Development of a rapid, pan fungal diagnostic assay

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ABSTRACT Diagnosing fungal infections faces a number of challenges including a decline in expertise needed for identifying fungi and a reduced number of instruments and assays specific for fungal identification compared to bacteria and viruses. These problems are exacerbated by the fact that patients with fungal infections are often immunosuppressed, which predisposes them to infections from both common and rarely seen fungi. Historically, molecular diagnostic assays for invasive infections have been labor intensive and required a high degree of technical expertise. Developing an efficient assay that could use technology as a substitute for classical mycology expertise has been difficult. While ribosomal ITS sequencing is the gold standard of fungal molecular diagnostics, it is slow, requires high capital costs, and for many fungi, is not specific enough. This study will rely on two innovative components that will enable the identification of almost any fungus to the species level quickly. The first component is a new sequencing technology called nanopore sequencing, which is fast and inexpensive. The second component will consist of a novel sequencing target called the intergenic spacer sequence (IGS), which is located within the fungal ribosomal repeat. The IGS is too long to be covered by traditional Sanger sequencing in a diagnostic assay because multiple reads generated from a primer walk would be required, so it is not used for fungal identification. However, nanopore sequencing can easily cover the IGS region in a single sequencing run. To achieve the goal of developing an efficient pan fungal diagnostic strategy, three aims will test the hypothesis that sequencing a little used, but highly informative region of the fungal rDNA repeat locus using nanopore sequencing will provide the greatest specificity and quickest turnaround time to date for fungal identification. These aims are: 1) Generation of a fungal intergenic spacer sequence reference sequence database within the GenBank RefSeq (reference sequence) Targeted Loci Project, called the Fungal IGS RefSeq database 2), Development of a rapid assay for fungal identification based on nanopore sequencing of the intergenic spacer sequence, 3) Comparison of a nanopore sequence diagnostic assay against current laboratory fungal diagnostic methods. The long-term goal that this project will enable after its completion will be the future development of a rapid, inexpensive, assay that is pan fungal in capability.