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Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole.

Posteraro B, Sanguinetti M, Sanglard D, La Sorda M, Boccia S, Romano L, Morace G, Fadda G.

Istituto Microbiologia, Universita Cattolica del S. Cuore, L. go F. Vito, 1, 00168 Rome, Italy.

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12519188&dopt=Abstract fluconazole Diflucan



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Emergence of nosocomial candidemia at a teaching hospital in Taiwan from 1981 to 2000: increased susceptibility of Candida species to fluconazole.

Hsueh PR, Teng LJ, Yang PC, Ho SW, Luh KT.

Department of Laboratory Medicine, National Taiwan University Hospital, Taipei 100, Taiwan. hsporen ha.mc.ntu.edu.tw

The incidence of nosocomial Candida fungemia increased 36-fold from 1981 (0.8/10,000 discharges) to 2000 (28.8/10,000 discharges) at the National Taiwan University Hospital, a 2000-bed teaching hospital in northern Taiwan. To understand the current status of resistance to available antifungal agents among Candida species causing invasive infections, the in vitro susceptibilities of 222 isolates (collected from July, 1999-June, 2001) were determined. Among all of the Candida species tested, 6% and 7% were resistant to fluconazole and itraconazole, respectively. The MIC90 values of voriconazole and amphotericin B were 0.5 and 1 microg/ml, respectively, although some isolates of C. krusei (amphotericin B and voriconazole MIC, >64 microg/ml) and C. tropicalis and C. glabrata (voriconazole MICs, >64 microg/ml) were less susceptible to voriconazole or amphotericin B. About one-half of the C. glabrata isolates belonged to susceptible dose-dependent (SDD, 36%) or resistant (12%) categories for fluconazole and 96% belonged to SDD (56%) or resistant (40%) category for itraconazole. When compared with fluconazole susceptibility data of blood Candida isolates recovered from patients treated at the same hospital (NTUH) from two different time periods (January, 1994, to June, 1995, and January, 1997, to June, 1999 described in previous reports), the incidence of increased susceptibility of non-krusei Candida isolates to fluconazole was evident. This trend of increasing susceptibility for fluconazole did not correlate to the increasing use of this agent in the hospital. None of the random amplified polymorphic DNA patterns generated by arbitrarily primed PCR using four random oligonucleotide primers for 14 isolates, which exhibited fluconazole MICs of > or = 16 microg/ml, were identical, indicating an absence of clonal dissemination among these isolates in the hospital.

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Efficacy of CS-758, a novel triazole, against experimental fluconazole-resistant oropharyngeal candidiasis in mice.

Kamai Y, Kubota M, Fukuoka T, Kamai Y, Maeda N, Hosokawa T, Shibayama T, Uchida K, Yamaguchi H, Kuwahara S.

Biological Research Laboratories, Laboratory Animal Science and Toxicology Research Laboratories, Sankyo Co., Ltd., Shinagawa-ku, Tokyo 140-8710, Japan. ykamai shina.sankyo.co.jp

The therapeutic efficacy of CS-758, a novel triazole, was evaluated against experimental murine oropharyngeal candidiasis induced by Candida albicans with various susceptibilities to fluconazole. Against infections induced by strains with various susceptibilities to fluconazole, the efficacy of fluconazole was strongly correlated with the MIC of fluconazole, as measured by the NCCLS method, and agreed with the NCCLS interpretive breakpoints, suggesting that the efficacies of new drugs could be predicted by using this model. The results of the fungal burden study corresponded with the results of the histopathological study. CS-758 exhibited potent in vitro activity (MICs, 0.004 to 0.06 micro g/ml) against the strains used in this murine model including fluconazole-susceptible dose-dependent and fluconazole-resistant strains (fluconazole MICs, 16 to 64 micro g/ml). CS-758 exhibited excellent efficacy against the infections induced by all the strains including a fluconazole-resistant strain, and the reductions in viable cell counts were significant at 10 and 50 mg/kg of body weight/dose. Fluconazole was not effective even at 50 mg/kg/dose against infections induced by a fluconazole-resistant strain (fluconazole MIC, 64 micro g/ml). These results suggest that CS-758 is a promising compound for the treatment of oropharyngeal candidiasis including fluconazole-refractory infections.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12543666&dopt=Abstract fluconazole Diflucan



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In vitro activities of caspofungin compared with those of fluconazole and itraconazole against 3,959 clinical isolates of Candida spp., including 157 fluconazole-resistant isolates.

Pfaller MA, Diekema DJ, Messer SA, Hollis RJ, Jones RN.

Department of Pathology, University of Iowa College of Medicine, Iowa City 52242, USA. michael-pfaller uiowa.edu

Caspofungin is an echinocandin antifungal agent with broad-spectrum activity against Candida and Aspergillus spp. The in vitro activities of caspofungin against 3,959 isolates of Candida spp. obtained from over 95 different medical centers worldwide were compared with those of fluconazole and itraconazole. The MICs of the antifungal drugs were determined by broth microdilution tests performed according to the NCCLS method using RPMI 1640 as the test medium. Caspofungin was very active against Candida spp. (MIC at which 90% of the isolates were inhibited [MIC(90)], 1 micro g/ml; 96% of MICs were < or =2 micro g/ml). Candida albicans, C. dubliniensis, C. tropicalis, and C. glabrata were the most susceptible species of Candida (MIC(90), 0.25 to 0.5 micro g/ml), and C. guilliermondii was the least susceptible (MIC(90), >8 micro g/ml). Caspofungin was very active against Candida spp., exhibiting high-level resistance to fluconazole and itraconazole (99% of MICs were < or =1 micro g/ml). These results provide further evidence for the spectrum and potency of caspofungin activity against a large and geographically diverse collection of clinically important isolates of Candida spp.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12604543&dopt=Abstract fluconazole Diflucan



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Distribution and antifungal susceptibility of Candida species causing candidemia from 1996 to 1999.

Cheng MF, Yu KW, Tang RB, Fan YH, Yang YL, Hsieh KS, Ho M, Lo HJ.

Department of Microbiology, Veterans General Hospital, Kaohsiung, Taiwan.

Susceptibilities to amphotericin B and fluconazole of 383 Candida species isolated from blood were determined. Candida albicans was the most common species (55.6%), followed by Candida parapsilosis (17.5%), Candida tropicalis (16.5%), Candida glabrata (5.2%), Candida guilliermondii (2.3%), and others (2.9%). All but three isolates, Candida ciferrii, C. tropicalis, and C. glabrata, one each, were susceptible to amphotericin B. A total of 367 (95.8%) and 15 (4.2%) isolates were susceptible and susceptible-dose dependent to fluconazole, respectively. Only one isolate, a C. glabrata, was resistant to fluconazole. Few patients (13%) having prior fluconazole treatments may explain the low rate of resistance to fluconazole in this study.

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Clinical resistance of recurrent Candida albicans vulvovaginitis to fluconazole in the presence and absence of in vitro resistance.

MacNeill C, Weisz J, Carey JC.

Division of Women's Health, Department of Obstetrics and Gynecology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA. cmacneill psu.edu

OBJECTIVE: To determine if failure of recurrent Candida albicans vulvovaginitis to respond clinically to fluconazole is related to in vitro mycologic resistance. STUDY DESIGN: We compared clinical response to fluconazole with culture and sensitivity data in all cases of recurrent C albicans vulvovaginitis referred to our clinic over an 18-month period. RESULTS: Of 52 patients referred to us with recurring vulvovaginitis, 10 were C albicans culture positive. All 10 had previously responded to fluconazole but subsequently failed fluconazole therapy. All were euglycemic and HIV negative. In 3 of the 10 isolates, the mean inhibitory concentration for fluconazole was > 64 micrograms/mL. The history of response to fluconazole in the 7 patients with susceptible isolates was indistinguishable from that of the 3 with resistant isolates. Five of the 10 patients were given multiagent antifungal therapy. Of 4 patients available for long-term follow-up in this group, all had negative fungal cultures. In contrast, 4 evaluable patients who received maintenance azole therapy were C albicans culture positive at long-term follow-up. CONCLUSION: Recurrent C albicans vulvovaginitis can display clinical resistance to fluconazole that correlates with in vitro resistance in only some cases. We postulate that aberrant host response may play a role in the failure to control fungal colonization with a single fungistatic agent.

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Preparation and crystal characterization of a polymorph, a monohydrate, and an ethyl acetate solvate of the antifungal fluconazole.

Caira MR, Alkhamis KA, Obaidat RM.

Department of Chemistry, University of Cape Town, P.D. Hahn Building, Room B 201, Private Bag, Rondebosch, Western Cape 7701, South Africa. xraymino science.uct.ac.za

The preparation and solid-state characterization of three crystalline modifications of the antifungal agent fluconazole [2-(2,4-difluorophenyl)-1,3-bis-(1H-124-triazol-1-yl)-propan-2-ol] are reported. Recrystallization of fluconazole from propan-2-ol yielded a polymorph (Form III), whereas the solvents water and ethyl acetate yielded the solvated products fluconazole monohydrate and fluconazole. (ethyl acetate)(0.25), respectively. These species were analyzed by thermogravimetry (TGA), differential scanning calorimetry (DSC), FTIR spectroscopy, powder X-ray diffractometry (PXRD), and single crystal X-ray diffraction. Availability of the hitherto unknown crystal structures facilitated interpretation of the thermal data and clarified previous findings relating to the polymorphism of this compound. Fluconazole was found to exist as a centrosymmetric hydrogen bonded dimer in Form III. For the solvated phases, the solvent locations within the drug host matrices were established as isolated sites for water molecules and constricted channels for ethyl acetate molecules. Desolvation of the monohydrate and ethyl acetate solvate yielded polymorphic Form I. Reference PXRD patterns computed from the refined single-crystal X-ray data for the title compounds are presented. Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association

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On-line determination of fluconazole in blood and dermal rat microdialysates by microbore high-performance liquid chromatography.

Mathy FX, Vroman B, Ntivunwa D, De Winne AJ, Verbeeck RK, Preat V.

Unite de Pharmacie Galenique, Universite catholique de Louvain, Av. E. Mounier 73, UCL 73.20, 1200 Brussels, Belgium.

To study the distribution of fluconazole in the dermis of the rat, on-line microdialysis using double-site sampling coupled with a microbore HPLC system was developed. The chromatographic conditions consisted of a mobile phase of 20 mM diammonium phosphate-acetonitrile (75:25, v/v, pH 7.0) pumped through a microbore C(18) column at 40 microl/min. The eluent was monitored with UV detector with UZ flow cell (30 mm path length) at 210 nm. A microbore 10-port pneumatic valve fitted with two loops of 1 microl was used to collect and directly inject microdialysates from jugular and dermal probes. The retention time was 5.8 min for fluconazole and 10.1 min for its fluorinated analog, UK-54373 used as a retrodialysis marker. The assay was precise, with inter- and intra-assay relative standard deviation values of 0.64 and 0.71%, respectively, and with a good linearity (r=0.999) in the range of 0.15-20 microg/ml with only 1 microl injected onto the column. The LOD and LOQ values for fluconazole were 0.100 and 0.150 microg/ml, respectively. The applicability of the method was demonstrated by studying the disposition of fluconazole in blood and dermis following i.v. bolus at a dose of 10 mg/kg.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12650755&dopt=Abstract fluconazole Diflucan