Widely dispersed clonal expansion of multi-fungicide-resistant Aspergillus fumigatus limits genomic epidemiology prospects

Abstract

BackgroundAspergillus fumigatus is a ubiquitous fungus that causes a range of diseases in animals, including humans. The most lethal manifestation is invasive aspergillosis for which treatment relies on triazoles. Triazole-resistant A. fumigatus can be recovered from decaying plant material and so-called hotspots containing triazole fungicide residues. Although observations have shown clonal isolates between the environment and clinical samples, a direct link between a specific environment and cases of triazole-resistant invasive aspergillus disease in an individual patient has not yet been demonstrated. MethodsTo understand where patients acquire A. fumigatus isolates causing disease, we used a genomic epidemiology approach with 157 Dutch A. fumigatus isolates, based on whole genome sequencing. Isolates were from three well-characterized environmental hotspots and two hospitals between 2016 and 2019. FindingsIn the Dutch dataset, A. fumigatus isolates from six patients showed near-identical genomes compared to five environmental isolates. One environmental isolate matched three probable cases of triazole-resistant invasive aspergillosis, including one fatal case. Patient isolates were recovered up to 34 months later than near-identical environmental isolates. Comparison to over 1{middle dot}2K global publicly available A. fumigatus genomes showed hundreds of clonal groups spread across three continents. In addition, finding variants associated with resistance to non-triazole fungicides such as benzimidazole, succinate dehydrogenase inhibitor and quinone outside inhibitor classes, strongly suggests an exposure history to multiple agricultural fungicides in these environmental hotspots. InterpretationEnvironmental hotspots represent highly selective habitats for multi-fungicide-resistant A. fumigatus, which we can now directly link to probable cases of aspergillus disease, including a triazole-resistant case. However, geographically widely dispersed clonal expansion limits the utility of genomic epidemiology to identify the source of a particular patients isolate. Furthermore, reducing a single class of fungicides in agriculture may not effectively reduce resistance selection when other classes are still in use. FundingNWO.Groen2019.002 Research in contextO_ST_ABSEvidence before this studyC_ST_ABSTriazole fungicides that exhibit activity against Aspergillus fumigatus have been shown to be a major source of resistant aspergillus disease in humans. However, the route of transmission from environmental hotspot to human remains poorly understood. Isolates of A. fumigatus can be recovered from both environmental samples and clinical specimens that harbour the same resistance signature haplotypes, e.g., TR34/L98H and TR46/Y121F/T289A, in the cyp51A-gene. We searched the literature for evidence using high resolution whole genome sequencing (WGS) to link environmental isolates to human infection. We searched PubMed for articles using the search terms Aspergillus fumigatus AND azole resistance AND whole genome sequencing on 15 April 2024. This search retrieved 32 articles describing different evolutionary routes to select for triazole-resistant A. fumigatus or population structure of whole-genome sequenced isolates. Twenty-six articles used whole-genome sequencing, but none focused on identifying clonal groups to identify direct cases of transmission between the environment and clinical cases of aspergillus disease. By using the additional search term transmission, no other records were retrieved. Added value of this studyOur study links triazole-resistant A. fumigatus isolates cultured from three environmental hotspots to cases of aspergillus disease in two hospitals in the Netherlands. Genome comparisons of isolates from environmental hotspots and patients showed multiple near-identical linked genotypes, consistent with a route of transmission from the environment to patients. Although a naive expectation may be a higher probability of matches of the hotspots located in the northwest of the Netherlands with the hospital located in the west of the country, in fact, more patient isolates from the far southeast were linked to the hotspots. Integrating the Dutch data set into a global data set showed 205 clonal groups spread across the Netherlands, Germany, the United Kingdom (UK), the United States of America (USA) and Japan. Our demonstration of a large number of geographically dispersed clonal groups suggests that current sampling is insufficient to definitively identify the source of an individual patients infection. A genetically highly diverse population combined with a wide global distribution of clones can make it impossible to definitively identify the source of an individual patients infection even with much more sampling. Implications of all the available evidenceOur study provides evidence that triazole-resistant A. fumigatus isolates with multi-fungicide resistance profiles cause aspergillus disease in at-risk patients and may contribute to treatment failure and mortality. The risk of infection due to these triazole-resistant isolates is not confined to the geographic vicinity of the environmental hotspot since clonal spread can be detected across great distances. The finding of linked cases without clear transmission routes limits epidemiological studies and underscores the need to better understand the ecology and environmental niches of this fungus. As it is highly unlikely that each patient visited the rural agricultural areas where a hotspot was located, research should address the complex and long-distance transmission routes of resistant isolates, which involves airborne dispersal of conidia or habitats of this fungus outside the agricultural environment. Furthermore, because of the multi-fungicide resistance phenotype of the triazole-resistant A. fumigatus, involving several classes of fungicides, reducing one class of fungicides in the environment may not effectively reduce resistance selection. Effective interventions should instead aim to reduce the burden of environmental resistance by modifying environments that currently favour the massive outgrowth of fungicide-resistant A. fumigatus to limit the escape of aerial spores from these environmental hotspots.