-->PONE-D-26-13662-->-->Multi-District Molecular Surveillance of Dengue Virus in Pakistan Reveals Serotype Cycling and Recurrent DENV-2 Dominance, 2021–2025-->-->PLOS One

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Additional Editor Comments:

I have received the reviews of your manuscript. While your paper addresses an interesting question, the reviewers stated several concerns about your study and did not recommend publication in its present form.  Please see reviewers’ insightful comments below. These comments need to be addressed carefully.  In addition, there are a few confusing points in the manuscript that need to be clarified, please see specific comments below.

Specific comments

1.To avoid confusion with NIH from other countries, it will be better to indicate that NIH in this manuscript is referring to National Institute of Health (NIH) Pakistan see line 21, 98, 269, 272, 285, & 293.

2.Line 71:  …twin cities:  Please clarify this as (Rawalpindi and Islamabad), since this term could have different meaning in different country.  For USA, twin cities mean  Minneapolis–Saint Paul.

3.Line 89,… between 2021 and 2024,… In the abstract and material and methods it is between 2021 and 2025. Which is the correct timeframe?

4.Line 112:  It will be helpful to the readers if the authors referred to Fig. 1 here.

5.Lin 112: Include a sentence indicate how many samples were excluded from analysis.

6.Line 127:  Please add detail of qRT-PCR condition.

7.Line 137:  I counted 1171 samples from the description below, please clarify.

8.Line 155:  This should be Fig. 2 instead of Fig. 1. Also, change “Month wise” to “Monthly”

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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?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

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 requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: • In the abstract it is stated that “Gender-Age distribution showed majority of cases in males (64.5%)” but in the results it is stated that “Overall, dengue infections were more frequently observed in males (66%)”. One of these figures is incorrect and must be corrected consistently throughout.

• In the abstract it is stated that DENV-3 is 0.08% “DENV-3 (0.08%)” but in the results it is stated that “DENV-3 remained sporadic, representing only 1.0% of cases in 2023 and was absent in other years”. These figures are contradictory. Add the number of cases along with percentages.

• The title, abstract, and methods all state the study duration from 2021-2025 while the introduction states the study covers "2021 and 2024".

• Both Figure 1 and Figure 2 labeled as “Fig 1”.

• For 2025, the data is available till October, 2025 not December, 2025. Correct the study duration in methods and results section.

• Of 1,644 NS1-positive samples, only 1,162 (70.7%) were confirmed positive by qRT-PCR. There is almost 30% discordance between NS1 antigen and qRT-PCR. The reason for this discrepancy and how it impacts the serotype distribution should be discussed.

• Clinical data available only for 604 patients but in Table 2, the total samples of the study (n=249 for DENV-1 and n=912 for DENV-2) used as denominators. This could inflate the sample size and impact the p-value. Re-run the analysis with only the samples for which the clinical data available.

• Serotype proportions are reported differently in the Abstract and Results. The author should provide a detailed table with numbers and percentages of serotype distribution over the years.

• Line 112 states that samples with "inappropriate conditions" or "incomplete clinical information" were excluded, but does not report how many samples were excluded for each reason.

• Figure 3 is not clear. Cannot determine the absolute numbers for the figure. Re-draw the figure with clarity.

Reviewer #2: I read with interest the manuscript by Umair et al. Multi-District Molecular Surveillance of Dengue Virus in Pakistan Reveals Serotype Cycling and Recurrent DENV-2 Dominance, 2021–2025. The dataset is clearly valuable, and the geographic scope is impressive. That said, I think the manuscript would benefit from some important clarifications before it is ready for publication. I have tried to be specific about what I would like to see addressed.

1. Abstract, Lines 35–36.

I appreciate the authors' transparency about referral bias, but the Abstract is not the right place for this disclaimer in its current form. As written, it reads like a footnote rather than useful information. I would suggest moving this point to the Discussion, where the authors can actually unpack what it means. For example, do referral patterns skew toward more severe cases, and if so, does that inflate the apparent dominance of DENV-2? If the authors prefer to keep some version of this in the Abstract, it needs to be more concrete—something like: "Because samples were referral-based, DENV-2 may be overrepresented, given this serotype infection is often associated with severe dengue fever and may prompt patients to visit hospitals or healthcare more than DF patients infected with DENV1 or DENV3.

2. Lines 53–55.

Secondary DENV infection is a risk factor for DHF. Given the well-established role of antibody-dependent enhancement and the fact that DENV-2 dominance with sporadic DENV-1 circulation in late 2022 and 2023, and rare DENV3 infection occurs in some districts. However, this doesn’t mean that DENV1 and DENV3 are rare in the communities because the peripheral healthcare-based or community-wide genomics surveillance had not been done to capture mild or subclinical cases, most likely with infection with DENV3 or DENV1. A brief paragraph framing this would help. If the authors do not have primary/secondary infection data for these samples, that is completely understandable, but it should be stated explicitly as a limitation rather than omitted entirely.

3. Lines 59–60 and Line 195. Minor point, but "dengue virus" is defined at Line 41. Line 59 expanded again. On the other hand, line 195 still has "Dengue virus" written out in full. DENV was already expanded in line 41 and throughout thereafter, DENV should have been used. Same for DHF.

4. Lines 120–121: Missing detail on the kit.

Could the authors add the city for Qiagen? I believe it is Hilden, but the manuscript should specify for reproducibility.

5. Figure 1: The pie charts representing the different representations of different DENV serotypes across the Pakistan districts

I found the current visualization difficult to interpret in a meaningful way. All pies are the same size, so a district with 500 samples looks identical to one with 20. That makes it impossible to judge whether the serotype proportions reflect genuine epidemiology or simply small-sample noise. I would strongly encourage the authors to scale the pie areas to the sample size and to report absolute counts rather than percentages. Additionally, it would help to readers if authors label each district with sampling years, since Figure 1 currently gives the impression of a uniform temporal snapshot when the text makes clear that sampling years differ by district.

6. Relationship to Jama et al. (2025).

I noticed a very recent paper by some of the same authors in Infection, Genetics and Evolution covering dengue in Pakistan during 2024. I am not suggesting anything improper, but as a reader, I genuinely could not tell whether the two papers analyze overlapping datasets, completely separate datasets, or some combination. For the readers, as they may encounter it as I did, it would be good if the authors clarify: (a) whether any sequences or data in this manuscript were published in the Jama et al. paper; (b) whether the geographic or temporal scope differs; and (c) whether the conclusions are consistent between the two studies. This kind of up-front transparency helps readers (and reviewers) understand what is genuinely new here.

7. The section for strengths and limitations is missing

I found no section on the strengths and limitations in the manuscript. I believe the section is important for a surveillance study that relies on convenience sampling. I would like to read how the authors address this. Specifically, how might referral patterns distort the serotype map? What clinical variables are missing, and how does that constrain interpretation? Are the apparent shifts in serotype dominance real epidemiological turnover, or could they reflect uneven sampling intensity across years and districts?

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Reviewer #1: No

Reviewer #2: Yes: Tesfaye Gelanew

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Multi-district molecular surveillance of dengue virus in Pakistan reveals serotype cycling and recurrent DENV-2 dominance, 2021–2025

PONE-D-26-13662R1

Dear Dr. Umair,

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,

Baochuan Lin, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: (No Response)

Reviewer #2: No major comments. All are addressed satisfactorily. However, please work on the quality of the figures.

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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: No

Reviewer #2: Yes: Tesfaye Gelanew

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