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.

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

Rationale: Acute cellular rejection (ACR) after lung transplantation 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. Objective: 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 timepoints. Measurements and Main Results: Among 103 lung transplant recipients, 25 (24.3%) developed ACR. In comparing samples acquired one month after transplant, subjects who never developed ACR demonstrated lower airway enrichment with several oral commensals (e.g., Prevotella and Veillonella spp.) compared to those with current or future (beyond one month) ACR. However, a subgroup analysis of those who developed ACR beyond one month revealed delayed enrichment with oral commensals occurring at the time of ACR diagnosis compared to baseline, when enrichment with more traditionally pathogenic taxa was present. In longitudinal models, dynamic changes in alpha 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. Conclusion: 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.

Pulmonary microbiome and transcriptome signatures reveal distinct pathobiologic states associated with mortality in two cohorts of pediatric stem cell transplant patients.

Lung injury is a major determinant of survival after pediatric hematopoietic cell transplantation (HCT). A deeper understanding of the relationship between pulmonary microbes, immunity, and the lung epithelium is needed to improve outcomes. In this multicenter study, we collected 278 bronchoalveolar lavage (BAL) samples from 229 patients treated at 32 children's hospitals between 2014-2022. Using paired metatranscriptomes and human gene expression data, we identified 4 patient clusters with varying BAL composition. Among those requiring respiratory support prior to sampling, in-hospital mortality varied from 22-60% depending on the cluster (p=0.007). The most common patient subtype, Cluster 1, showed a moderate quantity and high diversity of commensal microbes with robust metabolic activity, low rates of infection, gene expression indicating alveolar macrophage predominance, and low mortality. The second most common cluster showed a very high burden of airway microbes, gene expression enriched for neutrophil signaling, frequent bacterial infections, and moderate mortality. Cluster 3 showed significant depletion of commensal microbes, a loss of biodiversity, gene expression indicative of fibroproliferative pathways, increased viral and fungal pathogens, and high mortality. Finally, Cluster 4 showed profound microbiome depletion with enrichment of Staphylococci and viruses, gene expression driven by lymphocyte activation and cellular injury, and the highest mortality. BAL clusters were modeled with a random forest classifier and reproduced in a geographically distinct validation cohort of 57 patients from The Netherlands, recapitulating similar cluster-based mortality differences (p=0.022). Degree of antibiotic exposure was strongly associated with depletion of BAL microbes and enrichment of fungi. Potential pathogens were parsed from all detected microbes by analyzing each BAL microbe relative to the overall microbiome composition, which yielded increased sensitivity for numerous previously occult pathogens. These findings support personalized interpretation of the pulmonary microenvironment in pediatric HCT, which may facilitate biology-targeted interventions to improve outcomes.

Inflammation in the tumor-adjacent lung as a predictor of clinical outcome in lung adenocarcinoma.

Approximately 30% of early-stage lung adenocarcinoma patients present with disease progression after successful surgical resection. Despite efforts of mapping the genetic landscape, there has been limited success in discovering predictive biomarkers of disease outcomes. Here we performed a systematic multi-omic assessment of 143 tumors and matched tumor-adjacent, histologically-normal lung tissue with long-term patient follow-up. Through histologic, mutational, and transcriptomic profiling of tumor and adjacent-normal tissue, we identified an inflammatory gene signature in tumor-adjacent tissue as the strongest clinical predictor of disease progression. Single-cell transcriptomic analysis demonstrated the progression-associated inflammatory signature was expressed in both immune and non-immune cells, and cell type-specific profiling in monocytes further improved outcome predictions. Additional analyses of tumor-adjacent transcriptomic data from The Cancer Genome Atlas validated the association of the inflammatory signature with worse outcomes across cancers. Collectively, our study suggests that molecular profiling of tumor-adjacent tissue can identify patients at high risk for disease progression.

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.

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.

Use of digital measurement of medication adherence and lung function to guide the management of uncontrolled asthma (INCA Sun): a multicentre, single-blinded, randomised clinical trial.

BACKGROUND: The clinical value of using digital tools to assess adherence and lung function in uncontrolled asthma is not known. We aimed to compare treatment decisions guided by digitally acquired data on adherence, inhaler technique, and peak flow with existing methods. METHODS: A 32-week prospective, multicentre, single-blinded, parallel, randomly controlled trial was done in ten severe asthma clinics across Ireland, Northern Ireland, and England. Participants were 18 years or older, had uncontrolled asthma, asthma control test (ACT) score of 19 or less, despite treatment with high-dose inhaled corticosteroids, and had at least one severe exacerbation in the past year despite high-dose inhaled corticosteroids. Patients were randomly assigned in a 1:1 ratio to the active group or the control group, by means of a computer-generated randomisation sequence of permuted blocks of varying sizes (2, 4, and 6) stratified by fractional exhaled nitric oxide (FeNO) concentration and recruitment site. In the control group, participants were masked to their adherence and errors in inhaler technique data. A statistician masked to study allocation did the statistical analysis. After a 1-week run-in period, both groups attended three nurse-led education visits over 8 weeks (day 7, week 4, and week 8) and three physician-led treatment adjustment visits at weeks 8, 20, and 32. In the active group, treatment adjustments during the physician visits were informed by digital data on inhaler adherence, twice daily digital peak expiratory flow (ePEF), patient-reported asthma control, and exacerbation history. Treatment was adjusted in the control group on the basis of pharmacy refill rates (a measure of adherence), asthma control by ACT questionnaire, and history of exacerbations and visual management of inhaler technique. Both groups used a digitally enabled Inhaler Compliance Assessment (INCA) and PEF. The primary outcomes were asthma medication burden measured as proportion of patients who required a net increase in treatment at the end of 32 weeks and adherence rate measured in the last 12 weeks by area under the curve in the intention-to-treat population. The safety analyses included all patients who consented for the trial. The trial is registered with ClinicalTrials.gov, NCT02307669 and is complete. FINDINGS: Between Oct 25, 2015, and Jan 26, 2020, of 425 patients assessed for eligibility, 220 consented to participate in the study, 213 were randomly assigned (n=108 in the active group; n=105 in the control group) and 200 completed the study (n=102 in the active group; n=98 in the control group). In the intention-to-treat analysis at week 32, 14 (14%) active and 31 (32%) control patients had a net increase in treatment compared with baseline (odds ratio [OR] 0·31 [95% CI 0·15-0·64], p=0·0015) and 11 (11%) active and 21 (21%) controls required add-on biological therapy (0·42 [0·19-0·95], p=0·038) adjusted for study site, age, sex, and baseline FeNO. Three (16%) of 19 active and 11 (44%) of 25 control patients increased their medication from fluticasone propionate 500 µg daily to 1000 µg daily (500 µg twice a day; adjusted OR 0·23 [0·06-0·87], p=0·026). 26 (31%) of 83 active and 13 (18%) of 73 controls reduced their medication from fluticasone propionate 1000 µg once daily to 500 µg once daily (adjusted OR 2·43 [1·13-5·20], p=0·022. Week 20-32 actual mean adherence was 64·9% (SD 23·5) in the active group and 55·5% (26·8) in the control group (between-group difference 11·1% [95% CI 4·4-17·9], p=0·0012). A total of 29 serious adverse events were recorded (16 [55%] in the active group, and 13 [45%] in the control group), 11 of which were confirmed as respiratory. None of the adverse events reported were causally linked to the study intervention, to the use of salmeterol-fluticasone inhalers, or the use of the digital PEF or INCA. INTERPRETATION: Evidence-based care informed by digital data led to a modest improvement in medication adherence and a significantly lower treatment burden. FUNDING: Health Research Board of Ireland, Medical Research Council, INTEREG Europe, and an investigator-initiated project grant from GlaxoSmithKline.

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.

Pleural fluid microbiota as a biomarker for malignancy and prognosis.

Malignant pleural effusions (MPE) complicate malignancies and portend worse outcomes. MPE is comprised of various components, including immune cells, cancer cells, and cell-free DNA/RNA. There have been investigations into using these components to diagnose and prognosticate MPE. We hypothesize that the microbiome of MPE is unique and may be associated with diagnosis and prognosis. We compared the microbiota of MPE against microbiota of pleural effusions from non-malignant and paramalignant states. We collected a total of 165 pleural fluid samples from 165 subjects; Benign (n = 16), Paramalignant (n = 21), MPE-Lung (n = 57), MPE-Other (n = 22), and Mesothelioma (n = 49). We performed high throughput 16S rRNA gene sequencing on pleural fluid samples and controls. We showed that there are compositional differences among pleural effusions related to non-malignant, paramalignant, and malignant disease. Furthermore, we showed differential enrichment of bacterial taxa within MPE depending on the site of primary malignancy. Pleural fluid of MPE-Lung and Mesothelioma were associated with enrichment with oral and gut bacteria that are commonly thought to be commensals, including Rickettsiella, Ruminococcus, Enterococcus, and Lactobacillales. Mortality in MPE-Lung is associated with enrichment in Methylobacterium, Blattabacterium, and Deinococcus. These observations lay the groundwork for future studies that explore host-microbiome interactions and their influence on carcinogenesis.

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.

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.

A clinicians' review of the respiratory microbiome.

The respiratory microbiome and its impact in health and disease is now well characterised. With the development of next-generation sequencing and the use of other techniques such as metabolomics, the functional impact of microorganisms in different host environments can be elucidated. It is now clear that the respiratory microbiome plays an important role in respiratory disease. In some diseases, such as bronchiectasis, examination of the microbiome can even be used to identify patients at higher risk of poor outcomes. Furthermore, the microbiome can aid in phenotyping. Finally, development of multi-omic analysis has revealed interactions between the host and microbiome in some conditions. This review, although not exhaustive, aims to outline how the microbiome is investigated, the healthy respiratory microbiome and its role in respiratory disease. Educational aims: To define the respiratory microbiome and describe its analysis.To outline the respiratory microbiome in health and disease.To describe future directions for microbiome research.

Exhaled Breath Condensate (EBC) analysis of circulating tumour DNA (ctDNA) using a lung cancer specific UltraSEEK oncogene panel.

INTRODUCTION: Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. METHODS: In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). RESULTS: There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p < 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10-3, p = 3.14 × 10-5, p = 1.95 × 10-3) and EBC (p = 2.18 × 10-3, p = 2.28 × 10-4,p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. CONCLUSIONS: The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.

Modified Brixia chest X-ray severity scoring system and correlation with intubation, non-invasive ventilation and death in a hospitalised COVID-19 cohort.

INTRODUCTION: There are few existing severity scoring systems in the literature, and no formally widely accepted chest X-ray template for reporting COVID-19 infection. We aimed to modify the chest X-ray COVID-19 severity scoring system from the Brixia scoring system with placement of more emphasis on consolidation and to assess if the scoring tool could help predict intubation. METHODS: A severity chest X-ray scoring system was modified from the Brixia scoring system. PCR positive COVID-19 positive patient's chest X-rays admitted to our hospital over 3 months were reviewed and correlated with; non-invasive ventilation, intubation and death. An analysis was performed using a receiver operating curve to predict intubation from all admission chest X-rays. RESULTS: The median score of all 325 admission chest X-rays was 3 (Interquartile range (IQR) 0-6.5). The median score of admission chest X-rays of those who did not require ICU admission and survived was 1.5 (IQR 0-5); and 9 (IQR 4.75-12) was median admission score of those requiring intubation. The median scores of the pre-intubation ICU chest X-rays was 11.5 (IQR 9-14.125), this increased from a median admission chest X-ray score for this group of 9 (P-value < 0.01). A cut-off score of 6 had a sensitivity of 77% and specificity of 73% in predicting the need for intubation. CONCLUSION: Higher chest X-ray severity scores are associated with intubation, need for non-invasive ventilation and death. This tool may also be helpful in predicting intubation.

Microbial signatures in the lower airways of mechanically ventilated COVID-19 patients associated with poor clinical outcome.

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.

Anaerobe-enriched gut microbiota predicts pro-inflammatory responses in pulmonary tuberculosis.

BACKGROUND: The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood. METHODS: To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB. FINDINGS: Cases and SCs each had similar α- and ß-diversities in oral washes and sputum, however, ß-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways. INTERPRETATION: TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB. FUNDING: European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases.

Microbial signatures in the lower airways of mechanically ventilated COVID19 patients associated with poor clinical outcome.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

Microbial signatures in the lower airways of mechanically ventilated COVID19 patients associated with poor clinical outcome.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

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.