Birgitta Versluijs

82 Materials and methods older, according to American Thoracic Society/European Respiratory Society criteria, using calibrated pneumotachometer systems (Jaeger, Hochberg, Germany). Values are expressed as percentage of predicted values for age, race, sex, and height-matched con- trols (The Utrecht data set, Koopman 6 ). Forced expiratory volume in 1 second, forced vital capacity, total lung capacity, and lung diffusion capacity for CO, corrected for hemoglobin and alveolar volume < 80% of pre- dicted values are considered to be abnormal. Residual volume of >25% of total lung capacity is considered to be abnormal and suggestive for trapped air. HRCT scans are acquired using a 16-detector row scanner (Philips Medical Systems, Best, Netherlands). For infants and young children, scans are obtained at 25-cm H2O pressure (inspiration) and 0-cmH2O pressure (expiration). For older children, who were able to cooperate with breath hold instruction, scans were obtained at full inspiration and at end of exhalation. Inspiration images are obtained using fixed 90 kVp and 18 to 60 mAs (depending on bodyweight). For expiration images, we used 90 kVp and 11 mAs. Acquisition was volu- metric thin-slice for both inspiratory and expiratory computed tomography. All HRCT scans were assessed by a pediatric radiologist. Fleischner Society terms for thoracic imaging were used. 7 All abnormalities, as stated in the radiology report, were registered. Those abnormalities with clinical implications, such as antimicrobial treat- ment, guided lung biopsy or diuretics, were defined as clinically significant. BAL was performed under general anesthesia. BAL fluid was cultured and processed in accordance with standard microbiological procedures. Galactomannan (GM) tests are performed using BioRad Platelia Aspergillus EIA. Any positive culture or GM levels > .5 was considered to be abnormal. Nucleic acids are extracted using the total nucleic acid protocol with the MagNA pure LC nucleic acid isolation system (Roche Diagnostics, Basel, Switzerland). For detection of RNA-viruses cDNA is synthesized by using MultiScribe reverse transcriptase and ran- dom hexamers (Applied Bio-systems, Foster City, CA). Detection of viral and atypical pa- thogens was performed in parallel, using real-time PCR assays specific for the following viruses: bocavirus, human herpesvirus 6, respiratory syncytial virus, influenzavirus A and B, parainfluenzavirus 1 to 4, rhinoviruses, adenoviruses, human coronavirus OC43, NL63 and 229E, human metapneumovirus, and Mycoplasma pneumoniae . Real-time PCR procedures were performed as described previously. 8 Any positive PCR is considered to be abnormal. The total costs for the pulmonary screening were approximately 900 euro. Chest HRCT costs 300 euro, RV panel PCR 495, bacterial cultures 11 euro, GM 12 euro, PFT (com- plete) cost 48 euro. 5