Characterization of human exposure to Anopheles and Aedes bites using antibody-based biomarkers in rural zone of Cameroon

Idriss Nasser Ngangue-Siewe; Paulette Ndjeunia-Mbiakop; André Barembaye Sagna; Abdoul-Aziz Mamadou Maïga; Roland Bamou; Antoine Sanon; Jeannette Tombi; Jean Arthur Mbida Mbida; Christophe Antonio-Nkondjio; Franck Remoue; Athanase Badolo

doi:10.1371/journal.pone.0314709

Peer Review History

Original SubmissionAugust 3, 2024
4 Sep 2024 Decision Letter - Nazarudin Safian, Editor PONE-D-24-31144Characterization of human exposure to Anopheles and Aedes bites using antibody-based biomarkers in rural zone of CameroonPLOS ONE Dear Dr. Ngangue-Siewe, 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 Oct 19 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'. 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. We look forward to receiving your revised manuscript. Kind regards, Nazarudin Safian, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In the ethics statement in the Methods, you have specified that verbal consent was obtained. Please provide additional details regarding how this consent was documented and witnessed, and state whether this was approved by the IRB. 3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. 4. Thank you for stating the following financial disclosure: This work received financial support from the Bill & Melinda Gates Foundation, Grant ID: OPP1210340 to C.A.-N and Pan African Mosquito Control (PAMCA) association for the field work and from “Appui à la Formation, la Recherche et à l’Innovation pour le Développement Intra-Afrique” (AFRIDI) fellowship under Intra-Africa Academic Mobility Scheme of the European Union for the laboratory analysis to I.N.N.-S. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 5. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. 6. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an ""Other"" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. The following resources for replacing copyrighted map figures may be helpful: USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/ The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/ Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/ Landsat: http://landsat.visibleearth.nasa.gov/ USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/# Natural Earth (public domain): http://www.naturalearthdata.com/ 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] 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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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: No Reviewer #2: Yes ****** 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: No Reviewer #2: Yes ****** 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: PONE-D-24-31144 Review comments for the author. The objective of this study was to utilize a serological approach to determine the level of exposure and heterogeneity of Anopheles and Aedes bites in four villages in Cameroon. This study is relevant since mosquito borne diseases are a public health concern in Cameroon. Experimental approach • The study utilized ELISA to draw its conclusion. An alternative tool should have been used alongside this method in order to compare and validate these findings. In the current state, the data provided does not provide reasonable information to conclude whether this method is better than conventional methods that have been previously used (refer to introduction section lines 76–81). • The authors did not use appropriate controls in their assay. Although the reported values are changes in OD levels, samples from positive and negative individuals (exposed and non-exposed individuals) should have been included in the assay. Similarly, the cut-off value was not determined or indicated. • The study observed variation in OD values with regards to LLIN use. These antigens can last up to 40 days before they begin to wane. A follow-up study would be significant in determining this variation and would strengthen the conclusion. • Additional confounding factors were not factored in. For example, the authors do not show how they controlled for the migration of individuals across villages during the sampling period. The use of other vector control strategies like coils, indoor residual spraying, level of education/awareness of malaria control strategies, etc., at the household level was not considered, and this might influence the outcome of the study. Data analysis • The authors should justify why they opted for a non-parametric test instead of a parametric test. • The authors should consider using a violin plot or box plot in Figure 2. • What statistical test is represented in Table 2? Is it the post-hoc test or Kruskal-Wallis test? This should be reflected in line 182. • Supplementary File 1 should also include a comparison of the peptides with other variables such as age, gender, and the condition of bed nets. • The correlation between the two peptides should be compared in each village, as opposed to clumping everything together (Figure 3). • There is no evidence of the data reported on the correlation of village-specific analysis (lines 224–29). • Supplementary data with all OD levels would be useful. Study conclusion. The study, though relevant, is not novel. The correlation between these findings and the infection rate would be more relevant. qPCR can be used to determine parasitemia levels in these samples. Reviewer #2: In general, I am supportive of the manuscript "Characterization of human exposure to Anopheles and Aedes bites using antibody-based biomarkers in rural zone of Cameroon". I believe that such field studies that try to understand that general exposure of populations to vectors of disease are vital pieces of information in the effort of implementation of control measures. I appreciate the author's effort to collect demographic information about their study groups and to inform the reader about topographic differences between locations, which are vital pieces of information to the study. However, I do not think that the authors made the most of this data. 1.) The demographic data presented in table 1 offers itself for comparative statistics by chi-square or Fisher's Exact test to determine whether the distribution of subject categories between locations a comparable or not. However, according to the methods, no such tests were applied. As far as I can tell, the author's merely did an observational comparison of their tabled data, which is insufficient. 2.) The authors stick to non-parametric sample comparison by demographic category, which is not sufficient to understand the impact of each demographic on the IgG level outcome. The authors should make an effort to apply regression analysis models to determine which demographic categories are in fact significant in the description and prediction of the outcome variable, IgG level. In this context, the authors must explain, if the level of antigen-specific IgG reflects how recent and/or frequently an individual was exposed to vector species, or if this is an artifact of the outbred model, humans, where massive data variability is to be expected due to difference in host generics. 3.) Further, the authors need to make it clear that it is MEDIAN IgG responses that are higher or lower between study sites and not IgG responses per se. E.g., Njombe has more extreme high responders than Kekem, but also more low responders. Thus, in principal the Njombe data has much higher variance than the Kekem data, which may mean something, and perhaps a slightly higher median IgG response. However, the majority of individuals are within the same range between both sites. This information is of greater interest rather than just statistically significant differences between group medians. 4.) The authors must undertake a careful analysis of their correlation plot. Eyeballing the graph suggests that the weak correlation is dependent on the weight of the 5 data points farthest to the right of the graph. A careful statistical analysis should be able to establish this. If true, this would mean that their is in fact no relationship between Anopheles and Aedes responses in the majority of individuals. Also, it would be good to know if these five data points come from the same sample site or represent the most extreme responders from all sites. Etc. 5.) There are no known negative samples on the graphs, which means that I do not know what the cutoff would be between positive samples, true responders, and negative samples, which may still have signal over background. At the moment, I must assume that everything above 0 is considered positive, meaning that every individual must have been bitten by species of both genera, assuming that the provided antigens are genera specific rather than species specific. Is that a realistic observation? It may be helpful to establish what negative samples look like. Do they provide signal above the background, too? I understand that negative samples are hard to come by considering the global distribution of both genera and the likelihood that virtually everyone has been expose to either genera at some point. I appreciate that the authors attempted to circumnavigate that problem by presenting delta ODs. Either way, it would be useful to know, if the authors, thus, interpreted anything above zero as an indicator of exposure, which brings me back to my previous comment whether the delta OD is an indicator of how recent and/or frequently someone was bitten. Addressing these points, I believe, will significantly enrich this manuscript and improve its impact. Thus, I hope the authors will take these points under consideration when revising their manuscript as I hope to see their work published. ****** 6. 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 #1: Yes: FAITH ONDITI Reviewer #2: Yes: Johannes S. P. Doehl ******** [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.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0314709.r001
Revision 1
10 Nov 2024 Author Response PONE-D-24-31144 The Journal Editorial Office PLOS ONE Dear Editor-in-chief, Enclosed please find our revised manuscript, “Characterization of human exposure to Anopheles and Aedes bites using antibody-based biomarkers in rural zone of Cameroon” with the requested amendments (in red) for publication as a research article in the Journal PLOS ONE. Editorial Requirements for Revisions Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors affiliations.pdf Answer: Thanks, the manuscript has been revised accordingly. 2. In the ethics statement in the Methods, you have specified that verbal consent was obtained. Please provide additional details regarding how this consent was documented and witnessed, and state whether this was approved by the IRB. Answer: Thanks for the question. We noted in the manuscript (line 114-121) that the verbal consent was noted on each survey in the field if the person accepted to participate in the study or not. 3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why. Answer: Thanks for the comment. We added in the manuscript that an authorization of each local divisional officer was obtained (line 114-121). File of all signed authorization was uploaded. 4. Thank you for stating the following financial disclosure: This work received financial support from the Bill & Melinda Gates Foundation, Grant ID: OPP1210340 to C.A.-N and Pan African Mosquito Control (PAMCA) association for the field work and from “Appui à la Formation, la Recherche et à l’Innovation pour le Développement Intra-Afrique” (AFRIDI) fellowship under Intra-Africa Academic Mobility Scheme of the European Union for the laboratory analysis to I.N.N.-S. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Answer: Dear Editor, we edited the manuscript and noted that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. (see line 341-348). 5. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. Answer: We thank the Editor for the point addressing above. The manuscript has been revised 6. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. Answer: We removed Figure 1 from the manuscript and used the adjusted form of the study site figure published in our 2022 paper (Ngangue-Siewe et al., 2022). 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Answer: We thank the Editor for the point addressing above. The manuscript has been revised and we are taken into account all Editorial Requirements in revised MS. Responses to reviewer’s comments: Please find below the responses to each reviewer comments or suggestions. Additionally, requested changes were directly introduced in red text in the revised manuscript (MS). Reviewer reports: Reviewer 1: 1. The study utilized ELISA to draw its conclusion. An alternative tool should have been used alongside this method in order to compare and validate these findings. In the current state, the data provided does not provide reasonable information to conclude whether this method is better than conventional methods that have been previously used (refer to introduction section lines 76–81). Answer: Thank you for your insightful comments. We agree that using entomological data, such as human landing catches, could have helped compare exposure to Aedes and Anopheles vectors in these villages. However, the purpose of this study was not to validate these biomarkers but to use them as validated tools to evaluate spatial heterogeneity of exposure to mosquito bites in rural Cameroon. Indeed, these biomarkers of human exposure to Anopheles and Aedes mosquitoes have been developed, compared and validated in different epidemiological contexts over the past two decades (see lines 83-91, references 13-15). All these results pointed out the reliability of this immunological tools as epidemiological biomarkers of Anopheles and Ades bites. In addition, ELISA (in contrast to their immunological technic as multiplex) was the validated technology for assessing biomarkers of exposure to mosquito bites since 20 years by the IRD team, the international reference team in this topic (> 65 articles) as indicated in MS (reference 17 as review). 2. The authors did not use appropriate controls in their assay. Although the reported values are changes in OD levels, samples from positive and negative individuals (exposed and non-exposed individuals) should have been included in the assay. Similarly, the cut-off value was not determined or indicated. Answer: We thank the reviewer for this comment. We did use a positive control to monitor plate to plate variations and make sure our experiments effectively work. This has been added in the M&M section (lines 154-158). However, we were not able to use a negative control to calculate the cut-off due to the difficulty to get serum from unexposed individuals in Africa. Indeed, the biomarkers tool is presented only in terms of specific IgG levels/intensity and not as immune responders (positive/negative). By consequences, we do not estimate the cut-off value was necessary to be determine. 3. The study observed variation in OD values with regards to LLIN use. These antigens can last up to 40 days before they begin to wane. A follow-up study would be significant in determining this variation and would strengthen the conclusion. Answer: We totally agree with the reviewer comment. Unfortunately, this was a cross-sectional study. While we did not have a full picture of the variation over time, we did have a picture at the time of the study (rainy season). Follow-up studies made with these biomarkers have shown that IgG responses to these peptides varied according to the season with an increase during the wet season compared to the dry one (Elanga et al., 2012 and Doucoure et al., 2013 for Aedes; Sagna et al., 2013, Poinsignon et al., 2009, and Dramé et al., 2013 for Anopheles). Follow-up could be challenging as the individual/participant could receive another bite before the 40 days. 4. Additional confounding factors were not factored in. For example, the authors do not show how they controlled for the migration of individuals across villages during the sampling period. The use of other vector control strategies like coils, indoor residual spraying, level of education/awareness of malaria control strategies, etc., at the household level was not considered, and this might influence the outcome of the study. Answer: We appreciate your concern regarding the potential confounding factors that were not addressed in our study. We acknowledge that factors such as the migration of individuals across villages, the use of other vector control strategies (e.g., coils, indoor residual spraying), and the level of education/awareness of malaria control strategies at the household level could influence the study’s outcomes. Before starting our survey, we asked if the participant was in the study site for more than one month and concerning other vector control strategies, unfortunately, due to data availability, we were unable to control for these variables in the current study. In future research, we plan to incorporate these additional factors to provide a more comprehensive analysis, as indicated in discussion in the revised MS (lines 310-315). 5. Data analysis • The authors should justify why they opted for a non-parametric test instead of a parametric test. Answer: Thanks for your question: Our data did not meet the normality assumption required for parametric tests, and the data we analyzed were ordinal in nature, which is more appropriately handled by non-parametric tests, as indicated in line 162-163. • The authors should consider using a violin plot or box plot in Figure 2. Answer: We appreciate your concern regarding the type of our graphics but we preferred to use this scatter plot to better show results of specific IgG at populational level by studied group and their variation according to villages. • What statistical test is represented in Table 2? Is it the post-hoc test or Kruskal-Wallis test? This should be reflected in line 182. Answer: Thanks for the remark it was a Kruskal-Wallis test and we noted it in line 186 as recommended. Also, the Mann-Whitney test was used when there were only two groups to compare. • Supplementary File 1 should also include a comparison of the peptides with other variables such as age, gender, and the condition of bed nets. Answer: We thank the reviewer for the suggestion but we could not compare the IgG responses to peptides with other variables such as age, gender and bed nets condition because we did not have enough data per village regarding these different variables mentioned above. For example, regarding the condition of the mosquito nets and the age of the individuals, for some villages this was not relevant because we only had 2 or 3 mosquito nets with holes, the rest being without holes and we also had few individuals in certain age groups. So, it was difficult to do a statistical analysis with this • The correlation between the two peptides should be compared in each village, as opposed to clumping everything together (Figure 3). Answer: A village-by-village correlation between IgG levels to the two peptides has been made and results are described in lines 241-249 in the revised manuscript. But only the Figure combining all villages is presented. • There is no evidence of the data reported on the correlation of village-specific analysis (lines 224–29). Answer: Dear reviewer, we have 4 villages and have used 2 peptides. A village-specific correlation will give us 8 Figures. Since many correlations by village were not significant, we decided to only provide Figure 3 representing the correction between the 2 peptides when all villages are gathered and just provide a description for the village-specific correlation (line 241 to line 249). • Supplementary data with all OD levels would be useful. Answer: Thank you. The OD values have been provided as supplementary file as requested. 6. Study conclusion. The study, though relevant, is not novel. The correlation between these findings and the infection rate would be more relevant. qPCR can be used to determine parasitemia levels in these samples. Answer: We appreciate your insights and feedback. Our research provides a unique perspective by joining evaluation of human exposure to both Anopheles and Aedes bites in the same individual and it provides new methodology about human exposure to Aedes bites which has never been done in my country. Previous studies suggested that more mosquito bites elicit higher anti-gSG6-P1 antibodies and that higher antibody levels are also associated with a higher probability of being exposed to a bite from a Plasmodium-infected mosquito (Londono-Renteria et al., 2015 and Sagna et al., 2019). The measurement of real human-vector contact through antibody-based biomarkers could be used as a proxy to evaluate the risk of disease transmission. This is the reason why, without including information about infection status (arboviruses or Plasmodium) of the participant in this present study, we assumed that villages with children presenting high antibody responses to mosquito salivary peptides were where the risk of disease transmission were the highest. Reviewer 2: 1. The demographic data presented in table 1 offers itself for comparative statistics by chi-square or Fisher's Exact test to determine whether the distribution of subject categories between locations a comparable or not. However, according to the methods, no such tests were applied. As far as I can tell, the author's merely did an observational comparison of their tabled data, which is insufficient. Answer: We thank the reviewer for this for your insightful feedback. We acknowledge that the initial submission did not include the application of comparative statistical tests, such as the chi-square, to determine the comparability of subject categories between locations. In response to your valuable suggestion, we have now applied the chi-square test to the demographic data in Table 1. The Table 1 reflects the p-values and Chi-squared values obtained from this test, providing a more rigorous comparison of the demographic data. 2. The authors stick to non-parametric sample comparison by demographic category, which is not sufficient to understand the impact of each demographic on the IgG level outcome. The authors should make an effort to apply regression analysis models to determine which demographic categories are in fact significant in the description and prediction of the outcome variable, IgG level. In this context, the authors must explain, if the level of antigen-specific IgG reflects how recent and/or frequently an individual was exposed to vector species, or if this is an artifact of the outbred model, humans, where massive data variability is to be expected due to difference in host generics. Answer: We cannot do logistic regression for the simple reason that we don’t have binary data (positive vs negative) concerning the biomarker results. If we had a negative control and calculated the positivity threshold, we could have done it. 3. Further, the authors need to make it clear that it is MEDIAN IgG responses that are higher or lower between study sites and not IgG responses per se. E.g., Njombe has more extreme high responders than Kekem, but also more low responders. Thus, in principal the Njombe data has much higher variance than the Kekem data, which may mean something, and perhaps a slightly higher median IgG response. However, the majority of individuals are within the same range between both sites. This information is of greater interest rather than just statistically significant differences between group medians. Answer: Thank for your comments. We have revised the manuscript to emphasize that it is well the median IgG responses that differ between Njombe and Kekem, rather than th Attachments Attachment Submitted filename: PONE-D-24-31144 -18_October_responses to reviewers - Nasser.docx https://doi.org/10.1371/journal.pone.0314709.r002
15 Nov 2024 Decision Letter - Nazarudin Safian, Editor Characterization of human exposure to Anopheles and Aedes bites using antibody-based biomarkers in rural zone of Cameroon PONE-D-24-31144R1 Dear Dr. Ngangue-Siewe, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. 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, Nazarudin Safian, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0314709.r003
Formally Accepted
26 Nov 2024 Acceptance Letter - Nazarudin Safian, Editor PONE-D-24-31144R1 PLOS ONE Dear Dr. Ngangue-Siewe, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Nazarudin Safian Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0314709.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .