Development and performance evaluation of a clinical metagenomics approach for identifying pathogens in the whole blood from patients with undifferentiated fever

Jan Slunečko¹Rok Kogoj¹Samo Zakotnik¹Alen Suljič¹Nataša Knap¹Martin Bosilj¹Franc Strle²Tatjana Avsic Zupanc¹Petra Bogovič^2*Misa Korva^1*

• ¹Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
• ²Department of Infectious Diseases, University Medical Center Ljubljana, Ljubljana, Slovenia

Introduction: Blood culture is the cornerstone of microbiological diagnostics for patients with acute undifferentiated fever and no obvious localization of infection; however, up to 50% of cases remain undiagnosed. Infections caused by arboviruses, fastidious or even uncultivable bacteria, or parasites often go undiagnosed without the use of target-specific molecular methods. These are typically per-formed in a stepwise manner, increasing cost and delaying results. Metagenomic next-generation se-quencing (mNGS) has recently gained recognition as a potential universal pathogen detection tool for such cases. Our study aimed to develop a streamlined mNGS workflow for simultaneous detection of intracellular and cell-free pathogens within a single sequencing library. Methods: Total nucleic acid was isolated separately from 200 EDTA blood samples. The plasma iso-late was processed with DNase, followed by the depletion of host ribosomal and messenger RNA, reverse transcription, and sequence-independent single primer amplification (SISPA). The whole blood isolate was only reverse transcribed, with no other pre-processing manipulation. Finally, the two fractions were combined prior to library preparation and sequencing using either Oxford Nanopore Technologies or Illumina. Following established bioinformatics analysis, we developed a mathemati-cal ranking approach (ClinSeq score) that enabled quick identification of relevant pathogens in ap-proximately one hour. Results: The mNGS workflow reached 79.5% (159/200) overall sensitivity. For bacteria the sensitivi-ty was 88.6% (70/79), DNA viruses, 66.7% (10/15) and for RNA viruses 73.8% (76/103). Pathogen detections by individual sequencing methods showed overall sensitivity of Illumina and ONT to be 80.0% (76/95) and 79.1% (83/105) respectively. The ClinSeq score correctly highlighted the pathogen in 126/200 (63.0%) samples effectively with a Cohen’s kappa (κ) agreement of 0.61 with manual analysis. Conclusion: Developed comprehensive mNGS workflow detects a wide range of pathogens in patients with acute undifferentiated fever. The unified workflow improves sensitivity for intracellular bacteria and RNA viruses, reduces time, cost and complexity by eliminating the need for separate library preparations, enabling faster turnaround suitable for clinical settings. The ClinSeq score effectively differentiates true pathogen signals from background noise, reducing false positives and manual interpretation time. Overall, the workflow demonstrates flexible, and efficient pathogen detection, supporting its potential for clinical diagnostics and improved patient management.