PONE-D-20-21492

HIV-associated nephropathy: Protocol and rationale for an exploratory genotype-phenotype study in a sub-Saharan African population

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

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

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

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

Reviewer #1: The report protocol “HIV-associated nephropathy: Protocol and rationale for an exploratory genotype-phenotype study in a sub-Saharan African population” by Aminu Abba Yusuf et al., describes an exploratory study which intend to associate the potential differential expression biomarkers of HIV infection with chronic kidney disease using the next generation sequencing technique, RNASeq, and subsequently validate the altered gene expressions using real-time PCR technique. The authors described the rationale of the study well and I believe that the study has an important impact, which potentially help in understanding the physiological development of chronic kidney disease in people living with HIV. However, there are few concerns that I wish to clarify with the authors regarding the study design, which listed as below:

1. The authors mentioned that 150 of HIV-positive adults will be screened for Chronic Kidney Disease, and 32 individuals from the same cohort will be selected for the downstream analysis. Of the 32 individuals, 16 of them will be grouped into the discovery group, where sample of these subjects will be used for RNASeq analysis, while the other 16 samples will be grouped into the validation group, which their RNA samples will be subjected to PCR analysis. I wish to ask the authors, is there a reason why the subjects are separated in such a way? Why can’t the validation to be performed on the same subjects of the discovery cohort?

2. 16 patients from the discovery group and validation group will be further assigned equally into non-ckd, mild-ckd, moderate-ckd, and severe-ckd where in each group would only consist of four patients. Would such a low sample size in each group able to achieve a valid statistical significant? I do think that the sample size is rather small. The authors should consider to increase the number of samples in order to achieve a valid statistical difference.

3. Would there be any plan of exploring the proteins that expressed by the deferential expressed gene in plasma samples? I think this would have added value to the finding as circulating plasma proteins may be a better predictor for the disease compared to RNA sample.

Reviewer #2: This is a well written manuscript. The study is important and an area where further exploratory work needs to be done

Just a few minor comments

Introduction

1. Line 72 - in addition to reference 3 [ would suggest reference 5 as well

2. Please check reference 6 : it does not seem appropriate to the information for this reference. This reference is not a genetic based study

3. Line 84 : this last section needs a reference

Specific aims

line 119 : need to remove the word "of"

Inclusion criteria

- It is not clear whether all patients need to be on ARVs or not and not clear in methods that patients do not need to be virally suppressed. It is mentioned later on in the text regarding viral suppression however this should be clearly stated in the methods. I needed to go back and forth to review and try and figure this out and would assist if it was more specifically mentioned in the methods. Plus line 146 ARV has not been written out in full earlier in the manuscript

Exclusion criteria

- Not sure whether necessary to clearly state that < 18 years is an exclusion criteria --> it is in the inclusion

Figure 2 : This diagram does not stand alone : It would help if you stated in the recruitment ...the population you are recruiting

Figure 4 : It would also assist to state the population you are working with and whether they are on ART or not * This is not clear in the methods

These are very minor additions however I feel that they would assist the reader with clarity

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Reviewer #1: No

Reviewer #2: Yes: Prof Nicola Wearne

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HIV-associated nephropathy: Protocol and rationale for an exploratory genotype-phenotype study in a sub-Saharan African population

PONE-D-20-21492R1

Dear Dr. Yusuf,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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

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Kind regards,

Nicole Endlich, Prof

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

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

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

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

Reviewer #1: The authors have addressed all the queries and explained them well. I am satisfied with the answers provided by the authors.

Reviewer #2: Thank you for the feedback given. I am happy that all my concerns have been addressed. Concerns from other authors have also been reviewed and I am happy that their requirements have been met

Reviewer #3: In their article “HIV-associated nephropathy: Protocol and rationale for an exploratory genotypephenotype study in a sub-Saharan African population” the authors describe a planned study by which they try to identify differential expressed gene expression biomarkers in HIV associated nephropathy. The topic is very interesting and there is a lack of available data so far. In their point-by-point response to the earlier reviewer comments they answer in detail all questions and concerns raised sufficiently.

The authors know about the limitation of the study due to the low number of patients which will be included in the study. Due to the high costs for RNAseq it is not possible for this study to include more patients. I like the idea of separating the cohort into two groups (one RNAseq and one validation group) but the authors might consider instead of dividing their patients in 4 groups, to reduce the number of groups to two or three (no, moderate and severe ckd) to get higher patient numbers per group.

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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 #2: No

Reviewer #3: No