Sputum was collected within 72 hours of patient admission. Nebulisation was used for younger children. Induced sputum samples from the inhalation of hypertonic saline solution were collected by trained personnel according to standard operating procedures, as published previously (Lahti et al., 2009 (link); Honkinen et al., 2012 (link)).
A total of 2 mL of sputum was obtained from each patient with sterility sputum aspiratory tubes and stored at -80°C. The nucleic acids of the viruses were extracted using QIAamp MinElute Virus Spin kits (QIAGEN, Hilden, Germany), and cDNA was synthesized using SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To extract bacterial DNA, 200 μL of sputum were centrifuged at 5000 × g for 10 min, and pellets resuspended in 150 μL enzyme cocktail containing 6 mg lysozyme, 30 U lysostaphin, 37.5 U mutanolysin, and 30U lyticase in lysis buffer of 20 mM Tris-HCl (pH 8), 2 mM EDTA, and 1.2% Triton. The mixture was incubated at 37°C for 30 min to lyse the cell walls of Gram-positive bacteria. DNA was then purified using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Purified DNA was eluted in 200 μL nuclease-free water.
A total of 2 mL of sputum was obtained from each patient with sterility sputum aspiratory tubes and stored at -80°C. The nucleic acids of the viruses were extracted using QIAamp MinElute Virus Spin kits (QIAGEN, Hilden, Germany), and cDNA was synthesized using SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To extract bacterial DNA, 200 μL of sputum were centrifuged at 5000 × g for 10 min, and pellets resuspended in 150 μL enzyme cocktail containing 6 mg lysozyme, 30 U lysostaphin, 37.5 U mutanolysin, and 30U lyticase in lysis buffer of 20 mM Tris-HCl (pH 8), 2 mM EDTA, and 1.2% Triton. The mixture was incubated at 37°C for 30 min to lyse the cell walls of Gram-positive bacteria. DNA was then purified using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Purified DNA was eluted in 200 μL nuclease-free water.
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