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

Award Number: R44AI152796
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/03/2020
PERIOD OF PERFORMANCE END DATE: 04/30/2026

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Improving diagnostic sensitivity for difficult-to-lyse microbial samples with nanodroplet technology - Abstract Infectious diseases are a leading cause of global morbidity and mortality, accounting for 29% of worldwide deaths. Next-generation sequencing (NGS) is a useful tool in pathogen detection, strain identification, and drug susceptibility testing (among other applications). A primary issue for NGS for rapid pathogen genomic analysis is that raw patient samples typically have a low bacterial load, requiring culturing that can take weeks to months before a sufficient microbial load is generated. However, culturing is economically and logistically unsustainable and presents with other biological issues that may confound results. Additionally, enrichment of the pathogen- specific genes is highly dependent on sample extraction efficiency. Using nucleic acid testing (NAT) and NGS methods, efficient DNA extraction is essential for the successful and accurate identification of microorganisms or populations of microbes. Poor DNA extraction when analyzing clinical and environmental samples consisting of resilient microbes leads to inconclusive or inaccurate diagnostic results. There is a need for high-efficiency extraction of nucleic acids from hard-to-lyse microorganisms in direct patient samples to facilitate reliable clinical diagnostic workflows. Triangle Biotechnology (Triangle Bio) is developing a novel and proprietary technology for efficient, high-throughput, reproducible, and unbiased microbial lysis, based on a cavitation-enhancing nanodroplet reagent for use with low-cost sonication devices. The proposed nanodroplets preferentially target to microbes with resilient cell walls and deliver focused mechanical shear forces. In Phase I, Triangle Bio demonstrated a 6-100x and 2-5x improvement in DNA extraction from Mycobacterium smegmatis (a model for Mycobacterium tuberculosis [Mtb]) and Enterococcus faecalis (a Gram- positive bacteria), respectively, compared to commonly used commercial kits. In Phase II, the company will establish a platform of nanodroplet formulations applicable to a wide range of infectious pathogens with significant clinical impact. Triangle Bio will accomplish this research through the following three aims: 1) Identify targeting ligand candidates compatible with 12 representative microbial species and three clinical sample matrices (Y1), 2) Validate binding and cavitation performance of candidate formulations and optimize workflow conditions for clinical sample matrices spiked with four target microbial species (Y2-Y3), and 3) Evaluate workflows by demonstrating improved performance of targeted NGS for diagnosis of drug-resistant Mtb (Y3). Successful implementation of this technology could have significant impacts on a wide range of applications requiring reliable microbial lysis techniques, including but not limited to NGS for infectious disease detection and diagnosis, NGS based food safety testing for infectious pathogens, and clinical and environmental microbiome studies where resilient microbes can be underrepresented in metagenomic analysis.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	TRIANGLE BIOTECHNOLOGY, INC	303 W BARBEE CHAPEL RD	CHAPEL HILL	NC	27517	ORANGE	USA	Allergy and Infectious Diseases Research	000	4	2/12/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $997,367 )
2024	2024	TRIANGLE BIOTECHNOLOGY, INC	303 W BARBEE CHAPEL RD	CHAPEL HILL	NC	27517	ORANGE	USA	Allergy and Infectious Diseases Research	000	4	4/30/2024	NON-COMPETING CONTINUATION	$997,367
														Subtotal = $997,367

Issue Date FY: 2023 ( Subtotal = $998,364 )
2023	2023	TRIANGLE BIOTECHNOLOGY, INC.	303 W BARBEE CHAPEL RD	CHAPEL HILL	NC	27517	ORANGE	USA	Allergy and Infectious Diseases Research	000	3	4/6/2023	NON-COMPETING CONTINUATION	$998,364
														Subtotal = $998,364

Issue Date FY: 2022 ( Subtotal = $1,000,000 )
2022	2022	TRIANGLE BIOTECHNOLOGY, INC.	303 W BARBEE CHAPEL RD	CHAPEL HILL	NC	27517	ORANGE	USA	Allergy and Infectious Diseases Research	000	2	5/25/2022	COMPETING CONTINUATION	$1,000,000
														Subtotal = $1,000,000

Grand Total All Awards = $2,995,731

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

Award Number: R44AI152796

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 08/03/2020

PERIOD OF PERFORMANCE END DATE: 04/30/2026

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