Aspergillosis is a spectrum of diseases and a major cause of morbidity and mortality. To treat these diseases, there are a few classes of antifungal drugs approved for clinical use.

Respiratory infections caused by fungal pathogens present a growing global health concern and are a major cause of death in immunocompromised patients. Worryingly, coronavirus disease-19 (COVID-19) resulting in acute respiratory distress syndrome has been shown to predispose some patients to airborne fungal co-infections. These include secondary pulmonary aspergillosis and mucormycosis. Aspergillosis is most commonly caused by the fungal pathogen Aspergillus fumigatus and primarily treated using the triazole drug group, however in recent years, this fungus has been rapidly gaining resistance against these antifungals. This is of serious clinical concern as multi-azole resistant forms of aspergillosis have a higher risk of mortality when compared against azole-susceptible infections. With the increasing numbers of COVID-19 and other classes of immunocompromised patients, early diagnosis of fungal infections is critical to ensuring patient survival. However, time-limited diagnosis is difficult to achieve with current culture-based methods. Advances within fungal genomics have enabled molecular diagnostic methods to become a fast, reproducible, and cost-effective alternative for diagnosis of respiratory fungal pathogens and detection of antifungal resistance. Here, we describe what techniques are currently available within molecular diagnostics, how they work and when they have been used.

Aspergillosis, in its various manifestations, is a major cause of morbidity and mortality. Very few classes of antifungal have been approved for clinical use to treat these diseases and resistance to the first line therapeutics is increasing. A new class of antifungals, the orotomides, are currently in development with the first compound in this class olorofim in late-stage clinical trials. In this study, we characterise a network of genes that govern olorofim response in A. fumigatus. We reveal that the number of transcription factors that regulate olorofim susceptibility are far fewer than we have previously observed for the azoles and the change in sensitivity observed in these isolates is less extreme. Intriguingly, loss of function in two higher order transcriptional regulators, HapB a member of the heterotrimeric HapB/C/E (CBC) complex or the regulator of nitrogen metabolic genes AreA, leads to cross resistance to both the azoles and olorofim. However, a clinical azole resistant isolate with a point mutation in HapE (hapEP88L) retains sensitivity to olorofim. Our transcriptomic analysis suggests that altered sensitivity to olorofim may emerge via modification of genes involved in the production of pyrimidine biosynthetic precursors. Finally, we also show that the action of the azoles are antagonistic to olorofim in vitro.

Aspergillosis, in its various manifestations, is a major cause of morbidity and mortality. Very few classes of antifungal have been approved for clinical use to treat these diseases and resistance to the first line therapeutics is increasing. A new class of antifungals, the orotomides, are currently in development with the first compound in this class olorofim in late-stage clinical trials. In this study, we characterise a network of genes that govern olorofim response in A. fumigatus. We reveal that the number of transcription factors that regulate olorofim susceptibility are far fewer than we have previously observed for the azoles and the change in sensitivity observed in these isolates is less extreme. Intriguingly, loss of function in two higher order transcriptional regulators, HapB a member of the heterotrimeric HapB/C/E (CBC) complex or the regulator of nitrogen metabolic genes AreA, leads to cross resistance to both the azoles and olorofim. However, a clinical azole resistant isolate with a point mutation in HapE (hapEP88L) retains sensitivity to olorofim. Our transcriptomic analysis suggests that altered sensitivity to olorofim may emerge via modification of genes involved in the production of pyrimidine biosynthetic precursors. Finally, we also show that the action of the azoles are antagonistic to olorofim in vitro.