Real-time sequencing-based diagnostics of bacteremia in septic patients

Background: The increasing incidence of bloodstream infections including sepsis is a major challenge in intensive care units worldwide. To improve patient's outcome, the causative pathogens need to be identified rapidly allowing for an immediate initiation of a targeted antimicrobial therapy. However, current diagnostics for pathogen identification mainly depend on culture- and molecular-based approaches, which are not satisfactory regarding specificity, sensitivity and time to diagnosis. Recently, a highly specific and sensitive diagnostic workflow for bloodstream infections based on unbiased sequence analyses of free circulating DNA (cfDNA) from plasma by next-generation sequencing (NGS) have been developed. However, time to diagnosis still took approximately 24-36 hours.Methods: In the present work we established a complete diagnostic workflow for real-time high-throughput sequencing based on nanopore sequencing. Accordingly, we optimized wetlab protocols and bioinformatic procedures in order to allow a more rapid identification of pathogens without losing specificity and sensitivity. This workflow was then applied to the analyses of samples from eight septic patients and three healthy controls and compared to standard NGS results.Results: We initially optimized standard library preparation protocols for short fragments with low input amounts and could achieve a 3.5-fold increase in sequencing throughput. With tailored bioinformatic workflows, all eight septic patient samples were found to be positive for the relevant pathogens. When considering time to diagnosis, depending on the microbial burden for seven of the eight samples first pathogens were already identified within minutes to one hour after start of sequencing. Moreover, a statistical extrapolation of real-time sequencing performance on a cohort with 239 septic patient samples validated by standard NGS diagnostic workflows revealed that more than 90 % of the pathogen hits would have also been detected with the optimized MinION workflow despite its currently still limited throughput and sequencing quality.Conclusions: Reliable identification of pathogens based on cfDNA sequencing using optimized workflows and real-time nanopore-based sequencing can be accomplished within 5-6 hours after blood draw. Therefore this approach might provide therapy-relevant results in a clinically critical timeframe.

背景：包括脓毒症在内的血流感染发病率持续攀升，是全球重症监护病房面临的重大临床挑战。为改善患者预后，需快速鉴定致病病原体，以便及时启动靶向抗菌治疗。然而，当前病原体鉴定诊断主要依赖培养法与分子生物学方法，在特异性、灵敏度及诊断耗时方面均不尽如人意。近年来，研究人员基于血浆游离循环DNA（free circulating DNA, cfDNA）的无偏倚测序分析，借助下一代测序（next-generation sequencing, NGS）技术开发出针对血流感染的高特异性、高灵敏度诊断流程，但该流程的诊断耗时仍约为24~36小时。
方法：本研究构建了一套基于纳米孔测序的实时高通量测序完整诊断流程。据此，我们对湿实验方案与生物信息学流程进行优化，在不损失特异性与灵敏度的前提下，实现了病原体的更快速鉴定。随后将该流程应用于8例脓毒症患者与3例健康对照的样本分析，并与标准NGS检测结果进行对比。
结果：我们首先针对低起始量的短片段优化了标准文库制备方案，使测序通量提升了3.5倍。借助定制化的生物信息学流程，8例脓毒症患者样本均成功检出目标病原体。就诊断耗时而言，根据微生物载量差异，8例样本中的7例可在测序启动后数分钟至1小时内完成首次病原体鉴定。此外，针对经标准NGS诊断流程验证的239例脓毒症患者队列进行实时测序性能的统计外推分析显示，尽管优化后的MinION测序流程当前的通量与测序质量仍存在局限，但仍可检出超过90%的病原体阳性样本。
结论：基于优化后的实验流程与实时纳米孔测序的cfDNA测序技术，可在采血后5~6小时内完成病原体的可靠鉴定。因此，该方法可在临床救治的关键时间窗内提供与治疗决策相关的检测结果。