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.