Birgitta Versluijs

45 Association between respiratory virus and alloimmune lung syndromes Respiratory viral Before August 2005, nasal pharyngeal aspirate (NPA) samples were obtained for PCR only in the presence of symptoms of aURTI or LRTI, and then only up to day +100 post- transplantation. From August 2005 onward, we performed surveillance studies onNPA- samples of all patients admitted to our HSCT unit. Reversetranscriptase (RT)-PCR was done for all common RVs (see later).We repeated the NPA weekly in patients negative for RV and twice weekly in patients positive for RV. Real-time PCR for respiratory viruses Nucleic acids were 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 was synthesized using MultiScribe RT and random hexamers (Ap- plied Biosystems, Foster City, CA). Detection of viral and atypical pathogens was perfor- med in parallel, using real-time PCR assays specific for the following viruses: CMV; EBV; HHV-6; respiratory syncytial virus A and B; influenzavirus A and B; parainfluenzavirus 1-4; rhinoviruses; adenoviruses; human coronaviruses OC43, NL63, and 229E; human metapneumovirus; Mycoplasma pneumoniae ; and Chlamydia pneumoniae . Real-time PCR procedures were performed as described previously. 20 In brief, samples were assayed in duplicate in a 25-mL reaction mixture containing 10 mL of cDNA, 12.5 mL of TaqMan Universal PCR Master Mix (Applied Biosystems), 300-900 nmol/L of the forward and reverse primers, and 75-200 nmol/L of each probe. All samples had been spiked before extraction with an internal control virus (murine encephalomyocarditis vi- rus [RNA virus] and porcine herpesvirus [DNA virus]) to monitor for efficient extraction and amplification, essentially as described previously. 21 The cycle of threshold (Ct) gives an impression of the quantity of the viral load (ie, a semiquantitative value). Recording pulmonary complications All patients were observed for signs of respiratory disease early and late after transplanta- tion. All clinical symptoms were recorded. In patients with URTI symptoms, NPA sam- ples were obtained and tested for RV infection by PCR (see earlier). In patients with signs of LRTI, chest X-rays were obtained. Other tests, performed as indicated, included bronchoalveolar lavage (BAL) for broad infectious screening with bacterial/fungal cul- tures, viral PCR, and galactomannan, as well as high-resolution computed tomography (HRCT) scans. In cases of suspected allo-LS, HRCT and BAL were always performed (see definitions of disease shortly). 3