In Vitro Evaluation of Combination of Fluconazole and Flucytosine against Cryptococcus neoformans var. neoformans

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1995, p Vol. 39, No /95/$ Copyright 1995, American Society for Microbiology In Vitro Evaluation of Combination of Fluconazole and Flucytosine against Cryptococcus neoformans var. neoformans M. HONG NGUYEN, 1,2 FRANCESCO BARCHIESI, 3,4 DEANNA A. MCGOUGH, 3 VICTOR L. YU, 1,2 * AND MICHAEL G. RINALDI 3,5 Department of Medicine, University of Pittsburgh Medical Center, 1 and VA Medical Center, 2 Pittsburgh, Pennsylvania; Department of Pathology, Fungus Testing Laboratory, University of Texas Health Science Center, 3 and Audie L. Murphy Memorial Veterans Hospital, 5 San Antonio, Texas; and Istituto di Malattie Infecttive e Medicina Pubblica, Universita degli Studi di Ancona, Ancona, Italy 4 Received 20 March 1995/Returned for modification 12 May 1995/Accepted 30 May 1995 Amphotericin B and fluconazole are current acceptable therapies for cryptococcal meningitis; however, their effect remains suboptimal. The combination of fluconazole and flucytosine has yielded encouraging clinical results in human immunodeficiency virus patients with cryptococcal meningitis. To investigate the biological basis of this finding, we performed in vitro combination testing of fluconazole and flucytosine against 50 clinical strains of Cryptococcus neoformans var. neoformans. Synergy (fractional inhibitory concentration index of <1.0) was observed in 62% of cases, while antagonism (fractional inhibitory concentration index of >2.0) was not observed. For cases in which synergy was not achieved (autonomous or additive effects), the beneficial effect of the combination was still seen (i.e., there was still a decrease, although not as dramatic, in the MIC of one or both drugs when used in combination). The in vitro inhibitory action of flucytosine was greatly enhanced by the addition of fluconazole; the flucytosine MICs for Cryptococcus isolates were markedly decreased to concentrations which were severalfold lower than the achievable cerebrospinal fluid flucytosine concentration. On the other hand, the addition of flucytosine did not greatly enhance the in vitro activity of fluconazole if the initial fluconazole MIC for the isolate was >8 g/ml. Controlled clinical studies are warranted to further elucidate the potential utility of fluconazole-flucytosine combination therapy. Cryptococcal meningitis is a life-threatening opportunistic infection in immunocompromised hosts, especially in human immunodeficiency virus-infected patients and transplant recipients. Although amphotericin B and fluconazole are current acceptable therapies for patients with cryptococcal meningitis, the success of these therapeutic modalities remains suboptimal (13). The combination of amphotericin B and flucytosine appeared to provide the highest rate of clinical success in one study (7). This combination, however, is frequently associated with toxic side effects, and the use of amphotericin B requires close laboratory monitoring with long-term catheters for vascular access. In one pilot study, the combination of fluconazole and flucytosine yielded a clinical success rate of 63%, which is higher than that of previously reported experiences with either amphotericin B or fluconazole (6, 13); however, to date, in vitro synergistic testing to examine the biological basis for this empiric finding has not been performed. We therefore investigated the in vitro interaction of fluconazole with flucytosine against Cryptococcus neoformans var. neoformans. MATERIALS AND METHODS Fifty clinical isolates of C. neoformans var. neoformans submitted to the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio, were studied. Forty-four isolates were recovered from cerebrospinal fluid (CSF), and six were recovered from blood of human immunodeficiency virusinfected patients. In addition, two reference C. neoformans strains, ATCC and 90113, were used as controls. Antifungal agents. Fluconazole (Pfizer Inc., New York, N.Y.) and flucytosine * Corresponding author. Mailing address: University of Pittsburgh Medical Center, Division of Infectious Diseases, W931 Montefiore University Hospital, Pittsburgh, PA Phone: (412) Fax: (412) (Hoffman-La Roche Laboratory Inc., Nutley, N.J.) were tested singly and in combination. Stock solutions of fluconazole and flucytosine (each at 2,560 g/ml) were prepared in distilled water. Serial twofold drug dilutions were also prepared in water. Antifungal susceptibility testing. Drug interaction was assessed with a checkerboard titration, adhering to the recommendations of the National Committee for Clinical Laboratory Standards (11). Briefly, susceptibility testing was performed in RPMI 1640 medium (American Biorganics, Inc., Niagara Falls, N.Y.), with L-glutamine, without bicarbonate, and buffered at ph 7.0 with M morpholinepropanesulfonic acid (MOPS). Aliquots of 50 l of each drug (and in the case of the single-drug control, 50 l of that drug and 50 l of sterile water) at a concentration of 20 times the targeted final concentration were dispersed in polystyrene plastic tubes (12 by 75 mm; Falcon 2054; Becton Dickonson, Lincoln Park, N.J.). Yeast inocula (0.9 ml), prepared spectrophotometrically and further diluted as described elsewhere (11), were added to the tubes containing drugs. The final drug concentrations ranged from to 128 g/ml for both fluconazole and flucytosine. All tubes were incubated without agitation at 35 C, and the readings were made at 72 h. Before the readings, each tube was vortexed, and its turbidity was compared with that of the growth control (drug-free) tube. The MIC of both drugs, alone or in combination, was defined as the lowest drug concentration in a tube which produced a visual turbidity of 80% inhibition compared with that of the growth control. Definitions. Drug interaction was classified as synergistic, additive, autonomous, or antagonistic on the basis of the fractional inhibitory concentration (FIC) index. The FIC index is the sum of the FICs for each of the drugs, which in turn is defined as the MIC of each drug when used in combination divided by the MIC of the drug when used alone. The interaction was defined as synergistic if the FIC index was 1.0, additive if the FIC index was 1.0, autonomous if the FIC index was between 1.0 and 2.0, and antagonistic if the FIC index was 2.0 (2, 4, 14). Synergy was further subclassified as marked (FIC index of 0.50) and weak (FIC index of between 0.50 and 1.0) (8). The achievable serum drug concentrations are between 40 and 100 g/ml for flucytosine and 10 g/ml for fluconazole (3). Since the CSF flucytosine and fluconazole concentrations are at least 80% of those in serum, the achievable CSF drug concentrations were estimated to be 32 g/ml for flucytosine and 8 g/ml for fluconazole. Statistical analysis. The MIC data were logarithmically transformed to approximate a normal distribution prior to statistical analysis. Continuous variables were compared with Student s t test or the Mann-Whitney test. Differences were considered significant if P was less than

2 1692 NGUYEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. Distribution of fluconazole MICs for 50 clinical isolates of C. neoformans. FIG. 3. Flucytosine MICs for Cryptococcus isolates when flucytosine was given alone ( ) and after combination with fluconazole ( ). Note that the addition of fluconazole greatly enhances the activity of flucytosine; flucytosine MICs were all reduced to 8 g/ml after combination with fluconazole. Values in parentheses are numbers of isolates tested. RESULTS All organisms tested produced detectable growth after 72 h of incubation. Fluconazole and flucytosine MICs for both of the control (American Type Culture Collection) isolates were within the expected range (11). The combination of fluconazole and flucytosine had a synergistic effect against the ATCC strain and an autonomous effect against the ATCC strain. The MIC distributions of fluconazole and flucytosine are presented in Fig. 1 and 2. Fluconazole MICs ranged from to 32 g/ml, with a MIC at which 50% of the isolates are inhibited (MIC 50 ) and a MIC 90 of 2 and 16 g/ml, respectively. Flucytosine MICs ranged from to 128 g/ml, with a MIC 50 and a MIC 90 of 2 and 16 g/ml, respectively. The fluconazole MIC for 70% (35 of 50) of the isolates was 8 g/ml, and the flucytosine MIC for 92% (46 of 50) was 32 g/ml. The fluconazole MIC for 64% (32 of 50) of the isolates was 8 g/ml, and the flucytosine MIC was 32 g/ ml. When fluconazole and flucytosine were given in combination, there were significant reductions in the geometric mean of the fluconazole MIC (from 5 to 1 g/ml; P 0.001) and of the flucytosine MIC (from 12 to 0.1 g/ml; P ). Sixty-two percent (31 of 50) of the interactions were synergistic, 24% (12 of 50) were autonomous, and 6% (3 of 50) were additive (Table 1). In four instances, the interaction could not be defined because the isolates were extremely susceptible to fluconazole (MIC of g/ml [one isolate]) or to flucytosine (MIC of g/ml [three isolates]); therefore, further reductions in MIC were not evaluated. Antagonism was not observed. Synergy. When synergy was documented, the median reductions in MIC were 4-fold (range of 2- to 16-fold) for fluconazole and 4-fold (range of 2- to 1,000-fold) for flucytosine. The flucytosine MICs were all reduced to 8 g/ml after combination with fluconazole (Table 2 and Fig. 3). On the other hand, only 40% (4 of 10) of the isolates for which the fluconazole MIC was 8 g/ml actually exhibited a MIC reduction to less than 8 g/ml after combination with flucytosine (Fig. 4). Autonomy. For 24% (12 of 50) of the isolates, the combina- FIG. 2. Distribution of flucytosine MICs for 50 clinical isolates of C. neoformans. FIG. 4. Fluconazole MICs for Cryptococcus isolates when fluconazole was given alone ( ) and after combination with flucytosine ( ). Note that the addition of flucytosine failed to reduce the fluconazole MIC for 53% (8 of 15) of the isolates for which the fluconazole MIC was 8 g/ml to the level below the achievable CSF fluconazole level (8 g/ml). Values in parentheses are numbers of isolates tested.

3 VOL. 39, 1995 COMBINED FLUCONAZOLE-FLUCYTOSINE AGAINST C. NEOFORMANS 1693 Interaction TABLE 1. Mode of interaction between fluconazole and flucytosine a Definition % (no. interacting/no. tested) of isolates showing interaction Synergy FIC Flu FIC 5FC (31/50) Additivism FIC Flu FIC 5FC (4/50) Autonomy FIC Flu FIC 5FC (12/50) Antagonism FIC Flu FIC 5FC (0/50) a In four isolates, the interaction could not be assessed because the organisms were extremely susceptible (MIC of g/ml) to both fluconazole (Flu [one isolate]) and flucytosine (5FC [three isolates]). tion demonstrated an autonomous interaction. Note that for all of these 12 isolates except 1, their initial fluconazole MIC was maintained despite combination with flucytosine (Table 3). On the other hand, 92% (11 of 12) of the isolates showed a decrease in flucytosine MICs of at least 16-fold (from 16- to 1,000-fold) upon combination with fluconazole. Additivism. For three isolates, the combination demonstrated additivism (Table 3). The fluconazole and flucytosine MICs were each reduced twofold after combination for these isolates. DISCUSSION Although most infections respond satisfactorily to a single antimicrobial agent, there are clinical circumstances in which a combination of antimicrobial agents is advantageous. Achievement of synergy is one of the major theoretical justifications for combination therapy. Synergism is of particular importance in cases in which the infecting organisms are relatively resistant to all available antimicrobial agents or in which the enhancement of drug activity is needed, as in infections involving immunocompromised hosts (9). Cryptococcal meningitis is an example in which synergistic combination therapy might be useful: the infection often involves patients with inadequate host defenses and carries a low cure rate (40%) with either amphotericin B or fluconazole alone (13). In addition, although fluconazole was proven to be as efficacious as amphotericin B in a randomized trial of cryptococcal meningitis therapy in AIDS patients, fluconazole did not appear to be as rapidly fungicidal as amphotericin B for TABLE 2. Synergistic action of fluconazole and flucytosine in 31 Cryptococcus isolates MIC ( g/ml) of b : Synergy for isolate a Fluconazole Flucytosine FIC index Alone Combined FIC Flu Alone Combined FIC 5FC Marked (FIC index, 0.5) Weak (FIC index, ) a Note that 39% (12 of 31) of the combinations were markedly synergistic (FIC index of 0.50) and 61% (19 of 31) were weakly synergistic (FIC index of 0.5 to 1.0). b Flu, fluconazole; 5FC, flucytosine.

4 1694 NGUYEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Autonomous or additive actions of fluconazole and flucytosine in 15 Cryptococcus isolates Action for isolate a MIC ( g/ml) of b : Fluconazole Flucytosine Alone Combined FIC Flu Alone Combined FIC 5FC FIC index (FIC Flu FIC 5FC ) Autonomous (FIC index, ) Additive (FIC index, 1.0) a Note that for 12 isolates, the combination demonstrated autonomy. The addition of flucytosine failed to enhance the activity of fluconazole; The initial fluconazole MIC for all isolates except one (isolate 46) was retained despite the addition of flucytosine. Despite the indifference of fluconazole toward the addition of flucytosine, the addition of fluconazol greatly enhanced the activity of flucytosine; 92% (11 of 12) of flucytosine MICs were reduced at least 16-fold after combination with fluconazole. Also note that for three isolates, the combination demonstrated additivism; the fluconazole and flucytosine MICs were each reduced twofold after combination. b Flu, fluconazole; 5FC, flucytosine. Cryptococcus neoformans in CSF (13). Given the dearth of available systemic antifungal agents, one option for treatment at this point is combination therapy. The combination of fluconazole and flucytosine is an attractive therapeutic option, given the benign side effects associated with fluconazole and the ease of oral administration of both agents. In addition, the differing mechanisms of action of these two drugs suggest that synergy is theoretically possible: fluconazole acts by damaging the fungal cell membrane, thereby allowing greater intracellular penetration of flucytosine. A recent prospective, open-label clinical trial with the combination of fluconazole and flucytosine against cryptococcal meningitis yielded encouraging results. A clinical success rate of 63% was observed, which was higher than that experienced with amphotericin B or fluconazole alone (6, 13). In addition, the median time required for the sterilization of the CSF with the fluconazole-flucytosine combination was 23 days, which was less than the previously reported sterilization rate with amphotericin B (42 days) or fluconazole (64 days) alone (6, 13). Despite the optimistic results of this study, to date, in vitro combination testing of fluconazole-flucytosine to investigate the biologic basis for this clinical finding has not been performed. The definition of synergy is methodology dependent and, to some degree, arbitrary (10). Our definition of synergy was adapted from that of Berenbaum and Elion et al. (FIC index of 0.1) (2, 4). Since the degree of synergy is inversely related to the FIC index, we used the further subclassifications marked (FIC index of 0.50) and weak (FIC index of between 0.50 and 1.0). Krogstad and Moellering defined antagonism as an FIC index of 1.0 (5). However, situations in which the MIC of one drug remains the same (therefore, FIC 1 of 1.0) while the MIC of the second drug is markedly reduced after combination (FIC 2 of 1.0; therefore, FIC index of 1.0) occur; it appears erroneous to classify this interaction as antagonistic, since this implies that the two agents act against each other. Therefore, for nonsynergistic interaction, we adapted the definitions autonomy (FIC index of between 1.0 and 2.0) and antagonism (FIC index of 2.0) (14). To our knowledge, our study is the first to report the in vitro combination of fluconazole-flucytosine with a large number of Cryptococcus isolates for which the range of MICs was wide (Fig. 1 and 2). The finding that 62% of the interactions were synergistic and that antagonism was not observed was encouraging. This implies that combination therapy with fluconazole and flucytosine might prove superior to single therapy with either agent alone. However, it should be pointed out that although experiments with bacteria have shown that an improved outcome is more likely when synergy is demonstrated in vitro than when it is not (9), the in vitro antifungal susceptibility and in vivo correlations for fungal infection have not been well studied. Therefore, clinical evaluation of this combination therapy in patients is needed. Our data further demonstrated that even when only autonomy was achieved (24% in this study), the combination of fluconazole and flucytosine still showed a beneficial effect; the in vitro inhibitory action of flucytosine on the isolates for which the MIC was 32 g/ml was greatly enhanced by the addition of fluconazole, as demonstrated by the reduction of flucytosine MICs from 128 g/ml to at least 1 g/ml, a concentration which is severalfold lower than the achievable serum and CSF flucytosine concentrations (Table 3 and Fig. 3). This finding illustrates the second advantage of combination therapy, i.e., the ability of combination therapy to reduce the amount of drug required for therapy and, thus, potentially reduce doserelated toxicities. Our data raise the possibility that the current recommended flucytosine dosage of 150 mg/kg of body weight per day might not be necessary for clinical efficacy when flucy-

5 VOL. 39, 1995 COMBINED FLUCONAZOLE-FLUCYTOSINE AGAINST C. NEOFORMANS 1695 tosine is used in combination with fluconazole, since the flucytosine MIC achieved with the combination is at least 10 times below the achievable level in CSF attained with this recommended dosage. Reduction of the flucytosine dose might alleviate some of the more serious side effects (especially pancytopenia and gastrointestinal intolerance) associated with this agent, since these effects are often dose related. Again, this hypothesis requires validation by in vivo animal studies. In contrast to the MICs of flucytosine, which dropped to concentrations well below the achievable level in CSF with the addition of fluconazole (Fig. 3), the addition of flucytosine did not greatly enhance the in vitro activity of fluconazole if the fluconazole MIC for the isolate was initially 8 g/ml. For example, only 47% (7 of 15) of isolates for which the fluconazole MIC was 8 g/ml exhibited a reduction in MIC to less than 8 g/ml when fluconazole was combined with flucytosine (Fig. 4). This finding suggests that fluconazole, even in combination with flucytosine, might be suboptimal for therapy against Cryptococcus isolates for which the MIC is 8 g/ml or greater. Combined therapy with fluconazole and flucytosine was shown to be superior to single-drug therapy in one experimental murine model of Cryptococcus meningitis (1) but not in another (12). The reason for the discrepancy in these results is unclear. We recommend that the in vivo combination of these two agents should be studied with isolates for which the range of MICs is different, since our data demonstrated the differential benefit of the in vitro fluconazole-flucytosine combination, depending on the initial fluconazole MIC for the isolates (Fig. 3). In addition, consideration should be given to evaluating lower dosages of flucytosine and to determining if the clinical efficacy can still be maintained at these lower dosages when used in combination with fluconazole. In summary, the in vitro results with combination fluconazole-flucytosine therapy against C. neoformans var. neoformans are encouraging. Antagonism was not observed for this combination, and synergy was achieved in 62% of cases. For the remaining cases, in which synergism was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when used in combination. Animal model and clinical studies are warranted to further elucidate the potential utility of fluconazole-flucytosine combination therapy. ACKNOWLEDGMENTS F. Barchiesi is a recipient of a scholarship from Istituto Superiore di Sanita, Rome, Italy. We thank Janet Stout and Robert R. Muder for critical review of the manuscript and Jennifer D. 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