Fungal infections affect more than a billion people worldwide, with a mortality rate that matches that of malaria or tuberculosis. A growing concern is Aspergillus fumigatus, a globally prevalent environmental mould that can cause multiple clinical diagnoses. Among these, invasive aspergillosis (IA) can occur in at-risk populations, such as patients with severe neutropenia, haematopoietic stem cell or solid organ transplants, patients receiving immunosuppressive drugs and, increasingly, patients with influenza and coronavirus disease 2019. Patients with cystic fibrosis (CF) are also at risk of chronic infections, with 30% developing Aspergillus-related bronchitis and 19% developing allergic bronchopulmonary aspergillosis4. With over 2.25 million individuals suffering from infections caused by A. fumigatus in the European Union alone5, this is a global concern. Unfortunately, recent studies have also reported an emerging worldwide resistance to azole antifungal drugs in both clinical and environmental isolates, which have long proven effective against A. fumigatus9.
Azole drug resistance has serious clinical implications, with retrospective studies of patients with drug-resistant IA showing a 25% increase in mortality at day 90 compared to patients with wild-type (WT) infections. While in vivo resistance emergence during extended azole therapy is well documented, more recent studies postulate an ex vivo evolution of resistance in the environment as a result of exposure to agricultural chemicals—particularly sterol 14α-demethylation inhibitor fungicides developed in the 1970s. Broadly, environmentally occurring azole resistance in A. fumigatus is characterized by signature mechanisms involving expression-upregulating tandem repeats (TRs) in the promoter region of cyp51A accompanied by point mutations within this gene, which decrease the affinity of azoles for the target protein; the most commonly occurring alleles are known as TR34/L98H and TR46/Y121F/T289A and are associated with high-level itraconazole and voriconazole resistance, respectively both inside and outside the clinic. The spatially widespread occurrence of these alleles alongside increasing reports of more complex cyp51A resistance-associated polymorphisms underpin the hypothesis that the broad application of agricultural azole fungicides is driving natural selection, amplification and ultimately acquisition of azole-resistant airborne A. fumigatus conidia by susceptible patients. Furthermore, the potential for global spread of these resistance mechanisms through floriculture products, especially plant bulbs, has been demonstrated, while the global dispersal of conidia on air currents is impossible to contain.
Modern genomic epidemiological methods further indicate a potential link between the increasing clinical incidence of azole-resistant IA and the increasingly broad range of azole-resistant genotypes that are being reported in the environment. The rate at which environmental resistance develops will be determined by natural selection on beneficial mutations. This is in turn influenced by recombination, gene flow and dispersal, which leave their characteristic signatures in the genome. Evidence for these expectations comes from a recent global study from our laboratory demonstrating the non-random distribution of azole resistance in multilocus microsatellite genotypes.
In this study, we used whole-genome sequencing (WGS) of 218 A. fumigatus isolates (n = 65 environmental isolates and n = 153 clinical isolates) to interrogate the molecular epidemiology of this fungus and determine whether acquisition of drug-resistant isolates by at-risk patient groups occurs. We also leveraged the power of these data to perform genome-wide association studies (GWAS) and pan-genome analyses to identify the variation associated with itraconazole drug resistance, revealing potentially new mechanisms of resistance.