-->PONE-D-25-65592-->-->Integrated Laboratory Protocol for the Diagnosis of Sexually Transmitted Infections (STIs): Standardized Pre-Analytical Procedures, Rapid Screening, Hemagglutination, and ELISA Methods for Use in Resource-Limited Settings-->-->PLOS One

Dear Dr. Ramamonjiarisoa,

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 three experts in the field. Please consider carefully the attached comments and provide point-by-point responses

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Please submit your revised manuscript by Apr 12 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.. 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.

Please include the following items when submitting your revised manuscript:-->

• 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'.
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-->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 . 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..

We look forward to receiving your revised manuscript.

Kind regards,

Yury E Khudyakov, PhD

Academic Editor

PLOS One

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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. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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-->3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.-->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->4. If the manuscript contains new data, have the authors made this data 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.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

Reviewer #3: N/A

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-->5. Is the article 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 highlight any specific errors that need correcting in the box below. -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: Dear editor and authors

The manuscript defines a lab protocol provides a harmonized, integrated laboratory workflow that was operationally validated during a population-based study of adolescent STIs in Madagascar.

This protocol offers a validated, integrated laboratory workflow combining: rapid immunochromatographic screening, non-treponemal and treponemal confirmatory testing, hemagglutination assays,and multi-pathogen ELISA panels (HBsAg, HCV IgG, HSV2 IgG, CT IgG), with pre-analytical guidance, QC requirements, and confirmatory pathways adapted to LMICs. The protocol is distinct from routine clinical algorithms because It has been operationally validated in an adolescent population.

The manuscript and lab protocol was well defined and written however I have some minor suggestions;

1- HSV2 IgG and CT IgG positivity can be used to assess the seroprevalence of past infections, not for diagnostic purposes. As the authors point out as a limitation, it is not meaningful for active infection screening without an IgM result. It can be used for surveillance purpose.

2- The authors state that the protocol offers a cost-effective and scalable diagnostic strategy. However they do not give any cost effectivenes data. Please add if there is any data on it.

3- The protocol was well defined but I think it is important to give the sensitivity and specifity of the test to predict false negatives and positives results.

4- Abbreviations such as QC, SOPs etc. should be given in their full form the first time they appear in the manuscript.

5- The entire article should be reviewed for spelling errors and any typos should be corrected. Punctuation marks should be added without leaving a space after the word, and then a space should follow. For example ‘Keysword :’ should be written as ‘Keywords:’ , ‘DOI :’ as ‘DOI:’ etc.

Reviewer #2: For Chlamydia trachomatis IgG antibodies (7.6): What is the risk of a false-positive result?, Are there other conditions that can mimic a positive Chlamydia IgG test? What is the delay (incubation period) between infection and a positive IgG result?

Can asymptomatic Chlamydia carriage lead to positive IgG antibodies?

What is your management for these chlamydia positive patients: Do you inform them? Do you perform PCR testing? Do you treat them as asymptomatic Chlamydia carriers?

Reviewer #3: This is an important piece of work that is relevant in LMICs. While the authors have clearly provide the workflow for the integrated analysis of the 6 STIs, there are some revisions regarding the process and cost analysis justification that they need to provide or revise the manuscript accordingly.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: Yes: Aliye BastugAliye Bastug

Reviewer #2: No

Reviewer #3: No

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

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Attachments

Attachment

Submitted filename: Plos one review.docx

Attachment

Submitted filename: PONE-D-25-65592_my comments.pdf

Integrated Laboratory Protocol for the Diagnosis of Sexually Transmitted Infections (STIs): Standardized Pre-Analytical Procedures, Rapid Screening, Hemagglutination, and ELISA Methods for Use in Resource-Limited Settings

PONE-D-25-65592R1

Dear Dr. Ramamonjiarisoa,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Yury E Khudyakov, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?-->

Reviewer #3: Yes

**********

-->2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.-->

Reviewer #3: Yes

**********

-->3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.-->

Reviewer #3: Yes

**********

-->4. If the manuscript contains new data, have the authors made this data 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.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 #3: N/A

**********

-->5. Is the article 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 highlight any specific errors that need correcting in the box below. -->

Reviewer #3: Yes

**********

-->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 #3: The authors have improved the clarity of the protocol and revisions were satisfactory based on my previous comments.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #3: No

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