Division of Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Isehara, Japan.
Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan.
Infect Dis (Lond). 2021 Jun;53(6):450-459. doi: 10.1080/23744235.2021.1892178. Epub 2021 Mar 9.
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).
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.
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 sp. ( = 6), sp. ( = 5), sp. ( = 5), and sp. ( = 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 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.
Combining NGS with semi-quantitative PCR can facilitate the diagnosis of pleural empyema/parapneumonic effusion and its causal bacteria.
尽管微生物学诊断在临床上对胸腔感染很重要，但由于先前的抗生素治疗和/或某些细菌种类的培养困难，通常会使诊断变得复杂。因此，我们旨在通过结合 16S 核糖体 RNA（rRNA）基因扩增和下一代测序（NGS）来鉴定胸腔积脓/肺炎旁胸腔积液中的可能致病细菌。
从 19 例感染性胸腔积液和 9 例非感染性恶性胸腔积液患者中收集胸腔积液。我们使用 Genome Search Toolkit 和 GenomeSync 数据库，通过 NGS 分析从胸腔积液上清液中提取的 DNA，或者直接分析，或者在扩增 16S rRNA 基因后进行分析。通过 16S rRNA 基因的半定量 PCR 区分感染性和非感染性积液。
仅 8 例（42%）积液为培养阳性，然而，16S rRNA 基因扩增子的 NGS 在所有患者中均鉴定出 14 种厌氧菌和 7 种需氧菌/兼性厌氧菌，包括 sp.（ = 6）、sp.（ = 5）、sp.（ = 5）和 sp.（ = 4），占总基因组的>10%。8 例中有 3 例培养和 NGS 结果不一致，这 3 例患者均有先前的抗生素治疗史。16S rRNA 基因半定量 PCR 的总（2 值）和特定（总细菌负荷乘以 NGS 中主要细菌的比例）细菌负荷可有效区分脓胸/肺炎旁胸腔积液和非感染性胸腔积液。
将 NGS 与半定量 PCR 相结合可以促进胸腔积脓/肺炎旁胸腔积液及其致病细菌的诊断。
