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Combination Treatment of Invasive Fungal Infections

Center for Medical Mycology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio,¹ Pfizer Global Pharmaceuticals, Pfizer Inc., New York, New York,² Exploratory Development, Pfizer Global Research & Development, Sandwich Laboratories, Sandwich, United Kingdom³

SUMMARY

INTRODUCTION

METHODS TO DETERMINE THE IN VITRO EFFICACY OF ANTIFUNGAL AGENTS IN COMBINATION

Checkerboard Method

Fractional inhibitory concentration index.

Response surface-modeling method.

Time-Kill Method

Epsilometer Strip Test (E-test)

METHODS TO DETERMINE THE IN VIVO EFFICACY OF ANTIFUNGAL AGENTS IN COMBINATION

COMBINATION THERAPY IN PRACTICE GUIDELINES FOR TREATMENT OF FUNGAL INFECTIONS

Dual Combinations in the Practice Guidelines for Candidiasis

Dual Combinations in the Practice Guidelines for Cryptococcosis

Dual Combinations in the Practice Guidelines for Aspergillosis and Coccidioidomycosis

ANTIFUNGAL COMBINATIONS AGAINST CANDIDA SPECIES

Established Antifungal Agents in Combination

Amphotericin B plus fluconazole against candidiasis. (i) In vitro studies.

(ii) In vivo studies.

(iii) Clinical studies.

Amphotericin B plus 5-fluorocytosine: in vitro studies.

Amphotericin B plus 5-fluorocytosine plus fluconazole: in vitro studies.

Amphotericin B plus fluconazole in sequential combinations against candidiasis.

Fluconazole plus 5-fluorocytosine against candidiasis.

(i) In vitro studies.

(ii) In vivo studies.

(iii) Case reports.

Newer Antifungals in Combination

Voriconazole combinations. (i) In vitro studies.

(ii) In vivo studies.

(iii) Combination with 5-fluorocytosine or amphotericin B against systemic candidiasis in immunocompetent guinea pigs.

Caspofungin combinations.

Fluconazole plus terbinafine combination against candidiasis: case report.

ANTIFUNGAL COMBINATIONS AGAINST CRYPTOCOCCUS SPECIES

Established Antifungal Agents in Combination

Fluconazole plus 5-fluorocytosine against cryptococcosis. (i) In vitro studies.

(ii) In vivo and clinical studies.

Amphotericin B plus fluconazole against cryptococcosis

(i) In vitro studies.

(ii) In vivo studies.

Newer Antifungals in Combination

Triazoles in combination. (i) In vitro studies.

(ii) In vivo studies.

Echinocandins in combination.

ANTIFUNGAL COMBINATIONS AGAINST ASPERGILLUS AND FUSARIUM SPECIES

Established Antifungals in Combination

Amphotericin B, triazoles, and 5-fluorocytosine against Aspergillus and Fusarium: in vitro and in vivo studies.

Amphotericin B plus itraconazole against aspergillosis: clinical studies.

Fluconazole plus itraconazole compared with amphotericin B against aspergillosis.

Newer Antifungals in Combination against Aspergillosis

Voriconazole plus echinocandins and other antifungals. (i) In vitro studies.

(ii) In vivo studies.

(iii) Clinical studies.

Terbinafine plus triazoles and other antifungals against Aspergillus.

ANTIFUNGAL COMBINATIONS AGAINST SCEDOSPORIUM SPECIES

ANTIFUNGAL COMBINATIONS AGAINST OTHER FUNGI

MISCELLANEOUS COMBINATIONS

Combinations of Antifungal, Antibacterial, and Anticancer Agents

Antifungals Combined with Immunomodulators

GENERALIZATIONS REGARDING ANTIFUNGAL COMBINATIONS

Antagonistic Interactions

Interpretation of FICI Values

Potentially Useful Combinations

CONCLUSIONS

REFERENCES

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INTRODUCTION

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High morbidity and mortality persist for systemic fungal infections due to pathogenic yeast (47, 55, 91, 191) and molds (42, 119, 169). The Food and Drug Administration has approved several antifungal agents belonging to different chemical classes (polyenes, pyrimidines, azoles, and echinocandins) as therapeutic options for fungal infections (reviewed in references 50 and 79). However, treatment is often complicated by high toxicity, low tolerability, or narrow spectrum of activity. These difficulties have driven recent efforts to determine the efficacy of combination therapy in the treatment and management of invasive infections. The most common rationales behind the studies focused on combination therapy are based on (i) mechanisms of action, combining agents with complementary targets within the fungal cells (polyenes plus azoles or echinocandins, antifungals plus immune factors, etc.), (ii) spectrum of action (combining agents potent against different organisms), and (iii) stability and pharamacokinetic/pharmacodynamic characteristics. Of these three main rationales, most combination therapy studies are based on the rationale of combining agents that have complementary mechanisms of action (Fig. 1). Potential benefits of using combination therapy include broad spectrum of efficacy, greater potency than either of the drugs used in monotherapy, improved safety and tolerability, and reduction in the number of resistant organisms (127).

View larger version (39K): [in this window] [in a new window]   FIG. 1. Schematic description of sites of action of different antifungal agents. Candins cause disruption of cell wall, allowing other antifungals (polyenes, azoles, and 5-FC) to enter. Azoles and polyenes can inhibit or bind to ergosterol, leading to cell lysis and allowing 5FC to enter the cell and inhibit nucleic acid synthesis. Dashed arrows indicate the site of action for each antifungal class. The mechanisms of action for each class of antifungal agent are depicted in panels A through D.

Interactions between different drugs are described variously as synergistic, indifferent, additive, or antagonistic. Assessments of in vitro drug interactions are usually based on the "no interaction" theory, which assumes that drugs in combination do not interact with each other. When the observed effect of the drug combination is more than that predicted from the "no interaction" theory, synergy is claimed. On the other hand, antagonism is claimed when the observed effect is less than that predicted (88). Although several categories including "additive," "subadditive," and "indifferent" have been used to describe intermediate drug-drug interactions, the emerging consensus has been to group all of them under the "no interaction" category (163).

Inherent problems associated with susceptibility testing of single antifungal agents are also relevant to testing antifungal combinations. Evaluation of drug-drug interactions against filamentous fungi is further problematic for a number of reasons: (i) in spite of concerted efforts which resulted in a published reference method for the evaluation of susceptibility of conidia-forming filamentous fungi, in vitro and in vivo correlation are not yet well-defined (68, 90, 176, 180, 235); (ii) susceptibility testing of the candins is known to be influenced by different factors (48, 155, 175), and, despite recent attempts, a standard method for this class has not been developed so far; (iii) spectrophotometric reading, which is successfully used to measure the drug MICs for yeast, is less useful for filamentous fungi; and (iv) response to treatment (damage, recovery, or viability) varies differentially for apical and subapical hyphal components (155).

In this article, we present an overview of methods commonly applied to assess the effects of combination therapy, historical and current uses of combination therapy, and potential limitations of antifungal combination therapy. This review demonstrates the increased interest in antifungal combination and clearly illustrates the dynamic nature of this field, with all its complexities.

METHODS TO DETERMINE THE IN VITRO EFFICACY OF ANTIFUNGAL AGENTS IN COMBINATION

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The methods for determining antifungal susceptibilities have been extensively reviewed, especially for single antifungal agents (189), and recommendations for determination of the MIC of single antifungals for yeasts and molds are currently available (65, 66, 158, 159, 189). The MIC of an antifungal agents is defined as the minimum concentration of the drug resulting in 80% (or 50%, in some cases) inhibition of fungal growth relative to the control (with no drug exposure). Antifungal susceptibility testing of individual agents is based on the following principles: (i) MIC is not a physical or chemical measure, (ii) host factors are often more important than susceptibility test results in determining clinical outcome, (iii) susceptibility of a microorganism in vitro does not predict successful therapy, and (iv) resistance in vitro should often predict therapeutic failure (186, 187).

In vitro susceptibility assays are highly dependent on the fungal species under investigation and on the testing conditions of exposure time, incubation temperature, media, and other method-specific factors. Many in vitro and in vivo modifications have been devised to better approximate human disseminated disease (130) and to examine the influence of immunosuppression (149) or neutropenia (106) on treatment success. Another complexity is derived from the fact that the activity of antifungals in combination is dependent not only on the drug-drug concentration but also the absolute ratio of the drugs (74, 76, 78). The majority of published results from studies using combination treatments tend to focus on two-drug combinations, although this limit is likely to be revised as methodological and analytical techniques evolve that will allow evaluation of three or more drugs in combination. Additionally, these methods are based on static drug concentrations, and interaction is determined at a single time point; therefore, direct correlation between in vitro and in vivo interactions is not always possible. These principles are also applicable to susceptibility testing of antifungals in combination, resulting in considerably more complex problems. The MICs of different drugs in combination can be determined using the checkerboard method, time-kill method, or Epsilometer test (E-test) (105, 123, 173, 174, 187). In this section, we briefly present an overview of the application of these methods to determine the interactions between antifungal agents used in combination.

Fractional inhibitory concentration index. Most studies investigating the in vitro efficacy of antifungal agents in combination interpret results in terms of the FICI, which is defined by the following equation:

where MIC_A and MIC_B are the MICs of drugs A and B, respectively. Over the years, several investigators have categorized interactions between antifungal agents by using the FICI in different terms, leading to sometimes confusing nomenclature and interpretations of results. Recently, for the sake of uniformity in interpretation, Odds (163) proposed that an FICI of <0.5 should be considered synergy, a FICI of >4 should be considered antagonism, and a FICI of 0.5 to 4 should be considered no interaction. The interpretation of these criteria has also been discussed in detail in a recent review (103). We feel that additivity or indifference implies that the drugs in combination do not have a detrimental effect on the response, even though they are not synergistic. In agreement with the proposed system of interpretation of FICI values, we suggest that all future studies should follow this system while interpreting FICI data.

Ease of use, simplicity, and feasibility of performance make FICI the method of choice for analyses of drug-drug interactions in most clinical laboratories (57). However, this method also has some significant disadvantages: (i) it is dependent on dilution-based determination of MICs and hence may lead to interexperimental errors; (ii) it does not differentiate between the possibility that at some concentrations in the checkerboard there may be synergy while at other dilutions there may be indifference or antagonism; (iii) for some antifungal combinations, the choice of MIC end point is not clear, leading to difficulties in calculating the FICI; (iv) it is not amenable to statistical analyses; and (v) the definition of FICI varies greatly between different studies (Table 1) (146-148, 223). Attempts to overcome the difficulty of statistical analyses of the FICI include determining the median and range from several replicates of MIC determinations (31), concordant FICI values from several replicates (147), and establishment of consensus guidelines regarding interpretation of the FICI values (163).

where D_A and D_B represent the MICs of drugs A and B, respectively, IC is the interaction coefficient, IC₅₀ represents the drug concentrations resulting in 50% inhibition, m_A and m_B are the slope parameters for drugs A and B, E is the measured response (optical density [OD]) and E_max is the control response. Interactions with an IC of >0 indicate synergy, while an IC of <0 indicates antagonism. Additivity is claimed when IC equals zero. IC is considered statistically significant if the associated CI does not overlap zero. Although the RSM approach is increasingly being advocated by different studies to be more robust than the FICI method (146-148, 223), this model is also not without some major drawbacks: (i) the model involves complicated mathematical data-fitting and modeling steps; (ii) although specialized software for these analyses have been reported, they are not commonly accessible to clinical laboratories; (iii) since the model relies on regression analysis based on data fitting, results generated are dependent on factors such as initial parameters, ways of calculating sum of squares, variance, and weighting parameters; and (iv) it may give erroneous results (e.g., very high interaction parameters) when IC₅₀ is not known, i.e., no combination results in 50% inhibition (147).

The RSM method has been used in a number of in vitro studies in an attempt to better characterize antifungal drug-drug interactions as a function of specific drugs, by taking into account the absolute and relative concentrations of drugs in combination (76). RSM methods are especially useful for investigations of double and triple drug combinations over a very wide range of doses. Drug concentration and interaction data must be analyzed mathematically to generate descriptions of fungal growth response over a range of drug concentrations and ratios; the results can be visualized using three-dimensional contour or surface response plots.

Although RSM has a place when an in-depth analysis of drug-drug interactions is required, the testing will likely be undertaken by specialized laboratories having the appropriate expertise. Some investigators have suggested using a combination of the FICI and RSM approach and have shown that results obtained from the two correlate well (2, 222, 223). However, the inherent complexity of the RSM method makes it less attractive as the primary method to evaluate the interactions between antifungal agents in combination, and the FICI method has remained the method of choice for most studies.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Schematic representation of the criteria used to interpret results from time-kill studies of drug-drug interactions. The following criteria are commonly followed: synergy, a 2-log10 decrease in CFU/ml compared to the most active constituent; antagonism, a 2-log10 increase in CFU/ml compared to the least active agent; additivity, a <2- but >1-log10 decrease in CFU/ml compared to the most active agent; and indifference, a <2- but >1-log10 increase in CFU/ml compared to the least active agent.

Although simple to perform, the E-test method has drawbacks: (i) it has not undergone extensive testing using different organisms, with at least one report suggesting a species-dependent variation in MIC determination for Candida (203); (ii) it has not been tested against different antifungal agents, (iii) the effect of different growth media needs to be ascertained (RPMI-based agars generally appearing most useful) (177); (iv) growth of the fungal lawn tends to be nonuniform in some cases; and (v) the presence of a feathered or trailing growth edge can make the MIC determination confusing.

In spite of its limitations, checkerboard-based methodols remain the most popular approach for evaluating antifungal drug-drug interactions, evident in the fact that 75% of the drug interaction papers published from 1998 to 2002 in the Journal of Antimicrobial Chemotherapy used the checkerboard method in their analyses (163). Criteria commonly used to interpret in vitro methods to evaluate antifungal combinations are summarized in Table 2.

METHODS TO DETERMINE THE IN VIVO EFFICACY OF ANTIFUNGAL AGENTS IN COMBINATION

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In vivo assessment of combination therapy is based on animal studies followed by clinical trials, case series, or anecdotal reports. Most animal studies evaluate the efficacy of combination therapy based on tissue fungal burden of target organs (liver, kidneys, brain, etc.), tissue histopathology (sterilization of tissue), and/or survival studies. Several animal models have been developed, and involve mice (97, 98, 130, 154, 156, 211), rats (140), or rabbits (172). Factors that need to be considered while performing in vivo studies include variable drug absorption, distribution, and metabolism among animal species. Clinical trials evaluating the interactions between different antifungal agents have been very limited. This is not a surprise due to the costs involved and the fact that many companies that market these antifungal agents often do not encourage the idea of combining drugs. Clinical trials of combination therapy have been performed with nonneutropenic patients, and recent trials determined the efficacy of FLU plus AmB combination therapy compared to monotherapy with AmB (185). This clinical trial showed no detectable antagonism when using the FLU plus AmB combination and was instrumental in demonstrating that in vitro antagonism seen in combination therapy may not always be present in the clinical setting (see below).

COMBINATION THERAPY IN PRACTICE GUIDELINES FOR TREATMENT OF FUNGAL INFECTIONS

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The Practice Guidelines for the Treatment of Fungal Infections also suggest the use of specific drug combinations in certain situations (207). These guidelines addressed each pathogen separately and provided a guide for the use of single and combination therapy. The following sections provide a brief review of these guidelines, focusing on the recommendations to use combination therapy to treat fungal infections.

Abel-Horn et al. (1) demonstrated site-dependent variation in activity of the same antifungal combination. These investigators performed a prospective, randomized study to compare FLU monotherapy with the combination of AmB plus 5FC for treatment of 72 nonneutropenic intensive-care patients with systemic candidiasis. One arm (n = 36) received FLU 400 mg on day 1 followed by 200 mg/day for 14 days, while the other arm (n = 36) received AmB (1.0 to 1.5 mg/kg of body weight every other day) and 5FC (2.5 g three times a day) for 14 days. For pneumonia and sepsis, treatment success was comparable between the two groups: 18 of 28 FLU patients and 17 of 27 combination patients (P > 0.05, not significant). For peritonitis, however, the combination was more effective than FLU monotherapy with respect to both cure rate (55 and 25%, respectively) and pathogen eradication (86 and 50%, respectively). Such site-specific differences in the activity of antifungal combinations may be due to differences in tissue distribution of antifungal agents and to different effects of the in situ environments on the pharmacodynamic and pharmacokinetic characteristics of the drugs.

An open, prospective randomized trial compared combination therapy with AmB plus 5FC to FLU monotherapy in 40 surgical patients with deep-seated candidal infection (113). Most infections were due to gastrointestinal perforations; C. albicans was the most frequent isolate. Patients received either AmB (0.5 mg/kg of body weight) in combination with 5FC 2.5 g (three times a day; n = 20) or monotherapy with FLU (400 mg on the first day followed by 300 mg daily; n = 20). Although there was no difference between regimens with respect to cure rates, patients receiving combination therapy showed earlier elimination of fungal pathogens than did patients receiving monotherapy with FLU (median elimination time, 8.5 days in the FLU arm and 5.5 days in the AmB-plus-5FC group). Additional studies in support of AmB-plus-5FC and FLU-plus-5FC combinations include those performed by Levine et al. (122), Smego et al. (206), and Marr et al. (142), indicating the clinical usefulness of these combinations.

Among non-HIV patients with cryptococcal meningitis, combination therapy with AmB plus 5FC for 4 to 6 weeks is effective (19, 51). However, adverse yet non-life-threatening reactions to 5FC were reported in 32% of the patients (11 of 34) (19). Due to the toxicity issues associated with this regimen, a frequently used alternative treatment for cryptococcal meningitis in immunocompetent patients has been induction with AmB (0.5 to 1 mg/kg/day) plus 5FC (100 mg/kg/day) for 2 weeks, followed by consolidation therapy with FLU (400 mg/day) for an additional 8 to 10 weeks (167). Lower doses of FLU (200 mg/day) for longer periods (6 to 12 weeks) have also been suggested as maintenance therapy (195). For immunocompromised patients with low tolerance for 5FC, a similar therapy of induction, consolidation, and suppression has been suggested: AmB (0.7 to 1 mg/kg/day) for 2 weeks followed by FLU (400 to 800 mg/day) for 8 to 10 weeks followed by 6 to 12 months of lower-dose FLU (200 mg/day) (166, 195).

For patients with severe form of AIDS-related cryptococcal pneumonia, a combination of FLU (400 mg/day) plus 5FC (100 to 150 mg/day) for 10 weeks is suggested (166, 195). However, toxicity issues have been reported for this regimen (195). For HIV-associated cryptococcal meningitis, the primary treatment suggested is induction with AmB (0.7 to 1 mg/kg/day) plus 5FC (100 mg/kg/day) for 2 weeks followed by FLU (400 mg/day) for a minimum of 10 weeks (184, 227). If the severity of disease abates, the FLU dose can be subsequently reduced (200 mg/day) but should be continued for life. An alternative regimen is AmB (0.7 to 1 mg/kg/day) plus 5FC (100 mg/kg/day) for 6 to 10 weeks followed by FLU as a maintenance therapy (117, 118, 196, 236). Larsen et al. (117) showed that combination therapy with FLU (400 to 800 mg/day) plus 5FC (100 to 150 mg/kg/day for 6 weeks) is an effective regimen for cryptococcal meningitis in AIDS patients but reported toxicity in 28% of these patients (117). In a separate randomized trial, Mayanja-Kizaa et al. (145) determined the efficacy of combination therapy with low-dose FLU (200 mg/day for 2 months) + short-term 5FC (150 mg/kg/day for the first 2 weeks) followed by FLU maintenance therapy (200 mg three times per week for 4 months) against cryptococcal meningitis. These investigators showed that this combination therapy prevented death within 2 weeks and significantly increased the survival rate, with no serious adverse reactions (145).

The only other organism for which the guidelines mention combination therapy is coccidiodomycoses (71). AmB is often selected for treatment of patients with respiratory failure due to Coccidioides immitis or rapidly progressive coccidioidal infections. With other more chronic manifestations of coccidioidomycosis, additional treatment with FLU, ITRA, or ketoconazole (KETO) is common. The duration of therapy often ranges from many months to years; for some patients, chronic suppressive therapy is needed to prevent relapses (71). In cases of chronic fibrocavity pneumonia caused by C. immitis, the Practice Guidelines suggest an initial treatment with an azole (usually FLU) followed by higher doses of the azole. If the infection persists, adding AmB therapy is suggested. The guidelines also suggest that severe cases of coccidiodal meningitis and hydrocephalus be treated with a combination of AmB and azoles. These guidelines did not address combination therapy with the new agents due to the limited data available at the time these guidelines were being written. Studies focusing on these agents are described below.

ANTIFUNGAL COMBINATIONS AGAINST CANDIDA SPECIES

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In a separate study, Lewis et al. (123) used checkerboard dilution, E-test, and time-kill methods to determine the activities of FLU plus AmB, FLU plus 5FC, and AmB plus 5FC against six Candida isolates (three C. albicans isolates and one isolate each of C. glabrata, C. krusei, and C. tropicalis). The checkerboard method showed that all three combinations were indifferent or synergistic against all three C. albicans isolates, while the E-test showed that AmB plus FLU was antagonistic for two of the three C. albicans isolates and indifferent for the third isolate. Similar differences in interpretation between checkerboard and E-test methods were observed for exposure of C. glabrata to the three combinations, which were synergistic by checkerboard but indifferent by E-test. In general, there was better agreement between results from the E-test and time-kill methods.

These wide variations in results can often be traced to different types of strains, drug concentrations, method of evaluation, and the criteria of interpretation, and they underscore the necessity of a uniform set of guidelines (similar to the NCCLs guidelines for in vitro susceptibility testing of antifungals tested singly) for testing of antifungal combinations before results from these studies can be widely correlated.

(ii) In vivo studies. The first study to examine whether antagonism in vitro was pertinent to experimental candidiasis in vivo compared AmB plus FLU in three murine models of invasive candidiasis: acute and subacute infections in immunocompetent mice, and subacute infection in immunosuppressed mice (220). This study showed that the order of administration of FLU with respect to AmB, using low-dose and high-dose regimens for each drug, was not a factor in rate of animal survival. In acute infection in immunocompetent mice, the combination was not antagonistic. On day 9 postinfection, the survival of mice treated with the antifungal combination was significantly increased compared to those treated with FLU alone (P < 0.01). In contrast, survival when the combination was used was as effective as that when AmB was used alone (75 and 90%, respectively; P > 0.1). For treatment of subacute infection in immunocompetent mice, the results were similar: no antagonism was noted, and survival rates were comparable (P > 0.1) for groups of mice receiving the combination or AmB alone. For treatment of less acute infection in immunocompromised mice, the combination was more effective on day 30 than was FLU alone (100 and 10% survival, respectively; P < 0.01), and was as effective as AmB alone (100 and 70% survival, respectively; P > 0.1).

In a separate in vivo study, Sanati et al. (198) demonstrated no antagonism between AmB and FLU in combination therapy in two candidiasis models: a neutropenic-mouse model of hematogenously disseminated candidiasis and a rabbit model of infective endocarditis. In the former model, the combination was responsible for significantly increased survival and reduced fungal densities in both the kidneys and the brain; in the latter model, significant reductions of fungal densities in cardiac vegetations were observed. Louie et al. (134) further evaluated the efficacy of FLU plus AmB in a murine model of systemic candidiasis for one FLU-susceptible strain (MIC, 0.5 µg/ml), two strains with intermediate FLU resistance (FLU-midresistant strains) (MIC, 64 and 128 µg/ml), and one highly FLU-resistant strain (MIC, 512 µg/ml) of C. albicans. Comparison parameters were survival and fungal densities in the kidneys of infected mice. For the FLU-susceptible and FLU-midresistant strains, the FLU-plus-AmB combination was antagonistic, as shown by both quantitative culture results and survival. Interestingly, for the highly FLU-resistant strain (which is where combination therapy is most likely to be used), no antagonism was observed (134). These results need to be verified by further studies. Although the breakpoints defining FLU susceptibility and resistance are arbitrary and do not follow the NCCLS breakpoints, these studies indicates that antagonism between AmB and FLU in animal models of candidiasis may also be influenced by the azole resistance phenotype of C. albicans.

(iii) Clinical studies. A crucial question was whether the pattern seen in animal models will also be present in the clinical setting. A recent clinical trial attempted to address this issue, in nonneutropenic patient populations (185). These authors compared high-dose FLU monotherapy and high-dose FLU in combination with conventional AmB in a prospective clinical study of nonneutropenic patients with candidemia. Patients with positive blood culture for Candida spp. other than C. krusei and with fever or hypotension within 4 days or signs of local candidal infection within 2 days were randomized to receive either FLU (800 mg/day plus placebo), or FLU plus AmB (0.7 mg/kg/day). In both regimens, placebo or AmB was given only for the first 3 to 8 days while FLU was given for 14 days after symptom resolution. Analysis of 211 patients (FLU, n = 104; AmB plus FLU, n = 107) defined clinical success as blood culture clearance and symptom resolution. Infections identified at baseline were due to C. albicans (60%), C. glabrata (15%), C. parapsilosis (12%), C. tropicalis (9%), or other species (4%). Patient groups were comparable at baseline, except that those receiving FLU monotherapy had higher mean (± standard error) acute physiology and chronic health evaluation II (APACHE II) scores (16.9 ± 0.6 versus 15.1 ± 0.7; P = 0.046). Both overall success rates and blood clearance rates were statistically higher for the combination. Success rates were 68% for the combination and 56% for FLU monotherapy (P = 0.045); blood clearance failure rates were 17 and 7% for FLU monotherapy and the combination, respectively (P = 0.02). Additionally, the success rate by species was the same between regimens. Mortality rates at 30 and 90 days were not significantly different between groups. Results were also comparable between groups for treatment failures secondary to both renal toxicity and hepatotoxicity; patients receiving combination treatment had expectedly higher elevations in creatinine levels (16%) compared with those receiving monotherapy (6%; P = 0.021). The odds of treatment failure were decreased by having an infection attributable to C. parapsilosis (odds ratio [OR] = 0.208; P = 0.018) or by receiving combination treatment (OR = 0.681, 0.22); the odds of treatment failure were increased for patients with higher APACHE II scores (OR = 1.090 per point; P = 0.0006) or a requirement for total parenteral nutrition (TPN) (OR = 3.00; P = 0.0008). Treatment response was independent of prestudy antifungal therapy. The authors concluded that combination treatment with FLU plus AmB was not antagonistic compared with FLU monotherapy. Although the analysis was qualified by differences in baseline APACHE II scores, combination treatment overall trended toward better success and more rapid blood culture clearance, similar overall mortality for both regimens, but more episodes of renal toxicity for the combination (185). This is a seminal study because it adds significant input to the antagonism debate, at least in this patient population.

Amphotericin B plus 5-fluorocytosine: in vitro studies. Keele et al. (105) reported that no antagonistic or additive effects were observed in vitro for the combination of AmB and 5FC against C. albicans and C. neoformans. Using time-kill analysis, three isolates each of C. albicans and Cryptococcus neoformans were tested with three monotherapy and two combination drug regimens of 5FC and AmB. Monotherapy regimens included 5FC (50 µg/ml), low-dose AmB (0.125 µg/ml), and high dose AmB (2.4 µg/ml). Combinations used a fixed dose of 5FC with either low-dose AmB (5FC at 50 µg/ml plus AmB at 0.125 µg/ml) or high-dose AmB (5FC at 50 µg/ml plus AmB at 2.4 µg/ml). None of the interactions were antagonistic. There were no differences between combination regimens with respect to either 5FC preexposure or timing, i.e., staggered versus simultaneous administration. In both the low-dose and high-dose combination regimens, drug-drug interactions were indifferent. Furthermore, regardless of the AmB concentration, no antagonism or additive effects were observed. The authors stated the importance of their finding of a lack of any antagonistic interaction between the two drugs in combination, reflecting its potential for improved clinical treatment of certain fungal infections based on the different mechanisms of action, distinct toxicity profiles, and complementary pharmacokinetic profiles (105).

Amphotericin B plus 5-fluorocytosine plus fluconazole: in vitro studies. Using a microdilution plate technique, Ghannoum et al. (76) studied one-, two-, and three-drug regimens of AmB, FLU, and 5FC against three isolates each of C. albicans and C. neoformans. Results of two-drug combinations against both C. albicans and C. neoformans showed that inhibition by AmB plus FLU was greater than inhibition by either drug alone. At low concentrations of AmB, addition of 5FC enhanced the growth-inhibitory effect against C. albicans, but antagonism was noted at higher concentrations of AmB. Data for the three-drug pairs (AmB plus FLU, AmB plus 5FC, and FLU plus 5FC) were presented as contour plots, which showed distinct upward or downward contour plots for C. neoformans and C. albicans (Fig. 3). Results of the three-drug combinations for C. neoformans showed inhibition with AmB plus different concentrations of FLU plus a single fixed dose of 5FC. In the presence of 5FC, the combined effects of AmB and FLU on the growth of C. neoformans remained indifferent; when the AmB concentration was greater than approximately 1 to 1.2 µg/ml, addition of 5FC had no further effect on growth. These investigators suggested that the effects of a drug combination on in vitro fungal growth depends on the ratios and concentrations of the drugs used, as well as the fungal strains tested, apart from other differences related to variations in study design, pathogens, drug conditions, and regimens.

View larger version (41K): [in this window] [in a new window]   FIG. 3. Stacked contour plots showing the combined effects of AmB, FLU, and 5-FC on the growth of C. neoformans. Susceptibilities of two- and three-drug combinations were determined using a microdilution method. The range of concentrations of drugs used for the combinations was based on the individual MICs and included concentrations both above and below the MICs. Analysis of antifungal drug concentrations and interactions affecting fungal growth was performed using multifactorial design models. Eighty data points were generated describing growth responses to a wide range of antifungal concentrations and ratios. The data were used to generate contour plots and surface response plots and to develop mathematical equations with coefficients providing the best empirical fit of the experimental data. The numerical values on the contours indicate the percentages of maximal fungal growth in the presence of the various combined antifungal concentrations. The points on the lower contour plane indicate the concentrations of AmB and FLU used at each of the three levels of 5-FC shown to develop the contour plots. Contour plots with generally upward concave form suggest favorable interactions, while those with concave downward form suggest less favorable interactions. The equation describing the best fit of the growth data was as follows: growth = 101 – 27(FLU) – 33(AmB) – 173(5FC) + 7(FLU)(AmB) + 19(FLU)(5FC) + 36(AmB)(5FC) – 7(AmB)(FLU)(5FC) + 2(FLU)2 + 78(5FC)2. These data demonstrate that at AmB concentrations of >1.0 to 1.2 µg/ml, addition of Flu had no further effect on growth. Reprinted from reference 76 with permission.

Fluconazole plus 5-fluorocytosine against candidiasis. Since FLU and 5FC act on the fungal cell by completely different routes, it is expected that a combination of these two drugs will not be antagonistic and should be synergistic or noninteractive. The efficacy of this combination has been tested both in vitro and in vivo, as described below.

(i) In vitro studies. Only a limited number of studies have been performed with the FLU-plus-5FC combination against Candida infections. In a recent study, Te Dorsthorst et al. (223) evaluated the in vitro efficacy of FLU plus 5FC against 27 Candida species including C. albicans (n = 9), C. glabrata (n = 9), and C. krusei (n = 9). These investigators defined drug interactions as synergy if the FICI was <1, additive if the FICI was 1, and antagonistic if the FICI was >1. Synergism and antagonism were observed for five and four C. albicans isolates, respectively. For C. krusei, synergy was observed for only one isolate, and antagonism was observed for eight isolates. Notably, this combination was antagonistic for all the C. glabrata isolates tested (223).

(ii) In vivo studies. In vivo studies with 5FC-plus-FLU combinations have shown both synergistic and antagonistic interactions. Graybill et al. (86) evaluated the in vivo efficacy of combining 5FC with azoles in a murine model of disseminated C. tropicalis infection. Survival and tissue fungal burden of the spleen and kidneys were used to evaluate the efficacy of antifungal therapy. These studies showed that combining 5FC with FLU did not increase efficacy against C. tropicalis infection (86). In a separate study, Atkinson et al. (7) established an immunosuppressed-mouse model of C. glabrata infection to evaluate the efficacy of combinations of AmB, 5FC, and FLU treatments in vivo. Treatment with FLU, 5FC, AmB, or a combination was begun 1 day after infection. Kidneys and spleen CFU counts following 5 days of treatment revealed that the FLU-plus-5FC combination was superior to these agents alone in reducing the tissue fungal burden in the kidneys for one isolate of C. glabrata. High doses of FLU alone produced modest reductions in kidneys counts but did not reduce spleen tissue fungal burden. Moreover, there was a poor correlation between in vitro MICs and in vivo results (7). In a subsequent study, the efficacies of FLU plus 5FC and FLU plus AmB were evaluated in a rabbit model of C. albicans endocarditis, endophthalmitis, and pyelonephritis (136). In all the treatment groups except 5FC monotherapy, 93% of animals appeared well and survived until they were sacrificed. On day 5, the relative decreases in CFU per gram in the vitreous humor were greater in groups receiving FLU alone and in combination with 5FC or AmB than in groups receiving AmB or 5FC monotherapies (P < 0.005). However, the results were similar thereafter. In the choroid retina. 5FC was the least active drug. However, there were no differences in choroidal fungal densities between the other treatment groups. Both 5FC plus FLU and 5FC plus AmB demonstrated enhanced killing in cardiac vegetations compared with monotherapies (P < 0.03). In contrast, AmB plus FLU demonstrated antagonism in the treatment of C. albicans endocarditis and in reduction of kidney CFU (136).

(iii) Case reports. Although studies using animal models suggested mixed interactions for 5FC-plus-FLU combinations, some clinical case reports have indicated successful treatment with this regimen. Scheven et al. (201) reported successful treatment of C. albicans sepsis with FLU-plus-5FC combination therapy. Recently, Girmenia et al. (82) reported two cases of a 46-year-old patient (with C. krusei infection) and a 10-year-old child (with C. glabrata infection) who were successfully treated with FLU-plus-5FC regimens. In vitro susceptibility testing of both strains showed resistance to FLU (MIC, >64 µg/ml). Combination therapy included FLU (600 mg/day) plus 5FC (4 g three times a day [tid], 150 mg/kg/day) for C. krusei; and a regimen of FLU (300 mg/day) plus 5FC (2 g/tid, 150 mg/kg/day) for C. glabrata infection. Combination therapy resulted in clearing of symptoms within 1 to 2 weeks. Subsequent in vitro susceptibility testing using FICI and time-kill methods revealed no antagonistic interactions (FICI = 1.0019) against both isolates.

Therefore, the in vitro and animal model results for the FLU-plus-5FC combination are not accurately predictive for clinical application; this may be due to differences in drug availability and tissue distribution of the drug in the in vivo setting or to differences in strains used in the in vitro animal experiments and clinical isolates. Although these studies suggest that combining FLU and 5FC may have utility in the treatment of azole-resistant Candida, further evaluation is necessary.

Voriconazole combinations. (i) In vitro studies. Checkerboard methods and visual and colorimetric end points were used to assess drug-drug interactions of 5FC combined in vitro with either AmB or VORI against C. albicans, C. glabrata, C. krusei, and C. tropicalis isolates (M. A. Ghannoum and N. Isham, Abstr. 29th Annu. Meet. Eur. Group Blood Marrow Transplant. abstr. P671, 2003). Interactions of 5FC with either VORI or AmB were synergistic or additive against 50% of C. albicans isolates tested. Interactions against C. glabrata isolates were additive for 5FC plus AmB (60%), with no antagonism noted; however, VORI-plus-5FC interactions were indifferent (50%) or antagonistic (50%). Additionally, 5FC-plus-AmB interactions were additive against 70% of the C. krusei isolates, which are innately resistant to FLU. Finally, VORI-plus-5FC exhibited synergistic or additive interactions against 40% of C. tropicalis. In another study, Ghannoum et al. (M. A. Hossain, M. A. Ghannoum, and D. J. Sheehan, Abstr. Trends Invasive Fungal Infect., abstr. P-59, 2001) determined the efficacy of VORI in combination with conventional AmB, liposomal AmB (LAmB), 5FC, FLU, micafungin (FK 463, MICAF), or CAS against isolates of Candida and Cryptococcus. No antagonism was observed for any combination evaluated. Across all combinations of VORI and another agent, 74% of the interactions were indifferent while 26% were synergistic or additive. Combinations of VORI and MICAF were synergistic against C. albicans and non-albicans species.

A separate study by Ghannoum et al. (M. A. Ghannoum, N. Isham, M. A. Hossain, and D. J. Sheehan, Abstr. Int. Immunocompromised Host Soc. Conf. abstr. 65, 2002) supported the results of an earlier trial using the same methods and definitions against nine Candida isolates. All the isolates were susceptible to VORI plus AmB and to CAS plus MICAF. Overall, VORI combinations were 33% additive, 4% synergistic, and 63% indifferent, with no observed antagonism. VORI-plus-AmB combinations were 17% additive and 83% indifferent. VORI-plus-FLU combinations were 42% additive and 58% indifferent. VORI-plus-MICAF combinations were 17% synergistic, 17% additive, and 58% indifferent. As before, the interactions were strain specific and correlations with clinical outcome were not determined. These studies emphasize the notion that VORI is unlikely to be antagonistic when combined with other antifungal agents.

Ernst et al. (63) used broth microdilution and E-test methods, and time-kill studies to determine the antifungal activities of AmB, FLU, 5FC, and VORI alone and in combination against 11 isolates of C. lusitaniae. AmB demonstrated fungicidal activity against most isolates tested, whereas FLU, VORI, and 5FC demonstrated primarily fungistatic activities. For AmB-resistant isolates (MIC, 3µg/ml), a trend toward shorter times taken to reach the fungicidal end point was observed in cells exposed to AmB plus 5FC, compared to those exposed to AmB alone (9.3 and 14.5 h, respectively; P = 0.068). These isolates (3 of 4) also showed a 0.5-log-greater reduction in fungal growth in the presence of AmB plus 5FC, indicating a greater extent of activity by this combination (63). Other combinations did not produce improvement in activity compared to single agents (63).

(ii) In vivo studies. Recently, VORI has been studied in combination with 5-FC or AmB against systemic candidiasis in immunocompetent guinea pigs (C. A. Hitchcock, R. J. Andrews, B. G. H. H. Lewis, G. W. Pye, G. P. Oliver, and P. F. Troke, Int. J. Antimicrob. Agents 19(SI):S74, 2002; 4th Eur. Congr. Chemother. Infect. 2002), and the results are reviewed below.

(iii) Combination with 5-fluorocytosine or amphotericin B against systemic candidiasis in immunocompetent guinea pigs. When VORI (0.1, 1, and 5 mg/kg p.o. twice daily [bid] for 5 days) was combined with 5-FC (5 mg/kg p.o. bid for 5 days), there was no evidence of antagonism in terms of survivors or reductions in kidney fungal loads. At 0.1 and 1 mg of VORI per kg, addition of 5-FC caused significantly greater decreases in fungal loads compared with the same VORI doses alone (P < 0.01). When combined with AmB (1 mg/kg intraperitoneally [i.p.]) VORI (0.1 and 1 mg/kg p.o. bid for 5 days) showed no antagonism and was significantly more effective in reducing fungal loads than was the corresponding VORI monotherapy (P < 0.001). The mean fungal load in animals treated with a combination of the highest dose of VORI (5 mg/kg p.o. bid for 5 days) and AmB (1 mg/kg i.p.) was significantly higher (P < 0.05) than that in animals receiving VORI monotherapy. Parallel groups of animals dosed with FLU (0.1, 1, and 5 mg/kg p.o. bid for 5 days) were included for comparison with VORI in these studies. As expected, when combined with AmB, FLU demonstrated a similar trend to that observed for VORI. Thus, addition of AmB (1 mg/kg i.p. once a day for 5 days) significantly (P < 0.01) increased the reduction in kidney fungal load at the lowest doses of FLU (0.1 and 1 mg/kg p.o. bid for 5 days). At the highest dose of FLU (5 mg/kg p.o. bid for 5 days), there was no significant difference (P < 0.01) in fungal load between the monotherapy and combination therapy groups. In general, combinations with VORI, when tested against Candida, did not display any antagonism. Clinical data are needed to determine the validity of these interpretations.

Caspofungin combinations. The candins, which inhibit cell wall synthesis through disruption of 1,3-ß-D-glucan synthesis and possibly 1,6-ß-D-glucan synthesis (45, 85, 121, 165), may enhance the activity of either the azoles or AmB by increasing the rate or degree of their access to the cell membrane (Fig. 1 and 4). Hossain et al. (98) recently evaluated in vitro and in vivo efficacies of CAS plus AmB against an azole-resistant strain of C. albicans isolated from a patient who failed to respond to FLU therapy. In vitro antifungal susceptibility testing was performed based on the M27-A method and drug interactions assessed using the checkerboard technique. Interactions were defined as synergy (FICI < 0.5), positive (FICI < 1), negative (FICI > 1), or antagonism (FICI > 4.0). These investigators showed that combination of CAS and AmB resulted in a two- and fourfold reduction in MIC of AmB and CAS, respectively, and exhibited a positive in vitro interactive effect (FICI = 0.75). Furthermore, in vivo studies showed that the combination of CAS (0.002 mg/kg) plus AmB (0.016 mg/kg) significantly prolonged animal survival compared with untreated controls (P = 0.006). The proportions of mice treated with CAS plus AmB survived longer (72%) than those treated with the single drugs alone (50% for AmB, 22% for CAS), although this difference was not statistically significant (P = 0.36) compared to the effect of AmB alone. The CAS-plus-AmB combination was the only treatment that resulted in significant reduction in kidney CFU counts (P = 0.05). Compared to untreated controls, CFU counts in the brains of infected mice were significantly reduced when the animals were treated with CAS plus AmB (P = 0.005) or CAS alone (P = 0.05). However, no significant difference was observed between animals treated with CAS alone and CAS plus AmB (P = 0.094). Interestingly, no antagonistic interactions were observed between the two agents, which tended to result in favorable interactions in vivo and in vitro (98). Graybill et al. (87) recently investigated the in vivo efficacy of the CAS-plus-FLU combination in a murine model of candidiasis. FLU was administered at doses ranging from 0.06 to 5 mg/kg, while CAS doses ranged from 0.0005 to 5 mg/kg/day. Kidney tissue fungal burden was used as a measure of efficacy. These studies showed that none of the CAS-plus-FLU combinations led to any benefit over using the individual drugs alone.