Journal of Medical and Veterinary Mycology (1992), 30,197-206
Synergistic postantifungal effect of flucytosine and fluconazole on Candida albicans Y. MIKAMI 1, G. M. SCALARONE 1"2, N. KURITA 1, K. YAZAWA 1,J. UNO 1 AND M .
MIYAJI 1
1Department of Experimental Chemotherapy, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chiba, Japan; and 2Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA Med Mycol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
(Accepted 22 January 1992) The in vitro efficacy of flucytosine and ftuconazole, separately and in combination, with respect to induction of a postantifungal effect (PAFE) on Candida albicans was studied. PAFE refers to the persistent suppression of fungal cell growth following a short period of exposure to an antifungal agent. A turbidometric method was used to measure cell growth and to quantitate the PAFE following exposure of C. albicans yeast cells to different concentrations of the two agents for 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the drug by dilution. Minimum (MIC) and fractional inhibitory concentration determinations were made and the data used for selecting the concentrations used in the PAFE evaluations. A synergistic interaction of the two drugs at concentrations well below their individual MICs was evidenced. Flucytosine:fluconazole ratios of 1:16-1:32 at concentrations ranging from 0.024-0.098/.tg ml 1 and from 0.78-1.56/~g ml 1 with flucytosine and fluconazole, respectively, induced PAFEs which persisted for 2.5 h longer than those achieved when each of the two agents was assayed separately.
Over the past several years considerable progress has been made with regard to the development of antifungal agents for the treatment of candidiasis, but the search continues for agents that may be used alone or in combination with other drugs to provide successful therapy. The difficulties associated with treatment of fungal infections in patients with impaired immune defense mechanisms are of major concern at the present time. The water-soluble agent flucytosine (5-fluorocytosine) has been widely used alone or in combination with amphotericin B for treatment of infections caused by Candida albicans and Cryptococcus neoformans [5]. Flucytosine is a synthesized fluorinated pyrimidine analog that interferes with pyrimidine metabolism and ultimately RNA and protein synthesis [2]. Treatment with flucytosine, however, has produced severe side effects in some patients including neutropenia resulting from bone marrow suppression, hepatic dysfunction and gastrointestinal toxicity problems [3, 8, 9, 27]. Recently another antifungal compound, fluconazole, was licensed for use in treating candidal and cryptococcal infections. This agent has generated a lot of interest because of its unique pharmacologic properties. Fluconazole, a fluorinated bis-triazole whose mode of action is inhibition of ergosterol biosynthesis, differs markedly from other antifungal azole compounds. It is water-soluble, can be administered both orally and parenterally, exhibits a low level of protein binding, its absorption after oral dosing is good and it readily penetrates into CSF [8, 12, 25]. Enthusiasm has been generated Correspondence address: Dr Y. Mikami, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chiba, Japan (280). 197
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concerning the efficacy and potential of this drug for use in the treatment of a variety of fungal infections including candidiasis, coccidioidomycosis, blastomycosis, cryptococcosis, aspergillosis and histoplasmosis [12, 25]. However, there are a few recent reports that fluconazole was not as effective as other agents such as amphotericin B, Sch39304 and a combination of amphotericin B and flucytosine for the treatment of disseminated candidiasis and endophthalmitis [10], cryptococcosis [16, 21], coccidioidomycosis [7] and histoplasmosis [15] in animal models and human studies. These findings have led investigators to consider and evaluate the potential beneficial effects of combined therapy [14, 24]. In a recent study, Allendoerfer et al. [1] showed that combined therapy with fluconazole and flucytosine was superior to single drug therapy in murine cryptococcal meningitis. Other studies have indicated that the combinations of flucytosine and amphotericin B [3, 9, 22, 24, 27] and flucytosine plus imidazole or triazole derivatives [24] were synergistic. Such studies have indicated that it is of considerable importance not only to evaluate the efficacy of single antifungal agents, but also to consider the potential benefits that may result from synergistic interactions of different agents. Various in vitro and in vivo procedures have been used to assess the effectiveness of antifungal agents against particular organisms. The steady proliferation of new agents, however, will inevitably necessitate more in vitro testing to provide reliable results to the clinician [11]. We have recently been conducting in vitro studies in our laboratory to determine if persistent suppression of fungal growth can be induced following limited exposure of C. albicans to different antifungals. This phenomenon, termed the postantibiotic effect (PAE), has been studied extensively with bacteria during the past several years [6, 13, 17, 30, 31]. We have been able to demonstrate this effect with different concentrations of flucytosine, amphotericin B and miconazole on C. albicans following limited exposure of the yeast cells to the agent and subsequent removal of the agent by dilution [26]. Viable counts as well as turbidometric methods [19] were used to determine organism regrowth. We have termed this phenomenon the postantifungal effect (PAFE). In the present study we investigated the PAFE produced by the synergistic interaction of flucytosine and fluconazole on different strains of C. albicans. A turbidometric method was used to evaluate growth of the yeast cells following limited exposure to different concentrations of flucytosine, fluconazole and combinations of both drugs. METHODS Organisms Three strains of C. albicans were used in this study. Clinical isolates IFM 1001, which is used as a reference strain for antifungal sensitivity testing in our laboratory, 9760-H85 and 3532-K31, were maintained on Sabouraud glucose agar (Eiken Chemical Co. Ltd., Tokyo, Japan) slants. The cultures were transferred to yeast nitrogen base broth (Difco Laboratories, Detroit, MI) containing 1% glucose (YNB-G) and incubated for approximately 24 h prior to use in the assays. Hemacytometer counts were made on the cell suspensions and the cell number was adjusted as required for each assay described below.
Antifungal agents Flucystosine was generously provided by Nippon Roche, K.K. (Tokyo, Japan). An in-
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itial stock solution (100 ~g m1-1) was made in saline and the solution filter sterilized by passage through a 0-22 ~m membrane filter (Nucleopore, Pleasanton, CA). Fluconazole was obtained from Pfizer Pharmaceuticals Inc. (Aichi, Japan). A stock solution containing 10 mg m1-1 was prepared in methanol which served to sterilize the preparation. Dilutions (two-fold dilution scheme) of each drug ranging from 6.25-0-006 /~g ml-1 (flucytosine) and from 50-0-39/.tg ml 1 (fluconazole), in a two dilution series from 50 ~tg ml-1 were prepared in saline for use in the assay.
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Minimum (MIC) and fractional (FIC) inhibitory concentration determinations Initial susceptibility studies were conducted in order to determine if a synergistic interaction could be induced by combinations of flucytosine and fluconazole and to determine approximate concentrations of each agent to use in the PAFE assays. A broth dilution method described previously was used for MIC determinations with yeast cells from the three strains of C. albicans [20]. A checkerboard style titration [4, 14, 22, 28], in which each agent was evaluated both alone and in combination, was performed. Two-fold serial dilutions of flucytosine (6.25-0.006 ~tg m1-1) and fluconazole (50--0.78 /~g m1-1) were added to YNB-G broth in microdilution plates. Yeast cells (approximately 2.5 × 104 cells m1-1) were added and the plates incubated for 48 h at 30°C. This assay and the PAFE determinations were performed at 30°C since previous studies in our laboratory and by others [23] have indicated that this temperature was optimal when using YNB-G for susceptibility studies with C. albicans. Endpoints were determined visually and by a spectrophotometric method (490 nm, Toyo ETY III plate analyser, Tokyo, Japan). The MIC was defined as the lowest concentration of drug that inhibited multiplication of the yeast cells, as indicated by the absence of turbidity. A FIC [4, 22] index was calculated from the MIC values. The FIC is a measure of synergy or antagonism and is equal to the MIC of each drug alone divided into its concentration in a particular combination that inhibits growth. The index is equal to the sum of the two FICs. Sums of 1 indicate synergy, additivity and antagonism, respectively [4, 22]. PAFE assay Approximately 1 × 107 yeast cells m1-1 from each of the three strains were added to tubes containing 1.6 ml of YNB-G broth. Varying concentrations of flucytosine (0.78--0.024 ~g ml-1), fluconazole (25-0.39/tg m1-1) or combinations of both drugs were added (0.1 ml) to the tubes, as detailed further in the results section. Control tubes containing only yeast cells were also used in each comparative assay. The tubes were incubated at 30°C with shaking for 2 h. Earlier studies indicated that these conditions were optimal for PAFE determinations [6] with our system. Following the exposure period the drugs were removed by dilution in which the cultures were diluted 102-fold in sterile saline. Aliquots (100 ¢tl) were added to duplicate wells of a microdilution plate containing 150/,tl of YNB-G, placed in a Bioscreen C instrument (Labsystems, Tokyo, Japan) and incubated for 72 h at 30°C. Growth of the cells was monitored by measuring turbidity (absorbance at 600 nm) at 20 min intervals. The data were recorded by a computer interfaced with the analyser and growth curves were generated from each cell preparation. Mean values were determined for each of the cultures from the duplicate growth curve data.
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The duration of the PAFE was calculated by modifying a method used previously for PAE determinations with bacteria [6, 13] and used in initial PAFE studies in our laboratory [6]. The formula PAFE = T - C was used where T was the time (h) required for the turbidity of the drug-exposed yeast cell suspension to reach the 0.5 absorbance level after dilution/removal of drug, and C was the time required for the turbidity of the drug-free control culture to reach the same level. We determined previously that approximately 1 × 10 7 viable cells m1-1 w e r e present when the turbidity of the cell suspension reached the 0-5 absorbance level. Thus T - C expressed the time interval during which an antifungal agent is able to induce a PAFE on fungal growth.
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RESULTS Susceptibility determinations indicated that individual MIC values of flucytosine and fluconazole were considerably greater than the concentrations required for growth inhibition when the two drugs were used in combination against yeast cells from the three strains of C. albicans (Table 1). Fluconazole MICs ranged from 50-12.5/lg m1-1 and an MIC value of 1-56/~g ml -a was obtained with flucytosine when the two agents were used alone. In contrast, MICs as low as 1-56 pg ml -t (fluconazole) and 0.049/lg m1-1 (flucytosine) were evidenced when the two drugs were used in combination. FIC index determinations, using the above MIC data, revealed that the two agents showed a marked degree of synergism. FIC index values ranged from 0.06-0.56 when calculations were made from three different drug concentration combinations that inhibited growth with each strain (Table 2). Thus the values were well below the index value of 1 (additive or indifferent interaction), denoting a synergistic interaction. The lowest FIC index values were obtained when the flucytosine:fluconazole concentration ratio was 1:32. Since synergy between flucytosine and fluconazole was noted in the above susceptibility studies, it was decided to investigate the potential of the two agents in combination with regard to PAFE induction. Several initial PAFE studies were performed using a wide range of concentrations of flucytosine and fluconazole at ratios ranging from 1:0.6-1:100 of flucytosine to fluconazole, respectively. The data from these studies indicated that the agents interacted synergistically and produced optimal PAFEs when combinations of the two were tested at flucytosine:fluconazole ratios ranging from 1:16-1:64. T A B L E 1. MICs of flucytosine and fluconazoIe alone and in combination against three strains of C. albicans C. albicans
MIC
strain
5-FC
FLCZ
1001 9760-H85 3532-K31
1-56 1.56 1.56
50 12.5 12.5
(#g ml- t) 5-FC
& FLCZ
0-049 - 1.56 0-098 - 3.12 0.049 - 1.56
5-FC = flucytosine; F L C Z = fluconazole. MICs were determined by liquid microdilution m e t h o d using yeast nitrogen base (1% glucose) medium. MICs for the 5 - F C - F L C Z combination represent the concentrations of each agent which gave the lowest FIC index value.
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T A B L E 2. FIC indices for flucytosine and fluconazole against three strains of C. albicans FIC index
C. albicans strain
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1001 9760-H85 3532-K31
A
B
C
0.06 0.31 0.16
0.08 0.50 0.26
0.51 0.56 0.50
FIC index values calculated for three different drug concentration combinations with each strain as follows: (concentrations expressed as/lg ml-~; 5-FC = flucytosine, F L C Z = fluconazole). Strain 1001: A = 5-FC (0.049) & F L C Z (1-56); B = 5-FC (0.024) & F L C Z (3-12); C = 5-FC (0-012 & F L C Z (25). Strain 9760-H85: A = 5-FC (0.098) & F L C Z (3.12); B = 5-FC (0-006) & F L C Z (6-25): C = 5-FC (0.78) & F L C Z (0-78). Strain 3532-K31: A = 5-FC (0.049) & F L C Z (1.56); B = 5-FC (0.012) & F L C Z (3.12): C = 5-FC (0-006) & F L C Z (6.25).
Comparative evaluations were then performed using two-fold serial dilutions of flucytosine (0.78-0.024/.tg m1-1) and fluconazole (25-0.39/,tg ml -t) in combination at ratios of 1:16, 1:32 and 1:64 against yeast cells from the three strains of C. albicans. The agehts were also tested alone at the above concentrations. The results of these studies are shown in Tables 3, 4 & 5. Flucytosine alone induced PAFEs of 6.8-24.8 h (strain 1001; Table 3), 3.8-14-5 h (strain 9760-H85; Table 4) and 1.5-6 h (strain 3532-K31; Table 5). On
T A B L E 3. P A F E following exposure of C. albicans strain 1001 to various concentrations of flucytosine and fluconazole
Ratio
Conc. (//g m1-1)
5-FC: F L C Z
5-FC
1:64
0.39 0-195 0.098 0.049 0.024
1:32
1:16
FLCZ
P A F E (h) 5-FC
FLCZ
5-FC & F L C Z
25 12-5 6.25 3.13 1.56
21-3 15-3 11.5 9-5 6-8
2.3 1.8 1.3 1 0.8
21 15-8 14 9.8 7
0.78 0.39 0.195 0.098 0.049 0-024
25 12.5 6.25 3.13 1-56 0-78
24-8 21.3 15-3 11-5 9-5 6-8
2-3 1.8 1.3 1 0.8 0.5
25.5 20.8 17.5 13 11.5 8
0.78 0.39 0.195 0.098 0.049 0-024
12.5 6.25 3.13 1.56 0-78 0-39
24-8 21-3 15-3 11-5 9-5 6-8
1.8 1.3 1 0.8 0.5 0-1
25.3 23-8 16 14 11-5 7
5-FC = flucytosine; F L C Z = fluconazole.
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TABLE 4. PAFE following exposure of C. albicans strain 9760-H85 to various concentrations of flucytosine and fluconazole
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Ratio 5-FC: FLCZ
Conc. (,ug ml 1) 5-FC
FLCZ
PAFE (h) 5-FC
FLCZ
5-FC & FLCZ
1:64
0.39 0.195 0.098 0.049 0.024
25 12-5 6-25 3-13 1-56
11.3 9-3 7-3 7 3.8
2 1.5 1 0-5 0-5
12.3 12 6.8 7.3 4
1:32
0.78 0-39 0.195 0.098 0.049 0.024
25 12-5 6.25 3.13 1.56 0.78
14.5 11-3 9.3 7.3 7 3-8
2 1-5 1 0-5 0-5 0.1
11.5 10-5 8.3 7-3 9 5-5
1:16
0.78 0.39 0.195 0.098 0.049 0.024
12-5 6.25 3.13 1.56 0.78 0.39
14-5 11-3 9-3 7-3 7 3-8
1-5 1 0-5 0.5 0-1 0
31.5 12-3 9.5 9.8 9 3.8
5-FC = flucytosine; FLCZ = fluconazole.
TABLE 5. PAFE following exposure of C. albicans strain 3532-K31 to various concentrations of flucytosine and fluconazole Ratio 5-FC: FLCZ
Conc. (pg ml 1) 5-FC
FLCZ
PAFE (h) 5-FC
FLCZ
5-FC & FLCZ
1:64
0.39 0.195 0.098 0.049 0.024
25 12.5 6.25 3.13 1.56
5.8 5 3.8 3 1.5
1.5 1.5 1 0.5 0.5
5.8 7.3 6 5 1.5
1:32
0-78 0.39 0.195 0.098 0-049 0-024
25 12.5 6.25 3.13 1.56 0-78
6 5-8 5 3.8 3 1-5
1.5 1-5 1 0.5 0.5 0
8.5 4.5 4 4 3.5 4
1:16
0-78 0.39 0.195 0.098 0-049 0.024
12.5 6.25 3.13 1.56 0-78 0.39
6 5.8 5 3.8 3-0 1.5
1.5 1 0-5 0.5 0 0
4.3 6.5 2.5 4.5 4-5 1.8
5-FC = flucytosine; FLCZ = fluconazole.
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TABLE 6. Lowest concentrations of flucytosine and fluconazole that produced the optimal synergistic PAFE C albicans strain
PAFE (h) 5-FC & FLCZ
Conc. 5-FC
(pg ml- l) FLCZ
Ratio 5-FC:FLCZ
1001 9760-H85 3532-K31
14 9.8 4
0-098 0.098 0.024
1-56 1.56 0-78
1:16 1:16 1:32
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5-FC = flucytosine; FLCZ = fluconazole. These concentration combinations produced PAFEs that persisted for 2.5 h longer than those produced by either agent separately.
the other hand, considerably shorter PAFEs were induced with fluconazole with values ranging from 0.5-2.3 h, 0-2 h and 0-1-5 h with the above three strains, respectively. When the two drugs were used in combination, synergism was noted with several of the combinations with PAFEs ranging from 0.2-2-5 h longer than those achieved with either drug alone (Tables 3, 4 & 5). The lowest concentration combinations that produced optimal synergistic effects are summarized in Table 6. Flucytosine:fluconazole concentration ratios of 1:16 (0-098 and 1-56 pg ml-1; strain 1001 and strain 9760 H85) and 1:32 (0.024 and 0.78 pg ml-1; strain 3532-K31) produced PAFEs of 14, 9.8 and 4 h, respectively. The PAFEs persisted for 2.5 h longer than those induced by either agent alone. DISCUSSION An increasing amount of interest has been generated recently with regard to combination therapy for the systemic mycoses. Various studies have been concerned with attempts to improve the efficacy and broaden the spectrum of the antifungal agents, particularly in situations where opportunistic organisms produce infections in immunocompromised patients. Toxicity problems associated with some of the available agents, the necessity for high dose therapy with others and the proven inefficiency of some of the newer antifungals, have been of major concern to clinicians in recent years. Thus many in vitro and in vivo investigations are being performed in an effort to improve the situation. In relation to this, numerous in vitro laboratory procedures have been used to determine susceptibility profiles of fungi and to determine dosing schedules of certain drugs, but they are vulnerable to the numerous in vitro test variables. Such procedures have suffered from a lack of a universally accepted standardized testing methodology and in many instances there has been a lack of correlation of in vitro results with the outcome of in vivo therapy [5]. MIC determinations have been performed by many laboratories, but one must recognize that the data from such procedures can vary appreciably depending upon testing parameters including media composition, exposure time, incubation temperature and type of assay [11, 18, 23]. Thus a great deal of effort will be required before antifungal susceptibility testing reaches the level of reliability that is associated with antibacterial susceptibility procedures. Another factor to consider is that in vivo the organisms are exposed to fluctuating levels of a drug instead of constant levels as is the case with in vitro MIC assays. With this in mind we began investigations on a phenomenon we have termed the PAFE in an effort to determine if exposure of an organism to a drug for a short period of time might
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produce persistent suppression of growth. It seems reasonable to assume that such a short period of exposure rather than continual exposure, as with standard susceptibility assays, might approximate the in vivo situation. Numerous PAE studies have been done with bacteria [6, 13, 17, 30, 31] and such studies have indicated that antibiotics exert suppressive effects on bacterial growth after a short period of exposure. Such an assay thus provides information on the drug-organism interaction that standard susceptibility tests do not provide. In previous studies we used both a viable count method (unpublished data) and a turbidometric technique [26] to measure C. albicans cell regrowth following exposure to flucytosine, amphotericin B and miconazole for short periods of time (0.5-2 h). Profound suppressive effects were evidenced and generally the length of the PAFE was dependent upon the drug concentration and the period of exposure. In the present study we used the turbidometric method to investigate the synergistic interaction of flucytosine and fluconazole on three strains of C. albicans. The aim of the study was to determine if a PAFE could be induced by combinations of the drugs each at concentrations considerably lower than their individual MIC values. We were indeed able to demonstrate a synergistic effect with the two drugs at concentrations well below their individual MICs. Of interest is the fact that flucytosine induced PAFEs, both alone and to a greater extent in combination with fluconazole, at concentrations (0-024-0.78 pg m1-1) considerably lower than optimal peak serum levels (50-100 ~tg ml a) as reported previously [8, 27]. A major concern has been flucytosine toxicity associated with peak serum levels of 100/.tg ml- t or greater [27]. PAFEs were also induced with concentrations of fluconazole (0-78-1-56 pg ml-t), in combination with flucytosine, that were also less than optimal serum levels of 2/lg ml i [12]- 45/lg m1-1 [16] reported in recent studies. PAFE values varied according to the strain of C. albicans being assayed. These results are encouraging, but one must bear in mind that the PAFE studies have been performed in vitro and further studies must be performed to assess the utility of this assay with regard to providing clinicians with information necessary to determine optimal dosing regimens. Theoretically the present or absence of a PAFE should provide a rationale for less frequent or more frequent administration of the antifungal agent. ACKNOWLEDGEMENTS Financial support was provided by the Japanese Ministry of Education for Dr Gene M. Scalarone as a guest professor (foreign researcher) at Chiba University, Research Center for Pathogenic Fungi and Microbial Toxieoses. The authors are grateful for the excellent technical assistance provided by Yuri Ichihara and Shinji Ohashi.
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4. BERENBAUM, M. C. 1978. A method for testing for synergy with any number of agents. Journal of Infectious Diseases, 137, 122-130. 5. BODEY, G. P. 1988. Topical and systemic antifungal agents. Medical Clinics of North America, 72, 637-659. 6. BUNDTZEN,R. W.. GERBER, A. U., COHN, D. L. & CRAIG, W. A. 1981. Postantibiotic suppression of bacterial growth. Reviews of lnfectious Disease, 3, 28-37. 7. CLEMONS, K. V., HANSON, L. H., PERLMAN, A. M. & STEVENS, D. A. 1990. Efficacy of SCH39304 and fluconazole in a murine model of disseminated coccidioidomycosis. Antimicrobial Agents and Chemotherapy, 34, 928-930. 8. DISMUKES,W. E. 1988. Azole antifungal drugs: old and new. Annals of Internal Medicine, 109, 177-179. 9. DISMUKES,W. E., CLOUD, G., GALLIS, H. A., KERKERING,T. M., MEDOFF, G., CRAVEN,P. C., KAPLOWITZ, L. G., FISHER, J. F., GREGG, C. R., BOWELS,C. A., SHADOMY,S., STAMM,A. M., DIASIO, R. B., KAUFMAN, L., SOONG, S. & BLACKWELDER, W. C. 1987. Treatment of cryptococcal meningitis with combination amphotericin B and flucytosine for four as compared to six weeks. New England Journal of Medicine, 317, 334-341. 10. FILLER, S. G., CRISL1P, M. A., MAYER, C. L. & EDWARDS, J. E. 1991. Comparison o£ fluconazole and amphotericin B for treatment of disseminated candidiasis and endophthalmitis in rabbits. Antimicrobial
Agents and Chemotherapy, 35,288-292. 11. GALGIANI, J. N. 1987. Antifungal susceptibility tests. Antimicrobial Agents and Chemotherapy, 31, 1867-1870. 12. GALGIANI,J. N. 1990. Fluconazole, a new antifungal agent. Annals oflnternal Medicine, 113, 117-179. 13. GERBER, A. U. & CRAIG, W. A. 1981. Growth kinetics of respiratory pathogens after short exposures to ampicillin and erythromycin in vitro. Journal of Antimicrobial Chemotherapy, 8, 81-91. 14. HANSON, L. H., PERLMAN, A. M., CLEMONS, K. V. & STEVENS, D. A. 1991. Synergy between cilofungin and amphotericin B in a routine model of candidiasis. Amtimicrobial Agents and Chemotherapy, 35, 1334-1337. 15. KOBAYASHI,G. S., TRAVIS, S. J., RINALDI, M. G. & MEDOFF, G. 1990. In vitro and in vivo activities of Sch 39304, fluconazole, and amphotericin B against Histoplasma capsulatum. Antimicrobial Agents and Chemotherapy, 34, 524-528. 16. LARSEN, R. A., LEAL, M. A. E. & CHAN, L. S. 1990. Fluconazole compared with amphotericin B plus flucytosine for cryptococcal meningitis in AIDS. Annals of Internal Medicine, 113, 183-187. 17. McDONALD, P. J., CRAIG, W. A. & KUN1N, C. M. 1977. Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. Journal of Infectious Diseases, 135, 217-227. 18. MIKAMI, Y., HOUR-LONG, C., YAZAWA,K., UNO, J., ARAI, T. & KANNO, H. 1987. Some new approaches to the evaluation of antifungal activities of amphotericin B. Japandse Journal of Medical Mycology, 28, 273-384. 19. MIKAMI,Y., UNO, J., YAZAWA,K. & ARAI, T. 1989. Turbidometric characterization of growth inhibition by aztreonam, a synthetic monolactam antibiotic. Chemotherapy (Tokyo), 37, 723-730. 20. MIKAMI, Y., YAZAWA,K. & MATSUMAE,A. 1991. Evaluation of in vitro antifungal activities of amphotericin B, fluconazole, itraconazole and miconazole on seven different antifungal assay media, Chemotherapy (Tokyo), 39, 761-770. 21. NEGRONI, R., DE ELIAS COSTA, M. R. I., F1NQUELIEVICH,J. L , IOVANN1TTI,C., AGORIO, I., TIRABOSCHI, I. N. & LOEBENBERG, D. 1991. Treatment of experimental cryptococcosis with SCH 39304 and fluconazole. Antimicrobial Agents and Chemotherapy, 35, 1460-1463. 22. ODDS, F. C. 1982. Interactions among amphotericin B, 5-fluorocytosine, ketoconazole, and miconazole against pathogenic fungi in vitro. Antimicrobial Agents and Chemotherapy, 22, 763-770. 23. PFALLER, M. A., RINALDI, M. G., GALG1ANLJ. N., BARTLETF, M. S., BODY, B. A., ESPINEL-INGROFF, A., FROMTL1NG, R. A., HALL, G. S., HUGHES, C. E., ODDS, F. C. & SUGAR, A. M. 1990. Collaborative investigation of variables in susceptibility testing of yeasts. Antimicrobial Agents and Chemotherapy, 34, 1648-1654. 24. POLAK, A. 1989. Combination therapy for systemic mycosis. Infection, 17, 203-209. 25. SAAG, M. S. & DISMUKES,W. E. 1988. Azole antifungal agents: emphasis on new triazoles. Antimicrobial Agents and Chemotherapy, 32, 1-8. 26. SCALARONE, G. M., MIKAMI, Y., KURITA, N., ICHIBARA, Y., YAZAWA, K. & MIYAJI, M. 1992. Turbidometrie characterization of the postantifungal effect: comparative studies with amphotericin B, 5-fluorocytosine and miconazole on Candida albicans. Mycoses (in press). 27. STAMM,A. M., DIASIO, R. B., DISMUKES, W. E., SHADOMY, S., CLOUD, G. A., BOWLES, C. A., KARAM,
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G. H. & ESPINEL-INGROFF,A. 1987. Toxicity of amphotericin B plus flucytosine in 194 patients with cryptococcal meningitis. American Journal of Medicine, 83,236-242. 28. STEVENS, D. A. & Vo, P. T. 1982. Synergistic interaction of trimethoprim and sulfamethoxazole on
Paracoccidioides brasiliensis. Antimicrobial Agents and Chemotherapy, 21,852-854.
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29. SUGAR, A. M. 1991. Interactions of amphotericin B and SCH 39304 in the treatment of experimental murine candidiasis: lack of antagonism of a polyene-azole combination. Antimicrobial Agents and Chemotherapy, 35, 1669-1671. 30. VOGELMAN,B. S. & CRAIG, W. A. 1985. Postantibiotic effects. Journal of Antimicrobial Chemotherapy, 15, 37--46. 31. VOGELMAN, B. S., GUDMUNDSON, S., TURNIDGE, J., LEGGE'Vr, J. & CRAIG, W. m. 1988. In vivo postantibiotic effect in a thigh infection in neutropenic mice. Journal of Infectious Diseases, 157, 287-298.
Synergistic postantifungal effect of flucytosine and fluconazole on Candida albicans Y. MIKAMI 1, G. M. SCALARONE 1"2, N. KURITA 1, K. YAZAWA 1,J. UNO 1 AND M .
MIYAJI 1
1Department of Experimental Chemotherapy, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chiba, Japan; and 2Department of Biological Sciences, Idaho State University, Pocatello, Idaho, USA Med Mycol Downloaded from informahealthcare.com by University of Otago on 01/04/15 For personal use only.
(Accepted 22 January 1992) The in vitro efficacy of flucytosine and ftuconazole, separately and in combination, with respect to induction of a postantifungal effect (PAFE) on Candida albicans was studied. PAFE refers to the persistent suppression of fungal cell growth following a short period of exposure to an antifungal agent. A turbidometric method was used to measure cell growth and to quantitate the PAFE following exposure of C. albicans yeast cells to different concentrations of the two agents for 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the drug by dilution. Minimum (MIC) and fractional inhibitory concentration determinations were made and the data used for selecting the concentrations used in the PAFE evaluations. A synergistic interaction of the two drugs at concentrations well below their individual MICs was evidenced. Flucytosine:fluconazole ratios of 1:16-1:32 at concentrations ranging from 0.024-0.098/.tg ml 1 and from 0.78-1.56/~g ml 1 with flucytosine and fluconazole, respectively, induced PAFEs which persisted for 2.5 h longer than those achieved when each of the two agents was assayed separately.
Over the past several years considerable progress has been made with regard to the development of antifungal agents for the treatment of candidiasis, but the search continues for agents that may be used alone or in combination with other drugs to provide successful therapy. The difficulties associated with treatment of fungal infections in patients with impaired immune defense mechanisms are of major concern at the present time. The water-soluble agent flucytosine (5-fluorocytosine) has been widely used alone or in combination with amphotericin B for treatment of infections caused by Candida albicans and Cryptococcus neoformans [5]. Flucytosine is a synthesized fluorinated pyrimidine analog that interferes with pyrimidine metabolism and ultimately RNA and protein synthesis [2]. Treatment with flucytosine, however, has produced severe side effects in some patients including neutropenia resulting from bone marrow suppression, hepatic dysfunction and gastrointestinal toxicity problems [3, 8, 9, 27]. Recently another antifungal compound, fluconazole, was licensed for use in treating candidal and cryptococcal infections. This agent has generated a lot of interest because of its unique pharmacologic properties. Fluconazole, a fluorinated bis-triazole whose mode of action is inhibition of ergosterol biosynthesis, differs markedly from other antifungal azole compounds. It is water-soluble, can be administered both orally and parenterally, exhibits a low level of protein binding, its absorption after oral dosing is good and it readily penetrates into CSF [8, 12, 25]. Enthusiasm has been generated Correspondence address: Dr Y. Mikami, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chiba, Japan (280). 197
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concerning the efficacy and potential of this drug for use in the treatment of a variety of fungal infections including candidiasis, coccidioidomycosis, blastomycosis, cryptococcosis, aspergillosis and histoplasmosis [12, 25]. However, there are a few recent reports that fluconazole was not as effective as other agents such as amphotericin B, Sch39304 and a combination of amphotericin B and flucytosine for the treatment of disseminated candidiasis and endophthalmitis [10], cryptococcosis [16, 21], coccidioidomycosis [7] and histoplasmosis [15] in animal models and human studies. These findings have led investigators to consider and evaluate the potential beneficial effects of combined therapy [14, 24]. In a recent study, Allendoerfer et al. [1] showed that combined therapy with fluconazole and flucytosine was superior to single drug therapy in murine cryptococcal meningitis. Other studies have indicated that the combinations of flucytosine and amphotericin B [3, 9, 22, 24, 27] and flucytosine plus imidazole or triazole derivatives [24] were synergistic. Such studies have indicated that it is of considerable importance not only to evaluate the efficacy of single antifungal agents, but also to consider the potential benefits that may result from synergistic interactions of different agents. Various in vitro and in vivo procedures have been used to assess the effectiveness of antifungal agents against particular organisms. The steady proliferation of new agents, however, will inevitably necessitate more in vitro testing to provide reliable results to the clinician [11]. We have recently been conducting in vitro studies in our laboratory to determine if persistent suppression of fungal growth can be induced following limited exposure of C. albicans to different antifungals. This phenomenon, termed the postantibiotic effect (PAE), has been studied extensively with bacteria during the past several years [6, 13, 17, 30, 31]. We have been able to demonstrate this effect with different concentrations of flucytosine, amphotericin B and miconazole on C. albicans following limited exposure of the yeast cells to the agent and subsequent removal of the agent by dilution [26]. Viable counts as well as turbidometric methods [19] were used to determine organism regrowth. We have termed this phenomenon the postantifungal effect (PAFE). In the present study we investigated the PAFE produced by the synergistic interaction of flucytosine and fluconazole on different strains of C. albicans. A turbidometric method was used to evaluate growth of the yeast cells following limited exposure to different concentrations of flucytosine, fluconazole and combinations of both drugs. METHODS Organisms Three strains of C. albicans were used in this study. Clinical isolates IFM 1001, which is used as a reference strain for antifungal sensitivity testing in our laboratory, 9760-H85 and 3532-K31, were maintained on Sabouraud glucose agar (Eiken Chemical Co. Ltd., Tokyo, Japan) slants. The cultures were transferred to yeast nitrogen base broth (Difco Laboratories, Detroit, MI) containing 1% glucose (YNB-G) and incubated for approximately 24 h prior to use in the assays. Hemacytometer counts were made on the cell suspensions and the cell number was adjusted as required for each assay described below.
Antifungal agents Flucystosine was generously provided by Nippon Roche, K.K. (Tokyo, Japan). An in-
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itial stock solution (100 ~g m1-1) was made in saline and the solution filter sterilized by passage through a 0-22 ~m membrane filter (Nucleopore, Pleasanton, CA). Fluconazole was obtained from Pfizer Pharmaceuticals Inc. (Aichi, Japan). A stock solution containing 10 mg m1-1 was prepared in methanol which served to sterilize the preparation. Dilutions (two-fold dilution scheme) of each drug ranging from 6.25-0-006 /~g ml-1 (flucytosine) and from 50-0-39/.tg ml 1 (fluconazole), in a two dilution series from 50 ~tg ml-1 were prepared in saline for use in the assay.
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Minimum (MIC) and fractional (FIC) inhibitory concentration determinations Initial susceptibility studies were conducted in order to determine if a synergistic interaction could be induced by combinations of flucytosine and fluconazole and to determine approximate concentrations of each agent to use in the PAFE assays. A broth dilution method described previously was used for MIC determinations with yeast cells from the three strains of C. albicans [20]. A checkerboard style titration [4, 14, 22, 28], in which each agent was evaluated both alone and in combination, was performed. Two-fold serial dilutions of flucytosine (6.25-0.006 ~tg m1-1) and fluconazole (50--0.78 /~g m1-1) were added to YNB-G broth in microdilution plates. Yeast cells (approximately 2.5 × 104 cells m1-1) were added and the plates incubated for 48 h at 30°C. This assay and the PAFE determinations were performed at 30°C since previous studies in our laboratory and by others [23] have indicated that this temperature was optimal when using YNB-G for susceptibility studies with C. albicans. Endpoints were determined visually and by a spectrophotometric method (490 nm, Toyo ETY III plate analyser, Tokyo, Japan). The MIC was defined as the lowest concentration of drug that inhibited multiplication of the yeast cells, as indicated by the absence of turbidity. A FIC [4, 22] index was calculated from the MIC values. The FIC is a measure of synergy or antagonism and is equal to the MIC of each drug alone divided into its concentration in a particular combination that inhibits growth. The index is equal to the sum of the two FICs. Sums of 1 indicate synergy, additivity and antagonism, respectively [4, 22]. PAFE assay Approximately 1 × 107 yeast cells m1-1 from each of the three strains were added to tubes containing 1.6 ml of YNB-G broth. Varying concentrations of flucytosine (0.78--0.024 ~g ml-1), fluconazole (25-0.39/tg m1-1) or combinations of both drugs were added (0.1 ml) to the tubes, as detailed further in the results section. Control tubes containing only yeast cells were also used in each comparative assay. The tubes were incubated at 30°C with shaking for 2 h. Earlier studies indicated that these conditions were optimal for PAFE determinations [6] with our system. Following the exposure period the drugs were removed by dilution in which the cultures were diluted 102-fold in sterile saline. Aliquots (100 ¢tl) were added to duplicate wells of a microdilution plate containing 150/,tl of YNB-G, placed in a Bioscreen C instrument (Labsystems, Tokyo, Japan) and incubated for 72 h at 30°C. Growth of the cells was monitored by measuring turbidity (absorbance at 600 nm) at 20 min intervals. The data were recorded by a computer interfaced with the analyser and growth curves were generated from each cell preparation. Mean values were determined for each of the cultures from the duplicate growth curve data.
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The duration of the PAFE was calculated by modifying a method used previously for PAE determinations with bacteria [6, 13] and used in initial PAFE studies in our laboratory [6]. The formula PAFE = T - C was used where T was the time (h) required for the turbidity of the drug-exposed yeast cell suspension to reach the 0.5 absorbance level after dilution/removal of drug, and C was the time required for the turbidity of the drug-free control culture to reach the same level. We determined previously that approximately 1 × 10 7 viable cells m1-1 w e r e present when the turbidity of the cell suspension reached the 0-5 absorbance level. Thus T - C expressed the time interval during which an antifungal agent is able to induce a PAFE on fungal growth.
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RESULTS Susceptibility determinations indicated that individual MIC values of flucytosine and fluconazole were considerably greater than the concentrations required for growth inhibition when the two drugs were used in combination against yeast cells from the three strains of C. albicans (Table 1). Fluconazole MICs ranged from 50-12.5/lg m1-1 and an MIC value of 1-56/~g ml -a was obtained with flucytosine when the two agents were used alone. In contrast, MICs as low as 1-56 pg ml -t (fluconazole) and 0.049/lg m1-1 (flucytosine) were evidenced when the two drugs were used in combination. FIC index determinations, using the above MIC data, revealed that the two agents showed a marked degree of synergism. FIC index values ranged from 0.06-0.56 when calculations were made from three different drug concentration combinations that inhibited growth with each strain (Table 2). Thus the values were well below the index value of 1 (additive or indifferent interaction), denoting a synergistic interaction. The lowest FIC index values were obtained when the flucytosine:fluconazole concentration ratio was 1:32. Since synergy between flucytosine and fluconazole was noted in the above susceptibility studies, it was decided to investigate the potential of the two agents in combination with regard to PAFE induction. Several initial PAFE studies were performed using a wide range of concentrations of flucytosine and fluconazole at ratios ranging from 1:0.6-1:100 of flucytosine to fluconazole, respectively. The data from these studies indicated that the agents interacted synergistically and produced optimal PAFEs when combinations of the two were tested at flucytosine:fluconazole ratios ranging from 1:16-1:64. T A B L E 1. MICs of flucytosine and fluconazoIe alone and in combination against three strains of C. albicans C. albicans
MIC
strain
5-FC
FLCZ
1001 9760-H85 3532-K31
1-56 1.56 1.56
50 12.5 12.5
(#g ml- t) 5-FC
& FLCZ
0-049 - 1.56 0-098 - 3.12 0.049 - 1.56
5-FC = flucytosine; F L C Z = fluconazole. MICs were determined by liquid microdilution m e t h o d using yeast nitrogen base (1% glucose) medium. MICs for the 5 - F C - F L C Z combination represent the concentrations of each agent which gave the lowest FIC index value.
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T A B L E 2. FIC indices for flucytosine and fluconazole against three strains of C. albicans FIC index
C. albicans strain
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1001 9760-H85 3532-K31
A
B
C
0.06 0.31 0.16
0.08 0.50 0.26
0.51 0.56 0.50
FIC index values calculated for three different drug concentration combinations with each strain as follows: (concentrations expressed as/lg ml-~; 5-FC = flucytosine, F L C Z = fluconazole). Strain 1001: A = 5-FC (0.049) & F L C Z (1-56); B = 5-FC (0.024) & F L C Z (3-12); C = 5-FC (0-012 & F L C Z (25). Strain 9760-H85: A = 5-FC (0.098) & F L C Z (3.12); B = 5-FC (0-006) & F L C Z (6-25): C = 5-FC (0.78) & F L C Z (0-78). Strain 3532-K31: A = 5-FC (0.049) & F L C Z (1.56); B = 5-FC (0.012) & F L C Z (3.12): C = 5-FC (0-006) & F L C Z (6.25).
Comparative evaluations were then performed using two-fold serial dilutions of flucytosine (0.78-0.024/.tg m1-1) and fluconazole (25-0.39/,tg ml -t) in combination at ratios of 1:16, 1:32 and 1:64 against yeast cells from the three strains of C. albicans. The agehts were also tested alone at the above concentrations. The results of these studies are shown in Tables 3, 4 & 5. Flucytosine alone induced PAFEs of 6.8-24.8 h (strain 1001; Table 3), 3.8-14-5 h (strain 9760-H85; Table 4) and 1.5-6 h (strain 3532-K31; Table 5). On
T A B L E 3. P A F E following exposure of C. albicans strain 1001 to various concentrations of flucytosine and fluconazole
Ratio
Conc. (//g m1-1)
5-FC: F L C Z
5-FC
1:64
0.39 0-195 0.098 0.049 0.024
1:32
1:16
FLCZ
P A F E (h) 5-FC
FLCZ
5-FC & F L C Z
25 12-5 6.25 3.13 1.56
21-3 15-3 11.5 9-5 6-8
2.3 1.8 1.3 1 0.8
21 15-8 14 9.8 7
0.78 0.39 0.195 0.098 0.049 0-024
25 12.5 6.25 3.13 1-56 0-78
24-8 21.3 15-3 11-5 9-5 6-8
2-3 1.8 1.3 1 0.8 0.5
25.5 20.8 17.5 13 11.5 8
0.78 0.39 0.195 0.098 0.049 0-024
12.5 6.25 3.13 1.56 0-78 0-39
24-8 21-3 15-3 11-5 9-5 6-8
1.8 1.3 1 0.8 0.5 0-1
25.3 23-8 16 14 11-5 7
5-FC = flucytosine; F L C Z = fluconazole.
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TABLE 4. PAFE following exposure of C. albicans strain 9760-H85 to various concentrations of flucytosine and fluconazole
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Ratio 5-FC: FLCZ
Conc. (,ug ml 1) 5-FC
FLCZ
PAFE (h) 5-FC
FLCZ
5-FC & FLCZ
1:64
0.39 0.195 0.098 0.049 0.024
25 12-5 6-25 3-13 1-56
11.3 9-3 7-3 7 3.8
2 1.5 1 0-5 0-5
12.3 12 6.8 7.3 4
1:32
0.78 0-39 0.195 0.098 0.049 0.024
25 12-5 6.25 3.13 1.56 0.78
14.5 11-3 9.3 7.3 7 3-8
2 1-5 1 0-5 0-5 0.1
11.5 10-5 8.3 7-3 9 5-5
1:16
0.78 0.39 0.195 0.098 0.049 0.024
12-5 6.25 3.13 1.56 0.78 0.39
14-5 11-3 9-3 7-3 7 3-8
1-5 1 0-5 0.5 0-1 0
31.5 12-3 9.5 9.8 9 3.8
5-FC = flucytosine; FLCZ = fluconazole.
TABLE 5. PAFE following exposure of C. albicans strain 3532-K31 to various concentrations of flucytosine and fluconazole Ratio 5-FC: FLCZ
Conc. (pg ml 1) 5-FC
FLCZ
PAFE (h) 5-FC
FLCZ
5-FC & FLCZ
1:64
0.39 0.195 0.098 0.049 0.024
25 12.5 6.25 3.13 1.56
5.8 5 3.8 3 1.5
1.5 1.5 1 0.5 0.5
5.8 7.3 6 5 1.5
1:32
0-78 0.39 0.195 0.098 0-049 0-024
25 12.5 6.25 3.13 1.56 0-78
6 5-8 5 3.8 3 1-5
1.5 1-5 1 0.5 0.5 0
8.5 4.5 4 4 3.5 4
1:16
0-78 0.39 0.195 0.098 0-049 0.024
12.5 6.25 3.13 1.56 0-78 0.39
6 5.8 5 3.8 3-0 1.5
1.5 1 0-5 0.5 0 0
4.3 6.5 2.5 4.5 4-5 1.8
5-FC = flucytosine; FLCZ = fluconazole.
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TABLE 6. Lowest concentrations of flucytosine and fluconazole that produced the optimal synergistic PAFE C albicans strain
PAFE (h) 5-FC & FLCZ
Conc. 5-FC
(pg ml- l) FLCZ
Ratio 5-FC:FLCZ
1001 9760-H85 3532-K31
14 9.8 4
0-098 0.098 0.024
1-56 1.56 0-78
1:16 1:16 1:32
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5-FC = flucytosine; FLCZ = fluconazole. These concentration combinations produced PAFEs that persisted for 2.5 h longer than those produced by either agent separately.
the other hand, considerably shorter PAFEs were induced with fluconazole with values ranging from 0.5-2.3 h, 0-2 h and 0-1-5 h with the above three strains, respectively. When the two drugs were used in combination, synergism was noted with several of the combinations with PAFEs ranging from 0.2-2-5 h longer than those achieved with either drug alone (Tables 3, 4 & 5). The lowest concentration combinations that produced optimal synergistic effects are summarized in Table 6. Flucytosine:fluconazole concentration ratios of 1:16 (0-098 and 1-56 pg ml-1; strain 1001 and strain 9760 H85) and 1:32 (0.024 and 0.78 pg ml-1; strain 3532-K31) produced PAFEs of 14, 9.8 and 4 h, respectively. The PAFEs persisted for 2.5 h longer than those induced by either agent alone. DISCUSSION An increasing amount of interest has been generated recently with regard to combination therapy for the systemic mycoses. Various studies have been concerned with attempts to improve the efficacy and broaden the spectrum of the antifungal agents, particularly in situations where opportunistic organisms produce infections in immunocompromised patients. Toxicity problems associated with some of the available agents, the necessity for high dose therapy with others and the proven inefficiency of some of the newer antifungals, have been of major concern to clinicians in recent years. Thus many in vitro and in vivo investigations are being performed in an effort to improve the situation. In relation to this, numerous in vitro laboratory procedures have been used to determine susceptibility profiles of fungi and to determine dosing schedules of certain drugs, but they are vulnerable to the numerous in vitro test variables. Such procedures have suffered from a lack of a universally accepted standardized testing methodology and in many instances there has been a lack of correlation of in vitro results with the outcome of in vivo therapy [5]. MIC determinations have been performed by many laboratories, but one must recognize that the data from such procedures can vary appreciably depending upon testing parameters including media composition, exposure time, incubation temperature and type of assay [11, 18, 23]. Thus a great deal of effort will be required before antifungal susceptibility testing reaches the level of reliability that is associated with antibacterial susceptibility procedures. Another factor to consider is that in vivo the organisms are exposed to fluctuating levels of a drug instead of constant levels as is the case with in vitro MIC assays. With this in mind we began investigations on a phenomenon we have termed the PAFE in an effort to determine if exposure of an organism to a drug for a short period of time might
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produce persistent suppression of growth. It seems reasonable to assume that such a short period of exposure rather than continual exposure, as with standard susceptibility assays, might approximate the in vivo situation. Numerous PAE studies have been done with bacteria [6, 13, 17, 30, 31] and such studies have indicated that antibiotics exert suppressive effects on bacterial growth after a short period of exposure. Such an assay thus provides information on the drug-organism interaction that standard susceptibility tests do not provide. In previous studies we used both a viable count method (unpublished data) and a turbidometric technique [26] to measure C. albicans cell regrowth following exposure to flucytosine, amphotericin B and miconazole for short periods of time (0.5-2 h). Profound suppressive effects were evidenced and generally the length of the PAFE was dependent upon the drug concentration and the period of exposure. In the present study we used the turbidometric method to investigate the synergistic interaction of flucytosine and fluconazole on three strains of C. albicans. The aim of the study was to determine if a PAFE could be induced by combinations of the drugs each at concentrations considerably lower than their individual MIC values. We were indeed able to demonstrate a synergistic effect with the two drugs at concentrations well below their individual MICs. Of interest is the fact that flucytosine induced PAFEs, both alone and to a greater extent in combination with fluconazole, at concentrations (0-024-0.78 pg m1-1) considerably lower than optimal peak serum levels (50-100 ~tg ml a) as reported previously [8, 27]. A major concern has been flucytosine toxicity associated with peak serum levels of 100/.tg ml- t or greater [27]. PAFEs were also induced with concentrations of fluconazole (0-78-1-56 pg ml-t), in combination with flucytosine, that were also less than optimal serum levels of 2/lg ml i [12]- 45/lg m1-1 [16] reported in recent studies. PAFE values varied according to the strain of C. albicans being assayed. These results are encouraging, but one must bear in mind that the PAFE studies have been performed in vitro and further studies must be performed to assess the utility of this assay with regard to providing clinicians with information necessary to determine optimal dosing regimens. Theoretically the present or absence of a PAFE should provide a rationale for less frequent or more frequent administration of the antifungal agent. ACKNOWLEDGEMENTS Financial support was provided by the Japanese Ministry of Education for Dr Gene M. Scalarone as a guest professor (foreign researcher) at Chiba University, Research Center for Pathogenic Fungi and Microbial Toxieoses. The authors are grateful for the excellent technical assistance provided by Yuri Ichihara and Shinji Ohashi.
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