title: Rapid susceptibility testing and microcolony analysis of Candida spp. cultured and imaged on porous aluminum oxide.
abstract: BACKGROUND
  Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results.
  
  
  METHODS
  Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture).
  
  
  CONCLUSIONS
  Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications.
authors.affiliation: Jeroen Bosch Hospital, 's-Hertogenbosch, The Netherlands. colinutrecht@gmail.com
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Ingham
authors.forename: Sjoukje
authors.initials: S
authors.lastname: Boonstra
authors.forename: Suzanne
authors.initials: S
authors.lastname: Levels
authors.forename: Marit
authors.initials: M
authors.lastname: de Lange
authors.forename: Jacques F
authors.initials: JF
authors.lastname: Meis
authors.forename: Peter M
authors.initials: PM
authors.lastname: Schneeberger
mesh_terms: D000537:Aluminum Oxide; D000935:Antifungal Agents / Q000494:pharmacology; D002175:Candida / Q000187:drug effects* / Q000254:growth & development* / Q000472:pathogenicity; D015169:Colony Count, Microbial; D025141:Drug Resistance, Fungal; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008828:Microbiological Techniques; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D016062:Porosity
chemical_list: D000935:Antifungal Agents; D000537:Aluminum Oxide