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ToolsMoragues-Solanas, Lluis, Le-Viet, Thanh, McSorley, Elinor, Halford, Carl, Lockhart, Daniel S., Aydin, Alp, Kay, Gemma L., Elumogo, Ngozi, Mullen, William, O'Grady, Justin and Gilmour, Matthew W. (2024) Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection. BMC Medical Genomics, 17. ISSN 1755-8794
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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.
| Item Type: | Article |
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| Additional Information: | Availability of data and materials: All the sequencing data supporting the conclusions of this article are available at the European Nucleotide Archive (ENA) repository under accession PRJJEB64522 (https://www.ebi.ac.uk/ena/browser/view/PRJEB64522). The accession and run numbers for each sample can be found on Additional file 2: Table S5. Funding Information: LMS is funded by the MRC Doctoral Antimicrobial Research Training (DART) Industrial CASE Programme Project grant number MR/R015937/1. TLV was supported by the Quadram Institute Bioscience BBSRC funded Core Capability Grant (project number BB/CCG1860/1). CH is funded by the UK Ministry of Defence. This research was funded by the BBSRC Institute Strategic Programme Microbes in the Food Chain BB/R012504/1 and its constituent project BBS/E/F/000PR10352 (Theme 4, Research Infrastructure), the BBSRC Institute Strategic Programme Microbes and Food Safety BB/X011011/1 and its constituent project BBS/E/F/000PR13636 (Theme 3, Flexible capabilities to reduce food safety threats and respond to national needs) and Innovate UK project TS/S00887X/1. |
| Uncontrolled Keywords: | bacteraemia,bacterial infection,clinical diagnostics,long-read sequencing,metagenomics,sepsis,genetics,genetics(clinical) ,/dk/atira/pure/subjectarea/asjc/1300/1311 |
| Faculty \ School: | Faculty of Medicine and Health Sciences > Norwich Medical School |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 23 Oct 2024 09:30 |
| Last Modified: | 27 Nov 2024 10:45 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/97125 |
| DOI: | 10.1186/s12920-024-01835-5 |
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