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Problems in the design and reporting of trials of antifungal agents encountered during meta-analysis.

Johansen HK, Gotzsche PC.

The Nordic Cochrane Centre, Rigshospitalet, Copenhagen, Denmark.

Meta-analyses may become biased if the reported data in the individual trials are biased and if overlap among trials cannot be identified. We describe the unanticipated problems we encountered in collecting data for a meta-analysis comparing a new antifungal agent, fluconazole, with amphotericin B in patients with cancer complicated by neutropenia. In 3 large trials that comprised 43% of the patients identified for the meta-analysis, results for amphotericin B were combined with results for nystatin in a "polyene" group. Because nystatin is recognized as an ineffective drug in these circumstances, this approach creates a bias in favor of fluconazole. Furthermore, 79% of the patients were randomized to receive oral amphotericin B, which is poorly absorbed and not an established treatment, in contrast to intravenous amphotericin B, which was administered in 4 of 5 placebo-controlled trials, or 86% of patients. It was unclear whether there was overlap among the "polyene" trials, and it is possible that results from single-center trials were included in multicenter trial reports. We were unable to obtain information to clarify these issues from the trial authors or the manufacturer of fluconazole. Two of 11 responding authors replied that the data were with the drug manufacturer and two indicated that they did not have access to their data because of change of affiliation. In the meta-analyses, fluconazole and amphotericin B (mostly given orally) had similar effects (13 trials), whereas nystatin was no better than placebo (3 trials). Since individual trials are rarely conclusive, investigators, institutions, and pharmaceutical companies should provide essential details about their work to ensure that meta-analyses can accurately reflect the studies conducted and that patients will realize maximum benefits from treatments. We recommend that investigators keep copies of their trial data to help facilitate accurate and unbiased meta-analyses.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10568648&dopt=Abstract fluconazole Diflucan



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In vitro antifungal activity of BMS-207147 and itraconazole against yeast strains that are non-susceptible to fluconazole.

Fung-Tomc JC, White TC, Minassian B, Huczko E, Bonner DP.

Department of Microbiology, Bristol-Myers Squibb Company, Wallingford, Connecticut 06492, USA.

The activities of itraconazole and the new triazole BMS-207147 were determined against Candida strains that were susceptible-dose dependent (fluconazole MICs 16 to 32 micrograms/mL) or resistant (MICs > or = 64 micrograms/mL) to fluconazole. These strains included clinical isolates of Candida krusei, Candida glabrata, and Candida albicans. In addition, 16 isogenic, genetically characterized isolates of C. albicans, with progressively decreased susceptibility to fluconazole, were tested. BMS-207147 MICs to C. krusei, a species considered intrinsically resistant to fluconazole, were at 0.13 to 0.5 microgram/mL. The population distribution of the fluconazole-nonsusceptible C. glabrata was bimodal with BMS-207147/itraconazole MICs at 0.5 to 2 micrograms/mL and > or = 16 micrograms/mL. The BMS-207147 MICs to the majority of fluconazole-nonsusceptible C. albicans strains tested were < or = 1 microgram/mL. The activity of BMS-207147 was minimally affected by overexpression of the gene encoding the efflux pump MDR1, but MIC increases were observed with changes in ERG11 and with overexpression of the CDR transporter gene. Nonetheless, BMS-207147 can be active against C. albicans mutants containing cumulative resistance mechanisms to azoles. In other words, fluconazole-resistant candidal strains may be susceptible to BMS-207147.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10579098&dopt=Abstract fluconazole Diflucan



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Efficacies of high-dose fluconazole plus amphotericin B and high-dose fluconazole plus 5-fluorocytosine versus amphotericin B, fluconazole, and 5-fluorocytosine monotherapies in treatment of experimental endocarditis, endophthalmitis, and pyelonephritis due to Candida albicans.

Louie A, Liu W, Miller DA, Sucke AC, Liu QF, Drusano GL, Mayers M, Miller MH.

Divisions of Infectious Diseases, Albany Medical College, Albany, New York 12208, USA. arnold_louie_at_amc01-3 ccgateway.amc.edu

We compared the efficacies of fluconazole (Flu), amphotericin B (AmB), and 5-fluorocytosine (5FC) monotherapies with the combination of Flu plus 5FC and Flu plus AmB in a rabbit model of Candida albicans endocarditis, endophthalmitis, and pyelonephritis. The dose of Flu used was that which resulted in an area under the concentration-time curve in rabbits equivalent to that seen in humans who receive Flu at 1,600 mg/day, the highest dose not associated with central nervous system toxicity in humans. Quantitative cultures of heart valve vegetations, the choroid-retina, vitreous humor, and kidney were conducted after 1, 5, 14, and 21 days of therapy. All untreated controls died within 6 days of infection; animals treated with 5FC monotherapy all died within 18 days. In contrast, 93% of animals in the other treatment groups appeared well and survived until they were sacrificed. At day 5, the relative decreases in CFU per gram in the vitreous humor were greater in groups that received Flu alone and in combination with 5FC or AmB than in groups receiving AmB or 5FC monotherapies (P < 0. 005) but 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. On days 5 and 14 of therapy, fungal densities in kidneys of AmB recipients were lower than those resulting from the other therapies (P < 0.001 and P < or = 0.038, respectively) and AmB-plus-Flu therapy was antagonistic; however, all therapies for fungal pyelonephritis were similar by treatment day 21. While fungal counts in cardiac valves of Flu recipients were similar to those of controls on day 5 of therapy and did not change from days 1 to 21, AmB therapy significantly decreased valvular CFUs versus Flu at days 5, 14, and 21 (P < 0.005 at each time point). 5FC plus Flu demonstrated enhanced killing in cardiac vegetations compared with Flu or 5FC as monotherapies (P < 0. 03). Similarly, the combination of AmB and Flu was more active than Flu in reducing the fungal density in cardiac vegetations (P < 0.03). However, as in the kidney, AmB plus Flu demonstrated antagonism versus AmB monotherapy in the treatment of C. albicans endocarditis (P < 0.05, P = 0.036, and P < 0.008 on days 5, 14, and 21, respectively).

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10582868&dopt=Abstract fluconazole Diflucan



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Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole.

Marr KA, Seidel K, White TC, Bowden RA.

Fred Hutchinson Cancer Research Center Program in Infectious Diseases, University of Washington Department of Medicine, Seattle, Washington, USA. kmarr fhcrc.org

The prophylactic use of fluconazole is common in blood and marrow transplant (BMT) recipients. To evaluate how fluconazole has influenced the development of azole resistance and candidemia, weekly mouthwashings were done, and fluconazole susceptibility was determined for 1475 colonizing and invasive isolates obtained from patients undergoing BMT. Of 585 patients, 256 (44%) were colonized with Candida species during the course of BMT. Of these, 136 patients (53%) had at least 1 mouthwashing sample that yielded Candida species other than C. albicans on culture. Only 4.6% of patients developed candidemia. Overall, C. albicans was the most common colonizing isolate, but it caused only 7% of cases of candidemia. About 5% of colonizing C. albicans strains and 100% (2 of 2) invasive C. albicans strains were fluconazole-resistant. Colonization, cytomegalovirus disease, and bacteremia are risk factors for the development of candidemia. The use of prophylactic fluconazole is associated with a low incidence of candidemia and attributable mortality, despite colonization with azole-resistant Candida species in BMT recipients.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10608780&dopt=Abstract fluconazole Diflucan



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Drug resistance genes and trailing growth in Candida albicans isolates.

Lee MK, Williams LE, Warnock DW, Arthington-Skaggs BA.

Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, N.E., Mailstop G-11, Atlanta, Georgia 30333, USA.

OBJECTIVES: To investigate possible molecular mechanisms of azole resistance among fluconazole-susceptible bloodstream isolates of Candida albicans that displayed the trailing growth phenomenon, and to compare these isolates with bloodstream and mucosal isolates that showed reduced susceptibilities to fluconazole. METHODS: Twelve C. albicans isolates-seven trailing and five susceptible dose dependent (SDD) or resistant (R)-were screened for ERG11 mutations by DNA sequencing and quantification of ERG11, CDR1 and MDR1 expression by RT-PCR using the LightCycler high-speed PCR system. RESULTS: SDD and R isolates possessed more homozygous ERG11 mutations than did the trailing isolates. Two of these, V404I and V509M, have not been described previously and were found exclusively in fluconazole SDD and R isolates. Quantification of ERG11 expression revealed that both trailing and SDD and R isolates were capable of ERG11 up-regulation in response to fluconazole, although the SDD and R isolates showed maximal up-regulation at higher fluconazole concentrations. Quantification of CDR1 and MDR1 revealed that all isolates, regardless of in vitro fluconazole response, were capable of CDR1 and MDR1 up-regulation following fluconazole exposure. Furthermore, the SDD and R isolates expressed higher constitutive levels of CDR1 and MDR1 or CDR1, respectively, in the absence of drug compared with trailing isolates. CONCLUSIONS: Trailing isolates, although susceptible to fluconazole, express the same molecular mechanisms as SDD and R isolates following fluconazole exposure but regulate them differently.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14688046&dopt=Abstract fluconazole Diflucan



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Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant, clinical Candida albicans strains is caused by mutations in a trans-regulatory factor.

Wirsching S, Michel S, Kohler G, Morschhauser J.

Zentrum fur Infektionsforschung, Universitat Wurzburg, Rontgenring 11, D-97070 Wurzburg, Germany.

Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA(R) developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive MDR1 expression.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10629186&dopt=Abstract fluconazole Diflucan



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Effect of fluconazole on the growth and adhesion of Candida albicans in the presence of antineoplastic agents.

Fekete-Forgacs K, Kis B, Nagy G, Lenkey B.

Department of Microbiology and Biotechnology, Kossuth Lajos University, Debrecen, Hungary.

The effect of fluconazole and the antineoplastic agents etoposide and methotrexate on the growth and adhesion of Candida albicans were studied. All the tested chemicals inhibited the growth and the adhesion of the yeast to buccal epithelial cells, while fluconazole and etoposide inhibited the adhesion to acrylate surface as well. Our experiments also demonstrated that etoposide and methotrexate interfered with the inhibitory effect of fluconazole on both the growth and cell adhesion. While etoposide strengthened the inhibitory effect of fluconazole, in the presence of methotrexate fluconazole showed lower inhibition on both the growth and adhesion.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10629971&dopt=Abstract fluconazole Diflucan



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Cross-resistance between fluconazole and ravuconazole and the use of fluconazole as a surrogate marker to predict susceptibility and resistance to ravuconazole among 12,796 clinical isolates of Candida spp.

Pfaller MA, Messer SA, Boyken L, Rice C, Tendolkar S, Hollis RJ, Diekema DJ.

Department of Pathology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA. michael-pfaller uiowa.edu

Cross-resistance within a class of antimicrobial agents is a problem that is often encountered with antibacterial agents, and it is also an issue with antifungal agents. A current example is ravuconazole, a new triazole antifungal with an expanded spectrum and potency against Candida spp., Aspergillus spp., and other opportunistic fungal pathogens. The present study addresses the issue of cross-resistance between fluconazole and ravuconazole and the use of fluconazole as a surrogate marker to predict the susceptibility of Candida spp. to ravuconazole. Reference broth microdilution MIC results for 12,796 strains of Candida spp. isolated from more than 200 medical centers worldwide were used. Ravuconazole MICs and tentative interpretive categories (susceptible, </=1 microg/ml; resistant, >/=2 microg/ml) were compared with those of fluconazole by using regression statistics and error rate bounding analyses. For all 12,796 isolates, the absolute categorical agreement rate was 92.5% (rate of false-susceptible results, or very major errors [VME], 0.1%). Ravuconazole was active (MIC, </=1 microg/ml) against 99.9% of the fluconazole-susceptible isolates, 96% of the fluconazole-susceptible dose-dependent isolates, and 49% of the fluconazole-resistant isolates, including 99% of the Candida krusei isolates. Since ravuconazole is 16- to 32-fold more potent than fluconazole, the performance of fluconazole as a surrogate marker for ravuconazole susceptibility was improved by designating those isolates with fluconazole MICs of </=32 microg/ml susceptible to ravuconazole, resulting in a categorical agreement rate of 98.3%, with a VME rate of 0.3% (99 and 0.4%, respectively, when C. krusei was omitted). Cross-resistance between fluconazole and ravuconazole applies most directly to fluconazole-resistant Candida glabrata and is variable among other species of Candida. Fluconazole may serve as a surrogate marker to predict the susceptibility of Candida spp. to ravuconazole.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15243072&dopt=Abstract fluconazole Diflucan