Microbiological evaluation
Viral infections due to influenza A (Flu A), influenza B (Flu B), human rhinovirus (HRV), adenovirus (AdV), bocavirus, coronavirus (CoV), parainfluenza (PIV), respiratory syncytial virus (RSV), enterovirus(EV) and human metapneumovirus (HMPV) were confirmed using the xTAG Respiratory Viral Panel (Luminex Molecular Diagnostics Corporation, Toronto, ON, Canada) [24] via qualified sputum (defined as >25 leukocytes and <10 epithelial cells per 100× magnified field [18]), endotracheal aspirate (ETA), bronchoalveolar lavage fluid (BALF) samples, or nasopharyngeal (NP) swabs. If there was discordance between two tests, the result was confirmed by monoplex PCR. Since Jan 22, 2020, SARS-CoV-2 was further detected in respiratory specimens by real-time PCR methods (Zhijiang, Shanghai, China).
Low respiratory tract specimens and pleural fluid underwent routine Gram staining and quantitative culture for bacterial pathogens. Blood cultures were obtained from patients with temperature 38.5℃ within 48 h on admission. A bacterial pathogen was determined to be present if detected in a blood sample, sputum, ETA, BALF, or pleural fluid through culture or PCR assay (Seegene Inc. Seoul, Korea), selected bacteria were considered to be contaminants or colonization [25]; if C. pneumonia or M. pneumonia or L. pneumophila was detected in low respiratory tract specimens using PCR assay; or if L. pneumophila or S. pneumonia was detected in urine employing antigen detection (Binax now, Trinity Biotech, Bray, Ireland) [26].
Fungal pathogens were determined per clinical guidelines [27]. We used culture and microscopic morphological characteristics to distinguish different types of fungal pathogens. A “probable” fungal diagnosis was based on the presence of acute pulmonary infiltrates of the lungs with the positive determination of galactomannan (GM) antigen in the serum or BALF and/or filamentous fungi isolates in BALF, but not necessarily need the presence of immunocompromised disorder [11]. Filamentous fungi isolate in respiratory samples but the absence of newly pulmonary infiltrates or with a negative GM test was described as colonization.Pneumocystis jirovecii was determined to be present if detected microscopically in sputum, ETA, or BALF using conventional or immunofluorescence staining or PCR assay (Zhuochenghuisheng, Beijing, China) [28], and supplemented by 1,3-β-D-glucan tests (G tests) in the serum or BALF. Candida spp. isolated from low respiratory tract specimens were not considered to be contaminants rather than pathogens.