Review 4: "A New Method Using Rapid Nanopore Metagenomic Cell-free DNA Sequencing to Diagnose Bloodstream Infections: A Prospective Observational Study"

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: Study presented information to help suggest that culture independent NGS needs further evaluation as a feasible alternative for diagnostics in patients with a severe infection or sepsis. Design of study was practical given the issue with availability of technology to conduct real time-set up and testing and the difficulty in adjudicating negative NGS results with patient samples. Results were presented in a concise manner with analysis including much of the variables to consider for technology used for diagnosis of BSI. Concordance between NGS and blood culture results helped establish accuracy in patients with confirmed BSI. Additionally, the data from patients with high likelihood of BSI but not confirmed via conventional standards further supported the author’s claims. Presentation of cases series within the analyzed categories aided the readers in better appreciation of the categorization used for initial analysis. Reporting on GPM data was seen to correlate with the trend of confirmed and likely – adding to the growing data available about the use of NGS and how to interpret its results.

Overall, the study did a great job of presenting data to support its claim despite of the limitation noted by the authors and additional concerns raised below. It would have been helpful for author to provide more clarity on use of donor blood samples as a control. The lack of analysis on patients in the BSI-unlikely category possibly hindered the ability to show performance characteristic in the other side of the spectrum for infections where BSI is of lower likelihood. Therefore, the author’s claim may be better suited to include use of pathogen identification for patient’s with “high likelihood of BSI” rather than those with severe infection or sepsis as not all sepsis necessarily presents with bacteremia (though understandably this is a difficult distinction to make in real time). More description of the specific characteristics that were associated with subgrouping would help inform the applicability of the technology to specific limited. Study certainly adds to the growing literature on the use of NGS technology for infectious disease diagnostics as a first line approach. More elaboration is needed regarding the clinical impact and adjudication discordant results and the potential impact of positive NGS with negative culture/corroborative microbiological data is needed to understand how this could practically be used in a real life setting. In a real life setting, interpretation may not be done by clinical microbiologist who are versed in the organisms and relevance of them but rather emergency room doctors who might not yet have all the corroborative information to determine the relevance of positive results