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This dissertation focuses on the detection of pathogens using cell-free DNA (cfDNA) Next-Generation Sequencing (NGS), an innovative technology for identifying infections. Pathogens such as bacteria, viruses, and fungi can cause severe diseases, particularly in vulnerable and immunocompromised patients. Rapid and accurate identification of the causative agent is essential for targeted and effective treatment. Traditional diagnostic methods, such as culture or PCR, are often slow or insufficiently sensitive, especially in complex infections among immunocompromised populations.
Genetic sequencing, and cfDNA NGS in particular, offers new opportunities for early and broadly applicable pathogen detection. In this approach, cfDNA is isolated from body fluids such as blood or lung fluid, analyzed, and compared to known genetic profiles of microorganisms. This dissertation explores how this technology can be further optimized, with special attention to both laboratory procedures (such as library preparation) and advanced computational data analysis.
Chapter 2 applies cfDNA NGS to immunocompromised children with suspected fungal infections. Elevated levels of Aspergillus fumigatus cfDNA were successfully detected, particularly in lung fluid. Chapter 3 investigates methods to further enrich Aspergillus cfDNA using specific physical characteristics of DNA fragments. Chapter 4 explores the application of this technology in newborn foals with sepsis, where bacterial cfDNA was successfully identified.
This dissertation highlights the potential of cfDNA NGS as a rapid, sensitive, and non-invasive diagnostic tool for infectious diseases and provides directions for future research and clinical implementation.