Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection.

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.

Rapid metagenomics for diagnosis of bloodstream and respiratory tract nosocomial infections: current status and future prospects.

Introduction: Nosocomial infections represent a major problem for the health-care systems worldwide. Currently, diagnosis relies on microbiological culture, which is slow and has poor sensitivity. While waiting for a diagnosis, patients are treated with empiric broad spectrum antimicrobials, which are often inappropriate for the infecting pathogen. This results in poor patient outcomes, poor antimicrobial stewardship and increased costs for health-care systems.Areas covered: Clinical metagenomics (CMg), the application of metagenomic sequencing for the diagnosis of infection, has the potential to become a viable alternative to culture that can offer rapid results with high accuracy. In this article, we review current CMg methods for the diagnosis of nosocomial bloodstream (BSI) and lower respiratory-tract infections (LRTI).Expert opinion: CMg approaches are more accurate in LRTI compared to BSI. This is because BSIs are caused by low pathogen numbers in a high background of human cells. To overcome this, most approaches focus on cell-free DNA, but, to date, these tests are not accurate enough yet to replace blood culture. The higher pathogen numbers in LRTI samples make this a more suitable for CMg and accurate approaches have been developed, which are likely to be implemented in hospitals within the next 2-5 years.