PONE-D-20-30776

A double-blind randomized control trial to examine the effect of Moringa Oleifera leaf powder supplementation on the anthropometric and immune status of adult HIV patients on antiretroviral therapy in a resource- limited setting

PLOS ONE

Dear Dr. Gambo,

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 manuscript has been evaluated by three reviewers, and their comments are available below. You will see the reviewers have commented on the importance of your work. However, the reviewers have also raised critical concerns and the manuscript will need significant revision before it can be considered for publication – you should anticipate that the reviewers will be re-invited to assess the revised manuscript, so please ensure that your revision is thorough. I have outlined some of the key concerns noted by the reviewers below, but you should respond all concerns mentioned by the reviewers in your response-to-reviewers document. 

The key concerns noted by the reviewers relate to the study intervention and analysis. Specifically, the reviewers have suggested alternate approaches to the statistical analysis and presentation of results. Additionally, Reviewer 2 noted that the distinguishing taste of MOG may be considered a source of potential bias in this study. Finally, Reviewer 3 noted that inclusion was limited to participants on one ARV regimen, which limits the generalizability of the findings. These issues have limitations for the interpretation of the results and should be explored.

Please submit your revised manuscript by Mar 02 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Danielle Poole

Staff Editor

PLOS ONE

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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: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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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: No

Reviewer #2: No

Reviewer #3: No

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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: No

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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: *** General comments: ***

The manuscript presents interesting data from what appears to have been a well-run study.

However, the statistical analysis is quite idiosyncratic and fails to control for multiple comparisons. It should be redone.

In addition, the manuscript suffers from a variety of English syntactic and usage errors. These should be corrected. There are relatively few in the results section, they are more frequent in the discussion section, and they are numerous in the remaining sections. There are too many to list.

*** Specific comments: ***

1) The protocol is quite weak in its description of the statistical methods to be used for the data, as well as the hypotheses of interest and the specific tests or contrasts that were to be employed. This weakness appears to have carried over to the actual statistical analysis. The study design is fairly straightforward, so it is unclear why a better framework for evaluation could not be specified.

Having said that, the most important test specified in the protocol is the t-test of the two treatments at the last time point, so this should be presented in any case.

2) The current layout of the statistical analysis is quite idiosyncratic and simplistic. The following approach or equivalent should be used instead:

Test baseline differences via the t-test as already performed. This is analogous to a two-factor analysis of variance (ANOVA) with one fixed effect --- that is, treatment.

Assess the differences between groups in changes over time by using a linear mixed effect model framework. The fixed effects are treatment, time, and the treatment by time interaction. Include a correlation structure to account for the dependence structure caused by observing the same patient over time. Do not use a compound symmetry structure (equivalent to "classical" repeated measures) --- this is an unrealistic assumption here. Instead, either use an autoregressive correlation structure such as AR(1) (since time points are equally spaced) or use a completely unstructured correlation matrix.

The test of the interaction of treatment by time provides the answer to the question of whether the response profile over time differs between treatments. If there is no interaction effect, that is not necessarily the end of the road, but it is arguably of primary interest.

Next, perform post hoc comparisons using an adjustment for multiple comparisons such as the Tukey-Kramer adjustment. It is up to the authors, they can perform an analysis of either simple effects (comparison between treatments at each time point or comparison among time points within each treatment) or an analysis of all pairwise comparisons. Note that the test of the interaction effect is not technically required to be statistically significant for these tests to be performed, though this is the usual procedure.

All linear models should be assessed for fit using graphical analysis of the residuals to check for symmetry, homoscedasticity, and independence. The Shapiro-Wilk or Kolmogorov-Smirnov test can be used, but is probably of less importance.

If transformations are required, please report all transformations attempted. Even if fit is marginal on the original scale, if the results are qualitatively similar to a fit on a transformed scale then it may be best to report analysis on the original scale for interpretation.

For completeness's sake, since the protocol specifies it, also include the t-test of the last endpoints. However, this is probably less powerful than the result that will be obtained from the linear mixed effects model approach.

3) The evaluation of socio-demographic characteristics (Line 270 forward) should be redone. This should be done in the framework described above, as an exploratory analysis. These covariates can be entered as main effects in linear mixed effects model described above. This will yield answers to the question of whether the treatment effect remains if the effect of the covariate is statistically corrected, which is probably the question that the authors have in mind.

4) In some cases, it is difficult to understand what exactly is being presented. For example, what does Table 5 present? What are the "differences" here? Each table and figure should have a caption that clearly describes its contents.

5) Figure 2 is really terrible and should be replaced with a standard figure that shows the behavior of the expected marginal means over time for each group.

6) Lines 317-318: This conclusion seems far too strong for this paper. Has it really been proved to this degree?

7) Lines 338-344: The authors do not disclose whether similar claims were made by COG patients. This paragraph seems really close to misinformation as it is currently worded. If patients from both treatments reported these claims, then how can they be attributed to Moringa oleifera? All such claims in the discussion need to be backed up by data analysis in the results. Where is the presentation of these results?

8) The methods section lacks critical details. As an example, on Line 175 the authors state that viral loads were measured "by the Laboratory scientist using Real-time PCR machine" (sic). Which machine? What was the protocol? Which laboratory? The same issue pertains to the CD4 counts.

9) Lines 403-405 overstates the results.

10) The conclusion and recommendation in Lines 417-121 do not seem to be fully supported by the manuscript.

Reviewer #2: 1. Moringa leaf powder packaged in sachets as used in the MOG potentially had a taste distinguishable from COG. This most likely violated the blinding process especially in a community where there was a “high awareness” level of Moringa’s usefulness as a nutraceutical. This should be considered as significant source of bias enough to dispute the conclusion of a causal relationship between the MOG intervention and the observed increase in CD4 count. A more firm conclusion could have been justified if the powder had been packaged in gelatin capsules which could mask the taste and also ensure more accurate and consistent dosing of the product.

2. While literature references were made, the claims that Moringa leaf samples contained essential and non-essential amino acids, wide range of vitamins, minerals, carotenoids, polyphenols, phenolic acids, flavonoids, alkaloids, glucosinolates, isothiocyanates, tannins and saponins were unsubstantiated in this particular study. Perhaps a description of whether the manufacturer Prime Global Agricultural Industries Limited routinely analyzed the samples for the claimed ingredients would have been useful.

3. Addition of the discussion of the potential of interactions between Moringa and antiretroviral therapy, particularly with efavirenz via alteration of liver metabolism would enrich the manuscript.

4. It is more scientifically acceptable to use the terms such as “study participants” or “HIV infected individuals” instead of “HIV patients”.

5. Sentences on line 174 and line 176 need reconstruction

6. There may still be more work before a causal relationship can be claimed between the consumption of Moringa leaf powder and observation of increased CD4 count. Review recommends that the conclusion should limited to an association between the intervention and increased CD4 count, siting the observed shortcomings of the study design.

Reviewer #3: The manuscript reports findings from a randomised controlled double blinded trial assessing the effect of Moringa oleifera (MO) leaf powder supplementation on weight, BMI, MUAC, CD4 count and viral load. This is an important study given that MO is widely consumed and is an important herb among people living with HIV. Based on the data, the authors conclude that only the CD4 cell counts improve significantly (p=0.03) after 6 months concurrent MO leaf supplementation and ART. The manuscript however has some technical issues in some aspects of the trial design and statistical analysis.

p11 Selection criteria

L105 - Inclusion of patients on only one regimen limits generalisability of the effect on CD4 counts to patients on other regimen. Why was this necessary?

L113 - Typically, randomisation follows screening and indicates a participant is enrolled into the study. However, under exclusion criteria authors state that patients taking micronutrient or natural health product supplements within 30 days of randomisation were excluded. Please clarify.

p12 Sample size

L122 - In the abstract the authors talk about significance of the mean increase of CD4 count from baseline at the 99% CI which is inconsistent with sample size justification based on a 5% margin of error, i.e. 95% CI.

p12 - 13 Intervention

L135 - How were the MO leaves positively identified? Was a botanist consulted?

L156 - What seive size was used to prepare the intervention? 5g weight seems too heavy for 1 teaspoon of MO leaf powder.

L158 - Specify that MO from other sources is what was disallowed.

p14

L174, L177 - specify the type and volume of blood samples collected and a brief explanation or reference to the details of the laboratory tests.

p15

L202 - It is the participants who provide consent to participant in the study, not the researchers. This presents a significant ethics issue for this study.

p18-19

Why was the change in anthropometric and immmunological parameters within group important to analyse statistically? Given the objectives of the study, it is the difference between the intervention and control groups that is relevant. The results section is crowded with results that may not be important. In addition, the change from baseline at the end of each month may be more useful than the change between each month. Consider revising the results section and highlighting only the difference in change from baseline between the intervention and control group. One figure and one table, with legends, and focusing on the key results will further enhance the section.

p24

L334 - There is no data on which the discussion on 'good adherence' is based. The strategy the authors describe on p13 L161 was to monitor adherence, rather than enhance or optimise it. No results are presented on the level of adherence. In fact a large portion of the discussion refers to perceptions from participants and other operational and implementation aspects that are core to the study and not presented in the results and are appearing for the first time as discussion.

p21

L407 - From the power calculation, the study was adequately powered, perhaps a more diverse population of PLWH rather than a larger sample size could be recommended? The recommendations should be based on the effect on MO on CD4 and not extend to issues of food security and nutrition which have not been demonstrated by the current study.

Several challenges and limitations are cited. To what extend were participants reluctant to keep clinic appointments? What was the effect on study outcomes and how was that accounted for in the analysis?

The write up also has some grammatical errors and could benefit from revision by an English editor. Some comments that may be helpful

Page 1

L1 - 'oleifera' is always written in lower case and itilised. Correct throughout the manuscript.

L2 - Be consistent with the term used for study population. Ideally, use people first language such as 'people living with HIV (PLWH)' rather than HIV patients or HIV infected patients.

Page 9

L46 - Change accountable to accounts

L48 - revise grammar

L71 - 'leaf' powder

L72 - Sub-Saharan

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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.

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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.]

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PONE-D-20-30776R1

A double-blind randomized control trial to examine the effect of Moringa Oleifera leaf powder supplementation on the anthropometric and immune status of adult HIV patients on antiretroviral therapy in a resource- limited setting

PLOS ONE

Dear Dr. Gambo,

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 after your first revision. Therefore, we invite you to submit a further revised version of the manuscript that addresses the outstanding statistical issues raised during the current review process.

Please submit your revised manuscript by Jul 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,

Tsitsi Grace Monera-Penduka, PhD, MSc, MPhil, BPharm Hons

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.

Additional Editor Comments (if provided):

The authors have addressed the grammatical and other issue raise satisfactorily save the statistical issues. They should revisit additional comments from the current review and address them.

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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)

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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

**********

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

Reviewer #1: I Don't Know

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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

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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

**********

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 authors appear to have at least partially implemented some changes to the statistical analysis. However, it is not possible to tell whether the analysis was performed correctly due to the lack of detail in the description of the statistical analysis and in the results shown. From what can be seen, it appears that the analysis was not specified correctly; or, if it was specified correctly, it was not reported correctly.

Also, the authors should put some energy into evaluating whether the different types of analysis performed produce similar results. My suggestion to keep the planned analysis was because as a rule all pre-planned analyses should be presented unless completely inappropriate. However, that means that there will be potentially conflicting results, perhaps due to the effects of statistical correction, perhaps due to increased or decreased power, or perhaps due to different assumptions.

In fact, this might result in relegating some part of the current work into a supplementary appendix on the grounds that it is less germane.

The covariate analysis that is presented is also difficult to assess. The model structure (as in the other case) is not clearly specified. If transformations were used, they are not described. Note that "transformation" does not mean changing data format from wide to long format. Instead, it means such things as logarithmic transformation or other functional transformations, aggregation, or recoding. Simple manipulation of data shape is irrelevant to the manuscript because it is only a technical requirement of the data analysis software.

As initially mentioned in the first review, all linear model analysis must be subjected to goodness of fit evaluation before interpretation. This is described in brief in the first review and does not appear to have been performed.

My current recommendation is to obtain the services of a consulting statistician to work through the data analysis and review comments and to put everything onto a solid and consistent footing.

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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

[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.

A double-blind, randomized controlled trial to examine the effect of Moringa oleifera leaf powder supplementation on the immune status and anthropometric parameters of adult HIV patients on antiretroviral therapy in a resource-limited setting.

PONE-D-20-30776R2

Dear Dr. Gambo,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Tsitsi G. Monera-Penduka

Guest Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: