Abstract
Flow cytometry has found wide applications in areas like haematology and immunology, but also presents great potential in microbiology. The susceptibility of clinical isolates of Candida and Cryptococcus to antifungal compounds can be assayed by flow cytometry using fluorescent probes like FUN-1, propidium iodide and JC-1, with several advantages. Following 1 or 2 h of incubation, depending on the antifungal compound, versus 48 h of the classical methods, it is possible to establish the different susceptibility profiles. Additionally, it provides information regarding the mechanisms of action and might infer about resistance mechanisms.
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References
Álvarez-Barrientos A, Arroyo J, Cantón R, Nombela CA, Sánchez-Pérez M (2000) Applications of flow cytometry to clinical Microbiology. Clin Microbiol Rev 13:167–195
Pina-Vaz C, Costa-Oliveira S, Rodrigues AG, Salvador A (2004) Novel method using a laser scanning cytometer for detection of mycobacteria in clinical samples. J Clin Microbiol 42:906–908
Pina-Vaz C, Costa-de-Oliveira S, Rodrigues AG (2005) Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16. J Med Microbiol 54:77–81
Barbosa JM, Costa-de-Oliveira S, Rodrigues AG, Hanscheid T, Shapiro H, Pina-Vaz C (2008) A flow cytometric protocol for detection of Cryptosporidium spp. Cytometry A 73:44–47
Barbosa J, Costa-de-Oliveira S, Rodrigues AG, Pina-Vaz C (2008) Optimization of a flow cytometry protocol for detection and viability assessment of Giardia lamblia. Travel Med Infect Dis 6:234–239
Pina-Vaz C, Sansonetty F, Rodrigues AG, Costa-Oliveira S, Tavares C, Martinez-de-Oliveira J (2001) Cytometric approach for a rapid evaluation of susceptibility of Candida strains to antifungals. Clin Microbiol Infect 7:609–618
Pina-Vaz C, Sansonetty F, Rodrigues AG, Costa-de-Oliveira S, Martinez-de-Oliveira J, Fonseca AF (2001) Susceptibility to fluconazole of Candida clinical isolates determined by FUN-1 staining with flow cytometry and epifluorescence microscopy. J Med Microbiol 50:375–382
Pina-Vaz C, Costa-de-Oliveira S, Rodrigues AG, Espinel-Ingroff A (2005) Comparison of two probes for testing susceptibilities of pathogenic yeasts to voriconazole, itraconazole, and caspofungin by flow cytometry. J Clin Microbiol 43:4674–4679
National Commitee for Clinical for Laboratory Standards (2002) Reference method for broth dilution antifungal susceptibility of yeasts. Approved Standard M27-A2, 2nd edn. CLSI, Wayne, PA, USA
Hospenthal DR, Murray CK, Rinaldi MG (2004) The role of antifungals susceptibility testing in the therapy of candidiasis. Diagn Microbiol Infect Dis 48:153–160
Pina-Vaz C, Rodrigues AG, Sansonetty F, Martinez-De-Oliveira J, Fonseca AF, Mårdh P-A (2000) Antifungal activity of local anesthetics against Candida species. Infect Dis Obstet Gynecol 8:124–137
Pina-Vaz C, Rodrigues AG, Costa-de-Oliveira S, Ricardo E, Mårdh P-A (2005) Potent synergic effect between ibuprofen and azoles on Candida resulting from blockade of efflux pumps as determined by FUN-1 staining and flow cytometry. J Antimicrob Chemother 56:678–685
Pina-Vaz C, Rodrigues GA, Pinto E, Costa-de-Oliveira S, Tavares C, Salgueiro L, Cavaleiro C, Gonçalves MJ, Martinez-de-Oliveira J (2004) Antifungal activity of Thymus oils and their major compounds. J Eur Acad Dermatol Venereol 18:73–78
Pina-Vaz C, Sansonetty F, Rodrigues AG, Martinez-De-Oliveira J, Fonseca AF, Mårdh P-A (2000) Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species. J Med Microbiol 49:831–840
Millard PJ, Roth BL, Truong YH-P, Yue ST, Haugland RP (1997) Development of the FUN-1 Family of Fluorescent Probes for vacuole labeling and viability testing of yeasts. Appl Environ Microbiol 63:2897–2905
Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C (1993) A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the JC-aggregate forming lipophilic cation 5, 5′, 6, 6′-tretrachoro-1, 1′, 3, 3′-tetraethylbenzidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 197: 40–45
Phillips AJ, Sudbery I, Ramsdale M (2003) Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. Proc Natl Acad Sci U S A 100:14327–14332
Chaturvedi V, Ramani R, Pfaller MA (2004) Collaborative study of the NCCLS and flow cytometry methods for antifungal susceptibility testing of Candida albicans. J Clin Microbiol 42:2249–2251
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Pina-Vaz, C., Rodrigues, A.G. (2010). Evaluation of Antifungal Susceptibility Using Flow Cytometry. In: Sharon, A. (eds) Molecular and Cell Biology Methods for Fungi. Methods in Molecular Biology, vol 638. Humana Press. https://doi.org/10.1007/978-1-60761-611-5_21
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DOI: https://doi.org/10.1007/978-1-60761-611-5_21
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