Improving detection and diagnosis of antimicrobial resistance in Aspergillus fumigatus

Aspergillus fumigatus is a ubiquitous mould that plays a critical role breaking down plant material in the environment. However, A. fumigatus may cause a range of diseases when inhaled, including life-threatening infections in humans with certain immune defects. We can treat patients with azole drugs, but increasingly these drugs have become less effective due to resistance. Resistance usually does not develop during therapy but is already present in the fungus before infection is established. Researchers have found that the fungus becomes resistant in the environment when it grows in organic waste containing residues of azole fungicides. Resistance is caused by DNA changes in the fungus. These need to be detected when the fungus causes an infection so treatment can be adjusted accordingly. A major problem is that it is very difficult to determine if a patient has a resistant infection because current tests are not very good. The problem is worsened by an increasing range of DNA changes associated with resistance, indeed for 20% of resistant fungi the DNA changes are unknown. In this proposal we aim to confirm the presence of existing DNA changes and find new DNA changes that cause resistance, and then use this knowledge to develop better diagnostic tests. We will also improve a screening test by enabling it to detect resistance against an important newly developed drug. The development of better diagnostic tests will benefit patients, will help establish how often patients are infected with resistant A. fumigatus, and improve understanding of how patients become infected.

Azole antimicrobial resistance is an emerging concern in Aspergillus fumigatus, a fungus that may cause life- threatening infections in humans and animals. A. fumigatus is listed as one of 4 fungal pathogens in the 2022 WHO critical priority pathogen group, which is the first global effort to systematically prioritize fungal pathogens, considering their unmet research needs and perceived public health importance. Resistance in A. fumigatus is mainly caused by mutations in the cyp51A-gene, which commonly causes a pan-azole- resistant phenotype. Alternative treatment options to azoles are very limited although new compounds are under development, most notably olorofim (OLO). Detection of azole resistance is challenging as current tests have limited sensitivity. In addition, the variation in cyp51A resistance genotypes that impact on azole MIC is increasing and a large subset of ca. 20% of isolates with high azole MICs have unknown resistance mechanisms. Our proposal aims to IMPROVE current diagnostic tools by developing reverse complement (RC)-PCR, characterising critical single nucleotide polymorphisms (SNPs) and identifying new resistance mechanisms in azole-resistant A. fumigatus. In addition, we will IMPROVE current agar-based screening tools to allow rapid screening for azole resistance and add OLO resistance screening. Our studies will provide improved reference diagnostics as well as low cost screening tools benefiting patients globally with Aspergillus diseases. These tools and insights will also benefit international One Health resistance surveillance programs.