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Review 1: "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 13, 2024
Review 1: "A New Method Using Rapid Nanopore Metagenomic Cell-free DNA Sequencing to Diagnose Bloodstream Infections: A Prospective Observational Study"
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key-enterThis Pub is a Review of
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

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.


Review: This work describes the set up of a metagenomic nanopore sequencing method applied to the diagnosis of bloodstream infections. In their prospective study, the authors confirmed the results of 11 positive blood cultures and identified another 12 patients with negative blood cultures who displayed positivity for at least one microorganism. The methodology is overall adequate for the study, even if I would like to have more technical details. In particular:

  • Can you expand the methods section on the SRSLY kit? Can you briefly describe the method/workflow specifying that SRSLY kit allows sample multiplexing (UMI barcodes)?

  • Which were the N50, mean read length and mean quality achieved? Have you measured the percentage of human reads over microbial reads? Was the sequencing depth sufficient to run further analysis such as Antimicrobial Resistance Genes detection or genome assembly (usually associated to improved microbial taxonomy)?

  • Please specify the MinKNOW version used.

  • Which Bowtie2 parameters/settings were used?

  • Please provide additional information on the “proprietary refinement algorithm” used.

Other points:

  1. I do not particularly agree with the statement at lines 51-53 that blood cultures miss more than 50% of patients with clinical sepsis, since "clinical sepsis" is based on evaluation of some clinical parameters that are not totally specific for an actual bloodstream infection. Also, the referenced paper does not seem so rigorous to me.

  2. The choice of using two aerobic and just one anaerobic bottles (methods lines 103-104) could decrease the recovery of anaerobic/fastidious bacteria such as S. pneumoniae or H. influenzae.

  3. There is no statistical analysis on the influence of antibiotic therapy on microbial recovery in blood culture vs metagenomic sequencing (even if numbers are small).

  4. I would like more details on p092 who was positive for H. pylori: is there any record that the patient has gastritis or resulted positive for H. pylori with fecal antigen testing? 

  5. The polymicrobic samples p019 and p173 also look interesting: the former seems to indicate translocation of respiratory tract bacteria in blood, while the latter contains microbes potentially coming from gut/skin. Is there a technical problem with the samples hinting at a misclassification? 

Minor points:

  • For the sake of clarity I would refer to, e.g., figure S1 instead of appendix p.3.

  • I would reverse the sentence at lines 195-198: "we found that the CRP level was significantly higher in patients from BSI-confirmed (n=11, median: 216 mg/L, p=0·047 and p=0·002, Wilcoxon-Mann-Whitney) and BSI-suspected (n=25, median: 204 mg/L, p=0·035 197 and p<0·0001, Wilcoxon-Mann-Whitney) compared to BSI-absent (n=4, median: 1·3 mg/L) and BSI-unlikely (n=141, median: 67 mg/L)".

  • In supplementary figures, the unit of measure is missing in all the y axes.

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