Background Fungal co-infection is a recognised complication of respiratory virus infections, increasing morbidity and mortality, but can be readily treated if diagnosed early. An increasing number of small studies describing aspergillosis in COVID-19 patients with severe respiratory distress are being reported, but comprehensive data is lacking. The aim of this study was to determine the incidence, risk factors and impact of invasive fungal disease in adult COVID-19 patients with severe respiratory distress. Methods An evaluation of a national, multi-centre, prospective cohort evaluation of an enhanced testing strategy to diagnose invasive fungal disease in COVID-19 intensive care patients. Results were used to generate a mechanism to define aspergillosis in future COVID-19 patients. Results One-hundred and thirty-five adults (median age: 57, M/F: 2·2/1) were screened. The incidence was 26.7% (14.1% aspergillosis, 12·6% yeast infections). The overall mortality rate was 38%; 53% and 31% in patients with and without fungal disease, respectively (P: 0.0387). The mortality rate was reduced by the use of antifungal therapy (Mortality: 38·5% in patients receiving therapy versus 90% in patients not receiving therapy (P: 0.008). The use of corticosteroids (P: 0.007) and history of chronic respiratory disease (P: 0.05) increased the likelihood of aspergillosis. Conclusions Fungal disease occurs frequently in critically ill, mechanically ventilated COVID-19 patients. The survival benefit observed in patients receiving antifungal therapy implies that the proposed diagnostic and defining criteria are appropriate. Screening using a strategic diagnostic approach and antifungal prophylaxis of patients with risk factors will likely enhance the management of COVID-19 patients.

No abstract

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.

Author Contributions LL was involved in data acquisition, analysis & drafting manuscript. TPH was involved in data acquisition, analysis and revising the manuscript. PLW was involved in data analysis, interpretation and revising the manuscript. ACM and RP were involved in data acquisition, analysis and revising the manuscript. MR, DWD and DFMcA were involved in conception and design of study and revising the manuscript. AJS and RMcM were involved in conception and design of study, drafting and revising the manuscript.

bThe commercially developed PathoNostics AsperGenius species assay is a multiplex real-time PCR capable of detecting aspergillosis and genetic markers associated with azole resistance. The assay is validated for testing bronchoalveolar lavage fluids, replacing the requirement for culture and benefiting patient management. Application of this assay to less invasive, easily obtainable samples (e.g., serum) might be advantageous. The aim of this study was to determine the analytical and clinical performance of the AsperGenius species and resistance assays for testing serum samples. For the analytical evaluations, serum samples were spiked with various concentrations of Aspergillus genomic DNA for extraction, following international recommendations. For the clinical study, 124 DNA extracts from 14 proven/probable invasive aspergillosis (IA) cases, 2 possible IA cases, and 33 controls were tested. The resistance assay was performed on Aspergillus fumigatus PCR-positive samples when a sufficient fungal burden was evident. The limits of detection of the species and resistance assays for A. fumigatus DNA were 10 and >75 genomes/ sample, respectively. Nonreproducible detection at lower burdens was achievable for all markers. With a positivity threshold of 39 cycles, the sensitivity and specificity of the species assay were 78.6% and 100%, respectively. For 7 IA cases, at least one genetic region potentially associated with azole resistance was successfully amplified, although no resistance markers were detected in this small cohort. The AsperGenius assay provides good clinical performance with the added ability to detect azole resistance directly from noninvasive samples. While the available burden will limit application, it remains a significant advancement in the diagnosis and management of aspergillosis. Several recent clinical studies have shown that PCR testing can aid in the diagnosis of invasive aspergillosis (IA), particularly when combined with antigen testing (1-3). Reductions in empirical therapy and an earlier diagnosis, leading to improved patient outcomes, have been shown (1-3). PCR testing has been hampered by the lack of commercially manufactured assays, resulting in limited acceptance and exclusion by disease-defining criteria (4). The development of commercial Aspergillus PCR systems will help standardize methodology, increase accessibility, and provide quality control assurance through commercial manufacture.Several commercial PCR assays with the capacity to detect Aspergillus have been developed (MycAssay Aspergillus, Renishaw Fungiplex, Roche SeptiFast, AdemTech MycoGenie, and Abbott Plex-ID) (5-9). Many assays focus on detection in respiratory specimens, and while clinical performance looks favorable, data are compromised by limited numbers and sample types (7-9).While potentially fatal, IA is a low-incidence disease, and different testing strategies have been applied. High-risk patients can be screened using sensitive biomarker assays, performed on easy-to-obtain specimens such as serum or plasma t...

Background/Objectives: Azole resistance in Aspergillus fumigatus associated with the TR34/L98H mutations in the cyp51A gene have been increasingly reported. Determining the environmental resistance rate has been deemed important when considering front-line therapy for invasive aspergillosis. The aim of the study was to determine prevalence of azole resistance in environmental A. fumigatus isolates across South Wales.Methods: Over 5 months in 2015, 513 A. fumigatus isolates were cultured from 671 soil and 44 air samples and were screened for azole resistance using VIPcheck™ agar plates containing itraconazole, voriconazole and posaconazole. Resistance was confirmed by the CLSI M38-A2 methodology. The mechanism of resistance was investigated using the PathoNostics AsperGenius® Assay.Results: Screening by VIPcheck™ plate identified azole-resistance in 30 isolates, most of which (28/30) harbored the TR34/L98H mutation, generating a prevalence of 6.0%. Twenty-five isolates had a MIC of ≥2 mg/L with itraconazole, 23 isolates had a MIC of ≥2 mg/L with voriconazole and seven isolates had a MIC ≥0.25 mg/L with posaconazole. All isolates deemed resistant by VIPcheck™ plates were resistant to at least one azole by reference methodology.Conclusions: There is significant environmental azole resistance (6%) in South Wales, in close proximity to patients susceptible to aspergillosis. Given this environmental reservoir, azole resistance should be routinely screened for in clinical practice and environmental monitoring continued.

Management of invasive aspergillosis has been improved by biomarker assays, but limited accessibility and batch testing limit the impact. Lateral flow assays (LFA) are a simple method for use outside specialist centers, provided performance is acceptable. The objective of this study was to determine the performance of the recently released IMMY sona Aspergillus LFA when testing serum samples. The study took the form of a retrospective, anonymous case/control study comprising 179 serum samples from 136 patients with invasive fungal disease, previously documented using recently revised internationally accepted definitions. The LFA was performed following the manufacturer’s instructions using a cube reader to generate a galactomannan index (GMI). Performance parameters were determined, and receiver operator characteristic (ROC) analysis was used to identify an optimal threshold. Concordance with the Bio-Rad Aspergillus Ag assay (GM-EIA) was performed. At the recommended positivity threshold (GMI ≥ 0.5), LFA sensitivity and specificity were 96.9% (31/32) and 98% (98/100), respectively. ROC analysis confirmed the optimal threshold and generated an area under the curve of 0.9919. Qualitative agreement between LFA and GM-EIA was 89.0%, generating a Kappa statistic of 0.698, representing good agreement, with most discordance arising due to false-negative GM-EIA samples that were positive by LFA. The median GMI generated by the LFA was significantly greater than that generated by the GM-EIA. The IMMY sona Aspergillus LFA, when used with a cube reader, provides a rapid alternative to the well-established GM-EIA, potentially detecting more GM epitopes and enhancing sensitivity.

With the proposal to include Aspergillus PCR in the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) definitions for fungal disease, commercially manufactured assays may be required to provide standardization and accessibility. The PathoNostics AsperGenius assay represents one such test that has the ability to detect a range of Aspergillus species as well as azole resistance in Aspergillus fumigatus. Its performance has been validated on bronchoalveolar lavage (BAL) fluid and serum specimens, but recent evidence suggests that testing of plasma may have enhanced sensitivity over that with serum. We decided to evaluate the analytical and clinical performances of the PathoNostics AsperGenius assay for testing of plasma. For the analytical evaluations, plasma was spiked with various concentrations of Aspergillus genomic DNA before extraction following international recommendations, using two automated platforms. For the clinical study, 211 samples from 10 proven/probable invasive aspergillosis (IA) and 2 possible IA cases and 27 controls were tested. The limits of detection for testing of DNA extracted using the bioMérieux EasyMag and Qiagen EZ1 extractors were 5 and 10 genomes/0.5-ml sample, respectively. In the clinical study, true positivity was significantly greater than false positivity (P Ͻ 0.0001). The sensitivity and specificity obtained using a single positive result as significant were 80% and 77.8%, respectively. If multiple samples were required to be positive, specificity was increased to 100%, albeit sensitivity was reduced to 50%. The AsperGenius assay provided good clinical performance, but the predicted improvement of testing with plasma was not seen, possibly as a result of target degradation attributed to sample storage. Prospective testing is required to determine the clinical utility of this assay, particularly for the diagnosis of azole-resistant disease.