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      Airway bacteria measured by quantitative polymerase chain reaction and culture in patients with stable COPD: relationship with neutrophilic airway inflammation, exacerbation frequency, and lung function

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          Abstract

          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 (>10 6 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 >10 6 copies/mL. Samples that had qPCR copy numbers >10 6/mL, whether culture-positive or not, had increased sputum neutrophil counts. H. influenzae qPCR copy numbers correlated with sputum neutrophil counts ( r=0.37, P<0.001), were repeatable within subjects, and were >10 6/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 (FEV 1), 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.

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          Most cited references 20

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          Defective macrophage phagocytosis of bacteria in COPD.

          Exacerbations of chronic obstructive pulmonary disease (COPD) are an increasing cause of hospitalisations and are associated with accelerated progression of airflow obstruction. Approximately half of COPD exacerbations are associated with bacteria and many patients have lower airways colonisation. This suggests that bacterial infection in COPD could be due to reduced pathogen removal. This study investigated whether bacterial clearance by macrophages is defective in COPD. Phagocytosis of fluorescently labelled polystyrene beads and Haemophillus influenzae and Streptococcus pneumoniae by alveolar macrophages and monocyte-derived macrophages (MDM) was assessed by fluorimetry and flow cytometry. Receptor expression was measured by flow cytometry. Alveolar macrophages and MDM phagocytosed polystyrene beads similarly. There was no difference in phagocytosis of beads by MDM from COPD patients compared with cells from smokers and nonsmokers. MDM from COPD patients showed reduced phagocytic responses to S. pneumoniae and H. influenzae compared with nonsmokers and smokers. This was not associated with alterations in cell surface receptor expression of toll-like receptor (TLR)2, TLR4, macrophage receptor with collagenous structure, cluster of differentiation (CD)163, CD36 or mannose receptor. Budesonide, formoterol or azithromycin did not suppress phagocytosis suggesting that reduced responses in COPD MDM were not due to medications. COPD macrophage innate responses are suppressed and may lead to bacterial colonisation and increased exacerbation frequency.
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            Persistent colonization by Haemophilus influenzae in chronic obstructive pulmonary disease.

            Nontypeable Haemophilus influenzae colonizes the respiratory tract of adults with chronic obstructive pulmonary disease (COPD) and causes intermittent exacerbations. Isolates of H. influenzae collected monthly in a prospective study were subjected to molecular typing. During a 7-year study spanning 345 patient-months of observation, 122 episodes of negative cultures lasting 1 month or more, and that were preceded and followed by isolation of an apparently identical strain of H. influenzae, were found. Seventeen such episodes of negative cultures, lasting 6 months or more and spanning 203 patient-months, were studied in detail to test the hypothesis that these periods of negative cultures represented continuous colonization by the same strain of H. influenzae. Molecular typing by three independent methods established that the strains preceding and following the episodes of negative cultures were indeed identical. Strain-specific H. influenzae DNA was detected in some of the sputum samples that had yielded negative cultures. These results indicate that some patients with COPD are persistently colonized with H. influenzae and that sputum cultures underestimate the frequency of colonization of the respiratory tract by H. influenzae in COPD. This observation has a significant impact on understanding bacterial colonization in COPD.
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              Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis.

              Viable bacteria are often isolated from airway secretions in clinically stable patients with chronic bronchitis. We hypothesized that the number of organisms and bacterial species might be important modulators of airway inflammation. We performed quantitative sputum cultures in 160 stable patients [55 with chronic obstructive pulmonary disease (COPD) and normal serum alpha(1)-antitrypsin levels, 62 with COPD and severe alpha(1)-antitrypsin deficiency (PiZ), and 43 with idiopathic bronchiectasis]. The results were related to several indicators of the mechanisms and severity of airway inflammation. Airway bacterial load correlated with sputum myeloperoxidase level, an indirect measure of neutrophil activation and number (r = 0.50, P<0. 001); sputum neutrophil chemoattractants [interleukin-8 level (r = 0. 68, P<0.001) and leukotriene B4 level (r = 0.53, P<0.001)]; sputum leukocyte elastase activity (r = 0.55, P<0.001); and albumin leakage from serum to sputum (r = 0.26, P<0.01). Markers of inflammation increased at bacterial loads of 10(6) to 10(7) colony-forming units per milliliter, and increased progressively with increasing bacterial load. For example, the median (interquartile range) sputum myeloperoxidase level was 0.3 U/mL (0.1 to 0.5 U/mL) for patients who were not colonized or who had mixed normal oropharyngeal flora alone; 0.5 U/mL (0.2 to 0.7 U/mL) for patients with 10(5) to 10(6) colony-forming units per milliliter (P = 0.07); 0.5 U/mL (0.3 to 1.2 U/mL) for patients with 10(6) to 10(7) colony-forming units per milliliter (P<0.01); 0.7 U/mL (0.3 to 1.2 U/mL) for patients with 10(7) to 10(8) colony-forming units per milliliter (P <0.005); and 2.4 U/mL (0.7 to 4.8 U/mL) for patients with 10(8) or greater colony-forming units per milliliter (P<0.0001). The bacterial species influenced airway inflammation; for example, sputum myeloperoxidase activity was greater (P<0.005) in patients colonized with Pseudomonas aeruginosa [median 32 U/mL (interquartile range, 20 to 65 U/mL)] than those colonized with nontypeable Hemophilus influenzae [4 U/mL (2 to 31 U/mL)], which in turn was greater (P = 0.01) than among those colonized with Moraxella catarrhalis [1.1 U/mL (0.6 to 1.8 U/mL)]. We did not find a relation between bacterial load and lung function. The bacterial load and species contribute to airway inflammation in patients with stable chronic bronchitis. Further studies are required to determine the consequences of bacterial colonization on patient morbidity and decline in lung function.
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                Author and article information

                Journal
                Int J Chron Obstruct Pulmon Dis
                Int J Chron Obstruct Pulmon Dis
                International Journal of COPD
                International Journal of Chronic Obstructive Pulmonary Disease
                Dove Medical Press
                1176-9106
                1178-2005
                2015
                09 June 2015
                : 10
                : 1075-1083
                Affiliations
                [1 ]Respiratory Medicine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK
                [2 ]Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK
                [3 ]Institute for Lung Health, National Institute for Health Research Respiratory Biomedical Research Unit, Glenfield Hospital, University of Leicester, Leicester, UK
                [4 ]Department of Clinical Microbiology, University Hospitals of Leicester NHS Trust, Leicester, UK
                Author notes
                Correspondence: Mona Bafadhel, Respiratory Medicine Unit, NDM Research Building, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus, Oxford, OX3 7FZ, UK, Tel +44 18 65 61 2898, Email mona.bafadhel@ 123456ndm.ox.ac.uk
                Article
                copd-10-1075
                10.2147/COPD.S80091
                4468933
                © 2015 Bafadhel et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License

                The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.

                Categories
                Original Research

                Respiratory medicine

                h. influenzae, qpcr, sputum

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