Development of azole resistance during fluconazole maintenance therapy for AIDS-associated cryptococcal disease

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Five sequential Cryptococcus neoformans isolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-␣ lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S.Mechanisms of azole resistance already described for yeasts include altered affinity of lanosterol 14-␣ demethylase (ERG11) to azole drugs due to target site mutation or its overexpression and decreased accumulation of drugs due to enhanced energydependent drug efflux (5, 13). Changes in the azole affinity of the lanosterol 14-␣ demethylase have already been related to low-level fluconazole resistance in Cryptococcus neoformans isolates (15). In addition, the decreased affinity of lanosterol 14-␣ demethylase for azole derivatives due to mutations that contributes to the increase in the MICs of fluconazole has been described for sequential clinical isolates of Candida albicans (5, 6). To elucidate if this mechanism could also be implicated in the resistance of C. neoformans to azole, we compared the ERG11 genomic sequence in five sequential isolates recovered from recurrent episodes of cryptococcal meningitis.Clinical case. The five strains of C. neoformans were isolated from a 33-year-old male patient who had been positive for human immunodeficiency virus since 1990 and was presenting advanced AIDS (CD4 ϩ count, Ͻ100 cells/mm 3 ) and recurrent cryptococcosis. The first episode of cryptococcosis was diagnosed in August 1997 at Hospital Fernandez, Buenos Aires, Argentina. During the following 15 months, four more episodes of cryptococcal meningitis were detected and documented by cultures recovered from cerebrospinal fluid. In the first episode the patient was treated with amphotericin B (AMB), and for the rest of the episodes a fluconazole therapy at different doses was always established, reaching a cumulative dose of 336 g at the moment of the fifth episode. The five isolates from each episode were identified as C. neoformans var. grubii by the following parameters: morphology, assimilation and fermentation of carbon and nitrogen compounds, and molecular taxonomy.Susceptibility testing was performed by microdilution and E-test methods. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control strains throughout the experiments (7). Microdilution method. The susceptibility testing followed the NCCLS recommendations (7) but included some modifications as previously described (11). Briefly, the susceptibility testing included RPMI medium supplemented with 2% glucose as the assay medium (RPMI-2% glucose), an inoculum size of 10 5 CFU/ml, flat-bottomed trays, spectrophotometric reading at 530 nm, incubation at 30°C, and shaking at 350 rpm for 48 h (11). The antifungal agents used in the study were as follows: AMB (Sigma Aldrich Quimica S.A., Madrid, Spain), 5-flucytosine (5FC) (Sigma Aldrich Quimica), fluconazole ...

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confidence: 95%

G484S Amino Acid Substitution in Lanosterol 14-α Demethylase ( ERG11 ) Is Related to Fluconazole Resistance in a Recurrent Cryptococcus neoformans Clinical Isolate

Rodero

Mellado

Rodriguez

et al. 2003

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112

“…Improvements in the treatment of cryptococcal meningitis have come primarily from the use of combination antifungal chemotherapy and attention to raised intracranial pressure (21,25,26). However, the frequent need to use toxic agents, particularly the combination of amphotericin B and flucytosine, and the recent emergence of fluconazole-resistant isolates of C. neoformans highlight the need for improved treatment regimens and the use of combination therapy to forestall the emergence of resistant organisms (1,2,5,13). This study evaluated the combinations of fluconazole, flucytosine, and amphotericin B in a murine model of cryptococcal meningitis.…”

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confidence: 99%

Amphotericin B and Fluconazole, a Potent Combination Therapy for Cryptococcal Meningitis

Larsen

Bauer

Thomas

et al. 2004

We evaluated the antifungal activities of amphotericin B, fluconazole, and flucytosine, alone and in combination, in a murine model of cryptococcal meningitis. The objectives were to determine the greatest antifungal effects achievable with these drugs alone or in combination. Meningitis was established in male BALB/c mice weighing 23 to 25 g by intracerebral injection of Cryptococcus neoformans. Treatment was started on day 2. Amphotericin B was tested at 0.3 to 1.3 mg/kg of body weight/day by slow intravenous injection. Fluconazole at 10 to 40 mg/kg/day and flucytosine at 20 to 105 mg/kg/day were administered in the sole source of drinking water. The mice were killed at 16 days, and the numbers of fungal colonies in the brain were quantified. The association between the response and the dose combination was evaluated by local nonparametric response surface methods; 99% confidence intervals were used to evaluate the antifungal effects. Ninety-five percent of the mice treated with amphotericin B at 0.5 mg/kg survived to the end of the experiment, regardless of the fluconazole or flucytosine dose used. The greatest activity was seen with amphotericin B plus fluconazole with or without flucytosine. However, the addition of flucytosine did not increase the antifungal activity. Given the widespread availability of amphotericin B and fluconazole and the relative safety profile of fluconazole compared to that of flucytosine, the full potential of this two-drug combination deserves further evaluation.

“…Voriconazole is not currently licensed for use in cryptococcosis and no clinical trials have evaluated its efficacy for cryptococcal disease. There is limited published information regarding the clinical use of voriconazole for cryptococcosis (12,30). In a study by Perfect et al, voriconazole therapy resulted in a 39% response rate in 18 patients with refractory cryptococcal meningoencephalitis (30).…”

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confidence: 99%

Effects of Voriconazole on Cryptococcus neoformans

Duin

Cleare

Zaragoza

et al. 2004

Voriconazole is a broad-spectrum triazole that offers extended activity against molds and yeasts that are not susceptible to earlier azole-type drugs. Recent studies indicate that melanization can severely reduce the susceptibility of certain antifungal drugs, but there is no information as to whether voriconazole is vulnerable to this effect. The activity of voriconazole on C. neoformans was assessed by MIC analysis and time-kill assays for melanized and nonmelanized cells. Cell morphology, capsule release, and phagocytosis of C. neoformans were studied in the presence or absence of subinhibitory concentrations of voriconazole. Voriconazole was fungicidal at concentrations of >8 g/ml in vitro against the strains of C. neoformans examined, and its efficacy was not diminished by melanization. Cells grown in subinhibitory concentrations of voriconazole had smaller cellular and capsular volumes than cells grown in the absence of drug. The induction of the capsule by serum was not affected by voriconazole. Cells grown in subinhibitory concentrations of voriconazole released their capsule and were phagocytosed at rates comparable with yeast grown without the antifungal. The high activity of voriconazole against both melanized and nonmelanized cells results suggest that voriconazole may be a particularly valuable drug for cryptococcosis.Cryptococcus neoformans is a relatively frequent cause of serious fungal infections in immunocompromised patients. The prevalence in the United States of cryptococcal meningoencephalitis in patients with AIDS receiving retroviral therapy is currently estimated to be Ͻ2% (23) but is Ͼ30% in areas of South East Asia and Sub-Saharan Africa (29). Patients with AIDS complicated by cryptococcosis often respond poorly to treatment and, in the setting of continued immunosuppression, require lifelong maintenance therapy since currently available antifungal agents seldom eradicate these fungal pathogens in the setting of severe immune suppression (16,41).Voriconazole, a synthetic derivative of fluconazole, is a broad-spectrum triazole antifungal that inhibits cytochrome P450-dependent 14␣-lanosterol demethylation, which is a critical step in fungal cell membrane ergosterol synthesis. Voriconazole demonstrates excellent in vitro activity against C. neoformans (17, 31) and achieves good levels in cerebrospinal fluid (35). Voriconazole is not currently licensed for use in cryptococcosis and no clinical trials have evaluated its efficacy for cryptococcal disease. There is limited published information regarding the clinical use of voriconazole for cryptococcosis (12, 30). In a study by Perfect et al., voriconazole therapy resulted in a 39% response rate in 18 patients with refractory cryptococcal meningoencephalitis (30).Given recent evidence that melanization can significantly reduce the efficacy of certain antifungal drugs against C. neoformans (36), we evaluated the activity of voriconazole against both melanized and nonmelanized yeast cells. Although we previously did not see a protective effect...