Real-Time Unbiased Pathogen Detection in Febrile Illnesses by Nanopore Sequencing Acute febrile illness is one of the most common presenting symptoms in clinical medicine, with a broad differential that includes a variety of infectious etiologies. Conventional laboratory testing can take days to a result, and most rapid detection technologies such as PCR are limited to the detection of a single or narrow range of targets. Unbiased diagnosis of all pathogens in a single test by metagenomic next-generation sequencing has proven feasible, although still requires >24 hours due to long sequencing times, lack of portable instrumentation, and/or the complexity of bioinformatics analysis. Here we propose to use the Oxford Nanopore, a USB-sized, portable sequencing instrument with current read capacity of 500 Gb data and >100,000 reads, to develop a diagnostic assay to rapidly diagnose patients with acute febrile illness by screening for all potential pathogens in under 2 hours. We have already demonstrated in preliminary data that metagenomic detection of Chikungunya and Ebola virus performed by real-time nanopore sequencing and analysis is possible with a 6-hour sample-to-answer timeframe. In years 1-2 (R21 phase), we will optimize the assay, validate it in a CLIA-certified laboratory by developing standard operating procedures, establish its performance characteristics including sensitive, specificity, and limits f detection, and develop standalone and cloud-based software pipelines for real-time sequencing analysis. In years 3-5 (R33 phase), we use it to test positive and negative control clinical samples from patients with acute febrile illness. We will also use it to investigate clinical samples from patients with febrile respiratory and systemic illnesses and infected by a variety of pathogens, including influenza virus, Ebola virus, Lassa virus, Chikungunya virus, enterovirus D68, and Plasmodium falciparum (malaria). We will also test the platform at point-of-care field sites in California (California Department of Public Health), Central America (American Red Cross) and Democratic Republic of the Congo, Africa (Institut National de Recherches Medicales). Ultimately, the goal of this 5-year project is implementation of a field-ready, real-time sequencing assay for unbiased pathogen diagnosis of acute febrile illness by metagenomic nanopore sequencing.
|Castro-Wallace, Sarah L; Chiu, Charles Y; John, Kristen K et al. (2017) Nanopore DNA Sequencing and Genome Assembly on the International Space Station. Sci Rep 7:18022|
|Sardi, Silvia I; Somasekar, Sneha; Naccache, Samia N et al. (2016) Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing. J Clin Microbiol 54:2348-53|
|Naccache, Samia N; Thézé, Julien; Sardi, Silvia I et al. (2016) Distinct Zika Virus Lineage in Salvador, Bahia, Brazil. Emerg Infect Dis 22:1788-92|