Evaluation of multiple extraction and inactivation methods for the rapid identification of filamentous fungi using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) offers high sensitivity for the rapid identification of microbial isolates. While predominantly used in bacterial and yeast identification within the clinical laboratory, recent advancements have prompted interest in expanding MALDI-TOF MS applications for the rapid identification of filamentous fungi, particularly clinically relevant molds. However, the implementation of mold identification protocols introduces biosafety concerns due to the potential for spore aerosolization during sample handling and inoculation, posing occupational hazards to laboratory personnel. To evaluate the feasibility and safety of MALDI-TOF MS-based mold identification, we conducted a prospective study comparing an extraction-free approach to two different rapid extraction methods across routine media and specialized fungal plates. Each method was assessed for spectral quality, reproducibility, and identification accuracy using a validated reference library. Importantly, we performed a biosafety validation study to assess the efficacy of the extraction-free approach in inactivating mold spores and hyphal elements. The extraction-free approach generally outperformed the rapid extraction methods, demonstrating more accurate fungal identifications (90.5% vs. 61.9% and 66.7%) and a higher average spectra score; accuracy and spectra scores were further enhanced using specialized fungal media (92.9% vs. 81.0% and 52.4%). Our findings support the use of this extraction-free approach as both a safe and effective method to rapidly identify filamentous fungi using MALDI-TOF MS within the clinical laboratory. IMPORTANCE This study overviews the implementation of identifying clinically relevant filamentous fungi using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a clinical laboratory. The methodology applied in this study produced both highly sensitive and specific results with significantly reduced turnaround time compared to traditional standard of care culture-based workup. Faster time to detection has the potential to impact the healthcare treatment of millions of patients each year without requiring significant changes to clinical workflow. This study used multiple approaches to improve the clinical utility and performance of MALDI-TOF MS for fungal identification. Importantly, it also highlights the effectiveness of a developed inactivation method to ensure laboratory personnel safety. This method and workflows are of interest to support clinical microbiology diagnostics and to help aid in infection identification to enhance timely treatment.