Diagnosis of pleural empyema/parapneumonic effusion by next-generation sequencing
Diagnosis of pleural empyema/parapneumonic effusion by next-generation sequencing
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DOI：
10.1080/23744235.2021.1892178
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发表时间：
2021-02-18
影响因子：
5.8
通讯作者：
Asano, Koichiro
中科院分区：
文献类型：
--
作者：
Shiraishi, Yoshiki;Kryukov, Kirill;Asano, Koichiro
背景尽管胸膜感染的微生物诊断在临床上很重要，但它通常因先前的抗生素治疗和/或某些细菌培养困难而变得复杂。因此，我们的目的是确定可能的病原菌在胸膜脓胸/胸膜旁积液相结合的16 S核糖体RNA（rRNA）基因扩增和下一代测序（NGS）。Methods胸膜液收集19例感染性积液和9例非感染性恶性积液。我们使用基因组搜索工具包和GenomeSync数据库，直接或在16 S rRNA基因的PCR扩增后，通过NGS分析了从胸膜液上清液中提取的DNA。结果仅8例（42%）胸腔积液培养阳性，但16 S rRNA基因扩增产物的NGS在所有患者中鉴定出14种厌氧菌和7种需氧菌/兼性厌氧菌，包括链球菌属（n = 6）、梭杆菌属（n = 5）、卟啉单胞菌属（n = 5）、和普雷沃氏菌（Prevotella sp.）（n = 4），占总基因组的10%以上。8例患者中有3例的培养和NGS结果不一致，所有患者之前均接受过抗生素治疗。16 SrRNA基因半定量PCR的总菌量（Δ CT值为2）和特异性菌量（总菌量乘以NGS中原发菌的比例）可有效区分脓胸/胸膜旁积液与非感染性积液。
BackgroundAlthough 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).MethodsPleural 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.ResultsOnly 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(Delta CT) 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.ConclusionCombining NGS with semi-quantitative PCR can facilitate the diagnosis of pleural empyema/parapneumonic effusion and its causal bacteria.