SignificanceLower respiratory tract infections (LRTIs) are the leading cause of infectious disease-related deaths worldwide yet remain challenging to diagnose because of limitations in existing microbiologic tests. In critically ill patients, noninfectious respiratory syndromes that resemble LRTIs further complicate diagnosis and confound targeted treatment. To address this, we developed a metagenomic sequencing-based approach that simultaneously interrogates three core elements of acute airway infections: the pathogen, airway microbiome, and host response. We studied this approach in a prospective cohort of critically ill patients with acute respiratory failure and found that combining pathogen, microbiome, and host gene expression metrics achieved accurate LRTI diagnosis and identified etiologic pathogens in patients with clinically identified infections but otherwise negative testing.
The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.
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