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Review 5: "A New Method Using Rapid Nanopore Metagenomic Cell-free DNA Sequencing to Diagnose Bloodstream Infections: A Prospective Observational Study"

Reviewers agree that the methodology is well-designed and the evidence is reliable. However, they suggest including more technical details and caution against overestimating the method's capabilities while emphasizing its supplementary role alongside blood cultures.

Published onJun 27, 2024
Review 5: "A New Method Using Rapid Nanopore Metagenomic Cell-free DNA Sequencing to Diagnose Bloodstream Infections: A Prospective Observational Study"
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A new method using rapid Nanopore metagenomic cell-free DNA sequencing to diagnose bloodstream infections: a prospective observational study
A new method using rapid Nanopore metagenomic cell-free DNA sequencing to diagnose bloodstream infections: a prospective observational study
Description

Abstract Background Bloodstream infections (BSIs) remain a major cause of mortality, in part due to many patients developing sepsis or septic shock. To survive sepsis, it is paramount that effective antimicrobial therapy is initiated rapidly to avoid excess mortality, but the current gold-standard to identify the pathogen in BSIs, blood culturing, has great limitations with a long turnaround time and a poor sensitivity. This delay to correct empiric broad-spectrum antimicrobial treatments leads to excess mortality and antimicrobial resistance development.Methods In this study we developed a metagenomic next-generation sequencing (mNGS) assay utilizing the Oxford Nanopore Technologies platform to sequence microbial cell-free DNA from blood plasma. The method was evaluated in a prospective observational clinical study (n=40) in an emergency ward setting, where a study sample was taken from the same venipuncture as a blood culture sample from patients with a suspected BSI.Findings Nanopore mNGS confirmed all findings in patients with a positive blood culture (n=11), and identified pathogens relevant to the acute infection in an additional 11 patients with a negative blood culture. In an analysis of potential impact on the antibiotic treatment, we found that 59% (n=13) of mNGS positive answers could have impacted the treatment, with five cases of a change from ineffective to effective therapy.Interpretation This study demonstrates that culture-independent Nanopore mNGS directly on blood plasma could be a feasible alternative to blood culturing for infection diagnostics for patients admitted with a severe infection or sepsis. The method identified a relevant pathogen in patients with a broad range of etiologies including urinary tract infections and lower respiratory tract infections. With a turnaround time of 6 hours the method could provide unprecedented speed and sensitivity in BSI diagnostics.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: Nanopore sequencing on cell-free DNA provides high sensitivity identification of pathogens responsible for bloodstream infections. Compared to conventional blood culture, the method detected 11/11 cases that were positive by culture (100% sensitivity), and it identified organisms that were not detected by culture in 12/29 cases (59%), with 11 of these cases being likely true detections. If these results were used for treatment decisions, the method could have informed decisions in about half of the cases.

While this is a preliminary observational study, the results suggest the potential for very high clinical impact. Sensitivity compared to blood culture is excellent. Specificity compared to blood culture is reported as 59%, however the "false positives" were in nearly every case due to organisms that were in fact very likely to have existed in these patients - specifically, the organisms were plausible infectious agents and in most cases were also identified by culture of a sample from the primary infection site. The authors are commended for reporting the results conservatively and with appropriate caveats, but the evidence is convincing that most of the NGS results were likely correct. No infectious organisms were detected in patients with negative culture and no suspected bloodstream infection, which further supports that NGS-detected cases in patients with negative blood culture were true detection events. However, there are some relevant deficiencies: 1) infections could potentially result in cell-free DNA in the bloodstream that is not actually due to organisms growing in the bloodstream (where blood culture would be expected to be negative, but cell-free DNA might be detected by NGS), 2) patients with negative blood culture but low to medium suspicion of bloodstream infection were not analyzed - this could be the most challenging patient group so could result in lower sensitivity if these patients were included in the study. There are some deficiencies in disclosure of methods: 1) Primers used prior to sequencing were not described, 2) genetic analysis was done with "proprietary refinement algorithms" - both omissions prevent assessment of bias in the methods, but it is unlikely that either of these would affect the main conclusions. 

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