BackgroundBloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases. Due to the lack of timely diagnostic approaches with sufficient sensitivity, mortality rates of sepsis are still unacceptably high. However a prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bloodstream infections. Although various targeted molecular tests for blood samples are available, time-consuming blood culture-based approaches still represent the standard of care for the identification of bacteria.MethodsHere we describe the establishment of a complete diagnostic workflow for the identification of infectious microorganisms from seven septic patients based on unbiased sequence analyses of free circulating DNA from plasma by next-generation sequencing.ResultsWe found significant levels of DNA fragments derived from pathogenic bacteria in samples from septic patients. Quantitative evaluation of normalized read counts and introduction of a sepsis indicating quantifier (SIQ) score allowed for an unambiguous identification of Gram-positive as well as Gram-negative bacteria that exactly matched with blood cultures from corresponding patient samples. In addition, we also identified species from samples where blood cultures were negative. Reads of non-human origin also comprised fragments derived from antimicrobial resistance genes, showing that, in principle, prediction of specific types of resistance might be possible.ConclusionsThe complete workflow from sample preparation to species identification report could be accomplished in roughly 30 h, thus making this approach a promising diagnostic platform for critically ill patients suffering from bloodstream infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0326-8) contains supplementary material, which is available to authorized users.

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The increasing incidence of bloodstream infections including sepsis is a major challenge in intensive care units worldwide. However, current diagnostics for pathogen identification mainly depend on culture-and molecular-based approaches, which are not satisfactory regarding specificity, sensitivity, and time to diagnosis. Herein, we established a complete diagnostic workflow for real-time highthroughput sequencing of cell-free DNA from plasma based on nanopore sequencing for the detection of the causative agents, which was applied to the analyses of eight samples from four septic patients and three healthy controls, and subsequently validated against standard next-generation sequencing results. By optimization of library preparation protocols for short fragments with low input amounts, a 3.5-fold increase in sequencing throughput could be achieved. With tailored bioinformatics workflows, all eight septic patient samples were found to be positive for relevant pathogens. When considering time to diagnosis, pathogens were identified within minutes after start of sequencing. Moreover, an extrapolation of real-time sequencing performance on a cohort of 239 septic patient samples revealed that more than 90% of pathogen hits would have also been detected using the optimized MinION workflow. Reliable identification of pathogens based on circulating cell-free DNA sequencing using optimized workflows and real-time nanopore-based sequencing can be accomplished within 5 to 6 hours following blood draw. Therefore, this approach might provide therapy-relevant results in a clinically critical timeframe. (J Mol Diagn 2020, 22: 405e418; https://doi.Bloodstream infections, in particular sepsis, represent one of the main causes of death, with a mortality rate of up to 30% to 50% in intensive care units worldwidedand this trend is rising. 1e4 To provide an appropriate treatment for sepsis patients, the major challenge is not only the timely diagnosis of sepsis, but also the identification of the sepsis-causing agents. 4 A rapid detection of the causative pathogens enables early targeted antimicrobial therapy, which significantly increases survival rate, prevents subsequent complications, and reduces drugrelated side effects as well as medical expenses. 4e8 Despite extensive medical research in the field of sepsis, there are to date no fully satisfactory diagnostic approaches for a rapid, reliable, and sensitive identification of pathogens in the bloodstream. 9e12 Therefore, time-consuming, error-and contamination-prone blood cultures (BC) are still considered the standard of care for sepsis diagnostics, frequently leading to an inappropriate and delayed targeted therapy. 11,13

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