Abstract
Background
The prompt 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 sepsis and febrile and infectious conditions, sample preparation for
cell-free DNA (cfDNA) analysis in decentralised settings presents 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 cfDNA isolation from
human plasma samples. Device performance was evaluated by comparing cfDNA yield
against a reference (QIAGEN QIAamp Circulating Nucleic Acid Kit). cfDNA eluates from ten
non-cultured blood samples from hospital patients were sequenced on a nanopore
sequencer, and results compared to blood cultures.
Results
Extraction of cfDNA using the CNASafe device was completed in 40 minutes, compared to
the 2-hour reference protocol. The device achieved an average relative cfDNA recovery of
100.5% over 333 unique extractions encompassing all parameter variations, demonstrating
a performance equivalent to the reference kit. From the patient samples, a sufficient quantity
of microbial cfDNA was extracted to either 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
The prompt 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 sepsis and febrile and infectious conditions, sample preparation for
cell-free DNA (cfDNA) analysis in decentralised settings presents 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 cfDNA isolation from
human plasma samples. Device performance was evaluated by comparing cfDNA yield
against a reference (QIAGEN QIAamp Circulating Nucleic Acid Kit). cfDNA eluates from ten
non-cultured blood samples from hospital patients were sequenced on a nanopore
sequencer, and results compared to blood cultures.
Results
Extraction of cfDNA using the CNASafe device was completed in 40 minutes, compared to
the 2-hour reference protocol. The device achieved an average relative cfDNA recovery of
100.5% over 333 unique extractions encompassing all parameter variations, demonstrating
a performance equivalent to the reference kit. From the patient samples, a sufficient quantity
of microbial cfDNA was extracted to either 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
| Original language | English |
|---|---|
| Publisher | medRxiv |
| Pages | 1-21 |
| Number of pages | 21 |
| DOIs | |
| Publication status | E-pub ahead of print - 31 Dec 2024 |