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Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resistance to azole antifungals in Candida glabrata.

Sanglard D, Ischer F, Bille J.

Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland. Domique.Sanglard chuv.hospvd.ch

Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753-2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 microg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 x 10(-4) to 4 x 10(-4)). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of CgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 microg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, called CgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2 suppressed the development of HFAR in a medium containing fluconazole at 5 microg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11257032&dopt=Abstract fluconazole Diflucan



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In vitro susceptibility of 137 Candida sp. isolates from HIV positive patients to several antifungal drugs.

Magaldi S, Mata S, Hartung C, Verde G, Deibis L, Roldan Y, Marcano C.

Instituto de Medicina Tropical, U.C.V., Caracas, Venezuela. caphart telcel.net.ve

Oropharyngeal candidiasis caused by various species of Candida is one of the most common infections in HIV seropositive or AIDS patients. Drug resistance among these yeasts is an increasing problem. We studied the frequency of resistance profile to fluconazole, itraconazole, ketoconazole, amphotericin B and terbinafine of 137 isolates of Candida sp. From HIV positive or AIDS patients with oropharyngeal candidiasis at Instituto de Inmunologia, U.C.V. and the Hospital "Jose Ignacio Baldo", Caracas Venezuela, using the well diffusion susceptibility test (Magaldi et al.). We found that nearly 10% of C. albicans isolates were primarily fluconazole resistant, 45% of C. albicans isolates from patients with previous treatment were resistant to fluconazole, of which 93% showed cross-resistance to itraconazole, and even about 30% of C. tropicalis (n = 13) were resistant to fluconazole and/or itraconazole. To this respect, several recent reports have been described antifungal cross-resistance among azoles. Therefore, we consider that C. tropicalis should be added to the growing list of yeast in which antifungal drug resistance is common. This report could be useful for therapeutic aspect in AIDS patients with oral candidiasis.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11265163&dopt=Abstract fluconazole Diflucan



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Pharmacokinetics of sequential intravenous and enteral fluconazole in critically ill surgical patients with invasive mycoses and compromised gastro-intestinal function.

Buijk SL, Gyssens IC, Mouton JW, Verbrugh HA, Touw DJ, Bruining HA.

Department of Surgical Intensive Care, Erasmus University Medical Center, Rotterdam, The Netherlands. buijk rdgg.nl

OBJECTIVES: (1) To determine the pharmacokinetics of sequential intravenous and enteral fluconazole in the serum of surgical intensive care unit (ICU) patients with deep mycoses. (2) To determine the concentrations of fluconazole reached at the site of infection. (3) To determine if enteral administration of fluconazole, which has an important pharmaco-economic advantage, is justified in this specific patient group. DESIGN: Descriptive, sequential study as a part of a therapeutic drug monitoring programme. SETTING: Eighteen-bed surgical ICU in a referral centre. PATIENTS: Fourteen critically ill surgical patients with recent gastro-intestinal (GI) surgery and deep mycosis caused by a fluconazole-susceptible fungus and a calculated creatinine clearance of more than 40 ml/min. INTERVENTIONS: Fluconazole dosage regimen: 400 mg i. v. every 24 h with an extra dose of 400 mg i.v. after 12 h on day 1. If the clinical condition allowed enteral administration on day 4, the content of two capsules of 200 mg was given via the feeding tube with concomitant enteral feeds. MEASUREMENTS AND MAIN RESULTS: Serum, exudate from the site of infection and urine samples collected at assumed steady state ( after > or = 5 doses). Fluconazole concentrations were determined by high-performance liquid chromatography (HPLC). The mean area under the concentration curve (AUC0-24 h) in serum after enteral administration did not significantly differ from the AUC0-24 h during intravenous treatment. The elimination half-life was longer compared to healthy volunteers. The mean (95% CI) estimated bioavailability was 124 (90-158)%. The mean (95% CI) area under the concentration time curves (AUCs) achieved in the exudate from the site of infection were 67 (55-79)% of the AUCs reached in serum for both regimens. CONCLUSIONS: In critically ill patients with recent GI surgery and/or peritonitis the bioavailability of enteral fluconazole was adequate. The concentrations of fluconazole reached in exudate were lower than those in serum for both regimens, but adequate to treat most cases of deep mycoses in this specific patient group.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11280621&dopt=Abstract fluconazole Diflucan



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Fluconazole and voriconazole multidisk testing of Candida species for disk test calibration and MIC estimation.

Kronvall G, Karlsson I.

Department of Microbiology and Tumor Biology--MTC, Clinical Microbiology, Karolinska Institute, Karolinska Hospital, Stockholm SE-17176, Sweden. goran.kronvall ks.se

Fluconazole and voriconazole MICs were determined for 114 clinical Candida isolates, including isolates of Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropicalis. All strains were susceptible to voriconazole, and most strains were also susceptible to fluconazole, with the exception of C. glabrata and C. krusei, the latter being fully fluconazole resistant. Single-strain regression analysis (SRA) was applied to 54 strains, including American Type Culture Collection reference strains. The regression lines obtained were markedly different for the different Candida species. Using an MIC limit of susceptibility to fluconazole of < or =8 microg/ml, according to NCCLS standards, the zone breakpoint for susceptibility for the 25-microg fluconazole disk was calculated to be > or =18 mm for C. albicans and > or =22 mm for C. glabrata and C. krusei. SRA results for voriconazole were used to estimate an optimal disk content according to rational criteria. A 5-microg disk content of voriconazole gave measurable zones for a tentative resistance limit of 4 microg/ml, whereas a 2.5-microg disk gave zones at the same MIC level for only three of the species. A novel SRA modification, multidisk testing, was also applied to the two major species, C. albicans and C. glabrata, and the MIC estimates were compared with the true MICs for the isolates. There was a significant correlation between the two measurements. Our results show that disk diffusion methods might be useful for azole testing of Candida isolates. The method can be calibrated using SRA. Multidisk testing gives direct estimations of the MICs for the isolates.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11283066&dopt=Abstract fluconazole Diflucan



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[Fluconazole resistance in Candida albicans assayed by PCR fingerprinting with M13 prime]

[Article in Chinese]

Wang YB, Wang H, Guo HY, Zhao YZ, Luo SQ.

Department of Cell Biology, First Military Medical University, Guangzhou 510515, China. wangybgz 163.net

OBJECTIVE: To understand the molecular and genetic mechanism underlying fluconazole resistance in Candida albicans by PCR fingerprinting with M13 primer. METHODS: Paper disc diffusion method was employed for assay of fluconazole resistance in 41 clinical isolates of Candida albicans, followed by PCR fingerprinting with M13 primer to study the gel patterns with cluster analysis using neighbor joining (NJ) method performed with RAPD200 software. RESULTS: Of the 41 clinical isolates, 11 strains (26.8%) were fluconazole-sensitive, 8 (19.5%) fluconazole-dependent and 22 (53.7%) fluconazole-resistance. Two to twelve bands could be observed among these strains, and the gel patterns revealed by cluster analysis were associated with the reactions of the strains against fluconazole and the location of infection. CONCLUSION: There is high prevalence of fluconazole resistance in clinical Candida albicans isolates, and PCR fingerprinting with M13 primer is convenient for assay of fluconazole resistance and molecular epidemiological study of Candida albicans.

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Fungal peritonitis in peritoneal dialysis patients: effect of fluconazole treatment and use of the twin-bag disconnect system.

Chen CM, Ho MW, Yu WL, Wang JH.

Division of Infectious Disease, Department of Internal Medicine, China Medical University Hospital, No. 2 Yuh Der Road, Taichung, Taiwan 404, ROC.

Fungal peritonitis is an uncommon but potentially life-threatening complication for patients undergoing continuous ambulatory peritoneal dialysis. This retrospective study evaluated the efficacy of fluconazole in fungal peritonitis treatment and the incidence of fungal peritonitis in different peritoneal dialysis disconnect systems. Fungal peritonitis was caused by Candida species in 67% of episodes. The most common pathogen in this series was Candida parapsilosis (29%), followed by Candida albicans (14%). One patient (5%) died within 1 month after admission for treatment of fungal peritonitis. Only 1 patient (5%) in this series could resume peritoneal dialysis. Treatment with fluconazole alone has an effect comparable to intraperitoneal (IP) amphotericin B alone or IP amphotericin B combined with oral or intravenous fluconazole. The incidence of fungal peritonitis in patients who used the spike, Y-set, and UV antiseptic systems was 5.69, 6.20, and 2.93 times, respectively, as frequent as that of fungal peritonitis in patients who used the twin-bag disconnect system.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15181494&dopt=Abstract fluconazole Diflucan



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Mechanisms of fluconazole resistance in Candida albicans isolates from Japanese AIDS patients.

Maebashi K, Niimi M, Kudoh M, Fischer FJ, Makimura K, Niimi K, Piper RJ, Uchida K, Arisawa M, Cannon RD, Yamaguchi H.

Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192-0395, Japan. Kazunori_Maebashi meiji.co.jp

Four Candida albicans isolates, TIMM 3163, TIMM 3164, TIMM 3165 and TIMM 3166, with reduced fluconazole susceptibility were obtained from three AIDS patients in Japan, and the mechanisms of their drug resistance were studied. All isolates showed lower levels of intracellular accumulation of fluconazole than ATCC 10231, a susceptible control strain of C. albicans. Increased amounts of CDR1 and CDR2 mRNA encoding putative ATP binding cassette (ABC) transporters were associated with the azole resistance of all TIMM isolates, apart from TIMM 3164. In addition, increased Cdr1p levels were immunodetected in the cell membrane fractions of all the TIMM strains except for TIMM 3164. Gene amplification was not responsible for CDR1 overexpression and there were no significant differences in the mRNA levels of CDR3 or CDR4 (ABC transporters) in the azole-susceptible and -resistant cells. CaMDR1 (a major facilitator superfamily) gene expression was not observed in any of the resistant isolates or the control strain. These results suggest that energy-dependent drug efflux associated with increased expression of CDR1 and CDR2 is involved in the fluconazole resistance mechanisms in two of the four isolates, TIMM 3165 and TIMM 3166. TIMM 3164 demonstrated energy-dependent drug efflux without overexpression of CDR1-4 or CaMDR1, indicating that some other pump may be operating. Despite showing low levels of drug efflux and overexpression of CDR1 and CDR2, efflux in TIMM 3163 was not energy dependent, suggesting that the expressed Cdr1p non-functional Cdr1p and that other resistance mechanisms may operate in this strain.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11328762&dopt=Abstract fluconazole Diflucan



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Role of aprotinin in the management of experimental fungal keratitis.

Biswas NR, Das H, Satpathy G, Mohanty S, Panda A.

Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.

In an experimentally induced Aspergillus fumigatus fungal keratitis in 20 rabbits, aprotinin, an antiplasmin agent, was studied to find out its role as an adjuvant when given along with other established antifungal agents like natamycin and fluconazole. The 20 rabbits included in this study were randomly divided into four equal treatment groups. Once the ulcer was produced after intrastromal injection of 0.03 ml of A. fumigatus (5.5 x 10(4) spores/ml), different drugs/agents in combination were used in a randomized manner. These were natamycin (5%) + placebo, natamycin + aprotinin (40 IU/ml), fluconazole (0.3%) + placebo and fluconazole + aprotinin. The rabbits were followed up every day to note the signs of healing which included subsidence of corneal infiltration, disappearance of stromal abscess and subsidence of corneal oedema till complete healing. Results showed that the average healing time was 28.2, 28.4, 49.8 and 49.0 days for natamycin + placebo, natamycin + aprotinin, fluconazole + placebo and fluconazole + aprotinin, respectively. It suggests that aprotinin as an adjuvant has no definite role in the management of fungal keratitis. The plasminogen activator-plasmin system which is inhibited by aprotinin may not be the pathway through which filamentous fungi like A. fumigatus cause tissue destruction. Copyright 2001 S. Karger AG, Basel

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11340405&dopt=Abstract fluconazole Diflucan