738 - A Novel Molecular Diagnostic Assay for Identification of Fungal Pathogens

Background: A rapid and accurate diagnostic method for invasive fungal infections remains a critical clinical need. We recently reported a rapid molecular method for bacterial species identification directly from clinical samples that targets highly abundant ribosomal RNA on a multiplexed hybridization platform called NanoString. Here we report an adaptation of this assay that accurately distinguishes common fungal pathogens with limit of detection at a single yeast cell.

Methods: Building on our bacterial approach, we computationally designed specific hybridization probes targeting species-specific variable regions of fungal 18S and 28S rRNA from 12 clinically relevant fungi: Aspergillus fumigatus, Cryptococcus neoformans, and 10 Candida species, including Candida auris. Following mechanical lysis of crude specimens, fungi were detected from laboratory culture or artificial cerebrospinal fluid via multiplexed hybridization on a NanoString (Seattle, WA) instrument which yielded results within 7 hours from sample collection. Assay sensitivity was probed using serial dilutions of lysed C. albicans in culture, and cell-equivalents were confirmed by plating.

Results: Our hybridization probes targeting fungal rRNA specifically recognized all species tested to date: A. fumigatus, C. neoformans, and C. albicans with no cross-reactivity (Fig 1a). Serial dilutions of C. albicans lysate demonstrated a limit of detection around 0.1 cell equivalents without rRNA amplification (Fig 1b), capitalizing on the intrinsic abundance of rRNA in fungal cells.Figure 1.


Conclusion: We adapted a rapid, ultrasensitive hybridization-based diagnostic assay that has proven successful in bacteria, to fungi. Here we show the accurate detection of Aspergillus, Cryptococcus, and Candida species, including a computational design that will enable the distinction of 10 different Candida species, including C. auris, within hours from clinical specimen collection.