BackgroundChronic airway infection contributes to the underlying pathogenesis of non-cystic fibrosis bronchiectasis (NCFBr). In contrast to other chronic airway infections, associated with COPD and CF bronchiectasis, where polymicrobial communities have been implicated in lung damage due to the vicious circle of recurrent bacterial infections and inflammation, there is sparse information on the composition of bacterial communities in NCFBr. Seventy consecutive patients were recruited from an outpatient adult NCFBr clinic. Bacterial communities in sputum samples were analysed by culture and pyrosequencing approaches. Bacterial sequences were analysed using partial least square discrimination analyses to investigate trends in community composition and identify those taxa that contribute most to community variation.ResultsThe lower airway in NCFBr is dominated by three bacterial taxa Pasteurellaceae, Streptococcaceae and Pseudomonadaceae. Moreover, the bacterial community is much more diverse than indicated by culture and contains significant numbers of other genera including anaerobic Prevotellaceae, Veillonellaceae and Actinomycetaceae. We found particular taxa are correlated with different clinical states, 27 taxa were associated with acute exacerbations, whereas 11 taxa correlated with stable clinical states. We were unable to demonstrate a significant effect of antibiotic therapy, gender, or lung function on the diversity of the bacterial community. However, presence of clinically significant culturable taxa; particularly Pseudomonas aeruginosa and Haemophilus influenzae correlated with a significant change in the diversity of the bacterial community in the lung.ConclusionsWe have demonstrated that acute exacerbations, the frequency of exacerbation and episodes of clinical stability are correlated, in some patients, with a significantly different bacterial community structure, that are associated with a presence of particular taxa in the NCFBr lung. Moreover, there appears to be an inverse relationship between the abundance of P. aeruginosa and that of of H. influenzae within the NCFBr lung bacterial community. This interaction requires further exploration.

Hepatitis C virus (HCV) particles found in vivo are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of HCV in human serum. Previously, our group has found that HCV circulates in blood in association with very-low-density lipoprotein (VLDL). Our aim in this study was to characterize HCV RNA-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with VLDL occurs. HCV was purified by density using iodixanol gradients and by size using gel filtration. Both positive-strand HCV RNA (present in virus particles) and negative-strand HCV RNA (an intermediate in virus replication) were found with densities below 1.08 g ml−1. Viral structural and non-structural proteins, host proteins ApoB, ApoE and caveolin-2, as well as cholesterol, triglyceride and phospholipids were also detected in these low density fractions. After fractionation by size with Superose gel filtration, HCV RNA and viral proteins co-fractionated with endoplasmic reticulum proteins and VLDL. Fractionation on Toyopearl, which separates particles with diameters up to 200 nm, showed that 78 % of HCV RNA from liver was >100 nm in size, with a positive-/negative-strand ratio of 6 : 1. Also, 8 % of HCV RNA was found in particles with diameters between 40 nm and 70 nm and a positive-/negative-strand ratio of 45 : 1. This HCV was associated with ApoB, ApoE and viral glycoprotein E2, similar to viral particles circulating in serum. Our results indicate that the association between HCV and VLDL occurs in the liver.

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Methods Adult patients were recruited from specialist bronchiectasis and Cystic Fibrosis clinics. The gold standard for diagnosing P. aeruginosa infection was positive sputum cultures. 72 sputum samples were analysed. A sputum sample was kept in a glass vial with a cap containing septum. The septum was pierced with a solid phase microextraction (SPME) fibre allowing sampling of the headspace for 50 min at 378C before transferring the fibre into gas chromatography mass spectrometry. AnalyzerPro software (automated peak capture software) and manual identification were used to identify relevant to P. aeruginosa specific compounds in the headspace of sputum. Results 32 samples grew P. aeruginosa either on its own or mixed with other species. 2-nonanone was a marker of P. aeruginosa in sputum headspace gas with sensitivity of 72% and specificity of 88%. Cyanide was not detected. However, a combination of manually identified 2-nonanone with 17 other volatile compounds as identified by AnalyzerPro, increased sensitivity in detection of P. aeruginosa to 91% with specificity of 88%. Conclusion Optimal sampling and capture protocols still need refinement: we were unable to detect the prior noted biomarker Cyanide. These data however demonstrate the potential for rapid and accurate diagnosis of P. aeruginosa infection from sputum samples. In contrast to the 48+ hour turnaround for standard microbiological culture, these results were available within 1e2 h. It also provides a library of compounds as targets to validate in a future study of breath testing. Introduction Lung Clearance Index (LCI) is a measure of lung gas mixing derived from the Multiple Breath Washout (MBW) test. We present LCI, FEV 1 and CT data from the Run-In Study, a longitudinal study in preparation for a multi-dose trial of nebulised gene therapy for CF. Methods MBW, spirometry and low-dose HRCT chest were performed as part of the first Run-In Study visit. LCI was reported as the mean result from at least two technically acceptable sulphur hexafluoride MBW tests performed using a modified Innocor gas analyser. Spirometry was performed to ERS standards. CT scans were assessed by two independent radiologists for extent and severity of bronchiectasis, wall thickening, presence of small and large airway plugs, and gas trapping. Results 191 patients attended visit 1, mean (range) age 22.6 (10e59.1) years. Validated LCI, FEV 1 and CT results were available for 167, 191 and 150 patients, respectively. Mean (SD) FEV 1 was 72 (19)% predicted. Mean (SD) LCI was 10.7 (2.7), with mean intravisit coefficient of variation of 4.9%. LCI correlated negatively with FEV 1 (r¼À0.68, p<0.001), but was abnormally elevated in 72% of participants with normal FEV 1 (see Abstract S22 Figure 1; triangles indicate FEV 1 >80% and LCI >7.5). 95% CI for LCI in normal subjects 5.9 to 7.5. Both FEV 1 and LCI correlated with all CT measures (p<0.001), most strongly with extent of bronchiectasis. LCI correlated better than FEV 1 with extent of bronchiectasis, r¼0.72 (p<0.001) vs r¼À0.61...

Introduction Bacterial infections are a well-known trigger for exacerbations of COPD. A variety of antibiotics are regularly prescribed for this group of patients but the risk and frequency of antibiotic resistance in the COPD population is less understood. Routine culture data can be evaluated to establish resistance prevalence and patterns. Methods Culture data were collected from the sputum samples of 293 patients in the London COPD cohort over a period of 5 years (01/01/2006e31/12/2010) mean (6SD) months in study 28.4 (619.9); age 69.9 years (68.9); predicted FEV 1 47.8% (616.5); male gender 58%; exacerbation samples 48.9%; sputum producers 77.5%. Identification of bacterial presence was established and where clinically indicated drug sensitivity tests (DSTs) were performed. A resistant sample was reported as any bacterial isolate resistant to at least one antimicrobial agent. Results 92/293 (31.4%) patients had at least one bacteria positive sample over the study period. 87/92 (94.6%) patients had samples where DSTs were performed on bacteria positive samples. Resistance was observed in 69/87 (79.3%) patients. 30/293 (10.2%) patients were resistant to all samples where DSTs were performed. 227/293 (77.5%) of patients were sputum producers. There was no significant relationship between predicted FEV 1 and antibiotic resistance frequency in this cohort (X 2 -test; p¼0.577). Patients who were classified as regular sputum producers were more likely to exhibit resistance in culture positive bacteria (p¼0.048). Conclusion Results from this analysis conclude that an estimated 23.5% of COPD patients will develop resistance to an antimicrobial agent within 28.4 months of follow-up with sputum producers being at a higher risk. This study highlights the importance of investigating sputum samples with determination of resistance patterns. Information on resistance patterns and transmission of resistance in COPD can allow more appropriate and targeted antibiotic therapy for COPD exacerbations with improved outcomes. Methods Quantitative PCR was utilised, measuring prevalence and load on paired baseline and exacerbation samples, with baseline samples obtained within 1 year prior to its paired exacerbation. SP, HI and MC gene targets were Spn9082; Haemophilus influenzae P4 lipoprotein gene; copB outer-membrane-protein gene, respectively. The baseline state was defined as being at least 6 weeks after, and 2 weeks before, an exacerbation. Exacerbation was defined as two consecutive days of at least two increased symptoms (Anthonisen criteria), at least one of which is a major symptom (dyspnoea; sputum purulence; sputum volume). Results Sixty-nine paired baseline and exacerbation sputum samples were obtained from 56 patients: mean (6SD) age 71.0 years (68.4); predicted FEV 1 46.4% (617.0); male gender 60.4%; current smoker 30.2%. Bacteria were detected at significantly higher rate at exacerbation, being seen in 36/69 (52.2%) exacerbations, and 19/69 (27.5%) baseline samples (c 2 -test; p¼0.003). Mean bacterial load was signi...