Rapid diagnostics of CNS infectious diseases from formalin-fixed paraffin-embedded specimens using nanopore sequencing

Routine neuropathology diagnostic methods are limited to histological staining techniques or directed PCR for pathogen detection and microbial cultures of brain abscesses are negative in one-third of the cases. Fortunately, due to improvements in technology, metagenomic sequencing of a conserved bacterial gene could provide an alternative diagnostic method. For histopathological work up, formalin-fixed paraffin-embedded (FFPE) tissue with highly degraded nucleic acids is the only material being available.
Innovative amplicon-specific next-generation sequencing (NGS) technology has the capability to identify pathogens based on the degraded DNA within a few hours. This approach significantly accelerates diagnostics and is particularly valuable to identify challenging pathogens. This ensures optimal treatment for the patient, minimizing unnecessary health damage.
Within this project, highly conserved primers in a universal PCR will be used, followed by determining the nucleotide sequence. Based on the obtained data, it is then precisely determined which microorganism(s) is/are responsible for the infection, even in cases of co-infection with multiple pathogens. This project will focus to answer the following research question; how can a new form of rapid molecular diagnostics contribute to the identification of microbial pathogens in CNS infections?
The SME partner Molecular Biology Systems B.V. (MBS) develops and sells equipment for extremely rapid execution of the commonly used PCR. In this project, the lectorate Analysis Techniques in the Life Sciences (Avans) will, in collaboration with MBS, Westerdijk Institute (WI-KNAW) and the Institute of Neuropathology (Münster, DE) establish a new molecular approach for fast diagnosis within CNS infections using this MBS technology. This enables the monitoring of infectious diseases in a fast and user-friendly manner, resulting in an improved treatment plan.
 

Eindrapportage

This project aims to develop and validate a rapid molecular diagnostic approach for the identification of microbial pathogens in central nervous system (CNS) infections, using formalin-fixed paraffin-embedded (FFPE) tissue. Traditional diagnostic methods—such as histological staining, targeted PCR, and microbial cultures—are often time-consuming and may fail to detect causative pathogens, particularly in cases with degraded nucleic acids or negative cultures.The consortium, consisting of Avans University of Applied Sciences (Lectorate Analysis Techniques in the Life Sciences), Molecular Biology Systems B.V. (MBS), the Westerdijk Institute (WI-KNAW), and the Institute of Neuropathology Münster, combines expertise in rapid PCR technology, microbiology, neuropathology, and bioinformatics. Central to the project is the application of MBS’s ultra-fast PCR platform, enabling amplicon-based next-generation sequencing (NGS) of conserved microbial genes, even from highly fragmented DNA in FFPE samples.The results demonstrate that this rapid amplicon sequencing approach is highly effective for the detection and identification of bacterial pathogens in CNS infections, significantly reducing diagnostic turnaround time while maintaining accuracy. This enables faster clinical decision-making and more targeted treatment strategies, ultimately improving patient outcomes.A major innovation from the project is the development of a dedicated bioinformatics tool, GermGenie, which allows automated and precise identification of microbial sequences, including the detection of co-infections involving multiple bacterial species.However, the detection of fungal pathogens using this amplicon-based approach remains challenging. Limitations include the fragmented nature of FFPE-derived DNA and the relatively small and variable internal transcribed spacer (ITS) regions used for fungal identification, which complicate full taxonomic resolution. The findings suggest that a metagenomic sequencing approach may be more suitable for reliable detection of fungi in CNS infections.Overall, this project establishes a fast, user-friendly diagnostic workflow that significantly enhances the detection of bacterial pathogens in CNS infections and provides a strong foundation for further optimization toward broader pathogen coverage.