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Risk factors for fluconazole-resistant candidiasis in human immunodeficiency virus-infected patients.

Maenza JR, Keruly JC, Moore RD, Chaisson RE, Merz WG, Gallant JE.

Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-6220, USA.

In a case-control study to identify risk factors for fluconazole-resistant oroesophageal candidiasis in human immunodeficiency virus-infected patients, 25 patients with clinical and in vitro fluconazole-resistant candidiasis were paired with controls who had treatment-responsive candidiasis and who had been observed for similar time periods. After their first episode of candidiasis, patients who later developed fluconazole resistance had more treated episodes than did matched controls (cases, 3.1; controls, 1.8; P = .004), lower median CD4 cell counts (11/mm3 vs. 71/mm/3; P = .004), and greater median durations of all antifungal therapy (419 vs. 118 days; P < .001) and of systemic azole therapy (272 vs. 14 days; P < .001). When paired with a second set of controls matched by CD4 cell count as well as first diagnosis of candidiasis, cases continued to show greater median exposure to azoles (272 vs. 88 days; P = .005). These data indicate that advanced immunosuppression and exposure to oral azoles are risk factors for the development of fluconazole resistance.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8537662&dopt=Abstract fluconazole Diflucan



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[Fluconazole level in aqueous humor after oral drug administration in humans]

[Article in German]

Aust R, Kruse FE, Wildfeuer A, Pfaff G, Rohrschneider K, Volcker HE.

Universitats-Augenklinik Heidelberg.

For 2 years fluconazole, a triazole antimycotic, has been available for treatment of systemic mycosis. Compared to amphotericin B fewer severe side effects have been reported. So far, no data have been published as to its penetration into the human eye. In the present study, 20 cataract patients were given 200 mg fluconazole (0.5 to 8 h preoperatively. During the cataract operation 0.1 ml of the aqueous was removed as well as 10 ml serum. With the help of high-pressure liquid chromatography (HPLC), the concentration of fluconazole in each of the samples was determine. If the aqueous humor was removed at least 2h after fluconazole application, concentrations between 2.7 and 5.4 micrograms/ml were reached (mean 3.7 +/- 2.17) In these cases the concentration in the aqueous humor was 80% of the concentration found in the serum at the same time. If the sample of the aqueous humor was collected only 1 h after application, 40% of the concentration in the serum was found in the aqueous humor. These data prove that fluconazole shows an extremely good penetration through the blood-aqueous barrier. After a single dose of 200 mg, a concentration is reached in the eye that surmounts the minimal inhibiting concentration found for Candida species sensitive to fluconazole. Therefore, fluconazole seems to be a good alternative to amphotericin B for the treatment of infections caused by such fungi.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8563432&dopt=Abstract fluconazole Diflucan



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Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters.

Sanglard D, Kuchler K, Ischer F, Pagani JL, Monod M, Bille J.

Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

Azole antifungal agents, and especially fluconazole, have been used widely to treat oropharyngeal candidiasis in patients with AIDS. An increasing number of cases of clinical resistance against fluconazole, often correlating with in vitro resistance, have been reported. To investigate the mechanisms of resistance toward azole antifungal agents at the molecular level in clinical C. albicans isolates, we focused on resistance mechanisms related to the cellular target of azoles, i.e., cytochrome P450(14DM) (14DM) and those regulating the transport or accumulation of fluconazole. The analysis of sequential isogenic C. albicans isolates with increasing levels of resistance to fluconazole from five AIDS patients showed that overexpression of the gene encoding 14DM either by gene amplification or by gene deregulation was not the major cause of resistance among these clinical isolates. We found, however, that fluconazole-resistant C. albicans isolates failed to accumulate 3H-labelled fluconazole. This phenomenon was reversed in resistant cells by inhibiting the cellular energy supply with azide, suggesting that resistance could be mediated by energy-requiring efflux pumps such as those described as ATP-binding cassette (ABC) multidrug transporters. In fact, some but not all fluconazole-resistant clinical C. albicans isolates exhibited up to a 10-fold relative increase in mRNA levels for a recently cloned ABC transporter gene called CDR1. In an azole-resistant C. albicans isolate not overexpressing CDR1, the gene for another efflux pump named BENr was massively overexpressed. This gene was cloned from C. albicans for conferring benomyl resistance in Saccharomyces cerevisiae. Therefore, at least the overexpression or the deregulation of these two genes potentially mediates resistance to azoles in C. albicans clinical isolates from AIDS patients with oropharyngeal candidiasis. Involvement of ABC transporters in azole resistance was further evidenced with S. cerevisiae mutants lacking specific multidrug transporters which were rendered hypersusceptible to azole derivatives including fluconazole, itraconazole, and ketoconazole.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8585712&dopt=Abstract fluconazole Diflucan



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Different components in human serum inhibit multiplication of Cryptococcus neoformans and enhance fluconazole activity.

Nassar F, Brummer E, Stevens DA.

Department of Medicine, Santa Clara Valley Medical Center, San Jose, California 95128-2699, USA.

The inhibitory effect of human serum on the multiplication of Cryptococcus neoformans and the interaction with fluconazole were studied. Compared with cryptococcal multiplication in RPMI 1640 medium alone, 5% human serum in medium inhibited multiplication by 76% +/- 6% (n = 8). The inhibitory effect of human serum was donor independent, [corrected] heat stable (56 degrees C, 30 min), and not due to albumin or globulin. Bovine and murine sera were not inhibitory at that concentration. A fungistatic concentration of fluconazole (5.0 micrograms/ml) in medium plus 5% human serum resulted in 40% +/- 5% (n = 8) killing (reduction of inoculum CFU) in a 24-h assay. Bovine or murine sera did not have the enhancing effect, and this human serum activity was heat stable and donor independent. At 2.5 micrograms of fluconazole per ml, fungistasis by fluconazole plus human serum was significantly greater than with either alone. Higher serum concentrations [corrected] potentiated fluconazole more. At higher fluconazole concentrations (e.g., 20 micrograms/ml) fluconazole alone could kill, but serum potentiated this. A fluconazole-resistant isolate (MIC, 100 micrograms/ml) was not killed by fluconazole (5.0 micrograms/ml) in 5% human serum, but human serum potentiated the partial fluconazole inhibition. When human serum was dialyzed (molecular weight cutoff, 6,000 to 8,000) against phosphate-buffered saline, it lost the ability to synergize with fluconazole for killing Cryptococcus organisms but not the capacity to inhibit multiplication. Filtration of serum suggested the filtrate with a molecular weight of < 10,000 could interact synergistically with fluconazole for killing but could not inhibit cryptococcal multiplication. These findings indicate that human serum has two components, one (macromolecular) with a unique ability to inhibit C. neoformans and a low-molecular-weight component that enhances fluconazole anticryptococcal activity.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8585731&dopt=Abstract fluconazole Diflucan



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Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans.

Espinel-Ingroff A, Rodriguez-Tudela JL, Martinez-Suarez JV.

Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0049, USA.

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by the visual and subjective inspection of growth inhibition after 48 h of incubation. Two alternative microdilution procedures for MIC endpoint determination, a spectrophotometric MIC endpoint test that evaluates 80% growth inhibition by the drug and a colorimetric method with an oxidation-reduction indicator (Alamar Blue), were compared with the M27-P method for fluconazole susceptibility testing of 45 susceptible and resistant isolates of Candida albicans. The spectrophotometric method was performed with RPMI 1640 medium with 2% glucose, and the other two tests were performed with plain RPMI 1640 medium. All tests were incubated at 35 degrees C. Excellent agreement was demonstrated between the M27-P method and both 24-h microdilution tests (97.7%) as well as between the two microdilution tests (95.5%). Also, there was agreement in the detection in vivo of fluconazole resistance by the three methods. These preliminary data indicate that both microdilution methods may serve as less subjective alternatives to the M27-P method for the determination of fluconazole MIC endpoints.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8586692&dopt=Abstract fluconazole Diflucan



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Itraconazole susceptibilities of fluconazole susceptible and resistant isolates of five Candida species.

Johnson EM, Davey KG, Szekely A, Warnock DW.

PHLS Mycology Reference Laboratory, Public Health Laboratory, Kingsdown, Bristol, UK.

The in-vitro susceptibilities of 1380 isolates of five Candida species were determined in order to establish whether isolates resistant to fluconazole were cross-resistant to itraconazole. IC50 values were determined by a broth microdilution method. 690 Candida albicans isolates, seven Candida glabrata isolates, seven Candida krusei isolates, 120 Candida parapsilosis isolates and 37 Candida tropicalis isolates were susceptible to both fluconazole (IC50 < or = 32 mg/L) and itraconazole (IC50 < or = 4 mg/L). Twenty eight of 160 C. albicans isolates (17.5%), 180 of 293 C. glabrata isolates (61.4%), six of 48 C. krusei isolates (12.5%), and 10 of 18 C. tropicalis isolates (55.5%) resistant to fluconazole (IC50 > or = 64 mg/L) were also resistant to itraconazole (IC50 > or = 8 mg/L). In contrast, drug-specific resistance to itraconazole was not observed in any of the isolates tested. However, the itraconazole IC50s for fluconazole susceptible isolates were lower than those for fluconazole resistant isolates, which suggests that patients who fail fluconazole treatment might require itraconazole at higher dosages than usual.

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Treatment of experimental cryptococcal meningitis with fluconazole: impact of dose and addition of flucytosine on mycologic and pathophysiologic outcome.

Kartalija M, Kaye K, Tureen JH, Liu Q, Tauber MG, Elliott BR, Sande MA.

Infectious Diseases Laboratory, San Francisco General Hospital, CA 94143-0811, USA.

Fluconazole is effective in the therapy of cryptococcal meningitis in patients with AIDS. The optimal dosage of fluconazole and the impact of combination with flucytosine are not known. In this study, rabbits with experimental cryptococcal meningitis were given fluconazole at low, intermediate, or high dose or in combination with a low or intermediate dose of flucytosine. Serial cerebrospinal fluid (CSF) examinations showed that all three doses of fluconazole and low-dose fluconazole in combination with intermediate-dose flucytosine were effective in reducing CSF cryptococcal titer, lactate, white blood cell count, and cryptococcal antigen (CRAG) titers. The intermediate and high doses of fluconazole reduced CSF fungal (P < .05) and CRAG (P < .001) titers earlier than low-dose fluconazole alone or in combination with flucytosine. Only the highest dose of fluconazole reduced brain edema after 7 days. In this model of cryptococcal meningitis, there was evidence of a dose response with fluconazole but no in vivo synergism with flucytosine.

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Correlation between in vitro resistance to fluconazole and clinical outcome of oropharyngeal candidiasis in HIV-infected patients.

Quereda C, Polanco AM, Giner C, Sanchez-Sousa A, Pereira E, Navas E, Fortun J, Guerrero A, Baquero F.

Department of Microbiology, Ramon y Cajal Hospital, National Institute of Health (INSALUD), Madrid, Spain.

Fifty episodes of oropharyngeal candidiasis in HIV-infected patients were analyzed prospectively in order to evaluate the clinical response to fluconazole. The minimum inhibitory concentrations (MICs) of fluconazole for the Candida strains isolated from the pharynx were correlated with the clinical response. Treatment with fluconazole (100 mg/day) was successful in 86% of the cases. A good clinical outcome followed in 97% of the cases when a strain sensitive to fluconazole was isolated. This figure fell to 22% when the strain was resistant to fluconazole (p < 0.001). The rate of post-treatment colonization was high (87%), and selection of non-albicans Candida species occurred in 23% of the cases. In conclusion, fluconazole treatment for oropharyngeal candidiasis of HIV-infected patients was useful in most cases, but less sensitive non-albicans species can be selected. Most treatment failures were associated with increased MICs of fluconazole for the strains isolated before treatment; therefore, susceptibility testing is recommended as an aid in clinical decision-making for the use of the azole group of drugs.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8641300&dopt=Abstract fluconazole Diflucan