PONE-D-21-39842Diagnostic performance of GENEDIA W and ActiveXpress+ COVID-19 antigens tests among symptomatic individuals in Peru and The United KingdomPLOS ONE

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Reviewer #1: Partly

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

Reviewer #1: No

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Reviewer #1: No

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Reviewer #1: No

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5. Review Comments to the Author

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Reviewer #1: The authors present a study to evaluate clinical performance of two antigen-based rapid diagnostics tests (Ag-RDTs) for diagnosing SARS-CoV-2 virus: GENEDIA and ActiveXpress+. The study is performed independently in two testing locations- Peru and the United Kingdom. The performance of each of the tests is measured against the RT-qPCR test using sensitivity and specificity as the metrics. The authors conclude that the overall sensitivity for both the tests is lower than what is reported by the manufacturers and lie below the recommended values by WHO. Although the study is important to evaluate the benefits of these tests in controlling the spread of SARS-CoV-2, I am not convinced by the interpretation of some of the data from the study and the corresponding discussion. Additionally, the presentation of key results/comparisons in the current form is difficult to comprehend because of the lack of graphical figures. I recommend that authors should address my following comments in the revision-

1. In lines 204-207, the authors mention that the sensitivity of ActiveXpress+ test in the samples collected at 0-3 days from symptom onset is significantly lower than in the samples collected at 4-7 days from symptom onset for the UK cohort. The sensitivity values presented in the text are extremely low and do not match the data presented in the “sensitivity” column of table 4 for the UK ActiveXpress+ cohort. Additionally, authors mention 161 PCR positive tests for this cohort, which doesn’t match with the data reported in the table 3 (11 PCR positive tests). The authors should address this discrepancy and modify the interpretation of data in the text accordingly.

2. In paragraph 199-211, the authors present the results from the subgroup analysis for the UK cohort but do not compare it (or present) the subgroup analysis for the Peru cohort, especially for days from symptom onset. This should be included in the clinical evaluation section.

3. In lines 264-266, the authors claim that both brands showed better sensitivity when Ct<25 and in the group of participants with <7 days of symptom onset. The authors should clearly specify what these sensitives are being compared to. For example, for the GENEDIA cohort (Table 2), are the individual sensitives for Ct<25 for Peru (87.9%) and UK (59.0%) being compared to the overall sensitivity (60.4%)? If that is the case, the authors’ claim is not valid for the GENEDIA UK Ct<25 cohort. Perhaps, authors should calculate the combined “overall” sensitives for Ct<25 (Peru+UK cohort), which according to my calculation is 67.5%, and compare it to the overall sensitivity (60.4%). On the other hand, if the individual sensitives for Ct<25 for Peru (87.9%) and UK (59.0%) are being compared to the individual clinical sensitivity for Peru (72.2%) and UK (53.7%), an argument should be provided for why such comparison is justified over comparing overall sensitives. Either way, the statistical significance of the comparisons should be provided. Additionally, the same applies to the analysis of sensitives for participants with <7 days of symptom onset.

4. In line 270, it is not clear why the differences in the type of samples used for RT-PCR could be a reason for differences between Peru’s and UK’s sensitives. A clear explanation for this argument should be provided. Furthermore, in line 268, a statistical analysis for significant differences should be provided.

5. In lines 269-270, the authors briefly mention the influence of the low number of RT-PCR positive cases in ActiveXpress+ UK cohort (11) on the sensitivity values. However, the authors should discuss the limitations of this low number and its broad implication on the analysis and the interpretation, especially when making the claims about ActiveXpress+ meeting the minimum sensitivity requirement of WHO in the UK cohort (in line 213).

6. In line 285-287, it is claimed that the decreasing trend of sensitives with the increasing median days from symptom onset is not observed in the UK cohort. It is also claimed that in the UK ActiveXpress+ evaluation, there is a significant difference in the sensitivity at <0-3 days vs 4-7 days. However, this is not supported by the data in table 4 and claims in lines 207-209, as both the sensitives are 100%. Perhaps, the authors intend to make the “significant difference” comparison for the GENEDIA UK cohort. If that is the case, however, lines 210-211 claim that the time from symptom onset has no discernible impact on sensitivity for the GENEDIA UK cohort (p>0.05). Although the first claim in line 285 is still valid based on the data presented in lines 207-211, the language and discrepancy about significant difference between <0-3 days and 4-7 days should be corrected.

7. In paragraph 292-298, the authors discuss that the circulation of several variants at different times during the study could lead to different performances of the tests between the two locations. This suggests that the timeline for the participant recruitment for each test is important for the contextual interpretation of the data. Although the authors mention the overall timeline of the study in each country, it is not clear if the individual timeline for GENEDIA and ActiveXpress+ testing is different or if both the tests were conducted throughout the entire timeline. The authors should specify each of these timelines for both the locations and discuss the performance in context of those timelines.

8. While the discussion is expansive, and probably not all required, the section on limitations is insufficient (paragraph 331-334). This study is limited by:

- Specimens taken at a single time-point; it is, for example, entirely possible that a second Ag test would significantly recuperate assay sensitivity.

- The fact that not every specimen was tested on both platforms. The authors should also explain why this was not done.

- A likely heterogenous group of participants when it comes to vaccination, including vaccine types administered, number of doses, and specimen testing relative to the last vaccine dose.

- While outside the scope of this article, it would have been interesting to compare select specimens with viral culture to determine the proportion of replication-competent SARS-CoV-2 isolates that tested negative by Ag-RDTs. This is another limitation of the study.

9. With the available sensitivity and specificity data, the authors can calculate + and - LR and are encouraged to do so to better contextualize the operating parameters of these assays.

10. How do the authors define vaccinated individuals? Any # of doses? The type of vaccines?

11. What were the median CT values among the vaccinated individuals? Is it possible that vaccinated individuals had, on average, higher CT values? This is important because it could explain the lower sensitivity in this group.

12. Did any of the participants have previously confirmed SARS-CoV-2 infections?

13. In line 183, the authors describe that 212 participants were registered with ActiveXpress+ test in the UK. However, table 3 shows that 211 participants were tested. This discrepancy should be discussed.

Moreover, table 1 also reports 403 patients tested with the GENEDIA, yet table 2 only reports 399 confirmed by RT-PCR. While I recognize that 4 participants did not have paired data, these should be excluded from all analyses.

14. The authors should provide the data (at least in Supplementary Information) from the dilution experiments described in section 2.2 to determine the limit of detection.

15. In addition to the tables, the authors should present the key data in graphical format along with p-values and CI.

In addition to the above, I have minor comments I hope the authors consider:

16. Line 24, the introductory sentence of the abstract is long and would benefit from a grammatical revision.

17. Line 36, similarly, the first two sentences of the results section of the abstract would benefit from a grammatical correction.

18. The grammar should be revised throughout the manuscript. Particular attention should be paid to the following sentences:

- Lines 50, 64, 108

19. The authors are encouraged to have the manuscript reviewed by colleagues who are fluent in the English language to improve the readability of the manuscript.

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Reviewer #1: No

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Diagnostic performance of GENEDIA W and ActiveXpress+ COVID-19 antigens tests among symptomatic individuals in Peru and The United Kingdom

PONE-D-21-39842R1

Dear Dr. Palomino-Padilla,

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