To Örebro University

Bloodstream infection (BSI) is a life-threatening condition associated with high mortality. Pathogen identification is essential for patient management, yet blood culture (BC), the diagnostic gold standard, may require 2–5 days and has reduced sensitivity after antimicrobial treatment. This thesis investigates modern molecular DNA sequencing approaches for detecting bacterial DNA as a diagnostic alternative. In Study I, a cohort of 484 patients with suspected BSI was described, and prediction tools were applied to identify patients at high risk of BSI for subsequent studies. Study II used whole blood samples from 51 patients to detect bacterial DNA with short-read sequencing and showed low concordance with routine BC results. In Study III, a shotgun metagenomic workflow using the Nanopore platform was developed, and DNA extraction efficiency was evaluated in contrived samples. Bacterial DNA recovery was slightly higher in whole blood than in plasma, but no firm conclusion regarding the optimal sample matrix could be drawn. We also observed that extraction efficiency differed between bacterial species. Based on these methodological challenges and limited sensitivity, Study IV evaluated bacterial DNA enrichment prior to sequencing through short BC incubation (4 h) followed by targeted 16S gene sequencing in 161patients selected using the criteria defined in Study I. This workflow showed increased diagnostic performance, with sensitivity of 56.8% and specificity of 46.7% compared with BC. Additional bacteria with plausible clinical relevance were detected in a few patients with negative routine BC. Overall, the sequencing protocols evaluated in this thesis provided limited additional diagnostic value for BSI compared to BC. In comparison with the literature, we highlight methodological challenges to guide future research in this area.