PONE-D-21-08255

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

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?

 

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

 Reviewer #1: 

Banik et. al. describes the use of a commercially available buffer to inactivate SARS-CoV-2 virus in saliva sample which is compatible with downstream RT-PCR applications. The research question is clear, and the methods used and results are adequate to answer the research question. The paper is well written in general. There are some minor comments

Reviewer #2: 

This manuscript demonstrates eNAT transport buffer inactivates infectious SARS-CoV-2 virus in contrived swab and saliva samples. The authors also quantify SARS-CoV-2 RNA stability in eNAT for various lengths of time and at different temperatures. Overall the manuscript is technically sound and the data support the conclusions. However, not all data described is presented by the authors — the manuscript will be ready for publication following clarification of the following points:

* Lines 116-119 and 251-252 refer to data not shown in this manuscript, which is not permitted by PLoS journals. Please remove this text or include the referenced data.

* Lines 123-124 of the Methods state: “For the first approach, 100µl of the SARS-CoV-2 virus culture (8X106 PFU/ml) was added to 2ml of eNAT”. However, lines 174-174 of the Results state: “The virus was added directly to eNAT at the final concentration of 8X105 PFU/ml”. If 100ul of 8e6 PFU/mL was added to 2mL, the final concentration would be ~4e5 PFU/mL. Please clarify.

* Lines 185-186 describe the “>5.6 log10 PFU/ml reduction in viral load” following mock swab inactivation with eNAT. 5.6 log10 PFU/ml is roughly equivalent to 4e5 PFU/mL so this statement is accurate if this was the viral treatment titer (see previous point). However, line 199 repeats that the inactivation efficiency is >5.6 log10 PFU/mL for virus-spiked saliva, even though the authors used a less concentrated viral stock to generate the contrived saliva samples (100ul 3e6 PFU/mL into 1ml saliva = ~3e5 PFU/ml). How can the inactivation efficiency be the same for mock swab and saliva samples when the titer of the mock saliva samples was lower? Please update the statement about saliva sample inactivation (or elaborate on how these calculations were performed).

* Lines 245-247 state: “Both dialysis and filtration using Pierce DRSC spin columns resulted in complete removal of basal cytotoxicity from eNAT and did not result in any viral loss”. Additionally, lines 135-137 state: “Positive control samples were also assessed before and after DRSC purification to determine viral loss during this process.” However, this manuscript does not present data demonstrating viral titers are equivalent pre and post dialysis/filtration. Please amend the claim in 245-247 and remove lines 135-137 (or include equivalency data supporting this claim).

Reviewer #3: 

The manuscript is technically sound, with appropriate controls. It is well written and clear, easy to understand. The results have useful implicatios for sample testing as explained in the intro/discussion.

A few minor points:

1. In intro, lines 38-39: suggest define sars-cov-2 and covid19 upon first use.

2. lines 135-137 explain that a positive control was included to show no loss from DRSC (which is excellent, sometimes this is overlooked so great to include), can these data be added/explained to show there's no loss? (currently it seems results just say there was high CPE, but an actual titer comparison would be useful, as if there is some loss of infectivity from DRSC, then a lower concentration of virus [e.g. if only partial inactivation happened] would look completely negative due to the DRSC, rather than due to the eNAT).

3. method lines 148-150 explain the RT-PCR assays, but can it be more clearly stated in results what is being measured in each section (e.g. "live" infectious virus in the inactivation sections, vs. just RNA copies later on?). The PCR results are used to show inactivation, for example in Table 2, so negative on PCR means virus is inactivated, but one would expect RNA to still be present (which is shown later in stability section), so different things are being measured via RT-PCR in the different sections. But if this can be briefly, clearly described that might be beneficial for some readers.

Reviewer #4: 

Review Summary

The manuscript titled, “Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays” is concise and well-written. It clearly demonstrates the effectiveness of eNAT, a guanidium thiocyanate containing buffer, at rapidly inactivating live SARS-CoV-2 virus when used at a ratio of 2:1 with saliva or as a transport medium for swab specimen. eNAT supported detection of low concentrations of SARS-CoV-2 RNA for at least 7 days at a range of storage temperatures with almost no loss of sensitivity. The manuscript suggests in passing that the eNAT buffer also preserves the specimen, although this is not explicitly addressed by the data. There are some minor inconsistencies in the reported concentrations of SARS-CoV-2 virus used in the manuscript, but they do not affect the conclusions of the paper. Overall, the research presented here is of use to the public health and clinical testing communities and represents a useful addition to the available arsenal of specimen collection mechanisms, but the scope of the findings is necessarily narrow.

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

Partly: The manuscript is technically sound, but the reported viral concentrations and dilutions don’t totally add up. The data support the main conclusion that eNAT can inactivate SARS-CoV-2 in saliva at a ratio of 1:2 (Saliva:eNat) in as little as 5 minutes.

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

Yes.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Partly: The data from the CPE and TCID50 dilution series are only presented in a final summary. I don’t know if it is customary to present the entirety of this type of data, but I have seen it presented in other manuscripts.

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

Yes, the manuscript is well written and easy to read.

Important Points

1. In the ”Inactivation of SARS-CoV-2 with eNAT” results section it states that SARS-CoV-2 is diluted to a final concentration of 8x10^5 PFU/mL in eNAT. The methods section describes diluting 100uL of 8x10^6 PFU/mL in 2mL of eNAT which should give you a final concentration of ~4x10^5 PFU/mL.

a. The results describe a “100uL positive control containing 8x10^6 PFU/mL of SARS-CoV-2” but the methods suggest that this control was further diluted in 2mL of DMEM to the same concentration as the eNAT test samples (~4x10^5 PFU/mL).

b. Ideally the concentrations of virus should be reported consistently throughout the manuscript as either the final diluted concentration or the stock concentration into a known dilution volume.

c. The 8x10^5 PFU/mL diluted concentration does not make sense given the stock concentration and dilution volumes listed

2. Although the SARS-CoV-2 titers used in the manuscript are reasonably high, it is not uncommon to see quantities of genomic fragments in saliva on the order of 10^8-10^10 copies/mL (~10% of cases) and >10^10 copies/mL in a smaller number of cases (~2%) based on the preprint below, using qRT-PCR. I see similar results using ddPCR for quantification. Although these PCR-based methods do not quantify infectious virions, is an inactivation efficacy >5.6 log10 PFU/ml an appropriate benchmark for SARS-CoV-2 inactivation of saliva samples?

a. Yang Q, Saldi TK, Lasda E, Decker CJ, Paige CL, Muhlrad D, Gonzales PK, Fink MR, Tat KL, Hager CR, Davis JC, Ozeroff CD, Meyerson NR, Clark SK, Fattor WT, Gilchrist AR, Barbachano-Guerrero A, Worden-Sapper ER, Wu SS, Brisson GR, McQueen MB, Dowell RD, Leinwand L, Parker R, Sawyer SL. Just 2% of SARS-CoV-2-positive individuals carry 90% of the virus circulating in communities. medRxiv [Preprint]. 2021 Mar 5:2021.03.01.21252250. doi: 10.1101/2021.03.01.21252250. PMID: 33688663; PMCID: PMC7941634.

3. The evaluation of stability of SARS-CoV-2 RNA in eNAT shows that eNAT does not interfere with qRT-PCR detection of viral RNA over a course of 7 days, but there is no data showing that eNAT improves the stability of viral RNA compared to a similar specimen without an inactivating substance. My experience is that raw saliva is stable for at least 7 days after heat inactivation without the need for preservatives, and this is documented in the SalivaDirect protocol.

a. In the discussion the authors conclude, “eNAT, a guanidinium thiocyanate-based buffer can be used as a viral inactivation and preservation media for SARS-CoV-2 specimens.”

b. I don’t think the data show that eNAT “preserves” the specimen. This may be a matter of nomenclature, where any media that doesn’t degrade the sample is considered to preserve the sample, but in my mind a preservative prevents normal degradation, and there is no evidence in this manuscript that eNAT reduces the normal degradation of RNA in saliva.

c. My experience with heat-inactivated saliva is that the RNA is severely degraded but still detectable by RT-PCR. Any data or citation showing a reduction in degradation of RNA in saliva mixed with eNAT would be strong evidence that eNAT does stabilize/preserve the specimen.

Minor Points

1. From a practical standpoint, it is difficult to see how eNAT would be implemented in a saliva collection aid, which would need to come pre-aliquoted with a generous amount of eNAT to account for the variable amount of saliva collected. This does not affect the findings of the manuscript, but I am curious how the authors envision the use of this product.

2. There is only a brief description of the Xpert Xpress SARS-CoV-2 test. Since this test was designed and validated for upper respiratory specimen such as nasopharyngeal swabs but is being applied to saliva specimen perhaps a more detailed explanation is appropriate. For instance, from what I could gather the volume of sample loaded into the Xpert Xpress cartridge is ~300uL. This is a significant deviation from standard RT-PCR protocols which use on the order of 20uL total for a single reaction.

a. The authors only report results for the N2 target gene in the manuscript. This is certainly adequate to monitor for the presence of viral RNA, but is there a reason the results for the E target were omitted? Was there any interference from the eNAT on the amplification of the E target?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Philip D Fox

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Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

PONE-D-21-08255R1

Dear Dr. Banada,

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Kind regards,

Ruslan Kalendar, PhD

Academic Editor

PLOS ONE