PONE-D-22-00630Saliva molecular testing bypassing RNA extraction is suitable for monitoring and diagnosing SARS-CoV-2 infection in childrenPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Reviewer #1: 

This work investigates if saliva is an effective sample for detecting SARS-CoV-2 RNA and antibodies, especially in children. They selected a group of 85 children under 10 years old, and found that 63% have at least one COVID-19 symptom, 54.1% are positive cases. From where, they concluded that Saliva is effective for children under 10. This topic is helpful for recent covid-19 omicron variant detection. The writing style is scientific and easy to follow. However, the topic is not new, since it has been talked about for a while and many other research groups have done similar works in this area. Besides, there are several details I would like authors to address.

Given these unsolved questions above, a major revision of this manuscript is required.

Major concerns:

The adults over 18 have been chosen if they have at least one symptom, while for children under 10 there is not a such restriction. Why did author use those two data?

Minor concern:

Figure resolution. Not sure if pictures shown are from their original resolution, but the resolution in my end is really low. I barely can see details clearly. Please double check the submission of figures. Some other concerns about figures:

• Figure2 involves four different colors in panel B and D, but cannt see clear differences between blue green and pink red (I guessed the color). Maybe due to the resolution, but I highly recommend the authors to change four different colors or shapes with high contrasts.

• Figure S2 has a x-scale of 10 – 10000. Whats the reason for doing so? Did you did any log transformation? Please clarify.

Reviewer #2: 

Alenquer et al. evaluated an alternative method respect to the nasopharyngeal swab for the detection and quantification of SARS-CoV-2. In detail, they investigated whether saliva is an effective sample for detecting RNA in children, and assessed a possible association between viral RNA load and infectivity. Focusing on the detection of SARS-CoV-2 RNA, the authors devised two methods: one involving RNA extraction and the other not. They demonstrated that the saliva molecular test bypassing RNA extraction is a valid and useful method for surveillance.

The manuscript is interesting, even if some statements need to be clarified and proven.

These are my main concerns:

- Lines 133-135: “The limit of detection (LOD) of the saliva assay was performed by serial dilution of IDT synthetic copies (20,000 to 20) of SARS-CoV-2 in fresh saliva samples (non-positives) (S1 Fig)”. In detail, how was the detection limit of the assay evaluated? Which kind of tests were applied to calculate this limit? Have the authors performed a probit regression analysis? In addition, the coefficient of variation and the lower limit of blank (LoB) should be evaluated to better define the accuracy and limit of detection.

- Table 1- Adults: in the table it is reported that a negative swab sample was positive in saliva both with and without extraction. Were these assays performed on the same sample? How can the authors explain these different results? Have further swabs been performed on the patient to see if it is a really false positive in saliva?

- Table 1- children <1y and <10y: Did the samples that tested positive to swab but negative to saliva belong to the same patient? For these samples is there a correlation between the result in saliva and the CT value in the swab?

- Lines 211-214: “The differences in CT values between saliva (with or without RNA extraction) and NP swab were not statistically significant (Fig 1A), but CT values in saliva with vs. without RNA extraction are statistically different, with extraction of RNA consistently decreasing the CT values obtained.” To better understand the data, it would be useful to report the median values (IQR) of the Ct values obtained by the 3 detection methods.

- How can the authors explain the fact that samples with RNA extraction had lower labours than those without extraction?

- Figure 1: Please report sample size

- Lines 228-230: “Next, we analyzed the stability of saliva samples stored for 3 days at 4ºC or 7 days at -20ºC, prior to processing (Fig 1B). We detected viral RNA in all saliva samples kept at 4ºC and -20ºC, regardless of RNA extraction, demonstrating they are stable under the conditions studied.” The detection of the virus has not changed, but if you look at the figure 1B, a clear increase in CT in samples not extracted and stored at -20 for 7 days can be appreciated. Have the authors defined if this increase is statistically significant?

Moreover, this may affect the outcome of the result. For example, if the sample processed immediately had 35 Ct there is a high risk that it will be negative after a -20 storage 7 days long.

- Table 2: Please report the % of Concurrent conditions.

- Lines 257-260: “It is important to notice that there was a time interval between NP swab and saliva collection that could go up to 48 h and, therefore, these differences may not reflect a lower sensitivity of our method but rather a decrease in the patient viral load.” Stratifying the samples as: collected within 24 hours of TNF and collected 24-48 hours later, did you see differences in terms of CT values? And if so, did the former have values more similar to those obtained in TNF?

- Figure 1 e 2: “Saliva stability at 4ºC and -20ºC: comparison of CT values from paired saliva samples (with and without RNA extraction) processed immediately, after 3 days at 4ºC or 7 days at -20ºC.” Non-extracted samples do not seem similar between adults and children. Is there an explanation for this?

- Table 3: please define the populations used to calculate p-values.

- No symptoms concerning the lower respiratory tract (pneumonia, bronchiolitis) were described in the sampled population. This does not allow to define the specificity and sensitivity of the salivary method in detecting SARS-CoV-2 in subjects developing lower respiratory tract infections respect to those developing upper respiratory tract symptoms only. This limitation should be mentioned in the discussion section.

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Reviewer #1: No

Reviewer #2: No

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Saliva molecular testing bypassing RNA extraction is suitable for monitoring and diagnosing SARS-CoV-2 infection in children

PONE-D-22-00630R1

Dear Dr. Amorim,

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,

Ruslan Kalendar

Academic Editor

PLOS ONE