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The culture and antifungal sensitivity testing of pathogenic fungi on a highly porous ceramic

Abstract number: P1344

Ingham C., Boonstra S., Lange M., Schneeberger P.

Objective: To develop a fast and widely applicable, culture-based antifungal sensitivity testing (AST) assay developed around the growth and in situ imaging of fungi on a porous ceramic support.

Methods: Sterile highly porous ceramic (PAO) strips (Anopore, an inert nanoporous ceramic derived from aluminium oxide) were placed upon agar plates (Sabauroud media or RPMI) containing defined concentrations of antifungals. The strips were then inoculated with the fungi (yeast, Trichophyton) to be tested, incubated, stained with fluorogenic dyes and imaged by fluorescence microscopy. Digital mages were processed in ImageJ software [1] to determine the effect of the antifungal agent.

Figure: Examples from AST of Candida albicans. Upper row shows a raw image of the yeast cultured on PAO showing growth inhibition with increasing concentrations of amphotericin B (AmpB). Lower section shows processed image used to calculate microcolony areas.

Results: A ssignificant effect of ampB on Candida was demonstrated within 90 min. MIC values could be derived reliably with 4 h culture (amphotericin B and caspofungin) or 7 h (voriconazole, itraconazole). For a total of 74 assays, in 65 cases the assignment by PAO (sensitive [S], resistant [R], intermediate [I]) was identical to the E-test. Where differences occurred these were always single category disagreements. Testing of Trichophyton rubrum and other filamentous fungi was possible with 16 h culture time.

Conclusions: Microcolony imaging is a rapid (c. 5× faster than E-test) and accurate approach to AST testing. Fungi grow well on PAO, adhere to the material and the images are easy to process. Performing these tests on PAO facilitates staining and imaging in situ and has allowed rapid assessment of whether an antifungal was inhibitory to growth. The method could be improved by multiplexing (more than one antibiotic/strip), reinforcing the PAO to facilitate handling and using cheaper dyes. Given that the method is also applicable to bacteria [1] including TB [2] PAO may be generally usable for antimicrobial testing and for applications outside clinical microbiology [3]. The PAO approach may also be useful for the study of heterogeneity of response to antimicrobials and other research applications.

References

1. Ingham CJ, van den Ende M, Wever PC, Schneeberger PM. J Med Microbiol. 2006 11:1511–9.

2. Ingham CJ, Ben Ayad A, Mulder B. IJTLD submitted.

3. Ingham CJ, Sprenkels A, Bomer J, Molenaar D, van den Berg A, van Hylckama Vlieg JE, de Vos WM. Proc Natl Acad Sci U S A. 2007 46:18217–22.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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