RESUMO
Using a citizen science approach, we identify a country-wide exposure to aerosolized spores of a human fungal pathogen, Aspergillus fumigatus, that has acquired resistance to the agricultural fungicide tebuconazole and first-line azole clinical antifungal drugs. Genomic analysis shows no distinction between resistant genotypes found in the environment and in patients, indicating that at least 40% of azole-resistant A. fumigatus infections are acquired from environmental exposures. Hotspots and coldspots of aerosolized azole-resistant spores were not stable between seasonal sampling periods. This suggests a high degree of atmospheric mixing resulting in an estimated per capita cumulative annual exposure of 21 days (±2.6). Because of the ubiquity of this measured exposure, it is imperative that we determine sources of azole-resistant A. fumigatus to reduce treatment failure in patients with aspergillosis.
Assuntos
Aspergilose , Ciência do Cidadão , Humanos , Aspergillus fumigatus/genética , Farmacorresistência Fúngica/genética , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Antifúngicos/farmacologia , Azóis/farmacologiaRESUMO
Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.
Assuntos
Anti-Infecciosos , Aspergillus fumigatus , Aspergillus fumigatus/genética , Azóis/farmacologia , Farmacorresistência Fúngica/genética , Humanos , Metagenômica , Testes de Sensibilidade MicrobianaRESUMO
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.
Assuntos
COVID-19 , Pneumologia , Aspergillus fumigatus , Genômica , Humanos , SARS-CoV-2RESUMO
Aspergillus fumigatus has widely evolved resistance to the most commonly used class of antifungal chemicals, the azoles. Current methods for identifying azole resistance are time-consuming and depend on specialized laboratories. There is an urgent need for rapid detection of these emerging pathogens at point-of-care to provide the appropriate treatment in the clinic and to improve management of environmental reservoirs to mitigate the spread of antifungal resistance. Our study demonstrates the rapid and portable detection of the two most relevant genetic markers linked to azole resistance, the mutations TR34 and TR46, found in the promoter region of the gene encoding the azole target cyp51A. We developed a lab-on-a-chip platform consisting of: (i) tandem-repeat loop-mediated isothermal amplification; (ii) state-of-the-art complementary metal-oxide-semiconductor microchip technology for nucleic acid amplification detection; and (iii) a smartphone application for data acquisition, visualization, and cloud connectivity. Specific and sensitive detection was validated with isolates from clinical and environmental samples from 6 countries across 5 continents, showing a lower limit of detection of 10 genomic copies per reaction in less than 30 min. When fully integrated with a sample preparation module, this diagnostic system will enable the detection of this ubiquitous fungus at the point-of-care, and could help to improve clinical decision making, infection control, and epidemiological surveillance.
Assuntos
Aspergilose , Aspergillus fumigatus , Antifúngicos/farmacologia , Aspergillus fumigatus/genética , Azóis/farmacologia , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Humanos , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade Microbiana , Técnicas de Diagnóstico Molecular , Mutação , Técnicas de Amplificação de Ácido NucleicoRESUMO
The global emergence of azole resistance in Aspergillus fumigatus is resulting in health and food security concerns. Rapid diagnostics and environmental surveillance methods are key to understanding the distribution and prevalence of azole resistance. However, such methods are often associated with high costs and are not always applicable to laboratories based in the least-developed countries. Here, we present and validate a low-cost screening protocol that can be used to differentiate between azole-susceptible "wild-type" and azole-resistant A. fumigatus isolates. © 2020 The Authors. Basic Protocol 1: Preparation of Tebucheck multi-well plates Basic Protocol 2: Inoculation of Tebucheck multi-well plates.
Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Testes de Sensibilidade Microbiana/métodos , Triazóis/farmacologia , Aspergillus fumigatus/isolamento & purificação , Azóis/farmacologia , Farmacorresistência Fúngica , GenótipoRESUMO
Azole resistance in the opportunistic pathogen Aspergillus fumigatus is increasing, dominated primarily by the following two environmentally associated resistance alleles: TR34/L98H and TR46/Y121F/T289A. By sampling soils across the South of England, we assess the prevalence of azole-resistant A. fumigatus (ARAf) in samples collected in both urban and rural locations. We characterize the susceptibility profiles of the resistant isolates to three medical azoles, identify the underlying genetic basis of resistance, and investigate their genetic relationships. ARAf was detected in 6.7% of the soil samples, with a higher prevalence in urban (13.8%) than rural (1.1%) locations. Twenty isolates were confirmed to exhibit clinical breakpoints for resistance to at least one of three medical azoles, with 18 isolates exhibiting resistance to itraconazole, 6 to voriconazole, and 2 showing elevated minimum inhibitory concentrations to posaconazole. Thirteen of the resistant isolates harbored the TR34/L98H resistance allele, and six isolates carried the TR46/Y121F/T289A allele. The 20 azole-resistant isolates were spread across five csp1 genetic subtypes, t01, t02, t04B, t09, and t18 with t02 being the predominant subtype. Our study demonstrates that ARAf can be easily isolated in the South of England, especially in urban city centers, which appear to play an important role in the epidemiology of environmentally linked drug-resistant A. fumigatus.