PONE-D-20-04369

Risk factors for sero-prevalence of Toxoplasma infection and the role of CD4+ T-cell count as a determinant of acute infection amongst HIV patients in the Bamenda Health District, Cameroon.

PLOS ONE

Dear Dr. Enah,

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.

There are several major problems in the understanding of this study, the way the statistical analysis has been performed and presented, as well in the interpretation of the findings. I urge you to carefully consider the best course of action for you with regards to this manuscript. If you feel it is possible to undertake a re-analysis and re-write of the manuscript in the required time frame, ensuring all of the areas of concern raised by all Reviewers have been thoroughly addressed, a revised manuscript will be reviewed and considered. If you do not feel it is possible to address all of these issues thoroughly, you may wish to consider taking the time necessary to do this and ensure the manuscript is technically sound and appropriately interpreted, before re-submitting here or elsewhere as a new manuscript.

Please submit your revised manuscript by 12th July 2020. 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Leanne Joy Robinson

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

The manuscript does not currently meet the requirements for publication in PLoS One. There are several major problems in the understanding of this study, the way the statistical analysis has been performed and presented, as well in the interpretation of the findings. The authors need to consider the best course of action. If it is possible to undertake a full re-analysis and re-write of the manuscript in the required time frame, ensuring all of the areas of concern have been thoroughly addressed, a revised manuscript will be reviewed and considered. If all of the above requirements cannot be thoroughly addressed, the authors may wish to consider taking the time to do this and ensure their manuscript is technically sound and appropriately interpreted, before re-submitting here or elsewhere as a new manuscript.

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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: No

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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: The manuscript titled "Risk factors for sero-prevalence of Toxoplasma infection and the role of CD4+ T-cellcount as a determinant of acute infection amongst HIV patients in the Bamenda Health

District, Cameroon" was well written and contains good information. But it could not present the results in a tight manner. For example, the demographic, economic, and nutritional variables are related to each other and affected each other and should have been examined together. And it was necessary to determine the basis on which the variable is included for the multivariate examination.

Also, with respect to the numerical variables, the authors did not reveal the nature of the distribution of those variables and whether they followed the normal distribution in order to use Anova test and why not use T-Test?.

In addition, the authors treat the Odd ratio test on the basis that it quantites the association between  the variables and this is incorrect.  Odd ratio quantifies the strength of the association between two events. There are other tests to do this, such as a risk ratio (RR). On the other hand, we cannot say that the risk increases by 2.4 (table 2 etc..), in fact, the increase is only 1.4 because one is not calculated, as it means that there is no difference.

Other minor corrections include:

It would be better for the author to indicate the average temperatures in the study area. For the phrase "Toxoplama infection" throughout the manuscript, the  the genus Toxoplasma should be written in Italic. The age group 40 and more should be corrected (Table 1).

Reviewer #2: This paper analyzed the seroprevalence of toxoplasmosis in HIV-infected patients in the DHB, Cameroon. Then the authors searched for risk factors for toxoplasmosis. The technical part is well described and the paper is well written. However, it suffers from insufficiencies and inadequate data analysis, which flaws the conclusion.

There are several major problems in the understanding of this study. First of all, the level of CD4+ T cells is well-known as a determinant factor favoring Toxoplasma reactivation, it is not “hypothesized” (line 72). Why do the authors think that “it is necessary to determine the relationship between CD4+ T-cell count and antibody titer to current Toxoplasma infection” (line 75-76) to guide patient management? After primary infection, the residual level of specific IgG is very different from one patient to another, whatever their HIV status. This seems the announced goal of the study (which does not make real sense), but despite that, the main part of the paper is dedicated to antibody screening and risk factor analysis.

Regarding the serological analysis, the most faithful seroprevalence should be calculated on the detection of IgG, regardless the presence of IgM. Indeed, the sole presence of IgM does not necessarily means that it is an acute infection, as non-specific IgM can be detected with many assays. As it is a cross-sectional study, we don’t have the result of a serological follow-up confirming primary-acquired infection by the rise of specific IgG. Thus seroprevalence should be 164/325. Besides, it is incorrect to state that IgM-positive patients have “acute” infection (line 185), as IgM can persist for months or years after primary infection (or be non-specific). Only IgG avidity testing would help confirming possible recent infection. What is really missing here is the context of patient inclusion. Where they consulting for routine checkup and delivery of antiretroviral therapy, or because of clinical signs? This would make a difference in the interpretation of serological results. Anyway, as expected, the seroprevalence increases with age, this is no novelty. There is no need to re-analyze age categories in Table 2. Tables 2, 3 and 4 should be merged.

The analysis of CD4+ T cell counts among Toxoplasma-seropositive and Toxoplasma-seronegative patients does not make sense. Do the authors expect that it influences the contamination with the parasite, which is environmental and foodborne ? it only influences the reactivation of past infection, which is not addressed here. Not surprisingly, there is no correlation between IgG or IgM titers and CD4+ cell counts. Comments about a trend (lines 248-51 and 255-57) are overstated and should be removed.

In the Discussion, it would be interesting to focus on prevalence data obtained in other African countries on the same patient population (HIV+), and to search for possible explanations for this discrepancy with previous results from Cameroon: is the DHB particular in terms of demography, climate, cultural behavior…? This part is well discussed, but the setting of the studies used for comparison should be more precisely addressed (HIV? Pregnant women? Randomly selected population?).

The sentence: “Contrary to other studies, which have shown that CD4+ T cell count threshold of ≤200cells/mL was indicative of onset of clinical manifestations of toxoplasma infection, in this study, it was observed that there was no significant difference between the mean CD4+ T-cell count of non-infected, chronically infected and acutely infected patients of toxoplasma infection” demonstrates that the authors poorly understand the pathophysiology of the disease and mix up serological and clinical findings. Again, the population study (not described here) is probably not the same as the studies cited. Most patients included, I guess, had no clinical signs and visited the health care facility for routine checkup, not because of clinical signs. Additionally, the authors considered that the diagnosis relied on their serological results (i.e. IgG+ = chronic infection, IgM+= acute infection), which is perfectly wrong. Indeed, acute toxoplasmosis in HIV+ patients mostly results from reactivation, thus all patients with clinical signs AND specific IgG (NOT IgM) should considered at risk for reactivation.

Altogether, this paper relies mainly on a seroprevalence study and associated risk factors and should only present the related results.

Reviewer #3: This study has been done to evaluate the seroprevalence of T. gondii infection in 325 HIV positive patients and the risk factors associated with the infection. Authors investigated the threshold of CD4+ T- cell count associated with acute T. gondii infection in the Bamenda Health District of the North West Region of Cameroon.

Comments to authors:

Introduction

Line 58: please write the full scientific name, Toxoplasma gondii, when you mention the name for the first time in the manuscript

Lines 67-69: it would be useful to present data regarding the seroprevalence of T. gondii from different areas of the world, to better put this study in the big picture, and to make this study more interesting to international readers.

Line 69: reference 4 is a poster presentation, please provide full length papers as references

Line 71: reference 7 is a poster presentation of a case report. Authors should provide full length papers as references

Lines 99-100: authors to rephrase the sentence, it is not clear

Lines 120-153: Methods, Serological diagnosis of T. gondii infection, sensitivity and specificity would be good to provide for the serological tests

Lines 120, 128, 130, 179, 198, 203, 204, 206, 212, 216, 219 aso in the paper: “Toxoplasma infection”, please use full scientific name in scientific writing, “T. gondii infection”

Line 200: please write toxoplasmosis, with lower case t

Lines 320-321: the journal is not BMC Veterinary Research, please check and provide journal’s name

Line 377, list of abbreviation: “Toxoplasmosis gondii”, please write “Toxoplasma gondii”

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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: No

Reviewer #2: No

Reviewer #3: No

[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.

PONE-D-20-04369R1

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

PLOS ONE

Dear Dr. Enah,

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.

The authors write the article in a good way. However, they need to address some concerns related to the statistical analysis as requested by reviewer 1. Also, they need to enhance the discussion part. The results contradict most of the previously published articles. Thus, the authors need to discuss the results critically. There are other minor corrections required to be addressed to enhance the quality of the article.

Please submit your revised manuscript by May 08 2021 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: http://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,

Mohammed Abdelfatah Mosa Alhoot, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Authors write the article in good way but need to address the some concerns related to the statistical analysis as requested by the reviewer 1. Also they need to enhance the discussion part. The results come in contradiction with most of previously published articles and the author need to give explanation for the results. there are another minor correction required to improve the quality of the article.

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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: (No Response)

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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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: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: 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: The manuscript titled "Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon" was well written and contains good information. However, Demographic, socioeconomic and nutritional variables have a relationship with each other and affect each other, and they should be included in the multivariate statistical analysis together.

And it was necessary to determine the basis on which the variable is included for the multivariate examination.

In addition, the authors treat the Odd ratio test on the basis that it quantites the association between the variables and this is incorrect. Odd ratio quantifies the strength of the association between two events. There are other tests to do this, such as a risk ratio (RR). On the other hand, we cannot say that the risk increases by 2.4 (table 2 etc..), in fact, the increase is only 1.4 because one is not calculated, as it means that there is no difference.

Other minor corrections include:

It would be better for the author to indicate the average temperatures in the study area. For the phrase "Toxoplama infection" throughout the manuscript, the the genus Toxoplasma should be written in Italic. The age group 40 and more should be corrected (Table 1). Use three decimal digits for P value results.

Reviewer #3: Lines 67-69: Authors to rephrase the paragraph and provide references regarding the seroprevalence and risk factors associated with T. gondii infection among individuals with human immunodeficiency virus (HIV) from different areas of the world.

Lines 326-327: please delete BMC, the journal is Parasite Vectors

Reviewer #4: please check the uploaded file for corrections.

For the discussion need to find researches to support your findings

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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 #1: No

Reviewer #3: No

Reviewer #4: No

[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.

Attachments

Attachment

Submitted filename: PONE-D-20-04369_R1_reviewer.pdf

PONE-D-20-04369R2

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

PLOS ONE

Dear Dr. Enah,

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.

There are few grammatical/typo errors that need to be addressed before proceed to the next step as suggested by the reviewers.

Please submit your revised manuscript by Aug 27 2021 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: http://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,

Mohammed Abdelfatah Mosa Alhoot, PhD

Academic Editor

PLOS ONE

Journal Requirements:

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 #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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 #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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: Comments to authors:

The manuscript was improved. However, minor edits should be made.

Lines 69-71: please delete “It would be useful to present data regarding the seroprevalence and risk factors associated with the infection amongst human immunodeficiency virus (HIV) of T. gondii from different areas of the world.”

I suggest to replace with this statement:

“Knowledge of T. gondii seroprevalence and its associated risk factors is important to predict the risk of infection in humans.”

Reviewer #4: The authors have adequately addressed comments raised in previous review. This time only 2 corrections in text (typing errors)

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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 #3: No

Reviewer #4: No

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Attachments

Attachment

Submitted filename: PONE-D-20-04369_R2_reviewer.pdf

Investigating the risk factors for seroprevalence and the correlation between CD4+ T-cell count and humoral antibody responses to Toxoplasma gondii infection amongst HIV patients in the Bamenda Health District, Cameroon.

PONE-D-20-04369R3

Dear Dr. Enah,

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Mohammed Abdelfatah Mosa Alhoot, PhD

Academic Editor

PLOS ONE

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