tropicalis [4%], 3 Geotrichum candidum [1%], and 2 Saccharomyces cerevisiae [0.5%]). The last two strains were isolated from two different patients in serial samples. C. albicans could be isolated from all patients with OPC before FCZ therapy and from up to 75% of all patients after clinical cure of the signs and symptoms of OPC.. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. production and, finally, by using the API 20C AUX identification system (bio-Merieux, Freiburg, Germany). After the final identification, isolates were stored at -20°C in sterile skim milk (no. 0032-01; Difco Laboratories, Detroit, Mich.) until susceptibility tests were performed. Drugs. Five antifungal agents were used for susceptibility. FLUCONAZOLE-RESISTANT C. ALBICANS.
(3) 2094. RUHNKE ET AL.. J. CLIN. MICROBIOL.. 15. z. 10. 10. 5. 5. 0. 0 g0.19. 0.39. 0.78. 1.55. 3.12. 6.25. 12.5. 25. >,50. 40.19. IL 0.39. 0.78. 1.56. [pig/mi]. and AD (U) assays were compared.. Non-C. albicans strains were detected only after FCZ therapy either as a single organism or together with C. albicans in patients with recurrent OPC repeatedly treated with FCZ. For two patients with typical signs and symptoms of OPC who were both treated with FCZ before (>3-g total dose), only non-C. albicans strains (one patient infected with T. glabrata; one patient infected with C. krusei) could be isolated as a single organism from several samples. At least two Candida species were isolated from 38 samples (in each of four samples from three patients, three different species: in two samples, C. albicans, C. krusei, and T. glabrata; in another two samples, C. albicans, C. krusei, and C. tropicalis). MICs of antifungal agents for C. albicans. At the beginning of the study, MIC testing was done on 63 C. albicans isolates (recovered between October 1989 and July 1991) obtained from 11 patients with recurrent OPC by a broth microdilution (MD2) and an AD method. As shown in Fig. 1, 53 C. albicans isolates were FCZ susceptible (MICs, <6.25 ,ug/ml) by the MD2 method and 51 C. albicans isolates were FCZ susceptible by the AD method. Two (MD2) and five (AD) isolates of C. albicans showed intermediate susceptibilities to FCZ (MICs, -6.25 to < 25 ,ug/ml). Five (AD) were FCZ resistant (MICs, .25 ,ug/ml) in vitro. The MICs obtained by the AD method tended to be greater by one or two end point readings than by MD2 for FCZ-susceptible isolates, but the differences between the two methods were not statistically significant (P >0.1). FCZ-resistant C. albicans isolates were recovered from the same two patients by the MD2 and AD methods, but were also recovered from another two patients by the MD2 method only. In vitro susceptibility testing was repeated by the MD1 method for all isolates recovered up to July 1991 and was performed for all isolates recovered after July 1991. The microdilution method in a modified form (MD1) showed intralaboratory reproducibility within 2 doubling dilutions for 92% (n = 58) of all tested isolates (Fig. 2). The differences were not statistically significant (P > 0.1). In vitro FCZ resistance correlated with the in vivo findings in three of four cases. By August 1993, 201 C. albicans strains were isolated from 65 patients, and the MICs of FCZ, ketoconazole, itraconazole, and 5-FC for 160 C. albicans strains were determined by the MD1 method (Fig. 3). As shown in Fig. 3, 97 C. albicans isolates were FCZ susceptible by the MD1 method, 30 C. albicans isolates showed intermediate susceptibility to FCZ, and 33 were FCZ resistant (MICs, .25 ,ug/ml). Clinical FCZ resistance was observed in 6 of 65 patients (9%). Another three patients with clinical FCZ resistance were referred to our hospital from other institutions, where no pretreatment isolates were presenred. For isolates. 12.5. >50. 25. [pg/mil. FIG. 2. MICs of FCZ for 63 C. albicans isolates (recovered up to July 1991) from 11 patients with HIV infection and OPC performed by the broth microdilution method in a comparison of two assay preparations (MD1 [E] and MD2 [X]).. from all nine patients, in vitro FCZ resistance could be proven (26 C. albicans isolates for which MICs were .25 ,ug/ml). Seven C. albicans isolates for which FCZ MICs were .25 ,ug/ml were recovered from four patients who were either lost to follow-up or for whose isolates no in vivo resistance was documented. Correlations of in vitro and in vivo results refer to the MD1 method. As shown in Fig. 3, the MICs of three other antifungal agents (ketoconazole, itraconazole, and 5-FC) were determined. Of 160 C. albicans isolates, 156 were 5-FC susceptible (MICs -3.12 ,ug/ml). The MICs of ketoconazole and itraconazole were found to be within the same range as those of FCZ for all isolates together, but individual MICs varied markedly in comparison with those of FCZ. Only two of nine C. albicans isolates for which itraconazole MICs were .25 ,ug/ml were recovered from patients with FCZ-resistant OPC. Table 1 shows the individual MICs of five antifungal agents for isolates from two patients who developed FCZ-resistant OPC. These two subjects were selected from among nine patients with FCZ-resistant OPC and who were frequently treated with FCZ for recurrent OPC. Patient A. In August 1989, the patient experienced the first documented episode of OPC (ketoconazole therapy). In October 1990, the patient received the first successful therapy. Fluconazole. Ketoconazol. Itraconazole. 5-FC. 70 60. -40. 30 20. 10. 0. -<O. 10.19. 0.39. 0.78. 1.56. 3.12. 6.25. 12.5. 25. 4O0. [pg/mi]. FIG. 3. MICs of FCZ, ketoconazole, itraconazole, and 5-FC for 160 C. albicans isolates from 65 patients with HIV infection and OPC tested by the MD1 method.. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. FIG. 1. MICs of FCZ for 63 C. albicans isolates (recovered up to July 1991) from 11 patients with HIV infection and OPC; the MD1 (U). 6.25. 3.12.
(4) 2095. FLUCONAZOLE-RESISTANT C. ALBICANS. VOL. 32, 1994. TABLE 1. In vitro. susceptibilities of C. albicans isolates to five antifungal agents from patients with HIV infection and clinical FCZ-resistant OPC. Patient. Date of infection. (mo. yr). MIC (,ug/ml). Isolate FCZ. Ketoconazole. 5-FC. Itraconazole. Saperconazole. 10.90 08.91 10.92 02.93 03.93 05.93 06.93 07.93 08.93. HP1 HP2 HP3 HP4 HP5 HP6 HP7 HP8 HP9. 1.56 3.12 12.5 25 25 12.5 25 25 25. 0.19 0.19 1.56 3.12 3.12 0.78 1.56 1.56 0.78. 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39. 0.78 0.78 1.56 1.56 1.56 1.56 1.56 1.56 1.56. 0.78 1.56 6.25 6.25 6.25 12.5 12.5 12.5 12.5. B. 01.90 02.90 08.90 10.90 11.90 04.91 05.91 07.91. MG1 MG2 MG3 MG4 MG5 MG6 MG7 MG8. 0.78 0.78 1.56 0.78 1.56 6.25 25 >50. 0.05 0.05 0.05 0.1 0.05 0.05 0.19 0.19. 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1. 0.19 0.19 0.19 0.19 0.39 0.78 0.78 0.78. 0.39 0.39 0.39 0.39 0.39 0.78 25 25. with FCZ for OPC. In April 1992, disseminated Mycobacterium avium complex disease was treated with multiple antimicrobial agents. Up to April 1993, recurrent OPC was successfully treated with FCZ or ketoconazole. From June 1993 daily dosages of 100 mg of FCZ were no longer seen effective. Even an increase in the FCZ dose to 400 mg/day was no longer effective. From July 1993 the patient was treated with itraconazole (400 mg of itraconazole solution per day) and was then given maintenance therapy of 200 mg of itraconazole (capsules); the patient showed a marked response, with cure of candidosis, and the patient was free of recurrences until November 1993. When the patient was unable to take the itraconazole solution because of severe vomiting and nausea caused by treatment for M. avium complex disease, recurrent OPC could be suppressed with 800 mg of FCZ per day (given intravenously) without apparent toxicity. The patient died in February 1994. Patient B. In May 1988 the patient experienced the first documented episode of OPC (against which nystatin solution was effective). Up to October 1989 the patient experienced single episodes of OPC that were effectively treated with ketoconazole, miconazole gel, or nystatin solution. In October 1989 the patient received the first treatment with FCZ for recurrent OPC; this was treated effectively with FCZ until April 1991 (each episode was treated with 100 mg orally for 5 to 10 days). From November 1990, 100 mg of fluconazole was given prophylactically once per week to prevent the recurrence of OPC. From May 1991 clinical resistance to FCZ occurred after treatment with 400 mg of FCZ per day for 7 days. From May 1991, effective therapy with itraconazole (300 mg/day, capsules) was given until the death of patient in July 1991 because of progressive Kaposi's sarcoma. C. albicans isolates could be serially isolated from patients A and B over a period of more than 12 months. The data showed an almost identical increase in the MICs of FCZ and saperconazole, but the increases in the MICs of ketoconazole and itraconazole were less (up to 1 log unit). There was no major increase in the MICs of 5-FC for the C. albicans isolates from these patients. The MICs for the isolates from both patients indicated that they were resistant to FCZ, but the MICs of itraconazole were apparently lower (<1.56 ,ug/ml). Similar. patterns of the development of resistance, with increasing MICs, could be documented for the isolates from two other patients as well (data not shown).. DISCUSSION. Antifungal susceptibility tests with FCZ and other azoles were performed in large collaborative studies (8, 29, 36, 41) which showed good inter- and intralaboratory reproducibilities for certain testing methods (e.g., the macrodilution technique with HR medium (27). The major sources of antifungal susceptibility test variation for FCZ and the other azoles arise from differences in the manner in which the test is conducted (e.g., the pH of the medium, medium composition, inoculum size, incubation time, and incubation temperature) (35). An increasing number of reports of in vitro and in vivo resistance to FCZ have been published recently, but serial isolates have usually not been saved and changes in FCZ susceptibility among C. albicans isolates obtained from HIV-positive subjects with recurrent OPC are not well documented (1, 2, 7, 10, 18, 25, 31, 32, 37, 46). Four questions were addressed in the present study. First, do two different methods of FCZ antifungal susceptibility testing yield comparable results? Second, do in vitro data correspond to the in vivo outcome? Third, does cross-resistance exist between different azoles? Fourth, how frequently does FCZresistant OPC occur in a cohort of HIV-infected patients who receive therapy with FCZ for OPC. The broth microdilution method with the chemically defined HR medium was described by Troke and Pye (42) and was shown to give reproducible data in multicenter studies (27, 41). This method appeared to be more easy to perform than the broth dilution methods recommended by the National Committee for Clinical Laboratory Standards (23, 24). The AD method described by Gordon et al. (13) was chosen as the second assay because this technique, as well as the broth microdilution method, could be done in microtiter plates. Therefore, both assays are suitable for application to the routine testing of Candida isolates on a larger scale. The microdilution method, which was used initially, was slightly modified later in the study by using a 0.2 M. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. A.
(5) 2096. RUHNKE ET AL.. ages of up to 400 mg/day (6, 18, 32, 37) but did not provide the MICs for pre- and posttreatment isolates. Bart-Delabesse et al. (1) presented data on the MICs for the DNA typing of C. albicans strains from four patients with AIDS who developed clinical and mycological resistance to FCZ. Even if this group did not clearly define their understanding of clinical FCZ resistance in these patients, they noted that the genotype of the C. albicans isolate for which the MIC was low (.3.12 ,ug/ml) was similar to the genotype of the C. albicans strain for which the MIC was high (.12.5 ,ug/ml) recovered later in the course of HIV disease from three (of four) patients with HIV infection. These findings support the data of Schmid et al. (33) and Miyasaki et al. (22), who found that the same C. albicans strain persisted through recurrent infections in the majority of patients with AIDS, as demonstrated by restriction fragment length polymorphism-DNA fingerprinting (with the Ca3 probe). Pfaller et al. (28) concluded in their observations that the introduction or selection of strains with a more resistant DNA subtype during the course of FCZ therapy was not uncommon, but they were not able to link those data with clinical FCZ resistance, because all of their patients responded to FCZ (up to 400 mg). In our study, all isolates recovered from patients with FCZ-resistant OPC were further characterized by DNA fingerprinting with restriction enzyme digestion. Preliminary data did not reveal differences in serial C. albicans strains from these patients (unpublished data). Clinical resistance to FCZ occurred in patients after repeated treatment with FCZ for OPC but, nevertheless, has been observed only in severely compromised patients (CD4 count, <20/,l) with end-stage AIDS disease and patients with concomitant bacterial or malignant diseases such as disseminated M. avium complex infection or progressive Kaposi's sarcoma, which require antimicrobial or antitumor chemotherapy. The isolation of non-C. albicans yeasts (e.g., T glabrata, C. krusei, and C. tropicalis) was common during the study period, concomitant with the frequent use of FCZ for the treatment of OPC. Cameron et al. (2) did not find a significant selection of non-C. albicans strains in patients with HIV infection after azole therapy, but these findings may have been due to the small group of patients treated with FCZ and the limited observation period of their study. However, FCZ is still very effective for the treatment of the first episodes of chronic recurrent OPC in HIV-infected patients. FCZ-resistant oral candidosis occurred in 9% of the cohort of HIV-infected patients described here and may become a major issue, e.g., with the general use of FCZ for continuous prophylaxis of fungal infections and the prolonged life expectancies of these patients (26). According to our experiences, clinical FCZ resistance correlated with the in vitro results for C. albicans when the MICs of FCZ were at least .25 ,ug/ml by certain testing procedures. In HIV-positive patients with FCZ-resistant OPC, other azoles may still serve as effective therapeutic agents before more toxic drugs such as intravenously administered amphotericin B are needed. REFERENCES 1. Bart-Delabesse, E., P. Boiron, A. Carlotti, and B. Dupont. 1993. Candida albicans genotyping in studies with patients with AIDS developing resistance to fluconazole. J. Clin. Microbiol. 31:29332937. 2. 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. 3. Chave, J. P., A. Cajot, J. Bille, and M. P. Glauser. 1989. Single oral dose therapy for oral candidiasis with fluconazole in HIV-infected. Downloaded from http://jcm.asm.org/ on May 15, 2020 by guest. phosphate buffer instead of MOPS. The intralaboratory reproducibility of the microdilution method appeared to be good (92%) when performed on separate occasions by different laboratory workers. Comparison of the results of the AD method with those of the MD2 method indicated that the differences were not statistically significant (P > 0.1) for the distribution of the tested isolates in three susceptibility ranges (susceptible, <6.25 ,ug/ml; intermediate, .6.25 to <25 ,ug/ml; resistant, -25 pLg/ml) for 86% of all tested isolates for which MICs were within 2 doubling dilutions. However, for almost all isolates of C. albicans for which FCZ MICs were <6.25 ,ug/ml by the AD method, MICs tended to be 1 or 2 doubling dilutions higher than those obtained by the MD1 method. This trend toward higher MICs by the AD method has been described earlier by Shawar et al. (36), who compared the AD method of Gordon et al. (13) with a broth microdilution test done with a medium different from the one used in the present study. The definition of FCZ-resistant C. albicans isolates used in the present study was adopted from that of Troke et al. (41), who defined resistant isolates as strains for which the MIC is -25 ,ug/ml. However, the classification of C. albicans isolates as susceptible, intermediate, or resistant to FCZ is based on preliminary breakpoints developed at Pfizer (Sandwich, United Kingdom) and does not necessarily reflect the clinical response to treatment with FCZ. This definition is still the only classification for the in vitro susceptibility testing of FCZ, but it has not yet been correlated with the clinical results in patients treated with FCZ. In recent studies by Bart-Delabesse et al. (1) and Sandven et al. (31), the breakpoint for FCZresistant isolates of C. albicans was even lower (greater than 12.5 ,ug/ml), but no further explanations of why this breakpoint was chosen were given. Thirty-three C. albicans isolates (16 strains for which FCZ MICs were 25 ,ug/ml, 17 strains for which FCZ MICs were 250 ,ug/ml) which were resistant in vitro were recovered from 13 patients (Fig. 3). For 9 patients with clinical FCZ resistance (not responsive to 300 to 400 mg of FCZ), the in vitro findings could be clearly correlated with the in vivo findings. All patients received repeated therapy with FCZ because of oral or esophageal candidosis (a total dose of >5 g of FCZ was given to all patients). Interestingly, two patients (Table 1) with FCZ-resistant candidosis could be effectively treated with itranconazole (300 to 400 mg/day, capsules or solution). The MICs of azoles (ketoconazole and itraconazole) other than FCZ increased moderately, suggesting the occurrence of azole cross-resistance. At least two of nine patients were successfully treated with itraconazole, which may correlate with the low MICs for the isolates from these patients. However, when itraconazole and ketoconazole were tested with a larger group of C. albicans isolates (Fig. 3), MICs gave highly variable results in comparison with those of FCZ (MIC range, <25 ,ug/ml). Whether the lipophilic character of itraconazole and ketoconazole, in contrast to the hydrophilic character of the drugs FCZ and saperconazole, is a critical issue or the microdilution method used in the present study is not appropriate for MIC testing was not further evaluated. Primary resistance to FCZ has not been observed in vivo or in vitro. The very low number of 5-FC-resistant C. albicans isolates found in the present study is in contrast to the number described in reports by other authors, but might be explained by our failure to detect heterotypic resistance or the very infrequent use of 5-FC in our patient population (4, 35, 45). Some recent reports described the clinical observation that FCZ failed to cure oropharyngeal or esophageal candidosis in AIDS patients (so-called FCZ-resistant candidosis) with dos-. J. CLIN. MICROBIOL..
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