Dear Dr. Kostka,

Thank you very much for submitting your manuscript "Absence of Anti-<i>Babesia microti<i> Antibody in Commercial Intravenous Immunoglobulin (IVIG)" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

  

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Angela Monica Ionica, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: all aspects are acceptable.

Reviewer #2: The study was designated appropriate and all analysis were performed correctly. I have one comment to clarify – why were only a portion of IVIG samples tested using ELISA, while 57 commercial samples were tested using IFA?

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: No problem.

Reviewer #2: I have no comments to Result section.

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: It is ok

Reviewer #2: I have no comments to Conclusions section.

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: In my opinion manuscript may be accepted for publication.

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: The manuscript entitled "Absence of anti-Babesia microti antibody in commercial Intravenous immunoglobulin (IVIG)” deals with a very important aspect of the therapeutic effect of IVIG as adjunctive therapy in babesiosis treatment, especially in immunocompromised patients. Overall this is a well-conceived, relevant and rigorously implemented study, and I believe the results contained would further knowledge in this area. Therefore, I congratulate the authors on well-conducted research and valuable results. However, I have one comment to clarify – why were only a portion of IVIG samples tested using ELISA, while 57 commercial samples were tested using IFA? I think this short explanation should be added to the text.

Reviewer #3: This manuscript demonstrated the anti-Babesia microti antibody titers of commercial IVIG and can provide useful insight into the potential therapeutic efficacy of IVIG for babesiosis.

However, due to the several defects, it was determined that this manuscript was insufficient to publish.

1) Lack of information about a 66-year-old male donor

The donor information used for the data in Fig. 1 should be explained in the Materials and Methods section, not in the Results section.

There was no information on whether informed consent was obtained from this donor.

The information on the IVIG administered to this donor should be presented, along with a detailed list of the 57 samples tested in this study.

Authors stated that this donor had B-cell lymphoma, but did not write when he received rituximab.

The description of rituximab in the Discussion section should be presented in the Introduction section. In the Discussion section, the authors should discuss the results of the donor's serum IgG levels in relation to the history of rituximab administration, IVIG administration, etc.

Since clindamycin was administered at the same time as the IVIG in this study, the efficacy of IVIG should be discussed in considering of antibiotics, and should be compared with the results with antibiotics alone.

In addition, it would be interesting to determine if anti-Babesia microti antibodies are present in the serum of this recovered donor.

2) Lack of detailed information about IVIG

More detailed information on all 57 IVIG samples, including name, lot, and collection site, should be presented in a supplemental table.

3) No IFA results

There were no figures or discussion of the IFA results for the 57 samples of IVIG.

For IFA, a comparison with the results of non-infected RBCs as controls is needed, but was not stated.

4) ELISA problems

The product name of ELISA assay kit should be presented.

Even though the manufacturer's positive control was used, the PC results in Fig. 2 showed a large difference (> 1.0 OD) between the two replicates. It should be re-tested.

--------------------

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

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Kostka,

Thank you very much for submitting your manuscript "Absence of Anti-<i>Babesia microti<i> Antibody in Commercial Intravenous Immunoglobulin (IVIG)" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The manuscript has been improved according to the reviewer's suggestions. Some minor revisions are still required.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Angela Monica Ionica, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

Section Editor

PLOS Neglected Tropical Diseases

***********************

The manuscript has been improved according to the reviewer's suggestions. Some minor revisions are still required.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: I have no comments.

Reviewer #3: Need to check the number of IVIG samples again in the full text. The number of IVIG lots appears to be 58 in Table S1, not 57 (line 108). In addition, was the data for 24 (or 28? according to the response to Reviewer 2) additional IVIG samples added?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: I have no comments.

Reviewer #3: In the Methods section, ELISA was explained after IFA, so the same order should be used in the Results section (lines 159-176).

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: I have no comments.

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: I have no comments.

Reviewer #3: Regarding the duplicate statement that rituximab depletes B cells in lines 73 and 77, delete one of them.

The differences between A, B, and C attached to the sample numbers in Table S1 should be explained in the legend. Alternatively, they should be described as one lot with one sample number.

If possible, the results of IFA and the ELISA OD450 value used for Fig 2 should be added in Table S1. This would make it easier for the reader to understand which samples were tested in IFA and ELISA.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: All comments of both reviewers were carefully considered and answered step by step. In my opinion, the manuscript is now in a form to be published.

Reviewer #3: (No Response)

--------------------

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

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

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.

Dear Dr. Kostka,

We are pleased to inform you that your manuscript 'Absence of Anti-Babesia microti Antibody in Commercial Intravenous Immunoglobulin (IVIG)' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Angela Monica Ionica, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************