PONE-D-21-27242Evaluation of GENECUBE® HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocolPLOS ONE

Dear Dr. Suzuki,

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Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: 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

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: As a whole this is a good and convincing paper, but I have some concerns – both minor and major.

Journal policy concern: Authors state that there will be some restrictions on data sharing, without specifying what those are. They later state that all relevant data are in the manuscript. This seems internally contradictory.

Minor concern: Line 69: Authors state that “This reagent was approved in October, 2021.” Who was the approving body?

Major concern: Authors note that if discrepancies were found between GeneCube analysis and the RTPCR reference method, a third method was employed to resolve the discrepancy. This procedure, known as “discrepant analysis” is biased. [1–7]. Bias results from the fact that testing of non-discrepant samples (based on two assays) may, in principle, result in a discrepancy when the “resolver” test is applied; however, there is no chance to detect this discrepancy because the resolver is never applied to the non-discrepant tests. This may give the appearance that both of the initial test methods are better than they actually are. What the authors did was not as bad as it could have been since they apparently did not use the discrepant analysis to create or adjust sensitivity numbers. However, use of a composite reference standard for assessing the performance of methods does not suffer from this bias [8,9], and gives a more accurate portrayal of the relative sensitivity of both methods. While the impact of this inappropriate technique on the conclusions is negligible, its appearance in the literature promotes its further use. I think the paper would be significantly improved (and shortened somewhat) if none of the discrepant analysis is included.

Minor concern: Sampling from a single nares is to be avoided, as there is sufficient data on respiratory infections to show that sampling from both nares improves sensitivity. I include one reference, but there are many others I haven’t taken the time to look up [10]. I think the authors should note in their discussion the fact that sampling both nares is preferable.

Minor concern: The number of samples assessed in the LOD study is too small to draw strong conclusions about the LOD (see CLSI document EP7 and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556583/). Pooling of sample types (UTM, nasal swab, saliva) to achieve N is not appropriate, since these samples may have differing sources of assay interference. I think it is fair to say the authors have approximated the LOD, however.

1. Miller WC. Bias in discrepant analysis: When two wrongs don’t make a right. Journal of Clinical Epidemiology. 1998;51: 219–231. doi:10.1016/S0895-4356(97)00264-3

2. Hadgu A. Discrepant analysis: A biased and an unscientific method for estimating test sensitivity and specificity. Journal of Clinical Epidemiology. 1999;52: 1231–1237. doi:10.1016/S0895-4356(99)00101-8

3. Hadgu A. The discrepancy in discrepant analysis. Lancet. 1996;348: 592–593. doi:10.1016/S0140-6736(96)05122-7

4. McAdam AJ. Discrepant analysis and bias: A micro-comic strip. Journal of Clinical Microbiology. American Society for Microbiology; 2017. pp. 2878–2879. doi:10.1128/JCM.00969-17

5. Hadgu A, McAdam AJ. Discrepant analysis is an inappropriate and unscientific method (multiple letters) [4]. Journal of Clinical Microbiology. 2000. pp. 4301–4302.

6. Miller WC. Can we do better than discrepant analysis for new diagnostic test evaluation? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 1998. pp. 1186–1193. doi:10.1086/514996

7. Green TA, Black CM, Johnson RE. Evaluation of bias in diagnostic-test sensitivity and specificity estimates computed by discrepant analysis. Journal of Clinical Microbiology. 1998;36: 375–381. doi:10.1128/jcm.36.2.375-381.1998

8. Baughman AL, Bisgard KM, Cortese MM, Thompson WW, Sanden GN, Strebel PM. Utility of composite reference standards and latent class analysis in evaluating the clinical accuracy of diagnostic tests for pertussis. Clinical and Vaccine Immunology. 2008;15: 106–114. doi:10.1128/CVI.00223-07

9. Tang S, Hemyari P, Canchola JA, Duncan J. Dual composite reference standards (dCRS) in molecular diagnostic research: A new approach to reduce bias in the presence of imperfect reference. Journal of Biopharmaceutical Statistics. 2018;28: 951–965. doi:10.1080/10543406.2018.1428613

10. van Wesenbeeck L, Meeuws H, D’Haese D, Ispas G, Houspie L, van Ranst M, et al. Sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring. Virology Journal. 2014;11. doi:10.1186/s12985-014-0233-9

Reviewer #2: The manuscript submitted by Naito et al entitled “Evaluation of GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol” details a study that evaluates a molecular assay for detection of SARS-CoV-2. The assay was previously evaluated for NP specimens, but this assay evaluated the use of both NS, saliva, and saliva with a rapid processing protocol. In total 359 NS were evaluated and 240 saliva samples. Overall, the results demonstrated that the assay was accurate for all specimen types tested; However, there are some discrepancies in numbers that need to be clarified to ensure the proper comparisons were made. There are also a few additional clarifications needed prior to publication listed below:

Major Comments

For the enriched positive patients that had confirmed COVID-19 results, how was this detected. Did patients come back in after the results came through or was it a rapid test. Also, by cherry picking these patients you mess with the pre-test probability and would likely improve the assay performance, especially as the LoD appears to be better for GENECUBE vs RT-PCR assay. I would suggest breaking out the results as Total, prospective, call back subjects to be clear and see effects on true prospective testing.

The definition of the rapid method is lacking in specifics. What steps were modified in the maglead extraction process that reduced the method by 20 minutes. As this is written it would not be possible to replicate the study or adopt the new method for a clinical lab to validate and use for faster TAT.

Ln223-227: This is a bit unclear. There are 3 FP results based on rapid. 1 was the FP found in 4a, but the other 2 are new. Why were these 2 specimens tested 8 times on the lightmix test? One was picked up 50% of the time and the other was 12.5%.

Ln245-250: Why is table 4a and 4b not using the NP PCR result as the reference method. This analysis was done is ST2, which should be the results used for the evaluation and not comparing saliva tested on the two platforms (Is the reference PCR validated for saliva?). If you use the presentation of data in table 4a and 4b this would suggest that saliva with GENECUBE is more sensitive, but if you compare it to the PCR from NP (gold standard) then it seems like it would be slightly less sensitive of a sample type. I would delete 4a and 4b and replace with data comparing to NP. This is the same for NS comparison data as ln 257 indicates that PPA was actually 88.1% compared to the 98.1% you present in table 3.

Minor Comments

Ln 73: Was the pos and negative concordance both 99.7% in the NP study as you only give 2 values for the overall, pos-, neg concordance.

Were discordant specimens tested on the Xpert run as package insert (i.e. testing directly from VTM).

Table 2: The current layout is a bit confusing at first with the spacing for “Standard method with magLead…”

LoD study: The standard method is to perform 20 replicates at each concentration in pooled negatives and the LoD is then defined as the lowest concentration that was 19/20. What was the rationale for using multiple matrices and at the end having 24 tests for GENECUBE and 12 for the reference method.

Ln197: Was the copies/mL determined based on the CT value and the LoD of the specimen? If so what was the CT value for the reference test and the Xpert assay.

Why were the saliva samples frozen initially? Could this have skewed results as there is some data that a freeze thaw can help sensitivity of saliva samples.

Reviewer #3: Thank you for inviting me to peer review this manuscript. The authors have studied the GENECUBE ® HQ SARS-CoV-2 using anterior nasal samples and saliva samples by using a new protocol. This study aims to evaluate the detection of SARS-CoV-2 with saliva samples for the first time and using a rapid protocol. Here are some comments which could be considers to improve the manuscript:

1. Line 69 “This reagent was approved in October, 2021.”. The date needs to be corrected.

2. Line 86, the situation of the infected cases is not explained. How did the authors select them and in which phase of disease they were? Did they have symptoms or they were asymptomatic cases?

3. It seems that the authors have collected the anterior nasal samples and saliva samples from different cases. It was better to take both sample types from each studied case to be able to compare them and investigate the accuracy of the samples to detect the infection. (It seems that such work is mentioned very briefly in the Discussion)

4. There is no explanation for positive and negative controls.

5. New Method and Standard Method are not explained in the Materials and Methods. In Line 133, “For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened to approximately 10 minutes.” How this happened and what is the main difference making the novel method this short?

6. The obtained resulted are not discussed deeply. As an example, Line 181 “The detection rate was 100% down to 1000 copies/mL for pooled …” by considering these results, is this method useful for early detection or it is just useful for the chronic cases? How could a Dr. decide this strategy is a good choice for a specific case (like asymptomatic cases, accurate or chronic infections)?

7. Line 186. Compared with the standard test, how could the Rapid Strategy decrease the LoD of the nasopharyngeal samples from 1000 copies/mL to 500 copies/mL? (not discussed again).

8. Line 245 “In our current study, we used 60 saliva samples with positive nasopharyngeal sample results for SARS-CoV-2 (Supplementary Table 2), and the rapid protocol detected SARS-CoV-2 in most samples (98.3%; 59/60)”. It is the first time that the authors are refer to performing some tests on the nasopharyngeal and saliva samples taken from one case. It is better to be explained (materials and methods) and reported (results) in the previous sections before the Discussion.

9. The authors could also add graph(s) to report the results in a more useful way.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Tina Shaffaf

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PONE-D-21-27242R1The evaluation of the utility of the GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocolPLOS ONE

Dear Dr. Suzuki,

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 Jan 13 2022 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

Journal Requirements:

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.

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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 #2: All comments have been addressed

Reviewer #3: 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 Response)

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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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: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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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: (No Response)

Reviewer #2: Thank you for the changes to the manuscript, which improves the clarity of study. One minor suggestion I would have is to add a limitation about the lack of low viral load samples tested in the evaluation. In ln 202 only 3 samples were CT >30. This is a small proportion of your positives and that could have improved concordance as too few were near the limit of detection.

Reviewer #3: (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.

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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

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The evaluation of the utility of the GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol

PONE-D-21-27242R2

Dear Dr. Suzuki,

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,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: 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 #2: Yes

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

Reviewer #2: Yes

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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 #2: Yes

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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 #2: Yes

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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 #2: (No Response)

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Reviewer #2: No