Abstract Details

Title Illuminating Tuberculosis Meningitis with Metagenomics and FLASH Enrichment

Topic Infectious Disease

Presentation(s) S45 - Neuroinfectious Disease: Treatments, Diagnostics, and Outcomes (1:44 PM-1:55 PM)

Poster/Presentation
Number 005

Background

TBM diagnosis is difficult. mNGS permits broad detection of CNS pathogens in a single assay. Further, targeted enrichment of low abundance genes by up to 10⁵-fold is possible with Finding Low Abundance Sequences by Hybridization (FLASH), which is a novel CRISPR-Cas9 technology. Combining these technologies could improve detection of TB and associated AMR mutations.

Objective

To enhance detection of Mycobacterium tuberculosis meningitis (TBM) and anti-microbial resistance (AMR) mutations in HIV-infected Ugandan adults with meningitis, using metagenomic next generation sequencing (mNGS) of CSF coupled with CRISPR-Cas9 enrichment technology.

Design/Methods

Cohort: 15 patients with subacute meningitis: 6 definite TBM, 4 Cryptococcus (1 meningitis, 3 symptomatic antigenemia), 1 potential Cryptococcus-immune reconstitution inflammatory syndrome (IRIS), and 4 unknown. Method: DNA was extracted from CSF, and unbiased cDNA libraries were sequenced on an Illumina NextSeq. In parallel, FLASH enrichment was performed by incubating phosphatase blocked-DNA fragments with recombinant Cas9 and 314 computationally designed guide RNAs targeting 46 TB-AMR genes, allowing adapter ligation of the exposed ends and preferential amplification.

Results

Unbiased mNGS: Low abundance (0.03-6.41 rpm) TB reads were identified in 100% of the definite TB samples (n=6), and Cryptococcus neoformans was detected in 100% of cryptococcal cases (n=4). Among the 4 unknowns, Cryptococcus (n=1) and Toxoplasma gondii (n=1) were detected. The first FLASH experiment demonstrated up to a 100-fold increase in TB read abundance in definite TBM cases and detection of an additional case of TBM that had initially been mis-classified as Cryptococcus paradoxical IRIS.

Conclusions

We report the development of a novel TBM and AMR CSF diagnostic protocol combining the agnostic ability of mNGS with FLASH, a CRISPR-Cas9 tool that enriches pathogen sequences of interest. These promising preliminary data suggest the possibility of developing an “all-in-one” assay to detect TB, any associated AMR genes and any additional or alternate infections.

Authors/Disclosures
Prashanth S. Ramachandran, Jr., MBBS (Royal Melbourne Hospital) PRESENTER	Dr. Ramachandran has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Biogen. Dr. Ramachandran has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Merk.
Fiona Cresswell	No disclosure on file
Charles Langelier	No disclosure on file
	No disclosure on file
	No disclosure on file
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	No disclosure on file
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	No disclosure on file
Joseph DeRisi, PhD	Joseph DeRisi, PhD has received personal compensation for serving as an employee of Chan Zuckerberg Biohub. Joseph DeRisi, PhD has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Allen & Co. Joseph DeRisi, PhD has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Public Health Company, Inc. Joseph DeRisi, PhD has received personal compensation in the range of $0-$499 for serving as a Consultant for Delve Bio. Joseph DeRisi, PhD has received personal compensation in the range of $0-$499 for serving as a Consultant for VeriPhi Inc. The institution of Joseph DeRisi, PhD has received research support from Chan Zuckerberg Biohub. Joseph DeRisi, PhD has received intellectual property interests from a discovery or technology relating to health care.
David R. Boulware, MD, MPH (University of Minnesota)	The institution of David R. Boulware, MD, MPH has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sfunga. David R. Boulware, MD, MPH has received publishing royalties from a publication relating to health care. David R. Boulware, MD, MPH has received personal compensation in the range of $0-$499 for serving as a Study Section with NIH.
	No disclosure on file
Michael R. Wilson, MD, FAAN (University of California San Francisco)	Dr. Wilson has received personal compensation in the range of $10,000-$49,999 for serving as an officer or member of the Board of Directors for Delve Bio. Dr. Wilson has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Cambridge Medical Experts. Dr. Wilson has stock in Delve Bio. The institution of Dr. Wilson has received research support from Genentech / Roche. The institution of Dr. Wilson has received research support from NIH. The institution of Dr. Wilson has received research support from Novartis. The institution of Dr. Wilson has received research support from National Multiple Sclerosis Society. The institution of Dr. Wilson has received research support from Fanconi Anemia Research Foundation. The institution of Dr. Wilson has received research support from Department of Defense. The institution of Dr. Wilson has received research support from Chan Zuckerberg Initiative. Dr. Wilson has received intellectual property interests from a discovery or technology relating to health care. Dr. Wilson has received intellectual property interests from a discovery or technology relating to health care. Dr. Wilson has received personal compensation in the range of $10,000-$49,999 for serving as a Expert Witness with US Dept of Justice.

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