PONE-D-23-24305COVID-19 IgG Seropositivity and its determinants in occupational groups of varying infection risks in two Andean cities of Ecuador before mass vaccinationPLOS ONE

Dear Dr. Leon-Rojas,

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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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: This manuscript describes COVID-19 IgG Seropositivity and factors contributing to seropositivity in occupational groups of varying infection risks in Ecuador before mass vaccination. The authors have compared three different occupational risk groups based on an expected level of exposure to SARS-CoV-2: high, median, and low risk (HR, MR and LR, respectively). Several factors including specific symptoms were studied to investigate if they were associated with being seropositive for antibodies against SARS-CoV-2.

I have several questions and also think that the authors should have focused more on the age (and sex) difference between the groups, regions and time of sampling in the discussion.

I wonder why the group expected to have median exposure risk had significantly higher seropositivity than the other two groups. Why did the high-risk group have such a low seropositivity? Was it because they were mostly health care workers using personal protective equipment (PPE)? This could have been discussed further, se more below.

Does Ecuador have a registry of infection based on confirmed COVID-19? This would have been interesting to know. Also, if such a register exists, did it show a difference between different regions of Ecuador? (as this manuscript focuses on two cities).

Abstract

Where is the number of infections and deaths reported in the first line of the abstract reported and when (reference)? These numbers are not repeated and referenced in the introduction. The numbers also seem a little high. According to worldometer on the 30th of August 2023, the numbers for South America was 69 million cases and 1.4 million deaths.

In the objectives, the part about studying determinants potentially associated with seropositivity is not mentioned.

In the conclusion, I would suggest using another word than “significant” to describe the proportions as this term if commonly used when discussing statistical significance.

Introduction

lines 38-43 is one long sentence and the meaning is difficult to understand. I suggest that you rephrase.

Lines 48-52, a bit detailed with all the different world regions, it is perhaps better to focus more on South/Latin America.

Line 58-59 states that it is limited information on seropositivity in occupational groups and associated factors. I guess you mean in Ecuador as line 59 and 56 have many references studying this.

Materials and methods

I wonder if it is a “problem” that the participants came from different regions of Ecuador? What is known about the spread of the pandemic between different regions and time points?

In line 85-86 it states that the recruitment in the HR group started in January 2021 and that the LR and MR groups in July 2021, but how long did it the sampling continue for the different groups? What was the range of the sampling time? If the LR and MR groups were sampled 6 months later, could that explain why there were more seropositive samples among LR than HR? Update: The recruitment dates are given in Table 1. The HR group was sampled between January and February 2021 and the MR group was sampled between 21 July and 27 September. This is unfortunate. How did the pandemic develop between January and September 2021 in Ecuador?

Line 118-119: the numbers of participants in each group add up to 882, not 873.

The LR group was much younger than the other two groups. Did you try to age-match the participants? Younger individuals could be less worried of getting COVID-19 and behave differently than older individuals. According to Table 1, there was also a sex difference between the groups with HR and MR being 66.4% and 61.4%, respectively, but the LR groups was only 37.2% women.

The individuals in each group should ideally have been more similar in age, samples collected in the same region and at the same time point to optimize the comparison between the groups.

Was the access to PCR-testing similar in the different regions? Or was it easier access to testing in the HR group?

Table 1: in the HR risk, 38.4% reported to have been positive. This is very close the number of positive samples in this group (39.9%). I think it is surprising that there has not been more undetected disease in this group. Usually, seropositive numbers are higher than reported cases. Is it because this group was tested very frequently?

Table 1: The MR group had a shorter time from COVID-19 diagnosis than the other two groups (95% CI did not overlap for the HR and MR groups). Could this have influenced the seropositivity with waning of antibodies?

Table 1- age: I don’t understand the Age category. Is it the SD or the range of age that is shown? Is age given only for positive cases? I think it should be given for all participants in the group with the mean/median and range. And possible between infected/non-infected if there was an age difference between these groups.

Results

Line 144. It seems that the MR group was significantly different from the other two groups based on the 95% CI.

Line 151, a questionnaire is mentioned with 20 symptoms. Is this questionnaire added supplementary data? There are only 5 symptoms included in table 2. How very these 5 chosen from the 20 in the questionnaire?

Lines 160-165 mentions a subgroup analysis of the HR group between those with direct patient contact versus minimal patient contact. How many participants were included (overall and in the two groups)? This finding is also mentioned in the conclusion, lines 277-278 and more information about these sub-analyses should be included.

Table 3 – was age a continuous variable? I don’t understand the entry.

Table 2 and 3: There is a line dividing the myalgia row that is not in the other symptom categories. I think this line should be removed.

Discussion

First paragraph: are there any other seroprevalence studies from Ecuador?

Line 185: The 44.6% estimate (overall?) is not mentioned in results.

Reference 38 in line 203, from Colombia, measured seroprevalence using antibodies against the nucleocapsid and not spike/RBD. I found it strange that seropositivity was only 35% after completion of vaccination as most vaccines at that time were based on spike/RBD. I would rephrase this sentence.

Lines 221-231: about the restrictions in different districts of Ecuador. What were the differences in restrictions between the regions included here?

I think the authors could have speculated further on why the seropositivity of the MR group was so high, such as different sampling periods etc.

Line 143: levels of IgG in relation to loss of taste and smell. These levels are not mentioned or shown in results only positive/negative results.

Lines 251-254: I don’t understand what the authors want to say here. The LR group had several symptoms associated with seropositivity, but this was the youngest group. Less severe infections have been more common in younger individuals. The groups with the highest seroprevalence and less positive test, was the MR group (suggesting higher asymptomatic infections. But asymptomatic infections also could give lower levels of antibodies and less seroconversion). Could it be that the MR group was too small to detect associations?

Reviewer #2: The work in the present manuscript by Leon-Rojas et al. addresses the COVID-19 IgG Seroposittivity and its determinats in occupational groups of varying infection risks in two Andean cities of Ecuador before mass vaccination. Thus, this manuscript addresses an urgent medical problem – The presence of anti-SARS-CoV-2 IgG antibodies that can be a protector effect against COVID-19 . The idea, implementation, presentation of results and discussion are convincingly presented. However, a few minor things need to be addressed:

1. Materials and Methods: The study is well-done but suffers lack of information on immune responses after immunization. Since immune responses were not evaluated after vaccination and boost, the following sentences should be revised: “analyse changes in antibody levels over a period one year following vaccination with several COVID-19 vaccines”.

2. Please, in line 78, clarify “ICUs” acronym.

3. Please, in line 107, substitute the “Analyses were conducted on each of three samples” by “Analyses were conducted on each of the samples from three diferent groups”.

4. line 151-154: Whats the difference between symptoms described in your questionnaire and the individual COVID-19 symptomatology? Please, Clarify.

5. Please, to all tables, show the significant difference found with a sign and describe it the table footer.

6. Please, write the questionnaire used in the study during recruitment period and post it as supplementary material.

Reviewer #3: This in an interesting and valuable study about the epidemiology of COVID-19 in Ecuador that reports a high seropositivity values in the population after 1 year and a half of COVID-19 pandemic and prior to the introduction of vaccines.

However, there are some major concerns that the authors should address to improve the quality of the manuscript:

1. The period of analysis is from January to September 2021. Is is possible to show the values for each risk group and for the whole month for each month of the study, or at least bi-monthly? My impression is that community transmission was happening from the beginning of the pandemic so perhaps as early as January 2021, the authors will find the same high seropositivity values.

2. These results endorsed that SARS-CoV-2 community transmission was happening in Ecuador since early stages of the pandemic. This is why no matter the risk group considered, the values are really high. I am missing important references and a wider discussion of this topic in this manuscript. There are several articles done in Ecuador during the first semester of COVID-19 pandemic that even using RT-PCR testing, a non cummulative approach like serology to address prevalence of infection, were able to detect really high attack rates:

- Interestingly, three of this manuscript refers to risk occupational groups like police, food delivery riders or funeral home workers (See the DOI for this papers: DOI: 10.3389/fpubh.2022.1012434; DOI: 10.3389/fmed.2021.735821; DOI: 10.1016/j.scitotenv.2021.145225). This papers should be discussed in the context of this manuscript as clearly endorse the same findings that the authors get on this study: a high prevalence of SARS-CoV-2 infection on occupational risk groups.

- Also, few more manuscripts also endorse extended community transmission in general population even in rural and remote locations in Ecuador. These studied should be also discussed in the context of this manuscript as they endorse the findings of the authors: even in low risk group the prevalence is really high. This is expected if those results are put on the context of the findins of these mentioned articles that show extensive community transmission all over Ecuador (See the DOI to revise and cite: DOI: 10.3389/fmed.2023.1001679; DOI: 10.4269/ajtmh.21-0582; DOI: 10.2471/BLT.20.283028; DOI: 10.22605/RRH7643).

3. The authors should provide more details about the clinical performance of the serological test used. It is important to know the sensitivity of this test as this would impact the true values of seropositivity. For instance, the authors cite papers done in communities from Santa Elena and Esmeraldas, where rapid antigen test were used. That means a lost of sensitivity compared to a good ELISA test and probably an underestimation of prevalence of easily 20% or more. I would expect that the ELISA used in this study has a good sensitivity but no matter that the authors should address the estimated true values considering the sensitivity (and also specificity) of the text used.

4. Those results are quite worrying and means that COVID-19 pandemic was really out of control in Ecuador. I am missing an strong discussion about this, contextualizing this results with the mortality in Ecuador associated to COVID-19 (there are also several papers addressing this topic in Ecuador that the authors should consider to cite). Morevover, the scenario described for Ecuador was happening in other countries in Europe or Asia much more later and basically after omicron wave. It is important to discuss this topic as that means that COVID-19 pandemic would have been poorly managed in Ecuador.

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Reviewer #1: No

Reviewer #2: Yes: Bernardes, WPOS

Reviewer #3: Yes: Miguel Angel Garcia Bereguiain

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PONE-D-23-24305R1COVID-19 IgG Seropositivity and its determinants in occupational groups of varying infection risks in two Andean cities of Ecuador before mass vaccinationPLOS ONE

Dear Dr. Leon-Rojas,

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.

Please submit your revised manuscript by Aug 16 2024 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal 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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• 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.

We look forward to receiving your revised manuscript.

Kind regards,

Mariana Lourenço Freire, Ph.D

Academic Editor

PLOS ONE

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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

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 #3: 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 #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #3: Yes

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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 #3: 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 #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 #1: I am satisfied with the authors’ answers to my questions/comments and the updated manuscript. I support that this manuscript now should be accepted.

Reviewer #3: The authors have improved the manuscript with a better contextualization of their results.

However, there is one of my comments that I believe it was not fully understood. I ask the authors to provide the values for seropositivity for each month of the study, or bi-monthly at least. This is possible to do no matter if only one sample was collected for each individual.

My idea with this comment is to see if there was any trend in seropositivity of if those high values found by the authors were already happening as early as January 2021, what would mean that COVID-19 was spreading massively along 2020 in the Ecuadorian population.

I believe this analysis will make the manuscript even more interesting.

It is up to the authors to include this final recommendation in the final version.

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Reviewer #1: No

Reviewer #3: Yes: Miguel Angel Garcia-Bereguiain

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COVID-19 IgG Seropositivity and its determinants in occupational groups of varying infection risks in two Andean cities of Ecuador before mass vaccination

PONE-D-23-24305R2

Dear Dr. Leon-Rojas,

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,

Mariana Lourenço Freire, Ph.D

Academic Editor

PLOS ONE