PONE-D-22-31157LOCUS (LOng Covid – Understanding Symptoms, events and use of services in Portugal): A three-component study protocolPLOS ONE

Dear Dr. Dinis Teixeira,

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Additional Editor Comments:

Thank you for submitting this manuscript for consideration. Please find below the comments from the reviewers, which require your attention. In particular, please note point 1 from reviewer 2 with regards to the second objective of your study (Physical and Mental symptoms following Covid-19).

In addition to the comments from the reviewers, please clarify:

1. Your definition for symptom persistence in the second objective of your study - is this symptoms present at 9 and 12 months? If so, please justify why these timepoints were chosen, they seem rather arbitrary.

2. Please explain why you are choosing to use the background rate of cardiovascular and respiratory events to compare to those that occur in Covid-19 patients, rather than estimating these rates from Covid-19 negative patients who are hospitalised at the same centres and over the same time period as the Covid-19 positive patients. Given the impact of Covid-19 on health system functioning generally it would seem important to use contemporaneous rather than historical data for the Covid-19 negative patients. This is potentially a limitation that requires acknowledging.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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: No

Reviewer #2: Yes

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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

********** 

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well written protocol with clear objectives/components and the procedure. I have few comments for clarification.

Page 7, can you explain who is the data owner who will acquire data from SINAVE?

Providing names and contact number of a person with RT-PCR status bears ethical issues, please add what safeguards have you placed to ensure that the patient’s identification is not disclosed outside the research team. Identification can also lead to potential stigmatization, please add lines on how you will prevent those?

Clarify why you are conducing FGDs? And also add lines on why semi-structured interviews? And why on top of FGDs?

Reviewer #2: My compliments on your study design, as a mixed methods approach to long COVID is essential to furthering our understanding of the condition and involving patients themselves in setting research priorities. I look forward to reading about the results of your research.

I have several comments/suggestions, that you may wish to consider:

1. For the “Physical and mental symptoms following COVID-19 component, you will recruit individuals who tested positive and individuals with a negative test-result. It is unclear to me whether you will match these individuals on age and sex, or even other important confounders such as BMI? If not, why not? This could help in later data analysis.

2. The current state of the pandemic that we are in is vastly different from last year, and I am concerned that the people who are now tested for COVID-19 at an official testing centre are not representative of the general population. How will you go about correcting for this?

3. I am very intrigued by your survival analysis of cardiovascular and respiratory events following COVID-19. How will you determine what proportion of these events are attributable to COVID-19 as opposed to other pre-existing comorbidities? It may be useful to consider COVID-19 as an effect modifier in the relationship between comoborbidities and an event, as this could be of great clinical importance.

4. An important (and generally poorly understood) risk factor for long COVID is immunity resulting from previous vaccination and/or infection. Given that most individuals will have had at least one previous infection and may have been vaccinated multiple times, how do the authors aim to characterise different combinations of this hybrid immunity?

********** 

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.

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Reviewer #1: No

Reviewer #2: No

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LOCUS (LOng Covid – Understanding Symptoms, events and use of services in Portugal): A three-component study protocol

PONE-D-22-31157R1

Dear Dr Jose Teixeira,

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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Kind regards,

Dirceu Henrique Paulo Mabunda, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Congratulations you did all reviewers comments.

Reviewers' comments:

Reviewer 1

This is a well written protocol with clear objectives/components and the procedure. I have few comments for clarification.

Page 7, can you explain who is the data owner who will acquire data from SINAVE?

Providing names and contact number of a person with RT-PCR status bears ethical issues, please add what safeguards have you placed to ensure that the patient’s identification is not disclosed outside the research team. Identification can also lead to potential stigmatization, please add lines on how you will prevent those?

Clarify why you are conducing FGDs? And also add lines on why semi-structured interviews? And why on top of FGDs?

Reviewer 2 

My compliments on your study design, as a mixed methods approach to long COVID is essential to furthering our understanding of the condition and involving patients themselves in setting research priorities. I look forward to reading about the results of your research.

I have several comments/suggestions, that you may wish to consider:

1. For the “Physical and mental symptoms following COVID-19 component, you will recruit individuals who tested positive and individuals with a negative test-result. It is unclear to me whether you will match these individuals on age and sex, or even other important confounders such as BMI? If not, why not? This could help in later data analysis.

2. The current state of the pandemic that we are in is vastly different from last year, and I am concerned that the people who are now tested for COVID-19 at an official testing centre are not representative of the general population. How will you go about correcting for this? 

3. I am very intrigued by your survival analysis of cardiovascular and respiratory events following COVID-19. How will you determine what proportion of these events are attributable to COVID-19 as opposed to other pre-existing comorbidities? It may be useful to consider COVID-19 as an effect modifier in the relationship between comoborbidities and an event, as this could be of great clinical importance.

4. An important (and generally poorly understood) risk factor for long COVID is immunity resulting from previous vaccination and/or infection. Given that most individuals will have had at least one previous infection and may have been vaccinated multiple times, how do the authors aim to characterise different combinations of this hybrid immunity?

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 #2: Yes

Reviewer #3: 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 #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Thank you for responding to my previous comments.

In your response to my second point, you discuss the difficulty in case-finding individuals with a negative test. I have two main concerns about your response.

Firstly, even during high-transmission/high-prevalence periods of time, the majority of individuals with respiratory symptoms who are tested will not have a SARS-CoV-2 infection, therefore the statement that finding negative cases will be difficult, does not make sense. I suggest that you reword the statement.

Second, my initial comment was actually concerning the sampling bias introduced due to changing regulations for testing, not about recruitment. Following the availability of self-tests, for instance, not everyone will continue to visit a formal testing centre to receive their diagnosis. My question therefore was whether the authors take into account that the type of person to be tested (your sampling frame) may change over time. A supplementary table comparing baseline characteristics of individuals tested (negative or positive) during difference phases of the pandemic could be very insightful here.

Reviewer #3: The authors have addressed most of the reviewers comments. Where this was not possible, the authors acknowledge the limitations. It is a strength of this project to bring together three studies that address different questions around PCC. This is very valuable for increasing the knowledge base.

The only comment I would make at this point is the collaboration and communication to health care professionals, representatves of persons living with PCC and health authorities. It would be good to have a plan for science to public and to policy makers communication so that the results of this study contribute to the development of a common understand of PCC and how to best support persons living with PCC.

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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 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 #2: No

Reviewer #3: Yes: Milo Puhan

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