Dear Dr. Kailankangas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Your manuscript was reviewed by two experts in the field. Both identified many important problems in your submission which require your careful attention. Please respond to all comments point-by-point.

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Please submit your revised manuscript by May 21 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Yury E Khudyakov, PhD

Academic Editor

PLOS One

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VK has received coverage for congress travel/accommodation expenses from Pfizer. JO has been a scientific advisor (advisory committee) to Astra-Zeneca, GlaxoSmithKline, MSD Finland, and Pfizer; received lecture honoraria from Advanz Pharma, Biocodex, GlaxoSmithKline, Pfizer, Professio, Roche, and Tillotts; and received coverage for congress travel/accommodation expenses from Gilead and Unimedic Pharma.

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5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Comments

• Only four patients experienced delayed death (after timepoint B), yet multiple gene modules, individual genes, and HLA types are evaluated in relation to this outcome. The authors themselves cite Peduzzi et al. and acknowledge that the events-per-variable ratio required for robust modeling is not met. In its current form, the risk of overfitting and chance associations is very high. Please substantially temper all causal and predictive claims regarding delayed mortality, explicitly frame findings as exploratory/hypothesis-generating, and avoid language implying that these modules “differentiate delayed death” with clinical reliability. The ROC/ AUC values of 0.95–1.00 for individual genes/modules with only four events are not credible as stable estimates. These require clear reporting of CI, emphasis on overfitting and optimism bias and removal of any suggestion that these are ready for clinical risk stratification.

• Patients who died after timepoint B were significantly older and had markedly higher Charlson comorbidity scores than survivors (mean age 78 vs 53 years, Charlson 7.5 vs 2.4). These factors are strong predictors of delayed mortality and may drive both the clinical outcome and aspects of the transcriptome. The current analysis appears largely unadjusted for these key confounders.

• The Abstract and Conclusions state that gene expression “was better at differentiating delayed death from survival than C-reactive protein level.” Yet there is no formal comparison of discrimination (e.g., AUCs with CIs for module vs CRP, or net reclassification) and CRP differences are described only descriptively and non-significant.

• The Discussion notes that prior work in chronic critical illness found upregulation of many HLA genes, whereas this study finds downregulation of multiple HLA genes at timepoint B associated with severity and death. This apparent discrepancy deserves deeper exploration.

• A more nuanced, mechanistic discussion would improve the translational value and help avoid overinterpretation.

• The authors acknowledge that whole-blood RNA-seq cannot distinguish per-cell expression from changes in leukocyte populations. This is particularly relevant because modules linked to delayed death at timepoint B relate to neutrophil regulation, necroptosis, and HLA expression and are associated with leukocyte count and right shift.

• Explore whether simple measures such as neutrophil and lymphocyte counts (or NLR) correlate with the key modules and whether modules remain associated with outcome after adjusting for these counts in a univariable or limited multivariable framework.

• Emphasize in the Discussion that some transcriptomic signals may primarily reflect shifts in cell populations rather than intrinsic reprogramming.

• Provide thresholds for inclusion of genes (filtering criteria for low expression/variance) and the soft-thresholding power used in WGCNA, including rationale.

• Clarify module preservation/overlap between timepoints A and B; the text states that genes were reassigned at each timepoint, but some quantitative measure of overlap would help interpret the “markedly different” profiles.

• Multiple testing: clarify how multiple comparisons were controlled across modules, traits, and genes (beyond the -log10(P)*sign(r) metric). Were adjusted p-values or FDRs calculated for module–trait associations and GO analyses?

• For the Mann–Whitney U tests comparing laboratory values between survivors and delayed deaths, provide exact p-values and 95% CIs where possible.

• Please clarify:

• The distribution of time from symptom onset and from admission to sampling at timepoints A and B (medians and IQRs).

• How deaths occurring “later in hospital” and “over a month after onset” are allocated into early vs delayed categories.

• Whether any patients were lost to follow-up between discharge and 90 days, and how this was handled.

• For Table 1, report 95% CIs for the AUCs and sample sizes used.

• For Table 2, briefly indicate how GO enrichment p-values were adjusted (e.g., Benjamini–Hochberg).

Recommendation

In summary, the study raises an interesting and clinically relevant hypothesis about distinct transcriptomic patterns associated with delayed mortality in iGAS sepsis, but the very limited number of delayed death events and lack of robust adjustment for confounding restrict the strength of the conclusions. I would support publication in PLOS ONE after major revision that (1) reframes the work as exploratory, (2) clearly presents the limitations and potential biases, and (3) strengthens methodological transparency and interpretative restraint as outlined above.

Reviewer #2: This is an interesting exploratory study of possible associations between gene expression as determined by transcriptomics in peripheral blood and outcome, specifically delayed mortality, in patients with iGAS. The sample collection was made as part of a prospective observational study of iGAS, which is a commendable effort. 45 included patients is a good sample size considering the relative rarity of iGAS, at least for a nordic setting, although it restricts the possibilities for multivariable analysis. The deceased patients were, as expected, significantly older and with more co-mordbidity. There were also indications of an increased and sustained systemic inflammatory response in patients with poor outcome. The results provide clues to the mechanisms linking advanced age and co-morbidity to worse prognosis through a dysregulated immune response. However, iGAS is an heterogenous disease and there are more potential factors that may also come into play, such as bacterial load, type of infection focus, patient's delay etc. The authors acknowledge this in their discussion. The results are presented clearly and conclusions are supported by the provided data.

Minor comments

1. I think the term "right shift" is somewhat ambiguous. Presumably, the authors mean low lymphocyte count relative to total leukocyte count. In my experience, right shift can also be related to the distribution of mature vs immature neutrophiles. I would advise to use a more precise term

2. The time point of sampling is stated as 2 days after admission and 5-7 days later. There is no data on time point in relation to first day of symptoms, although disease duration could potentially influence results and interpretation.

3. Were there any associations between infection focus and gene expression?

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Reviewer #1: Yes:  Atul Jindal

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Plos One Comments.docx

Dear Dr. Kailankangas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Your revised manuscript was reviewed by one original reviewer who identified some remaining issues in your submission. Please consider the attached comments and provide point-by-point responses.

==============================

Please submit your revised manuscript by Aug 29 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Yury E Khudyakov, PhD

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

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Reviewer #1: The revised manuscript is substantially improved, and the authors have addressed many of the major methodological and interpretative concerns raised in the first round. The study remains interesting as an exploratory transcriptomic analysis of delayed mortality in iGAS, but the small number of delayed-death events continues to limit the strength of inference and the scope for clinical prediction.

Major points

The authors have appropriately softened the language throughout the manuscript and now frame the findings more cautiously, which is a major improvement.

The added details on WGCNA filtering, soft-thresholding, multiple-testing handling, and confidence intervals improve methodological transparency.

The clarification of the timing of sampling relative to symptom onset and the distinction between early and delayed deaths is helpful and should reduce ambiguity.

The discussion of confounding by age, comorbidity, and leukocyte composition is more balanced, but the manuscript still cannot fully disentangle intrinsic transcriptomic reprogramming from changes in circulating cell populations.

Remaining concerns

Despite the revisions, the number of delayed deaths remains very small, so any predictive interpretation should remain strictly exploratory.

The reported discrimination statistics still need to be interpreted with caution because small-event ROC estimates can be unstable and optimistic.

The manuscript would benefit from even clearer acknowledgment that associations are not evidence of causality and should not be presented as clinically actionable biomarkers at this stage.

Minor points

The replacement of “right shift” with a more precise term is appropriate and improves clarity.

The updated description of infection foci is acceptable, and the clarification that no significant association was found is sufficient.

The revised data-availability and competing-interests statements appear more complete and consistent with PLOS ONE requirements.

Recommendation

Overall, the revision is much improved and acceptable in principle, provided the final version continues to emphasize its exploratory nature and avoids overstating prediction or clinical applicability

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what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes:  Atul Jindal

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Transcriptome profile of delayed mortality in patients with invasive group A Streptococcal disease

PONE-D-25-68006R2

Dear Dr. Kailankangas,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yury E Khudyakov, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

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