Improving diagnostic sensitivity for difficult-to-lyse microbial samples with nanodroplet technology

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Abstract Communicable diseases claim the lives of 29% of world's population and more than 15 million persons die as a result of bacterial and fungal diseases every year. Timely and accurate diagnosis can reduce debilitation and save lives, and nucleic acid testing (NAT) of biomedical samples is a powerful method for identifying microorganisms that can return results in just a few hours (or faster). However, NAT-based diagnostics have repeatedly failed to displace traditional tests that can take several days (or more) to deliver a definitive diagnosis. Diagnostic assays need to meet exacting standards of sensitivity, specificity, and repeatability; and for NAT- tests, the initial step of efficiently extracting high-quality nucleic acids is critical. Unfortunately, life-threatening diseases such as bloodstream infections and tuberculous meningitis often produce clinical samples with pathogen concentrations as low as 1 colony-forming unit per milliliter. Notably, Mycobacterium tuberculosis, the organism that causes TB, is notoriously difficult to lyse. In fact, many gram-positive bacteria, viruses and fungi are hard to break open using conventional approaches and, in roughly half of patients with bloodstream infections, the causative microbe falls into one of these categories. Physical and mechanical approaches used to improve microbial lysis efficiencies include bead beating, high-pressure homogenization, direct or indirect sonication, and freeze-thawing/boiling. These techniques commonly achieve only moderate success despite their need for specialized and expensive equipment or materials, making them unsuitable for routine point-of- care diagnostics. Indirect sonication – which works by initiating the formation and collapse of microscopic bubbles (a process called cavitation) – offers multiple benefits compared to alternative lysis methods including reduced cost and contamination, ease of use, and being scalable for portability. Nonetheless, like other approaches, indirect sonication struggles to achieve consistently high yields of nucleic acids from resilient microbes without sacrificing quality. Triangle Biotechnology is developing novel and proprietary sonication reagents that substantially improve the efficiency of nucleic acid extraction by reducing the acoustic energy required for cavitation. This Phase I SBIR will establish a proof-of-concept for our reagent-assisted sonication technology by identifying conditions that maximize microbial lysis efficiency (Aim 1) and demonstrating improved NAT results from DNA extracted using our approach compared to traditional methods (Aim 2). The non- pathogenic target microorganisms, M. smegmatis, E. faecalis, and B. subtilis are commonly used models for pathogenic mycobacteria, Gram-positive bacteria, and spore-forming bacteria that threaten public health and safety. The data collected in this project will provide the foundation for Phase II studies to validate our platform technology by establishing high sensitivity (low limits of detection) for multiple resilient microbes using NAT methods. Triangle's long-term objective is to productize the technology in the form of a kit (reagents, buffers) and integrate the platform with emerging applications for small and portable sonication devices, beginning with the generation of optimized protocols for use of the kit with existing, low-cost sonicators.