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Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the respiratory microbiome may be playing a role in disease progression, yet current studies report contradictory results. Here, we examine potential confounders in COVID-19 respiratory microbiome studies by analyzing the upper ( n  = 58) and lower ( n  = 35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We find that time in the intensive care unit and type of oxygen support, as well as associated treatments such as antibiotic usage, explain the most variation within the upper respiratory tract microbiome, while SARS-CoV-2 viral load has a reduced impact. Specifically, mechanical ventilation is linked to altered community structure and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomics of the lower respiratory tract of COVID-19 patients identifies specific oral bacteria in physical association with proinflammatory immune cells, which show higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients. Here, the authors profile the respiratory microbiome of COVID-19 patients and link clinical practices, such as mechanical ventilation, with vast changes in the microbiota. In the lungs, oral bacteria are found physically associated with proinflammatory immune cells, thus possibly contributing to exacerbated immune responses in severe disease

How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8 + resident-memory (T RM ) and CD4 + T-helper-17 (T H17 ) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4 + T-cells with T-helper-1 characteristics (T H1 -like) and CD8 + T-cells expressing exhaustion markers (T EX -like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

PurposeInvasive pulmonary aspergillosis is increasingly reported in patients with influenza admitted to the intensive care unit (ICU). Classification of patients with influenza-associated pulmonary aspergillosis (IAPA) using the current definitions for invasive fungal diseases has proven difficult, and our aim was to develop case definitions for IAPA that can facilitate clinical studies.MethodsA group of 29 international experts reviewed current insights into the epidemiology, diagnosis and management of IAPA and proposed a case definition of IAPA through a process of informal consensus.ResultsSince IAPA may develop in a wide range of hosts, an entry criterion was proposed and not host factors. The entry criterion was defined as a patient requiring ICU admission for respiratory distress with a positive influenza test temporally related to ICU admission. In addition, proven IAPA required histological evidence of invasive septate hyphae and mycological evidence for Aspergillus. Probable IAPA required the detection of galactomannan or positive Aspergillus culture in bronchoalveolar lavage (BAL) or serum with pulmonary infiltrates or a positive culture in upper respiratory samples with bronchoscopic evidence for tracheobronchitis or cavitating pulmonary infiltrates of recent onset. The IAPA case definitions may be useful to classify patients with COVID-19-associated pulmonary aspergillosis (CAPA), while awaiting further studies that provide more insight into the interaction between Aspergillus and the SARS-CoV-2-infected lung.ConclusionA consensus case definition of IAPA is proposed, which will facilitate research into the epidemiology, diagnosis and management of this emerging acute and severe Aspergillus disease, and may be of use to study CAPA.

Purpose Invasive pulmonary aspergillosis (IPA) is increasingly reported in patients with severe coronavirus disease 2019 (COVID-19) admitted to the intensive care unit (ICU). Diagnosis and management of COVID-19 associated pulmonary aspergillosis (CAPA) are challenging and our aim was to develop practical guidance. Methods A group of 28 international experts reviewed current insights in the epidemiology, diagnosis and management of CAPA and developed recommendations using GRADE methodology. Results The prevalence of CAPA varied between 0 and 33%, which may be partly due to variable case definitions, but likely represents true variation. Bronchoscopy and bronchoalveolar lavage (BAL) remain the cornerstone of CAPA diagnosis, allowing for diagnosis of invasive Aspergillus tracheobronchitis and collection of the best validated specimen for Aspergillus diagnostics. Most patients diagnosed with CAPA lack traditional host factors, but pre-existing structural lung disease and immunomodulating therapy may predispose to CAPA risk. Computed tomography seems to be of limited value to rule CAPA in or out, and serum biomarkers are negative in 85% of patients. As the mortality of CAPA is around 50%, antifungal therapy is recommended for BAL positive patients, but the decision to treat depends on the patients’ clinical condition and the institutional incidence of CAPA. We recommend against routinely stopping concomitant corticosteroid or IL-6 blocking therapy in CAPA patients. Conclusion CAPA is a complex disease involving a continuum of respiratory colonization, tissue invasion and angioinvasive disease. Knowledge gaps including true epidemiology, optimal diagnostic work-up, management strategies and role of host-directed therapy require further study.

All data generated or analyzed during this study are included in this published article.

Background Invasive pulmonary aspergillosis (IPA) is an increasingly recognized complication in intensive care unit (ICU) patients, especially those with influenza, cirrhosis, chronic obstructive pulmonary disease, and other diseases. The diagnosis can be challenging, especially in the ICU, where clinical symptoms as well as imaging are mostly nonspecific. Recently, Aspergillus lateral flow tests were developed to decrease the time to diagnosis of IPA. Several studies have shown promising results in bronchoalveolar lavage fluid (BALf) from hematology patients. We therefore evaluated a new lateral flow test for IPA in ICU patients. Methods Using left-over BALf from adult ICU patients in two university hospitals, we studied the performance of the Aspergillus galactomannan lateral flow assay (LFA) by IMMY (Norman, OK, USA). Patients were classified according to the 2008 EORTC-MSG definitions, the AspICU criteria, and the modified AspICU criteria, which incorporate galactomannan results. These internationally recognized consensus definitions for the diagnosis of IPA incorporate patient characteristics, microbiology and radiology. The LFA was read out visually and with a digital reader by researchers blinded to the final clinical diagnosis and IPA classification. Results We included 178 patients, of which 55 were classified as cases (6 cases of proven and 26 cases of probable IPA according to the EORTC-MSG definitions, and an additional 23 cases according to the modified AspICU criteria). Depending on the definitions used, the sensitivity of the LFA was 0.88–0.94, the specificity was 0.81, and the area under the ROC curve 0.90–0.94, indicating good overall test performance. Conclusions In ICU patients, the LFA performed well on BALf and can be used as a rapid screening test while waiting for other microbiological results.

Coronavirus disease 19 (COVID-19)-associated pulmonary aspergillosis (CAPA) is a severe fungal infection complicating critically ill COVID-19 patients. Numerous retrospective and prospective studies have been performed to get a better grasp on this lethal co-infection. We performed a qualitative review and summarized data from 48 studies in which 7047 patients had been included, of whom 820 had CAPA. The pooled incidence of proven, probable or putative CAPA was 15.1% among 2953 ICU-admitted COVID-19 patients included in 18 prospective studies. Incidences showed great variability due to multiple factors such as discrepancies in the rate and depth of the fungal work-up. The pathophysiology and risk factors for CAPA are ill-defined, but therapy with corticosteroids and anti-interleukin-6 therapy potentially confer the biggest risk. Sampling for mycological work-up using bronchoscopy is the cornerstone for diagnosis, as imaging is often aspecific. CAPA is associated with an increased mortality, but we do not have conclusive data whether therapy contributes to an increased survival in these patients. We conclude our review with a comparison between influenza-associated pulmonary aspergillosis (IAPA) and CAPA.

Pneumonia is the leading infectious disease killer worldwide and commonly requires admission to critical care. Despite its prevalence, the underpinning biology of severe pneumonia remains incompletely understood. Here we perform multifaceted assessments of bronchoalveolar transcriptome, cytokines, microbiology, and clinical features to biologically characterise a cohort of patients with suspected severe pneumonia. Our data implicate three lung-restricted transcriptionally defined severe pneumonia endotypes (termed ‘Pneumotypes’ (Pn)). All three Pneumotypes have comparable clinical presentations and severity of respiratory failure but experience divergent outcomes. Pn1, the most common, is characterised by low alveolar cytokines, expanded tolerogenic macrophages and epithelial damage. Pn3 is characterised by immature neutrophil infiltration, IL-6-STAT3 activation and longer duration of mechanical ventilation. Pn2 displays the fastest resolution, exhibiting a balanced immune response and epithelial-endothelial repair signatures. We identify and validate mechanistically distinct phenotypes in the lungs of patients with suspected pneumonia and acute lung injury, implicating targets for personalised therapy. Analysis of cell samples from pneumonia patients may indicate specific phenotypes associated with disease. Here the authors use a multi-omics approach to analyse transcriptional pneumotypes in severe pneumonia showing different features and inflammatory profiles in these different groups including differences in neutrophil phenotypes and cytokine responses.