Wednesday, December 3, 2014
IgE-Mediated Immune Responses and Airway Detection of Aspergillus
This prospective observational cohort study was carried out between October 2008 and February 2011. Approval was obtained from the South Manchester research ethics committee. Patients were invited to participate between October 2008 and February 2009 if they were aged > 18 years and given a diagnosis of CF confirmed by genetic or sweat testing. Patients were enrolled during routine outpatient appointments at the Manchester Adult Cystic Fibrosis Centre, and all gave written informed consent.
Patients were excluded at enrollment if they were unable to produce a > 2 mL sputum sample spontaneously or had an exacerbation of pulmonary symptoms requiring additional therapy. Baseline demographic and clinical details were collected from medical case records. Lung function (FEV1 and FVC % predicted) at enrollment and 2 years after enrollment was obtained by documenting the patient’s best lung function achieved within that year.
This method was chosen to minimize the wide variability in lung function measurements observed in patients with CF. All lung function was performed postbronchodilator by experienced clinical staff according to European Respiratory Society guidelines. Total days of IV antibiotics were prospectively monitored over 2 years to examine exacerbation rates. Each patient was given 10 mL of sterile water and asked to rinse his or her mouth for 30 s and return the water to a sterile universal container. A sputum sample was then collected without sputum induction.
This was done to differentiate oral cavity and lower respiratory tract colonization. Patients provided two sputum samples within 1 year. Sputum samples were homogenized with Sputasol, and culture was performed according to the UK Health Protection Agency National Standards Method BSOP but modified to plate 10 pL rather than 1 pL of sputum. Ten microliters of homogenized sputum was inoculated onto each of three Sabouraud dextrose with chloramphenicol agar plates and one CHROMagar Candida plate.
SABC plates were incubated at 30°C, 37°C, and 45°C for 72 h. CHROMagar plates were incubated at 37°C for 72 h. This culture method was repeated for the oral rinse sample but with no homogenization. Following culture, the remaining sputum sample underwent additional homogenization using sonication. Fungal DNA was extracted using the MycXtra DNA extraction kit.