Facile Method for Enhanced Pathogen Detection

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Abstract Leveraging microfluidic technologies for high-efficiency and high-precision cellular sample processing, Tico Biosciences is developing a culture-free direct-from-sputum method for high-efficiency host cell and host genomic DNA (gDNA) removal from clinical tuberculosis (TB) samples, enabling rapid, facile, and cost-effective whole-genome sequencing (WGS) for TB drug susceptibility diagnostics. Drug-resistant TB is a primary threat to global TB control, and strategies for treatment and medical intervention are heavily dependent on effective diagnosis and screening. However, current methods for sample processing and enrichment of Mycobacterium tuberculosis (Mtb) are severely inadequate. TB diagnostic methods currently involve either WGS directly from sputum samples, resulting in the overwhelming contamination of host genetic material, or culturing of patient samples, which is time consuming and leads to delayed patient treatment courses and loss of Mtb heterogeneity. Available strategies for host cell and host DNA removal are labor-intensive, have high skill barriers, and result in loss of up to 60% of the initial bacillary load. Consequently, the current state of the art results in poor patient outcomes, especially for those with mixed or drug resistant strains. To address these limitations, Tico Biosciences has licensed microfluidic technologies that allow for high-efficiency and high-precision cellular sample processing in order to remove host cell and gDNA in culture-free direct-from-sputum samples. This method will significantly reduce (1) total analysis turn-around time, (2) amount of sample handling, (3) user skill requirements, (4) resource requirements (e.g. laboratory space, electrical power, standard biology lab equipment, etc.), and (5) per-sample costs for reagents and sequencing by a factor of ten. This method results in the rapid removal of over 99% of mammalian DNA while retaining 85% of the initial bacillary load from a mixed sample of spiked human and E. coli cells. In this Phase I project, Tico plans to further optimize the performance of this microfluidic system with mock sputum and demonstrate the feasibility of this approach on pre-banked TB human clinical samples. This will be accomplished by: 1) Using Tico's system to recover over 95% of bacteria directly from sputum samples while removing over 99% of mammalian cells as quantified by fluorescence microscopy and qPCR; and 2) Demonstrating Tico's system to enable culture-free WGS of Mtb directly from sputum with fewer than 10% of total reads mapped to the human genome. This project will lead to an overall faster, easier-to-use, and cheaper method of processing clinical sputum samples for genetic analyses such as WGS or qPCR and will translate to increased adoption of otherwise prohibitively expensive approaches for early drug resistance screening, which is critically needed in many regions around the world.