Background
Fungi represent a kingdom of life that encompasses a diverse array of non-photosynthesising eukaryotic macro- and micro-organisms. Fungi are abundant globally and play an essential role in biological decomposition. Most fungi are incapable of causing infections in mammals and birds, including humans, due to relatively high host body temperatures. Although rare, certain microfungi can overcome this thermal barrier to cause opportunistic, life-threatening infections, particularly in people with suppressed immune systems, including those undergoing cancer treatment. As climate change becomes ever more present, microfungi are expected to adapt to the warming environment, leading to a broadening of regions with tropical and subtropical climates globally. Alongside this warming will be the emergence of previously unreported fungal infections in humans and other mammals.
The prevalence of fungal infections has increased in recent years due to an increasing population of immunocompromised patients, intensive immunosuppressive chemotherapy, increasing awareness of fungal infections, and the widespread use of broad-spectrum antibiotics and central venous catheters). Standard methods for the diagnosis of Candida and Aspergillus fungal infections include culture and histopathology, but these methods have limited sensitivity and specificity. For example, blood cultures are positive for fewer than 50% of patients with hepatosplenic candidiasis and are rarely positive for patients with invasive aspergillosis. In addition, cultures of bronchoalveolar lavage fluid are frequently negative for patients with pulmonary aspergillosis), and by the time that positive cultures are obtained, disease is usually advanced.
A PCR assay for the detection of fungal nucleic acids may be the optimal diagnostic approach because it offers the potential of (i) being more sensitive than current culture-based methods, (ii) encompassing multiple fungal genera, and (iii) being applied to a variety of specimen types. The design of fungal primers that can detect the appropriate range of fungal organisms has remained a challenge to the development of PCR assays for fungi. “PCR” is now a household term thanks to its crucial role in gold-standard diagnosis of COVID-19 infections.
Technology overview
On the surface, many existing panfungal (‘all fungi’) PCR assays appear to have sufficient specificity (i.e. they accurately identify all fungi) and sensitivity (i.e. they do not incorrectly identify non-fungi as fungi). However, we have examined the existing panfungal assays in detail using in silico methods, which has revealed numerous flaws that may affect the detection of certain fungi (i.e. lacking sensitivity). Unfortunately, single-nucleotide polymorphisms (SNPs) and small insertions-deletions (indels) present in crucial primer- and probe-binding sites in all existing panfungal assays, lead to potential or probable false-negative results. Another issue is that some existing assays suffer from oligo dimer issues; that is, the primers and/or probes tend to stick to themselves rather than the DNA template, a phenomenon that can be quantified using the ΔG metric (the lower the ΔG value, the higher the dimer tendency). Dimers are problematic in PCR as they lead to occasional false-positive results in samples that are truly fungal negative, thereby overestimating fungal presence.
To overcome these numerous issues with existing panfungal assays, we have designed a novel panfungal assay using all (n=13,386) non-redundant, publicly available fungal genomes; to our knowledge, it has 100% specificity and 100% sensitivity, thus surpassing the performance of existing panfungal assays. Our assay targets the highly conserved 5.8S region.
Benefits
- Reduced false-negative and false-positive results
- Our SYBR Green-based assay, which is lower cost and thus more accessible than probe-based formats, takes 47 min from start to finish; however, positive results can begin being seen within ~10min if fungal load is high
- Primers designed to amplify all fungi, based on our analysis of >13K fungal genomes
- The small amplicon size enables rapid thermocycling and faster fungal diagnosis
Applications
- Novel primer oligos for PCR molecular assay for panfungal detection for clinical/veterinary use
Commercial opportunity
UniSC is seeking a commercialisation partner for the development and deployment of the Technology for clinical/veterinary use.
The commercialisation strategy for this technology is to further demonstrate efficacy as a diagnostic test that could identify the broadest range of micro fungi i.e. fungi that aren't visible to the eye, but which cause infection in humans and animals.
Brief note on scientific founders
Associate Professor Erin Price– is a microbiologist, molecular geneticist, and co-lead of the Pathogen-Omics lab team at the University of the Sunshine Coast (UniSC). The Pathogen-Omics team investigates the transmission, antibiotic resistance potential, prevalence, and evolution of bacterial pathogens affecting humans, with a focus on pathogens associated with chronic respiratory diseases. Associate Professor Price has a keen interest in applying microbial genomics and transcriptomics to better understand, diagnose, and treat infectious diseases. She has extensive experience using comparative genomics to develop inexpensive, sensitive, and accurate real-time PCR diagnostic assays to target important microbes and their antimicrobial resistance potential.
Dr Derek Sarovich– is currently an Advance Queensland Fellow who joined UniSC in mid-2017 as a Senior Research Fellow. Derek is a molecular microbiologist and bioinformatician and is interested in antibiotic resistance, bacterial pathogenesis, molecular epidemiology, genomics and transcriptomics. Derek works closely with several clinically focused collaborators to bring next-generation sequencing technologies to the clinical realm, with a focus on improved treatment and diagnostics of antibiotic-resistant bacterial infections, particularly those with high morbidity and mortality.
Contact
For further information concerning the commercial opportunity please contact:
Michael Finney – Commercialisation Advisor