Microbiological assays

Each patient provided a sputum sample on day 1 (visit 1), day 10 ± 3 (visit 3), and day 36 ± 7 (visit 4). A blood sample was obtained on day 1 and day 36 ± 7. The sputum specimens containing group 4 or 5 in Gram stain score were used for bacterial culture and group 3 to 5 were considered suitable for PCR. Paired serum samples were used for serological tests for M. pneumoniae, C. pneumoniae, and L. pneumophila. Urine samples for the urinary antigen test were also collected on day 1 and examined for the presence of L. pneumophila and Streptococcus pneumoniae. All samples were sent to the central Seegene medical foundation reference laboratory (Seoul, Korea). Serological assays for M. pneumoniae were performed using the M. pneumoniae immunoglobulin G (IgG)/IgM enzyme-linked immunosorbent assay (ELISA) (Vircell, Granada, Spain). The assay for C. pneumoniae was performed using the SeroCP™ IgG/IgM kit (Savyon Diagnostics, Ashdod, Israel) and that for L. pneumophila was performed using the L. pneumophila serogroup 1 IgG/IgM ELISA (Vircell). All assays were performed according to the manufacturer’s protocols. A definite acute infection was defined as a 4-fold or greater increase of the IgG titer between the acute (day 1) and convalescent (day 36 ± 7) serum specimens. A probable acute infection was defined as a positive IgM result on day 1. Legionella antigen was detected in urine samples using the Binax NOW Legionella Urinary Antigen Test (Binax, Portland, ME, USA). Sputum samples were tested for atypical respiratory pathogens (M. pneumoniae, C. pneumoniae, and L. pneumophila) using a multiplex PCR assay (Seeplex PneumoBacter ACE Detection kit; Seegene), according to the manufacturer’s instructions. This multiplex PCR assay had a detection limit of 100 copies per reaction, and no cross-amplification of DNA from the 63 different pathogens was observed. The assay was also reproducible when PCR reactions were run on 5 different days. An acute atypical respiratory pathogenic infection was defined when serological tests, PCR, or the Legionella urinary antigen test was positive.

The study was approved by the Institutional Review Board of each hospital (2012-52), and all participants provided written informed consent. The study is registered with ClinicalTrials.gov (number {"type":"clinical-trial","attrs":{"text":"NCT01658020","term_id":"NCT01658020"}}NCT01658020) (Clinical Research Information Service http://ncrc.cdc.go.kr/cris;KCT0000532).