ABSTRACT
The in vitro activities of amphotericin B in combination with rifampin were assessed against 95 ocular fungal isolates. The interactions between amphotericin B and rifampin at 4, 8, 16, and 32 μg/ml were synergistic for 11.8%, 51.0%, 90.2%, and 94.1%, respectively, of Fusarium solani species complex isolates and for 13.6%, 45.5%, 93.2%, and 95.5%, respectively, of Aspergillus flavus species complex isolates. Antagonism was never observed for the amphotericin B-rifampin combinations.
KEYWORDS: amphotericin B, Aspergillus flavus species complex, Fusarium solani species complex, rifampin, synergistic activity, fungal keratitis
TEXT
Keratomycosis is a major cause of vision loss in developing countries like China because of higher incidence and the unavailability of effective antifungal agents (1–3). The Fusarium solani species complex (FSSC) and Aspergillus flavus species complex (AFSC) are two predominant ocular fungal pathogens and are thought to be particularly virulent, more resistant to antifungals, and have worse outcomes than other species of Fusarium and Aspergillus in China and in many other parts of the world (4–10). Keratomycosis is notoriously difficult to treat. Amphotericin B is one of the most commonly used topical agent to treat keratomycosis (11, 12); however, nonsusceptibility to amphotericin B has been recently reported for filamentous fungi (13–16), and some studies have shown that the response rates to amphotericin B for Fusarium keratitis and Aspergillus keratitis are 56% and 27%, respectively (11, 17, 18). Therefore, there is an urgent need for new approaches to manage amphotericin B-nonsusceptible filamentous fungi. One possible approach is to combine amphotericin B with other antimicrobial agents (12). Amphotericin B and natamycin are often combined in the treatment of keratomycosis. However, a study by Lalitha et al. has shown that amphotericin B and natamycin are not synergistic in vitro against Fusarium and Aspergillus species isolated from keratitis (19). Two small clinical studies have shown the potential of an amphotericin B-rifampin combination to improve outcomes in keratomycosis (20, 21). This combination therapy may be an option for patients with amphotericin B-nonsusceptible keratomycosis. The aim of this study was to investigate the potentiation of the antifungal activity of amphotericin B by rifampin with clinically relevant concentrations against FSSC and AFSC isolates in vitro.
Fifty-one FSSC and 44 AFSC strains isolated from patients with keratomycosis from the Henan Eye Institute in China were investigated. These isolates were identified based on morphology by standard methods (22). Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality controls for each test.
Antifungal susceptibility was assayed by the microdilution method standardized by CLSI M38-A2 (23). The isolates were tested for susceptibility to amphotericin B (Amresco E437, USA; 298 μg/mg) alone, rifampin (Alfa Aesar; 99%) alone, and amphotericin B-rifampin combinations. For antimicrobial agents alone, final concentrations ranged from 0.0313 to 16 μg/ml for amphotericin B and from 4 to 2,048 μg/ml for rifampin. Drug interactions were tested using a limited checkerboard microdilution method with combinations of rifampin at 4, 8, 16, and 32 μg/ml with 0.0313 to 16 μg/ml of amphotericin B (24, 25). The MIC was determined as the lowest concentration that prevented any discernible growth. The isolates were classified as wild type or non-wild type according to the CLSI epidemiological cutoff values (ECVs) (97.5%) proposed for amphotericin B against AFSC and FSSC, which are 4 and 8 μg/ml, respectively (26, 27). Interaction was determined by calculating the fractional inhibitory concentration index (FICI) with standard definitions: synergy FICI is ≤0.5, indifference FICI is >0.5 and ≤4, and antagonism FICI is >4 (24, 25).
The MIC50, MIC90, and MIC range for drugs alone and in combinations were calculated for the isolates with the SPSS statistical package (version 17.0). For calculations, any high off-scale MIC was converted to the next higher concentration.
The in vitro activities of amphotericin B alone, rifampin alone, and the amphotericin B-rifampin combinations against the ocular pathogens are summarized in Table 1. Ninety-eight percent of the FSSC and 97.7% of the AFSC isolates showed MICs of ≤8 μg/ml and MICs of ≤4 μg/ml, respectively, and were categorized as wild type for amphotericin B. A total of 66.7% of the FSSC and 77.3% of the AFSC isolates exhibited MICs of ≥2 μg/ml for amphotericin B. Upon combination with 4, 8, 16, or 32 μg/ml rifampin, the activity of amphotericin B against FSSC was 2 to 4 times, 4 to 8 times, 8 to 16 times, or 8 to 16 times greater, respectively, than that of amphotericin B alone, and the activity of amphotericin B against AFSC was 2 to 8 times, 4 to 16 times, 8 to 32 times, or 16 to 32 times greater, respectively, than that of amphotericin B alone. For a non-wild-type FSSC isolate, the MIC of amphotericin B was reduced from 16 μg/ml to 4, 1, 0.5, or 0.25 μg/ml in combination with 4, 8, 16, or 32 μg/ml rifampin, respectively. For a non-wild-type AFSC isolate, the MIC of amphotericin B was reduced from 32 μg/ml to 2, 1, 0.25, or 0.25 μg/ml in combination with 4, 8, 16, or 32 μg/ml rifampin, respectively. Table 2 summarizes the in vitro interactions determined by the FICI of the isolates for the amphotericin B-rifampin combinations. Antagonism was never observed for the amphotericin B-rifampin combinations.
TABLE 1.
In vitro susceptibilities of ocular Fusarium solani species complex and Aspergillus flavus species complex isolates to amphotericin B and rifampin alone and in combination
| Organism (no. of isolates) or agenta | MIC (μg/ml) |
||
|---|---|---|---|
| Range | MIC50 | MIC90 | |
| Fusarium solani species complex (51) | |||
| AMB | 0.5–16 | 2 | 4 |
| RIF | 4,096 | 4,096 | 4,096 |
| AMB-RIF(4)b | 0.25–4 | 1 | 2 |
| AMB-RIF(8)c | 0.125–2 | 0.5 | 2 |
| AMB-RIF(16)d | 0.063–1 | 0.25 | 0.5 |
| AMB-RIF(32)e | 0.063–1 | 0.25 | 0.5 |
| Aspergillus flavus species complex (44) | |||
| AMB | 1–32 | 2 | 4 |
| RIF | 4,096 | 4,096 | 4,096 |
| AMB-RIF(4)b | 0.5–4 | 1 | 2 |
| AMB-RIF(8)c | 0.125–2 | 1 | 2 |
| AMB-RIF(16)d | 0.063–1 | 0.25 | 0.5 |
| AMB-RIF(32)e | 0.031–1 | 0.25 | 0.5 |
AMB, amphotericin B; RIF, rifampin.
Amphotericin B at 10 concentrations in combination with 4 μg/ml rifampin.
Amphotericin B at 10 concentrations in combination with 8 μg/ml rifampin.
Amphotericin B at 10 concentrations in combination with 16 μg/ml rifampin.
Amphotericin B at 10 concentrations in combination with 32 μg/ml rifampin.
TABLE 2.
Interactions of amphotericin B-rifampin combinations against ocular Fusarium solani species complex and Aspergillus flavus species complex isolates
| Combination | FICI and % of isolates showing interactiona |
|||||
|---|---|---|---|---|---|---|
|
Fusarium solani species complex (n = 51) |
Aspergillus flavus species complex (n = 44) |
|||||
| FICI (mean ± SD) | Synergy | Indifference | FICI (mean ± SD) | Synergy | Indifference | |
| AMB-RIF(4)b | 0.58 ± 0.24 | 11.8 | 88.2 | 0.61 ± 0.27 | 13.6 | 86.4 |
| AMB-RIF(8)c | 0.38 ± 0.19 | 51.0 | 49.0 | 0.42 ± 0.21 | 45.5 | 54.5 |
| AMB-RIF(16)d | 0.23 ± 0.19 | 90.2 | 9.8 | 0.19 ± 0.20 | 93.2 | 6.8 |
| AMB-RIF(32)e | 0.19 ± 0.11 | 94.1 | 5.9 | 0.17 ± 0.20 | 95.5 | 4.5 |
Interactions: synergy, FICI of ≤0.5; indifference, FICI of >0.5 and ≤4; antagonism, FICI of >4.
Amphotericin B at 10 concentrations in combination with 4 μg/ml rifampin.
Amphotericin B at 10 concentrations in combination with 8 μg/ml rifampin.
Amphotericin B at 10 concentrations in combination with 16 μg/ml rifampin.
Amphotericin B at 10 concentrations in combination with 32 μg/ml rifampin.
This study has two major findings. (i) The interactions between amphotericin B and rifampin at 4, 8, 16, or 32 μg/ml were synergistic for 11.8%, 51.0%, 90.2%, or 94.1% of the FSSC isolates, respectively, and for 13.6%, 45.5%, 93.2%, or 95.5% of the AFSC isolates, respectively,. (ii) The amphotericin B-rifampin combinations may be more effective in the non-wild-type strains than in the wild-type strains against FSSC and AFSC isolates. The values of this interaction would be a potentiation of antifungal action, an effect against secondary bacterial infection, and a reduction in eye toxicity, i.e., (i) non-wild-type strains for amphotericin B alone may be susceptible to the drug combination; (ii) broad-spectrum antibacterial rifampin may be needed to combat secondary bacterial infection; and (iii) in the treatment of wild-type strains, the dosage of amphotericin B may be decreased, thereby avoiding toxicity.
In keratomycosis, Shapiro et al. demonstrated that a lower MIC is significantly associated with a good outcome (28). Lalitha et al. demonstrated that a higher MIC is associated with increased odds of perforation (9). Although the CLSI ECVs (97.5%) proposed for amphotericin B against AFSC and FSSC are 4 and 8 μg/ml, respectively, several documents indicate that isolates with MICs of ≥2 μg/ml are nonsusceptible to amphotericin B therapy and are, therefore, referred to as amphotericin B-nonsusceptible isolates (13, 15, 29). Our in vitro data support the clinical experience that the FSSC and AFSC are often refractory to amphotericin B. Indeed, 66.7% of FSSC isolates and 77.3% of AFSC isolates exhibited amphotericin B MICs of ≥2 μg/ml in this study. These levels are not reliably achieved in cornea in a bioactive form with the present dosage regimens of amphotericin B. The results from the pharmacokinetics of amphotericin B show that the peak concentrations of amphotericin B in debrided rabbit corneas are 6.13 μg/g (about 0.43 μg/g in a bioactive form) after one 20-μl drop of amphotericin B 0.15% is topically applied and 21.1 μg/g (about 1.48 μg/g in a bioactive form) after one 20-μl drop of amphotericin B 0.15% every 5 min for 13 applications (30). The amounts measured in the study are total drug levels. They do not indicate the quantity that is bioavailable. The studies in rabbit eyes have shown that only 7% of total amphotericin B measured in the corneas is in a bioactive form (31). For susceptible organisms, the peak concentration of amphotericin B in a bioactive form in corneas is still higher than the MIC of amphotericin B and this is still an adequate level; for nonsusceptible organisms, this level is lower than the MIC of amphotericin B and cannot exert its antifungal effect. Therefore, reducing the MIC of amphotericin B by amphotericin B-rifampin combination may be one of the keys to improving the efficacy of amphotericin B.
Rifampin is ineffective against fungi. Rifampin's mechanism of resistance in fungal cells may be related to its inability to penetrate the fungal cell membrane. Our findings suggested that the amphotericin B-rifampin combinations produced potently synergistic action against FSSC and AFSC in vitro. As amphotericin B acts mainly to bind to ergosterol in the fungal cell membrane and increase the membrane's permeability (32), we hypothesize for amphotericin B-susceptible strains that this increased permeability allows the intracellular substances to pass through the membrane, and this either inhibits the growth of the organism or kills it. However, for amphotericin B-nonsusceptible strains, this increased permeability of the cell membrane may not allow enough of the intracellular substances to pass through the membrane but may increase permeability enough to allow the penetration of rifampin. Once inside cells, rifampin can exert its antimicrobial effect by blocking the DNA-dependent RNA polymerase subunit B (RpoB) (33). By means of this mechanism, the activity of amphotericin B is potentiated by rifampin against fungi.
Two small clinical studies have indicated that compound amphotericin B, which mainly includes amphotericin B and rifampin, is used successfully to treat patients with keratomycosis (20, 21). Although such studies provide limited scientific evidence, they show clear clinical improvement in amphotericin B-rifampin combinations. Our findings of in vitro synergy between amphotericin B and rifampin support these observations. Since the concentrations of rifampin that are effective in in vitro combinations in this study may be achievable in cornea (34), the amphotericin B-rifampin combination may represent an attractive perspective for developing new management strategies for keratomycosis. Rifampin has been used in humans for the long-term treatment of trachoma with no serious adverse effects (35, 36). Considering that rifampin is a well-known secure drug and since no antagonism was seen between amphotericin B and rifampin in this study, we believe the amphotericin B-rifampin combination would be a well-tolerated and effective therapy form for FSSC and AFSC keratitis in humans.
In summary, the amphotericin B-rifampin combination significantly enhanced the antifungal activity of amphotericin B against FSSC and AFSC isolates. Our results suggest that an amphotericin B-rifampin combination therapy may also be beneficial for treating keratomycosis and deserves in vivo studies.
ACKNOWLEDGMENTS
Funding for this research was provided by the National Natural Science Foundation of China under grant 81241033 (Y.X., Y.H., L.H.), the Fund of the Bureau of Science and Technology of Henan Province under grant 142300410079 (Y.H., Y.X., L.Z., C.G., L.H.), and the Fund of the Bureau of Health of Henan Province under grant 201002012 (Y.X., C.G., L.Z.).
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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