PONE-D-20-11317

Sensitivity Optimisation of Tuberculosis Bioaerosol Sampling

PLOS ONE

Dear Dr. Patterson,

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.

Please submit your revised manuscript by Jul 26 2020 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,

Selvakumar Subbian, Ph.D.

Academic Editor

PLOS ONE

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We note that one or more of the authors are employed by a commercial company: Edge Embossing and Zeteo Tech.

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4. Please include a caption for figure 1.

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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: No

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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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: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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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: Yes

Reviewer #4: Yes

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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: The authors describe some changes to an aerosol sampling procedure that appears to improve the sensitivity in detecting sputum-positive TB patients who exhale infectious aerosols. There are some serious problems with the manuscript.

1) The paper compares data from the current study (RASC-2) with a previous study (RASC-1). Data from RASC-1 have already been published (Ref 14) and are repeated here.

2) The two studies cannot be compared. They were conducted 2-3 years apart with different patient groups. More importantly, two different measurements were used to determine the number of mycobacteria in the sampled aerosols – viable bacilli or cfu (RASC-1) and fluorescence microscopy (RASC-2). The authors provide no evidence that the fluorescently-labeled bacilli in RASC-2 were viable.

3) The authors admit (lines 143-144) that the fluorescence microscopy detection assay for detecting mycobacteria in RASC-2 was subjective and dependent upon the skill of the operator. Please provide some data or references to support the validity of this assay (e.g., repeated determinations from the same sample).

4) The overall conclusion of the study is trivial and can be summarized in a single sentence – “Increasing the amount of exhaled air sampled will increase the sensitivity of bacillary detection”. This is intuitively obvious to any intelligent person and does not require data or a manuscript to confirm.

5) The authors have grossly over interpreted their results. They speculate wildly about the possible implications of their aerosol sampling results with no supporting data.

6) The authors utilize jargon that is not defined for the reader. What, precisely, is an “aged” aerosol? What is the difference between “exhaled air” and “respiratory bioaerosol”?

Reviewer #2: In the present study, the authors have presented the method to sample bioaerosol from TB patients in order to determine the infectiousness. The method they described is an improvement of their previous work. However, I find following concerns,

1. Data for TB cases has been given for year 2018, instead of 2019.

2. The technical improvements in the design of RASC has not been elaborately explained. The focus has more been put on the collection outputs of aerosols in results section. I think, it will be better to present collection output in relation to modifications in the design of RASC.

3. In the method section, superficial information has been given. They should describe all the modifications in detail.

4. In the sample collection section, it is difficult to understand the technicalities. The authors should present these elaborately with proper diagrams and flowcharts.

5. In fig3, statistical significance and p value are missing.

6. Figure legends are not self explanatory, more details are required.

Reviewer #3: In the present study, authors employed modified version of Respiratory Aerosol Sampling Chamber (RASC) to incorporate liquid collection of bioaerosol using a high-flow wet-walled cyclone (RASC-2). Authors demonstrated this technical modification increased the utility of bioaerosol sampling and lowered the limit of Mtb detection in RASC-2 platform. Study result is interesting and has future application in characterizing exhaled air and bioaerosol from sputum negative and subclinical individuals. Authors have to address the following comments.

Major Comments

1. Authors estimated the Mtb count per 100 liters of exhaled air as Mtb-CFU for RASC and DMN-Tre stained Mtb bacilli. Since authors employed two different methodology to estimate the lower limit of detection, study result may vary. Please clarify this discrepancy in methodology.

2. This includes patients with and without cavitary TB. Earlier studies have shown that pulmonary TB patients with lung cavitation are the main source of disease transmission. Did you analyze the data such as Mtb count and lower limit of Mtb detection between the patients with and without cavitary TB in both RASC-1 and RASC-2.

3. Is it possible to study the nature of Mtb such as Mtb in the form of singles, small and large clumps in exhaled air and bioaerosol?

4. There is typo in the table 1; the number of patients with lung cavitation in RASC-2 is 6 (28.6%) not 46.2%. Correct the typo.

Reviewer #4: Authors have tried to improvise on a previously established protocol for aerosol sampling from the patients for possible bacterial count. The sensitivity can improvise the testing capability for possible MTB clearance.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: 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.

PONE-D-20-11317R1

Sensitivity Optimisation of Tuberculosis Bioaerosol Sampling

PLOS ONE

Dear Dr. Patterson,

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.

==============================

ACADEMIC EDITOR: The crux of this paper is that an improvised air sample collection system increased the sensitivity of Mtb detection. As Rev#1 mentioned in previous round of review, this could be done without comparing the two methods, since they cannot be compared head-to-head. As can be seen from the instrumentation picture, these two air collection systems are not comparable. The parameters used to collect samples, the pressure of the unit, and the Mtb detection methods are very different. As the authors noted, since multiple parameters are changed, it is hard to define which parameter contributed to the improved sensitivity. Then, a question is "why to compare these two in the first place ?". Thus, i would suggest the authors to present only the features of the latest model (RASC-2), its design, parameters and performance. The previous publication on the RASC-1 model can be discussed in the "Discussion" section, if/when relevant. Alternatively, if the authors still want to compare these two systems head-to-head, then add a table to explicitly mention all the minute differences between the two units on every parameter, such that the reader would understand what "optimization" happened from RASC-1 to become RASC-2. 

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

==============================

Please submit your revised manuscript by Sep 04 2020 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

We look forward to receiving your revised manuscript.

Kind regards,

Selvakumar Subbian, Ph.D.

Academic Editor

PLOS ONE

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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)

Reviewer #2: All comments have been addressed

Reviewer #4: All comments have been addressed

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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: No

Reviewer #2: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #4: Yes

**********

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

Reviewer #2: Yes

Reviewer #4: 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 #4: 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: Please see the previous review......................................................................

Reviewer #2: (No Response)

Reviewer #4: (No Response)

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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 #4: 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.

Sensitivity Optimisation of Tuberculosis Bioaerosol Sampling

PONE-D-20-11317R2

Dear Dr. Patterson,

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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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,

Selvakumar Subbian, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: