Efficacy of voriconazole in treatment of murine pulmonary blastomycosis.

We evaluated the efficacy of voriconazole, a new broad-spectrum triazole antifungal compound, in the treatment of murine pulmonary blastomycosis. Since mice metabolize voriconazole rapidly, we took advantage of our previous observation that administration of grapefruit juice to mice resulted in suitable serum voriconazole concentrations so that treatment studies with mice could be done (A. M. Sugar and X.-P. Liu, Med. Mycol. 38:209-121, 2000). Our results show that voriconazole prolonged survival in a dose-dependent fashion and that the fungal burden in the lungs was decreased by voriconazole administered at 40 mg/kg of body weight/day. Voriconazole should be studied in humans with blastomycosis.

Overview: antifungal combination therapy.

We have yet to realize the clinical benefits of combination antifungal therapy, but the literature is beginning to show the potential positive and negative aspects of this approach. Detailed laboratory-based study of each new antifungal compound will be required in order to define the interactions between the different drugs. Most important in this discussion is that a determination of the most appropriate experimental design and parameters to reflect the outcomes in the treatment of human mycoses is required. That will be the challenge for the next several years. In the meantime, combination antifungal therapy should be approached carefully and be initiated on a case-by-case basis following consideration of all the options available for the patient.

Effect of grapefruit juice on serum voriconazole concentrations in the mouse.

Voriconazole is a broad spectrum, triazole antifungal drug now well into the final phases of clinical trials in humans. During preclinical phases of development, it was found that when administered to mice, one of the more important animals used in the in vivo evaluation of antifungal compounds, serum voriconazole concentrations were very low at best and often undetectable. This was due to a combination of high clearance and extensive metabolism by cytochrome P450 enzymes. As a result, mice were abandoned as being suitable for further study of voriconazole and most subsequent work with voriconazole has been performed in the guinea pig. In this study, we show that the administration of grapefruit juice, a known inhibitor of cytochrome P450 enzymes, is effective in producing measurable serum concentrations of voriconazole in mice when the drug is administered once daily. Serum voriconazole concentrations were < 3 microg ml(-1) at all time points in mice not receiving grapefruit juice. In contrast, grapefruit juice administered by once daily gavage or continuously in lieu of water in the water bottle resulted in serum voriconazole concentrations ranging 0.4-2.6 and 1.8-5.8 microg ml(-1), respectively, with increasing concentrations observed over the 10-day evaluation period. Further studies to elucidate the precise mechanism of action and optimal dosing schedule in mice can now be performed to improve our understanding of the pharmacokinetics of voriconazole in the mouse.

Combination antifungal therapy in treatment of murine pulmonary mucormycosis: roles of quinolones and azoles.

Amphotericin B is the only recognized antifungal used in the treatment of mucormycosis. In this study, we evaluated various combinations of amphotericin B, fluconazole, and trovafloxacin or ciprofloxacin in the treatment of murine pulmonary mucormycosis. The combination of fluconazole and a quinolone has a marked effect on the outcome of murine pulmonary mucormycosis. Even though we did not optimize therapy with the drugs, these experiments suggest that azoles, especially fluconazole, in combination with either trovafloxacin or ciprofloxacin were effective in the treatment of this aggressive mycosis in the mouse model.

Prospective multicenter surveillance study of funguria in hospitalized patients. The National Institute for Allergy and Infectious Diseases (NIAID) Mycoses Study Group.

Although fungal urinary tract infections are an increasing nosocomial problem, the significance of funguria is still not clear. This multicenter prospective surveillance study of 861 patients was undertaken to define the epidemiology, management, and outcomes of funguria. Diabetes mellitus was present in 39% of patients, urinary tract abnormalities in 37.7%, and malignancy in 22.2%; only 10.9% had no underlying illnesses. Concomitant nonfungal infections were present in 85%, 90% had received antimicrobial agents, and 83.2% had urinary tract drainage devices. Candida albicans was found in 51.8% of patients and Candida glabrata in 15.6%. Microbiological and clinical outcomes were documented for 530 (61.6%) of the 861 patients. No specific therapy for funguria was given to 155 patients, and the yeast cleared from the urine of 117 (75.5%) of them. Of the 116 patients who had a catheter removed as the only treatment, the funguria cleared in 41 (35.3%). Antifungal therapy was given to 259 patients, eradicating funguria in 130 (50.2%). The rate of eradication with fluconazole was 45.5%, and with amphotericin B bladder irrigation it was 54.4%. Only 7 patients (1.3%) had documented candidemia. The mortality rate was 19.8%, reflecting the multiple serious underlying illnesses found in these patients with funguria.

Combined treatment: antifungal drugs with antibodies, cytokines or drugs.

To improve present results with antifungal drugs, modulation of the host immune response is being explored. Human phagocytes of various lineages work cooperatively in vitro with antifungal drugs to inhibit or kill fungal pathogens, and this activity is augmented by several recombinant cytokines. Monoclonal antibodies against the cryptococcal capsule have been shown to act as an adjunct in enhancing the outcome of cryptococcosis in animal models. This approach is now being pursued in systematic clinical trials. In experimental candidiasis, several manipulations of the immune system, via administration of cytokines, gene deletion or antibodies to cytokines, have been shown to significantly affect survival and fungal clearance in vivo. This approach has already been demonstrated to be of benefit with recombinant human granulocyte-colony stimulating factor adjunct therapy of human candidiasis. Combining antifungal drugs of different classes may enhance their therapeutic effect.

Fluconazole in transplant recipients: options and limitations.

Fluconazole is currently a first-line agent used for therapy of non-critically ill patients with candidal infection. Its efficacy, the availability of an oral formula, and its relatively low toxicity make it a very attractive drug for use in many clinical situations. The advisability of prophylaxis and empirical treatment in transplant patients is a difficult issue for the following reasons: the potential emergence of resistance to the azoles, the lack of solid data establishing its advantage over placebo and/or oral nonabsorbable antifungal agents in some of the clinical conditions encountered, its ineffectiveness against molds, and its cost. Judicious use of fluconazole where its efficacy has been well established would provide the best therapy for patients and would limit the emergence of potential pathogens. As new antifungal agents are approved for clinical use, appropriate clinical trials will need to be designed and conducted in order for clinicians to make rational decisions in selecting the most appropriate drug for the specific indication. Prophylaxis and treatment with fluconazole in various transplant situations is reviewed.

Interactions of itraconazole with amphotericin B in the treatment of murine invasive candidiasis.

The interactions of amphotericin B and itraconazole were studied in murine invasive candidiasis. Candida albicans-infected mice were treated for 10 consecutive days, 24 h after infection. Survival was monitored over 30 days and kidney cultures were done. Mice treated with amphotericin B (0.2 mg/kg/day intraperitoneally) or itraconazole (100 mg/kg/day by oral gavage in two divided doses/ day) had a 30-day survival of 20% or 40%. Concomitant administration of both drugs resulted in 100% mortality; 90% of mice treated with amphotericin B (1 mg/kg/day) survived. With the combination, 100% were dead by day 28 (P < or = .001 vs. amphotericin B). With sequential therapy (i.e., 5 days with one drug and then 5 days with the other), survival was inferior to that with amphotericin B alone but similar to that with itraconazole alone. Kidney culture results confirmed the antagonism of the combination compared with amphotericin B alone. In treatment of murine invasive candidiasis, the concomitant or sequential use of amphotericin B and itraconazole results in a negative interaction.

Antifungal activity of 3'-deoxyadenosine (cordycepin).

The antifungal activity of the nucleoside analog 3'-deoxyadenosine (cordycepin) was studied in a murine model of invasive candidiasis. When protected from deamination by either deoxycoformycin or coformycin, both of which are adenosine deaminase inhibitors, cordycepin exhibited potent antifungal efficacy, as demonstrated by prolongation of survival and a decrease in CFU in the kidneys of mice treated with cordycepin plus an adenosine deaminase inhibitor. The antifungal effect was seen with three different Candida isolates: Candida albicans 64, a relatively fluconazole-resistant clinical isolate of C. albicans (MIC, 16 micrograms/ml), and the fluconazole-resistant Candida krusei. Cordycepin and related compounds may provide another avenue for the discovery of clinically useful antifungal drugs.

Cloning and sequencing of a Candida albicans catalase gene and effects of disruption of this gene.

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.

Short report: use of the polymerase chain reaction to detect Paracoccidioides brasiliensis in murine paracoccidioidomycosis.

The polymerase chain reaction (PCR) was used to detect the presence of Paracoccidioides brasiliensis in a murine model of disseminated paracoccidioidomycosis. Using a previously identified P. brasiliensis-specific DNA sequence, P. brasiliensis DNA was detected in serum of five experimentally infected mice. The PCR method was able to detect as little as 10 pg of P. brasiliensis DNA in serum, and it was more sensitive than blood culture isolation (five of five were PCR positive versus two of five blood culture positive). There were no amplified fragments in serum from three noninfected control mice. Lung colony counts were similar in all infected mice and reflected a similar degree of P. brasiliensis infection at the time the samples were drawn. The relatively short processing time for the PCR, when compared with culture, its sensitivity, and the possibility of using serum samples for analysis, are important factors favoring this method for the diagnosis of paracoccidioidomycosis. Future studies should include the detection of P. brasiliensis in patients with different clinical forms of paracoccidioidomycosis.

Antifungal combination therapy: where we stand.

Amphotericin B has been used in combination, simultaneously or sequentially, with fluconazole and itraconazole. However, there are no data from clinical trials to suggest the most appropriate use of these drug combinations. In vitro work has consistently described antagonism of polyenes and azoles, but new work suggests that hydrophilic azoles (e.g. fluconazole) with amphotericin B may not result in antagonistic effects, whereas lipophilic azoles (e.g. ketoconazole, itraconazole) antagonize the antifungal effects of amphotericin B. Experimental studies in animals with invasive candidiasis show that fluconazole and amphotericin B are not antagonistic and may be additive in their effects. In contrast, itraconazole and amphotericin B are antagonistic in this same murine candidiasis model. In animals infected with Aspergillus, itraconazole plus amphotericin B seem to be antagonistic when used simultaneously and possibly when used sequentially. Nevertheless, initial therapy with amphotericin B followed by itraconazole in patients with aspergillosis is a common clinical strategy. Recent data and concepts concerning the combined use of available antifungal drugs are discussed in this paper.

Effectiveness of quinolone antibiotics in modulating the effects of antifungal drugs.

Quinolone antibacterial drugs inhibit DNA gyrase, a type 2 topoisomerase. Since topoisomerases are present in eukaryotic cells, it was of interest to evaluate the antifungal activities of two clinically available quinolones, ciprofloxacin and trovafloxacin, alone and in combination with amphotericin B or fluconazole, in vitro against Candida albicans and in a murine model of invasive candidiasis. The in vitro activity of trovafloxacin was also tested against other yeasts and molds. In vitro, trovafloxacin exhibited no antifungal activity against any of the fungi (MIC, >250 microg/ml). There was also no effect of the quinolone on the in vitro activity of either antifungal drug. Marked antifungal effects were seen, however, in the murine model of candidiasis. In all experiments, control mice infected intravenously with C. albicans were dead by day 24. While either quinolone had minimal effects on survival of mice when used alone in oral doses of up to 40 mg/kg twice daily, the combination of the quinolone with fluconazole (40 or 80 mg/kg given twice daily by oral gavage) was more effective in prolonging survival than was fluconazole alone. Colony counts of kidneys on days 12 and 30 showed similar reductions in C. albicans recovered from mice treated with fluconazole with or without trovafloxacin or amphotericin B with or without trovafloxacin. Survival of mice treated with a suboptimal dose of amphotericin B (0.2 mg/kg/day) was also improved when trovafloxacin (40 mg/kg) given twice daily was included (0 versus 27%, respectively; P < 0.05). While the mechanisms of action of the combination of trovafloxacin and amphotericin B or fluconazole are unclear, further work focused on fungal topoisomerase inhibition and the mechanism of the antifungal effect of quinolone antibacterial drugs is warranted.

In vitro and in vivo activities of SCH 56592 against Blastomyces dermatitidis.

The new triazole derivative SCH 56592 has been tested in a National Committee for Clinical Laboratory Standards-adapted in vitro susceptibility test, and its activity against 12 isolates of Blastomyces dermatitidis yeast-like forms has been compared with those of amphotericin B, itraconazole, and fluconazole. SCH 56592 was the most active of the four compounds, with an MIC at which 90% of the isolates are inhibited of 0.06 microgram/ml and a minimal fungicidal concentration at which 90% of the isolates are inhibited of 4 micrograms/ml. The results of the treatment of mice infected with B. dermatitidis with three different doses of SCH 56592 (25, 5, or 1 mg/kg of body weight), amphotericin B (1 mg/kg), or itraconazole (150 mg/kg) confirmed the potent activity of SCH 56592. Survival was prolonged at each dose of SCH 56592, and sterilization of the lungs occurred in the high-dose group but not in the groups treated with itraconazole or fluconazole. SCH 56592 is a promising new azole antifungal drug that should be studied in humans with blastomycosis.

Paracoccidioidomycosis and AIDS: an overview.

The scarcity of reported cases of paracoccidioidomycosis and AIDS remains unexplained. We review the details of the 27 cases reported in the medical literature. Paracoccidioidomycosis occurs in patients with advanced AIDS who are not receiving prophylaxis for Pneumocystis carinii pneumonia with trimethoprim-sulfamethoxazole, which is also effective against Paracoccidioides brasiliensis. Clinical manifestations include prolonged fever, weight loss, generalized lymphadenopathy, splenomegaly, hepatomegaly, and skin rash. Diagnosis can often be made by direct microscopic examination and culture of the fungus from skin and lymph node specimens and occasionally from sputum, blood, spinal fluid, and bone marrow specimens. Since antibodies to P. brasiliensis are occasionally detected, the diagnosis should not be ruled out for patients whose serology is negative. Despite specific therapy with different regimens, the overall mortality of paracoccidioidomycosis among patients with AIDS is high (30%). The prognosis can be improved by earlier diagnosis and aggressive therapy with amphotericin B, followed by lifelong immunosuppressive therapy with trimethoprim-sulfamethoxazole. Health care providers caring for human immunodeficiency virus-infected patients who live or have resided in areas in which paracoccidioidomycosis is endemic must be aware of the possibility that this systemic mycosis may occur and have potentially severe consequences.

Intravascular catheter exchange and duration of candidemia. NIAID Mycoses Study Group and the Candidemia Study Group.

During a comparative trial of amphotericin B vs. fluconazole for treatment of candidemia in nonneutropenic patients, data on the management of intravascular catheters were collected. Complete records were available for 91% of the 206 study patients. For the subset of patients with a catheter in place at the time of their first positive blood culture, removal and replacement of all intravascular catheters without exchange over a guidewire from a preexisting line on or before the first day the study drug was administered were associated with a reduction in the subsequent mean duration (+/- SE) of candidemia, from 5.6 +/ -0.8 days to 2.6 +/- 0.5 days (P < .001).