Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection
Abstract Background The timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteri...
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | BMC Medical Genomics |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12920-024-01835-5 |
| _version_ | 1797272934188843008 |
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| author | Lluis Moragues-Solanas Thanh Le-Viet Elinor McSorley Carl Halford Daniel S. Lockhart Alp Aydin Gemma L. Kay Ngozi Elumogo William Mullen Justin O’Grady Matthew W. Gilmour |
| author_facet | Lluis Moragues-Solanas Thanh Le-Viet Elinor McSorley Carl Halford Daniel S. Lockhart Alp Aydin Gemma L. Kay Ngozi Elumogo William Mullen Justin O’Grady Matthew W. Gilmour |
| author_sort | Lluis Moragues-Solanas |
| collection | DOAJ |
| description | Abstract Background The timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteria. Long-read sequencing technologies have successfully supported the use of clinical metagenomics for syndromes such as respiratory illness, and modified approaches may address two requisite factors for metagenomics to be used as a BSI diagnostic: depletion of the high level of host DNA to then detect the low abundance of microbes in blood. Methods Blood samples from healthy donors were spiked with different concentrations of four prevalent causative species of BSI. All samples were then subjected to a modified saponin-based host DNA depletion protocol and optimised DNA extraction, whole genome amplification and debranching steps in preparation for sequencing, followed by bioinformatical analyses. Two related variants of the protocol are presented: 1mL of blood processed without bacterial enrichment, and 5mL of blood processed following a rapid bacterial enrichment protocol—SepsiPURE. Results After first identifying that a large proportion of host mitochondrial DNA remained, the host depletion process was optimised by increasing saponin concentration to 3% and scaling the reaction to allow more sample volume. Compared to non-depleted controls, the 3% saponin-based depletion protocol reduced the presence of host chromosomal and mitochondrial DNA < 106 and < 103 fold respectively. When the modified depletion method was further combined with a rapid bacterial enrichment method (SepsiPURE; with 5mL blood samples) the depletion of mitochondrial DNA improved by a further > 10X while also increasing detectable bacteria by > 10X. Parameters during DNA extraction, whole genome amplification and long-read sequencing were also adjusted, and subsequently amplicons were detected for each input bacterial species at each of the spiked concentrations, ranging from 50–100 colony forming units (CFU)/mL to 1–5 CFU/mL. Conclusion In this proof-of-concept study, four prevalent BSI causative species were detected in under 12 h to species level (with antimicrobial resistance determinants) at concentrations relevant to clinical blood samples. The use of a rapid and precise metagenomic protocols has the potential to advance the diagnosis of BSI. |
| first_indexed | 2024-03-07T14:36:25Z |
| format | Article |
| id | doaj.art-fc3de77a33654a87a777998ef481a779 |
| institution | Directory Open Access Journal |
| issn | 1755-8794 |
| language | English |
| last_indexed | 2024-03-07T14:36:25Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Medical Genomics |
| spelling | doaj.art-fc3de77a33654a87a777998ef481a7792024-03-05T20:39:20ZengBMCBMC Medical Genomics1755-87942024-03-0117111810.1186/s12920-024-01835-5Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infectionLluis Moragues-Solanas0Thanh Le-Viet1Elinor McSorley2Carl Halford3Daniel S. Lockhart4Alp Aydin5Gemma L. Kay6Ngozi Elumogo7William Mullen8Justin O’Grady9Matthew W. Gilmour10Quadram Institute Bioscience, Norwich Research ParkQuadram Institute Bioscience, Norwich Research ParkMomentum Bioscience LtdUniversity of East AngliaMomentum Bioscience LtdQuadram Institute Bioscience, Norwich Research ParkQuadram Institute Bioscience, Norwich Research ParkQuadram Institute Bioscience, Norwich Research ParkMomentum Bioscience LtdUniversity of East AngliaQuadram Institute Bioscience, Norwich Research ParkAbstract Background The timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteria. Long-read sequencing technologies have successfully supported the use of clinical metagenomics for syndromes such as respiratory illness, and modified approaches may address two requisite factors for metagenomics to be used as a BSI diagnostic: depletion of the high level of host DNA to then detect the low abundance of microbes in blood. Methods Blood samples from healthy donors were spiked with different concentrations of four prevalent causative species of BSI. All samples were then subjected to a modified saponin-based host DNA depletion protocol and optimised DNA extraction, whole genome amplification and debranching steps in preparation for sequencing, followed by bioinformatical analyses. Two related variants of the protocol are presented: 1mL of blood processed without bacterial enrichment, and 5mL of blood processed following a rapid bacterial enrichment protocol—SepsiPURE. Results After first identifying that a large proportion of host mitochondrial DNA remained, the host depletion process was optimised by increasing saponin concentration to 3% and scaling the reaction to allow more sample volume. Compared to non-depleted controls, the 3% saponin-based depletion protocol reduced the presence of host chromosomal and mitochondrial DNA < 106 and < 103 fold respectively. When the modified depletion method was further combined with a rapid bacterial enrichment method (SepsiPURE; with 5mL blood samples) the depletion of mitochondrial DNA improved by a further > 10X while also increasing detectable bacteria by > 10X. Parameters during DNA extraction, whole genome amplification and long-read sequencing were also adjusted, and subsequently amplicons were detected for each input bacterial species at each of the spiked concentrations, ranging from 50–100 colony forming units (CFU)/mL to 1–5 CFU/mL. Conclusion In this proof-of-concept study, four prevalent BSI causative species were detected in under 12 h to species level (with antimicrobial resistance determinants) at concentrations relevant to clinical blood samples. The use of a rapid and precise metagenomic protocols has the potential to advance the diagnosis of BSI.https://doi.org/10.1186/s12920-024-01835-5Clinical diagnosticsMetagenomicsBacteraemiaSepsisBacterial infectionLong-read sequencing |
| spellingShingle | Lluis Moragues-Solanas Thanh Le-Viet Elinor McSorley Carl Halford Daniel S. Lockhart Alp Aydin Gemma L. Kay Ngozi Elumogo William Mullen Justin O’Grady Matthew W. Gilmour Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection BMC Medical Genomics Clinical diagnostics Metagenomics Bacteraemia Sepsis Bacterial infection Long-read sequencing |
| title | Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection |
| title_full | Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection |
| title_fullStr | Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection |
| title_full_unstemmed | Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection |
| title_short | Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection |
| title_sort | development and proof of concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection |
| topic | Clinical diagnostics Metagenomics Bacteraemia Sepsis Bacterial infection Long-read sequencing |
| url | https://doi.org/10.1186/s12920-024-01835-5 |
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