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PubMed2001

Effects of fluconazole and fluvoxamine on the pharmacokinetics and pharmacodynamics of glimepiride.

OBJECTIVE: Our objective was to study the effects of fluconazole and fluvoxamine on the pharmacokinetics and pharmacodynamics of glimepiride, a new sulfonylurea antidiabetic drug. METHODS: In this randomized, double-blind, three-phase crossover study, 12 healthy volunteers took 200 mg of fluconazole once daily (400 mg on day 1), 100 mg of fluvoxamine once daily, or placebo once daily for 4 days. On day 4, a single oral dose of 0.5 mg of glimepiride was administered. Plasma glimepiride and blood glucose concentrations were measured up to 12 hours. RESULTS: In the fluconazole phase, the mean total area under the plasma concentration-time curve of glimepiride was 238% (P <.0001) and the peak plasma concentration was 151% (P <.0001) of the respective control value. The mean elimination half-life of glimepiride was prolonged from 2.0 to 3.3 hours (P <.0001) by fluconazole. In the fluvoxamine phase, the mean area under the plasma concentration-time curve of glimepiride was not significantly different from that in the placebo phase. However, the mean peak plasma concentration of glimepiride was 143% (P <.05) of the control and the elimination half-life was prolonged from 2.0 to 2.3 hours (P <.01) by fluvoxamine. Fluconazole and fluvoxamine did not cause statistically significant changes in the effects of glimepiride on blood glucose concentrations. CONCLUSIONS: Fluconazole considerably increased the area under the plasma concentration-time curve of glimepiride and prolonged its elimination half-life. This was probably caused by inhibition of the cytochrome P-450 2C9-mediated biotransformation of glimepiride by fluconazole. Concomitant use of fluconazole with glimepiride may increase the risk of hypoglycemia as much as would a 2- to 3-fold increase in the dose of glimepiride. Fluvoxamine moderately increased the plasma concentrations and slightly prolonged the elimination half-life of glimepiride.

Adult↗
PubMed2006

Involvement of phenobarbital and SKF 525A in the hepatotoxicity of antifungal drugs itraconazole and fluconazole in rats.

Itraconazole and fluconazole are potent wide spectrum antifungal drugs. Both of these drugs induce hepatotoxicity clinically. The mechanism underlying the hepatotoxicity is unknown. The purpose of this study was to investigate the role of phenobarbital (PB), an inducer of cytochrome P450 (CYP), and SKF 525A, an inhibitor of CYP, in the mechanism of hepatotoxicity induced by these two drugs in vivo. Rats were pretreated with PB (75 mg/kg for 4 days) prior to itraconazole or fluconazole dosing (20 and 200 mg/kg for 4 days). In the inhibition study, for 4 consecutive days, rats were pretreated with SKF 525A (50 mg/kg) or saline followed by itraconazole or fluconazole (20 and 200 mg/kg) Dose-dependent increases in plasma alanine aminotransferase (ALT), gamma-glutamyl transferase (gamma-GT), and alkaline phosphatase (ALP) activities and in liver weight were detected in rats receiving itraconazole treatment. Interestingly, pretreatment with PB prior to itraconazole reduced the ALT and gamma-GT activities and the liver weight of rats. No changes were observed in rats treated with fluconazole. Pretreatment with SKF 525A induced more severe hepatotoxicity for both itraconazole and fluconazole. CYP 3A activity was inhibited dose-dependently by itraconazole treatment. Itraconazole had no effects on the activity of CYP 1A and 2E. Fluconazole potently inhibited all three isoenzymes of CYP. PB plays a role in hepatoprotection to itraconazole-induced but not fluconazole-induced hepatotoxicity. SKF 525A enhanced the hepatotoxicity of both antifungal drugs in vivo. Therefore, it can be concluded that inhibition of CYP may play a key role in the mechanism of hepatotoxicity induced by itraconazole and fluconazole.

Animals↗
PubMed2000

Intravitreal pharmacokinetics of plain and liposome-entrapped fluconazole in rabbit eyes.

The objective of our experiment was to study the pharmacokinetics of plain and liposome-encapsulated fluconazole after an intravitreal injection (100 microg/0.1 ml) in rabbit eyes. Equal concentrations of plain and liposome-entrapped fluconazole were injected intravitreally into albino rabbits through the pars plana after intravenous pentobarbitone anesthesia. The rabbits were sacrificed at various time intervals, and the concentration of fluconazole in vitreous, retina-choroid, aqueous humor and cornea was estimated using High Performance Liquid Chromatography (HPLC). Plain fluconazole showed a rapid vitreal clearance and a short half-life (3.08 hr), whereas liposome-entrapped fluconazole showed a longer half-life (23.40 hr). The terminal elimination constant (Ke) of the liposome-loaded drug from the vitreous was seven times less than the plain drug. In the plain fluconazole group, at 1 hr, a high concentration of the drug was found in the retina. To conclude, the elimination of fluconazole was primarily transretinal and was very rapid. Liposome encapsulation significantly increased the half-life of fluconazole in the vitreous cavity.

Animals↗
PubMed2004

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

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.

Animals↗
PubMed1995

Isolation of fluconazole-resistant Candida albicans from human immunodeficiency virus-negative patients never treated with azoles.

Isolation of fluconazole-resistant strains of Candida species from human immunodeficiency virus (HIV)-infected patients after repeated or continuous courses of treatment has been reported with increasing frequency. During 1991-1992, MICs of fluconazole for 139 Candida albicans isolates from our institution were bimodally distributed: 102 strains were susceptible (MICs, < or = 4 micrograms/mL) and 37 were resistant (MICs, > or = 8 micrograms/mL). There was incomplete cross-resistance between fluconazole and ketoconazole or miconazole, and there was no cross-resistance between azoles and amphotericin B or flucytosine. Twenty of the 37 fluconazole-resistant strains were isolated from 17 HIV-negative patients, some with systemic infections, who had never been treated with azoles. There were no differences in characteristics or risk factors for those patients as compared with those for an equal number of HIV-negative patients from whom fluconazole-susceptible strains were isolated. Among patients with systemic infection, 6 (50%) of 12 with infection caused by fluconazole-resistant strains survived and 11 (69%) of 16 with infection caused by fluconazole-susceptible strains survived (P = .54). Survival was not found to be related to treatment regimen, but the number of patients was small. The emergence of fluconazole-resistant C. albicans among HIV-negative patients never exposed to azoles is of concern.

Adolescent↗
PubMed1997

Infection due to fluconazole-resistant Candida in patients with AIDS: prevalence and microbiology.

A cross-sectional study was conducted to assess the prevalence and microbiology of oral infection due to fluconazole-resistant Candida in patients with AIDS. Oral swab specimens for fungal cultures were obtained from 100 consecutive outpatients with CD4 lymphocyte counts of < 200/mm3. At least one fungal organism demonstrating in vitro resistance to fluconazole (minimum inhibitory concentration, > or = 8 micrograms/mL) was isolated from 26 (41%) of 64 patients for whom cultures were positive. When fluconazole-resistant C. albicans was isolated, in vitro resistance correlated with clinical thrush. None of 10 patients from whom only non-albicans species of Candida were isolated had active thrush. The patients from whom fluconazole-resistant Candida albicans was isolated had lower CD4 cell counts (median, 9/mm3), a greater number of treated episodes of thrush (median, 4.5), and a greater median duration of prior fluconazole treatment (231 days) than did patients from whom fluconazole-susceptible C. albicans was isolated (median CD4 cell count, 58/mm3 [P = .004]; median number of treated episodes of thrush, 2.0 [P = .001]; and median duration of prior fluconazole treatment, 10 days [P = .01]; respectively). In a multivariate analysis, the number of episodes and duration of fluconazole therapy were independent predictors of resistance.

Acquired Immunodeficiency Syndrome↗
PubMed1991

The role of fluconazole in the early treatment and prophylaxis of experimental invasive aspergillosis.

The efficacy of fluconazole against Aspergillus fumigatus was assessed in an immunosuppressed temporarily leukopenic rabbit model of invasive aspergillosis. Therapy with fluconazole at 60 or 120 mg/kg/day was begun 24 h after lethal challenge and compared to that with amphotericin B at 1.5 mg/kg/day. Fluconazole reduced mortality compared with that in untreated controls and at 120 mg/kg/day reduced the tissue burden of A. fumigatus 10- to 100-fold in liver, kidney, and lung. However, amphotericin B was more effective in sterilizing tissues. Both fluconazole and amphotericin B dramatically decreased or eliminated circulating aspergillus antigen. Prophylaxis with fluconazole, begun 48 h before sublethal challenge, sterilized liver and kidney tissues and significantly reduced the tissue burden in lung. Early treatment with fluconazole reduced mortality and reduced antigenemia but did not sterilize tissues. Fluconazole prophylaxis at these doses prevented dissemination of invasive aspergillosis. Fluconazole was shown to have activity in the early treatment and prophylaxis of experimental invasive aspergillosis.

Animals↗
PubMed1996

Detection and significance of fluconazole resistance in oropharyngeal candidiasis in human immunodeficiency virus-infected patients.

The epidemiology and clinical significance of fluconazole resistance were assessed in a cohort of advanced human immunodeficiency virus (HIV)-infected patients with recurrent oropharyngeal candidiasis. Fifty patients were prospectively evaluated using a novel method of detecting fluconazole resistance with chromogenic media containing fluconazole; results were confirmed with macrobroth testing. Resistant yeasts, defined as MICs > or = 8 micrograms/mL, were detected in 16 (32%) of 50 patients: 7 (14%) had resistant Candida albicans, 7 (14%) had resistant non-C. albicans yeast, and 2 (4%) had mixed resistant yeasts. MICs were > or = 32 in 11 of 16 isolates. Previous fluconazole use and severe immunosuppression were risk factors for resistance. However, 5 of 26 patients had resistant isolates with no prior fluconazole use, and all were severely immunosuppressed. Despite the high prevalence of resistance, 48 patients clinically responded to fluconazole. Fluconazole-resistant C. albicans and non-C. albicans yeast infections are common in patients with advanced immunodeficiency, but clinical efficacy of fluconazole remains high.

AIDS-Related Opportunistic Infections↗
PubMed1997

Effect of continuous venovenous haemofiltration and haemodiafiltration on the elimination of fluconazole in patients with acute renal failure.

The elimination of fluconazole was studied in six patients with acute renal failure undergoing continuous venovenous haemofiltration (CVVH) for 24 h, continuous venovenous haemodiafiltration (CVVHD) 1 L/h for 24 h and CVVHD 2 L/h for 24 h. Fluconazole 200 mg once daily was given intravenously on three successive days and the concentrations of fluconazole in serum, ultrafiltrate/dialysate and urine were determined for 24 h after each dose. The half-life of fluconazole in patients during CVVH (83.5 +/- 30.1 h; mean +/- S.D.) was significantly (P < 0.05) longer than that during CVVHD 1 L/h (30.4 +/- 5.0 h) or CVVHD 2 L/h (21.8 +/- 3.5 h). The total fluconazole clearance was 0.57 +/- 0.16 L/h, 1.50 +/- 0.24 L/h and 1.85 +/- 0.17 L/h in CVVH, CVVHD 1 L/h and CVVHD 2 L/h, respectively, and there was a significant difference (P < 0.05) between all these treatments. Daily renal excretion of fluconazole was minimal, ranging from 0.002 mg to 11.2 mg in different patients with different treatment modes. The methods tested increased the elimination of the unchanged drug 20- to 400-fold in patients with acute renal failure. Patients undergoing CVVHD therapy with a dialysis flow rates of 1 or 2 L/h should be treated with a daily dose of at least 200 mg of fluconazole to maintain therapeutic drug concentrations. However, in patients on CVVH therapy smaller doses of fluconazole may be enough.

Acute Kidney Injury↗
PubMed1997

Fluconazole versus nystatin in the prevention of candida infections in children and adolescents undergoing remission induction or consolidation chemotherapy for cancer.

An open, prospective, randomized pilot study was performed to assess the efficacy and safety of oral fluconazole 3 mg/kg once daily compared with oral nystatin 50,000 units/kg/day in four divided doses in preventing candida infections in 50 children undergoing remission induction or consolidation therapy for cancer. In 21 of 25 fluconazole-treated and 20 of 25 nystatin-treated patients the overall outcome of prophylaxis was clearly successful. Mild and transient oropharyngeal candidosis was observed in two and three patients in the fluconazole and nystatin groups respectively. One patient randomized to fluconazole and two patients randomized to nystatin required empirical treatment with amphotericin B and one patient assigned to fluconazole developed tissue-proven candida colitis. Initially non-colonized patients remained yeast-free throughout treatment with no differences between the two study arms. Initially colonized patients stayed colonized throughout treatment although at the end of the study, more patients randomized to nystatin were still harbouring yeasts (P = 0.05). Almost exclusively, Candida albicans (95%) was isolated. A change in species was observed in one patient in each arm of the study. Candida krusei or Candida glabrata were not encountered. Transient elevations of hepatic transaminases were more common in the fluconazole group, although not statistically significant (28% vs 12%, P = 0.15). Reversible grade I gastrointestinal and skin symptoms were observed in four patients randomized to fluconazole (16 vs 0%, P < 0.05). Fluconazole was as safe and effective as nystatin in controlling yeast colonization and in preventing superficial and invasive candida infections and the empirical use of amphotericin B in children and adolescents undergoing intensive chemotherapy for cancer.

Adolescent↗
PubMed1998

Fluconazole susceptibility and strain variation of Candida albicans isolates from HIV-infected patients with oropharyngeal candidosis.

Over a 16 month period we conducted a prospective study in a cohort of 45 HIV-positive patients to detect the development of resistance to fluconazole and to analyse the epidemiology of oropharyngeal candidosis (OPC). Each episode was treated with fluconazole 100 mg/day po for 10 days. All yeast isolates were tested for their in-vitro susceptibility to fluconazole. Multiple strains of Candida albicans simultaneously isolated from a given patient were typed by electrophoretic karyotyping. Overall, 106 episodes of OPC were diagnosed among the 45 patients: 18/45 patients (40%) had only one episode, 11/45 (24%) had two episodes, and the remaining 16/45 (36%) had three or more episodes (range 3-7). Cure (complete resolution of signs and symptoms and negative post-treatment cultures) and improvement (complete resolution of signs and symptoms but positive post-treatment cultures) were observed in 30/106 (28%) and 69/106 (65%) episodes of OPC, respectively. Failure (absence of improvement or exacerbation of signs and symptoms) was observed in seven episodes (7%) from four patients. In two of these four patients a significant and progressive increase in fluconazole MICs was observed: from 0.25 to 16 mg/L in one patient, and from < or = 0.125 to 32 mg/L in the second one. Tests on multiple colonies from individual isolation plates showed that it was not unusual to obtain different fluconazole MICs, indicating that, in order to avoid misleading results, one should perform in-vitro susceptibility testing by using a multiple colony inoculum rather than an inoculum made from a single colony. A total of 213 strains of C. albicans isolated from seven patients who suffered from four or more episodes of OPC through the course of the study were typed by electrophoretic karyotyping. Five individuals (71%) were infected with yeasts with only one DNA type, while the other two patients showed the presence of two or three different DNA types. The simultaneous presence of multiple types was found only in one of the seven subjects. Our data confirm the efficacy of fluconazole 100 mg/day for the treatment of OPC in HIV patients. Isolation of fluconazole-resistant strains of C. albicans with this regimen is rare. The vast majority of HIV patients are infected with a unique strain of C. albicans throughout each episode of infection. A minority of patients, however, can harbour strains of C. albicans with variable patterns of fluconazole susceptibility simultaneously.

AIDS-Related Opportunistic Infections↗
PubMed2005

Enhanced pathogenicity of Candida albicans pre-treated with subinhibitory concentrations of fluconazole in a mouse model of disseminated candidiasis.

OBJECTIVES: To investigate the relative pathogenicity of Candida albicans treated with subinhibitory concentrations of fluconazole in a mouse model of disseminated candidiasis. Previous studies indicate that these cells secrete 10 times more farnesol than do untreated cells. In our usage, subinhibitory means a concentration which causes a prominent decrease in turbidity but still allows some cell growth. METHODS: C. albicans A72 cells were grown overnight in 0-5.0 microM fluconazole, washed, and inoculated in mice by tail vein injection. Groups of 15 or 16 mice were injected with 1.3 x 10(6) cells and mortality was recorded for 7 days post-inoculation. The levels of farnesol in control and treated C. albicans were determined by GC/MS. RESULTS: The MIC50 for strain A72 was 0.125 mg/L (0.4 microM). Mice administered C. albicans pre-treated with 0.5 to 1.0 microM fluconazole died 2.5 to 4 days earlier and had 2 to 4 times higher mortality rates than mice given untreated C. albicans. Fluconazole (0.5 to 1.0 microM) pre-treated cells were 4.2 to 8.5 times more lethal (P < 0.001) than untreated cells. The extracellular, membrane bound, and intracellular farnesol concentrations of cells pre-treated with 1.0 muM fluconazole were 12-, 2- and 6-times those of untreated cells. CONCLUSIONS: The effects of fluconazole on C. albicans are very concentration-dependent. The enhanced pathogenicity of fluconazole pre-treated C. albicans in mice should be relevant to the therapeutic and prophylactic use of fluconazole. Further research is needed to explore whether farnesol production by C. albicans is a virulence factor.

Animals↗
PubMed2005

Fluconazole dosing in continuous veno-venous haemofiltration (CVVHF): need for a high daily dose of 800 mg.

To cover intermediate sensitive Candida glabrata in ICU patients, fluconazole plasma peak levels at least in the range of 16-32 microg/ml appear necessary for treatment. Previous studies did not reach these fluconazole levels under continuous veno-venous haemofiltration (CVVHF) with dosages of 200-600 mg fluconzole daily. In the present study, nine patients simultaneously requiring CVVHF for treatment of acute oligoanuric renal failure and antimycotic therapy of Candida septicemia received fluconazole 800 mg/day. Fluconazole plasma levels were determined to evaluate whether this dosage is adequate to reach the advised fluconazole levels. Patients were dialysed on two consecutive days with an ultrafiltration rate (UF) of 1000 ml/h or 2000 ml/h, respectively, in a randomized order. The predilution was 800 ml/h and 1800 ml/h, respectively. The treatment was tolerated without adverse effects. All patients reached plasma fluconazole concentrations between 16 and 32 microg/ml, remaining in this range for a minimum of 1 up to 24 h with a mean of 9.6 h and a UF rate of 2000 ml/h, and 15.7 h with a UF rate of 1000 ml/h. So far, there are no in vivo data on the fluconazole plasma concentrations required for effective treatment. However, our data demonstrate, that at least the fluconazole concentrations desirable on the basis of in vitro susceptibility testing can be reached in critically ill patients on CVVHF in an ICU setting. However, in these patients, 800 mg fluconazole/day are necessary to achieve fungicidal drug concentrations.

Acute Kidney Injury↗
PubMed1996

Itraconazole cyclodextrin solution for fluconazole-refractory oropharyngeal candidiasis in AIDS: correlation of clinical response with in vitro susceptibility.

OBJECTIVE: To evaluate the efficacy of itraconazole cyclodextrin solution in fluconazole-refractory oropharyngeal candidiasis (OPC), and to correlate clinical outcome with in vitro susceptibility and serum azole levels. DESIGN: A prospective, open-label, intervention study. SETTING: A university hospital, which serves as the provincial HIV referral center. PATIENTS AND INTERVENTIONS: Thirty six HIV-infected individuals referred for fluconazole-refractory OPC were evaluated prospectively between May 1993 and March 1995, including clinical assessment, serum azole levels, and susceptibility testing of Candida spp, isolates. Itraconazole solution was administered orally at a daily dose of 200 mg for 14 days, followed by suppressive therapy. Thirty-four patients were evaluable. MAIN OUTCOME MEASURE: Resolution of oral pseudomembranous lesions. RESULTS: Initial isolates were Candida albicans (n = 33), C. glabrata (n = 1), C. krusei (n = 1), and mixed infection with C. albicans and C. krusei (n = 1). Fluconazole serum levels obtained at the time of failed therapy ranged from 4.7 to 40 mg/l (median, 12.9 mg/l). Itraconazole was generally well tolerated. Clinical responses were observed in 65% (22 out of 34) of evaluable cases. Among the responders, relapse had occurred within 2 months for four (36%) out of 11 cases who continued with follow-up. The median fluconazole minimal inhibitory concentration (MIC) was 64 mg/l for isolates from fluconazole-refractory cases, compared with a median of 0.5 mg/l for control isolates (P = 0.002). The median itraconazole MIC for isolates from fluconazole-refractory cases was 1.25 mg/l, compared with a median of 0.078 mg/l for controls (P = 0.011). CONCLUSION: A correlation between clinical response and in vitro susceptibility was clearly demonstrated for fluconazole, but not for itraconazole. Itraconazole cyclodextrin solution may be effective for fluconazole-refractory OPC and should be considered prior to salvage therapy with intravenous amphotericin B.

AIDS-Related Opportunistic Infections↗
PubMed1996

Fluconazole vs. amphotericin B for the treatment of neonatal fungal septicemia: a prospective randomized trial.

OBJECTIVE: Fungal septicemia is a devastating disease in the neonate, especially in the low birth weight preterm infant who is especially vulnerable to disseminated fungal sepsis. The objective of this study was to compare the efficacy, safety and overall convenience of fluconazole vs. amphotericin B for the treatment of disseminated fungal sepsis in neonates. DESIGN: A prospective, randomized, collaborative study conducted at two South African neonatal units. SUBJECTS: Twenty-four infants with proven fungal septicemia were treated from June, 1992, to June, 1993. Twelve received fluconazole, 11 received amphotericin B and 1 was excluded. Assessment of hepatic, renal and hematologic functions were performed before, during and after treatment. The two groups were comparable at the time of enrollment into the study. RESULTS: Infants receiving amphotericin B had significantly higher values of total and direct bilirubin and alkaline phosphatase values at the end of treatment, while the fluconazole group showed a significant increase in the platelet count. The cumulative total numbers of days receiving intravenous therapy for the administration of antifungal drugs were 57 for the fluconazole group and 162 for the amphotericin group; no central lines were needed in the fluconazole group, whereas 3 babies given amphotericin B had central catheters for a cumulative total of 27 days. The case fatality rate was 33% in the fluconazole group and 45% in the amphotericin B group; there was still proof of fungal septicemia at the time of death in 1 patient given amphotericin B and 2 given fluconazole. CONCLUSION: Fluconazole showed fewer side effects than amphotericin B and was more convenient to use.

Administration, Oral↗
PubMed2001

Susceptibility to fluconazole of Candida clinical isolates determined by FUN-1 staining with flow cytometry and epifluorescence microscopy.

The susceptibility of clinical Candida isolates to fluconazole was assayed by flow cytometry (FCM) and epifluorescence microscopy (EFM), with FUN-1 staining. In all, 25 clinical isolates of Candida spp. (12 sensitive, 3 dose-dependently sensitive and 10 resistant to fluconazole according to the NCCLS M27-A protocol) were treated with increasing concentrations of fluconazole during 1 or 2 h staining with FUN-1 for 30 min and analysed, respectively, by FCM at 575 nm (FL2) and by EFM. Fluconazole-susceptible strains showed an increased accumulation of FUN-1 in comparison with controls as determined by FCM and a reduced metabolic processing of the probe, confirmed by EFM. Conversely, resistant strains showed decreased FUN-1 staining and were able to process the probe. The fluconazole minimal inhibitory concentrations (MICs) determined by FCM or EFM after FUN-1 staining compared very well with the corresponding values determined by the M27-A protocol, indicating that FUN-1 staining can be used as an alternative to the conventional method. MIC values of resistant strains, with the exception of C. krusei, were lower when treatment with fluconazole followed pre-incubation with 0.1 mM sodium azide, a concentration known to inhibit the activity of efflux pumps. These results show that FUN-1 staining can be used as an alternative and rapid method for the assessment of susceptibility of Candida clinical isolates to fluconazole. Furthermore, the results suggest that resistance of Candida cells to fluconazole, with the exception of C. krusei strains, is likely to be due to the activity of efflux pumps.

Antifungal Agents↗
PubMed2003

Proliferation of intracellular structure corresponding to reduced affinity of fluconazole for cytochrome P-450 in two low-susceptibility strains of Candida albicans isolated from a Japanese AIDS patient.

Three Candida albicans isolates, TIMM 3164, 3165 and 3166 with reduced fluconazole susceptibility, were isolated from two Japanese AIDS patients. We earlier reported that a reduced intracellular accumulation of fluconazole in these isolates played an important role in the resistance mechanism of fluconazole, but we did not exclude the involvement of other factors. We here examined characteristics related to cytochrome P-450 (CYP), especially sterol 14alpha-demethylase encoded by the ERG11 gene which is the target molecule for fluconazole. In TIMM 3164 and 3165, the ergosterol synthesis by cell-free extracts was somewhat less susceptible to fluconazole, due to a decrease in fluconazole affinity for CYP. The nucleotide substitutions in the ERG11 gene were identified to result in three amino acid changes of K143R, E266D and V488I in TIMM 3164, and of E266D, V404L and V488I in TIMM 3165. These amino acid substitutions might contribute to the decreased affinity for CYP in both isolates. However, a single amino acid change, E266D, observed in TIMM 3166 was unrelated to the decreased affinity for CYP. The most prominent finding on the ultrastructure of TIMM 3164 and 3165 was the development of mesh membrane structures of the endoplasmic reticula, which is a location related to sterol synthesis. This phenomenon was not observed in the cells of TIMM 3166 or the susceptible control strains of ATCC 90028 and 10231. In addition to the reduced intracellular accumulation, the decreased affinity of fluconazole for CYP in TIMM 3164 and 3165 is assumed to be associated with the fluconazole-resistance phenotype.

AIDS-Related Opportunistic Infections↗
PubMed2004

Characterization of mechanisms of fluconazole resistance in a Candida albicans isolate from a Japanese patient with chronic mucocutaneous candidiasis.

We examined the mechanisms of fluconazole resistance in a fluconazole-resistant Candida albicans isolate from a Japanese patient with chronic mucocutaneous candidiasis. It was demonstrated that the highly resistant phenotype of this strain was associated with combined mechanisms of the energy-dependent reduced intracellular accumulation of fluconazole, presumably due to the increased expression of the ATP-binding cassette efflux pump CDR gene(s), and the reduced affinity of the target enzyme, Erg11p, to fluconazole. In particular, the reduced affinity of Erg11p was considered to contribute largely to the fluconazole resistance in the TIMM3209 strain. Biochemical studies indicated that the Erg11p from the TIMM3209 strain showed reduced susceptibility both to fluconazole and itraconazole of cell-free ergosterol biosynthesis, and cytochrome P-450 also showed reduced affinity to fluconazole in the carbon monoxidecytochrome P-450 complex formation assay. We identified two amino acid substitutions, Y132H and G448V, in Erg11p from the TIMM3209 strain. We found that the cytochrome P-450 from the TIMM3209 strain decayed during incubation at 37 C without fluconazole although it is unknown whether or not the phenomenon is linked to the resistant phenotype. These mutations are thought to confer the above-mentioned characteristics to Erg11p.

ATP Binding Cassette Transporter, Subfamily B, Mem↗