PONE-D-20-28601

The Accuracy of Healthcare Worker versus Self Collected (2-in-1) Oropharyngeal and Bilateral Mid-Turbinate (OPMT) Swabs and Saliva Samples for SARS-CoV-2

PLOS ONE

Dear Dr. Tan,

Thank you for submitting your manuscript to PLOS ONE.

Your manuscript has been reviewed by five experts in the field and their comments follow. All five reviewers like your work, but they also raised concerns and made specific suggestions. Addressing them in a revised paper will significantly improve the presentation and technical quality of your work. 

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We look forward to receiving your revised manuscript.

Kind regards,

Dong-Yan Jin

Academic Editor

PLOS ONE

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Additional Editor Comments:

Please make your best effort to address the reviewers' concerns.

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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: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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: No

Reviewer #5: 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: No

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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: In this study, Tan and colleagues present a comparison of self-collected swabs/ saliva vs. healthcare worker collected swabs. This is a controversial topic and worthy of exploration. Importantly, they find that self-collected options may be less sensitive than HCW collected swabs. The study is nicely performed and presented. It is particularly nice to see sample size analysis, which adds confidence in the conclusions. However, I do have a few recommendations to the authors to extract as much as possible from their data. I hope this helps them improve their manuscript further.

1. Please expand the abbreviation ‘OPMT’ when it first appears in the introduction.

2. Line 89: add a note on how patients in group 1 were confirmed to have COVID-19.

3. Can I confirm that the patient swabbed both mid-turbinates using a single swab stick and then put the same swab stick into their throat?

4. It is not clear what is meant by the ‘spitting’ method? Is this posterior oropharyngeal throat saliva collection? Collecting oral fluid, spitting out saliva, and collecting posterior oropharyngeal throat saliva are all likely to have different sensitivities for SARS-CoV-2 detection, so we need to define what exactly is being collected. Would actually be nice if the instructional videos could be uploaded as supplementary material.

5. Was there any particular timing of saliva collection? There is a tendency for early morning saliva to have higher viral loads (Hung DL et al, Open Forum Infect Dis, 2020).

6. Line 130 – 131: to clarify, the sample size was calculated based on a type I error rate of 1%?

7. Line 150: redundant ‘who’

8. Table 2 is not particularly useful and duplicates text in line 167 - 168. Could consider replacing with a contingency table of HCW swab vs saliva and HCW swab vs self-swab. This way, we can also check the % agreement and how many samples were detected by saliva/ self-swab, but not HCW swab.

9. Could add a McNemar test to compare sensitivities of saliva, self-swab and self-swab + saliva against the HCW-swab ‘gold standard’.

10. Table 3, 4: why is the total number of samples 336 (table 3), 335 (table 4)? The number of samples should be 401 – 27 (no. of negative samples) = 374? Are there missing data points?

11. Consider showing a scatterplot of the correlation of Ct values between self-swab and HCW swab and saliva and HCW swab.

12. Could include a column scatter plot comparing the RT-PCR Ct values of HCW swab, self-swab and saliva and statistically compare (? median) Cts of self-collected sample types to the HCW swab.

Reviewer #2: It is a great topic to compare the performance of self collected and HCW collected samples. But, the authors did not mention what clinical samples were used as gold standard for the diagnosis of COVID-19 in the 401 subjects. If HCW OP and MT swabs by HCW were used as gold standard, like procedure 2, listed under "test procedures", why the authors need to have a 2 stage design and include those previously tested positive for COVID-19 as subject and the second group of healthy volunteers as control? Why not include all the people when they were first tested for COVID-19 and test them with the three samples types at the very beginning?

The authors used the term 'detection rates' and 'negative correctness', are they referring to sensitivity and specificity which are more professional terms?

Reviewer #3: Tan SY and colleagues performed a cross-sectional study to investigate healthcare worker vs. self-collected OPMT swabs and saliva samples for the detection of SARS-CoV-2 among persons with a confirmed diagnosis of COVID-19 and healthy volunteers.

I have some concerns as follow:

Major comments:

1. Characteristics, e.g. age, the onset of symptoms, severity of the disease, of the study populations should be included to provide readers to understand the clinical setting of the study better. The different settings may associate with different sensitivity of each specimen. One study demonstrated the lower value of saliva for the diagnosis of COVID-19 in children (Chong CY, et al. Clin Infect Dis 2020; article in press). Many studies showed more testing agreement of saliva and nasopharyngeal swab at the earlier onset of the disease (Jamal AJ, et al. Clin Infect Dis 2020; article in press, Iwasaki S, et al. J Infect 2020; article in press).

2. The gene that was RT-PCR test should be described. Was a housekeeping gene included in the RT-PCR reaction? The presence of the housekeeping gene in the RT-PCR test could help to determine the adequacy of a specimen collection.

3. In the first paragraph of the discussion, the author stated: “Our findings corroborate with existing epidemiologic data which indicates that while viral RNA detection may persist in some patients, such persistent RNA detection likely represents non-viable virus and hence, such patients are noninfectious.” The author should demonstrate the results in their study that suggest the conclusion of this statement.

Minor comments:

1. The laboratory processing method that resulted in a lower yield of the saliva should be discussed. In this work, saliva samples were collected using the SAFER-Sample, which a saliva solubilizing solution was added to the samples. A preprint by Griesemer SB and coworkers demonstrated lower sensitivity to detect the virus when saliva stabilizing solution was added. Some studies showed a higher sensitivity of saliva when compare to nasopharyngeal swab. In these studies, the authors did not put either saliva stabilizing solution or viral transport media in the saliva specimen (Wylle AL, et al. N Engl J Med 2020; article in press; Rao M, et al. Clin Infect Dis 2020; article in press).

2. Please update the references. The preprints were accepted for publication.

3. The writing can be improved by reorganizing the content to increase the continuity of the idea and content in the manuscript.

Reviewer #4: The manuscript by Tan et al , compared the accuracy of healthcare worker versus self collected OPMT swabs and saliva samples for SARS-CoV-2. This is a comprehensive and important study during this COVID-19 pandemic.

I have the following minor comments:

- the authors may consider adding a flowchart for the workflow and sample size of the study

- the authors should include supplementary file for all single Ct values of different samples matching each patients

Reviewer #5: In the manuscript entitled “The Accuracy of Healthcare Worker versus Self Collected (2-in-1) Oropharyngeal and Bilateral Mid-Turbinate (OPMT) Swabs and Saliva Samples for SARS-CoV-2”, Seow Yen Tan et al compares the detection rate of SARS-C0V-2 by RT-PCR in self- collected samples and samples collected by HCW. In addition, they used saliva, OP swabs and both methods combined, and compared the sensitivity of each approach in 400 samples from patients diagnosed with COVID-19 and 100 negative subjects. They concluded that saliva and sel-collected samples are inferior to OP and HCW-collected ones, respectively. However, combining both self-collected samples provided a higher detection rate.

Some points to discuss:

1. How long it took to test saliva samples after collecting them? Although swab samples were preserved in transportation medium, saliva could be affected by time until testing;

2. Collecting saliva samples as the last procedure could affect results? Self-collected swab, followed by HCW collected swabs could interfere in the quantity and quality of saliva samples;

3. Line 167 “self-saliva” means “self-swab”?? Please, correct it;

4. In several parts of text authors refer to testing in “early phase” would provide better results: how early??In addition, there is no description on details of methodology, like time to test, preservation of samples, and characteristics of patients with positive and negative results. What about the time since diagnosis or duration of symptoms? Or severity of disease? These are important information to understand how samples were selected, and how such information could help explaining the results obtained;

5. Authors state at conclusion that self-collection would be preferable for use in low prevalence population? Why??

6. How author explain their results are so distinct of other reports, regarding sensitivity of saliva testing? It should be addressed in discussion. In limitations of study they cite the use of a very specific population (male migrants). How could this fact impact the results?

Minor comments: there are several typos and gramatical mistakes. A deeper English revision is warranted.

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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.

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: Yes: Siddharth Sridhar

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: 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-20-28601R1

The Accuracy of Healthcare Worker versus Self Collected (2-in-1) Oropharyngeal and Bilateral Mid-Turbinate (OPMT) Swabs and Saliva Samples for SARS-CoV-2

PLOS ONE

Dear Dr. Tan,

Thank you for submitting your revised manuscript to PLOS ONE.

We have now received comments from the original reviewers. Two of them carefully picked up some typos and errors in your paper that must be corrected in the final version. 

After careful consideration, we feel that your paper 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 Jan 21 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

We look forward to receiving your revised manuscript.

Kind regards,

Dong-Yan Jin

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: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: 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: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Yes

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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 #3: Yes

Reviewer #4: Yes

Reviewer #5: 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 #3: (No Response)

Reviewer #4: Yes

Reviewer #5: 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: Just a few minor points:

1. Correct grammar for this sentence: “Another study on 236 ambulatory, literate, mostly adult subjects the performance of self-collected nasal and throat swabs was at least equivalent to that of health worker collected swabs…”

2. Correct grammar for this sentence: “Self-collection of samples would reduce very significantly on the reliance of trained personnel to collect samples and ramp up testing capacity.”

3. Clarify in the methods that subjects were supervised during self-sample collection. This is hinted at in the discussion.

4. Line 155: inappropriately italicized ‘R’ in reverse-transcription.

5. Line 163: SARS-CoV-2, not SARS-Cov-2.

6. Line 265: Please elaborate on this statement. I can understand why the study findings would not be applicable to pediatric and very elderly populations, but the study findings should be broadly applicable to other sections of Singapore’s population?

Reviewer #3: My comments have been addressed.

However, some typos are still noted, e.g., Lines 246 and 247: the legends of figures 1 and 2 describe the same thing. The manuscript might be benifit from language editing.

In the first paragraph of the discussion, the authors stated the study on patients who are already known to be COVID-19 as the strength of the study. I think this is more likely to be the limitation, as the mean duration of the first positive swab to the study day and the mean duration between illness onset to study day were quite long. It is well known that Ct value correlated with days from symptom onset. Sensitivity of the virus detection is decreasing in saliva collected from later time of illness onset.

Ref: Jamal AJ, et al. Clin Infect Dis. 2020;ciaa848. Williams E, et al. J Clin Micriobiol. 2020;58(8):e00776-20.

Reviewer #4: The authors have addressed all my concerns, i have no further questions. The manuscript should be ready to published.

Reviewer #5: (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: Yes: Siddharth Sridhar

Reviewer #3: No

Reviewer #4: No

Reviewer #5: Yes: Carlos Brites

[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.

The Accuracy of Healthcare Worker versus Self Collected (2-in-1) Oropharyngeal and Bilateral Mid-Turbinate (OPMT) Swabs and Saliva Samples for SARS-CoV-2

PONE-D-20-28601R2

Dear Dr. Tan,

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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Kind regards,

Dong-Yan Jin

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: