Next-generation diagnostics of bloodstream infections enabled by rapid whole-genome sequencing of bacterial cells purified from blood cultures.

IF 9.7 1区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

EBioMedicine Pub Date : 2025-03-17 DOI:10.1016/j.ebiom.2025.105633

Vincenzo Di Pilato, Chiara Bonaiuto, Fabio Morecchiato, Alberto Antonelli, Tommaso Giani, Gian Maria Rossolini

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引用次数: 0

Abstract

Background: 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.

Methods: 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).

Findings: 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.

Interpretation: 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.

Funding: This study was partially supported by an investigator-initiated grant from Qvella Corporation.

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通过对血液培养物中纯化的细菌细胞进行快速全基因组测序，实现血液感染的下一代诊断。

背景：血培养（BC）仍然是血流感染（BSI）诊断的基础，但较长的周转时间（TAT）阻碍了及时选择合适的化疗方案。新的分子方法已经开发出来，提供更快的结果，但也受到限制的影响。我们开发了一种名为LC-WGS（全基因组测序的液体菌落）的分析工作流程，用于基于全基因组测序的BSI快速诊断，评估其在标准护理（SoC）诊断程序中的准确性。方法：共85例前瞻性收集的阳性BC与SoC（传代培养，MALDI-ToF鉴定，参考培养液微量稀释抗菌药敏试验，使用综合征面板）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 Corp.）， DNA纯化和Oxford Nanopore MinION实时测序。为病原菌鉴定（Kraken2）、抗性标记检测（KmerResistance、AMRFinderPlus）、病毒组分析（abricate、VFDB）、系统发育分析（snippy、IQ-TREE）和病原菌亚型分型（脑膜炎型）设计了简化的分析流程。发现:与SoC相比，LC-WGS对98%（65/66）的单微生物和88%（14/16）的多微生物bc进行了准确的物种水平鉴定，TAT短至2·6小时。在4·2小时内，对94%（58/62）的最具临床相关性的耐药谱进行了准确的抗性组谱分析（等位基因变异）。肺炎克雷伯菌)和比较系统基因组学的爆发调查（肺炎克雷伯菌）也证明是可行的。解释：在这项概念验证研究中，我们证明了基于实时测序的优化工作流程可以显著缩短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也被证明是公共卫生和感染控制应用的分子流行病学工具。经费：本研究部分由Qvella Corporation的研究者发起的拨款支持。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

EBioMedicine Biochemistry, Genetics and Molecular Biology-General Biochemistry,Genetics and Molecular Biology

CiteScore

17.70

自引率

0.90%

发文量

579

审稿时长

5 weeks

期刊介绍： eBioMedicine is a comprehensive biomedical research journal that covers a wide range of studies that are relevant to human health. Our focus is on original research that explores the fundamental factors influencing human health and disease, including the discovery of new therapeutic targets and treatments, the identification of biomarkers and diagnostic tools, and the investigation and modification of disease pathways and mechanisms. We welcome studies from any biomedical discipline that contribute to our understanding of disease and aim to improve human health.