Scientists tackle the threat of invasive fungal disease with cutting-edge diagnostics

A team of researchers at the Public Health Wales Mycology Reference Laboratory is working to advance the diagnosis and treatment of deadly invasive fungal disease (IFD) using insights from the QIAGEN next-generation digital PCR (dPCR) system, QIAcuity.

The team, led by Professor Lewis White, is building upon real-time PCR methods with dPCR to detect fungal DNA at lower concentrations and with greater accuracy – a pursuit they hope could lead to earlier IFD diagnosis and prevent the use of unnecessary antifungal therapy. In addition to detecting insidious fungal DNA, the team is also developing host (patient) biomarkers to be incorporated alongside fungal diagnostics to improve patient stratification and enable personalized treatment approaches.

An overlooked healthcare burden

Human fungal disease most commonly manifests as superficial infections of the skin, hair, nails, or mucosal membranes (thrush). “These infections are usually benign, and along with allergic syndromes, affect hundreds of millions of people on an annual basis,” explains Prof. White.

However, given the opportunity, fungi can also cause life-threatening invasive fungal disease (IFD), whereby the organism invades internal organs and can become systemic, spreading through the bloodstream. “IFD is associated with a mortality anywhere between 30% and 50% and occurs mostly in patients with an existing underlying health condition,” says Prof. White. “Fungi are readily overlooked as a significant cause of disease, but globally, more people die from fungal disease than they do from malaria and tuberculosis.”

Patients most at risk of invasive fungal disease include those who have undergone surgery or clinical intervention, had major trauma, or are immunocompromised. “For example, the breakdown of anatomical structures during surgery can allow commensal candida species to become invasive,” shares Prof. White. “We also see IFD in patients who’ve suffered major trauma, such as soldiers or survivors of extreme weather events, where wounds have become contaminated with fungi from the environment.”

One of the most significant predisposing risk factors for IFD is immunosuppression, which Prof. White warns is on the rise due to the increasing use of immunosuppressive medicines. “We're in a situation where we see an ever-growing population of immunosuppressed patients,” he says. “Historically, fungal disease has been associated with patients with HIV/AIDS, acute leukemia, or who’ve undergone stem cell transplantation, and thus have a weakened immune system. However, the increasing use of immunosuppressive medicines, such as anti-inflammatory corticosteroids and monoclonal antibodies, is increasing the number of patients at risk of fungal disease.”

dPCR outperforms real-time PCR in fungal DNA quantification

Given the high morbidity and mortality of IFD, the early identification of causative fungal pathogens is essential to optimize patient management and guide treatment. However, an IFD diagnosis can be challenging, as infective burdens can be very low, and the ubiquitous nature of fungi can lead to false positive results. Conventional methods, including histopathologic examination, fungal culture and serology, are often insufficient for diagnosis, even when used in combination.

In recent years, molecular methods, such as real-time PCR assays, have emerged as a suitable alternative to conventional methods for IFD diagnosis. “Real-time PCR assays, which enable the monitoring of PCR reactions using fluorescent dyes, eliminate the need to handle specimens’ post-amplification as in conventional PCR, minimizing the opportunity for contamination,” explains Prof. White. “The other benefit is that they are quantifiable, giving us an indication of the fungal burden in clinical specimens.”

However, the low concentration of fungal DNA in clinical samples can be close to the limits of detection of real-time PCR assays, while they are also prone to PCR inhibition. These limitations are overcome by dPCR, whereby samples are partitioned into thousands of individual reaction wells. “Because you've got tens of thousands of individual reactions, dPCR allows us to pick up lower concentrations of DNA compared to real-time PCR in a more precise manner,” Prof. White enthuses. “We can also determine the copies of fungal DNA in each of the partitions and, based on the number of positive partitions, obtain much more accurate quantification compared to real-time PCR with much greater sensitivity, which is useful for determining the clinical significance of a PCR result. Finally, due to the numerous individual PCR reactions, dPCR is less prone to inhibition.”

DNA insights served on a (nano)plate

For this work, Prof. White and his team are using the QIAGEN next-generation plate-based QIAcuity platform, which they found to have greatly streamlined their transition from real-time PCR assays to dPCR. “We could take our in-house or even commercial real-time PCR assays and, with minimal redesign, shift our assays onto the QIAcuity platform,” he shares.

“The quantification using the instrument’s software also made life a whole lot easier,” he continues. “We came across issues in terms of setting the threshold for determining a positive dPCR result, but because we could get access to the raw data, we were able to determine our own thresholds based on downstream analysis and clinical evaluation of performance.”

Host biomarkers to combat anti-fungal resistance

While dPCR has the potential to greatly advance fungal DNA detection in patient samples, Prof. White notes that more work is to be done before clinicians will be able to quickly and reliably diagnose IFD. “Through the combination of PCR with different tests, we can be pretty sure if all tests are negative, we can exclude fungal disease,” he asserts. “However, even with reliable DNA quantification combined with other biomarkers such as antigen tests, the probability of invasive fungal disease is not anywhere near 100%.”

For this reason, Prof. White is also turning his attention to patient DNA, looking to identify genetic markers in the innate immune system that could indicate a predisposition to fungal disease – another area where he sees great utility in the QIAcuity platform. “The ability of QIAcuity to pick up single nucleotide polymorphisms that may be within these innate genes would be a very useful application of the test,” he enthuses. “The plate setup of the QIAcuity would allow us to develop a plate with a number of different assays targeting different mutations within the host, and that would be a rapid way of screening patients for risk factors to fungal disease, but also genetic risk to a range of other pathogens outside of fungi.”

Prof. White sees similar potential in combining dPCR with other genomics approaches to screen for anti-fungal resistance, a significant emerging concern worldwide. “We are currently focusing on azole resistance in Aspergillus fumigatus,” he shares. “There are several commercial real-time assays that pick up the main causes of azole resistance in Aspergillus fumigatus, but the mechanisms associated with resistance are ever-increasing, so we need a platform that can allow us to pick up several genetic mutations associated with azole resistance.”

“The nanoplate-based format of the QIAcuity could allow us to do that in a quantifiable and rapid manner, and there's many other applications where we could combine dPCR with metagenomics or next-generation sequencing to screen for mutations that are associated with resistance, and potentially identify other, as of yet, unidentified resistance mechanisms.”

Disclaimer: The QIAcuity is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, monitoring, prediction, prognosis, treatment or alleviation of a disease or other conditions in human beings. For product instructions for use and limitations see the respective QIAGEN kit instructions for use or user operator manual.

Fiction to reality: Is ‘The Last of Us’ becoming a possible scenario?

In HBO’s The Last of Us, a parasitic fungal infection that turns human hosts into deadly zombies brings the world to its knees. While such an apocalyptic scenario is purely fictional, the real-life threat of fungal disease should not be underestimated.

“If you look back over the last 100 years, 74% of animal species that are extinct, close to extinction, or regionally extinct have been targeted by a fungal pathogen,” shares Prof. White. “Is it going to happen in humans? Probably not. But it is a concern that we're seeing an increase in the portion of the population that are immunosuppressed and susceptible to fungal disease.”

Another challenge presented by fungi is that they are opportunistic and do not rely on obligate pathogenicity. “When an obligate pathogen, such the SARS-CoV-2 virus, kills off the host, they kill off themselves,” explains Prof. White. “What you find in obligate pathogens is over time they tend to become less virulent, which allows them to survive longer periods and be transmitted between hosts. In contrast, fungi do not require a host to survive, so if they become pathogenic, there’s no selective pressure to not kill-off that food source.”

“Where we also need to be careful is the emergence of antifungal resistance, which can spread rapidly due to the fact fungi can reproduce sexually and asexually,” he warns. “There's no fitness cost in Aspergillus associated with resistance to many of the antifungal drugs that we use – they are basically as healthy as susceptible strains. This means they could become the predominant organism. If that were to happen broadly, we could struggle to treat patients for invasive fungal disease or even chronic fungal disease, if these became mainly associated with resistant antifungal pathogens.”

As the clinical burden of fungal disease becomes more apparent, continued efforts to improve diagnostic tools, enhance the surveillance of antifungal resistance, and develop novel therapeutics are imperative.