Massively Multiplexed dsDNA Invasion Arrays - PROJECT SUMMARY Septicemia, induced by an invasive microbial bloodstream infection (BSI), is a significant healthcare burden, accounting for over 1.7M cases annually just in the US. Characterized by high mortality rates (~20%), prognosis for septic patients deteriorates hourly in the absence of appropriate treatment. Timely and accurate pathogen identification is critical to match treatment to the infection. Unfortunately, current diagnostic approaches for BSIs rely on blood culture, which is slow (1-3 days) and unreliable in the presence of prior antimicrobial treatment. In the meantime, physicians must rely on broad-spectrum treatment, which often misses the infection, leads to complications, and drives antimicrobial resistance. New diagnostic approaches are needed which can quickly detect and identify the infectious agent directly from blood, without cultures To address this unmet need, HelixBind has developed a sample to answer process which can identify bacterial and fungal BSIs directly from blood in 3 hours. Utilizing a proprietary detection approach leveraging synthetic, duplex DNA invading, nucleic acids, the assay provides species level detail at single CFUs/ml sensitivity. dsDNA invasion-based detection (rather than ssDNA hybridization) affords single-bp specificity and the ability to reliably differentiate between closely related species and reduce false-positives due to random contaminations. To date, though, invasion has never been demonstrated in a format appropriate for massively parallel detection. In this proposal, HelixBind will develop, for the first time, a DNA invasion array which can detect and identify hundreds of pathogens simultaneously. This array will be incorporated into a sample-to- answer fluidic cassette operated on a benchtop instrument. The resulting test will provide clinicians with comprehensive coverage of essentially all pathogens associated with BSIs and unequivocal identification of an infection within hours of patient presentation, enabling early application of appropriate antimicrobials, improving care and saving countless lives. HelixBind has previously established the capability of fluid-based invasion to identify BSIs from clinical samples and in this application, we present preliminary data for solid-phase array-based invasion, allowing massively multiplexed detection. In this proposal we will address the biophysical challenges associated with creating a highly multiplexed dsDNA invasion array and the technical hurdles associated with integrating this array into a sample-to-answer fluidic device. Specific Aims, each with quantifiable deliverables, serve to address the key risks and progressive steps in the development process. Upon completion of this Phase II project, we will have a fully functional assay and a product appropriate for manufacturing scale up and clinical testing. We will also have developed a new, highly multiplexed assay format, with single-bp resolution, which can be adapted for a wide range of applications requiring sensitive and specific differentiation among closely related targets.
