Detection of Respiratory Pathogens in Parapneumonic Effusions by Hypothesis-free, Next-Generation Sequencing (NGS)

BACKGROUND: Species-specific polymerase chain reaction (PCR) testing of pleural fluid (PF) from children with parapneumonic effusion (PPE) has increased pathogen identification in pediatric PPE. However, a pathogen is not detected in 25–35% of cases. Hypothesis-free, next-generation sequencing (NGS) provides a more comprehensive alternative and has led to pathogen detection in PCR-negative samples. However, the utility of NGS in the evaluation of PF from children with PPE is unknown. METHODS: Archived PF (n = 20) from children younger than 18 years with PPE and hospitalized at Primary Children’s Hospital, Utah, in 2015 and previously tested by PCR were evaluated. Ten PCR-negative and 10 PCR-positive PF specimens were tested using RNA-seq at an average depth of 7.7×10(6) sequencing reads per sample. NGS data were analyzed with Taxonomer. We compared pathogens detected by blood and PF culture, PCR, and NGS. RESULTS: Overall, compared with blood/PF culture, PF PCR and PF NGS testing of PF increased bacterial identification from 15% to 50% (P < 0.05) and 65% (P = 0.003), respectively. Pathogen detection in PF by PCR and NGS were comparable (50 vs. 65%, p = NS) (Table). However, compared with PF PCR, NGS significantly increased detection of S. pyogenes (20% vs. 55%; P < 0.05), with 100% concordance when detected by PCR and culture. Detection of Fusobacterium spp. (10 vs. 10%) by PF NGS and PF PCR were comparable. In contrast, there was no detection of S. pneumoniae (15 vs. 0%) by PF NGS compared with PF PCR. CONCLUSION: PF NGS testing significantly improves bacterial identification and comparable to PF PCR testing, which can help inform antimicrobial selection. However there were differences in detection of S. pneumoniae and S. pyogenes. Further studies of NGS testing of PF of children with PPE are needed to assess its potential in the evaluation of PPE in children. DISCLOSURES: A. J. Blaschke, BioFire Diagnostics LLC: Collaborator, Have intellectual property in BioFire Diagnostics through the University of Utah and Investigator, Licensing agreement or royalty and Research support; R. Schlaberg, IDbyDNA: Co-founder, Consultant and Shareholder, Stock