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[Study on experimental induction of fluconazole resistance in Candida albicans]

[Article in Chinese]

Zhu YN, Lu SM.

Woman's Hospital, School of Medicine, Zhejiang University, Hangzhou.

To investigate the phenotype and genotype variation between the Fluconazole resistant C. albicans isolates and the corresponding susceptible ones, our research established a resistance-induction mode in vitro. Comparisons were done on drug resistance maintainability, metabolic profile and the doubling time in the logarithmic growth phase. Genotypes were determined by ERIC-PCR. The Fluconazole resistant isolates appeared in strain 435, A06, B07 and C01 from total 22 clinical Fluconazole susceptible isolates after being incubated for 45-80 days in YEPD broth with increasing Fluconazole concentration. The parent isolates had a same metabolic profile and a similar growth doubling time to their filial generation. The same ERIC-PCR profiles were also found between the susceptible parents and their resistant filial isolates. The resistant isolates maintained drug resistance for 24 days after growing on drug-free medium. It was supposed that candida albicans had a latent capacity to evolve resistance to azoles under a certain antifungal drug selective pressure, and the acquired resistance could maintain in drug-free media for a certain period. The resistant isolate with no adaptive cost may be prone to vogue among people. ERIC-PCR could be used in epidemiological study as a stable marker.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15636370&dopt=Abstract fluconazole Diflucan



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Prophylactic role of liposomized chloroquine against murine cryptococcosis less susceptible to fluconazole.

Khan MA, Jabeen R, Mohammad O.

Aligarh Muslim University, Aligarh 202002, India. alammasood1 rediffmail.com

PURPOSE: The prophylactic role of liposomized chloroquine (lip-CQ) has been assessed against less susceptible Cryptococcus neoformans infection in murine model. METHODS: In the current study, we investigated the antifungal activity of lip-CQ against C. neoformans in macrophages cell line (J 774) and murine model. Mice were pretreated with free as well as liposomized formulations of CQ at various doses. The anticryptococcal activity of fluconazole was compared in mice with or without CQ pretreatment. The efficacy of CQ prophylaxis was assessed by survival as well as colony forming units (cfu) in brain and lungs of treated mice. RESULTS: Fluconazole alone was not found significantly effective against C. neoformans in both in vitro and in vivo studies. However, the antifungal activity of fluconazole increases in chloroquine-pretreated mice. Lip-CQ was found to be more effective in comparison to the same dose of free chloroquine in reducing fungal burden from macrophages in vitro and lungs and brain of C. neoformans infected mice. CONCLUSIONS: The enhanced prophylactic activity of lip-CQ seems due to rapid uptake of drug-containing liposomes by macrophages. The liposome-mediated accumulation of CQ in macrophages makes the environment unfavorable (alkaline) for the intracellular multiplication of C. neoformans. Moreover, the increased incidence of multi-drug resistance and diversity of pathogenic microorganisms inhibited or killed by CQ makes it the drug of choice for prophylactic therapy.

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Vitrectomy with fluconazole infusion: retinal toxicity, pharmacokinetics, and efficacy in the treatment of experimental candidal endophthalmitis.

Cheng CK, Yang CH, Hsueh PR, Liu CM, Lu HY.

Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.

The aim of this study was to determine the retinal toxicity and intraocular pharmacokinetics of vitrectomy with fluconazole infusion in rabbit eyes and to study its efficacy in the treatment of experimental candidal endophthalmitis. The right eyes of 13 New Zealand White (NZW) rabbits were vitrectomized and infused with 20 mL of 0.2 mg/mL, 1 mg/mL, or 2 mg/mL of fluconazole. An electroretinogram (ERG) was performed on both eyes of each rabbit at different time points. The right eyes of 26 different NZW rabbits were vitrectomized and infused with 20 mL of 2 mg/mL of fluconazole. These rabbits were sacrificed, and their right eyes were enucleated at hours 2, 4, 8, and 24 after the operation, and the concentration of fluconazole in the vitreous was measured by highpressure liquid chromatography. Experimental candidal endophthalmitis was induced in the right eye of 42 other NZW rabbits. Twenty-six (26) of the eyes were then vitrectomized and infused with 20 mL of 2 mg/mL of fluconazole, and the other 16 rabbits served as control. Severity of ocular infection was graded from 0-4 at different time intervals, using an indirect ophthalmoscope. In the first group, ERG showed no significant difference between the experimental eyes and the control eyes--in all concentrations of fluconazole--for up to 3 months. In the second group, the intraocular concentration of fluconazole declined so rapidly that, as of 8 hours after operation, there was none in the vitreous cavity. In the third group, significantly less vitreous opacity was found in the treated eyes on days 3 and 6. However, the difference ceased to be apparent on day 15. Our study suggests that there is no retinal toxicity resulting from vitrectomy with a 2 mg/mL fluconazole infusion, and that it is temporally effective in the treatment of experimental candidal endophthalmitis.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15650518&dopt=Abstract fluconazole Diflucan



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Frequency of potential azole drug-drug interactions and consequences of potential fluconazole drug interactions.

Yu DT, Peterson JF, Seger DL, Gerth WC, Bates DW.

Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Division of General Medicine, Boston, MA, USA.

PURPOSE: To assess the frequency of potential azole-drug interactions and consequences of interactions between fluconazole and other drugs in routine inpatient care. METHODS: We performed a retrospective cohort study of hospitalized patients treated for systemic fungal infections with an oral or intravenous azole medication between July 1997 and June 2001 in a tertiary care hospital. We recorded the concomitant use of medications known to interact with azole antifungals and measured the frequency of potential azole drug interactions, which we considered to be present when both drugs were given together. We then performed a chart review on a random sample of admissions in which patients were exposed to a potential moderate or major drug interaction with fluconazole. The list of azole-interacting medications and the severity of interaction were derived from the DRUGDEX(R) System and Drug Interaction Facts. RESULTS: Among the 4185 admissions in which azole agents (fluconazole, itraconazole or ketoconazole) were given, 2941 (70.3%) admissions experienced potential azole-drug interactions, which included 2716 (92.3%) admissions experiencing potential fluconazole interactions. The most frequent interactions with potential moderate to major severity were co-administration of fluconazole with prednisone (25.3%), midazolam (17.5%), warfarin (14.7%), methylprednisolone (14.1%), cyclosporine (10.7%) and nifedipine (10.1%). Charts were reviewed for 199 admissions in which patients were exposed to potential fluconazole drug interactions. While four adverse drug events (ADEs) caused by fluconazole were found, none was felt to be caused by a drug-drug interaction (DDI), although in one instance fluconazole may have contributed. CONCLUSIONS: Potential fluconazole drug interactions were very frequent among hospitalized patients on systemic azole antifungal therapy, but they had few apparent clinical consequences. Copyright (c) 2005 John Wiley & Sons, Ltd.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15654717&dopt=Abstract fluconazole Diflucan



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Antifungal susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species from Taiwan: surveillance of multicenter antimicrobial resistance in Taiwan program data from 2003.

Hsueh PR, Lau YJ, Chuang YC, Wan JH, Huang WK, Shyr JM, Yan JJ, Yu KW, Wu JJ, Ko WC, Yang YC, Liu YC, Teng LJ, Liu CY, Luh KT.

Department of Laboratory Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Rd., Taipei 100, Taiwan. hsporen ha.mc.ntu.edu.tw

The susceptibilities of nonduplicate isolates to six antifungal agents were determined for 391 blood isolates of seven Candida species, 70 clinical isolates (from blood or cerebrospinal fluid) of Cryptococcus neoformans, and 96 clinical isolates of four Aspergillus species, which were collected in seven different hospitals in Taiwan (as part of the 2003 program of the study group Surveillance of Multicenter Antimicrobial Resistance in Taiwan). All isolates of Candida species other than C. glabrata and C. krusei were susceptible to fluconazole. Among the 59 C. glabrata isolates, 16 (27%) were not susceptible to fluconazole, and all were dose-dependently susceptible or resistant to itraconazole. For three (5.1%) C. glabrata isolates, voriconazole MICs were 2 to 4 microg/ml, and for all other Candida species isolates, voriconazole MICs were </=0.5 microg/ml. The proportions of isolates for which amphotericin B MICs were >/=2 microg/ml were 100% (3 isolates) for C. krusei, 11% (23 of 207 isolates) for Candida albicans, 3.0% (2 of 67 isolates) for Candida tropicalis, 20% (12 of 59 isolates) for C. glabrata, and 0% for both Candida parapsilosis and Candida lusitaniae. For three (4%) Cryptococcus neoformans isolates, fluconazole MICs were >/=16 microg/ml, and two (3%) isolates were not inhibited by 1 mug of amphotericin B/ml. For four (4.2%) of the Aspergillus isolates, itraconazole MICs were 8 microg/ml. Aspergillus flavus was less susceptible to amphotericin B, with the MICs at which 50% (1 microg/ml) and 90% (2 microg/ml) nsrsid417869\delrsid7301351 of isolates were inhibited being twofold greater than those for Aspergillus fumigatus and Aspergillus niger. All Aspergillus isolates were inhibited by </=1 microg of voriconazole/ml, including isolates with increased resistance to amphotericin B and itraconazole. This study revealed the emergence in Taiwan of decreased susceptibilities of Candida species to amphotericin B and of C. neoformans to fluconazole and amphotericin B. Voriconazole was the most potent agent against the fungal isolates tested, including fluconazole- and amphotericin B-nonsusceptible strains.

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Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance.

Sanguinetti M, Posteraro B, Fiori B, Ranno S, Torelli R, Fadda G.

Istituto di Microbiologia, Universita Cattolica del Sacro Cuore, Rome, Italy. msanguinetti rm.unicatt.it

The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15673750&dopt=Abstract fluconazole Diflucan



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Susceptibility of Candida albicans biofilms grown in a constant depth film fermentor to chlorhexidine, fluconazole and miconazole: a longitudinal study.

Lamfon H, Porter SR, McCullough M, Pratten J.

Division of Infection and Immunity, Eastman Dental Institute, University College London, 256 Gray's Inn Road, London WC1X 8LD, UK.

OBJECTIVES: The aim of this study was to assess the resistance of Candida albicans biofilms to both antifungal and antimicrobial agents in vitro. METHODS: Biofilms of C. albicans were grown on denture acrylic discs in a constant depth film fermentor and maintained with artificial saliva. The MIC of fluconazole, miconazole and chlorhexidine for C. albicans was first determined. Using these data, 72 h biofilms were exposed to these agents at different MIC levels. In order to assess growth, biofilms were removed from the fermentor, incubated in the test agent for various periods, the biofilms disrupted and the viable yeast cells present determined. The MIC for these cells was then also determined. In a separate experiment, biofilms of various ages (2-72 h) were exposed to sub-biofilm MIC concentrations for two different periods. RESULTS: C. albicans biofilms were found to be highly resistant to fluconazole and miconazole compared with the same cells grown in suspension (>/=1024 x MIC). In contrast, chlorhexidine inhibited the growth of C. albicans biofilms at a concentration up to 8 x MIC. When the susceptibility of biofilms over time was investigated, higher reductions were observed for chlorhexidine and miconazole than fluconazole for biofilms of 2 and 6 h. CONCLUSIONS: We have shown in this study that the susceptibility of C. albicans to antifungal and antimicrobial agents changes throughout biofilm development.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14729749&dopt=Abstract fluconazole Diflucan



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Rapid acquisition of stable azole resistance by Candida glabrata isolates obtained before the clinical introduction of fluconazole.

Borst A, Raimer MT, Warnock DW, Morrison CJ, 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 Rd., N.E., Mailstop G-11, Atlanta, GA 30333, USA.

Five azole-susceptible Candida glabrata isolates obtained before 1975 became resistant to fluconazole, itraconazole, and voriconazole within 4 days of in vitro fluconazole exposure. This cross-resistance was stable for at least 4 months after removal of fluconazole and was associated with increased CgCDR1 and CgCDR2 expression.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15673768&dopt=Abstract fluconazole Diflucan