20.500.12556/DiRROS-24618 Development and performance evaluation of a clinical metagenomics approach for identifying pathogens in the whole blood from patients with undifferentiated fever 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 performed in a stepwise manner, increasing cost and delaying results. Metagenomic next-generation sequencing (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 isolate 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 mathematical ranking approach (ClinSeq score) that enabled quick identification of relevant pathogens in approximately one hour. Results: The mNGS workflow reached 79.5% (159/200) overall sensitivity. For bacteria the sensitivity 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. mNGS clinical metagenomics molecular diagnostics universal pathogen detection enhanced RNA virus detection true false true Angleški jezik Ni določen Neznano 2025-12-09 11:58:46 2025-12-09 11:58:47 2025-12-10 03:45:43 0000-00-00 00:00:00 2025 0 0 Nasl. z nasl. zaslona; Opis vira z dne 2. 10. 2025; str. 1-11 no. [article no.] 1667422 Vol. 15 2025 0000-00-00 Zaloznikova Objavljeno apc 0000-00-00 0000-00-00 0000-00-00 616.9:579 2235-2988 10.3389/fcimb.2025.1667422 251524099 RAZ_Slunecko_Jan_2025.pdf RAZ_Slunecko_Jan_2025.pdf 1 3BAFE4B9156CA2C01C5CE3FF99CEF968 98634b402fa93c40bdb351b9413ecd7f705b108e56e112f1c1dec53a0b19b806 3584c87a-d4ee-11f0-94a7-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=36731 https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2025.1667422/full 1 12803b50-d4ee-11f0-94a7-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=36730 Univerzitetni klinični center Ljubljana 0 0 0