Abstract
Abstract
Purpose:
To compare in vitro susceptibility of amphotericin B (AMB) and amphotericin B methyl ester (AME) (a more soluble and less toxic formulation of AMB) against Candida albicans isolates recovered from human cases of endophthalmitis.
Methods:
The in vitro susceptibility of AMB and AME was determined for C. albicans isolates recovered from endophthalmitis (N=10) and for C. albicans ATCC reference strain 90028 using the Clinical and Laboratory Standards Institute M27-A2 (NCCLS/CLSI) broth dilution method. All isolates were obtained from samples of vitreous humor of patients with suspected endophthalmitis within the last 5 years at the Bascom Palmer Eye Institute, University of Miami Miller School of Medicine (Miami, FL).
Results:
The minimal inhibitory concentrations (MICs) of AME were equal to or lower than values for AMB in 7 of the 10 isolates; range: AME (0.125–1 μg/mL) versus (0.5–1 μg/mL) for AMB. The MIC90 value of both drugs was equal (1 μg/mL). Compared with AMB, the minimal fungicidal concentrations (MFCs) of AME were equal to or lower in 8 of 10 isolates; range: AME (0.125–2 μg/mL) versus AMB (0.25–4 μg/mL). MFC90 values of AME (1 μg/mL) was slightly superior to AMB (2 μg/mL). The MIC of the quality control strain (ATCC® 90028) was within an acceptable range.
Conclusions:
AME was equivalent to AMB in vitro against C. albicans. This formula may offer a slightly more efficient and less toxic formulation for the treatment of Candida endophthalmitis.
Introduction
AMB is a large molecular weight polyene antifungal that has highly insoluble intravenous and ocular form desoxycholate, a bile salt, that contributes considerable toxicity.7,8 Amphotericin B methyl ester (AME), a water-soluble derivative of AMB, has been shown to be significantly more soluble and less toxic compared with AMB.9–11 With respect to the antifungal efficacy, there are some conflicting evidence for susceptibility of AME against C. albicans. AME has also demonstrated slightly more effectiveness than AMB against Candida isolates, although some reports indicate similar activity compared with AMB in vitro.12,13 However, most strains were recovered from nonocular Candida infections. This is the first report to our knowledge establishing in vitro susceptibility of AME compared with AMB against C. albicans isolates recovered from human endophthalmitis.
Methods
Study design
The minimal inhibitory concentration (MIC) was determined by antifungal susceptibility testing by following the Clinical and Laboratory Standards Institute M27-A2 (NCCLS/CLSI) broth dilution guideline. After MIC determination, minimal fungicidal concentration (MFC) was evaluated for each isolates. All MICs and MFCs from each clinical isolate were compared between AMB and AME.
Clinical isolates
Ten isolates of C. albicans, obtained from vitreous humor specimens, were used in the study. All isolates recovered from patients with endogenous or traumatic endophthalmitis were part of the ocular isolate library of the Ocular Microbiology Laboratory at the Bascom Palmer Eye Institute (Miami, FL) within the last 5 years. Quality control was performed in accordance with CLSI document M27-A2 by using C. albicans ATCC reference strain 90028. All 10 isolates from the ocular isolate library, including 1 reference strain, were subcultured onto Sabouraud dextrose agar and incubated at 35°C for 48 h.
Inoculum preparation
After the incubation period, each isolate, including the reference strain, was suspended in 5 mL of sterile 0.145-mol/L saline, and then agitated via vortex for 15 s. The cell density of the suspensions was adjusted by a spectrophotometer (0.5 McFarland standard), yielding a stock suspension of 1×106 to 5×106 cells per mL. Subsequently, the final suspension is made by a 1:100 dilution followed by a 1:20 dilution of the stock suspension with an RPMI 1640 broth medium, which resulted in 5.0×102 to 2.5×103 cells per mL.
Antifungal dilution preparation
AMB powder was purchased from Sigma-Aldrich Co. (St Louis, CO). AME powder was supplied by Karykion Corp. (Princeton, NJ). A dry powder of AMB was dissolved in dimethyl sulfoxide (DMSO), yet AME was diluted with sterile distilled water to obtain the stock solutions at a concentration of 1,600 μg/mL.
Antifungal susceptibility testing
MIC testing
Based on previous studies (reference method for broth dilution antifungal susceptibility testing of yeasts, Approved Standard-Second Edition, NCCLS document M27-A2, National Committee for Clinical Laboratory Standards, Wayne, PA, 2002), the recommended AMB concentration range to be tested was 0.0313–16 μg/mL. Initially, the stock solutions that were diluted in DMSO for AMB and in sterile water for AME resulted in a concentration series from 3.13 to 1,600 μg/mL. To achieve the next concentration of antifungal agent, the RPMI 1640 broth was diluted in AMB solutions as well as AME solutions to prepare a concentration ranging from 0.313 to 160 μg/mL. Subsequently, 0.1 mL of various antifungal concentrations were placed in tubes together with 0.9 mL of the adjusted inoculums, which is a 1:10 dilution leading to a final antifungal concentration for each drug ranging from 0.0313 to 16 μg/mL.
With the growth control, 0.1 mL of dilution without antifungal agent was added to 0.9 mL of the adjusted inoculums.
Inoculated tubes of both antifungal agents, including growth control, were incubated at 35°C for 46–50 h in ambient air.
MFC testing
The minimal fungicidal concentrations (MFCs) were evaluated for each drug-isolate-medium combination. After at least 48 h of incubation period, all negative and the last positive tubes from inoculated solutions were subcultured onto Sabouraud dextrose agar plates. Subsequently, whole plates were incubated at 35°C until growth was observed.
End point determination
MIC determination
The amount of growth in each tube containing antifungal agents is compared visually with the amount of growth in the drug-free control tubes used in each set of tests. For interpretation of the result, the MIC is the lowest drug concentration that prevents any discernible growth, which is demonstrated by a clear solution without any turbidity. MIC90 is defined as the MIC required to inhibit the growth of 90% of strains.
MFC determination
The MFC is defined as the lowest drug concentration that showed no growth of the Candida colonies on the plates. MFC90 is defined as the MFC required to eradicate the growth of 90% of strains.
Results
The MICs and MFCs of AMB and AME against 10 strains of C. albicans are shown in Table 1. The MICs of AME were equal to or lower than the values for AMB in 7 of the 10 isolates. The MIC90 value of both drugs were equal (1 μg/mL); however, the MIC range of AME (0.125–1 μg/mL) is slightly greater than AMB (0.50–1 μg/mL).
MIC, minimal inhibitory concentration; MFC, minimal fungicidal concentration.
The MFCs of AME were equal to or lower than the values for AMB in 8 of the 10 isolates. Additionally, the MFC range of AME (0.125–2 μg/mL) is slightly greater than AMB (0.25–4 μg/mL). MFC90 values of AME were 1 μg/mL, which is lower than AMB (2 μg/mL).
The MIC of the quality control strain (ATCC® 90028) is 1 μg/mL, which is within an acceptable range (0.5–2.0 μg/mL) according to recommended 48-h MIC limits for quality control guidelines of the NCCLS/CLSI broth dilution procedure.
Discussion
The efficacy of AME against C. albicans recovered from ocular infection remains uncertain. Most studies demonstrate that AME has similar activity to AMB; however, these in vitro susceptibility studies have been performed in isolates obtained from systemic candidiasis.12–14 Very few studies have reported on ocular candidiasis models.
In vitro susceptibility testing of AME has been performed against various Candida strains, yet most of these isolates were recovered from nonocular infection. The average MICs and MFCs of AME against C. albicans isolates recovered from nonocular candidiasis were between 0.5–1.0 μg/mL and 0.5–1.0 μg/mL, respectively.12–14 Therefore, a similar potency of AME to parent AMB compound has been observed with the majority of studies with a limited number of reports, suggesting a superior activity.12–15 With our reported results, the MIC and MFC ranges of both drugs are comparable to previous studies. However, we found that the MIC range of AME is slightly superior to AMB, whereas the MIC90 is equal. Correspondingly, the MFC range of AME is slightly superior to AMB with a lower MFC90 value than AMB.
Evidence supporting the therapeutic potential of AME in the treatment of ocular candidiasis has been suggested with isolated clinical reports. Sekiya et al. found that AME affects the ergosterol-containing membranes of C. albicans more than AMB. 16 For in vivo testing, topical 1% AME administration demonstrated a potential role for the treatment of Candida keratitis in a limited rabbit model. 17 Furthermore, a single intravitreal injection of AME has been shown to resolve exogenous Candida fungal endophthalmitis in rabbit eyes.18,19
With respect to cytotoxicity, AME has been shown to be significantly less toxic than the parent compound (AMB). AME caused less membrane damage when compared with AMB in various types of tissue culture cells. 9 Furthermore, the AME was about one-eighth as nephrotoxic and one-fourth to one-half as hepatotoxic as the parent compound after a single intravenous injection in several kinds of animal models.10,11,20 The clinical use of AME has long been overshadowed by claims of neurotoxicity involving severe brain dysfunction and leukoencephalopathy observed among some patients with deep-seated systemic fungal infections who had received parenterally high doses of AME of unknown purity over an extended period of time. 21 Physical analysis of different lots of this AME revealed complex mixtures containing AME, AMB, and related derivatives. 22 Studies in dogs with these AME preparations produced confusing results where astrogliosis and pallor of the myelin were found in the AME-treated animals, but also in 5% glucose controls.23,24 Comparative studies employing pure AME and AMB in cell culture unquestionably revealed that AMB is potentially more neurotoxic than AME. This conclusion was based on the observed effect on glial cells (astrocytes and oligodendrocytes) and on the myelin sheath. 25 Intracisternal and intravenous administration of AMB, AME, and AME containing 10% AMB to rats again clearly revealed that AMB is significantly more neurotoxic than as revealed by behavior, central nervous system histopathology, and death. Of particular interest was the observation that AME containing 10% AMB was also more neurotoxic. 26 Similar more recent studies in mice further confirmed the greater neurotoxicity of AMB as compared to AME. 27 A number of clinical reports have revealed that AMB after parenteral administration to patients has resulted in neurotoxicologic changes involving behavior, demyelination, and leukoencephalopathy.28–30
Our report provides the preliminary data for susceptibility testing against a limited number of ocular C. albicans isolates causing endophthalmitis. Further studies with a larger number of isolates should be considered to provide definite MIC and MFC values. Pharmacokinetic as well as pharmacodynamic studies in ocular tissues are required to validate the role of this drug for the treatment of fungal endophthalmitis, including the aforementioned investigations of potential ocular toxicity.
In summary, AME is a more soluble formulation that may offer an equally or slightly more efficient activity, with less toxicity than the parent AMB compound for the treatment of ocular candidiasis.
Footnotes
Author Disclosure Statement
No competing financial interests exist.