ena-STUDY-FRAUNHOFER IGB-23-08-2018-12:42:25:473-13
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.
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.