Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

Background
Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI.
Methodology/Principal Findings
The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030).
Conclusions/Significance
Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination.

背景
细菌性血流感染（bacterial bloodstream infection, bBSI）是重症患者致死的主要病因之一，因此精准诊断具有至关重要的临床意义。本研究报道一种16S宏基因组学（16S metagenomics）方法，用于细菌性血流感染的诊断与病理机制解析。

研究方法与主要结果
本概念验证研究纳入布基纳法索75名罹患重症发热性疾病的儿童，其年龄中位数为15个月。受试者入院时均接受标准血培养与疟疾检测。随后对所有患者的血液样本进行回顾性、重复16S宏基因组学检测。从血液样本中提取总DNA，通过聚合酶链式反应（Polymerase Chain Reaction, PCR）扩增细菌16S核糖体RNA（16S ribosomal RNA, 16S rRNA）基因的V3-V4区域，并在Illumina MiSeq测序仪上进行深度测序。对双端测序读段进行质控整理、分类注释与过滤。血培养确诊12例细菌性血流感染，若联合血培养与16S宏基因组学检测结果，则确诊病例数增至22例。相较于标准血培养，16S宏基因组学不仅灵敏度更优，还为细菌性血流感染的病理特征提供了重要新见解。与近期无疟疾诊断史的患者相比，合并急性疟疾或正处于疟疾恢复期的患者发生多菌型血流感染的风险高7倍（p值=0.046）。已知疟疾会损伤肠道上皮功能，进而可能促进细菌从肠腔移位至血液。值得关注的是，此类多菌型血流感染患者死于发热性疾病的风险高9倍（p值=0.030）。

研究结论与意义
本研究数据表明，16S宏基因组学是诊断与解析细菌性血流感染的高效手段。本概念验证研究同时证实，设置恰当的对照样本对于消除环境污染导致的背景信号至关重要。