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Reviewers' comments:

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #1:  The manuscript addresses an important and clinically relevant topic regarding minimally invasive respiratory virus sampling methods.

The paired-sample design and use of RT-PCR strengthen the technical quality of the study.

The results demonstrate high concordance between Rhinoswabs and joint nose and throat swabs, supporting the potential utility of Rhinoswabs in outpatient settings.

The manuscript is generally well written and logically organized.

Statistical analysis is appropriate; however, inclusion of Cohen’s kappa statistics, sensitivity, specificity, and predictive values would provide a more comprehensive assessment of diagnostic agreement.

Additional discussion regarding applicability in asymptomatic individuals and unsupervised self-collection settings would strengthen the manuscript.

Reviewer #2:  The manuscript by Murphy et al addresses a practical question for clinical diagnostics. The authors provide a clear evaluation of a less invasive Rhinoswab swabbing method compared with standard practices. The manuscript is methodologically sound. The results are useful in clinical practice. I have several comments to improve the clarity and robustness before acceptance.

There were 18 discordant samples stated in Table 1. Please specify which swab yielded the positive result in these cases. It will be useful to know whether the Rhinoswab failed to detect viruses found by the NTS, or if the NTS missed viruses detected by the Rhinoswab. This also helps to address the next comment.

Negative cases are the majority here, which could skew the concordant value. For example, there are 32 positive SARS-CoV-2 cases, which means 456 negative cases. Let’s assume that Rhioswab couldn’t get any SARS-CoV-2 virus at all, thus it would only generate negative cases. In this scenario, the concordant value could still be as high as 456/488 = 93.4%. Please comment on this in the Discussion session.

1. Please consider using RT-qPCR since Ct values are obtained from qPCR.

2. “Written informed consent..” is stated twice at lines 64 and 67.

3. How is the Ct value determined? Baseline-Threshold or regression method?

4. Have any abnormal amplification curves been observed? If yes, how were the abnormal curves handled? This information should be included in the manuscript.

5. It will also be useful to detail the discordant cases (see above). Such as, how many have tested positive by Rhinoswab, but negative by joint NTS?

6. Figure 1 legend: Please use NTS for consistency. Please indicate that the r is Pearson’s correlation (I assume).

7. Figure 1: There are two linear regression lines, a solid and a dashed one, in the figure. What are they?

8. Figure 1: I assume in the figure only positive cases are plotted. Please make this clear in the legend.

9. Figure 1: Please consider indicating the discordant samples, maybe with a different color, to support the conclusion at line 114.

10. I found it interesting that the authors note that 43 participants opted out of providing paired samples, and 16 of those were unwilling after viewing the demonstration video. The Rhinoswab is marketed as a less invasive, patient-friendly alternative, but the video deterred some patients. No response for this comment is needed from the authors.

Reviewer #3:  Reviewer

It is a pleasure to review the manuscript entitled "Agreement of Rhinoswabs and joint nose and throat swabs for detecting influenza and SARS-CoV-2 among symptomatic ambulatory patients in Hong Kong” (PONE-D-26-15719). I have carefully reviewed this manuscript. I found that it is straightforward, the sample size is reasonable (488 paired samples), the statistics are appropriate for the objective, and the conclusions are generally supported by the data. This manuscript appears scientifically sound, clinically relevant, and appropriate for publication in PLOS ONE. In particular, the manuscript is concise and well written. Nevertheless, a few minor issues should be addressed to strengthen the manuscript. After this revision, I support accepting this manuscript in PLOS ONE.

Manor’s suggestion for revision

1. The study mainly reports “concordance” but does not provide sensitivity, specificity, positive predictive agreement, negative predictive agreement, and Cohen’s kappa. Because NTS is effectively treated as a reference standard, reporting these metrics would strengthen diagnostic interpretation. Thus, the study would be strengthened by providing additional reporting of the sensitivity, specificity, positive/negative agreement, and Cohen’s kappa statistics to provide a more comprehensive assessment of diagnostic performance.

2. The Rhinoswab was always collected AFTER rapid antigen testing and joint NTS sampling. This could reduce the amount of viral material remaining in the nose. The authors briefly mention this in the discussion. I think this deserves stronger acknowledgment. Because Rhinoswab collection was performed after other respiratory sampling procedures, the order of sampling may have influenced viral recovery. Please discuss this potential sampling-order bias in greater detail and clarify whether randomization of sampling order was considered.

3. I found a very important issue. The manuscript promotes Rhinoswab as suitable for easy or minimally invasive collection, yet all samples were collected by trained staff. Thus, the conclusions about usability and self-testing are somewhat overstated. Because all Rhinoswab specimens were collected under supervision by trained staff, the manuscript should avoid overgeneralizing the findings to unsupervised self-collection settings.

4. The authors used Ct ≤ 40 as the positivity cutoff. However, a Ct of 40 is relatively permissive. Please justify the use of a Ct of 40 as the positivity threshold and indicate whether equivocal results were observed near the cutoff.

5. The manuscript reports concordance percentages but only briefly presents confidence intervals. Please ensure that all concordance estimates include 95% confidence intervals in the main text and/or tables.

6. Please correct minor typographical and formatting errors throughout the manuscript.

Reviewer #4:  The manuscript addresses a practical question: whether a minimally invasive anterior nasal Rhinoswab can provide comparable RT-PCR detection of influenza A/B and SARS-CoV-2 to joint nose-and throat swabs in symptomatic outpatients. Overall, the study is clearly written, the sample size is reasonable, and the paired-sample design is appropriate for assessing diagnostic agreement. The reported concordance is high for all three viruses, and the analysis of Ct-value correlation and discordant samples supports the main conclusion that Rhinoswab may be a practical alternative in ambulatory settings.

However, the manuscript would benefit from a more careful framing of "agreement" versus diagnostic performance. Because neither specimen type is treated as an independent gold standard, term such as sensitivity or diagnostic accuracy should be avoided unless a composite reference standard is defined. The authors appropriately report concordance, but the Discussion occasionally implies broader diagnostic equivalence. This should be softened.

The statistical analysis is somewhat limited. Percent agreement alone maybe inflated by the large number of negative samples. The authors should consider adding positive percent agreement, negative percent agreement, and Cohen's kappa for each virus. Given the paired design, a McNemar test or equivalent comparison of discordant pairs would also strengthen the analysis. Confidence intervals should be provided consistently for all agreement metrics.

The sequential sampling order is another important limitation. Rhinoswab was collected after rapid antigen testing and joint NTS, which may affect available nasal material. The authors interpret the preserved performance as reassuring, but fixed order still prevents assessments of whether Rhinoswab performance would be similar if collected first. This should be stated more explicitly.

The manuscript also needs cleaner reporting of discordant results. A small table indicating which swab was positive, virus detected, Ct values, and symptom timing would help readers understand whether discordance reflects low viral load, sampling variability, or possible false positives.

Finally, the conclusion should be slightly narrower. The data support use in supervised collection among symptomatic outpatients early after symptom onset, but do not yet establish performance for self-collection, asymptomatic screening, later illness. or inpatient populations.

Overall, this is a concise and useful study with practical implications. i would support publication after minor revision, mainly to improve statistical reporting, clarify limitations, and avoid overinterpreting concordance as full diagnostic equivalence.

Reviewer #5:  Firstly, I truly appreciate for giving me this opportunity and selecting me to be apart of this work.

After carefully reviewing- Minor to moderate changes are required.

Clinically relevant study, highly suitable for publication.

Real time outpatient settings and patient population

Pairing of samples

Inclusion of three different respiratory viruses and large sample size study before conducting rhinoswab studies.

The topic is very interesting. Based on scientific reasoning- the conclusion is that ANS captures enough or sufficient viral RNA for reliable RT PCR in symptomatic patients is because respiratory viruses both SARS COV2 virus, RSV and influenza viruses-viral replication is more in the nasal cavity and helps in premature detection during infection and therefore, nasal swabs diagnostically works well during initial stages and thats why rhinoswabs are enough for molecular diagnostic detection of viruses.

Even MERS virus, other human coronovirus, human bocavirus, Adenoviruses, Human Parainfluenza Viruses, Human Metapneumovirus common rhinovirus can be detected-even though there is low viral load but still can be captured.

There fore, ANS is a friendly, easier to use and delivers the same results.

A great tool for detection leading to diagnosis of the viruses.

It has the same quality and accuracy that helps in faster outcomes and optimizes health outcomes.

Cost efficient

Flexible

Validation of care

Concise and well executed diagnostic concordance study.

The concept and comparison is excellent.

The findings from the study is a feasible and alternative specimen specially in outpatient settings particularly given the high concordance observed.

Prior to publication-several methodological and reporting issues should be clarified.

The reporting is more general and overall agreement of percentages.

Additional reporting includes- Negative and positive percent agreement and cohen Kappa statistics. These metrics will help in method comparison.

The exact distribution in terms of samples is unclear- Only 18 paired samples were discordant- Positive by NTS only vs Rhinoswab only must be provided.

Virus specific Discordance counts

Provision of CT values for both specimen types

To strengthen the interpretation summarizing all the discordant specimens.

Clarification of sampling order was fixed with participants, nostril sampling was repeatedly sampling.

Randomization order was considered

Since it is a self collection, the collection is lacking, self collection feasibility and evidence supporting diagnostic performance under supervised collection. The whole advantage of the rhinoswab is self collection and was collected by study staff.

There is a room for improvement on statistical reporting- Correlation alone does not assess concordance with agreements.

For the improvement use of Bland Altmann Analysis or report mean ct differences considering limitations.

Helps in systematic bias on specimens.

The title can be streamlined. Diagnostic concordance or agreement between Rhinoswab ans and jns for detection of influenza virus and SARS COV2.

Redundancy - Informed consent statement appears twice in the methods sections.

CT value <40 (Clarification of positivity threshold format)

Typographical issues

formatting issues

spacing

figure labelling

After interpretation - the overall study does not exactly show or talk about anterior nasal sampling captures higher viral loads that NTS.

It shows that ANS captures enough or sufficient viral RNA for reliable RT PCR in symptomatic patients and in outpatient settings.

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Reviewer #1: Yes:  DR LAKSHMI JYOTHI T

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: Yes:  MONISHA KANDALA

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Submitted filename: PONE-D-26-15719-2.pdf

Attachment

Submitted filename: Reviewer 1 recommendation.pdf

Diagnostic agreement between Rhinoswabs and joint nose and throat swabs for the detection of influenza and SARS-CoV-2 among symptomatic ambulatory patients in Hong Kong

PONE-D-26-15719R1

Dear Prof. Cowling,

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.

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Kind regards,

Hin Fung Tsang

Academic Editor

PLOS One

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