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Understanding asthma pathogenesis

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S109 PATHOGENIC BACTERIA IN INDUCED SPUTUM IN SEVERE ASTHMA

1B Green, 1V Kehagia, 1D Sammut, 2S Wiriyachaiporn, 1M Carroll, 2K Bruce, 1P Howarth. 1Southampton University Hospitals NHS Trust, Southampton, UK, 2King’s College London, London, UK

Introduction: The role of bacterial airway colonisation in chronic stable asthma is unclear. However, there is increasing evidence for the role of bacterial pathogen-associated molecular patterns such as lipopolysaccharide in the activation of the innate immune system, which may lead to the expression of a neutrophilic asthma phenotype.

Aims: We aimed to use terminal restriction fragment length polymorphism (T-RFLP) profiling to identify pathogenic bacteria in induced sputum from severe asthmatic patients and correlate findings with clinical characteristics and differential cell counts.

Methods: Induced sputa were obtained from chronic, stable, severe asthmatic patients (N  =  21). All patients were at BTS asthma treatment step 4 or 5 with a minimum of 6 weeks since last exacerbation. All patients were on high-dose inhaled corticosteroids and nine were on long-term maintenance oral prednisolone treatment. Sputa obtained were split for differential cell counts and 16S ribosomal DNA T-RFLP. Using this process bacterial nucleic acids are extracted from the sputum, 16S ribosomal RNA gene PCR products, specific to domain bacteria, are amplified then digested using a specific endonuclease. Ribosomal gene fragments are produced and separated by gel electrophoresis, forming a T-RFLP profile of the diversity of the colonising bacterial community.

Results: 18 out of 21 specimens contained at least one of either Haemophilus sp, Moraxella catarrhalis or Streptococcus pneumoniae. The presence of at least one of these species was associated with the earlier onset of disease (p = 0.039), duration of disease (p = 0.030) and increased peak flow variability over a 2-week period (p = 0.022). There was no significant association between inhaled corticosteroid dose, maintence oral prednisolone treatment, atopy, asthma symptom scores, exhaled …

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