New next-generation sequencing technique dramatically shortens diagnosis of sepsis

Availability of results within 5 to 6 hours of blood draw allows faster and more precise intervention when time is of the essence, reports The Journal of Molecular Diagnostics


Philadelphia, March 5, 2020

A report in The Journal of Molecular Diagnostics, published by Elsevier, describes a new technique that uses real-time next-generation sequencing (NGS) to analyze tiny amounts of microbial cell-free DNA in the plasma of patients with sepsis, offering the possibility of accurate diagnosis of sepsis-causing agents within a few hours of drawing blood. Current diagnostic tests are neither fast nor specific enough to provide timely, critically important information.

“With up to 50 million incident sepsis cases and 11 million sepsis-related deaths per year, sepsis represents a major cause of health loss,” explained co-lead-investigator Thorsten Brenner, MD, Vice Head of the Department of Anesthesiology, Heidelberg University Hospital, Heidelberg, Germany. “Reliable and early identification of the pathogen enables rapid and the most appropriate antibiotic intervention, thereby increasing the chance of better outcomes and patient survival. Currently, standard-of-care diagnostics still rely on microbiological culturing of the respective pathogens, which in most cases (70 to 90 percent) do not provide timely positive results.”

NGS encompasses several advanced processes used to sequence the nucleotides in DNA. In previous work, the investigators described a system that utilized NGS of microbial cell-free DNA to detect blood pathogens that was highly sensitive and faster than standard blood cultures (within 28 hours). However, their ultimate goal was to develop an even faster test to expedite treatment.

To overcome this limitation the investigators established a diagnostic workflow based on third-generation high-throughput (nanopore) sequencing of microbial DNA. Normally, nanopore sequencing is used to analyze long fragments in sufficient amounts. However, microbial cell-free DNA occurs in small fragments and low quantities in plasma. Nanopore sequencing offers the possibility of real-time analyses during sequencing, which dramatically reduces the time needed to obtain results. “We also had to create validated, specifically adapted bioinformatic procedures to reliably identify pathogens,” noted principal investigator Kai Sohn, PhD, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany.

This new technique relies on the use of a handheld nanopore sequencer known as the MinION. This device is portable, can read out ultra-long reads and immediately processes reads in real time.


Nanopore sequencer used for real-time microbial cell-free DNA analyses in septic patients. Courtesy of Fraunhofer IGBI.

In this proof-of-concept study, the investigators analyzed plasma from four septic patients and three healthy controls who were hospitalized in the ICU. Each sample’s DNA underwent sequencing using both technologies: the standard NGS (Illumina) and the new nanopore NGS technology. With nanopore sequencing, all septic patient samples were found to be positive for relevant pathogens, whether bacterial, viral, or fungal.

After additional refinements, the new technique was able to achieve a 3.5-fold increase in sequencing throughput, allowing pathogen identification within minutes after sequencing began. In fact, the highest quality results were generated within two or three hours of the beginning of sequencing. In contrast, with Illumina the final results are available only after the sequencing is finished. “This new system might facilitate same-shift adaption of antibiotic intervention at the ICU, which might, in turn, improve patient outcomes significantly,” commented Dr. Sohn.

The investigators also compared the probability of pathogen detection with the MinION system to the highly accurate and clinically validated Illumina NGS technique in a retrospective analysis of 239 samples taken from sepsis patients. Although the accuracy of nanopore sequencing was lower than with Illumina (approximately 85 percent vs. 99 percent), they found a strong correlation between the findings generated by MinION vs. Illumina.

“Time consuming, error- and contamination-prone blood cultures are still considered as the standard of care for sepsis diagnostics, frequently leading to an inappropriate and delayed targeted therapy,” said Prof. Dr. Brenner. “The nanopore sequencing platform sequences in real time and has the potential to reduce time to diagnosis to only a few hours.”

---

Notes for editors
The article is “Rapid Next-Generation Sequencing Based—Diagnostics of Bacteremia in Septic Patients,” by Christian Grumaz, Anne Hoffmann, Yevhen Vainshtein, Maria Kopp, Silke Grumaz, Philip Stevens, Sebastian O. Decker, Markus A. Weigand, Stefan Hofer, Thorsten Brenner, and Kai Sohn (https://doi.org/10.1016/j.jmoldx.2019.12.006). Also of interest “Commentary: Diagnosing Bacteremia in Real Time Using Next-Generation Sequencing—Based Technology,” by Charles W. Stratton and Yi-Wei Tang (https://doi.org/10.1016/j.jmoldx.2020.01.002). They appear in The Journal of Molecular Diagnostics, volume 22, issue 3 (March 2020) published by Elsevier.

Full text of this study and commentary is available to credentialed journalists upon request; contact Eileen Leahy at +1 732 238 3628 or jmdmedia@elsevier.com. Journalists wishing to interview the authors should contact Kai Sohn at kai.sohn@igb.fraunhofer.de.

This study was supported by the European Commission, Directorate-General for Research & Innovation (Grant Health-F3-2013-602125).

About The Journal of Molecular Diagnostics 
The Journal of Molecular Diagnostics, the official publication of the Association for Molecular Pathology, co-owned by the American Society for Investigative Pathology, and published by Elsevier, seeks to publish high quality original papers on scientific advances in the translation and validation of molecular discoveries in medicine into the clinical diagnostic setting, and the description and application of technological advances in the field of molecular diagnostic medicine. The editors welcome review articles that contain: novel discoveries or clinicopathologic correlations, including studies in oncology, infectious diseases, inherited diseases, predisposition to disease, or the description of polymorphisms linked to disease states or normal variations; the application of diagnostic methodologies in clinical trials; or the development of new or improved molecular methods for diagnosis or monitoring of disease or disease predisposition. jmd.amjpathol.org

About Elsevier
Elsevier is a global information analytics business that helps scientists and clinicians to find new answers, reshape human knowledge, and tackle the most urgent human crises. For 140 years, we have partnered with the research world to curate and verify scientific knowledge. Today, we’re committed to bringing that rigor to a new generation of platforms. Elsevier provides digital solutions and tools in the areas of strategic research management, R&D performance, clinical decision support, and professional education; including ScienceDirect, Scopus, SciVal, ClinicalKey and Sherpath. Elsevier publishes over 2,500 digitized journals, including The Lancet and Cell, 39,000 e-book titles and many iconic reference works, including Gray's Anatomy. Elsevier is part of RELX, a global provider of information-based analytics and decision tools for professional and business customers. www.elsevier.com

Media contacts
Eileen Leahy
Elsevier
+1 732 238 3628
jmdmedia@elsevier.com

Chhavi Chauhan, PhD, Director of Scientific Outreach
The Journal of Molecular Diagnostics
+1 240 283 9724
cchauhan@asip.org