JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1994, p. 2092-2098 Vol. 32, No. 90095-1137/94/$04.00+0Copyright © 1994, American Society for Microbiology

Emergence of Fluconazole-Resistant Strains of Candida albicansin Patients with Recurrent Oropharyngeal Candidosis and

Human Immunodeficiency Virus InfectionM. RUHNKE,l* A. EIGLER,1 I. TENNAGEN,1 B. GEISELER,1 E. ENGELMANN,2 AND M. TRAUTMANN3

Medizinische Klinik und Poliklinik, Abt. fOir Hamatologie/Onkologie Universitatsklinikum RudolfVirchowlCharlottenburg, Freie Universitat Berlin, 14050 Berlin, 1 Institut fOir Medizinische Mikrobiologie, FreieUniversitat Berlin, 12203 Berlin,2 and Institut fOir Mikrobiologie der Universitat Ulm, 89233 Ulm,3 Germany

Received 17 December 1993/Returned for modification 18 January 1994/Accepted 3 June 1994

After repeated use of fluconazole for therapy of oropharyngeal candidosis, the emergence of in vitrofluconazole-resistant Candida albicans isolates (MIC, 2 25 ,ig/ml) together with oral candidosis unresponsiveto oral dosages of up to 400 mg of fluconazole were observed in patients with human immunodeficiency virus(H1V) infection. Antifungal susceptibility testing was done by broth microdilution and agar dilution techniqueson C. albicans isolates recovered from a cohort of patients with symptomatic HIV infection who were treatedrepeatedly with fluconazole for oropharyngeal candidosis. In vitro findings did show a gradual increase in theMICs for C. albicans isolates recovered from selected patients with repeated episodes of oropharyngealcandidosis. Primary resistance of C. albicans to fluconazole was not seen. Cross-resistance in vitro occurredbetween fluconazole and other azoles (ketoconazole, itraconazole), but to a lesser extent. The results of thestudy suggest that the development of clinical resistance to fluconazole could be clearly correlated to in vitroresistance to fluconazole. Itraconazole may still serve as an effective antifungal agent in patients with HIVinfection and oropharyngeal candidosis nonresponsive to fluconazole.

Oropharyngeal candidosis (OPC) occurs in more than 80%of all patients during the course of human immunodeficiencyvirus (HIV) infection (11, 40). Disseminated manifestations ofinvasive fungal disease are rare with Candida albicans, butesophageal candidosis is seen in up to 20% of patients withHIV infection (11, 14). The new triazole fluconazole (FCZ)has proven to be highly effective against oral candidosis inclinical trials (3, 6, 9, 14, 16, 43), with only low levels of toxicitywhen given at various dosages and with even higher responserates to treatment than other antifungal agents, e.g., ketocon-azole and clotrimazole (5, 15, 19). Since 1989 fluconazole haswidely been used for the prevention and therapy of OPC inHIV-infected patients (14, 21, 25, 26, 39). Some earlier reportshave already described azole resistance in Candida species (17,30, 38, 44), but recently, concerns have been raised regardingthe development of resistance to FCZ in C. albicans strainsisolated from AIDS patients. However, in the majority of thosestudies, no correlations were established between in vitrosusceptibility data and in vivo treatment response (7, 10, 18, 25,30, 36, 46). Some studies have presented data on C. albicansisolates and compared the in vitro susceptibility findings withthe clinical findings from patients with HIV infection andso-called fluconazole-resistant OPC (1, 2, 31). Also, thosestudies failed to demonstrate clearly the development ofresistance by testing multiple isolates obtained from severalepisodes of infection from the same patients exposed to azoleantifungal agents. Furthermore, routine antifungal susceptibil-ity testing of azoles and other antifungal agents is still notdefinitely standardized and is not widely used because of theremarkable interlaboratory differences obtained with the sameisolates as well as with various media (e.g., broth microdilution,

* Corresponding author. Mailing address: Universitatsklinikum Ru-dolf Virchow/Charlottenburg, Abt. fur Innere Medizin und Poliklinik/Hamatologie-Onkologie, FU Berlin, Spandauer Damm 130, 14050Berlin, Germany. Phone: 49-30-3035-2269. Fax: 49-30-3035-3765.

broth macrodilution, and agar dilution [AD]). Several methodshave been published so far, but the correlations between invivo and in vitro findings are still limited. The experience fromthose studies is that at least the choice of medium, inoculumsize, the pH of the medium, incubation time, and incubationtemperature must be standardized to obtain reproducible data(8, 12, 13, 20, 23, 24, 29, 34, 35, 37, 41, 45). In the present study,antifungal in vitro susceptibility testing was done (by brothmicrodilution and AD assays) with C. albicans isolates fromAIDS patients with recurrent OPC to monitor changes inorganism susceptibilities to various azoles and flucytosine(5-FC). In vitro data from the study were correlated with theclinical findings.

MATERIALS AND METHODS

Patients. Between October 1989 and August 1993, eachpatient in a cohort of 65 patients with symptomatic HIVinfection (9 females and 56 males; Centers for Disease Controland Prevention stage IV A to E; CD4 count, 0 to 390/,Il;median CD4 count, 68/,l; leukocyte count 0.9 to 6.7/nl) wasfollowed prospectively for a period of up to 3 years. Thepatients suffered from at least one episode of OPC which hadbeen treated with FCZ (100 mg orally for 5 to 21 days). A totalof 305 samples recovered during this observation period wereobtained by oral washings with 10 ml of a 0.9% NaCl solution.Samples from the oral cavity (2 to 28 samples per patient) werecollected before and in many cases after repeated treatmentwith FCZ for OPC. Clinical resistance to FCZ was defined asfailure to respond to oral dosages of at least 7.5 mg of FCZ perkg of body weight (up to 400 mg) as treatment for OPC.

Diagnostic procedure and mycologic identification. Afterrinsing the mouth with tap water, oral washings obtained with10 ml of sterile 0.9% NaCl solution were collected fromHIV-positive patients with documented oral candidosis before

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and after (repeated) antifungal treatment with FCZ. Aftershaking the sample, 10 and 100 ,lI of washout fluid was platedonto two separate Sabouraud dextrose agar agar plates con-taining 4% dextrose (Becton Dickinson 011584362). By using atwo-step incubation procedure (20°C [room temperature] and37°C over 48 h), C. albicans colonies were counted after 48 hand, for non-C. albicans yeasts, after 120 h as CFU permilliliter, and one colony was transferred onto rice agar for thedetection of pseudomycelia and chlamydospores (diagnosticfor C. albicans). Detection of germ tube formation as anadditional diagnostic test for C. albicans was done in bovineserum after 4 h of incubation. All Candida isolates wereidentified on the basis of rice agar morphology and germ tubeproduction and, finally, by using the API 20C AUX identifica-tion system (bio-Merieux, Freiburg, Germany).

After the final identification, isolates were stored at -20°Cin sterile skim milk (no. 0032-01; Difco Laboratories, Detroit,Mich.) until susceptibility tests were performed.

Drugs. Five antifungal agents were used for susceptibilitytesting: FCZ, ketoconazole, itraconazole, saperconazole (someisolates only), and 5-FC. FCZ was obtained from Pfizer/Heinrich Mack Nachfolger (Illertissen, Germany), ketocon-azole (K 1003) was purchased from Sigma Chemical Co.(Deisenhofen, Germany), itraconazole was obtained fromJanssen Pharmaceutical (Neuss, Germany), saperconazole wasobtained from R. W. Johnson Pharmaceutical Research Insti-tute (Raritan, N.J.), and 5-FC (13321 CW) was purchased fromAldrich Chemie GmbH (Steinheim, Germany). FCZ and 5-FCpowders were dissolved in sterile water, while ketoconazole,itraconazole, and saperconazole powders were dissolved withdimethyl sulfoxid (D-5879; Sigma). The stock solutions wereprepared immediately prior to susceptibility testing.

Antifungal susceptibility testing. Two test methods wereapplied for antifungal susceptibility testing: a broth microdilu-tion technique and an AD technique. The broth microdilutionassay (MD1) was done with high-resolution (HR) antifungalassay medium (from Pfizer Central Research, Sandwich,United Kingdom) according to the instructions of the ongoingGerman collaborative study for standardized antifungal sus-ceptibility 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 earlierby Troke and Pye (42) (Pfizer Central Research). The MD1assay was done by the method by Troke and Pye (42) (MD2),but with 0.2 M phosphate buffer instead of MOPS (3-N-morpholinepropanesulfonic acid (no. M1254; Sigma). Briefly,for the MD1 test, the HR liquid medium for MIC (antifungalconcentration) testing was prepared with 0.2 M phosphatepuffer (pH 7.5) to reach a final pH of 7.2 and was mixed witha fresh Candida colony to give a density of 103 cells per ml.Separate dilution series of all antifungal agents were madefrom the stock dilution (400 ,ug/ml) by using the HR mediumto give final concentrations from 400 to 0.02 ,ug/ml. Finally, in24-well microtiter plates, 15 dilutions with different antifungalconcentrations (each of 100 pul) were mixed with 900 pd ofCandida suspension, the plates were incubated at 37°C, andMICs were determined at 24 and 48 h. The MIC end point wasdetermined by visual inspection by recording the lowest con-centration of the antifungal agents which prevented the ap-pearance of visible growth when viewed with the naked eye.Small spots of growth (fewer than five single spots) wereignored. The MIC was the. concentration in the first well inwhich there was no marked growth other than the small spotsof growth. All values were assessed in relation to the values forthe drug-free control. This method differed slightly from the

MD1 test established later in that we used 0.3 M MOPS bufferto adjust the HR medium to pH 7.0 with an inoculum of 103cells per ml.The AD method was also performed on the first 63 C.

albicans isolates only. The AD method was established byGordon et al. (13) by using a semisolid medium. In the presentstudy, dilution series of antifungal agents were made withsterile distilled water from the stock dilution (400 to 0.02p,g/ml), resulting in 15 different concentrations. A total of 1 mlof each antifungal dilution was added to 9 ml of freshlyprepared molten supplemented yeast nitrogen base (6.7 g ofyeast nitrogen base [no. 392-15-9; Difco Laboratories], 10 g ofD-glucose [no. 15.896-8; Aldrich Chemie], 1.5 g of L-asparagine[no. A4159; Sigma]) and 100 ml of distilled water as describedin detail elsewhere (13). The various agar-antifungal agentmixtures were pipetted into 15 wells of 24-well microtiterplates, and the plates were stored at 4°C until use for suscep-tibility testing. For in vitro testing, a yeast suspension wasprepared with sterile 0.9% NaCl. Finally, an inoculum of 2.0 x102 cells per ml was added to each well, which was filled withsemisolid agar. Incubation was done at 37°C for 48 h, withMICs read at 24 and 48 h. The MIC end point was obtainedfrom the well with the lowest concentration of azole in whichlittle or no growth was visually detected, with the control yeastculture used as the reference. A small, so-called ghost sedi-ment sometimes seen in wells containing azoles at inhibitoryconcentrations for C. albicans was considered no growth.

C. albicans ATCC 10231 (MIC, 0.313 to 0.625 ,ug ofFCZ perml) and C. albicans ATCC 76615 (MIC, 1.56 pug of FCZ perml) were used as reference cultures for all tests. Both referencestrains were kindly provided by A. Schmalreck Pfizer/HeinrichMack Nachfolger.Candida isolates were determined to be susceptible to FCZ

if MICs were <6.25 pug/ml, intermediately susceptible if MICswere .6.25 to <25 pug/ml, and resistant if MICs were .25pug/ml, according to previous recommendations (41, 42).

Statistical analysis. The distribution of values among twogroups (MD1 versus AD, MD1 versus MD2) was compared bythe Wilcoxon rank sum test for paired data when it wasassumed that the medium of the difference was zero or therewas no difference between MIC results in the two groups. A Pvalue of .0.05 was considered statistically significant. All Pvalues are two-tailed.

RESULTS

Isolation of Candida species. Between October 1989 andAugust 1993, 65 subjects with symptomatic HIV type 1 infec-tion were treated with FCZ at least once for OPC. Treatmentfor all episodes consisted of 100 mg of FCZ per day orally for5 to 21 days, which led in all cases to a rapid clinical cure of thesigns and symptoms of OPC unless resistance occurred. Of the65 subjects, 38 patients had one or two episodes of OPC thatwere treated with FCZ (two samples per patient). A total of 27patients had recurrent OPC with more than two recurrences ofOPC (3 to 28 samples per patient; mean, 4.5 samples perpatient). From 305 oral washouts obtained before and aftertherapy with FCZ, 302 Candida strains were isolated (201 C.albicans [66%], 50 Torulopsis [Candida] glabrata [16.5%], 35Candida krusei [12%], 11 Candida tropicalis [4%], 3 Geotri-chum candidum [1%], and 2 Saccharomyces cerevisiae [0.5%]).The last two strains were isolated from two different patients inserial samples. C. albicans could be isolated from all patientswith OPC before FCZ therapy and from up to 75% of allpatients after clinical cure of the signs and symptoms of OPC.

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z

10

5

0g0.19 0.39 0.78 1.55 3.12 6.25 12.5 25 >,50

[pig/mi]

FIG. 1. MICs of FCZ for 63 C. albicans isolates (recovered up toJuly 1991) from 11 patients with HIV infection and OPC; the MD1 (U)and AD (U) assays were compared.

Non-C. albicans strains were detected only after FCZ therapyeither as a single organism or together with C. albicans inpatients with recurrent OPC repeatedly treated with FCZ. Fortwo patients with typical signs and symptoms of OPC who wereboth treated with FCZ before (>3-g total dose), only non-C.albicans strains (one patient infected with T. glabrata; onepatient infected with C. krusei) could be isolated as a singleorganism from several samples. At least two Candida specieswere isolated from 38 samples (in each of four samples fromthree 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) obtainedfrom 11 patients with recurrent OPC by a broth microdilution(MD2) and an AD method. As shown in Fig. 1, 53 C. albicansisolates were FCZ susceptible (MICs, <6.25 ,ug/ml) by theMD2 method and 51 C. albicans isolates were FCZ susceptibleby 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 methodtended to be greater by one or two end point readings than byMD2 for FCZ-susceptible isolates, but the differences betweenthe two methods were not statistically significant (P >0.1).FCZ-resistant C. albicans isolates were recovered from thesame two patients by the MD2 and AD methods, but were alsorecovered from another two patients by the MD2 method only.In vitro susceptibility testing was repeated by the MD1 methodfor all isolates recovered up to July 1991 and was performedfor all isolates recovered after July 1991. The microdilutionmethod in a modified form (MD1) showed intralaboratoryreproducibility within 2 doubling dilutions for 92% (n = 58) ofall tested isolates (Fig. 2). The differences were not statisticallysignificant (P > 0.1). In vitro FCZ resistance correlated withthe in vivo findings in three of four cases. By August 1993, 201C. albicans strains were isolated from 65 patients, and theMICs 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 suscep-tible by the MD1 method, 30 C. albicans isolates showedintermediate susceptibility to FCZ, and 33 were FCZ resistant(MICs, .25 ,ug/ml). Clinical FCZ resistance was observed in 6of 65 patients (9%). Another three patients with clinical FCZresistance were referred to our hospital from other institutions,where no pretreatment isolates were presenred. For isolates

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5

0 IL40.19 0.39 0.78 1.56 3.12 6.25 12.5 25 >50

[pg/mil

FIG. 2. MICs of FCZ for 63 C. albicans isolates (recovered up toJuly 1991) from 11 patients with HIV infection and OPC performed bythe broth microdilution method in a comparison of two assay prepa-rations (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 lostto follow-up or for whose isolates no in vivo resistance wasdocumented. Correlations of in vitro and in vivo results refer tothe MD1 method.As shown in Fig. 3, the MICs of three other antifungal

agents (ketoconazole, itraconazole, and 5-FC) were deter-mined. Of 160 C. albicans isolates, 156 were 5-FC susceptible(MICs -3.12 ,ug/ml). The MICs of ketoconazole and itracon-azole were found to be within the same range as those of FCZfor all isolates together, but individual MICs varied markedlyin comparison with those of FCZ. Only two of nine C. albicansisolates for which itraconazole MICs were .25 ,ug/ml wererecovered from patients with FCZ-resistant OPC.

Table 1 shows the individual MICs of five antifungal agentsfor isolates from two patients who developed FCZ-resistantOPC. These two subjects were selected from among ninepatients with FCZ-resistant OPC and who were frequentlytreated with FCZ for recurrent OPC.

Patient A. In August 1989, the patient experienced the firstdocumented episode of OPC (ketoconazole therapy). In Oc-tober 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 for160 C. albicans isolates from 65 patients with HIV infection and OPCtested by the MD1 method.

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TABLE 1. In vitro susceptibilities of C. albicans isolates to five antifungal agents from patients with HIV infection and clinicalFCZ-resistant OPC

Date of infection MIC (,ug/ml)Patient Isolate(mo. yr) FCZ Ketoconazole 5-FC Itraconazole Saperconazole

A 10.90 HP1 1.56 0.19 0.39 0.78 0.7808.91 HP2 3.12 0.19 0.39 0.78 1.5610.92 HP3 12.5 1.56 0.39 1.56 6.2502.93 HP4 25 3.12 0.39 1.56 6.2503.93 HP5 25 3.12 0.39 1.56 6.2505.93 HP6 12.5 0.78 0.39 1.56 12.506.93 HP7 25 1.56 0.39 1.56 12.507.93 HP8 25 1.56 0.39 1.56 12.508.93 HP9 25 0.78 0.39 1.56 12.5

B 01.90 MG1 0.78 0.05 0.1 0.19 0.3902.90 MG2 0.78 0.05 0.1 0.19 0.3908.90 MG3 1.56 0.05 0.1 0.19 0.3910.90 MG4 0.78 0.1 0.1 0.19 0.3911.90 MG5 1.56 0.05 0.1 0.39 0.3904.91 MG6 6.25 0.05 0.1 0.78 0.7805.91 MG7 25 0.19 0.1 0.78 2507.91 MG8 >50 0.19 0.1 0.78 25

with FCZ for OPC. In April 1992, disseminated Mycobacteriumavium complex disease was treated with multiple antimicrobialagents. Up to April 1993, recurrent OPC was successfullytreated with FCZ or ketoconazole. From June 1993 dailydosages of 100 mg of FCZ were no longer seen effective. Evenan increase in the FCZ dose to 400 mg/day was no longereffective. From July 1993 the patient was treated with itracon-azole (400 mg of itraconazole solution per day) and was thengiven maintenance therapy of 200 mg of itraconazole (cap-sules); the patient showed a marked response, with cure ofcandidosis, and the patient was free of recurrences untilNovember 1993. When the patient was unable to take theitraconazole solution because of severe vomiting and nauseacaused by treatment for M. avium complex disease, recurrentOPC could be suppressed with 800 mg of FCZ per day (givenintravenously) without apparent toxicity. The patient died inFebruary 1994.

Patient B. In May 1988 the patient experienced the firstdocumented episode of OPC (against which nystatin solutionwas effective). Up to October 1989 the patient experiencedsingle episodes of OPC that were effectively treated withketoconazole, miconazole gel, or nystatin solution. In October1989 the patient received the first treatment with FCZ forrecurrent OPC; this was treated effectively with FCZ untilApril 1991 (each episode was treated with 100 mg orally for 5to 10 days). From November 1990, 100 mg of fluconazole wasgiven prophylactically once per week to prevent the recurrenceof OPC. From May 1991 clinical resistance to FCZ occurredafter treatment with 400 mg of FCZ per day for 7 days. FromMay 1991, effective therapy with itraconazole (300 mg/day,capsules) was given until the death of patient in July 1991because of progressive Kaposi's sarcoma.

C. albicans isolates could be serially isolated from patients Aand B over a period of more than 12 months. The data showedan almost identical increase in the MICs of FCZ and saper-conazole, but the increases in the MICs of ketoconazole anditraconazole were less (up to 1 log unit). There was no majorincrease in the MICs of 5-FC for the C. albicans isolates fromthese patients. The MICs for the isolates from both patientsindicated that they were resistant to FCZ, but the MICs ofitraconazole were apparently lower (<1.56 ,ug/ml). Similar

patterns of the development of resistance, with increasingMICs, could be documented for the isolates from two otherpatients as well (data not shown).

DISCUSSION

Antifungal susceptibility tests with FCZ and other azoleswere performed in large collaborative studies (8, 29, 36, 41)which showed good inter- and intralaboratory reproducibilitiesfor certain testing methods (e.g., the macrodilution techniquewith HR medium (27). The major sources of antifungalsusceptibility test variation for FCZ and the other azoles arisefrom differences in the manner in which the test is conducted(e.g., the pH of the medium, medium composition, inoculumsize, incubation time, and incubation temperature) (35). Anincreasing number of reports of in vitro and in vivo resistanceto FCZ have been published recently, but serial isolates haveusually not been saved and changes in FCZ susceptibilityamong C. albicans isolates obtained from HIV-positive sub-jects 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 testingyield comparable results? Second, do in vitro data correspondto the in vivo outcome? Third, does cross-resistance existbetween different azoles? Fourth, how frequently does FCZ-resistant OPC occur in a cohort of HIV-infected patients whoreceive therapy with FCZ for OPC. The broth microdilutionmethod with the chemically defined HR medium was describedby Troke and Pye (42) and was shown to give reproducible datain multicenter studies (27, 41). This method appeared to bemore easy to perform than the broth dilution methods recom-mended by the National Committee for Clinical LaboratoryStandards (23, 24). The AD method described by Gordon et al.(13) was chosen as the second assay because this technique, aswell as the broth microdilution method, could be done inmicrotiter plates. Therefore, both assays are suitable forapplication to the routine testing of Candida isolates on alarger scale. The microdilution method, which was used ini-tially, was slightly modified later in the study by using a 0.2 M

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phosphate buffer instead of MOPS. The intralaboratory repro-

ducibility of the microdilution method appeared to be good(92%) when performed on separate occasions by differentlaboratory workers. Comparison of the results of the ADmethod with those of the MD2 method indicated that thedifferences were not statistically significant (P > 0.1) for thedistribution 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 whichMICs were within 2 doubling dilutions. However, for almost allisolates of C. albicans for which FCZ MICs were <6.25 ,ug/mlby the AD method, MICs tended to be 1 or 2 doublingdilutions higher than those obtained by the MD1 method. Thistrend toward higher MICs by the AD method has beendescribed earlier by Shawar et al. (36), who compared the ADmethod of Gordon et al. (13) with a broth microdilution testdone with a medium different from the one used in the presentstudy.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 iso-lates as susceptible, intermediate, or resistant to FCZ is basedon preliminary breakpoints developed at Pfizer (Sandwich,United Kingdom) and does not necessarily reflect the clinicalresponse to treatment with FCZ. This definition is still the onlyclassification for the in vitro susceptibility testing of FCZ, butit has not yet been correlated with the clinical results inpatients treated with FCZ. In recent studies by Bart-Delabesseet al. (1) and Sandven et al. (31), the breakpoint for FCZ-resistant isolates of C. albicans was even lower (greater than12.5 ,ug/ml), but no further explanations of why this breakpointwas chosen were given.

Thirty-three C. albicans isolates (16 strains for which FCZMICs were 25 ,ug/ml, 17 strains for which FCZ MICs were 250,ug/ml) which were resistant in vitro were recovered from 13patients (Fig. 3). For 9 patients with clinical FCZ resistance(not responsive to 300 to 400 mg of FCZ), the in vitro findingscould be clearly correlated with the in vivo findings. Allpatients received repeated therapy with FCZ because of oralor esophageal candidosis (a total dose of >5 g of FCZ was

given to all patients). Interestingly, two patients (Table 1) withFCZ-resistant candidosis could be effectively treated withitranconazole (300 to 400 mg/day, capsules or solution). TheMICs of azoles (ketoconazole and itraconazole) other thanFCZ increased moderately, suggesting the occurrence of azolecross-resistance. At least two of nine patients were successfullytreated with itraconazole, which may correlate with the lowMICs for the isolates from these patients. However, whenitraconazole and ketoconazole were tested with a larger group

of C. albicans isolates (Fig. 3), MICs gave highly variableresults in comparison with those of FCZ (MIC range, <25,ug/ml). Whether the lipophilic character of itraconazole andketoconazole, in contrast to the hydrophilic character of thedrugs FCZ and saperconazole, is a critical issue or the microdi-lution method used in the present study is not appropriate forMIC testing was not further evaluated. Primary resistance toFCZ has not been observed in vivo or in vitro. The very lownumber of 5-FC-resistant C. albicans isolates found in thepresent study is in contrast to the number described in reportsby other authors, but might be explained by our failure todetect heterotypic resistance or the very infrequent use of 5-FCin our patient population (4, 35, 45).Some recent reports described the clinical observation that

FCZ failed to cure oropharyngeal or esophageal candidosis inAIDS patients (so-called FCZ-resistant candidosis) with dos-

ages of up to 400 mg/day (6, 18, 32, 37) but did not provide theMICs 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 developedclinical and mycological resistance to FCZ. Even if this groupdid not clearly define their understanding of clinical FCZresistance in these patients, they noted that the genotype of theC. albicans isolate for which the MIC was low (.3.12 ,ug/ml)was similar to the genotype of the C. albicans strain for whichthe MIC was high (.12.5 ,ug/ml) recovered later in the courseof HIV disease from three (of four) patients with HIVinfection. These findings support the data of Schmid et al. (33)and Miyasaki et al. (22), who found that the same C. albicansstrain persisted through recurrent infections in the majority ofpatients with AIDS, as demonstrated by restriction fragmentlength polymorphism-DNA fingerprinting (with the Ca3probe). Pfaller et al. (28) concluded in their observations thatthe introduction or selection of strains with a more resistantDNA subtype during the course of FCZ therapy was notuncommon, but they were not able to link those data withclinical FCZ resistance, because all of their patients respondedto FCZ (up to 400 mg). In our study, all isolates recoveredfrom patients with FCZ-resistant OPC were further character-ized by DNA fingerprinting with restriction enzyme digestion.Preliminary data did not reveal differences in serial C. albicansstrains from these patients (unpublished data). Clinical resis-tance to FCZ occurred in patients after repeated treatmentwith FCZ for OPC but, nevertheless, has been observed only inseverely compromised patients (CD4 count, <20/,l) withend-stage AIDS disease and patients with concomitant bacte-rial or malignant diseases such as disseminated M. aviumcomplex infection or progressive Kaposi's sarcoma, whichrequire 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 ofOPC. Cameron et al. (2) did not find a significant selection ofnon-C. albicans strains in patients with HIV infection afterazole therapy, but these findings may have been due to thesmall group of patients treated with FCZ and the limitedobservation period of their study. However, FCZ is still veryeffective for the treatment of the first episodes of chronicrecurrent OPC in HIV-infected patients. FCZ-resistant oralcandidosis occurred in 9% of the cohort of HIV-infectedpatients described here and may become a major issue, e.g.,with the general use of FCZ for continuous prophylaxis offungal infections and the prolonged life expectancies of thesepatients (26). According to our experiences, clinical FCZresistance correlated with the in vitro results for C. albicanswhen the MICs of FCZ were at least .25 ,ug/ml by certaintesting procedures. In HIV-positive patients with FCZ-resis-tant OPC, other azoles may still serve as effective therapeuticagents before more toxic drugs such as intravenously adminis-tered amphotericin B are needed.

REFERENCES1. Bart-Delabesse, E., P. Boiron, A. Carlotti, and B. Dupont. 1993.

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