Dear Dr. Mondal,

Thank you very much for submitting your manuscript "Dried Blood Spots (DBS): a suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era" 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.

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.

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[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.

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[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).

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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,

Travis J Bourret

Academic Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Section Editor

PLOS Neglected Tropical Diseases

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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: The hypothesis that continued surveillance must be done to preserve the gains made by the elimination campaign is widely accepted in the VL research community. However, the insistence that serology will be a good indicator of any

emerging foci needs to be clarified. Anti-rK39 and rK28 antibodies are found at high levels in South Asian endemic regions, compared to East Africa and are long lasting. Hence sero-surveys though useful in assessing endemicity, may not consistent predictors of new infections, which is the key need post- elimination. PCR may be a better predictor of that. These points need to be considered in the rewriting of the draft.

Reviewer #2: Methodology part need some improvements as suggested. The statement of invasive needs to change with appropriate word as DBS collection is as invasive as fresh sample collection i. e. use of needle.

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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: The results and analyses and their representations are sufficient for the serological tests. Statistical methods are appropriate. However for the PCR methods, more details must be include din the results section for clarity and completion.

Reviewer #2: Result is OK. P value of fig. 3 may be reevaluated.

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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: The conclusions are in line with the results mostly. However, the limitations of the analysis must be more clearly discussed. The sample size is mentioned, and the inclusion of a larger sample size is alluded to. This is to be expected. But, given the success of the elimination campaign, it is highly likely that larger a larger cohort of VL patients is not available. The authors must mention this and how it will impact any future studies.

Apart form that, the authors must also clearly state the advantages and dis advantages of using serology form surveillance in endemic foci. This has been a subject of extensive discussion in the VL field and how other techniques can fill the gap, namely PCR and antigen detection urine tests. Ihat is a clear limitation of the study and DBS utility, which has not been discussed in this manuscript.

Reviewer #2: Conclusion is well discussed but the importance of this study is not as much important as claimed. Need to address the point

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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: The authors must include the discussion on the limitations of sero surveillance that are suggested above in the revised manuscript.

The authors must include the Limit of detection of both the qPCR and RPA-PCR for whole blood and DBS in the results section. They only disclose the Ct and TT values, not the absolute levels of parasite genome equivalents or DNA concentrations that are detected by the methods. It is also necessary that the authors disclose the yield of DNA from DBS, and discuss its correlation to whole blood, since it is a highly likely contributor to the differences in sensitivity.

These details must be included in the revised manuscript.

Reviewer #2: 1. Which Whatman FTA card was used? There are various categories.

2. What was the basis of using two 5mm circles to elute DBS in 150 and 200 micro lit. elution buffer? Further, how the authors achieved 1:2000 dilutions from DBS? Previously Harith et al. 1989, Bimal et al. 2005 and many others had eluted single 5mm circle in 165 micro lit. elution buffer to get 1:50 dilution of antibodies. Quantification may be explained in the text.

3. After how long the samples collected were eluted? It also may be indicated in figure 7.

4. The sample was stored at -20oC till further use. How long it was stored? Prolonged storage in -20 deg. C loose Ab titer and protein. Definitely there is a value of deterioration. So, actual storage time must be recorded in methodology.

5. Statistical significance may be rechecked by an statistician for Fig. 3

6. Primers used may be provided in the text for consistency?

7. Invasive statement may be changed/ corrected. Isolation of DBS is not non invasive.

8. Fig. 1 and 2 must be reuploaded with good resolution and background.

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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: DBS is very promising for sero-surveillance and has been successfully implemented for other NTDSs such as filariasis that are also part of elimination campaigns. This is the first instance of a systematic study of DBS utility in the VL elimination campaign of South Asia. The authors have used the study to state the utility of DBS in Bangladesh clearly and precisely. The general execution, ethics, statistics and writing are very good and the authors clearly state the hypotheses and results. With the above suggested minor revisions, the manuscript is suitable for publication.

Reviewer #2: The manuscript “Dried Blood Spots (DBS): a suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era” has compared dried blood samples in comparison to whole blood NA for qPCR and RPA and serum for ELISA. Dried blood samples are being used to diagnose Leishmaniasis for years. In many areas, it was the sample of choice to diagnose Leishmaniasis because of comfortable transportation and storage. It was used mostly for sero-diagnosis (DAT and ELISA) and various molecular diagnostics. But now in the presence of rapid diagnostic kit; importance of DBS is not as much because the former needs only one drop blood from finger puncture and result comes within few minutes and don’t need much expertise and lab wares. Hence importance of this method is not as much in post RDK/RDT. However, it is a good effort to keep an alternative ready for the situations like non availability of RDK/RDT on the spot. Since the data shows compromised result in qPCR and RPA, the DBS cannot be an alternative of fresh blood sample. The proposed system is also as invasive as fresh blood collection.

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Reviewer #1: Yes: Aarthy C Vallur

Reviewer #2: Yes: Dr. Shubhankar Kumar Singh

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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.

Attachments

Attachment

Submitted filename: Review Report.docx

Dear Dr. Mondal,

We are pleased to inform you that your manuscript 'Dried Blood Spots (DBS): a suitable alternative to using whole blood samples for diagnostic testing of visceral leishmaniasis in the post-elimination era' 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,

Travis J Bourret

Academic Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

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: With the current revisions, the manuscript is acceptable for publication. The sample size question has been addressed by the authors along with the suitability of serology in this population.

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Yes

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

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

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

-Were correct statistical analysis used to support conclusions? Yes

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

**********

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: Yes, the analysis results and figures are appropriate. High- res figures are now included.

Reviewer #2: -Does the analysis presented match the analysis plan? Yes

-Are the results clearly and completely presented? Yes

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

**********

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: The conclusions hinge on their observations in this study and previous studies that have been used to support the use of DSBs. They are sufficient to justify the premise.

Reviewer #2: -Are the conclusions supported by the data presented? Yes

-Are the limitations of analysis clearly described? Yes

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

-Is public health relevance addressed? Yes

**********

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: Accept for publication

Reviewer #2: None

**********

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: DBS is very promising for sero-surveillance and has been successfully implemented

for other NTDSs such as filariasis that are also part of elimination campaigns. This is the first

instance of a systematic study of DBS utility in the VL elimination campaign of South Asia. The

authors have used the study to state the utility of DBS in Bangladesh clearly and precisely. The

general execution, ethics, statistics and writing are very good and the authors clearly state the

hypotheses and results.

Reviewer #2: (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: Yes: Dr. Shubhankar Kumar Singh