Lower Airway Samples Research Articles

Tobacco use, self-reported professional dental cleaning habits, and lung adenocarcinoma diagnosis are associated with bronchial and lung microbiome alpha diversity

RationaleThe lung microbiome is an inflammatory stimulus whose role in the development of lung malignancies is incompletely understood. We hypothesized that the lung microbiome associates with multiple clinical factors, including the presence of a lung malignancy.ObjectivesTo assess associations between the upper and lower airway microbiome and multiple clinical factors including lung malignancy.MethodsWe conducted a prospective cohort study of upper and lower airway microbiome samples from 44 subjects undergoing lung lobectomy for suspected or confirmed lung cancer. Subjects provided oral (2), induced sputum, nasopharyngeal, bronchial, and lung tissue (3) samples. Pathologic diagnosis, age, tobacco use, dental care history, lung function, and inhaled corticosteroid use were associated with upper and lower airway microbiome findings.Measurements and Main ResultsOlder age was associated with greater Simpson diversity in the oral and nasopharyngeal sites (p = 0.022 and p = 0.019, respectively). Current tobacco use was associated with greater lung and bronchus Simpson diversity (p < 0.0001). Self-reported last profession dental cleaning more than 6 months prior (vs. 6 or fewer months prior) was associated with lower lung and bronchus Simpson diversity (p < 0.0001). Diagnosis of a lung adenocarcinoma (vs. other pathologic findings) was associated with lower bronchus and lung Simpson diversity (p = 0.024). Last professional dental cleaning, dichotomized as ≤ 6 months vs. >6 months prior, was associated with clustering among lung samples (p = 0.027, R2 = 0.016). Current tobacco use was associated with greater abundance of pulmonary pathogens Mycoplasmoides and Haemophilus in lower airway samples. Self-reported professional dental cleaning ≤ 6 months prior (vs. >6 months prior) was associated with greater bronchial Actinomyces and lung Streptococcus abundance. Lung adenocarcinoma (vs. no lung adenocarcinoma) was associated with lower Lawsonella abundance in lung samples. Inhaled corticosteroid use was associated with greater abundance of Haemophilus among oral samples and greater Staphylococcus among lung samples.ConclusionsCurrent tobacco use, recent dental cleaning, and a diagnosis of adenocarcinoma are associated with lung and bronchial microbiome α-diversity, composition (β-diversity), and the abundance of several respiratory pathogens. These findings suggest that modifiable habits (tobacco use and dental care) may influence the lower airway microbiome. Larger controlled studies to investigate these potential associations are warranted.

SARS-CoV-2 infection increases airway bleeding risk in patients after tracheostomies

BackgroundAirway bleeding events are a rare incident in SARS-CoV-2-infected patients after tracheostomies. We aimed to explore the correlation between airway bleeding and SARS-CoV-2 infection and evaluate the consistency of SARS-CoV-2 RNA test results in the upper and lower airway samples from patients after tracheostomies.MethodsForty-four patients after temporary or permanent tracheostomy were divided into a positive group (29 patients) and a negative group (15 patients) based on the SARS-CoV-2 RNA test results of their oropharyngeal swabs. The oropharyngeal and tracheal swabs of the positive group were re-collected for SARS-CoV-2 RNA detection. Demographic and clinical characteristics and airway bleeding events were recorded for all enrolled patients.ResultsAirway bleeding was reported in eleven patients of the positive group (11/29), with seven displaying bloody sputum or hemoptysis, and four featuring massive sputum crust formation in the trachea that resulted in dyspnea, and only one patient in the negative group (1/15), with a significant difference in the airway bleeding rate (37.9% vs. 6.7%, p < 0.05). The SARS-CoV-2 RNA test results showed a statistical difference in cycle threshold (Ct) values between oropharyngeal swabs and tracheal swabs (p < 0.05).ConclusionsAfter tracheostomies, patients are more susceptible to airway bleeding if they are infected with SARS-CoV-2. The findings signify that in addition to droplet transmission through tracheostoma, SARS-CoV-2 may infect the oropharynx by airborne and close contact transmission, and that given the higher viral load and longer infection time in the trachea, tracheal swabs are more reliable for SARS-CoV-2 detection in these patients.

Cytokine signature clusters as a tool to compare changes associated with tobacco product use in upper and lower airway samples.

Inhalation exposure to cigarette smoke and e-cigarette aerosol is known to alter the respiratory immune system, particularly cytokine signaling. In assessments of health impacts of tobacco product use, cytokines are often measured using a variety of sample types, from serum to airway mucosa. However, it is currently unclear whether and how well cytokine levels from different sample types and the airway locations they represent are correlated, making comparing studies that utilize differing sample types challenging. To address this challenge, we compared baseline cytokine signatures in upper and lower airways and systemic samples and evaluated how groups of coexpressed cytokines change with tobacco product use. Matched nasal lavage fluid (NLF), nasal epithelial lining fluid (NELF), sputum, and circulating serum samples were collected from 14 nonsmokers, 13 cigarette smokers, and 17 e-cigarette users and analyzed for levels of 22 cytokines. Individual cytokine signatures were first compared across each sample type, followed by identification of cytokine clusters within each sample type. Identified clusters were then evaluated for potential alterations following tobacco product use using eigenvector analyses. Individual cytokine signatures in the respiratory tract were significantly correlated (NLF, NELF, and sputum) compared with randomly permutated signatures, whereas serum was not significantly different from random permutations. Cytokine clusters that were similar across airway sample types were modified by tobacco product use, particularly e-cigarettes, indicating a degree of uniformity in terms of how cytokine host defense and immune cell recruitment responses cooperate in the upper and lower airways. Overall, cluster-based analyses were found to be especially useful in small cohort assessments, providing higher sensitivity than individual signatures to detect biologically meaningful differences between tobacco use groups. This novel cluster analysis approach revealed that eigencytokine patterns in noninvasive upper airway samples simulate cytokine patterns in lower airways.

Microbiology, empiric therapy and its impact on the outcomes of nonventilated hospital-acquired, ventilated hospital-acquired, and ventilator-associated bacterial pneumonia in the United States, 2014-2019.

To explore whether microbiology profiles and the impact of inappropriate empiric treatment differ in the setting of hospital-acquired bacterial pneumonia that requires subsequent mechanical ventilation (vHABP) versus one that does not (nvHABP) versus ventilator-associated bacterial pneumonia (VABP). Multicenter retrospective cohort study within Premier Research database, 2014-2019. We identified cases based on a previously published International Classification of Disease, Ninth Revision/Tenth Revision Clinical Modification (ICD-9/ICD-10-CM) algorithm, and we compared the 3 groups with respect to the bacterial pathogens isolated from their blood, sputum, or lower airway samples, and their respective rates of exposure to inappropriate empiric treatment. Using regression modeling we computed the effect of inappropriate empiric treatment on outcomes. Among 17,819 patients who met enrollment criteria, 26.5% had nvHABP, 25.6% vHAPB, and 47.9% VABP. S. aureus (majority methicillin-susceptible) was the most frequently isolated organism, followed P. aeruginosa, K. pneumoniae, and E. coli with variations across the conditions. Rates of carbapenem resistance were highest in VABP (9.1%) and to third-generation cephalosporins in vHABP (14.9%). Patients with nvHABP were most likely to receive inappropriate empiric treatment (8.5%). Although inappropriate empiric treatment was associated with an increase in adjusted postinfection-onset hospital length of stay (2.3 days) and cost ($12,142), its greatest magnitude was in the nvHABP group (4.9 days, $13,147). Substantial microbiologic differences exist among populations who suffer nvHABP, vHABP, and VABP, and inappropriate empiric treatment significantly worsens utilization outcomes. Given the moderate rates of carbapenem resistance and third-generation cephalosporin resistance, all patients require empiric coverage for a range of bacteria, including those targeting extended-spectrum β-lactamase and carbapenem resistance where appropriate.

Dynamic Upper and Lower Airway Microbiotas in Paediatric Bronchiectasis Exacerbations: A Pilot Study.

IntroductionNon-cystic fibrosis bronchiectasis is a respiratory health condition with many possible aetiologies, some of which are potentially reversible in childhood with early diagnosis and appropriate treatment. It is important to understand factors which contribute to progression or potential resolution of bronchiectasis. It is evident that respiratory exacerbations are a key feature of bronchiectasis disease progression. In this pilot study we document how the microbiota of the upper and lower airways presents during the course of an exacerbation and treatment.MethodsWe recruited children (aged 1-15) undergoing antibiotic treatment for bronchiectasis exacerbations at Starship Children’s Hospital and outpatient clinics. Sputum and nasal swabs were taken before and after antibiotic treatment. Sample DNA was extracted, then bacterial 16S rRNA genes amplified and sequenced via Illumina MiSeq.ResultsThirty patients were recruited into this study with 81 samples contributing to the final dataset, including 8 patients with complete sets of upper and lower airway samples at both (before and after antibiotics) timepoints. Changes in alpha-diversity over the course of an exacerbation and treatment were non-significant. However, sample composition did alter over the course of an exacerbation, with most notably a reduction in the relative abundance of amplicon sequence variants assigned to Haemophilus.Discussion Haemophilus has been associated with more severe symptoms in respiratory infections and a reduction in its relative abundance may represent a positive shift in a patient’s microbiota. Current treatments for bronchiectasis may preserve bacterial diversity while altering microbiota composition.

Bacterial Signatures of Paediatric Respiratory Disease: An Individual Participant Data Meta-Analysis.

Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, but studies are often small. It remains unclear whether particular bacteria are associated with a given disease, or if a more general, non-specific microbiota association with disease exists, as suggested for the gut. We investigated overarching patterns of bacterial association with acute and chronic paediatric respiratory disease in an individual participant data (IPD) meta-analysis of 16S rRNA gene sequences from published respiratory microbiota studies.Methods: We obtained raw microbiota data from public repositories or via communication with corresponding authors. Cross-sectional analyses of the paediatric (<18 years) microbiota in acute and chronic respiratory conditions, with >10 case subjects were included. Sequence data were processed using a uniform bioinformatics pipeline, removing a potentially substantial source of variation. Microbiota differences across diagnoses were assessed using alpha- and beta-diversity approaches, machine learning, and biomarker analyses.Results: We ultimately included 20 studies containing individual data from 2624 children. Disease was associated with lower bacterial diversity in nasal and lower airway samples and higher relative abundances of specific nasal taxa including Streptococcus and Haemophilus. Machine learning success in assigning samples to diagnostic groupings varied with anatomical site, with positive predictive value and sensitivity ranging from 43 to 100 and 8 to 99%, respectively.Conclusion: IPD meta-analysis of the respiratory microbiota across multiple diseases allowed identification of a non-specific disease association which cannot be recognised by studying a single disease. Whilst imperfect, machine learning offers promise as a potential additional tool to aid clinical diagnosis.

Meteorological Factors Influence the Presence of Fungi in the Air; A 14-Month Surveillance Study at an Adult Cystic Fibrosis Center.

BackgroundCystic fibrosis is an inherited disease that predisposes to progressive lung damage. Cystic fibrosis patients are particularly prone to developing pulmonary infections. Fungal species are commonly isolated in lower airway samples from patients with cystic fibrosis. Fungal spores are prevalent in the air.MethodsWe performed environmental air sampling surveillance at the Manchester Adult Cystic Fibrosis Centre, UK (MACFC) over a 14-month period to assess fungal growth inside and outside the CF center.ResultsAirborne counts of fungal spores peaked from May to October, both in outdoor and indoor samples. Collection of meteorological data allowed us to correlate fungal presence in the air with elevated temperatures and low wind speeds. Additionally, we demonstrated patient rooms containing windows had elevated fungal counts compared to rooms not directly connected to the outdoors.ConclusionsThis study suggests that airborne Aspergillus fumigatus spores were more abundant during the summer months of the survey period, which appeared to be driven by increased temperatures and lower wind speeds. Indoor counts directly correlated to outdoor A. fumigatus levels and were elevated in patient rooms that were directly connected to the outdoor environment via an openable window designed for ventilation purposes. Further studies are required to determine the clinical implications of these findings for cystic fibrosis patients who are predisposed to Aspergillus related diseases, and in particular whether there is seasonal influence on incidence of Aspergillus related conditions and if screening for such complications such be increased during summer months and precautions intensified for those with a known history of Aspergillus related disease.

Complex Involvement of Interleukin-26 in Bacterial Lung Infection.

Pneumonia is a global cause of mortality, and this provides a strong incentive to improve the mechanistic understanding of innate immune responses in the lungs. Here, we characterized the involvement of the cytokine interleukin (IL)-26 in bacterial lung infection. We observed markedly increased concentrations of IL-26 in lower airway samples from patients with bacterial pneumonia and these correlated with blood neutrophil concentrations. Moreover, pathogen-associated molecular patterns (PAMPs) from both Gram-negative and -positive bacteria increased extracellular IL-26 concentrations in conditioned media from human models of alveolar epithelial cells, macrophages, and neutrophils in vitro. Stimulation with IL-26 inhibited the inherent release of neutrophil elastase and myeloperoxidase in unexposed neutrophils. This stimulation also inhibited the expression of activity makers in neutrophils exposed to Klebsiella pneumoniae. In addition, priming of human lung tissue ex vivo with exogenous IL-26 potentiated the endotoxin-induced increase in mRNA for other cytokines involved in the innate immune response, including the master Th17-regulator IL-23 and the archetype inhibitory cytokine IL-10. Finally, neutralization of endogenous IL-26 clearly increased the growth of Klebsiella pneumoniae in the macrophage culture. These findings suggest that IL-26 is involved in bacterial lung infection in a complex manner, by modulating critical aspects of innate immune responses locally and systemically in a seemingly purposeful manner and by contributing to the killing of bacteria in a way that resembles an antimicrobial peptide. Thus, IL-26 displays both diagnostic and therapeutic potential in pneumonia and deserves to be further evaluated in these respects.

Mucin Binding to Moraxella catarrhalis during Airway Inflammation Is Dependent on Sialic Acid.

Chronic obstructive pulmonary disease (COPD) is associated with colonization by bacterial pathogens and repeated airway infections, leading to exacerbations and impaired lung function. The highly glycosylated mucins in the mucus lining the airways are an important part of the host defense against pathogens. However, mucus accumulation can contribute to COPD pathology. Here, we examined whether inflammation is associated with glycosylation changes that affect interactions between airway mucins and pathogens. We isolated mucins from lower airway samples (n = 4-9) from long-term smokers with and without COPD and from never-smokers. The most abundant terminal glycan moiety was N-acetylneuraminic acid (Neu5Ac) among smokers with and without COPD and N-acetyl-hexoseamine among never-smokers. Moraxella catarrhalis bound to MUC5 mucins from smokers with and without COPD. M. catarrhalis binding correlated with inflammatory parameters and Neu5Ac content. M. catarrhalis binding was abolished by enzymatic removal of Neu5Ac. Furthermore, M. catarrhalis bound to α2,6 sialyl-lactose, suggesting that α2,6 sialic acid contributes to M. catarrhalis binding to mucins. Furthermore, we detected more M. catarrhalis binding to mucins from patients with pneumonia than to those from control subjects (n = 8-13), and this binding correlated with C-reactive protein and Neu5Ac levels. These results suggest a key role of inflammation-induced Neu5Ac in the adhesion of M. catarrhalis to airway mucins. The inflammation-induced ability of MUC5 mucins to bind M. catarrhalis is likely a host defense mechanism in the healthy lung, although it cannot be excluded that impaired mucociliary clearance limits the effectiveness of this defense in patients with COPD.

Fungal Infection and Inflammation in Cystic Fibrosis.

Fungi are frequently recovered from lower airway samples from people with cystic fibrosis (CF), yet the role of fungi in the progression of lung disease is debated. Recent studies suggest worsening clinical outcomes associated with airway fungal detection, although most studies to date are retrospective or observational. The presence of fungi can elicit a T helper cell type 2 (Th-2) mediated inflammatory reaction known as allergic bronchopulmonary aspergillosis (ABPA), particularly in those with a genetic atopic predisposition. In this review, we discuss the epidemiology of fungal infections in people with CF, risk factors associated with development of fungal infections, and microbiologic approaches for isolation and identification of fungi. We review the spectrum of fungal disease presentations, clinical outcomes after isolation of fungi from airway samples, and the importance of considering airway co-infections. Finally, we discuss the association between fungi and airway inflammation highlighting gaps in knowledge and future research questions that may further elucidate the role of fungus in lung disease progression.

Putative invasive pulmonary aspergillosis within medical wards and intensive care units: a 4-year retrospective, observational, single-centre study

Blot and colleagues have proposed putative invasive pulmonary aspergillosis (PIPA) definitions for troublesome diagnosis in suspected patients outside the classical criteria of immunosuppression. We retrospectively included in the study all admitted patients with an Aspergillus spp. positive culture within lower airway samples. Overall, Aspergillus spp. positivity in respiratory samples was 0.97 every 1000 hospital admissions (HA): 4.94 and 0.28/1000/HA, respectively, in intensive care units (ICUs) and medical wards (MW). 66.6% fulfilled PIPA criteria, and 33.4% were defined as colonized. 69.2% of PIPA diagnosis occurred in the ICU. Antifungal therapy was appropriate in 88.5% of subjects with PIPA and 37.5% of colonized, confirming the comparison between deads and lives. Patients with PIPA in the ICUs had more frequent COPD, sepsis or septic shock, acute kidney injury (AKI), needed more surgery, mechanical ventilation (MV), vasopressors, hemodialysis, blood or platelets transfusions. PIPA in MW had associated with a history of smoking, interstitial lung disease and inhaled steroid therapy. Overall mortality within 21 days was 50%: 54.2% in ICU, 36,8% in MW. Factors associated with death were length of hospitalization, influenza, pneumonia, liver transplant, AKI, ARDS, sepsis and septic shock. PIPA in the ICU had higher disease severity and needed more organ support than MW cases, despite that cases of PIPA in MW are emerging with trends difficult to demonstrate given the problematic diagnosis.

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

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. 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. 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. Functional characterisation of the lower airway microbiota through metatranscriptome data identifies metabolically active organisms capable of producing metabolites with immunomodulatory capacity, such as SCFAs.

Identification of Novel Genes and Biological Pathways That Overlap in Infectious and Nonallergic Diseases of the Upper and Lower Airways Using Network Analyses.

Previous genetic studies on susceptibility to otitis media and airway infections have focused on immune pathways acting within the local mucosal epithelium, and outside of allergic rhinitis and asthma, limited studies exist on the overlaps at the gene, pathway or network level between the upper and lower airways. In this report, we compared [1] pathways identified from network analysis using genes derived from published genome-wide family-based and association studies for otitis media, sinusitis, and lung phenotypes, to [2] pathways identified using differentially expressed genes from RNA-sequence data from lower airway, sinus, and middle ear tissues, in particular cholesteatoma tissue compared to middle ear mucosa. For otitis media, a large number of genes (n = 1,806) were identified as differentially expressed between cholesteatoma and middle ear mucosa, which in turn led to the identification of 68 pathways that are enriched in cholesteatoma. Two differentially expressed genes CR1 and SAA1 overlap in middle ear, sinus, and lower airway samples and are potentially novel genes for otitis media susceptibility. In addition, 56 genes were differentially expressed in both tissues from the middle ear and either sinus or lower airways. Pathways that are common in upper and lower airway diseases, whether from published DNA studies or from our RNA-sequencing analyses, include chromatin organization/remodeling, endocytosis, immune system process, protein folding, and viral process. Taken together, our findings from genetic susceptibility and differential tissue expression studies support the hypothesis that the unified airway theory wherein the upper and lower respiratory tracts act as an integrated unit also applies to infectious and nonallergic airway epithelial disease. Our results may be used as reference for identification of genes or pathways that are relevant to upper and lower airways, whether common across sites, or unique to each disease.

An observational study of the role of indoor air pollution in pets with naturally acquired bronchial/lung disease.

BackgroundIndoor air pollution (IAP) is an emerging issue for both human and veterinary patients under the concept of ‘One Health’. The association between IAP and respiratory disease in companion animals has been reported.ObjectivesThe present study investigated the relationship between quantifiable indoor air quality and clinical characteristics of naturally acquired bronchial/lung disease in pet dogs and cats.MethodsA total of 36 clinical cases (20 dogs and 16 cats) with naturally acquired bronchial/lung disease were prospectively recruited. Lower airway samples were collected and analysed, and clinical signs and the information from pulmonary function testing were examined. Indoor air quality was estimated by the average concentration of particles measuring ≤2.5 μm (PM2.5, μg/m3) and volatile organic compounds (VOC, ppm) in the animals’ domestic microenvironments.ResultsExposure to IAP was not found to be correlated with the severity of clinical signs, pulmonary function changes or bronchoalveolar lavage fluid cytology in cats with bronchial/lung disease. However, a hypercellular response in canine lower airways was found to be associated with poor indoor air quality, including unacceptable indoor PM2.5 levels (>35 μg/m3) or increases in VOC concentration (>1 ppm) in places most commonly frequented by the dogs in the home.ConclusionsPoor indoor air quality may exacerbate airway disease in pets and should not be ignored in modern society.

Coronavirus disease 2019: The story so far

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) emerged in China in late 2019 and became a global pandemic by March 2020. SARS-CoV-2 is a highly transmissible virus that causes a clinical illness known as Coronavirus Disease 2019. The clinical spectrum ranges from mild respiratory and generalized symptoms to severe pneumonia with multiple organ failure. Overall mortality is high in older patients and those with comorbidities such as obesity and cardiovascular disease. Diagnosis is usually confirmed by polymerase chain reaction on upper or lower airway samples. Clinical management is largely supportive; no specific therapeutic options are currently available. Public health interventions have thus far been centered around social distancing, large-scale testing, and isolation. An unprecedent global effort has been mounted for the rapid development of effective SARS-CoV-2 vaccines. Until such time, further waves of SARS-CoV-2 are likely, if the restrictive control measures are removed.

Laboratory contamination in airway microbiome studies

BackgroundThe low bacterial load in samples acquired from the lungs, have made studies on the airway microbiome vulnerable to contamination from bacterial DNA introduced during sampling and laboratory processing. We have examined the impact of laboratory contamination on samples collected from the lower airways by protected (through a sterile catheter) bronchoscopy and explored various in silico approaches to dealing with the contamination post-sequencing. Our analyses included quantitative PCR and targeted amplicon sequencing of the bacterial 16S rRNA gene.ResultsThe mean bacterial load varied by sample type for the 23 study subjects (oral wash>1st fraction of protected bronchoalveolar lavage>protected specimen brush>2nd fraction of protected bronchoalveolar lavage; p < 0.001). By comparison to a dilution series of know bacterial composition and load, an estimated 10–50% of the bacterial community profiles for lower airway samples could be traced back to contaminating bacterial DNA introduced from the laboratory. We determined the main source of laboratory contaminants to be the DNA extraction kit (FastDNA Spin Kit). The removal of contaminants identified using tools within the Decontam R package appeared to provide a balance between keeping and removing taxa found in both negative controls and study samples.ConclusionsThe influence of laboratory contamination will vary across airway microbiome studies. By reporting estimates of contaminant levels and taking use of contaminant identification tools (e.g. the Decontam R package) based on statistical models that limit the subjectivity of the researcher, the accuracy of inter-study comparisons can be improved.

Association of Antibiotics, Airway Microbiome, and Inflammation in Infants with Cystic Fibrosis

The underlying defect in the cystic fibrosis (CF) airway leads to defective mucociliary clearance and impaired bacterial killing, resulting in endobronchial infection and inflammation that contributes to progressive lung disease. Little is known about the respiratory microbiota in the early CF airway and its relationship to inflammation. To examine the bacterial microbiota and inflammatory profiles in bronchoalveolar lavage fluid and oropharyngeal secretions in infants with CF. Infants with CF from U.S. and Australian centers were enrolled in a prospective, observational study examining the bacterial microbiota and inflammatory profiles of the respiratory tract. Bacterial diversity and density (load) were measured. Lavage samples were analyzed for inflammatory markers (interleukin 8, unbound neutrophil elastase, and absolute neutrophil count) in the epithelial lining fluid. Thirty-two infants (mean age, 4.7 months) underwent bronchoalveolar lavage and oropharyngeal sampling. Shannon diversity strongly correlated between upper and lower airway samples from a given subject, although community compositions differed. Microbial diversity was lower in younger subjects and in those receiving daily antistaphylococcal antibiotic prophylaxis. In lavage samples, reduced diversity correlated with lower interleukin 8 concentration and absolute neutrophil count. In infants with CF, reduced bacterial diversity in the upper and lower airways was strongly associated with the use of prophylactic antibiotics and younger age at the time of sampling; less diversity in the lower airway correlated with lower inflammation on bronchoalveolar lavage. Our findings suggest modification of the respiratory microbiome in infants with CF may influence airway inflammation.

Utility of oropharyngeal real-time PCR for S. pneumoniae and H. influenzae for diagnosis of pneumonia in adults

A lack of sensitive tests and difficulties obtaining representative samples contribute to the challenge in identifying etiology in pneumonia. Upper respiratory tract swabs can be easily collected and analyzed with real-time PCR (rtPCR). Common pathogens such as S. pneumoniae and H. influenzae can both colonize and infect the respiratory tract, complicating the interpretation of positive results. Oropharyngeal swabs were collected (n = 239) prospectively from adults admitted to hospital with pneumonia. Analysis with rtPCR targeting S. pneumoniae and H. influenzae was performed and results compared with sputum cultures, blood cultures, and urine antigen testing for S. pneumoniae. Different Ct cutoff values were applied to positive tests to discern colonization from infection. Comparing rtPCR with conventional testing for S. pneumoniae in patients with all tests available (n = 57) resulted in: sensitivity 87 %, specificity 79 %, PPV 59 % and NPV 94 %, and for H. influenzae (n = 67): sensitivity 75 %, specificity 80 %, PPV 45 % and NPV 94 %. When patients with prior antimicrobial exposure were excluded sensitivity improved: 92 % for S. pneumoniae and 80 % for H. influenzae. Receiver operating characteristic curve analysis demonstrated for S. pneumoniae: AUC = 0.65 (95 % CI 0.51–0.80) and for H. influenzae: AUC = 0.86 (95 % CI 0.72–1.00). Analysis of oropharyngeal swabs using rtPCR proved both reasonably sensitive and specific for diagnosing pneumonia caused by S. pneumoniae and H. influenzae. This method may be a useful diagnostic adjunct to other methods and of special value in patients unable to provide representative lower airway samples.

Induction and Antagonism of Antiviral Responses in Respiratory Syncytial Virus-Infected Pediatric Airway Epithelium

Airway epithelium is the primary target of many respiratory viruses. However, virus induction and antagonism of host responses by human airway epithelium remains poorly understood. To address this, we developed a model of respiratory syncytial virus (RSV) infection based on well-differentiated pediatric primary bronchial epithelial cell cultures (WD-PBECs) that mimics hallmarks of RSV disease in infants. RSV is the most important respiratory viral pathogen in young infants worldwide. We found that RSV induces a potent antiviral state in WD-PBECs that was mediated in part by secreted factors, including interferon lambda 1 (IFN-λ1)/interleukin-29 (IL-29). In contrast, type I IFNs were not detected following RSV infection of WD-PBECs. IFN responses in RSV-infected WD-PBECs reflected those in lower airway samples from RSV-hospitalized infants. In view of the prominence of IL-29, we determined whether recombinant IL-29 treatment of WD-PBECs before or after infection abrogated RSV replication. Interestingly, IL-29 demonstrated prophylactic, but not therapeutic, potential against RSV. The absence of therapeutic potential reflected effective RSV antagonism of IFN-mediated antiviral responses in infected cells. Our data are consistent with RSV nonstructural proteins 1 and/or 2 perturbing the Jak-STAT signaling pathway, with concomitant reduced expression of antiviral effector molecules, such as MxA/B. Antagonism of Jak-STAT signaling was restricted to RSV-infected cells in WD-PBEC cultures. Importantly, our study provides the rationale to further explore IL-29 as a novel RSV prophylactic. Most respiratory viruses target airway epithelium for infection and replication, which is central to causing disease. However, for most human viruses we have a poor understanding of their interactions with human airway epithelium. Respiratory syncytial virus (RSV) is the most important viral pathogen of young infants. To help understand RSV interactions with pediatric airway epithelium, we previously developed three-dimensional primary cell cultures from infant bronchial epithelium that reproduce several hallmarks of RSV infection in infants, indicating that they represent authentic surrogates of RSV infection in infants. We found that RSV induced a potent antiviral state in these cultures and that a type III interferon, interleukin IL-29 (IL-29), was involved. Indeed, our data suggest that IL-29 has potential to prevent RSV disease. However, we also demonstrated that RSV efficiently circumvents this antiviral immune response and identified mechanisms by which this may occur. Our study provides new insights into RSV interaction with pediatric airway epithelium.

Assessment of airway microbiota and inflammation in cystic fibrosis using multiple sampling methods.

Oropharyngeal (OP) swabs and induced sputum (IS) are used for airway bacteria surveillance in nonexpectorating children with cystic fibrosis (CF). Molecular analyses of these airway samples detect complex microbial communities. However, the optimal noninvasive sampling approach for microbiota analyses and the clinical relevance of microbiota, particularly its relationship to airway inflammation, is not well characterized. The goals of this study were to compare molecular analyses of concurrently collected saliva, OP swabs, IS, and expectorated sputum (ES) from children with CF and to determine the association between microbiota, lung function, and airway inflammation. Saliva, OP swabs, IS, and ES were collected from 16 children with CF. Spirometry was performed. Respiratory and saliva samples (n = 61) were sequenced for bacterial microbial communities, and total and CF-specific bacterial quantitative PCR assays were performed. Airway samples underwent conventional culture for CF-specific pathogens. Neutrophil elastase, IL-1β, IL-1ra, IL-6, Il-8, TNF-α, and vascular endothelial growth factor were measured in ES and IS. Sequencing results from individual subjects were similar across samples, with greater between-subject than within-subject variation. However, Pseudomonas and Staphylococcus were detected in higher relative abundance from lower airways (ES and IS) compared with paired upper airway samples (OP and saliva). Pseudomonas, Staphylococcus, and Enterobacteriaceae correlated with increased airway inflammation. Divergence between microbiota in upper airway compared with lower airway samples, indicating greater differences between communities, was associated with increased sputum neutrophil elastase. Bacteria detected in IS samples resemble ES samples, whereas OP samples may underrepresent bacteria associated with airway inflammation. Divergence of lower airway communities from upper airway was associated with airway inflammation and may portend disease progression.