Citizen science reveals landscape-scale exposures to multiazole-resistant Aspergillus fumigatus bioaerosols.

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.

Does virtual reality technology reduce pain and anxiety during outpatient hysteroscopy? A randomised controlled trial.

OBJECTIVE: To evaluate the effectiveness of virtual reality technology in reducing pain and anxiety during outpatient hysteroscopy. DESIGN: A prospective randomised controlled trial. SETTING: A London University Teaching Hospital. POPULATION: Women aged 18-70 years undergoing outpatient hysteroscopy procedures. METHODS: An unblinded randomised controlled trial was performed between March and October 2022 comparing standard outpatient hysteroscopy care with standard care with the addition of a virtual reality headset playing a virtual reality immersive scenario as a distraction technique. MAIN OUTCOME MEASURES: Pain and anxiety numeric rating scores (NRS) from 0 to 11. RESULTS: Eighty-three participants were randomly allocated to the control (n = 42) and virtual reality groups (n = 41). The virtual reality group experienced significantly less anxiety during the procedure than the control group (mean NRS 3.29 versus 4.73, mean difference 1.50; 95% confidence interval [CI] 0.12-2.88; P = 0.03). There was no difference in reported average pain (mean NRS 3.73. versus 4.24, mean difference 0.51; 95% CI -1.76 to 0.64; p = 0.41) or maximum pain scores (mean NRS 5.32 versus 5.07, mean difference 0.25; 95% CI -1.05 to 1.55; P = 0.71). CONCLUSIONS: The use of virtual reality technology as an adjunct to standard care can reduce patient-reported anxiety but not pain during outpatient hysteroscopy procedures. Continued improvements in the technology and the development of increasingly immersive environments may continue to increase the potential to improve the patient experience in this setting.

Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment.

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.

Chytridiomycosis Outbreak in a Chilean Giant Frog (Calyptocephalella gayi) Captive Breeding Program: Genomic Characterization and Pathological Findings.

Emerging infectious diseases in wildlife are increasingly associated with animal mortality and species declines, but their source and genetic characterization often remains elusive. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has been associated with catastrophic and well-documented amphibian population declines and extinctions at the global scale. We used histology and whole-genome sequencing to describe the lesions caused by, and the genetic variability of, two Bd isolates obtained from a mass mortality event in a captive population of the threatened Chilean giant frog (Calyptocephalella gayi). This was the first time an association between Bd and high mortality had been detected in this charismatic and declining frog species. Pathological examinations revealed that 30 dead metamorphosed frogs presented agnathia or brachygnathia, a condition that is reported for the first time in association with chytridiomycosis. Phylogenomic analyses revealed that Bd isolates (PA1 and PA2) from captive C. gayi group with other Bd isolates (AVS2, AVS4, and AVS7) forming a single highly supported Chilean Bd clade within the global panzootic lineage of Bd (BdGPL). These findings are important to inform the strengthening of biosecurity measures to prevent the impacts of chytridiomycosis in captive breeding programs elsewhere.

Molecular Epidemiology of Azole-Resistant Aspergillus fumigatus in France Shows Patient and Healthcare Links to Environmentally Occurring Genotypes.

Resistance of the human pathogenic fungus Aspergillus fumigatus to antifungal agents is on the rise. However, links between patient infections, their potential acquisition from local environmental sources, and links to global diversity remain cryptic. Here, we used genotyping analyses using nine microsatellites in A. fumigatus, in order to study patterns of diversity in France. In this study, we genotyped 225 local A. fumigatus isolates, 112 azole susceptible and 113 azole resistant, collected from the Bourgogne-Franche-Comté region (Eastern France) and sampled from both clinical (n = 34) and environmental (n = 191) sources. Azole-resistant clinical isolates (n = 29) were recovered mainly from cystic fibrosis patients and environmental isolates (n = 84) from market gardens and sawmills. In common with previous studies, the TR34/L98H allele predominated and comprised 80% of resistant isolates. The genotypes obtained for these local TR34/L98H isolates were integrated into a broader analysis including all genotypes for which data are available worldwide. We found that dominant local TR34/L98H genotypes were isolated in different sample types at different dates (different patients and types of environments) with hospital air and patient's isolates linked. Therefore, we are not able to rule out the possibility of some nosocomial transmission. We also found genotypes in these same environments to be highly diverse, emphasizing the highly mixed nature of A. fumigatus populations. Identical clonal genotypes were found to occur both in the French Eastern region and in the rest of the world (notably Australia), while others have not yet been observed and could be specific to our region. Our study demonstrates the need to integrate patient, healthcare, and environmental sampling with global databases in order to contextualize the local-scale epidemiology of antifungal resistant aspergillosis.

Discriminating lineages of Batrachochytrium dendrobatidis using quantitative PCR.

The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage-specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates.

Cross-Disciplinary Genomics Approaches to Studying Emerging Fungal Infections.

Emerging fungal pathogens pose a serious, global and growing threat to food supply systems, wild ecosystems, and human health. However, historic chronic underinvestment in their research has resulted in a limited understanding of their epidemiology relative to bacterial and viral pathogens. Therefore, the untargeted nature of genomics and, more widely, -omics approaches is particularly attractive in addressing the threats posed by and illuminating the biology of these pathogens. Typically, research into plant, human and wildlife mycoses have been largely separated, with limited dialogue between disciplines. However, many serious mycoses facing the world today have common traits irrespective of host species, such as plastic genomes; wide host ranges; large population sizes and an ability to persist outside the host. These commonalities mean that -omics approaches that have been productively applied in one sphere and may also provide important insights in others, where these approaches may have historically been underutilised. In this review, we consider the advances made with genomics approaches in the fields of plant pathology, human medicine and wildlife health and the progress made in linking genomes to other -omics datatypes and sets; we identify the current barriers to linking -omics approaches and how these are being underutilised in each field; and we consider how and which -omics methodologies it is most crucial to build capacity for in the near future.

Rapid Detection of Azole-Resistant Aspergillus fumigatus in Clinical and Environmental Isolates by Use of a Lab-on-a-Chip Diagnostic System.

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.

A Low-Cost Tebuconazole-Based Screening Test for Azole-Resistant Aspergillus fumigatus.

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.

A New Lineage of Cryptococcus gattii (VGV) Discovered in the Central Zambezian Miombo Woodlands.

We discovered a new lineage of the globally important fungal pathogen Cryptococcus gattii on the basis of analysis of six isolates collected from three locations spanning the Central Miombo Woodlands of Zambia, Africa. All isolates were from environments (middens and tree holes) that are associated with a small mammal, the African hyrax. Phylogenetic and population genetic analyses confirmed that these isolates form a distinct, deeply divergent lineage, which we name VGV. VGV comprises two subclades (A and B) that are capable of causing mild lung infection with negligible neurotropism in mice. Comparing the VGV genome to previously identified lineages of C. gattii revealed a unique suite of genes together with gene loss and inversion events. However, standard URA5 restriction fragment length polymorphism (RFLP) analysis could not distinguish between VGV and VGIV isolates. We therefore developed a new URA5 RFLP method that can reliably identify the newly described lineage. Our work highlights how sampling understudied ecological regions alongside genomic and functional characterization can broaden our understanding of the evolution and ecology of major global pathogens.IMPORTANCECryptococcus gattii is an environmental pathogen that causes severe systemic infection in immunocompetent individuals more often than in immunocompromised humans. Over the past 2 decades, researchers have shown that C. gattii falls within four genetically distinct major lineages. By combining field work from an understudied ecological region (the Central Miombo Woodlands of Zambia, Africa), genome sequencing and assemblies, phylogenetic and population genetic analyses, and phenotypic characterization (morphology, histopathological, drug-sensitivity, survival experiments), we discovered a hitherto unknown lineage, which we name VGV (variety gattii five). The discovery of a new lineage from an understudied ecological region has far-reaching implications for the study and understanding of fungal pathogens and diseases they cause.

Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom.

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.

Nonrandom Distribution of Azole Resistance across the Global Population of Aspergillus fumigatus.

The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR34)/L98H and TR46/Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STRAf), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR34/L98H and TR46/Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of A. fumigatus isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus.IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection.

Rapid and Sensitive Detection of Azole-Resistant Aspergillus fumigatus by Tandem Repeat Loop-Mediated Isothermal Amplification.

Invasive fungal infections caused by multiazole-resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multiazole-resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single-nucleotide polymorphism, is associated with the occurrence of multiazole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double-stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.

Designing Probiotic Therapies With Broad-Spectrum Activity Against a Wildlife Pathogen.

Host-associated microbes form an important component of immunity that protect against infection by pathogens. Treating wild individuals with these protective microbes, known as probiotics, can reduce rates of infection and disease in both wild and captive settings. However, the utility of probiotics for tackling wildlife disease requires that they offer consistent protection across the broad genomic variation of the pathogen that hosts can encounter in natural settings. Here we develop multi-isolate probiotic consortia with the aim of effecting broad-spectrum inhibition of growth of the lethal amphibian pathogen Batrachochytrium dendrobatidis (Bd) when tested against nine Bd isolates from two distinct lineages. Though we achieved strong growth inhibition between 70 and 100% for seven Bd isolates, two isolates appeared consistently resistant to inhibition, irrespective of probiotic strategy employed. We found no evidence that genomic relatedness of the chytrid predicted similarity of inhibition scores, nor that increasing the genetic diversity of the bacterial consortia could offer stronger inhibition of pathogen growth, even for the two resistant isolates. Our findings have important consequences for the application of probiotics to mitigate wildlife diseases in the face of extensive pathogen genomic variation.

MARDy: Mycology Antifungal Resistance Database.

Summary: The increase of antifungal drug resistance is a major global human health concern and threatens agriculture and food security; in order to tackle these concerns, it is important to understand the mechanisms that cause antifungal resistance. The curated Mycology Antifungal Resistance Database (MARDy) is a web-service of antifungal drug resistance mechanisms, including amino acid substitutions, tandem repeat sequences and genome ploidy. MARDy is implemented on a Linux, Apache, MySQL and PHP web development platform and includes a local installation of BLASTn of the database of curated genes. Availability and implementation: MARDy can be accessed at http://www.mardy.net and is free to use. The complete database can be retrieved, ordered by organism, gene and drug. Missing or new mycological antifungal resistance data can be relayed to the development team through a contribute entry form. Updates and news will be publicized via a dedicated Twitter feed: @MARDYfungi.

The feasibility and safety of transvaginal bilateral salpingo-oophorectomy.

OBJECTIVE: To investigate the feasibility and safety of transvaginal bilateral salpingo-oophorectomy (BSO). METHODS: The present retrospective case series included consecutive women who underwent transvaginal BSO at a single general gynecology unit at Weston General Hospital, Weston-super-Mare, UK, between February 1, 2011, and July 31, 2014. Transvaginal BSO procedures were performed by an experienced surgeon. Feasibility and safety outcomes were reviewed from patient case notes. RESULTS: There were 127 patients included in the analysis. In all, 109 patients underwent transvaginal BSO at the time of vaginal hysterectomy, whereas 18 women underwent this procedure following a previous vaginal hysterectomy. Transvaginal BSO was successful in 126 (99.2%) patients; adverse events occurred among nine (7.1%) patients, including a single occurrence of ureteric injury that was detected and repaired intraoperatively. CONCLUSION: The present study demonstrated that transvaginal BSO was a feasible and safe procedure when conducted by an experienced surgeon.

Azole sensitivity in Leptosphaeria pathogens of oilseed rape: the role of lanosterol 14α-demethylase.

Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency change in crops. Here we used CYP51 protein modelling and heterologous expression to determine whether there are interspecific differences at the target-site level. Moreover, we provide an example of intrinsic sensitivity differences exhibited by both Leptosphaeria spp. in vitro and in planta. Comparison of homologous protein models identified highly conserved residues, particularly at the azole binding site, and heterologous expression of LmCYP51B and LbCYP51B, with fungicide sensitivity testing of the transformants, suggests that both proteins are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. Fungicide sensitivity testing on isolates shows that they sometimes have a minor difference in sensitivity in vitro and in planta. These results suggest that azole fungicides remain a useful component of integrated phoma stem canker control in the UK due to their effectiveness on both Leptosphaeria spp. Other factors, such as varietal resistance or climate, may be driving observed frequency changes between species.

A theoretical study of the relaxation of a phenyl group chemisorbed to an RDX freestanding thin film.

Energy relaxation from an excited phenyl group chemisorbed to the surface of a crystalline thin film of α-1,3,5-trinitro-1,3,5-triazacyclohexane (α-RDX) at 298 K and 1 atm is simulated using molecular dynamics. Two schemes are used to excite the phenyl group. In the first scheme, the excitation energy is added instantaneously as kinetic energy by rescaling momenta of the 11 atoms in the phenyl group. In the second scheme, the phenyl group is equilibrated at a higher temperature in the presence of static RDX geometries representative of the 298 K thin film. An analytical model based on ballistic phonon transport that requires only the harmonic part of the total Hamiltonian and includes no adjustable parameters is shown to predict, essentially quantitatively, the short-time dynamics of the kinetic energy relaxation (∼200 fs). The dynamics of the phenyl group for times longer than about 6 ps follows exponential decay and agrees qualitatively with the dynamics described by a master equation. Long-time heat propagation within the bulk of the crystal film is consistent with the heat equation.