Rapid susceptibility testing of fungi by flow cytometry using vital staining

Wenisch, Christoph; Linnau, Ken Floris; Parschalk, Bernhard; Zedtwitz-Liebenstein, Konstantin; Georgopoulos, Apostolos P.

doi:10.1128/jcm.35.1.5-10.1997

Cited by 55 publications

(25 citation statements)

References 28 publications

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“…These results have not been compared with the NCCLS broth macrodilution method. Wenisch et al (19) developed an antifungal assay measuring the metabolic activity with the dye FUN-1. Our method measures membrane integrity, which is the target of antifungal agents.…”

Section: Discussionmentioning

confidence: 99%

Rapid flow cytometric susceptibility testing of Candida albicans

Ramani

Wong

1997

J Clin Microbiol

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A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed by adapting the proposed reference method for broth macrodilution testing of yeasts. Membrane permeability changes caused by the antifungal agent were measured by flow cytometry using propidium iodide, a nucleic acid-binding fluorochrome largely excluded by the intact cell membrane. We determined the in vitro susceptibility of 31 Candida albicans isolates and two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) to amphotericin B and fluconazole. Amphotericin B MICs ranged from 0.03 to 2.0 g/ml, while fluconazole MICs ranged from 0.125 to 128 g/ml. This method results in clear-cut endpoints that were reproducible. Four-hour incubation was required for fluconazole, whereas a 2-h incubation was sufficient for amphotericin B to provide MICs comparable to the reference macrodilution method developed by the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Tests. Results of these studies show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of C. albicans.

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Section: Discussionmentioning

confidence: 99%

Rapid flow cytometric susceptibility testing of Candida albicans

Ramani

Wong

1997

J Clin Microbiol

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“…It was evaluated with Candida sp and C. neoformans. [54][55][56][57] The method measures permeability of the fungal cell membrane after contact with various concentrations of antifungal agent. A membrane-impermeable fluorescent DNA-intercalating dye is placed in the standardized yeast suspension 2-6 hours after exposure to the drug.…”

Section: Flow Cytometrymentioning

confidence: 99%

In Vitro Antifungal Susceptibility Testing

Hoffman

Pfaller

2001

Pharmacotherapy

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With the rising frequency of fungal infections, as well as increasing reports of resistance to antifungal agents, it is imperative that clinically applicable antifungal susceptibility testing be available. In 1997 the National Committee for Clinical Laboratory Standards published standard guidelines for antifungal susceptibility testing of Candida sp and Cryptococcus neoformans with amphotericin B, flucytosine, fluconazole, itraconazole, and ketoconazole. Although the methods are standard, they are time consuming, can be difficult to interpret, and are approved only for testing limited organisms and drugs. Modifications to the methods and alternative approaches have been proposed to make these tests more convenient and efficient, applicable to a greater number of species, and appropriate for performing in the clinical laboratory.

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“…We would also like to stress that the AO/PI staining method used here represents one way of measuring of cellular viability, and does not necessarily indicate cellular vitality and/or the ability to establish growth on solid medium in any given sample. Therefore, while our method provides a facile method of live/dead quantification of yeasts, it will also be interesting to investigate the use of image cytometry in combination with cellular metabolism indicators such as FUN-1 15 . Image cytometry software identifies and counts cells of interest based on size and shape, and therefore, it is absolutely critical that these parameters are set carefully during each experiment.…”

Section: Discussionmentioning

confidence: 99%

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Berkes

Chan

Wilkinson

et al. 2013

JoVE

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Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

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Rapid susceptibility testing of fungi by flow cytometry using vital staining

Cited by 55 publications

References 28 publications

Rapid flow cytometric susceptibility testing of Candida albicans

Rapid flow cytometric susceptibility testing of Candida albicans

In Vitro Antifungal Susceptibility Testing

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

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