Di Pilato Vincenzo, Bonaiuto Chiara, Morecchiato Fabio, Antonelli Alberto, Giani Tommaso, Rossolini Gian Maria
Department of Surgical Sciences and Integrated Diagnostics, University of Genoa, Genoa, Italy; Microbiology Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy.
Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; Clinical Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy.
EBioMedicine. 2025 Apr;114:105633. doi: 10.1016/j.ebiom.2025.105633. Epub 2025 Mar 17.
Blood culture (BC) remains the cornerstone for diagnosis of bloodstream infections (BSI), but the long turn-around time (TAT) hampers timely selection of appropriate chemotherapy. Novel molecular approaches have been developed to provide faster results but are also affected by limitations. We developed a analytical workflow named LC-WGS (Whole-Genome Sequencing of Liquid Colony) for rapid whole-genome sequencing-based diagnosis of BSI, evaluating its accuracy performance over standard of care (SoC) diagnostic procedures.
A total of 85 prospectively collected positive BC were processed in parallel with SoC (subculturing, identification by MALDI-ToF, antimicrobial susceptibility testing by reference broth microdilution, usage of syndromic panels) and LC-WGS, which relied on automated purification of microbial cells (Qvella FAST system, Qvella Corp.), DNA purification, and real-time sequencing with the Oxford Nanopore MinION. A streamlined analysis pipeline was designed for pathogen identification (Kraken2), detection of resistance markers (KmerResistance, AMRFinderPlus), virulome profiling (abricate, VFDB), phylogenetic analysis (snippy, IQ-TREE), and pathogen subtyping (Meningotype).
Compared with SoC, LC-WGS returned accurate species-level identification for 98% (65/66) of monomicrobial and 88% (14/16) of polymicrobial BCs, with a TAT as short as ∼2·6 h. Accurate resistome profiling (allelic variants) was achieved for 94% (58/62) of the most clinically-relevant resistance profiles in ∼4·2 h. In silico serotying (Neisseria meningitidis), virulotyping (Escherichia coli, Klebsiella pneumoniae) and comparative phylogenomics for outbreak investigation (K. pneumoniae) proved also feasible.
In this proof-of-concept study, we proved that diagnosis of BSI can be significantly shortened using an optimised workflow based on real-time sequencing, providing rapid, actionable clinical microbiological data in support of timely selection of appropriate chemotherapy. LC-WGS proved also useful as molecular epidemiology tool for public health and infection control applications.
This study was partially supported by an investigator-initiated grant from Qvella Corporation.
血培养（BC）仍然是血流感染（BSI）诊断的基石，但较长的周转时间（TAT）阻碍了及时选择合适的化疗方案。已开发出新型分子方法以提供更快的结果，但也受到局限性的影响。我们开发了一种名为LC-WGS（液体菌落全基因组测序）的分析工作流程，用于基于快速全基因组测序诊断BSI，并评估其相对于标准护理（SoC）诊断程序的准确性。
总共85份前瞻性收集的阳性血培养样本与SoC（传代培养、基质辅助激光解吸电离飞行时间质谱鉴定、参考肉汤微量稀释法进行抗菌药物敏感性测试、使用症状组合检测板）和LC-WGS并行处理，LC-WGS依赖于微生物细胞的自动纯化（Qvella FAST系统，Qvella公司）、DNA纯化以及使用牛津纳米孔MinION进行实时测序。设计了一个简化的分析流程用于病原体鉴定（Kraken2）、耐药标志物检测（KmerResistance、AMRFinderPlus）、毒力组分析（abricate、VFDB）、系统发育分析（snippy、IQ-TREE）以及病原体分型（Meningotype）。
与SoC相比，LC-WGS对98%（65/66）的单微生物血培养样本和88%（14/16）的多微生物血培养样本返回了准确的种水平鉴定结果，周转时间短至约2.6小时。在约4.2小时内，对94%（58/62）的最具临床相关性的耐药谱实现了准确的耐药组分析（等位基因变异）。计算机血清分型（脑膜炎奈瑟菌）、毒力分型（大肠杆菌、肺炎克雷伯菌）以及用于暴发调查的比较系统发育基因组学（肺炎克雷伯菌）也证明是可行的。
在这项概念验证研究中，我们证明了使用基于实时测序的优化工作流程可显著缩短BSI的诊断时间，提供快速、可操作的临床微生物学数据以支持及时选择合适的化疗方案。LC-WGS也被证明是用于公共卫生和感染控制应用的分子流行病学工具。
本研究部分得到了Qvella公司发起的研究者资助。
