Longitudinal Lower Airway Microbial Signatures of Acute Cellular Rejection in Lung Transplantation.

Rationale: Acute cellular rejection (ACR) after lung transplant is a leading risk factor for chronic lung allograft dysfunction. Prior studies have demonstrated dynamic microbial changes occurring within the allograft and gut that influence local adaptive and innate immune responses. However, the lung microbiome's overall impact on ACR risk remains poorly understood. Objectives: To evaluate whether temporal changes in microbial signatures were associated with the development of ACR. Methods: We performed cross-sectional and longitudinal analyses (joint modeling of longitudinal and time-to-event data and trajectory comparisons) of 16S rRNA gene sequencing results derived from lung transplant recipient lower airway samples collected at multiple time points. Measurements and Main Results: Among 103 lung transplant recipients, 25 (24.3%) developed ACR. In comparing samples acquired 1 month after transplant, subjects who never developed ACR demonstrated lower airway enrichment with several oral commensals (e.g., Prevotella and Veillonella spp.) than those with current or future (beyond 1 mo) ACR. However, a subgroup analysis of those who developed ACR beyond 1 month revealed delayed enrichment with oral commensals occurring at the time of ACR diagnosis compared with baseline, when enrichment with more traditionally pathogenic taxa was present. In longitudinal models, dynamic changes in α-diversity (characterized by an initial decrease and a subsequent increase) and in the taxonomic trajectories of numerous oral commensals were more commonly observed in subjects with ACR. Conclusions: Dynamic changes in the lower airway microbiota are associated with the development of ACR, supporting its potential role as a useful biomarker or in ACR pathogenesis.

Lower Airway Dysbiosis Augments Lung Inflammatory Injury in Mild-to-Moderate Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

More than Mycobacterium tuberculosis: site-of-disease microbial communities, and their functional and clinical profiles in tuberculous lymphadenitis.

BACKGROUND: Lymphadenitis is the most common extrapulmonary tuberculosis (EPTB) manifestation. The microbiome is important to human health but uninvestigated in EPTB. We profiled the site-of-disease lymph node microbiome in tuberculosis lymphadenitis (TBL). METHODS: Fine-needle aspiration biopsies were collected from 158 pretreatment presumptive TBL patients in Cape Town, South Africa. 16S Illumina MiSeq rRNA gene sequencing was done. RESULTS: We analysed 89 definite TBLs (dTBLs) and 61 non-TBLs (nTBLs), which had similar α- but different ß-diversities (p=0.001). Clustering identified five lymphotypes prior to TB status stratification: Mycobacterium-dominant, Prevotella-dominant and Streptococcus-dominant lymphotypes were more frequent in dTBLs whereas a Corynebacterium-dominant lymphotype and a fifth lymphotype (no dominant taxon) were more frequent in nTBLs. When restricted to dTBLs, clustering identified a Mycobacterium-dominant lymphotype with low α-diversity and non-Mycobacterium-dominated lymphotypes (termed Prevotella-Corynebacterium, Prevotella-Streptococcus). The Mycobacterium dTBL lymphotype was associated with HIV-positivity and features characteristic of severe lymphadenitis (eg, larger nodes). dTBL microbial communities were enriched with potentially proinflammatory microbial short-chain fatty acid metabolic pathways (propanoate, butanoate) vs nTBLs. 11% (7/61) of nTBLs had Mycobacterium reads BLAST-confirmed as Mycobacterium tuberculosis complex. CONCLUSIONS: TBL at the site-of-disease is not microbially homogeneous. Distinct microbial community clusters exist that, in our setting, are associated with different clinical characteristics, and immunomodulatory potentials. Non-Mycobacterium-dominated dTBL lymphotypes, which contain taxa potentially targeted by TB treatment, were associated with milder, potentially earlier stage disease. These investigations lay foundations for studying the microbiome's role in lymphatic TB. The long-term clinical significance of these lymphotypes requires prospective validation.

Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary T-Helper Cell Type 17 Response that Mitigates Susceptibility to Streptococcus pneumoniae.

Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen.Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.

Use of exhaled breath condensate (EBC) in the diagnosis of SARS-COV-2 (COVID-19).

False negatives from nasopharyngeal swabs (NPS) using reverse transcriptase PCR (RT-PCR) in SARS-CoV-2 are high. Exhaled breath condensate (EBC) contains lower respiratory droplets that may improve detection. We performed EBC RT-PCR for SARS-CoV-2 genes (E, S, N, ORF1ab) on NPS-positive (n=16) and NPS-negative/clinically positive COVID-19 patients (n=15) using two commercial assays. EBC detected SARS-CoV-2 in 93.5% (29/31) using the four genes. Pre-SARS-CoV-2 era controls (n=14) were negative. EBC was positive in NPS negative/clinically positive patients in 66.6% (10/15) using the identical E and S (E/S) gene assay used for NPS, 73.3% (11/15) using the N/ORF1ab assay and 14/15 (93.3%) combined.

Functional lower airways genomic profiling of the microbiome to capture active microbial metabolism.

BACKGROUND: Microbiome studies of the lower airways based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short-chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary. METHODS: Here we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole-genome shotgun (WGS) and RNA metatranscriptome sequencing. SCFAs were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed. RESULTS: Functional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome data were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFA levels were compared with WGS and metatranscriptome data. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing. CONCLUSIONS: Functional characterisation of the lower airway microbiota through metatranscriptome data identifies metabolically active organisms capable of producing metabolites with immunomodulatory capacity, such as SCFAs.

Characterization of the Inflammatory Response to Severe COVID-19 Illness.

Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness.Methods: Levels of IL-1ß, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVIDstable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAPICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.Measurements and Main Results: IL-1ß, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVIDstable patients, and demonstrated higher levels of IL-1ß, IL-6, and sTNFR1 but lower IL-10 than CAPICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (P < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (P < 0.0001).Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.

Evidence for Environmental-Human Microbiota Transfer at a Manufacturing Facility with Novel Work-related Respiratory Disease.

Rationale: Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease.Objectives: As part of a public health investigation of a manufacturing facility, we performed a cross-sectional study using paired environmental and human sampling to evaluate the cross-pollination of microbes between the environment and the host and possible effects on lung pathology present among workers.Methods: Workplace environmental microbiota were evaluated in air and MWF samples. Human microbiota were evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, in lung tissue samples from control subjects, and in skin, nasal, and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B cells were assessed.Measurements and Main Results: An increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared with control subjects. Among workers in different locations within the facility, those that worked in the machine shop area had skin, nasal, and oral microbiota more closely related to the microbiota present in the MWF samples. Lung samples from four index cases and skin and nasal samples from workers in the machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in the pathology of index cases.Conclusions: Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.

Sputum neutrophil elastase associates with microbiota and Pseudomonas aeruginosa in bronchiectasis.

INTRODUCTION: Neutrophilic inflammation is a major driver of bronchiectasis pathophysiology, and neutrophil elastase activity is the most promising biomarker evaluated in sputum to date. How active neutrophil elastase correlates with the lung microbiome in bronchiectasis is still unexplored. We aimed to understand whether active neutrophil elastase is associated with low microbial diversity and distinct microbiome characteristics. METHODS: An observational, cross-sectional study was conducted at the bronchiectasis programme of the Policlinico Hospital in Milan, Italy, where adults with bronchiectasis were enrolled between March 2017 and March 2019. Active neutrophil elastase was measured on sputum collected during stable state, microbiota analysed through 16S rRNA gene sequencing, molecular assessment of respiratory pathogens carried out through real-time PCR and clinical data collected. RESULTS: Among 185 patients enrolled, decreasing α-diversity, evaluated through the Shannon entropy (ρ -0.37, p<0.00001) and Pielou's evenness (ρ -0.36, p<0.00001) and richness (ρ -0.33, p<0.00001), was significantly correlated with increasing elastase. A significant difference in median levels of Shannon entropy as detected between patients with neutrophil elastase ≥20 µg·mL-1 (median 3.82, interquartile range 2.20-4.96) versus neutrophil elastase <20 µg·mL-1 (4.88, 3.68-5.80; p<0.0001). A distinct microbiome was found in these two groups, mainly characterised by enrichment with Pseudomonas in the high-elastase group and with Streptococcus in the low-elastase group. Further confirmation of the association of Pseudomonas aeruginosa with elevated active neutrophil elastase was found based on standard culture and targeted real-time PCR. CONCLUSIONS: High levels of active neutrophil elastase are associated to low microbiome diversity and specifically to P. aeruginosa infection.

Severe Obstructive Sleep Apnea Is Associated with Alterations in the Nasal Microbiome and an Increase in Inflammation.

RATIONALE: Obstructive sleep apnea (OSA) is associated with recurrent obstruction, subepithelial edema, and airway inflammation. The resultant inflammation may influence or be influenced by the nasal microbiome. OBJECTIVES: To evaluate whether the composition of the nasal microbiota is associated with obstructive sleep apnea and inflammatory biomarkers. METHODS: Two large cohorts were used: 1) a discovery cohort of 472 subjects from the WTCSNORE (Seated, Supine and Post-Decongestion Nasal Resistance in World Trade Center Rescue and Recovery Workers) cohort, and 2) a validation cohort of 93 subjects rom the Zaragoza Sleep cohort. Sleep apnea was diagnosed using home sleep tests. Nasal lavages were obtained from cohort subjects to measure: 1) microbiome composition (based on 16S rRNA gene sequencing), and 2) biomarkers for inflammation (inflammatory cells, IL-8, and IL-6). Longitudinal 3-month samples were obtained in the validation cohort, including after continuous positive airway pressure treatment when indicated. MEASUREMENTS AND MAIN RESULTS: In both cohorts, we identified that: 1) severity of OSA correlated with differences in microbiome diversity and composition; 2) the nasal microbiome of subjects with severe OSA were enriched with Streptococcus, Prevotella, and Veillonella; and 3) the nasal microbiome differences were associated with inflammatory biomarkers. Network analysis identified clusters of cooccurring microbes that defined communities. Several common oral commensals (e.g., Streptococcus, Rothia, Veillonella, and Fusobacterium) correlated with apnea-hypopnea index. Three months of treatment with continuous positive airway pressure did not change the composition of the nasal microbiota. CONCLUSIONS: We demonstrate that the presence of an altered microbiome in severe OSA is associated with inflammatory markers. Further experimental approaches to explore causal links are needed.

Airway Microbiota Is Associated with Upregulation of the PI3K Pathway in Lung Cancer.

RATIONALE: In lung cancer, upregulation of the PI3K (phosphoinositide 3-kinase) pathway is an early event that contributes to cell proliferation, survival, and tissue invasion. Upregulation of this pathway was recently described as associated with enrichment of the lower airways with bacteria identified as oral commensals. OBJECTIVES: We hypothesize that host-microbe interactions in the lower airways of subjects with lung cancer affect known cancer pathways. METHODS: Airway brushings were collected prospectively from subjects with lung nodules at time of diagnostic bronchoscopy, including 39 subjects with final lung cancer diagnoses and 36 subjects with noncancer diagnoses. In addition, samples from 10 healthy control subjects were included. 16S ribosomal RNA gene amplicon sequencing and paired transcriptome sequencing were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed. MEASUREMENTS AND MAIN RESULTS: The composition of the lower airway transcriptome in the patients with cancer was significantly different from the control subjects, which included up-regulation of ERK (extracellular signal-regulated kinase) and PI3K signaling pathways. The lower airways of patients with lung cancer were enriched for oral taxa (Streptococcus and Veillonella), which was associated with up-regulation of the ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus led to upregulation of these same signaling pathways. CONCLUSIONS: The data presented here show that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.

A randomised clinical trial of feedback on inhaler adherence and technique in patients with severe uncontrolled asthma.

In severe asthma, poor control could reflect issues of medication adherence or inhaler technique, or that the condition is refractory. This study aimed to determine if an intervention with (bio)feedback on the features of inhaler use would identify refractory asthma and enhance inhaler technique and adherence.Patients with severe uncontrolled asthma were subjected to a stratified-by-site random block design. The intensive education group received repeated training in inhaler use, adherence and disease management. The intervention group received the same intervention, enhanced by (bio)feedback-guided training. The primary outcome was rate of actual inhaler adherence. Secondary outcomes included a pre-defined assessment of clinical outcome. Outcome assessors were blinded to group allocation. Data were analysed on an intention-to-treat and per-protocol basis.The mean rate of adherence during the third month in the (bio)feedback group (n=111) was higher than that in the enhanced education group (intention-to-treat, n=107; 73% versus 63%; 95% CI 2.8%-17.6%; p=0.02). By the end of the study, asthma was either stable or improved in 54 patients (38%); uncontrolled, but poorly adherent in 52 (35%); and uncontrolled, but adherent in 40 (27%).Repeated feedback significantly improved inhaler adherence. After a programme of adherence and inhaler technique assessment, only 40 patients (27%) were refractory and adherent, and might therefore need add-on therapy.

Evaluation of the airway microbiome in nontuberculous mycobacteria disease.

Aspiration is associated with nontuberculous mycobacterial (NTM) pulmonary disease and airway dysbiosis is associated with increased inflammation. We examined whether NTM disease was associated with a distinct airway microbiota and immune profile.297 oral wash and induced sputum samples were collected from 106 participants with respiratory symptoms and imaging abnormalities compatible with NTM. Lower airway samples were obtained in 20 participants undergoing bronchoscopy. 16S rRNA gene and nested mycobacteriome sequencing approaches characterised microbiota composition. In addition, inflammatory profiles of lower airway samples were examined.The prevalence of NTM+ cultures was 58%. Few changes were noted in microbiota characteristics or composition in oral wash and sputum samples among groups. Among NTM+ samples, 27% of the lower airway samples were enriched with Mycobacterium A mycobacteriome approach identified Mycobacterium in a greater percentage of samples, including some nonpathogenic strains. In NTM+ lower airway samples, taxa identified as oral commensals were associated with increased inflammatory biomarkers.The 16S rRNA gene sequencing approach is not sensitive in identifying NTM among airway samples that are culture-positive. However, associations between lower airway inflammation and microbiota signatures suggest a potential role for these microbes in the inflammatory process in NTM disease.

Objective Assessment of Adherence to Inhalers by Patients with Chronic Obstructive Pulmonary Disease.

RATIONALE: Objective adherence to inhaled therapy by patients with chronic obstructive pulmonary disease (COPD) has not been reported. OBJECTIVES: To objectively quantify adherence to preventer Diskus inhaler therapy by patients with COPD with an electronic audio recording device (INCA). METHODS: This was a prospective observational study. On discharge from hospital patients were given a salmeterol/fluticasone inhaler with an INCA device attached. Analysis of this audio quantified the frequency and proficiency of inhaler use. MEASUREMENTS AND MAIN RESULTS: Patients with COPD (n = 244) were recruited. The mean age was 71 years, mean FEV1 was 1.3 L, and 59% had evidence of mild/moderate cognitive impairment. By combining time of use, interval between doses, and critical technique errors, thus incorporating both intentional and unintentional nonadherence, a measure "actual adherence" was calculated. Mean actual adherence was 22.6% of that expected if the doses were taken correctly and on time. Six percent had an actual adherence greater than 80%. Hierarchical clustering found three equally sized well-separated clusters corresponding to distinct patterns. Cluster 1 (34%) had low inhaler use and high error rates. Cluster 2 (25%) had high inhaler use and high error rates. Cluster 3 (36%) had overall good adherence. Poor lung function and comorbidities were predictive of poor technique, whereas age and cognition with poor lung function distinguished those with poor adherence and frequent errors in technique. CONCLUSIONS: These data may inform clinicians in understanding why a prescribed inhaler is not effective and to devise strategies to promote adherence in COPD.

The acoustic features of inhalation can be used to quantify aerosol delivery from a Diskus™ dry powder inhaler.

PURPOSE: Some patients are unable to generate the peak inspiratory flow rate (PIFR) necessary to de-agglomerate drug particles from dry powder inhalers (DPIs). In this study we tested the hypothesis that the acoustic parameters of an inhalation are related to the PIFR and hence reflect drug delivery. METHODS: A sensitivity analysis of the relationship of the acoustics of inhalation to simultaneously recorded airflow, in a cohort of volunteers (n = 92) was performed. The Next Generation Impactor (NGI) was used to assess in vitro drug delivery from salmeterol/fluticasone and salbutamol Diskus™ DPIs. Fine particle fraction, FPF, (<5 µm) was measured at 30-90 l/min for 2-6 s and correlated with acoustically determined flow rate (IFRc). In pharmacokinetic studies using a salbutamol (200 µg) Diskus™, volunteers inhaled either at maximal or minimal effort on separate days. RESULTS: PIFRc was correlated with spirometrically determined values (R (2) = 0.88). In in vitro studies, FPF increased as both flow rate and inhalation duration increased for the salmeterol/fluticasone Diskus™ (Adjusted R (2) = 0.95) and was proportional to flow rate only for the salbutamol Diskus™ (Adjusted R (2) = 0.71). In pharmacokinetic studies, blood salbutamol levels measured at 20 min were significantly lower when PIFRc was less than 60 l/min, p < 0.0001. CONCLUSION: Acoustically-determined PIFR is a suitable method for estimating drug delivery and for monitoring inhalation technique over time.

Pathobiological signatures of dysbiotic lung injury in pediatric patients undergoing stem cell transplantation.

Hematopoietic cell transplantation (HCT) uses cytotoxic chemotherapy and/or radiation followed by intravenous infusion of stem cells to cure malignancies, bone marrow failure and inborn errors of immunity, hemoglobin and metabolism. Lung injury is a known complication of the process, due in part to disruption in the pulmonary microenvironment by insults such as infection, alloreactive inflammation and cellular toxicity. How microorganisms, immunity and the respiratory epithelium interact to contribute to lung injury is uncertain, limiting the development of prevention and treatment strategies. Here we used 278 bronchoalveolar lavage (BAL) fluid samples to study the lung microenvironment in 229 pediatric patients who have undergone HCT treated at 32 children's hospitals between 2014 and 2022. By leveraging paired microbiome and human gene expression data, we identified high-risk BAL compositions associated with in-hospital mortality (P = 0.007). Disadvantageous profiles included bacterial overgrowth with neutrophilic inflammation, microbiome contraction with epithelial fibroproliferation and profound commensal depletion with viral and staphylococcal enrichment, lymphocytic activation and cellular injury, and were replicated in an independent cohort from the Netherlands (P = 0.022). In addition, a broad array of previously occult pathogens was identified, as well as a strong link between antibiotic exposure, commensal bacterial depletion and enrichment of viruses and fungi. Together these lung-immune system-microorganism interactions clarify the important drivers of fatal lung injury in pediatric patients who have undergone HCT. Further investigation is needed to determine how personalized interpretation of heterogeneous pulmonary microenvironments may be used to improve pediatric HCT outcomes.

The role of the respiratory microbiome in asthma.

Asthma is a common airways disease and the human microbiome plays an increasingly recognised role in asthma pathogenesis. Furthermore, the respiratory microbiome varies with asthma phenotype, endotype and disease severity. Consequently, asthma therapies have a direct effect on the respiratory microbiome. Newer biological therapies have led to a significant paradigm shift in how we treat refractory Type 2 high asthma. While airway inflammation is the generally accepted mechanism of action of all asthma therapies, including both inhaled and systemic therapies, there is evidence to suggest that they may also alter the microbiome to create a more functionally balanced airway microenvironment while also influencing airway inflammation directly. This downregulated inflammatory cascade seen biochemically, and reflected in improved clinical outcomes, supports the hypothesis that biological therapies may in fact affect the microbiome-host immune system dynamic and thus represent a therapeutic target for exacerbations and disease control.

A single-centre prospective cohort study: prone positioning in awake, non-intubated patients with covid-19 hypoxemic failure.

INTRODUCTION: A surge in critically ill patients with respiratory failure due to Covid-19 has overwhelmed ICU capacity in many healthcare systems across the world. Given a guarded prognosis and significant resource limitations, less invasive, inventive approaches such as prone positioning (PP) of non-intubated patients with hypoxemic respiratory failure were considered. AIMS AND OBJECTIVES: This is a prospective observational study and the aim is to evaluate the impact of awake PP at the ward level on the oxygenation levels of patients with COVID-19. We also are investigating as secondary outcomes, the risk factors for treatment failure among awake non-intubated patients who tolerated PP compared to those who did not. The primary outcome of this trial is the change in SpO2:FiO2 (SF) ratio from admission to discharge in the participants who tolerated PP compared to those that did not. Secondary outcomes included amongst others are ICU admission rate, in-hospital mortality, and length of stay. METHODS: A total of 63 patients admitted to Beaumont Hospital (BH), Dublin between January and February of 2021 with Covid-19 requiring supplemental oxygen were recruited. RESULTS: A total of 47 (74%) participants were reported as tolerating and 16 (26%) as non-tolerating PP. The mean rank in the primary endpoint in the tolerating group was 38 vs. 16 in the non-tolerating. This was statistically significant (P < 0.001). CONCLUSION: PP was associated with improvements in oxygenation parameters without any reported serious adverse events. A well-designed, randomised control trial, testing the efficacy of PP in non-intubated Covid-19 patients is needed, before the widespread adoption of this practice.

Tracheostomy insertion in COVID-19: insertion practice and factors leading to unplanned tube exchange.

Background: Tracheostomy insertion in patients with coronavirus disease 2019 (COVID-19) presents unique challenges. Patients frequently have high ventilatory requirements, and as an aerosol generating procedure, tracheostomy insertion creates the potential for staff transmission. Problems with tracheostomies contribute to morbidity and mortality, and tracheostomy changes may increase risks of staff transmission. We sought to quantify the incidence of clinically necessitated tracheostomy changes, establish the indications for change and investigate the incidence of staff transmission. Methods: We conducted a single institution, retrospective, observational cohort study of all intensive care unit (ICU) patients with COVID-19 who had a tracheostomy between March 2020 and April 2021. The institution is a large tertiary referral centre in Ireland. Results: Forty-three patients had a tracheostomy during the study period. All were a Shiley™ Flexible Adult Taperguard or Shiley™ XLT Tracheostomy. 14 patients (33%) required a tracheostomy change, with the majority (57%) involving a change from a standard size to an extended length tracheostomy. Persistent leak was the most common indication for change (71.6%). Other indications included patient-ventilator dyssynchrony, persistent cough and accidental decannulation. No staff transmission of COVID-19 occurred during this study. Conclusions: The incidence of tracheostomy change was 33%, highlighting the importance of selecting the right tracheostomy for each patient. We discuss how key characteristics of tracheostomies such as type, size, length and inner diameter may impact flow, resistance and work of breathing, leading to unplanned tracheostomy change. No staff transmission occurred arising from tracheostomy insertion, adding to increasing evidence that tracheostomy insertion in COVID-19 appears safe with adherence to guidelines describing the correct use of personal protective equipment.