Diagnosis of pleural empyema/parapneumonic effusion by next-generation sequencing

Abstract

Background

Although a microbiological diagnosis of pleural infection is clinically important, it is often complicated by prior antibiotic treatment and/or difficulties with culturing some bacterial species. Therefore, we aimed to identify probable causative bacteria in pleural empyema/parapneumonic effusions by combining 16S ribosomal RNA (rRNA) gene amplification and next-generation sequencing (NGS).

Methods

Pleural fluids were collected from 19 patients with infectious effusions and nine patients with non-infectious malignant effusions. We analysed DNA extracted from the pleural fluid supernatant by NGS using the Genome Search Toolkit and GenomeSync database, either directly or after PCR amplification of the 16S rRNA gene. Infectious and non-infectious effusions were distinguished by semi-quantitative PCR of the 16S rRNA gene.

Results

Only 8 (42%) effusions were culture-positive, however, NGS of the 16S rRNA gene amplicon identified 14 anaerobes and 7 aerobes/facultative anaerobes in all patients, including Streptococcus sp. (n = 6), Fusobacterium sp. (n = 5), Porphyromonas sp. (n = 5), and Prevotella sp. (n = 4), accounting for >10% of the total genomes. The culture and NGS results were discordant for 3 out of 8 patients, all of whom had previously been treated with antibiotics. Total (2^ΔCT value in semi-quantitative PCR of the 16S rRNA gene) and specific (total bacterial load multiplied by the proportion of primary bacteria in NGS) bacterial loads could efficiently distinguish empyema/parapneumonic effusion from non-infectious effusion.

Conclusion

Combining NGS with semi-quantitative PCR can facilitate the diagnosis of pleural empyema/parapneumonic effusion and its causal bacteria.

Keywords:

• Pleural empyema
• parapneumonic effusion
• next-generation sequencing
• malignant pleural effusion

Acknowledgments

We acknowledge the valuable technical support and comments regarding the NGS analysis provided by Keiko Yokoyama, Takuma Araki, Masayuki Tanaka, Tadayuki, Wang Ting, Tadayuki Satou, Hideki Hayashi, and Nobuo Watanabe. We are grateful to Naoki Hayama, Tsuyoshi Oguma, Takuya Aoki, Tomoe Takeuchi, Keito Enokida, and Shohei Obayashi for recruiting participants and providing clinical support.

Ethics approval and consent to participate

The Institutional Review Board for Clinical Research at Tokai University approved the study (Approval No: 14 R-220), which was conducted according to the Declaration of Helsinki (2013 amendment). The study was initiated after patients received written explanations of the study and its protocols. After ensuring all the details were fully understood, the patients provided written informed consent to participate.

Author contributions

YS and KA contributed to the conceptualisation and the design of the study and data interpretation.

KK analysed the NGS data. SN and TI supervised the bioinformatic analysis; KT, FS, and SI acquired the clinical data and the pleural effusion samples. YS drafted the manuscript and conducted statistical analyses. All authors contributed to the critical revision of the manuscript and approved its submission for publication.

Disclosure statement

The authors declare that they have no competing interests.

Data availability statement

The dataset used and/or analysed during the current study is available from the corresponding author on reasonable request.

Additional information

Funding

This study was supported by the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) of Japan Agency for Medical Research and Development (AMED) under Grant Number JP17fm0108023, and the Takeda Science Foundation.