Background The identification of pathogens in human circulation in a clinically deployable format remains an unmet clinical need. The established test for infection diagnostics remains blood culture, which typically takes 2-4 days and is positive in less than 15% of cases, with many prevalent pathogens difficult or impossible to culture. While microbial cfDNA in blood could facilitate the diagnosis of septic, febrile and infectious conditions, sample preparation for cell-free DNA (cfDNA) analysis in decentralised settings presents several challenges due to its complexity and the low concentration and fragmented nature of cfDNA in blood plasma. Methods. We developed a portable and automated platform (CNASafe) for the rapid isolation of cfDNA from human plasma samples. The performance of CNASafe was evaluated by comparing cfDNA yield obtained by quantitative PCR against a benchmark (QIAGEN QIAamp Circulating Nucleic Acid Kit). The device was combined with a real-time nanopore sequencing protocol to identify pathogens from microbial cfDNA from human plasma samples (n=10 septic patients). Results were compared to blood cultures. Results. Cell-free DNA extraction using the CNASafe device was completed in as little as 40 minutes, compared to the 2-hour manual protocol. The device yielded comparable DNA recovery to the benchmark. Extracted microbial cfDNA was extracted in sufficient quantity in septic patient samples to identify pathogens missed by blood cultures or confirm negative cultures. Conclusions. The CNASafe platform and real-time nanopore sequencing offer a promising solution for the rapid deployment of metagenomic diagnostics, enabling pathogen identification within a few hours in decentralised clinical environments.

Antonio Liga and Maiwenn Kersaudy-Kerhoas have granted and pending patents on elements of the cartridge design. Kazuhiro Horiba has a granted patent on the bioinformatic tool Pathdet.

This study was funded by the UK Research and Innovation Engineering and Physical Research Council EP/R00398X/1; the Royal Academy of Engineering FoDSF 1920 2 100018; Internal funds from Heriot-Watt University B19G1243; Scottish Enterprise Natantis HGSP Phases 1-2; Santander Universities - Santander X Award 2023

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Research Ethics Committees Northern Ireland (ORECNI) of UK NHS gave ethical approval for part of this work (Reference: 18/NI/0148) National Health Sciences Research Committee of Malawi gave ethical approval for part of this work (Reference: 2157) Research Ethics Committee of Liverpool School of Tropical Medicine gave ethical approval for part of this work (Reference: 18-062) Research Ethics Committee of Heriot-Watt University gave ethical approval for part of this work (Reference: 1285 -iSEP-SEQ_ARCS / 320361)

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