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Antimicrobial Agents and Chemotherapy, June 2009, p. 2629-2631, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01026-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Antifungal Combinations against Simulated Candida albicans Endocardial Vegetations

Manjunath P. Pai^*

College of Pharmacy, University of New Mexico, Albuquerque, New Mexico

Received 31 July 2008/ Returned for modification 13 November 2008/ Accepted 12 March 2009

Top ABSTRACT INTRODUCTION REFERENCES

 
The in vitro effects of flucytosine (5FC), liposomal amphotericin B (L-AmB), and micafungin (Mica) combinations against two Candida albicans strains that simulated 24-hour-old endocardial vegetations were studied. Mica was superior to 5FC or L-AmB, and the 5FC-L-AmB-Mica combination was superior to all other treatments for one strain but no different from the dual combination of L-AmB-Mica for the other strain.

Top ABSTRACT INTRODUCTION REFERENCES

 
Candida species can adhere to cell surfaces and form a three-dimensional community of microorganisms encased in an exopolymeric matrix known as biofilm (5). Biofilm can form on native and prosthetic heart valves to cause infective endocarditis. Candida albicans is the most common fungus linked to the development of infective endocarditis and is associated with a mortality rate in excess of 50% (6). Candida albicans organisms within biofilm have decreased cell membrane ergosterol content, have reduced expression of ergosterol biosynthetic genes, express higher levels of genes involved in amino acid and nucleotide metabolism, and upregulate efflux pumps (5, 9). These alterations may explain the poor activity of antifungals such as fluconazole and amphotericin that target ergosterol. Unlike these agents, the echinocandins target the cell wall of Candida species through inhibition of β-1,3-glucan synthase. A growing body of evidence suggests that echinocandins are active against Candida albicans biofilm (1, 4, 7, 8, 11, 13-15). However, cell wall integrity pathways and glucan-associated changes can also occur, which could limit echinocandin activity against Candida biofilm (10). As a consequence, combination antifungal therapy remains a rational approach to treatment of Candida endocarditis while possibly limiting emergence of resistance.

The effectiveness of combination antifungal therapy for Candida endocarditis is difficult to assess clinically. Animal models provide valuable data but are not always necessary for initial assessment. Replication of antifungal pharmacokinetics to mimic the human profile cannot be easily accomplished in animal models. Also, evaluation of the interaction of antifungals requires large sample sizes and can lead to unnecessary animal testing with limited information gain. In contrast, in vitro models provide an alternative, more rapid, and controlled environment for the assessment of antifungal combinations. My coworkers and I previously determined the activity of flucytosine (5FC), micafungin (Mica), and voriconazole as single agents and in combinations against Candida species by using an in vitro model of infective endocarditis (11). This work demonstrated the poor activity of voriconazole and the superior activity of 5FC and Mica against developing Candida biofilm. As a consequence, the present study was performed to test the interaction of 5FC, Mica, and liposomal amphotericin B (L-AmB) against mature (24-hour-old) C. albicans human platelet fibrin clots that simulated endocardial vegetations (SEVs).

Two C. albicans isolates, ATCC 14053 (CA1) and a clinical isolate (CA2) obtained from a septic patient with probable endocarditis collected from the University of New Mexico Hospital, were used for these experiments. Antifungal susceptibility testing was performed using the CLSI M27-A2 broth microdilution methodology for 5FC (Sigma, St. Louis, MO) and Mica (Astellas Pharma USA, Deerfield, IL). Both isolates were susceptible to 5FC with a MIC of 0.125 µg/ml. Amphotericin B (AmB) MICs were determined using the Etest methodology and were 0.25 µg/ml (CA1) and 1.0 µg/ml (CA2). The Mica MICs were 0.015 µg/ml (CA1) and 0.030 µg/ml (CA2). The C. albicans strains used in this study were selected because they reflected MIC₅₀s and MIC₉₀s of Mica from global surveillance data (12). A single colony of C. albicans obtained from a 24-h culture on Sabouraud dextrose agar (Cole-Parmer, Vernon Hills, IL) was grown in yeast nitrogen base medium (Difco Laboratories, Detroit, MI) supplemented with 2% dextrose at 27°C for 24 h. Infected fibrin clots were prepared as previously described but at a starting inoculum of 10⁴ CFU/g (11). The smaller inoculum permitted growth of C. albicans to 10⁶ CFU/g at 24 h just prior to exposure to antifungals.

A one-compartment infection model (250 ml) was utilized in duplicate as previously described (11). The experiments were conducted over 72 h, which included a 24-h biofilm development phase followed by a 48-h treatment phase. Fungal burden was determined at six time points: –24 (baseline), 0 (treatment initiation), 8, 24, 32, and 48 h. At every time point, two vegetations were removed from each model, weighed, placed in a 10-ml sterile test tube prefilled with normal saline, and homogenized. After serial dilution, a 20-µl aliquot was plated in triplicate onto Sabouraud dextrose agar (Cole-Parmer, Vernon Hills, IL) and incubated for 24 h at 35°C and the colonies were counted visually. The same model apparatus using yeast nitrogen base-2% dextrose as the reservoir medium and SEVs was used to compare the single, dual, and triple combination activities of the aforementioned agents. All antifungal agents were administered as bolus doses 24 h after retention of SEVs in the model to permit biofilm formation. Antifungals were administered to simulate (70-kg patient) doses of 37.5 mg/kg of body weight every 12 h (5FC), 5 mg/kg every 24 h (L-AmB), and 150 mg every 24 h (Mica). Peristaltic pumps were activated to mimic an elimination half-life of 6 h for 5FC and 12 h for L-AmB and Mica. Two fibrin platelet clots were removed from each model at each time point and handled as described above given that antifungal carryover was not demonstrated. The log₁₀ CFU/g over time (0 to 48 h) was also compared between the different regimens tested and growth controls. The maximum rate of kill (K_max), area under the rate of kill curve (AURKC), and area between the treatment and control time-kill curves (ABTKC) were calculated (11). The seven treatment groups were compared using one-way analysis of variance with post hoc comparisons using Bonferroni correction for multiple comparisons of significance. Consequently, a P value of <0.007 was considered significant.

Time-kill curves for both C. albicans isolates are illustrated in Fig. 1 by isolate. The use of Mica alone was associated with a significant reduction in fungal burden over 48 h compared to either 5FC or L-AmB for both isolates. The mean ± standard deviation (SD) change in log₁₀ CFU/g between time zero and 48 h was 1.05 ± 0.55, 2.05 ± 0.77, and –2.98 ± 0.35 for 5FC, L-AmB, and Mica, respectively (P < 0.001). The effect of L-AmB was greater against isolate CA1 than against CA2, which was consistent with the lower AmB MIC against CA1. However, no significant reduction of fungal burden was noted for either 5FC or L-AmB over time regardless of isolate. A summary of time-kill curve parameters is included in Tables 1 and 2 based on isolate. The use of Mica alone was superior to 5FC and L-AmB based on ABTKC, AURKC, and K_max against both isolates. The triple combination of 5FC-L-AmB-Mica was superior (<0.007) to all other treatments based on ABTKC and K_max for isolate CA1. In contrast, the dual combination of L-AmB-Mica was superior (P < 0.007) to all other treatments except the triple combination of 5FC-L-AmB-Mica against CA2. Despite, these superior effects of combination therapy, no synergistic combinations were identified for the triple combination based on regression. The mean interaction (95% confidence interval) coefficients for this triple combination were –12.8 (–47.5, 21.8) and 16.7 (–13.3, 46.8), indicating indifference against CA1 and CA2, respectively.

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FIG. 1. Effects of 5FC, L-AmB, and Mica on mean ± SD log10 CFU/g of Candida albicans (CA1 and CA2) SEV over time. Antifungals were introduced into the model at time zero to permit a 24-h biofilm maturity time.

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TABLE 1. Comparison of time-kill curve parameters of 5FC, L-AmB, and Mica combinations against C. albicans isolate CA1

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TABLE 2. Comparison of time-kill curve parameters of 5FC, L-AmB, and Mica combinations against C. albicans isolate CA2

The in vitro activity of echinocandins against C. albicans biofilm has been demonstrated by numerous investigators. Cateau and colleagues recently demonstrated that catheter lock solutions of either Mica or caspofungin significantly reduced the metabolic activity of C. albicans (4). These effects occurred irrespective of the maturity of biofilm, i.e., 12 h or 5 days old. Similarly, use of systemic caspofungin along with catheter lock therapy has been effective at sterilizing catheters in an animal model of a C. albicans intravascular catheter infection model (15). This unique activity of echinocandins such as Mica against mature Candida biofilm suggests a potential role for infective endocarditis. Most case series and reviews of Candida endocarditis were published prior to the introduction of the echinocandins (6). Baddley and colleagues recently queried the International Collaboration on Endocarditis-Prospective Cohort Study and identified 33 (1.2%) definitive cases of Candida endocarditis out of a cohort of 2,760 cases of infective endocarditis (2). Death occurred in 40% (6 of 15) of cases treated with monotherapy, 25% (2 of 8) of cases with sequential antifungal therapy, and 0% (0 of 2) of cases receiving combination therapy with an echinocandin. The low prevalence of Candida endocarditis limits the option of conducting a well-designed clinical trial to better define optimal treatment. The most recent guidelines from the American College of Cardiology and the American Heart Association provide no therapeutic strategy with the exception of surgical intervention for fungal endocarditis (3). Surgical removal of the infected valve is clearly the most effective management strategy to prevent embolic complications. However, not all patients qualify for surgery and time to surgical intervention can be delayed. The low incidence and high morbidity and mortality associated with this disease demand alternative research strategies to improve current care. As a result, in vitro and animal models will remain the most relevant approach for studying novel therapeutic agents for fungal endocarditis.

My coworkers and I previously demonstrated the limited activity of triazoles such as voriconazole relative to 5FC and Mica against developing C. albicans biofilm (11). The current study explored the activity of 5FC, L-AmB, and Mica alone and in combinations against mature (24-h-old) C. albicans SEVs. Our data suggest that Mica was uniquely active against mature C. albicans SEVs, while 5FC was not. These data are different from my group's previous report given that 5FC was markedly active against C. albicans SEVs that had not matured for 24 h (11). My group did not identify antifungal combinations that met definitions of synergy. However, synergy and antagonism are in vitro concepts that are difficult to translate clinically. As such, the primary goal of combination antimicrobial testing includes finding a combination that is different (positive or negative) in effect from the single agent. For that reason, my group approached this problem by compressing the time-kill data into area under the time-kill curves and tested to see if differences existed between treatments. The triple combination of 5FC-L-AmB-Mica was superior to all other treatments for one isolate (CA1) but no different from the dual combination of L-AmB-Mica for the other isolate (CA2). Although a clear pattern of interaction between these agents was not established, the superior activity of Mica against 24-h-old C. albicans SEVs relative to 5FC and L-AmB was apparent for both isolates. These data support previous reports that demonstrate marked activity of echinocandins when used alone or in combination with polyenes against C. albicans biofilm (8, 14). As a first step, translation of these in vitro findings to an animal model of C. albicans endocarditis could lead to improvements in current treatment of this high-morbidity and -mortality disease.

 
This study was supported in part through a grant from Astellas Pharma USA. I am supported through the University of New Mexico Clinical Translational Science Center funded in part through the National Institutes of Health (M01-0997).

I acknowledge the support of Renee-Claude Mercier for use of her in vitro endocarditis model.

 
* Mailing address: Institute for Clinical Pharmacodynamics, Ordway Research Institute, 43 British American Blvd., Latham, NY 12110. Phone: (518) 429-2707. Fax: (518) 429-2701. E-mail: apai-icpd{at}OrdwayResearch.org

Published ahead of print on 23 March 2009.

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Antimicrobial Agents and Chemotherapy, June 2009, p. 2629-2631, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.01026-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Pai, M. P. Search for Related Content PubMed PubMed Citation Articles by Pai, M. P.