title: Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment.
abstract: Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 μM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.
authors.affiliation: Laboratori de Microbiologia Sanitaria i Mediambiental (MSM-Lab) & Aquasost-UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya (UPC), Edifici Gaia, Pg. Ernest Lluch/Rambla Sant Nebridi, 08222, Terrassa, Barcelona, Spain.
authors.forename: Gemma
authors.initials: G
authors.lastname: Agustí
authors.forename: Mariana
authors.initials: M
authors.lastname: Fittipaldi
authors.forename: Jordi
authors.initials: J
authors.lastname: Morató
authors.forename: Francesc
authors.initials: F
authors.lastname: Codony
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001386:Azides / Q000378:metabolism; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D003022:Clotrimazole / Q000494:pharmacology; D015169:Colony Count, Microbial / Q000379:methods*; D015725:Fluconazole / Q000494:pharmacology; D031168:Matricaria / Q000737:chemistry; D050296:Microbial Viability / Q000187:drug effects*; D009822:Oils, Volatile / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D046930:Thymus Plant / Q000737:chemistry
chemical_list: D000935:Antifungal Agents; D001386:Azides; D009822:Oils, Volatile; C014535:8-azidoethidium; D015725:Fluconazole; D003022:Clotrimazole