10th International Aerosol Conference September 2 - September 7, 2018 America's Center Convention Complex St. Louis, Missouri, USA
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Capture and Characterization of Exhaled Bio-aerosols from Tuberculosis (TB) Patients
ROBIN WOOD, Carl Morrow, Benjamin Patterson, Wayne Bryden, Charles Call, David Silcott, Catherine Fenselau, D. Chen, R. Dinkele, S. Gessner, Digby Warner, IDM, University of Cape Town
Abstract Number: 1722 Working Group: Infectious Bioaerosol
Abstract The respiratory aerosol sampling chamber (RASC) was designed to create a safe, contained environment for bio-aerosol investigation of the production of Mycobacterium tuberculosis (MTB) bacilli by individual tuberculosis (TB) patients. Key design features include the small volume (1.4m3), HEPA filtration of inflowing air with continuous monitoring of temperature, relative humidity, carbon dioxide concentrations, airborne particle concentration, and cough frequency. Scanning electron microscopy studies of impacted particles revealed occasional MTB-like organisms, and solid medium culture produced colonies of confirmed MTB with detection efficiency of 43%. Here, we detail efforts to make further improvements on the sensitivity of the collection and detection systems.
Sampling system: The optimized collection of bio-aerosol uses a single, large air volume (250L/m) sampling cyclone (Bertin Coriolis ) and liquid collection. The liquid sample containing collected bio-aerosol is centrifuged followed by mass spectrometry of the supernatant and bacteriological assay of the pellet via fluorescence microscopy.
Mass spectrometry: The mass spectrometry (Thermo Fisher, Orbitrap LTQ) protocol developed in the pilot study resulted in a putative TB diagnosis from bio-aerosol sampling with a rapid (1-2 day) turnaround. Moreover, the diversity of lipids discovered to date indicates that correlates may be developed that provide more detailed information about the disease state. There is also the potential for discovery of specific diagnostic markers in human breath that could be measured more rapidly using less complex protocols. It is proposed that simplification of the process may lead to a readily fieldable diagnostic.
The liquid-capture pellet is re-suspended and distributed over a microscope slide containing multiple single nanoliter-sized wells. The spatially separated MTB within the nano-wells are then stained with a recently described fluorescent trehalose dye which is incorporated into the cell walls of viable, metabolically active bacteria. Quantitative detection of these organisms is enabled by fluorescence microscopy, with current efforts aimed at developing time-lapse imaging for additional phenotyping including growth rates.
Bio-aerosol from TB patients can be collected in the RASC. Viable MTB organisms can be rapidly imaged using a non-destructive assay and specific mycobacterial lipids detected by mass spectrometry.