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A comparison of dynamic characteristics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans using time-kill methodology
Diagnostic Microbiology and Infectious Disease 38 (2000) 87–93
www.elsevier.com/locate/diagmicrobio
Mycology
A comparison of dynamic characteristics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans using time-kill methodology David S. Burgess*, Rhonda W. Hastings College of Pharmacy, The University of Texas at Austin and Department of Pharmacology, The University of Texas Health Science Center at San Antonio Received 5 June 2000; accepted 27 June 2000
Abstract This study evaluated the in vitro pharmacodynamics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans. MICs were determined for three clinical isolates according to NCCLS guidelines (M27). Time-kill studies were performed using antifungal concentrations of 0.25-32xMIC and inocula of 103 and 105 CFU/ml. At predetermined time points over 72 hours, samples of each inoculum/drug combination were withdrawn and plated using a spiral plater. Colony counts were determined after incubation at 35°C for 48 hours. Area under the kill curves (AUKCs) were plotted versus the AUC/MIC ratios. Inoculum effect was evaluated by calculating an estimated AUKC for the low inoculum then comparing it to the measured low inoculum using the unpaired Student’s t-test. The MICs of fluconazole and itraconazole for isolate 97-1199, 97-1061, and 97-585 were 2, 4, 32g/ml and 0.03, 0.06, 0.5g/ml, respectively. For amphotericin B, the MIC was 0.25g/ml for each isolate. The triazoles demonstrated fungistatic activity against each isolate at both inocula with the exception of itraconazole against C. neoformans 97-585. Maximal suppression was noted at concentrations 8-16xMIC correlating with an AUC/MIC of 192 for both inocula. Conversely, amphotericin B was fungicidal and displayed concentration-dependent activity against each isolate at both inocula. Maximal killing was observed at concentrations ⬎4xMIC for the low inoculum and ⬎8xMIC for the high inoculum for each isolate. No statistically significant differences were detected between the measured and estimated AUKCs for each antifungal agent. In conclusion, our results suggest that the triazoles were most effective against C. neoformans when concentrations were maintained at 8-16xMIC. Amphotericin B, on the other hand, was concentration-dependent; thus, greater activity was exerted at higher concentrations. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Cryptococcus neoformans is an opportunistic fungus that often manifest itself clinically as meningitis in severely immunocompromised hosts (i.e., CD4 ⬍ 100 cells/mm3). Cryptococcal meningitis is one of the most common CNS infections in AIDS patients and has been associated with a mortality rate ranging from 10 to 30% (Pinner et al., 1995; Powderly 1993, 1996). The results of a recently published clinical trial conducted by van der Horst et al suggest that
* Corresponding author. Tel.: ⫹1-210-567-8329; fax: ⫹1-210-5678328. E-mail address: [email protected] (D.S. Burgess). Results of this study have been presented at Focus on Fungal Infections 8 in Orlando, Florida, March 5, 1998, and The American College of Clinical Pharmacy Clinical Practice and Research Forum in Palm Springs, California, April 7, 1998.
the optimal treatment regimen for AIDS patients with cryptococcus meningitis includes amphotericin B 0.7 mg/kg plus flucytosine 100 mg/kg/day for two weeks followed by fluconazole 400 mg daily for eight weeks (Van der Horst et al., 1997). In addition, maintenance therapy must be provided indefinitely because without such treatment, the probability of relapse approaches 50 to 60% (Dromer et al., 1996; Hood et al., 1996; Powderly et al., 1992; White et al., 1994). The toxicities of amphotericin B along with the inconvenience of parenteral administration limit its usefulness, particularly in chronic management of fungal infections (Powderly 1993; Dromer et al., 1996). Fluconazole and itraconazole are two systemic antifungal alternatives that can be administered orally and have improved side effect profiles in comparison to amphotericin B. However, the emergence of antifungal resistance is a potential concern associated with prolonged use of the triazoles. Although
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resistance has not been reported with Cryptococcus per se, it has been demonstrated with fluconazole and Candida albicans (Pinner et al., 1995; Rex et al., 1995). Until 1992, standardized procedures for antifungal susceptibility testing of yeast were not available. With the development of the NCCLS guidelines (M27), it is possible to compare activities of antifungals and correlate relationships between in vitro and in vivo activity. Furthermore, resistance can be monitored and pharmacodynamic effects of antifungals can be studied. The purpose of this study was to compare the dynamic characteristics of fluconazole, itraconazole, and amphotericin B against three clinical isolates of C. neoformans at two inocula and to determine if there was an inoculum effect for the triazoles.
2.4. Carryover effect A clinical isolate was used to determine if amphotericin B exhibited a carryover effect when using a spiral plater. Sixteen flasks containing 25 ml of Antibiotic Medium 3 were inoculated with suspensions of C. neoformans to yield an inoculum of approximately 103 and 105 CFU/ml (8 flasks per inoculum). Triplicate samples were withdrawn from each flask and plated onto Sabouraud dextrose agar using a spiral plater. Samples of 105 CFU/ml were diluted 1:100 with sterile water prior to plating. Next, amphotericin B was added to the flasks to achieve concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, and 32xMIC for both inocula. Immediately following, samples were withdrawn in triplicate and plated. All plates were incubated for 48 hours at 35°C, and colony counts were determined. 2.5. Time-kill studies
2. Methods 2.1. Antifungal agents Fluconazole (Pfizer Inc., New York, N.Y.), itraconazole (Janssen Pharmaceutica, Titusville, N.J.), and amphotericin B (Bristol-Myers Squibb, Princeton, N.J.) were provided by the manufacturers as standard powders. Stock solutions were prepared and stored at ⫺20°C. Fluconazole was dissolved in sterile water, itraconazole in polyethylene glycol 400 (Sigma Chemical Co., St. Louis, MO.), and amphotericin B in dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO.). 2.2. Yeast isolates Three clinical isolates of C. neoformans were selected for testing. The isolates were obtained from the Fungus Testing Laboratory, Department of Pathology, The University of Texas Health Science Center at San Antonio. 2.3. Susceptibility testing The MICs of fluconazole, itraconazole, and amphotericin B were determined in triplicate for each isolate using the broth macrodilution technique according to the NCCLS guidelines (M27) (NCCLS 1997). The media used for the triazoles was RPMI 1640 (American Biorganics, Inc., Niagara Falls, N.Y.) adjusted to a pH of 7.0 with 0.165M morpholinepropanesulfonic acid (MOPS). For amphotericin B, testing was accomplished in Antibiotic Medium 3. For the triazoles, the MIC was considered the lowest drug concentration that produced 80% inhibition of growth compared to the growth control. For amphotericin B, the MIC was defined as the lowest drug concentration that prevented any discernible growth.
All isolates were stored on Sabouraud dextrose agar at room temperature. Prior to testing, isolates were subcultured at least twice and grown for 48 hours at 35°C. Three to five 1 mm-diameter colonies from the 48-hour cultures were suspended in 5 mL of sterile water, vortexed for 15 seconds, and adjusted spectrophotometrically to match 0.5 and 1 McFarland standards. Appropriate volumes of the suspensions were diluted with 25 ml of RPMI 1640 or Antibiotic Medium 3 to obtain an inoculum of approximately 103 and 105 CFU/ml (8 flasks per inoculum for each antifungal). Fluconazole, itraconazole, and amphotericin B were added to the test solutions to achieve concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, and 32xMIC for each isolate. Test solutions and growth controls were then placed into a shaker and incubated at 35°C. Samples (500 l) were withdrawn at 0, 6, 12, 24, 36, 48, and 72 hours for the triazoles and 0, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hours for amphotericin B, serially diluted (when necessary), and plated onto Sabouraud dextrose agar using a spiral plater. The spiral plater dispenses 50 l in a logarithmically decreasing fashion from the center to the each of the plate. Colonies are enumerated from the outer edge, which has been shown to be unaffected by residual antibiotic (Yorassowsky et al., 1988). The lower limit of sensitivity for this method is 100 CFU/ ml. The plates were incubated at 35°C for 48 –72 hours, and colony counts were determined. All kill curves were performed in duplicate.
3. Data analysis 3.1. Carryover effect To determine a carryover effect, mean colony counts for each amphotericin B concentration were compared to mean colony counts of corresponding growth controls. Differences ⱖ25% were considered significant. The unpaired Stu-
D.S. Burgess, R.W. Hastings / Diagnostic Microbiology and Infectious Disease 38 (2000) 87–93
dent’s t-test was used to detect statistically significant differences between the amphotericin B and growth control samples.
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4.4. Fluconazole
Graphs of colony counts (log10 CFU/ml) versus time were constructed for each isolate and both inocula for all three antifungal agents. Area under the kill curves (AUKCs) was calculated using the trapezoidal rule, and graphs of 24 hour cumulative AUKCs (AUKC0 –24h) versus AUC/MIC ratios were graphed for each azole antifungal and isolate combination. The maximal activity was determined by visual examination of the graph.
Fluconazole concentrations ⬎4 x MIC exhibited fungistatic (⬍99.9% killing) activity against all three isolates of C. neoformans (Fig. 1). For both inocula, maximum suppression (i.e., lowest AUKC) occurred at concentrations 8-16xMIC for each isolate. (Data not shown.) Fluconazole exhibited ⬎90% killing at concentrations ⬎8xMIC against the most susceptible isolate 97-1199 (MIC ⫽ 2g/ml); however, for isolate 97-1061 (MIC ⫽ 4g/ml) and 97-585 (MIC ⫽ 32g/ml), fluconazole only provided suppression at concentrations ⱖ8xMIC. The AUC/MIC ranged from 6 – 678 with maximal activity being observed at 192 based on the AUKC0 –24h (Fig. 4).
3.3. Inoculum effect
4.5. Itraconazole
Low and high inoculum colony counts were further evaluated to determine a net inoculum effect as previously described (Firsov AA et al., 1997). Initial low inoculum colony counts (i.e., colony counts at time zero) were subtracted from initial high inoculum colony counts for every antifungal concentration for each isolate. Next, the calculated differences were subtracted from the respective high inoculum colony counts recorded at each timepoint. These newly calculated colony counts represented the estimated low inoculum colony counts. Estimated AUKCs were calculated, and statistical analyses were performed using the unpaired Student’s t-test comparing AUKC0 –24h for the measured and estimated low inoculum samples. A p-value ⱕ 0.05 was considered significant for all statistical analyses.
Itraconazole displayed fungistatic activity at concentrations ⬎2xMIC for each of the isolates except C. neoformans 97–585 (Fig. 2). Like fluconazole, itraconazole exhibited killing against the most susceptible isolate 97-1199 (MIC ⫽ 0.03g/ml). For isolate 97-1061, concentrations/MIC ⱖ4 prevented growth at both inocula and concentrations less than the MIC provided minimal activity; however, for the least susceptible isolate (97–585) itraconazole provided no activity at any drug concentration. The lowest AUKC (maximal effect) for both inocula of each isolate was noted at concentrations 4-8xMIC. Like fluconazole, maximal suppression occurred at an AUC/MIC of 192 (Fig. 4).
3.2. Time-kill studies
4. Results 4.1. Susceptibility testing The MICs of fluconazole and itraconazole for isolate 97-1199, 97-1061, and 97-585 were 2, 4, 32g/ml and 0.03, 0.06, 0.5g/ml, respectively. For amphotericin B, the MIC was determined to be 0.25g/ml for each isolate. 4.2. Carryover effect All amphotericin B samples had a difference of less than 25% for the mean colony counts. Overall, the median percent difference between the controls and amphotericin B samples was 10.5%. No statistically significant differences were detected between the colony counts with or without amphotericin B.
4.6. Amphotericin B Amphotericin B demonstrated concentration-dependent killing. For all 3 isolates at both inocula, concentrations at or below the MIC were fungistatic while concentrations ⱖ2xMIC were fungicidal (Fig. 3). The maximal effect for amphotericin B occurred at 4-8xMIC for each isolate. Regrowth was noted within 72 hours for concentrations ⱕ4xMIC. 4.7. Inoculum effect Plots of the measured and estimated low inoculum colony counts versus time showed similar patterns of killing or stasis for each concentration/MIC for all isolates. In addition, scatterplots of the measured versus estimated AUKC0 –24h for the low inoculum data revealed a strong linear relationship for each antifungal agent (r ⱖ 0.86). No statistically significant differences were detected between the measured and estimated AUKC0 –24h for each antifungal agent.
4.3. Time-kill studies 5. Discussion The time-kill curves for fluconazole, itraconazole, and amphotericin B versus C. neoformans are displayed in Figs. 1–3. (Data not shown for the low inocula.)
Applying pharmacodynamic principles of specific antimicrobial agents has improved the outcomes of infected
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Fig. 1. Time Kill Curves for Fluconazole versus C. neoformans. a. C. neoformans 97-1199, High inoculum. b. C. neoformans 97-1061, High inoculum. c. C. neoformans 97-585, High inoculum. ⫽ Growth Control; ....{.... ⫽ 0.25xMIC; ----E---- ⫽ 0.5xMIC; ----‚---- ⫽ 1xMIC; - - -µ- - - ⫽ 2xMIC; -.-. -.-. ⫽ 4xMIC; ---Q--- ⫽ 8xMIC; ---ƒ--- ⫽ 16xMIC; -.-m-.- ⫽ 32xMIC.
Fig. 2. Time Kill Curves for Itraconazole versus C. neoformans. a. C. neoformans 97-1199, High inoculum. b. C. neoformans 97-1061, High inoculum. c. C. neoformans 97-585, High inoculum. ⫽ Growth Control; ....{.... ⫽ 0.25xMIC; ----E---- ⫽ 0.5xMIC; ----‚---- ⫽ 1xMIC; - - -µ- - - ⫽ 2xMIC; -.-. -.-. ⫽ 4xMIC; ---Q--- ⫽ 8xMIC; ---ƒ--- ⫽ 16xMIC; -.-m-.- ⫽ 32xMIC.
D.S. Burgess, R.W. Hastings / Diagnostic Microbiology and Infectious Disease 38 (2000) 87–93
Fig. 3. Time Kill Curves for Amphotericin B versus C. neoformans 97-585, High inoculum. ⫽ Growth Control; ....{.... ⫽ 0.25xMIC; ----E---⫽ 0.5xMIC; ----‚---- ⫽ 1xMIC; - - -µ- - - ⫽ 2xMIC; -.-. -.-. ⫽ 4xMIC; ---Q--- ⫽ 8xMIC; ---ƒ--- ⫽ 16xMIC; -.-m-.- ⫽ 32xMIC.
patients (Drusano 1988; Forrest et al., 1993; Moore et al., 1987; Schentag et al., 1984). Based on pharmacodynamics, some antibacterial agents are classified as either concentration-dependent (e.g., aminoglycosides and fluoroquinolones) or time-dependent (e.g., beta-lactams). For concentration-dependent agents, higher drug concentrations correlate with faster rates of pathogen eradication. Therefore, the most important pharmacodynamic parameter for these agents is the peak serum concentration/MIC ratio. Clinical studies have demonstrated that for the aminoglycosides concentrations 8-12xMIC are needed for maximal effectiveness (Moore et al., 1987). Hence, the administration of one large
Fig. 4. AUC/MIC Ratio vs Area Under the Kill Curve for the High Inoculum with the Triazoles. 䊐 ⫽ Fluconazole; { ⫽ Itraconazole.
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daily dose of an aminoglycoside is preferable to smaller doses given multiple times a day. Alternatively, time-dependent agents are most effective when the duration of the serum concentration remains above the MIC at the site of infection for a significant proportion of the dosing interval. Accordingly, these agents are best administered using frequent dosing intervals. For antifungal agents, the most important pharmacodynamic parameters are unknown for any class of agents. Recently, Louie et al., published a murine model evaluating the pharmacodynamics of fluconazole in the treatment of systemic candidiasis. They studied 3 pharmacodynamic parameters (Cmax/MIC, time above the MIC, and AUC/ MIC). Through dose-fractionation studies, they showed that maximizing Cmax/MIC or time above the MIC did not affect the outcomes as determined by fungal densities in renal tissue. The AUC/MIC ratio remained the same regardless of the dosage regimen utilized; therefore, they concluded that the AUC/MIC ratio was the best predictor of outcome (Louie et al., 1998). Although our study was not designed to evaluate the AUC/MIC ratio, we did examine the relationship between AUC/MIC and AUKC0 –24h for C. neoformans. Our results suggest that the maximal effective AUC/ MIC ratio was 192 (correlating with a concentration of 8xMIC) for both fluconazole and itraconazole for each isolate except for isolate 97-585 and itraconazole. To our knowledge, this is the first in vitro study comparing the activity of fluconazole, itraconazole, and amphotericin B against C. neoformans. Isolates with a variety of MICs were selected in order to examine a wide range of susceptibility patterns for the triazoles. Our results suggest that fluconazole and itraconazole are time-dependent agents while amphotericin B is concentration-dependent. We have also noted similar findings for these antifungal agents against C. albicans (Burgess et al., 1997). Klepser et al have reported similar findings for fluconazole and amphotericin B against C. albicans and C. neoformans although differences in time-kill methodology in terms of media, incubation time, and inocula were utilized (Klepser et al., 1997). We used Antibiotic Medium 3 for amphotericin B instead of RMPI since Rex et al demonstrated a more pronounced difference in MICs for amphotericin B when using Antibiotic Medium 3 broth (Rex et al., 1995). In addition, we evaluated the antifungal activity over 72 hours against each Cryptococcal isolate in order to coincide with the NCCLS guidelines for in vitro susceptibility testing of C. neoformans. Finally, to determine whether an inoculum effect existed, we utilized both a high (105 CFU/ml) and low (103 CFU/ml) inoculum. In contrast to Klepser et al, a carryover effect was not demonstrated with amphotericin B in our study. Perhaps this is due to the differences in methodology. Klepser et al reported fungistatic activity for amphotericin B at concentrations ⬍0.5xMIC and fungicidal activity at concentrations ⬎0.5xMIC (Klepser et al., 1997). Our results suggest a similar pattern of activity in that concentrations ⱕ1xMIC were fungistatic while concentrations ⬎1xMIC
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were fungicidal. Interestingly, this pattern of activity was not maintained beyond 24 hours. In fact, regrowth was noted following 24, 36, and 48 hours for concentrations 0.25– 0.5, 1, and 2-4xMIC, respectively. This regrowth could be due to a number of facts including drug degradation or development of resistance. Since we did not retest these organisms to determine the MIC after exposure to amphotericin B, we are not sure if resistance occurred. However, the likelihood of drug degradation is low since the only difference between time-kill studies and in vitro susceptibility testing is the determination of colony count and the NCCLS recommends performing susceptibility testing over 72 hrs for C. neoformans. Finally, regrowth was not observed during the 72 hour time interval for amphotericin B concentrations ⬎4xMIC. Although this information has been determined in vitro, it is noteworthy that higher mortality and slower rates of cerebrospinal sterilization have been reported in clinical trials utilizing lower doses of amphotericin B (i.e., 0.3– 0.5 mg/kg) for the initial treatment of cryptococcus meningitis (Saag et al., 1992). More recent clinical trials have concluded that better outcomes (less mortality and faster CSF sterilization) are associated with higher doses of amphotericin B (i.e., 0.7–1 mg/kg) alone or in combination with flucytosine (Van der Horst et al., 1997; De Lalla et al., 1995). In a multivariate analysis, statistically significant lower rates of relapse have been reported with the addition of flucytosine to higher doses of amphotericin B therapy, regardless of the triazole prescribed for suppressive treatment (Saag et al., 1995). The in vitro data that we have reported illustrate that the duration of suppression is proportional to the concentration of amphotericin B; therefore, it is plausible that by combining flucytosine with amphotericin B the magnitude of response is enhanced thus extending the duration of suppression. The NCCLS has recently suggested tentative breakpoints for fluconazole and itraconazole against candida species. Isolates with a MIC ⱕ 8g/ml and 0.125g/ml are classified as susceptible to fluconazole and itraconazole, respectively. While those isolates with a MIC ⱖ 64g/ml and 1g/ml are considered resistant to fluconazole and itraconazole. A new category called susceptible-dose dependent (S-DD) was created for isolates responsive to higher doses or serum levels. For fluconazole, the S-DD category includes MICs of 16 –32g/ml whereas for itraconazole the S-DD category includes MICs of 0.25– 0.5g/ml (NCCLS 1997; Rex et al., 1997). It is important to remember that these breakpoints are proposed for candida only and are not intended for use with C. neoformans. In fact, our data would suggest that for C. neoformans a MIC of 0.5g/ml for itraconazole would be considered resistant since no activity was demonstrated against isolate 97-585 irrespective of the concentration of itraconazole. Interestingly, this same isolate (97-585) with a MIC ⫽ 32g/ml for fluconazole was susceptible to fluconazole depending on the drug concentration (i.e., concentrations ⱖ8xMIC were suppressive while concentrations ⬍8xMIC displayed minimal activity).
This data may in fact suggest that itraconazole and fluconazole should not be used interchangeably when treating infections due to C. neoformans. We have reported similar patterns of activity for itraconazole and fluconazole against C. albicans classified as S-DD (MIC ⫽ 0.5 and 32g/ml; itraconazole and fluconazole, respectively) (Burgess et al., 2000). In conclusion, the results of the fungal time-kill studies suggest that the triazoles may be most effective against C. neoformans when maintained at concentrations 8-16xMIC at the site of infection, while for amphotericin B effectiveness may increase with greater drug concentrations (i.e., maximizing peak concentration/MIC ratios). Clinical studies have compared lower doses of amphotericin B to fluconazole (400 mg daily) for the initial treatment of AIDSassociated cryptococcal meningitis (Saag et al., 1992) as well as higher doses of amphotericin B (administered weekly) to fluconazole (200 mg daily) for maintenance therapy (Powderly et al., 1992). What clinical studies have not yet evaluated are dosage regimens for initial and maintenance therapies, which apply these pharmacodynamic relationships, determined in vitro. Such studies are necessary to correlate in vitro and in vivo relationships.
Acknowledgment This work was support by the 1997–98 Merck-AFPE “Gateway” Research Scholarship and 1997 Summer Research Assignment from The University of Texas at Austin. We would like to thank The Fungus Testing Laboratory, Department of Pathology, The University of Texas Health Science Center at San Antonio for the clinical isolates of Cryptococcus neoformans and Dr. Thomas C. Hardin for his critical review of the manuscript.
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Mycology
A comparison of dynamic characteristics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans using time-kill methodology David S. Burgess*, Rhonda W. Hastings College of Pharmacy, The University of Texas at Austin and Department of Pharmacology, The University of Texas Health Science Center at San Antonio Received 5 June 2000; accepted 27 June 2000
Abstract This study evaluated the in vitro pharmacodynamics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans. MICs were determined for three clinical isolates according to NCCLS guidelines (M27). Time-kill studies were performed using antifungal concentrations of 0.25-32xMIC and inocula of 103 and 105 CFU/ml. At predetermined time points over 72 hours, samples of each inoculum/drug combination were withdrawn and plated using a spiral plater. Colony counts were determined after incubation at 35°C for 48 hours. Area under the kill curves (AUKCs) were plotted versus the AUC/MIC ratios. Inoculum effect was evaluated by calculating an estimated AUKC for the low inoculum then comparing it to the measured low inoculum using the unpaired Student’s t-test. The MICs of fluconazole and itraconazole for isolate 97-1199, 97-1061, and 97-585 were 2, 4, 32g/ml and 0.03, 0.06, 0.5g/ml, respectively. For amphotericin B, the MIC was 0.25g/ml for each isolate. The triazoles demonstrated fungistatic activity against each isolate at both inocula with the exception of itraconazole against C. neoformans 97-585. Maximal suppression was noted at concentrations 8-16xMIC correlating with an AUC/MIC of 192 for both inocula. Conversely, amphotericin B was fungicidal and displayed concentration-dependent activity against each isolate at both inocula. Maximal killing was observed at concentrations ⬎4xMIC for the low inoculum and ⬎8xMIC for the high inoculum for each isolate. No statistically significant differences were detected between the measured and estimated AUKCs for each antifungal agent. In conclusion, our results suggest that the triazoles were most effective against C. neoformans when concentrations were maintained at 8-16xMIC. Amphotericin B, on the other hand, was concentration-dependent; thus, greater activity was exerted at higher concentrations. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction Cryptococcus neoformans is an opportunistic fungus that often manifest itself clinically as meningitis in severely immunocompromised hosts (i.e., CD4 ⬍ 100 cells/mm3). Cryptococcal meningitis is one of the most common CNS infections in AIDS patients and has been associated with a mortality rate ranging from 10 to 30% (Pinner et al., 1995; Powderly 1993, 1996). The results of a recently published clinical trial conducted by van der Horst et al suggest that
* Corresponding author. Tel.: ⫹1-210-567-8329; fax: ⫹1-210-5678328. E-mail address: [email protected] (D.S. Burgess). Results of this study have been presented at Focus on Fungal Infections 8 in Orlando, Florida, March 5, 1998, and The American College of Clinical Pharmacy Clinical Practice and Research Forum in Palm Springs, California, April 7, 1998.
the optimal treatment regimen for AIDS patients with cryptococcus meningitis includes amphotericin B 0.7 mg/kg plus flucytosine 100 mg/kg/day for two weeks followed by fluconazole 400 mg daily for eight weeks (Van der Horst et al., 1997). In addition, maintenance therapy must be provided indefinitely because without such treatment, the probability of relapse approaches 50 to 60% (Dromer et al., 1996; Hood et al., 1996; Powderly et al., 1992; White et al., 1994). The toxicities of amphotericin B along with the inconvenience of parenteral administration limit its usefulness, particularly in chronic management of fungal infections (Powderly 1993; Dromer et al., 1996). Fluconazole and itraconazole are two systemic antifungal alternatives that can be administered orally and have improved side effect profiles in comparison to amphotericin B. However, the emergence of antifungal resistance is a potential concern associated with prolonged use of the triazoles. Although
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resistance has not been reported with Cryptococcus per se, it has been demonstrated with fluconazole and Candida albicans (Pinner et al., 1995; Rex et al., 1995). Until 1992, standardized procedures for antifungal susceptibility testing of yeast were not available. With the development of the NCCLS guidelines (M27), it is possible to compare activities of antifungals and correlate relationships between in vitro and in vivo activity. Furthermore, resistance can be monitored and pharmacodynamic effects of antifungals can be studied. The purpose of this study was to compare the dynamic characteristics of fluconazole, itraconazole, and amphotericin B against three clinical isolates of C. neoformans at two inocula and to determine if there was an inoculum effect for the triazoles.
2.4. Carryover effect A clinical isolate was used to determine if amphotericin B exhibited a carryover effect when using a spiral plater. Sixteen flasks containing 25 ml of Antibiotic Medium 3 were inoculated with suspensions of C. neoformans to yield an inoculum of approximately 103 and 105 CFU/ml (8 flasks per inoculum). Triplicate samples were withdrawn from each flask and plated onto Sabouraud dextrose agar using a spiral plater. Samples of 105 CFU/ml were diluted 1:100 with sterile water prior to plating. Next, amphotericin B was added to the flasks to achieve concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, and 32xMIC for both inocula. Immediately following, samples were withdrawn in triplicate and plated. All plates were incubated for 48 hours at 35°C, and colony counts were determined. 2.5. Time-kill studies
2. Methods 2.1. Antifungal agents Fluconazole (Pfizer Inc., New York, N.Y.), itraconazole (Janssen Pharmaceutica, Titusville, N.J.), and amphotericin B (Bristol-Myers Squibb, Princeton, N.J.) were provided by the manufacturers as standard powders. Stock solutions were prepared and stored at ⫺20°C. Fluconazole was dissolved in sterile water, itraconazole in polyethylene glycol 400 (Sigma Chemical Co., St. Louis, MO.), and amphotericin B in dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO.). 2.2. Yeast isolates Three clinical isolates of C. neoformans were selected for testing. The isolates were obtained from the Fungus Testing Laboratory, Department of Pathology, The University of Texas Health Science Center at San Antonio. 2.3. Susceptibility testing The MICs of fluconazole, itraconazole, and amphotericin B were determined in triplicate for each isolate using the broth macrodilution technique according to the NCCLS guidelines (M27) (NCCLS 1997). The media used for the triazoles was RPMI 1640 (American Biorganics, Inc., Niagara Falls, N.Y.) adjusted to a pH of 7.0 with 0.165M morpholinepropanesulfonic acid (MOPS). For amphotericin B, testing was accomplished in Antibiotic Medium 3. For the triazoles, the MIC was considered the lowest drug concentration that produced 80% inhibition of growth compared to the growth control. For amphotericin B, the MIC was defined as the lowest drug concentration that prevented any discernible growth.
All isolates were stored on Sabouraud dextrose agar at room temperature. Prior to testing, isolates were subcultured at least twice and grown for 48 hours at 35°C. Three to five 1 mm-diameter colonies from the 48-hour cultures were suspended in 5 mL of sterile water, vortexed for 15 seconds, and adjusted spectrophotometrically to match 0.5 and 1 McFarland standards. Appropriate volumes of the suspensions were diluted with 25 ml of RPMI 1640 or Antibiotic Medium 3 to obtain an inoculum of approximately 103 and 105 CFU/ml (8 flasks per inoculum for each antifungal). Fluconazole, itraconazole, and amphotericin B were added to the test solutions to achieve concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, and 32xMIC for each isolate. Test solutions and growth controls were then placed into a shaker and incubated at 35°C. Samples (500 l) were withdrawn at 0, 6, 12, 24, 36, 48, and 72 hours for the triazoles and 0, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hours for amphotericin B, serially diluted (when necessary), and plated onto Sabouraud dextrose agar using a spiral plater. The spiral plater dispenses 50 l in a logarithmically decreasing fashion from the center to the each of the plate. Colonies are enumerated from the outer edge, which has been shown to be unaffected by residual antibiotic (Yorassowsky et al., 1988). The lower limit of sensitivity for this method is 100 CFU/ ml. The plates were incubated at 35°C for 48 –72 hours, and colony counts were determined. All kill curves were performed in duplicate.
3. Data analysis 3.1. Carryover effect To determine a carryover effect, mean colony counts for each amphotericin B concentration were compared to mean colony counts of corresponding growth controls. Differences ⱖ25% were considered significant. The unpaired Stu-
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dent’s t-test was used to detect statistically significant differences between the amphotericin B and growth control samples.
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4.4. Fluconazole
Graphs of colony counts (log10 CFU/ml) versus time were constructed for each isolate and both inocula for all three antifungal agents. Area under the kill curves (AUKCs) was calculated using the trapezoidal rule, and graphs of 24 hour cumulative AUKCs (AUKC0 –24h) versus AUC/MIC ratios were graphed for each azole antifungal and isolate combination. The maximal activity was determined by visual examination of the graph.
Fluconazole concentrations ⬎4 x MIC exhibited fungistatic (⬍99.9% killing) activity against all three isolates of C. neoformans (Fig. 1). For both inocula, maximum suppression (i.e., lowest AUKC) occurred at concentrations 8-16xMIC for each isolate. (Data not shown.) Fluconazole exhibited ⬎90% killing at concentrations ⬎8xMIC against the most susceptible isolate 97-1199 (MIC ⫽ 2g/ml); however, for isolate 97-1061 (MIC ⫽ 4g/ml) and 97-585 (MIC ⫽ 32g/ml), fluconazole only provided suppression at concentrations ⱖ8xMIC. The AUC/MIC ranged from 6 – 678 with maximal activity being observed at 192 based on the AUKC0 –24h (Fig. 4).
3.3. Inoculum effect
4.5. Itraconazole
Low and high inoculum colony counts were further evaluated to determine a net inoculum effect as previously described (Firsov AA et al., 1997). Initial low inoculum colony counts (i.e., colony counts at time zero) were subtracted from initial high inoculum colony counts for every antifungal concentration for each isolate. Next, the calculated differences were subtracted from the respective high inoculum colony counts recorded at each timepoint. These newly calculated colony counts represented the estimated low inoculum colony counts. Estimated AUKCs were calculated, and statistical analyses were performed using the unpaired Student’s t-test comparing AUKC0 –24h for the measured and estimated low inoculum samples. A p-value ⱕ 0.05 was considered significant for all statistical analyses.
Itraconazole displayed fungistatic activity at concentrations ⬎2xMIC for each of the isolates except C. neoformans 97–585 (Fig. 2). Like fluconazole, itraconazole exhibited killing against the most susceptible isolate 97-1199 (MIC ⫽ 0.03g/ml). For isolate 97-1061, concentrations/MIC ⱖ4 prevented growth at both inocula and concentrations less than the MIC provided minimal activity; however, for the least susceptible isolate (97–585) itraconazole provided no activity at any drug concentration. The lowest AUKC (maximal effect) for both inocula of each isolate was noted at concentrations 4-8xMIC. Like fluconazole, maximal suppression occurred at an AUC/MIC of 192 (Fig. 4).
3.2. Time-kill studies
4. Results 4.1. Susceptibility testing The MICs of fluconazole and itraconazole for isolate 97-1199, 97-1061, and 97-585 were 2, 4, 32g/ml and 0.03, 0.06, 0.5g/ml, respectively. For amphotericin B, the MIC was determined to be 0.25g/ml for each isolate. 4.2. Carryover effect All amphotericin B samples had a difference of less than 25% for the mean colony counts. Overall, the median percent difference between the controls and amphotericin B samples was 10.5%. No statistically significant differences were detected between the colony counts with or without amphotericin B.
4.6. Amphotericin B Amphotericin B demonstrated concentration-dependent killing. For all 3 isolates at both inocula, concentrations at or below the MIC were fungistatic while concentrations ⱖ2xMIC were fungicidal (Fig. 3). The maximal effect for amphotericin B occurred at 4-8xMIC for each isolate. Regrowth was noted within 72 hours for concentrations ⱕ4xMIC. 4.7. Inoculum effect Plots of the measured and estimated low inoculum colony counts versus time showed similar patterns of killing or stasis for each concentration/MIC for all isolates. In addition, scatterplots of the measured versus estimated AUKC0 –24h for the low inoculum data revealed a strong linear relationship for each antifungal agent (r ⱖ 0.86). No statistically significant differences were detected between the measured and estimated AUKC0 –24h for each antifungal agent.
4.3. Time-kill studies 5. Discussion The time-kill curves for fluconazole, itraconazole, and amphotericin B versus C. neoformans are displayed in Figs. 1–3. (Data not shown for the low inocula.)
Applying pharmacodynamic principles of specific antimicrobial agents has improved the outcomes of infected
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Fig. 1. Time Kill Curves for Fluconazole versus C. neoformans. a. C. neoformans 97-1199, High inoculum. b. C. neoformans 97-1061, High inoculum. c. C. neoformans 97-585, High inoculum. ⫽ Growth Control; ....{.... ⫽ 0.25xMIC; ----E---- ⫽ 0.5xMIC; ----‚---- ⫽ 1xMIC; - - -µ- - - ⫽ 2xMIC; -.-. -.-. ⫽ 4xMIC; ---Q--- ⫽ 8xMIC; ---ƒ--- ⫽ 16xMIC; -.-m-.- ⫽ 32xMIC.
Fig. 2. Time Kill Curves for Itraconazole versus C. neoformans. a. C. neoformans 97-1199, High inoculum. b. C. neoformans 97-1061, High inoculum. c. C. neoformans 97-585, High inoculum. ⫽ Growth Control; ....{.... ⫽ 0.25xMIC; ----E---- ⫽ 0.5xMIC; ----‚---- ⫽ 1xMIC; - - -µ- - - ⫽ 2xMIC; -.-. -.-. ⫽ 4xMIC; ---Q--- ⫽ 8xMIC; ---ƒ--- ⫽ 16xMIC; -.-m-.- ⫽ 32xMIC.
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Fig. 3. Time Kill Curves for Amphotericin B versus C. neoformans 97-585, High inoculum. ⫽ Growth Control; ....{.... ⫽ 0.25xMIC; ----E---⫽ 0.5xMIC; ----‚---- ⫽ 1xMIC; - - -µ- - - ⫽ 2xMIC; -.-. -.-. ⫽ 4xMIC; ---Q--- ⫽ 8xMIC; ---ƒ--- ⫽ 16xMIC; -.-m-.- ⫽ 32xMIC.
patients (Drusano 1988; Forrest et al., 1993; Moore et al., 1987; Schentag et al., 1984). Based on pharmacodynamics, some antibacterial agents are classified as either concentration-dependent (e.g., aminoglycosides and fluoroquinolones) or time-dependent (e.g., beta-lactams). For concentration-dependent agents, higher drug concentrations correlate with faster rates of pathogen eradication. Therefore, the most important pharmacodynamic parameter for these agents is the peak serum concentration/MIC ratio. Clinical studies have demonstrated that for the aminoglycosides concentrations 8-12xMIC are needed for maximal effectiveness (Moore et al., 1987). Hence, the administration of one large
Fig. 4. AUC/MIC Ratio vs Area Under the Kill Curve for the High Inoculum with the Triazoles. 䊐 ⫽ Fluconazole; { ⫽ Itraconazole.
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daily dose of an aminoglycoside is preferable to smaller doses given multiple times a day. Alternatively, time-dependent agents are most effective when the duration of the serum concentration remains above the MIC at the site of infection for a significant proportion of the dosing interval. Accordingly, these agents are best administered using frequent dosing intervals. For antifungal agents, the most important pharmacodynamic parameters are unknown for any class of agents. Recently, Louie et al., published a murine model evaluating the pharmacodynamics of fluconazole in the treatment of systemic candidiasis. They studied 3 pharmacodynamic parameters (Cmax/MIC, time above the MIC, and AUC/ MIC). Through dose-fractionation studies, they showed that maximizing Cmax/MIC or time above the MIC did not affect the outcomes as determined by fungal densities in renal tissue. The AUC/MIC ratio remained the same regardless of the dosage regimen utilized; therefore, they concluded that the AUC/MIC ratio was the best predictor of outcome (Louie et al., 1998). Although our study was not designed to evaluate the AUC/MIC ratio, we did examine the relationship between AUC/MIC and AUKC0 –24h for C. neoformans. Our results suggest that the maximal effective AUC/ MIC ratio was 192 (correlating with a concentration of 8xMIC) for both fluconazole and itraconazole for each isolate except for isolate 97-585 and itraconazole. To our knowledge, this is the first in vitro study comparing the activity of fluconazole, itraconazole, and amphotericin B against C. neoformans. Isolates with a variety of MICs were selected in order to examine a wide range of susceptibility patterns for the triazoles. Our results suggest that fluconazole and itraconazole are time-dependent agents while amphotericin B is concentration-dependent. We have also noted similar findings for these antifungal agents against C. albicans (Burgess et al., 1997). Klepser et al have reported similar findings for fluconazole and amphotericin B against C. albicans and C. neoformans although differences in time-kill methodology in terms of media, incubation time, and inocula were utilized (Klepser et al., 1997). We used Antibiotic Medium 3 for amphotericin B instead of RMPI since Rex et al demonstrated a more pronounced difference in MICs for amphotericin B when using Antibiotic Medium 3 broth (Rex et al., 1995). In addition, we evaluated the antifungal activity over 72 hours against each Cryptococcal isolate in order to coincide with the NCCLS guidelines for in vitro susceptibility testing of C. neoformans. Finally, to determine whether an inoculum effect existed, we utilized both a high (105 CFU/ml) and low (103 CFU/ml) inoculum. In contrast to Klepser et al, a carryover effect was not demonstrated with amphotericin B in our study. Perhaps this is due to the differences in methodology. Klepser et al reported fungistatic activity for amphotericin B at concentrations ⬍0.5xMIC and fungicidal activity at concentrations ⬎0.5xMIC (Klepser et al., 1997). Our results suggest a similar pattern of activity in that concentrations ⱕ1xMIC were fungistatic while concentrations ⬎1xMIC
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were fungicidal. Interestingly, this pattern of activity was not maintained beyond 24 hours. In fact, regrowth was noted following 24, 36, and 48 hours for concentrations 0.25– 0.5, 1, and 2-4xMIC, respectively. This regrowth could be due to a number of facts including drug degradation or development of resistance. Since we did not retest these organisms to determine the MIC after exposure to amphotericin B, we are not sure if resistance occurred. However, the likelihood of drug degradation is low since the only difference between time-kill studies and in vitro susceptibility testing is the determination of colony count and the NCCLS recommends performing susceptibility testing over 72 hrs for C. neoformans. Finally, regrowth was not observed during the 72 hour time interval for amphotericin B concentrations ⬎4xMIC. Although this information has been determined in vitro, it is noteworthy that higher mortality and slower rates of cerebrospinal sterilization have been reported in clinical trials utilizing lower doses of amphotericin B (i.e., 0.3– 0.5 mg/kg) for the initial treatment of cryptococcus meningitis (Saag et al., 1992). More recent clinical trials have concluded that better outcomes (less mortality and faster CSF sterilization) are associated with higher doses of amphotericin B (i.e., 0.7–1 mg/kg) alone or in combination with flucytosine (Van der Horst et al., 1997; De Lalla et al., 1995). In a multivariate analysis, statistically significant lower rates of relapse have been reported with the addition of flucytosine to higher doses of amphotericin B therapy, regardless of the triazole prescribed for suppressive treatment (Saag et al., 1995). The in vitro data that we have reported illustrate that the duration of suppression is proportional to the concentration of amphotericin B; therefore, it is plausible that by combining flucytosine with amphotericin B the magnitude of response is enhanced thus extending the duration of suppression. The NCCLS has recently suggested tentative breakpoints for fluconazole and itraconazole against candida species. Isolates with a MIC ⱕ 8g/ml and 0.125g/ml are classified as susceptible to fluconazole and itraconazole, respectively. While those isolates with a MIC ⱖ 64g/ml and 1g/ml are considered resistant to fluconazole and itraconazole. A new category called susceptible-dose dependent (S-DD) was created for isolates responsive to higher doses or serum levels. For fluconazole, the S-DD category includes MICs of 16 –32g/ml whereas for itraconazole the S-DD category includes MICs of 0.25– 0.5g/ml (NCCLS 1997; Rex et al., 1997). It is important to remember that these breakpoints are proposed for candida only and are not intended for use with C. neoformans. In fact, our data would suggest that for C. neoformans a MIC of 0.5g/ml for itraconazole would be considered resistant since no activity was demonstrated against isolate 97-585 irrespective of the concentration of itraconazole. Interestingly, this same isolate (97-585) with a MIC ⫽ 32g/ml for fluconazole was susceptible to fluconazole depending on the drug concentration (i.e., concentrations ⱖ8xMIC were suppressive while concentrations ⬍8xMIC displayed minimal activity).
This data may in fact suggest that itraconazole and fluconazole should not be used interchangeably when treating infections due to C. neoformans. We have reported similar patterns of activity for itraconazole and fluconazole against C. albicans classified as S-DD (MIC ⫽ 0.5 and 32g/ml; itraconazole and fluconazole, respectively) (Burgess et al., 2000). In conclusion, the results of the fungal time-kill studies suggest that the triazoles may be most effective against C. neoformans when maintained at concentrations 8-16xMIC at the site of infection, while for amphotericin B effectiveness may increase with greater drug concentrations (i.e., maximizing peak concentration/MIC ratios). Clinical studies have compared lower doses of amphotericin B to fluconazole (400 mg daily) for the initial treatment of AIDSassociated cryptococcal meningitis (Saag et al., 1992) as well as higher doses of amphotericin B (administered weekly) to fluconazole (200 mg daily) for maintenance therapy (Powderly et al., 1992). What clinical studies have not yet evaluated are dosage regimens for initial and maintenance therapies, which apply these pharmacodynamic relationships, determined in vitro. Such studies are necessary to correlate in vitro and in vivo relationships.
Acknowledgment This work was support by the 1997–98 Merck-AFPE “Gateway” Research Scholarship and 1997 Summer Research Assignment from The University of Texas at Austin. We would like to thank The Fungus Testing Laboratory, Department of Pathology, The University of Texas Health Science Center at San Antonio for the clinical isolates of Cryptococcus neoformans and Dr. Thomas C. Hardin for his critical review of the manuscript.
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