During blood stream infection, pathogens are identified with blood culture as current reference method. As blood culture can take several days to identify bacteria or fungi, however, broad-spectrum antibiotics are initially administered to target a range of pathogens. This may, however, result in the selection of multidrug-resistant bacteria. Moreover, blood culture may yield inconclusive results due to concurrent antibiotic treatment or due to the presence of slow-growing or intracellular pathogens. Molecular diagnostic systems for pathogen identification, particularly polymerase chain reaction (PCR), provide short time-to-result, compatibility with antibiotic treatment, and absence of potentially pre-selecting culture steps, but may suffer from interference of sample matrix components. Additionally, bacterial or fungal DNA persisting in the blood stream after resolved infection may cause false positive results. Next-generation sequencing (NGS) is emerging as method for pathogen identification with the potential to cover the complete pathogen and resistance spectrum, but direct sequencing from patient samples holds multiple technical challenges, including a highly unfavorable ratio of pathogen and host DNA as well as the requirement for advanced bioinformatics algorithms to enable standardized and quality-controlled analysis of NGS raw data. With this project, we aim to substantially advance the detection of bacterial and fungal pathogens during blood stream infection by targeting the following objectives: (i) determination of the persistence of bacterial or fungal DNA in the circulation after resolved infection to allow for the discrimination of residual pathogen DNA from ongoing infections; (ii) identification of matrix components interfering with molecular diagnostics; (iii) identification of non-DNA markers indicative of blood stream infection, such as altered glycosylation of surface proteins associated with extracellular vesicles; (iv) proof-of-principle for the feasibility of pathogen identification by NGS directly from whole blood.