The recent rate of emergence of pathogenic fungi that are resistant to the limited number of commonly used antifungal agents is unprecedented. The azoles, for example, are used not only for human and animal health care and crop protection but also in antifouling coatings and timber preservation. The ubiquity and multiple uses of azoles have hastened the independent evolution of resistance in many environments. One consequence is an increasing risk in human health care from naturally occurring opportunistic fungal pathogens that have acquired resistance to this broad class of chemicals. To avoid a global collapse in our ability to control fungal infections and to avoid critical failures in medicine and food security, we must improve our stewardship of extant chemicals, promote new antifungal discovery, and leverage emerging technologies for alternative solutions.

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Worldwide emergence of resistance to antifungal drugs challenges human health and food security

The importance of aspergillosis in humans and various animal species has increased over the last decades. Aspergillus species are found worldwide in humans and in almost all domestic animals and birds as well as in many wild species, causing a wide range of diseases from localized infections to fatal disseminated diseases, as well as allergic responses to inhaled conidia. Some prevalent forms of animal aspergillosis are invasive fatal infections in sea fan corals, stonebrood mummification in honey bees, pulmonary and air sac infection in birds, mycotic abortion and mammary gland infections in cattle, guttural pouch mycoses in horses, sinonasal infections in dogs and cats, and invasive pulmonary and cerebral infections in marine mammals and nonhuman primates. This article represents a comprehensive overview of the most common infections reported by Aspergillus species and the corresponding diseases in various types of animals.

Aspergillus and aspergilloses in wild and domestic animals: a global health concern with parallels to human disease.

Agricultural industry uses pesticides to optimise food production for the growing human population. A major issue for crops is fungal phytopathogens, which are treated mainly with azole fungicides. Azoles are also the main medical treatment in the management of Aspergillus diseases caused by ubiquitous fungi, such as Aspergillus fumigatus. However, epidemiological research demonstrated an increasing prevalence of azole-resistant strains in A. fumigatus. The main resistance mechanism is a combination of alterations in the gene cyp51A (TR34/L98H). Surprisingly, this mutation is not only found in patients receiving long-term azole therapy for chronic aspergillosis but also in azole naive patients. This suggests an environmental route of resistance through the exposure of azole fungicides in agriculture. In this review, we report data from several studies that strongly suggest that agricultural azoles are responsible for medical treatment failure in azole-naive patients in clinical settings.

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Azole Resistance in Aspergillus fumigatus: A Consequence of Antifungal Use in Agriculture?

Fungal diseases are an increasing global burden. Fungi are now recognised to kill more people annually than malaria, whilst in agriculture, fungi threaten crop yields and food security. Azole resistance, mediated by several mechanisms including point mutations in the target enzyme (CYP51), is increasing through selection pressure as a result of widespread use of triazole fungicides in agriculture and triazole antifungal drugs in the clinic. Mutations similar to those seen in clinical isolates as long ago as the 1990s in Candida albicans and later in Aspergillus fumigatus have been identified in agriculturally important fungal species and also wider combinations of point mutations. Recently, evidence that mutations originate in the field and now appear in clinical infections has been suggested. This situation is likely to increase in prevalence as triazole fungicide use continues to rise. Here, we review the progress made in understanding azole resistance found amongst clinically and agriculturally important fungal species focussing on resistance mechanisms associated with CYP51. Biochemical characterisation of wild-type and mutant CYP51 enzymes through ligand binding studies and azole IC50 determinations is an important tool for understanding azole susceptibility and can be used in conjunction with microbiological methods (MIC50 values), molecular biological studies (site-directed mutagenesis) and protein modelling studies to inform future antifungal development with increased specificity for the target enzyme over the host homologue.

Resistance to antifungals that target CYP51

Aspergillus fumigatus, a ubiquitously distributed opportunistic pathogen, is the global leading cause of aspergillosis. Azole antifungals play an important role in the management of aspergillosis. However, over a decade, azole resistance in A. fumigatus isolates has been increasingly reported with variable prevalence worldwide and it is challenging the effective management of aspergillosis. The high mortality rates observed in patients with invasive aspergillosis caused by azole-resistant A. fumigatus (ARAF) isolates pose serious challenges to the clinical microbiologist for timely identification of resistance and appropriate therapeutic interventions. The majority of ARAF isolates contain alterations in the cyp51A gene; however, there have been increasing reports on non-cyp51A mutations contributing to azole resistant phenotypes. This review highlights the emergence and various mechanisms implicated in the development of azole resistance in A. fumigatus. We further present recent developments related to the environmental route in the emergence of ARAF isolates and discuss the therapeutic options available.

Exploring azole antifungal drug resistance in Aspergillus fumigatus with special reference to resistance mechanisms.

In recent years, triazole resistance in human Aspergillus diseases appears to have been increasing in several European countries. However, current data on the prevalence of resistance are based on a small number of studies which are only available from a few European countries. If present, triazole resistance can severely limit treatment options since alternatives, which are only available in intravenous form, have been shown to be associated with more side effects and poorer outcomes. Triazole resistance in Aspergillus spp. can evolve during therapy. Several point mutations, particularly in the cyp51A gene, have been associated with the development of resistance. Increasingly however, resistant isolates are also being detected in azole-naive patients. These isolates tend to have a particular genetic alteration consisting of a 34-base pair tandem repeat in the promoter coupled with a point mutation in the cyp51A target gene. This leads to an amino-acid substitution at codon 98 (TR34/L98H) causing multi-azole resistance. In patients whose Aspergillus isolates have developed resistance during azole therapy wildtype isolates, closely related genetically to the resistant isolates, have regularly been recovered from samples taken before the start of therapy or during an earlier phase. To date however, no isogenic isolate with a wild-type phenotype has been recovered from patients infected with an Aspergillus strain carrying the TR34/L98H genetic alteration. This suggests a possible environmental origin of the resistant fungus. This particular resistance mechanism has been observed most frequently in clinical isolates in the Netherlands where it has also been found in the environment. Moreover, the resistance mechanism has been demonstrated in clinical isolates in eight other European countries. Azole fungicides are widely used for crop protection and material preservation in Europe. They protect crops from disease, ensure yields and prevent fungal contamination of produce. It has been proposed that triazole resistance has evolved in the environment and could be driven by the selective pressure of azole fungicides. Although evidence supporting this hypothesis is growing, the link between the environmental use of azole fungicides and the development of triazole resistance in Aspergillus spp. is not yet proven. Triazole therapy has become the established treatment for invasive aspergillosis and is widely used in the treatment of allergic aspergillosis and chronic pulmonary aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis is usually prescribed for a minimum of 6–12 weeks, but often may need to be continued for months depending on the period of immunosuppression. Treatment of allergic aspergillosis and chronic pulmonary aspergillosis may need to continue for years or even throughout a patient’s lifetime. We estimated the burden of allergic, chronic and invasive aspergillosis using population statistics and published literature. Of the 733 million inhabitants in the European region1 [1], at any one time 2 100 000 patients may be suffering from allergic aspergillosis and 240 000 from chronic aspergillosis, that would be an indication for antifungal therapy. For invasive aspergillosis, we have estimated an annual incidence of 63 250 cases, complicating multiple underlying conditions including leukaemia, transplantation, chronic obstructive pulmonary disease (COPD) and medical intensive care. The inability to treat these patients with triazoles due to multi-azole resistance would have significant impact on patient management and associated health costs. Early and thorough investigation of this emerging public health problem is warranted in order to avoid the development and spread of resistance. This report examines current evidence for the environmental origin of resistance in Aspergillus spp. and makes recommendations for further steps to assess the risks and consequences of the environmental usage of azole derivatives. Improved surveillance of clinical isolates, including antifungal susceptibility testing, is the key to a better understanding of the magnitude of this emerging problem. Furthermore, the diagnosis of Aspergillus diseases needs to be improved and molecular methods allowing detection of resistance in culture-negative specimens must be further developed and implemented in laboratory practice. Finally, further environmental and laboratory studies are needed to confirm the environmental hypothesis

Risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species

In recent years, triazole resistance in human Aspergillus diseases appears to have been increasing in severalEuropean countries. However, current data on the prevalence of resistance are based on a small number of studieswhich are only available from a few European countries. If present, triazole resistance can severely limit treatmentoptions since alternatives, which are only available in intravenous form, have been shown to be associated withmore side effects and poorer outcomes. Triazole resistance in Aspergillus spp. can evolve during therapy. Severalpoint mutations, particularly in the cyp51A gene, have been associated with the development of resistance.Increasingly however, resistant isolates are also being detected in azole-naive patients. These isolates tend to havea particular genetic alteration consisting of a 34-base pair tandem repeat in the promoter coupled with a pointmutation in the cyp51A target gene. This leads to an amino-acid substitution at codon 98 (TR34/L98H) causingmulti-azole resistance. In patients whose Aspergillus isolates have developed resistance during azole therapy wild-type isolates, closely related genetically to the resistant isolates, have regularly been recovered from samples takenbefore the start of therapy or during an earlier phase. To date however, no isogenic isolate with a wild-typephenotype has been recovered from patients infected with an Aspergillus strain carrying the TR34/L98H geneticalteration. This suggests a possible environmental origin of the resistant fungus. This particular resistancemechanism has been observed most frequently in clinical isolates in the Netherlands where it has also been foundin the environment. Moreover, the resistance mechanism has been demonstrated in clinical isolates in eight otherEuropean countries.Azole fungicides are widely used for crop protection and material preservation in Europe. They protect crops fromdisease, ensure yields and prevent fungal contamination of produce. It has been proposed that triazole resistancehas evolved in the environment and could be driven by the selective pressure of azole fungicides. Althoughevidence supporting this hypothesis is growing, the link between the environmental use of azole fungicides and thedevelopment of triazole resistance in Aspergillus spp. is not yet proven.Triazole therapy has become the established treatment for invasive aspergillosis and is widely used in thetreatment of allergic aspergillosis and chronic pulmonary aspergillosis. Antifungal therapy for invasive pulmonaryaspergillosis is usually prescribed for a minimum of 6?12 weeks, but often may need to be continued for monthsdepending on the period of immunosuppression. Treatment of allergic aspergillosis and chronic pulmonaryaspergillosis may need to continue for years or even throughout a patient?s lifetime. We estimated the burden ofallergic, chronic and invasive aspergillosis using population statistics and published literature. Of the 733 millioninhabitants in the European region 1 [1], at any one time 2 100 000 patients may be suffering from allergicaspergillosis and 240 000 from chronic aspergillosis, that would be an indication for antifungal therapy. For invasiveaspergillosis, we have estimated an annual incidence of 63 250 cases, complicating multiple underlying conditionsincluding leukaemia, transplantation, chronic obstructive pulmonary disease (COPD) and medical intensive care.The inability to treat these patients with triazoles due to multi-azole resistance would have significant impact onpatient management and associated health costs.Early and thorough investigation of this emerging public health problem is warranted in order to avoid thedevelopment and spread of resistance. This report examines current evidence for the environmental origin ofresistance in Aspergillus spp. and makes recommendations for further steps to assess the risks and consequencesof the environmental usage of azole derivatives. Improved surveillance of clinical isolates, including antifungalsusceptibility testing, is the key to a better understanding of the magnitude of this emerging problem. Furthermore,the diagnosis of Aspergillus diseases needs to be improved and molecular methods allowing detection of resistancein culture-negative specimens must be further developed and implemented in laboratory practice. Finally, furtherenvironmental and laboratory studies are needed to confirm the environmental hypothesis.

Risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species (ECDC Technical Report)

N. Kleinkauf

Papers

Risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species

Risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species (ECDC Technical Report)