Next GeneSiS: cfDNA sequencing outperforms blood culture in sepsis.

Sepsis remains one of the most time-critical syndromes in medicine. Mortality rises by the hour when antimicrobials are delayed, yet microbiological diagnosis still relies on culture, a technology that too often delivers results only after treatment decisions are made. Even under optimal conditions, blood cultures identify pathogens in fewer than 40% of septic patients, and false negatives or contaminants frequently complicate interpretation¹. This diagnostic bottleneck has driven research interest in microbial cell-free DNA as a culture-independent window into infection biology.

When microbes are lysed by host defences or early antibiotic exposure, their DNA briefly enters the bloodstream and clears rapidly, with half-lives on the order of minutes to a few hours, depending on context². Detecting this microbial cell-free DNA through next-generation sequencing (NGS) provides an unbiased research approach to studying infection, enabling identification even when pathogens no longer grow in vitro³.

Evidence for microbial cfDNA in blood has accumulated for over two decades. Nikkari et al first detected bacterial 16S rDNA in the blood of healthy individuals in 2001, and later studies confirmed presence of low-level bacterial DNA even in healthy donors^4,5. In research using septic samples, cfDNA metagenomics has consistently outperformed culture, uncovering polymicrobial infections and identifying fastidious or anaerobic species often missed by standard methods. More recently, Blauwkamp et al⁶ and Chiu and Miller⁷ demonstrated that sequencing of plasma cfDNA could outperform culture for pathogen detection and established firmly that plasma metagenome is an actionable reflection of infection dynamics.

Until recently, however, most investigations were retrospective or carried out in a single center. The Next GeneSiS Trial (Next-Generation sequencing for Sepsis identification Study) whose results have been recently published, represent an important advancement in this area of research⁸. In the following paragraphs we will summarize the results of this important work.

The Next GeneSiS trial

Conducted across 17 German sites and involving nearly 500 participants, this study represents a comprehensive evaluation of plasma microbial cell-free DNA (mcfDNA) sequencing as a complement to blood culture. Using a standardized library prep workflow on Illumina® platforms, the study compared detection rates and investigated the potential research insights offered by NGS results in individuals with sepsis or septic shock.

Paired blood cultures and plasma samples were collected at sepsis onset and again after 72 h. Extracted cfDNA (1 ng input) was processed on a Biomek FXP workstation, using the NEXTFLEX™ Cell-Free DNA-Seq Kit and sequenced as 100 bp, single-end, ~30 million reads per sample. The workflow is notably reproducible and automatable with instruments found in most core-NGS laboratories. Analysis was performed with the Sepsis Indicating Quantifier (SIQ) statistical pipeline, which distinguishes true pathogen signals from background contamination.

Across both timepoints, mcfDNA sequencing identified pathogens in 70.5% of samples versus 19.4% by culture. An independent panel deemed 98.6% of NGS-positive results plausible. E. coli was the most frequent organism, but NGS detected it more than three times as often as culture and revealed extensive polymicrobial and anaerobic signals overlooked by standard blood culture methods. In 32.6% of cases, sequencing results were interpreted, in this research setting, as findings that could have influenced clinical decisions if applied prospectively; however, the study was not designed to assess clinical outcomes.

Overall, plasma mcfDNA sequencing emerged as a potential quantitative complement, not a replacement, to culture, providing broader microbial profiling and insight into infection dynamics. The ongoing DigiSep⁹ randomized trial will determine whether such implementation improves outcomes.

Conclusion

The Next GeneSiS trial underscores that next-generation sequencing of plasma microbial cell-free DNA shows promising potential as a complement to blood culture in research settings. The study demonstrates that metagenomic research approaches can reveal a far broader and more accurate picture of bloodstream infections, detecting non-viable organisms and those present at low abundance, capturing polymicrobial complexity.

The next challenge is to explore the integration of metagenomic data into real-time decision-making, where short turnaround would be critical for future applications. Achieving this will require development of streamlined sample-to-answer research workflows, validated interpretation frameworks, and close collaboration between research clinicians, microbiologists, and bioinformaticians. The ongoing DigiSep randomized trial will be critical in determining, within a study setting whether earlier, NGS-guided antimicrobial decisions could improve survival, reduce drug exposure, and shorten ICU stays.