Review 2: "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: The work described in this paper is a very well-designed prospective study aimed at applying a novel molecular diagnostic tool to detect blood-stream infections. Similar studies have been done previously but in a more limited framework. The novelty in this study is in stratification of patients into different category groups according to the presence or absence of infective agents in blood cultures (BC) as well as some additional clinical parameters.  The authors clearly and accurately present their findings in all patient groups with a focus on how well the new method detects bacterial DNA in comparison to the current gold standard BC that detects actively dividing bacteria. They use summarized data to promote their claim on the efficiency and benefit of the new mNGS assay over BC for an early and accurate bacterial detection. While this is partially correct, there is a limitation to this interpretation. This is a lack of recognition that these two methods detect two different things, which are: the presence of bacterial DNA (mNGS) and the presence of viable bacteria (BC). While both of these two methods have their own limitations, they are not interchangeable but rather complementary. So, I do not see mNGS as an ‘alternative’ but rather as a ‘supplement’ to BC.

To better illustrate the significance of the new diagnostic tool for blood-stream infections, the authors provide detailed medical histories for selected cases. This I find fascinating. Detailed description of these cases with an accurate and precise detection of microorganismal genomes provides a compelling insight into the power of mNGS in uncovering molecular signatures of all possible infections the patient has - acute, chronic, dominant, transient and dormant. You could see all of these infections represented quantitatively in blood plasma. The authors rightly comment on the clinical relevance of their findings and a need for validation. What is crucial to conclude is that mNGS cannot be used on its own as a single diagnostic tool. It can obviously impact clinical decisions about the treatments when combined with other findings in which BC remains a gold standard despite its limitations.

As for ethical approval and diversity, the authors provide sufficient evidence on applying the good practice rules and recognizing some of the limitations in their study.