Research on a Unified Metagenomic Method for Rapid Detection of Microorganisms in Clinical Samples

Background Introduction

The background of this research is based on the current limitations of clinical metagenomics. Clinical metagenomics involves genome sequencing of all microorganisms in clinical samples, ideally performed after depleting human DNA to enhance sensitivity and reduce turnaround time. However, current methods for human DNA depletion often only preferentially retain microorganisms containing DNA or RNA, but not both. This study aims to describe and demonstrate a practical and rapid mechanical host-depletion method that allows for the simultaneous detection of RNA and DNA microorganisms through nanopore sequencing.

Origin

This paper is collaboratively authored by Adela Alcolea-Medina, Christopher Alder, Luke B. Snell, Themoula Charalampous, and others, all from several academic and medical institutions in London, UK, including Synnovis, Guy’s and St. Thomas’ NHS Foundation Trust, King’s College London, Quadram Institute Bioscience, among others. The paper was published in the journal Communications Medicine in 2024.

Research Process

Research Process and Technical Methods

The research adopts a multi-step process:

1. Mechanical Lysis of Human Cells: Using 1.4mm zirconium-silicate microbeads to mechanically lyse human cells in respiratory samples.
2. Use of Nonspecific Endonuclease: Employing a nonspecific endonuclease to deplete human DNA.
3. Conversion of RNA to Double-Stranded DNA: Converting RNA into double-stranded DNA (dsDNA) to allow for simultaneous sequencing of DNA and RNA microorganisms.

Specific steps include:

• Sample Centrifugation: Centrifuging the sample at 1200g for 10 minutes to sediment human cells.
• Bead Beating Lysis: Performing bead beating lysis on 500μl of supernatant in a 2ml Lysis Matrix D at a speed of 50 oscillations/s for 3 minutes to lyse human cells.
• Nucleic Acid Digestion: Adding 10μl of HL-SAN nuclease and digesting at 37°C for 10 minutes to digest released human nucleic acids.
• Nucleic Acid Extraction: Extracting DNA and RNA using the MagNA Pure 24 system from Roche.

Main Experimental Data and Results

The method demonstrated excellent performance data in different trials:

1. Human DNA Depletion: Quantitative PCR measurement showed a median decrease of 7 cycles in Ct values before and after the human DNA depletion step in 29 samples.
2. Microbial Detection Performance: Detected a wide range of viruses, bacteria, and fungi, with a very fast turnaround time for generating preliminary reports.
3. Sensitivity and Specificity: Sensitivity for bacterial detection was 90%, specificity was 100%; sensitivity for viral detection was 92%, specificity was 100%, after 2 hours of sequencing.
4. Prospective Validation: Validation on 33 lower respiratory tract samples showed a 60% concordance rate with routine detection results, with 21% of samples detecting additional pathogens.

The experiment combined a mechanical host depletion method with nanopore sequencing, demonstrating a practical and rapid process capable of generating preliminary automated reports within a 7-hour end-to-end workflow. The performance data fully supports its potential for clinical application.

Research Conclusions

This study describes a rapid and unified metagenomic detection method, demonstrating its potential application in clinical laboratories. The scientific and application value of this method includes:

1. Rapid Identification and Characterization of Pathogens: Enables rapid identification and characterization of all pathogenic microorganisms in clinical samples during the early management of acute infections.
2. Resolve Existing Method Limitations: Existing methods are insufficient in retaining microorganisms with different physical and chemical properties and abundances. The new method overcomes this challenge through mechanical host depletion.
3. Broad Application Prospects: Demonstrated good detection results in samples containing multiple pathogens, showing potential for evaluation and application in routine microbiology laboratories.

Research Highlights and Innovations

1. Efficient Host DNA Depletion: The mechanical method reduced human DNA within 8 cycles, significantly improving the sensitivity of microbial sequence detection.
2. Unified Detection of RNA and DNA Microorganisms: Traditional methods often cannot effectively detect RNA and DNA microorganisms simultaneously. The new method enables the conversion of RNA to double-stranded DNA, making detection feasible.
3. Rapid Turnaround Time: The time from sample processing to preliminary report generation is only 7 hours, making it very suitable for the rapid needs of clinical laboratories.

Other Valuable Information

The study also mentions the necessity of further improving the workflow and validating it on a broader range of pathogenic samples. However, current data suggests that the method has the potential to become a clinically usable workflow, suitable for evaluation in routine microbiology laboratories. This research provides new ideas and solutions for future pathogen detection and management.

Through this study, we see the potential of clinical metagenomics in early-stage management of acute infections, offering a powerful tool for the rapid identification and characterization of pathogens, thereby improving diagnostic and treatment efficiency. In the context of continuous technological advancement, the practical application of metagenomics is poised for new development opportunities.