Dear Dr. Aminu,

Thank you very much for submitting your manuscript "Practical and effective diagnosis of animal anthrax in endemic low-resource settings" 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.

Both reviewers raise the question about the limitation of PCR facilities in developing countries - since there was no third reviewer, I also reviewed this manuscript and also had a similar question. I also agreed with the reviewer who suggested that figure S2 might be useful in the main manuscript.

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,

Brianna R Beechler, Ph.D., DVM

Guest Editor

PLOS Neglected Tropical Diseases

Ana LTO Nascimento

Deputy Editor

PLOS Neglected Tropical Diseases

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

Both reviewers raise the question about the limitation of PCR facilities in developing countries - since there was no third reviewer, I also reviewed this manuscript and also had a similar question. I also agreed with the reviewer who suggested that figure S2 might be useful in the main manuscript.

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: For the purposes of validating the method being investigate here (azure B), it would have been valuable to do culture diagnostics on the same samples. I realize this may not have been feasible in-country, but wonder if it would have been possible to ship the samples out for this analysis?

Reviewer #2: There are no major concerns about the methods.

Just three questions:

1.) The authors define "suspected cases of anthrax" (lines 182-185). Does this definition also apply for older carcasses where only skin or pieces of skin were available? For livestock, the owners might remember the circumstances of death, but what about wild animals which were found dead and probably widely decomposed? How can anthrax be assumed as cause of death in these cases?

2.) The scores for staining are indicated in S1 Fig. Which staining method is shown in this Fig?

3.) Did the authors use a balance to determine the weight of the skin portions (50 mg) or was this just estimated?

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

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: Results are clearly presented.

It would be interesting to know which PCR target was the most sensitive. From other publications, one could assume that this would be one of the plasmid targets because plasmids are present in more than one copy.

For completeness, a Table should be added to the supplementary material in which the results for each of the 367 suspected anthrax cases are summarized. This table will show the correlation between the PCR results for different sample materials of the same animal and also between staining and PCR of blood smears.

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

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: The conclusions are supported by the data and limitations were discussed. The public health aspects were well covered.

Staining and PCR seem to be reliable methods for confirmation of anthrax in low-resource settings. However, DNA extraction using a homogeniser might not be available everywhere. Are there any alternatives for DNA extraction from skin samples without using beads?

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

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: L80-83: It seems the problem of lack of infrastructure/consumables would also be a limitation for PCR. May need to add something here to strengthen the argument. Also, it seems the work here was conducted in a BSL3 facility (L248), so it does further raise the question about whether PCR based approaches alleviate the problem. The paper in general could use more discussion on previous use of PCR for anthrax diagnostics in clinical specimens (maybe not here, but in discussion).

L136: Possibly lead here by also indicating what is currently considered to be the “gold standard,” i.e., culture?

L157-158: Would it be possible to qualify the occurrence of anthrax in the NCA? For example, is it widespread and consistent, or does it occur sporadically? This would help support why the NCA may be a good system to validate the diagnostic approaches.

L220: Indicate here that the samples used for PMB staining were a subset of the total samples.

L225, 281, 283: There seems to be a problem with the references here.

L347: Write out “NTC” rather than using acronym, since it only appears a couple times.

L366-367: What proportion of samples had all 5 sample types?

L375-376: Does the time of sampling since death affect smear assay sensitivity? If approximate time since death is known, it may be helpful to assess whether there is a relationship between time since death and probability of being a false negative.

L407-409: This section could use a sentence that highlights which sample type resulted in a qPCR assay with the highest sensitivity/specificity. Also, change “very high” to “high” (L407).

L 446-449: It is not clear what level of observer experience is necessary to achieve the results presented here. This could use some discussion.

L491-500: Perhaps this is stating the obvious, but it may be important to explain why fly samples have low sensitivity. For example, flies found in association with carcasses may not all have been exposed and infected with B. anthracis (so are not true positives), and here you are evaluating their potential as indicator species for the infection status of the nearby carcass.

Table 1: I might suggest renaming the column heading to be “Three stain comparison (n =152)” and “Four stain comparison (n=102)”. But, really, I’m not sure the first column adds that much more, so could be omitted.

Table 2: Should it be 95% credible posterior intervals reported here?

S1 Fig: It would be helpful to include suspect samples that were determined to be negative, so with similar morphology, but lacking the pink capsule or demarcated rod ends.

S2 Fig: I found this Figure helpful to illustrate the approach used, as well as summarize the various methods being assessed. You may want to consider moving this into the main text. Also, would it be possible to breakdown the model into its component classes (e.g., positive/ negative classes)?

Reviewer #2: Some references or links seem to be missing (e.g. line 225)

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

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: This study evaluates the performance of microscopic and molecular-based methods for anthrax diagnostics from field-collected livestock and wildlife carcass samples. The authors’ main objective was to assess the use of a modified blood smear assay (using the azure B stain), which is more accessible in developing countries than culture-based methods. They found that the azure B method had a high sensitivity and specificity in comparison to 3 alternative microscopic staining methods, including the reference standard PMB method. Previous studies have tested the azure B method in the lab (e.g., Owen et al. 2013), but this appears to be the first (?) study to validate its application and performance in the field. Another goal of this study was to evaluate a qPCR-based assay for detecting Bacillus anthracis from a number of carcass sample types. The paper is well written, study design is robust, and conclusions appear to be valid. Ideally, the authors could have included culture data as the gold standard for estimating the sensitivity/specificity of these methods, but it appears there was not a facility in-country equipped for this (hence the need for this paper!). Also, it is not clear whether the study is novel with respect to the use of PCR diagnostics, which is already an accepted standard. Overall, these results provide support for the azure B blood smear method, and has the potential to improve surveillance and disease control in developing countries that lack infrastructure and resources.

Reviewer #2: Anthrax surveillance in endemic low-resource settings is a problem, and the data presented in this manuscript will be helpful to establish reliable methods for confirmation.

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

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

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, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Aminu,

We are pleased to inform you that your manuscript 'Practical and effective diagnosis of animal anthrax in endemic low-resource settings' 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,

Brianna R Beechler, Ph.D., DVM

Guest Editor

PLOS Neglected Tropical Diseases

Ana LTO Nascimento

Deputy Editor

PLOS Neglected Tropical Diseases

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

We thank the authors for completely addressing all the reviewer comments.

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 #2: (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 #2: (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 #2: (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 #2: (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 #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 #2: No