Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Molecular diagnostics development for rapid human fungal infection detection and identification (104212)

Tiana R Davey 1 2 , Erin P Price 1 2 , Derek S Sarovich 1 2 , Olusola S Olagoke 3 , Yu Pei Tan 4 , Kathryn Wilks 5 , Harrchun Panchalingam 6 , D. İpek Kurtböke 6 , Kay A Ramsay 7 , Timothy Baird 8 , Jane Neill 8 , Lucy D Burr 9 , Kellie R Strickland 1 2 , Scott C Bell 7 10 11
  1. Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, QLD, Australia
  2. Sunshine Coast Health Institute, Birtinya, QLD, Australia
  3. School of Public Health, University of California Berkeley , Berkeley, CA, USA
  4. Department of Agriculture and Fisheries, Queensland Plant Pathology Herbarium, Dutton Park, QLD, Australia
  5. Infectious Diseases Department, Sunshine Coast Hospital and Health Service, Birtinya, QLD, Australia
  6. School of Science, Technology, Engineering, and the Environment , University of the Sunshine Coast, Sippy Downs, QLD, Australia
  7. Child Health Research Centre, The University of Queensland, South Brisbane, QLD, Australia
  8. Respiratory Department, Sunshine Coast University Hospital, Birtinya, QLD, Australia
  9. Respiratory Department, Mater Hospital Brisbane, South Brisbane, QLD, Australia
  10. Adult Cystic Fibrosis Centre, The Prince Charles Hospital, Chermside, QLD, Australia
  11. Translational Research Institute, Woolloongabba, QLD, Australia

Long-neglected pathogens worldwide, fungi have gained greater recognition in recent years for their propensity for causing severe disease. Invasive fungal disease (IFD) is the most severe form of mycosis, and disproportionately affects immunocompromised people, such as those undergoing cancer treatment or stem cell transplantation, people with AIDS, and critically ill people with COVID-19 (1,2). A combination of insensitive or inaccurate tests, long turnaround times to confirmatory diagnosis (often taking up to 4-6 weeks), limited antifungal drugs, and undesirable drug side effects makes diagnosis and treatment of IFDs extremely difficult for clinicians (3-5). These factors likely contribute to increased mortality for those suffering from an IFD. With limited antifungal options currently available or on the horizon, the threat of antifungal resistance, which is already rising, is of great concern (6,7). Currently, IFD diagnosis relies heavily upon conventional culture methods, histopathology, and gene sequencing, both of which have long (>24h) turnaround times (3-5). There is an urgent need to develop and implement rapid, accurate, high-throughput, and economical tests for detecting the most prevalent fungi that cause IFDs. To meet this urgent need, we developed and validated 10 novel quantitative real-time PCR (qPCR) assays targeting 10 prevalent fungal pathogens affecting humans: Aspergillus flavus, Aspergillus fumigatus, Section Fumigati, Section Flavi, Nakaseomyces glabratus, Pichia kudriavzevii, Candida albicans, Candida dubliniensis, Candida parapsilosis, and Candida tropicalis. All 10 qPCR assays demonstrate excellent specificity towards the chosen targets and show good detection sensitivity (between 14 and 4841 genome equivalents) and quantitation capacity (between 621 and 12501 genome equivalents). To enable high-throughput screening of fungal cultures, these 10 assays have also been validated in duplex format, allowing for two assay targets to be simultaneously tested in a singular tube. We show that the turnaround time, cost, and accuracy of our qPCR assays are superior to commonly used conventional methods, such as phenotypic speciation, VITEK 2 identification, and internal transcribed spacer amplicon sequencing.

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