Diagnostic protocols for pneumonia etiologies

Gram stain and bacterial culture were performed on blood, PF, ET aspirates, and BAL specimens at each site using standard techniques; only high-quality ET aspirates and quantified BAL specimens were included (Supplementary Appendix).^{15 ,16 (link)} Real-time polymerase chain reaction (PCR) targeting Streptococcus pneumoniae (lyt-A) and Streptococcus pyogenes (spy) genes was performed on whole blood and PF at CDC.^{17 (link)} PF was also tested at the University of Utah for H. influenzae and other Gram-negative bacteria, Staphylococcus aureus, Streptococcus anginosus/mitis, S. pneumoniae, and S. pyogenes using PCR (Supplementary Appendix).^{18 (link),19 (link)} PCR was performed at the study sites on NP/OP swabs from children with pneumonia and controls using CDC-developed methods for detection of adenovirus (AdV); Chlamydophila pneumoniae; coronaviruses 229E, HKU1, NL63, and OC43 (CoV); human metapneumovirus (HMPV); human rhinovirus (HRV); influenza A/B viruses; Mycoplasma pneumoniae; parainfluenza viruses 1, 2, 3 (PIV); and respiratory syncytial virus (RSV).^{20 (link)-24} Quality assurance and monitoring protocols maintained standardization among sites.^{25 (link),26 (link)} Serology for AdV, HMPV, influenza A/B, PIV, and RSV was performed at CDC on available paired acute and convalescent sera (Supplementary Appendix).^{27 (link)-32}