Culture-free microbial enrichment for diagnosis and characterization of anti-microbial resistance

Abstract Timely identification of infectious agents enables targeted antibiotic administration and better health outcomes for patients. However, current methodologies take days to months, leading to the administration of broad- spectrum antibiotics when they may not be appropriate or effective. Drastic cost reductions of DNA sequencing technologies have led to increased demand for such workflows towards the identification of infectious agents. However, clinical samples often have extremely low levels of pathogens present. Additionally, depending upon the particular microorganism, the genetic payload is not always easy to access due to lysis-resilient cell wall structures. Finally, there is often overwhelming contamination from non-target DNA derived from the host or benign microorganisms that are part of the healthy microbiome. This project will establish the feasibility of a novel targeted-microbubble approach, Levios™, to enrich infectious pathogens directly from clinical samples and enable comprehensive diagnostics and strain identification for fast and personalized antimicrobial treatments. Enrichment of target microbes will enable the success of culture-free, molecular-based diagnostics via targeted enrichment of pathogenic microbial organisms. Triangle’s unique approach to microbial enrichment will address the clinical need for higher diagnostic sensitivity using direct clinical samples. In this Phase I proposal, we will demonstrate proof-of-concept of our Levios enrichment technology using Candida auris antimicrobial resistance (AMR) characterization as a real-world application for culture-free molecular-based assays. This is an ideal candidate to display the advantages of Levios as C. auris is an emerging multi-drug resistant pathogenic threat in the United States, is resilient to lysis, can live on hard surfaces for extended periods of time, and can colonize the skin. We will use our Levios microbial enrichment reagent to concentrate and enrich C. auris from swabs and blood samples to increase the sensitivity of detection for culture-free, molecular-based diagnostics. In alignment with our objectives, we will also develop digital PCR (dPCR)-based assays to identify the presence of pathogenic Candida strains (including C. auris) in patient samples and will include antimicrobial resistance information. We will also develop genomic sequencing workflows to characterize the strain of infectious Candida, which will provide more detailed strain information for epidemiology and AMR gene tracking. The proposed work will demonstrate that the Levios technology is capable of capturing target microbes from human samples, resulting in concentrated, high-quality samples for culture-free molecular-based diagnostics. The results will support commercialization of the platform in Phase II.