Early Diagnosis of Invasive Aspergillosis Based on Microbial Cell-Free DNA Derived from Invasive Aspergillus Species

다국어 초록 (Multilingual Abstract)

Invasive fungal diseases (IFDs) are opportunistic infections that predominantly affect immunocompromised or critically ill patients and are increasingly recognized as a major global health concern due to rising morbidity and mortality. According to the U.S. Centers for Disease Control and Prevention (CDC), the annual economic burden of fungal infections is estimated at approximately $10 billion. Among IFDs, invasive aspergillosis (IA) — primarily caused by Aspergillus niger, A. fumigatus, and A. flavus — remains one of the most life-threatening conditions due to challenges in early detection and limited diagnostic sensitivity. The present study aimed to develop a rapid, non-invasive, and species-specific molecular diagnostic assay using microbial cell-free DNA (mcfDNA) targeting the internal transcribed spacer (ITS) 2 region of Aspergillus species. The assay exhibited high analytical sensitivity, with all three species including A. niger, A. fumigatus, and A. flavus reliably detected down to 10¹ copies/µL. No cross-reactivity was observed in non-target microorganisms, confirming high specificity. In addition, consistent Ct and Tm values were obtained from 74 clinical isolates, supporting the assay’s diagnostic applicability. These findings indicate that mcfDNA-based qPCR represents a promising platform for early, non-invasive detection of IA, particularly in high-risk patient populations. Future validation using plasma-derived mcfDNA samples would further support its clinical implementation.

Keywords: Invasive aspergillosis (IA), Aspergillus spp., Microbial cell-free DNA (mcfDNA), Quantitative PCR (qPCR), Internal transcribed spacer (ITS) 2

목차 (Table of Contents)

• Abstract ⅰ
• List of Tables ⅳ
• List of Figures ⅳ
• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 6
• 1. Clinical and reference fungal strains
• 2. Fungal culture and lactophenol cotton blue stain
• 3. Genomic DNA extraction from fungal isolates
• 4. Quantification of fungal gDNA
• 5. Primer design and gradient PCR amplification
• 6. Quantitative PCR SYBR Green assay
• 7. Ethics approval
• Ⅲ. Results 15
• Ⅳ. Discussion 46
• References 48
• Abstract in Korean50