C J Ingham1, A B Ayad, K Nolsen, B Mulder. 1. Laboratory of Medical Microbiology, Twente Achterhoek, Enschede, The Netherlands. cingham2@mac.com
Abstract
BACKGROUND: Phenotypic, culture-based methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis are relatively simple and may be particularly appropriate for resource-limited settings where tuberculosis (TB) is most prevalent. However, these methods can be slow and generate significant amounts of infectious waste. Low-cost digital imaging and a unique porous ceramic support for cell culture (Anopore) may offer opportunities to improve this situation. OBJECTIVE: To test a rapid DST method based on fluorescence microscopy of mycobacteria grown for a few generations on Anopore. DESIGN: Mycobacteria were cultured with and without drugs, and the resulting microcolonies were heat-killed and stained with the fluorogenic dye Syto16. Microscopy, image-capture with a charge-coupled device camera and digital processing were used to quantify the inhibition of growth by drugs. Rapid DST for rifampicin and isoniazid was performed for clinical isolates. RESULTS: Mycobacteria could be cultured, killed, stained and imaged on Anopore. For DST, the Anopore method gave an accurate result in 3 days. CONCLUSION: This is an unprecedented speed for culture-based DST for this group of organisms and results in minimal infectious waste (<20,000 colony forming units). Analysis of mycobacteria by fluorescence and electron microscopy on Anopore also opens up research possibilities.
BACKGROUND: Phenotypic, culture-based methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis are relatively simple and may be particularly appropriate for resource-limited settings where tuberculosis (TB) is most prevalent. However, these methods can be slow and generate significant amounts of infectious waste. Low-cost digital imaging and a unique porous ceramic support for cell culture (Anopore) may offer opportunities to improve this situation. OBJECTIVE: To test a rapid DST method based on fluorescence microscopy of mycobacteria grown for a few generations on Anopore. DESIGN: Mycobacteria were cultured with and without drugs, and the resulting microcolonies were heat-killed and stained with the fluorogenic dye Syto16. Microscopy, image-capture with a charge-coupled device camera and digital processing were used to quantify the inhibition of growth by drugs. Rapid DST for rifampicin and isoniazid was performed for clinical isolates. RESULTS: Mycobacteria could be cultured, killed, stained and imaged on Anopore. For DST, the Anopore method gave an accurate result in 3 days. CONCLUSION: This is an unprecedented speed for culture-based DST for this group of organisms and results in minimal infectious waste (<20,000 colony forming units). Analysis of mycobacteria by fluorescence and electron microscopy on Anopore also opens up research possibilities.
Authors: Matthijn C Hesselman; Dorett I Odoni; Brendan M Ryback; Suzette de Groot; Ruben G A van Heck; Jaap Keijsers; Pim Kolkman; David Nieuwenhuijse; Youri M van Nuland; Erik Sebus; Rob Spee; Hugo de Vries; Marten T Wapenaar; Colin J Ingham; Karin Schroën; Vítor A P Martins dos Santos; Sebastiaan K Spaans; Floor Hugenholtz; Mark W J van Passel Journal: PLoS One Date: 2012-05-14 Impact factor: 3.240
Authors: Colin J Ingham; Sjoukje Boonstra; Suzanne Levels; Marit de Lange; Jacques F Meis; Peter M Schneeberger Journal: PLoS One Date: 2012-03-16 Impact factor: 3.240