Faculty Bibliography

RATIONALE/BACKGROUND:Lower airway enrichment with oral commensals has been previously associated with grade 3 severe primary graft dysfunction (PGD) after lung transplantation (LT). We aimed to determine whether this dysbiotic signature is present across all PGD severity grades, including milder forms, and whether it is associated with a distinct host inflammatory endotype. METHODS:Lower airway samples from 96 LT recipients with varying degrees of PGD were used to evaluate the lung allograft microbiota via 16S rRNA gene sequencing. Bronchoalveolar lavage (BAL) cytokine concentrations and cell differential percentages were compared across PGD grades. In a subset of samples, we evaluated the lower airway host transcriptome using RNA sequencing methods. RESULTS:Differential analyses demonstrated lower airway enrichment with supraglottic-predominant taxa (SPT) in both moderate and severe PGD. Dirichlet Multinomial Mixtures (DMM) modeling identified two distinct microbial clusters. A greater percentage of subjects with moderate-severe PGD were identified within the dysbiotic cluster (C-SPT) than within the no PGD group (48 and 29%, respectively) though this difference did not reach statistical significance (p=0.06). PGD severity associated with increased BAL neutrophil concentration (p=0.03) and correlated with BAL concentrations of MCP-1/CCL2, IP-10/CXCL10, IL-10, and TNF-α (p<0.05). Furthermore, microbial signatures of dysbiosis correlated with neutrophils, MCP-1/CCL-2, IL-10, and TNF-α (p<0.05). C-SPT exhibited differential expression of TNF, SERPINE1 (PAI-1), MPO, and MMP1 genes and upregulation of MAPK pathways, suggesting that dysbiosis regulates host signaling to promote neutrophilic inflammation. CONCLUSIONS:Lower airway dysbiosis within the lung allograft is associated with a neutrophilic inflammatory endotype, an immune profile commonly recognized as the hallmark for PGD pathogenesis. This data highlights a putative role for lower airway microbial dysbiosis in the pathogenesis of this syndrome.

BACKGROUND:The site-of-disease microbiome and predicted metagenome were evaluated in a cross-sectional study involving people with presumptive tuberculous pericarditis. We also explored the interaction between C-reactive protein (CRP) and the microbiome. METHODS:People with effusions requiring diagnostic pericardiocentesis (n=139) provided pericardial fluid for sequencing and blood for CRP measurement. RESULTS:Pericardial fluid microbiota differed in β-diversity among people with definite (dTB, n=91), probable (pTB, n=25), and non- (nTB, n=23) tuberculous pericarditis. dTBs were Mycobacterium-, Lacticigenium-, and Kocuria-enriched vs. nTBs. HIV-positive dTBs were Mycobacterium-, Bifidobacterium-, Methylobacterium-, and Leptothrix-enriched vs. HIV-negative dTBs. HIV-positive dTBs on ART were Mycobacterium- and Bifidobacterium-depleted vs. those not on ART. dTBs exhibited enrichment in short-chain fatty acid (SCFA) and mycobacterial metabolism pathways vs. nTBs. Additional non-pericardial involvement (pulmonary infiltrates) was associated with Mycobacterium-enrichment and Streptococcus-depletion. Mycobacterium reads were in 34 % (31/91) of dTBs, 8 % (2/25) of pTBs and 17 % (4/23) nTBs. People with CRP above (vs. below) the median value had different β-diversity (Pseudomonas-depleted). No correlation was found between enriched taxa in dTBs and CRP. CONCLUSIONS:Pericardial fluid microbial composition varies by tuberculosis status, HIV (and ART) status and dTBs are enriched in SCFA-associated taxa. The clinical significance, including mycobacterial reads in nTBs and pTBs, requires evaluation.

BACKGROUND:Risk of early-stage lung adenocarcinoma (LUAD) recurrence after surgical resection is significant, and post-recurrence median survival is approximately two years. Currently there are no commercially available biomarkers that predict recurrence. Here, we investigated whether microbial and host genomic signatures in the lung can predict recurrence. METHODS:In 91 early-stage (Stage IA/IB) LUAD-patients with extensive follow-up, we used 16s rRNA gene sequencing and host RNA-sequencing to map the microbial and host transcriptomic landscape in tumor and adjacent unaffected lung samples. RESULTS:23 out of 91 subjects had tumor recurrence over 5-year period. In tumor samples, LUAD recurrence was associated with enrichment with Dialister, Prevotella, while in unaffected lung, recurrence was associated with enrichment with Sphyngomonas and Alloiococcus. The strengths of the associations between microbial and host genomic signatures with LUAD recurrence were greater in adjacent unaffected lung samples than in the primary tumor. Among microbial-host features in the unaffected lung samples associated with recurrence, enrichment with Stenotrophomonas geniculata and Chryseobacterium were positively correlated with upregulation of IL-2, IL-3, IL-17, EGFR, HIF-1 signaling pathways among the host transcriptome. In tumor samples, enrichment with Veillonellaceae Dialister, Ruminococcacea, Haemophilus Influenza, and Neisseria were positively correlated with upregulation of IL-1, IL-6, IL17, IFN, and Tryptophan metabolism pathways. CONCLUSIONS:Overall, modeling suggested that a combined microbial/transcriptome approach using unaffected lung samples had the best biomarker performance (AUC=0.83). IMPACT/CONCLUSIONS:This study suggests that LUAD recurrence is associated with distinct pathophysiological mechanisms of microbial-host interactions in the unaffected lung rather than those present in the resected tumor.

OBJECTIVES/OBJECTIVE:To evaluate the clinical significance of low-field MRI lung opacity severity. METHODS:Retrospective cross-sectional analysis of post-acute Covid-19 patients imaged with low-field MRI from 9/2020 through 9/2022, and within 1 month of pulmonary function tests (PFTs), 6-min walk test (6mWT), and symptom inventory (SI), and/or within 3 months of St. George Respiratory Questionnaire (SGRQ) was performed. Univariate and correlative analyses were performed with Wilcoxon, Chi-square, and Spearman tests. The association between disease and demographic factors and MR opacity severity, PFTs, 6mWT, SI, and SGRQ, and association between MR opacity severity with functional and patient-reported outcomes (PROs), was evaluated with mixed model analysis of variance, covariance and generalized estimating equations. Two-sided 5 % significance level was used, with Bonferroni multiple comparison correction. RESULTS:81 MRI exams in 62 post-acute Covid-19 patients (median age 57, IQR 41-64; 25 women) were included. Exams were a median of 8 months from initial illness. Univariate analysis showed lung opacity severity was associated with decreased %DLCO (ρ = -0.55, P = .0125), and lung opacity severity quartile was associated with decreased %DLCO, predicted TLC, FVC, and increased FEV1/FVC. Multivariable analysis adjusting for sex, initial disease severity, and interval from Covid-19 diagnosis showed MR lung opacity severity was associated with decreased %DLCO (P < .001). Lung opacity severity was not associated with PROs. CONCLUSION/CONCLUSIONS:Low-field MRI lung opacity severity correlated with decreased %DLCO in post-acute Covid-19 patients, but was not associated with PROs.

DNA characterisation in people with tuberculosis (TB) is critical for diagnostic and microbiome evaluations. However, extracellular DNA, more frequent in people on chemotherapy, confounds results. We evaluated whether nucleic acid dyes [propidium monoazide (PMA), PEMAX] and DNaseI could reduce this. PCR [16S Mycobacterium tuberculosis complex (Mtb) qPCR, Xpert MTB/RIF] was done on dilution series of untreated and treated (PMA, PEMAX, DNaseI) Mtb. Separately, 16S rRNA gene qPCR and sequencing were done on untreated and treated sputa before (Cohort A: 11 TB-negatives, 9 TB-positives; Cohort B: 19 TB-positives, PEMAX only) and 24-weeks after chemotherapy (Cohort B). PMA and PEMAX reduced PCR-detected Mtb DNA for dilution series and Cohort A sputum versus untreated controls, suggesting non-intact Mtb is present before treatment-start. PEMAX enabled sequencing-based Mycobacterium-detection in 7/12 (58%) TB-positive sputa where no such reads otherwise occurred. In Cohort A, PMA- and PEMAX-treated versus untreated sputa had decreased α- and increased β-diversities. In Cohort B, β-diversity differences between timepoints were only detected with PEMAX. DNaseI had negligible effects. PMA and PEMAX (but not DNaseI) reduced extracellular DNA in PCR and improved pathogen detection by sequencing. PEMAX additionally detected chemotherapy-associated taxonomic changes that would otherwise be missed. Dyes enhance microbiome evaluations especially during chemotherapy.

BACKGROUND:Tuberculosis (TB), a major cause of disease and antimicrobial resistance, is spread via aerosols. Aerosols have diagnostic potential and airborne-microbes other than Mycobacterium tuberculosis complex (MTBC) may influence transmission. We evaluated whether PneumoniaCheck (PMC), a commercial aerosol collection device, captures MTBC and the aeromicrobiome of people with TB. METHODS:PMC was done in sputum culture-positive people (≥ 30 forced coughs each, n = 16) pre-treatment and PMC air reservoir (bag, corresponding to upper airways) and filter (lower airways) washes underwent Xpert MTB/RIF Ultra (Ultra) and 16S rRNA gene sequencing (sequencing also done on sputum). In a subset (n = 6), PMC microbiota (bag, filter) was compared to oral washes and bronchoalveolar lavage fluid (BALF). FINDINGS/RESULTS:54% (7/13) bags and 46% (6/14) filters were Ultra-positive. Sequencing read counts and microbial diversity did not differ across bags, filters, and sputum. However, microbial composition in bags (Sphingobium-, Corynebacterium-, Novosphingobium-enriched) and filters (Mycobacterium-, Sphingobium-, Corynebacterium-enriched) each differed vs. sputum. Furthermore, sequencing only detected Mycobacterium in bags and filters but not sputum. In the subset, bag and filter microbial diversity did not differ vs. oral washes or BALF but microbial composition differed. Bags vs. BALF were Sphingobium-enriched and Mycobacterium-, Streptococcus-, and Anaerosinus-depleted (Anaerosinus also depleted in filters vs. BALF). Compared to BALF, none of the aerosol-enriched taxa were enriched in oral washes or sputum. INTERPRETATION/CONCLUSIONS:PMC captures aerosols with Ultra-detectable MTBC and MTBC is more detectable in aerosols than sputum by sequencing. The aeromicrobiome is distinct from sputum, oral washes and BALF and contains differentially-enriched lower respiratory tract microbes.

IMPORTANCE/OBJECTIVE:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has evolved through multiple phases in the United States, with significant differences in patient centered outcomes with improvements in hospital strain, medical countermeasures, and overall understanding of the disease. We describe how patient characteristics changed and care progressed over the various pandemic phases; we also emphasize the need for an ongoing clinical network to improve the understanding of known and novel respiratory viral diseases. OBJECTIVES/OBJECTIVE:To describe how patient characteristics and care evolved across the various COVID-19 pandemic periods in those hospitalized with viral severe acute respiratory infection (SARI). DESIGN/METHODS:Severe Acute Respiratory Infection-Preparedness (SARI-PREP) is a Centers for Disease Control and Prevention Foundation-funded, Society of Critical Care Medicine Discovery-housed, longitudinal multicenter cohort study of viral pneumonia. We defined SARI patients as those hospitalized with laboratory-confirmed respiratory viral infection and an acute syndrome of fever, cough, and radiographic infiltrates or hypoxemia. We collected patient-level data including demographic characteristics, comorbidities, acute physiologic measures, serum and respiratory specimens, therapeutics, and outcomes. Outcomes were described across four pandemic variant periods based on a SARS-CoV-2 sequenced subsample: pre-Delta, Delta, Omicron BA.1, and Omicron post-BA.1. SETTING/METHODS:Multicenter cohort of adult patients admitted to an acute care ward or ICU from seven hospitals representing diverse geographic regions across the United States. PARTICIPANTS/METHODS:Patients with SARI caused by infection with respiratory viruses. MAIN OUTCOMES AND RESULTS/RESULTS:Eight hundred seventy-four adult patients with SARI were enrolled at seven study hospitals between March 2020 and April 2023. Most patients (780, 89%) had SARS-CoV-2 infection. Across the COVID-19 cohort, median age was 60 years (interquartile range, 48.0-71.0 yr) and 66% were male. Almost half (430, 49%) of the study population belonged to underserved communities. Most patients (76.5%) were admitted to the ICU, 52.5% received mechanical ventilation, and observed hospital mortality was 25.5%. As the pandemic progressed, we observed decreases in ICU utilization (94% to 58%), hospital length of stay (median, 26.0 to 8.5 d), and hospital mortality (32% to 12%), while the number of comorbid conditions increased. CONCLUSIONS AND RELEVANCE/CONCLUSIONS:We describe increasing comorbidities but improved outcomes across pandemic variant periods, in the setting of multiple factors, including evolving care delivery, countermeasures, and viral variants. An understanding of patient-level factors may inform treatment options for subsequent variants and future novel pathogens.

INTRODUCTION/UNASSIGNED:Mounting evidence indicates that an individual's humoral adaptive immune response plays a critical role in the setting of SARS-CoV-2 infection, and that the efficiency of the response correlates with disease severity. The relationship between the adaptive immune dynamics in the lower airways with those in the systemic circulation, and how these relate to an individual's clinical response to SARS-CoV-2 infection, are less understood and are the focus of this study. MATERIAL AND METHODS/UNASSIGNED:We investigated the adaptive immune response to SARS-CoV-2 in paired samples from the lower airways and blood from 27 critically ill patients during the first wave of the pandemic (median time from symptom onset to intubation 11 days). Measurements included clinical outcomes (mortality), bronchoalveolar lavage fluid (BALF) and blood specimen antibody levels, and BALF viral load. RESULTS/UNASSIGNED:While there was heterogeneity in the levels of the SARS-CoV-2-specific antibodies, we unexpectedly found that some BALF specimens displayed higher levels than the paired concurrent plasma samples, despite the known dilutional effects common in BALF samples. We found that survivors had higher levels of anti-spike, anti-spike-N-terminal domain and anti-spike-receptor-binding domain IgG antibodies in their BALF (p<0.05), while there was no such association with antibody levels in the systemic circulation. DISCUSSION/UNASSIGNED:Our data highlight the critical role of local adaptive immunity in the airways as a key defence mechanism against primary SARS-CoV-2 infection.

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.