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Leena M George,1,* Richard D Haigh,1,* Vijay Mistry,1 Koirobi Haldar,1 Michael R Barer,1 Marco R Oggioni,2 Christopher E Brightling1 1Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK; 2Department of Genetics, University of Leicester, Leicester, UK *These authors contributed equally tothis work Purpose: Moraxella catarrhalis is implicated in the pathogenesis of some COPD exacerbations. We sought to investigate whether the M. catarrhalis strain is variable between COPD subjects; that an exacerbation is associated with acquisition of a new strain and that certain strains are more commonly associated with exacerbations. Patients and methods: Sputum samples were collected at stable and exacerbation visits from COPD subjects from a single center as part of the COPDMAP consortium. Samples identified asM. catarrhalispositive by qPCR were recultured in liquid cultures grown to extract genomic DNA; underwent Illumina MiSeq and bacterial genome sequences were de novo assembled and Multi Locus Sequence Type (MLST) was determined. Results: Thirty-five samples were obtained from 18 subjects. These included 13 stable and 22 exacerbation samples. The diversity between samples was very large with 25 differentM. catarrhalisMLSTs being identified out of the 35 samples of which 12 MSLTs have not been described previously. Change and persistence ofM. catarrhalisstrain were observed between stable visits, from stable to exacerbation and vice-a-versa, and between exacerbation visits. Conclusion: SputumM. catarrhalisstrains exhibit marked diversity within and between COPD subjects. Acquisition of a new strain is common between stable and exacerbation events such that no strain is specifically associated with an exacerbation. Keywords: COPD, Moraxella catarrhalis, strain change, exacerbation

Leena George,1 Adam Wright,1 Vijay Mistry,1 Amanda Sutcliffe,1 Latifa Chachi,1 Koirobi Haldar,1 Mohammadali Yavari Ramsheh,1 Matthew Richardson,1 René van der Merwe,2 Ubaldo Martin,3 Paul Newbold,3 Christopher E Brightling11Department of Respiratory Sciences, Institute for Lung Health, National Institute for Health Research Biomedical Research Centre, University of Leicester, Leicester, UK; 2Clinical Respiratory Management, MedImmune Ltd., Cambridge, UK; 3Global Medical Affairs, AstraZeneca, Gaithersburg, MD, USAAbstract: We hypothesized whether the reduction in eosinophilic airway inflammation in patients with chronic obstructive pulmonary disease (COPD) following treatment with benralizumab, a humanized, afucosylated, monoclonal antibody that binds to interleukin-5 receptor α, increases the airway bacterial load. Analysis of sputum samples of COPD patients participating in a Phase II trial of benralizumab indicated that sputum 16S rDNA load and Streptococcus pneumoniae were reduced following treatment with benralizumab. However, in vitro, eosinophils did not affect the killing of the common airway pathogens S. pneumoniae or Haemophilus influenzae. Thus, benralizumab may have an indirect effect upon airway bacterial load.Keywords: COPD, benralizumab, IL-5, bacterial load, S. pneumoniae, H. influenzae

Background: Airway ecology is altered in asthma and chronic obstructive pulmonary disease (COPD). Anti‐microbial interventions might have benefit in subgroups of airway disease. Differences in sputum microbial profiles at acute exacerbation of airways disease are reflected by the γProteobacteria:Firmicutes (γP:F) ratio. We hypothesized that sputum microbiomic clusters exist in stable airways disease, which can be differentiated by the sputum γP:F ratio. Methods: Sputum samples were collected from 63 subjects with severe asthma and 78 subjects with moderate‐to‐severe COPD in a prospective single centre trial. Microbial profiles were obtained through 16S rRNA gene sequencing. Topological data analysis was used to visualize the data set and cluster analysis performed at genus level. Clinical characteristics and sputum inflammatory mediators were compared across the clusters. Results: Two ecological clusters were identified across the combined airways disease population. The smaller cluster was predominantly COPD and was characterized by dominance of Haemophilus at genus level (n = 20), high γP:F ratio, increased H influenzae , low diversity measures and increased pro‐inflammatory mediators when compared to the larger Haemophilus ‐low cluster (n = 121), in which Streptococcus demonstrated the highest relative abundance at the genus level. Similar clusters were identified within disease groups individually and the γP:F ratio consistently differentiated between clusters. Conclusion: Cluster analysis by airway ecology of asthma and COPD in stable state identified two subgroups differentiated according to dominance of Haemophilus . The γP:F ratio was able to distinguish the Haemophilus ‐high versus Haemophilus ‐low subgroups, whether the Haemophilus‐ high group might benefit from treatment strategies to modulate the airway ecology warrants further investigation.

Patients with COPD have a susceptibility to respiratory tract infections associated with increased pulmonary inflammation. Bacteria can reside within the host as cell-associated (attached to host cells via adhesins, pili or biofilm formation) or cell-dissociated bacteria. It is unclear how bacteria-to-cell interactions affect pulmonary inflammation and whether these levels differ over an exacerbation time course. We sought to investigate the effects of Haemophilius influenzae cell-interaction upon airway inflammation and whether the levels of H. influenzae bacteria and cell-dissociated bacteria differ over an exacerbation time course.

BACKGROUND: Exacerbations of asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous. OBJECTIVE: We sought to investigate the sputum cellular, mediator, and microbiome profiles of both asthma and COPD exacerbations. METHODS: Patients with severe asthma or moderate-to-severe COPD were prospectively recruited to a single centre. Sputum mediators were available in 32 asthma and 73 COPD patients assessed at exacerbation. Biologic clusters were determined using factor and cluster analyses on a panel of sputum mediators. Patterns of clinical parameters, sputum mediators, and microbiome communities were assessed across the identified clusters. RESULTS: The asthma and COPD patients had different clinical characteristics and inflammatory profiles, but similar microbial ecology. Three exacerbation biologic clusters were identified. Cluster 1 was COPD predominant, with 27 COPD and 7 asthma patients exhibiting elevated blood and sputum neutrophil counts, proinflammatory mediators (IL-1β, IL-6, IL-6R, TNFα, TNF-R1, TNF-R2, and VEGF), and proportion of the bacterial phylum Proteobacteria. Cluster 2 had 10 asthma and 17 COPD patients with elevated blood and sputum eosinophil counts, Type 2 (T2) mediators (IL-5, IL-13, CCL13, CCL17, and CCL26), and proportion of the bacterial phylum Bacteroidetes. Cluster 3 had 15 asthma and 29 COPD subjects with elevated Type 1 (T1) mediators (CXCL10, CXCL11, and IFN-ϒ) and proportions of phyla Actinobacteria and Firmicutes. CONCLUSIONS: A biologic clustering approach revealed three subgroups of asthma and COPD exacerbations each with different percentages of overlapping asthma and COPD patients. The sputum mediator and microbiome profiles were distinct between clusters. CLINICAL IMPLICATIONS: Sputum mediator and microbiome profiling can determine the distinct and overlapping asthma and COPD biologic exacerbation clusters, highlighting the heterogeneity of these exacerbations.

Mona Bafadhel,1 Koirobi Haldar,2 Bethan Barker,2,3 Hemu Patel,4 Vijay Mistry,2,3 Michael R Barer,2–4 Ian D Pavord,1 Christopher E Brightling2,3 1Respiratory Medicine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 2Department of Infection, Immunity and Inflammation, University of Leicester, 3Institute for Lung Health, National Institute for Health Research Respiratory Biomedical Research Unit, Glenfield Hospital, University of Leicester, 4Department of Clinical Microbiology, University Hospitals of Leicester NHS Trust, Leicester, UK Background: Potentially pathogenic microorganisms can be detected by quantitative real-time polymerase chain reaction (qPCR) in sputum from patients with COPD, although how this technique relates to culture and clinical measures of disease is unclear. We used cross-sectional and longitudinal data to test the hypotheses that qPCR is a more sensitive measure of bacterial presence and is associated with neutrophilic airway inflammation and adverse clinical outcomes.Methods: Sputum was collected from 174 stable COPD subjects longitudinally over 12 months. Microbial sampling using culture and qPCR was performed. Spirometry and sputum measures of airway inflammation were assessed.Findings: Sputum was qPCR-positive (>106 copies/mL) in 77/152 samples (Haemophilus influenzae [n=52], Moraxella catarrhalis [n=24], Streptococcus pneumoniae [n=19], and Staphylococcus aureus [n=7]). Sputum was culture-positive in 50/174 samples, with 49 out of 50 culture-positive samples having pathogen-specific qPCR bacterial loads >106 copies/mL. Samples that had qPCR copy numbers >106/mL, whether culture-positive or not, had increased sputum neutrophil counts. H. influenzae qPCR copy numbers correlated with sputum neutrophil counts (r=0.37, P106/mL three or more times in 19 patients, eight of whom were repeatedly sputum culture-positive. Persistence, whether defined by culture, qPCR, or both, was associated with a higher sputum neutrophil count, lower forced expiratory volume in 1 second (FEV1), and worsened quality of life.Interpretation: qPCR identifies a significant number of patients with potentially bacteria-associated neutrophilic airway inflammation and disease that are not identified by traditional culture-based methods. Keywords: H. influenzae, qPCR, sputum

BACKGROUND: Relationships between airway inflammation and respiratory potentially pathogenic microorganisms (PPMs) quantified using quantitative polymerase chain reaction (qPCR) in subjects with COPD are unclear. Our aim was to evaluate mediators of airway inflammation and their association with PPMs in subjects with COPD at stable state and during exacerbations. METHODS: Sputum from 120 stable subjects with COPD was analyzed for bacteriology (colony-forming units; total 16S; and qPCR targeting Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae), differential cell counts, and inflammatory mediators using the Meso-Scale Discovery Platform. Subjects were classified as colonized if any PPM was identified above the threshold of detection by qPCR. Symptoms were quantified using the visual analog scale. RESULTS: At stable state, 60% of subjects were qPCR positive for H influenzae, 48% for M catarrhalis, and 28% for S pneumoniae. Elevated sputum concentrations of IL-1β, IL-10, and tumor necrosis factor (TNF)-α were detected in samples qPCR positive for either H influenzae or M catarrhalis. Bacterial loads of H influenzae positively correlated with IL-1β, IL-8, IL-10, TNF-α, and symptoms; and M catarrhalis correlated with IL-10 and TNF-α. H influenzae qPCR bacterial load was an independent predictor of sputum TNF-α and IL-1β. In 55 subjects with paired exacerbation data, qPCR bacterial load fold change at exacerbation in M catarrhalis but not H influenzae correlated to changes in sputum TNF-α and IL-1β concentrations. CONCLUSIONS: At stable state, H influenzae is associated with increased airway inflammation in COPD. The relationship between bacterial load changes of specific pathogens and airway inflammation at exacerbation and recovery warrants further investigation.

Mullite (3Al2O3·2SiO2) samples doped with 0.4 M, 0.6 M, 0.8 M, 1.0 M and 1.2 M of iron, nickel and Copper, were prepared by a sol-gel process. Prepared gels were then dried, grinded, pressed into pellets and sintered at temperatures 1100°C and 1400°C for 4 h. Phase formation, densification behavior has been investigated as a function of the metal content and sintering temperature. The density of the sintered ceramics was measured using Archimedes method. The main intention is to study the role of metal ions in influencing mullitization behavior in the case of the sol-gel reaction process, in order to provide useful information of mullite. This paper deals with the effect of metal ions on mullite formation, microstructure and densification behavior in single-phase sol-gel derived mullite. The results showed with increase in concentration of metals (Fe2+, Ni2+, Cu2+), crystallization of mullite was enhanced which is evident from X-ray diffraction upto G3 and FESEM of the composites. The density of the doped samples increases with the increase of metal ion concentration as well as with the sintering temperature. Copper-doped mullite exhibits the highest density 2.46 g cm-3 at 1400°C.

Objective To determine the accuracy of interpretation of a non-contrast MRI protocol in characterizing adnexal masses. Methods and materials Two hundred ninety-one patients (350 adnexal masses) who underwent gynecological MRI at our institution between the 1st of January 2008 and the 31st of December 2018 were reviewed. A random subset (102 patients with 121 masses) was chosen to evaluate the reproducibility and repeatability of readers’ assessments. Readers evaluated non-contrast MRI scans retrospectively, assigned a 5-point score for the risk of malignancy and gave a specific diagnosis. The reference standard for the diagnosis was histopathology or at least one-year imaging follow-up. Diagnostic accuracy of the non-contrast MRI score was calculated. Inter- and intra-reader agreement was analyzed with Cohen’s kappa statistics. Results There were 53/350 (15.1%) malignant lesions in the whole cohort and 20/121 (16.5%) malignant lesions in the random subset. Good agreement between readers was found for the non-contrast MRI score (к = 0.73, 95% confidence interval [CI] 0.58–0.86) whilst the intra-reader agreement was excellent (к = 0.81, 95% CI 0.70–0.88). The non-contrast MRI score value of ≥ 4 was associated with malignancy with a sensitivity of 84.9%, a specificity of 95.9%, an accuracy of 94.2% and a positive likelihood ratio of 21 (area under the receiver operating curve 0.93, 95% CI 0.90–0.96). Conclusion Adnexal mass characterization on MRI without the administration of contrast medium has a high accuracy and excellent inter- and intra-reader agreement. Our results suggest that non-contrast studies may offer a reasonable diagnostic alternative when the administration of intravenous contrast medium is not possible. Key Points • A non-contrast pelvic MRI protocol may allow the characterization of adnexal masses with high accuracy. • The non-contrast MRI score may be used in clinical practice for differentiating benign from malignant adnexal lesions when the lack of intravenous contrast medium precludes analysis with the O–RADS MRI score.