Unified metagenomic method for rapid detection of microorganisms in clinical samples

Adela Alcolea-Medina; Christopher Alder; Luke B. Snell; Themoula Charalampous; Alp Aydin; Gaia Nebbia; Tom Williams; Simon Goldenberg; Sam Douthwaite; Rahul Batra; Penelope R. Cliff; Hannah Mischo; Stuart Neil; Mark Wilks; Jonathan D. Edgeworth

doi:10.1038/s43856-024-00554-3

Unified metagenomic method for rapid detection of microorganisms in clinical samples

Adela Alcolea-Medina^*, Christopher Alder, Luke B. Snell, Themoula Charalampous, Alp Aydin, Gaia Nebbia, Tom Williams, Simon Goldenberg, Sam Douthwaite, Rahul Batra, Penelope R. Cliff, Hannah Mischo, Stuart Neil, Mark Wilks, Jonathan D. Edgeworth

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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Abstract

Background: Clinical metagenomics involves the genomic sequencing of all microorganisms in clinical samples ideally after depletion of human DNA to increase sensitivity and reduce turnaround times. Current human DNA depletion methods preferentially preserve either DNA or RNA containing microbes, but not both simultaneously. Here we describe and present data using a practical and rapid mechanical host-depletion method allowing simultaneous detection of RNA and DNA microorganisms linked with nanopore sequencing. Methods: The human cells from respiratory samples are lysed mechanically using 1.4 mm zirconium-silicate spheres and the human DNA is depleted using a nonspecific endonuclease. The RNA is converted to dsDNA to allow the simultaneous sequencing of DNA and RNA. Results: The method decreases human DNA concentration by a median of eight Ct values while detecting a broad range of RNA & DNA viruses, bacteria, including atypical pathogens (Legionella, Chlamydia, Mycoplasma) and fungi (Candida, Pneumocystis, Aspergillus). The first automated reports are generated after 30 min sequencing from a 7 h end-to-end workflow. Sensitivity and specificity for bacterial detection are 90% and 100%, respectively, and viral detection are 92% and 100% after 2 h of sequencing. Prospective validation on 33 consecutive lower respiratory tract samples from ventilated patients with suspected pneumonia shows 60% concordance with routine testing, detection of additional pathogens in 21% of samples and pathogen genomic assembly achieve for 42% of viruses and 33% of bacteria. Conclusions: Although further workflow refinement and validation on samples containing a broader range of pathogens is required, it holds promise as a clinically deployable workflow suitable for evaluation in routine microbiology laboratories.

Original language	English
Article number	135
Journal	Communications Medicine
Volume	4
Issue number	1
Early online date	7 Jul 2024
DOIs	https://doi.org/10.1038/s43856-024-00554-3
Publication status	Published - Dec 2024

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Alcolea-Medina, A., Alder, C., Snell, L. B., Charalampous, T., Aydin, A., Nebbia, G., Williams, T., Goldenberg, S., Douthwaite, S., Batra, R., Cliff, P. R., Mischo, H., Neil, S., Wilks, M., & Edgeworth, J. D. (2024). Unified metagenomic method for rapid detection of microorganisms in clinical samples. Communications Medicine, 4(1), Article 135. https://doi.org/10.1038/s43856-024-00554-3

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title = "Unified metagenomic method for rapid detection of microorganisms in clinical samples",

abstract = "Background: Clinical metagenomics involves the genomic sequencing of all microorganisms in clinical samples ideally after depletion of human DNA to increase sensitivity and reduce turnaround times. Current human DNA depletion methods preferentially preserve either DNA or RNA containing microbes, but not both simultaneously. Here we describe and present data using a practical and rapid mechanical host-depletion method allowing simultaneous detection of RNA and DNA microorganisms linked with nanopore sequencing. Methods: The human cells from respiratory samples are lysed mechanically using 1.4 mm zirconium-silicate spheres and the human DNA is depleted using a nonspecific endonuclease. The RNA is converted to dsDNA to allow the simultaneous sequencing of DNA and RNA. Results: The method decreases human DNA concentration by a median of eight Ct values while detecting a broad range of RNA & DNA viruses, bacteria, including atypical pathogens (Legionella, Chlamydia, Mycoplasma) and fungi (Candida, Pneumocystis, Aspergillus). The first automated reports are generated after 30 min sequencing from a 7 h end-to-end workflow. Sensitivity and specificity for bacterial detection are 90% and 100%, respectively, and viral detection are 92% and 100% after 2 h of sequencing. Prospective validation on 33 consecutive lower respiratory tract samples from ventilated patients with suspected pneumonia shows 60% concordance with routine testing, detection of additional pathogens in 21% of samples and pathogen genomic assembly achieve for 42% of viruses and 33% of bacteria. Conclusions: Although further workflow refinement and validation on samples containing a broader range of pathogens is required, it holds promise as a clinically deployable workflow suitable for evaluation in routine microbiology laboratories.",

author = "Adela Alcolea-Medina and Christopher Alder and Snell, {Luke B.} and Themoula Charalampous and Alp Aydin and Gaia Nebbia and Tom Williams and Simon Goldenberg and Sam Douthwaite and Rahul Batra and Cliff, {Penelope R.} and Hannah Mischo and Stuart Neil and Mark Wilks and Edgeworth, {Jonathan D.}",

note = "Publisher Copyright: {\textcopyright} The Author(s) 2024. corrected publication 2024.",

year = "2024",

month = dec,

doi = "10.1038/s43856-024-00554-3",

language = "English",

volume = "4",

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T1 - Unified metagenomic method for rapid detection of microorganisms in clinical samples

AU - Alcolea-Medina, Adela

AU - Alder, Christopher

AU - Snell, Luke B.

AU - Charalampous, Themoula

AU - Aydin, Alp

AU - Nebbia, Gaia

AU - Williams, Tom

AU - Goldenberg, Simon

AU - Douthwaite, Sam

AU - Batra, Rahul

AU - Cliff, Penelope R.

AU - Mischo, Hannah

AU - Neil, Stuart

AU - Wilks, Mark

AU - Edgeworth, Jonathan D.

PY - 2024/12

Y1 - 2024/12

N2 - Background: Clinical metagenomics involves the genomic sequencing of all microorganisms in clinical samples ideally after depletion of human DNA to increase sensitivity and reduce turnaround times. Current human DNA depletion methods preferentially preserve either DNA or RNA containing microbes, but not both simultaneously. Here we describe and present data using a practical and rapid mechanical host-depletion method allowing simultaneous detection of RNA and DNA microorganisms linked with nanopore sequencing. Methods: The human cells from respiratory samples are lysed mechanically using 1.4 mm zirconium-silicate spheres and the human DNA is depleted using a nonspecific endonuclease. The RNA is converted to dsDNA to allow the simultaneous sequencing of DNA and RNA. Results: The method decreases human DNA concentration by a median of eight Ct values while detecting a broad range of RNA & DNA viruses, bacteria, including atypical pathogens (Legionella, Chlamydia, Mycoplasma) and fungi (Candida, Pneumocystis, Aspergillus). The first automated reports are generated after 30 min sequencing from a 7 h end-to-end workflow. Sensitivity and specificity for bacterial detection are 90% and 100%, respectively, and viral detection are 92% and 100% after 2 h of sequencing. Prospective validation on 33 consecutive lower respiratory tract samples from ventilated patients with suspected pneumonia shows 60% concordance with routine testing, detection of additional pathogens in 21% of samples and pathogen genomic assembly achieve for 42% of viruses and 33% of bacteria. Conclusions: Although further workflow refinement and validation on samples containing a broader range of pathogens is required, it holds promise as a clinically deployable workflow suitable for evaluation in routine microbiology laboratories.

AB - Background: Clinical metagenomics involves the genomic sequencing of all microorganisms in clinical samples ideally after depletion of human DNA to increase sensitivity and reduce turnaround times. Current human DNA depletion methods preferentially preserve either DNA or RNA containing microbes, but not both simultaneously. Here we describe and present data using a practical and rapid mechanical host-depletion method allowing simultaneous detection of RNA and DNA microorganisms linked with nanopore sequencing. Methods: The human cells from respiratory samples are lysed mechanically using 1.4 mm zirconium-silicate spheres and the human DNA is depleted using a nonspecific endonuclease. The RNA is converted to dsDNA to allow the simultaneous sequencing of DNA and RNA. Results: The method decreases human DNA concentration by a median of eight Ct values while detecting a broad range of RNA & DNA viruses, bacteria, including atypical pathogens (Legionella, Chlamydia, Mycoplasma) and fungi (Candida, Pneumocystis, Aspergillus). The first automated reports are generated after 30 min sequencing from a 7 h end-to-end workflow. Sensitivity and specificity for bacterial detection are 90% and 100%, respectively, and viral detection are 92% and 100% after 2 h of sequencing. Prospective validation on 33 consecutive lower respiratory tract samples from ventilated patients with suspected pneumonia shows 60% concordance with routine testing, detection of additional pathogens in 21% of samples and pathogen genomic assembly achieve for 42% of viruses and 33% of bacteria. Conclusions: Although further workflow refinement and validation on samples containing a broader range of pathogens is required, it holds promise as a clinically deployable workflow suitable for evaluation in routine microbiology laboratories.

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DO - 10.1038/s43856-024-00554-3

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SN - 2730-664X

VL - 4

JO - Communications Medicine

JF - Communications Medicine

IS - 1

M1 - 135

ER -

Unified metagenomic method for rapid detection of microorganisms in clinical samples

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