Rapid flow cytometry-based antifungal susceptibility testing method for Candida albicans

Vale Silva1, L.A., Pinto1, E., Queiroz2, M.J.

Author address: 

1Faculty of Pharmacy, University of Porto, PORTO, Portugal 2Chemistry Centre, University of Minho, BRAGA, Portugal

Abstract: 

The rising incidence of systemic fungal infections and, more recently, the expansion of the available armamentarium of antifungal drugs have been progressively stressing the need for development and implementation of antifungal susceptibility testing (AST) in the clinical mycology laboratory. However, the current standard broth dilution methods are still hindered by certain well known limitations. In this context, the objective of this work was the development of a flow cytometry-based test that could add to previously reported protocols as a potential alternative to more traditional approaches. We used the fluorescent dyes FUN® 1 and Acridine Orange (AO) in a 4-h incubation protocol to analyse the influence of serial two-fold dilutions of caspofungin, fluconazole and an experimental antifungal diheteroarylamine on Candida albicans. The minimum inhibitory concentrations (MICs) were determined in parallel using the reference M27-A2 protocol, published by the Clinical Laboratory Standards Institute (CLSI, formerly NCCLS). The flow cytometry method produced clear-cut results, presented as minimum fluorescence-enhancing concentrations (MFECs) and read according to criteria established from the comparison to the determined MICs. For each particular drug-dye pair, MFECs corresponded to the first test concentrations giving rise to a certain minimum increase in sample fluorescence. Overall, the flow cytometry results obtained with both dyes revealed very good performance in producing MFECs well associated to the determined MICs (0,5 µg/mL for caspofungin and fluconazole and 64 µg/mL for the experimental diheteroarylamine). When different, the MFECs showed a tendency to be lower than the correspondent MICs (within one or two dilutions), particularly in the case of caspofungin. The method seems, therefore, to perform well for drugs from different chemical groups and with different mechanisms of antifungal action. Further studies are now under way at our laboratory to investigate more thoroughly its definite sensitivity and reliability. According to these preliminary results, the protocol appears to be a promising model not only for rapid AST of C. albicans but also for research concerning the effect of commercial or experimental antifungal compounds on fungal cells.
2007

abstract No: 

P035

Full conference title: 

3rd Trends in Medical Mycology
    • TIMM 3rd (2011)