Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

Development of an improved real-time PCR assay for the detection of fungal DNA (#54)

Olusola Olagoke 1 2 , Timothy Baird 2 3 , Kathryn Wilks 4 5 , Jane Neil 3 , Harrchun Panchalingam 1 , Dilber Kurtboke 1 , Derek Sarovich 1 2 , Erin Price 1 2
  1. Genecology Research Centre, University of the Sunshine Coast, Sippy Downs, Queensland, Australia
  2. Sunshine Coast Health Institute, Birtinya, Queensland, Australia
  3. Respiratory Department, Sunshine Coast University Hospital, Birtinya, Queensland, Australia
  4. Pathology Queensland, Sunshine Coast University Hospital, Birtinya, Queensland, Australia
  5. Infectious Diseases Department, Sunshine Coast University Hospital, Birtinya, Queensland, Australia

Abstract

The ongoing COVID-19 pandemic has highlighted that invasive fungal infections remain a major cause of mortality in immunocompromised patients and those with secondary infections. For instance, mortality in mechanically-ventilated patients with COVID-19-associated aspergillosis is as high as 47% [1], thus making the need for early and effective detection of fungal pathogens essential. Unfortunately, current diagnostic platforms for pan-fungal detection continue to be problematic, especially in polymicrobial samples, with common problems encountered including poor sensitivity and specificity, and a need for fungal culture, which is time-consuming and results in delayed diagnosis [2].

Here, we performed comparative genomic analyses to identify a highly conserved target within the fungal internal transcribed spacer 5.8S rRNA gene, to the exclusion of all nonfungal organisms. We subsequently developed a Black Hole Quencher probe-based multiplexable real-time PCR assay to rapidly and accurately detect pan-fungal DNA in endotracheal aspirate (ETA), sputum and culture samples.

Using our assay, fungal DNA was detected in 100% of tested clinical fungal species (Aspergillus fumigatus, A. flavus, Candida albicans, C. tropicalis, C. krusei, C. dubliniensis, Rhizopus arrhizus, Cladophialophora carrionii, Scedosporium aurantiacum, Trichophyton rubrum, Lomentospora prolificans, Exophiala spinifera and Cryptococcus neoformans). Fungal DNA was also detected in 28 of 68 respiratory samples (ETA and sputum). The limits of detection and quantitation were ~0.5 fg/µl and 0.5 pg/µl, respectively, demonstrating excellent sensitivity. Additionally, this assay was optimized for 5 µl reactions, thus leading to savings in diagnostic costs.

Our accurate and cost-effective pan-fungal assay facilitates the rapid detection of fungal DNA in both cultures and clinical samples. We are now in the process of developing additional assays to identify clinically important fungal genera and species, especially in people with COVID-19, including pan-Aspergillus, A. fumigatus, A. flavus, pan-Candida, C. albicans, C. tropicalis, and C. krusei.

  1. Meijer EFJ, Dofferhoff ASM, Hoiting O, Meis JF. COVID-19-associated pulmonary aspergillosis: a prospective single-center dual case series. Mycoses. 2021 Apr;64(4):457-464. doi: 10.1111/myc.13254. Epub 2021 Feb 16. PMID: 33569857; PMCID: PMC7986084.
  2. Cuenca-Estrella M, Bernal-Martinez L, Buitrago MJ, Castelli MV, Gomez-Lopez A, Zaragoza O, Rodriguez-Tudela JL. Update on the epidemiology and diagnosis of invasive fungal infection. Int J Antimicrob Agents. 2008 Nov;32 Suppl 2:S143-7. doi: 10.1016/S0924-8579(08)70016-5. PMID: 19013339.