Aluminium Gauze Reduces SARS-CoV-2 Viral Load in Non-Woven Masks Worn by Patients with COVID-19

Round 1

Reviewer 1 Report

In this manuscript, the authors investigate the effect of adding an aluminium gauze to a mask on the SARS-CoV-2 RNA load. They showed that the Ct values of the nucleocapsid gene and envelope gene of SARS-CoV-2 were significantly higher in the aluminium gauze than in the cotton gauze. SARS-CoV-2 RNA was not detected in breath condensates collected behind both aluminium and cotton gauzes. The authors found that the addition of aluminium gauze increases the mask’s ability to capture viruses. The following points should be clarified.

The manuscript should include a statement indicating that the research was approved by ethics committee.

Is it possible for you to detect the virus in breath concentrates without a mask?

Author Response

Reviewer’s comment 1:

The manuscript should include a statement indicating that the research was approved by ethics committee.

Answer 1:

Thank you for your comments. We added an ethical statement as follows.

“Institutional Review Board Statement: The study protocol had been prepared in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Kishiwada City Hospital (Kishi-Byou-Rin 23, Kishi-Byou-Rin 24)."

Reviewer’s comment 2:

Is it possible for you to detect the virus in breath concentrates without a mask?

Answer 2:

Thank you for your instructive comments. There have been several studies on the collection of exhaled breath condensates. Turbo Deccs System® is a device to collect and study exhaled breath condensates. However, we have never used such a device and could not collect breath condensate without a mask.

Reviewer 2 Report

In this paper, Yasuda et al demonstrated whether adding an aluminium gauze to a face mask could reduce the SARS-CoV-2 RNA load in the mask and whether SARS-CoV-2 is detectable in the breath could permeate through this type of mask in clinical settings. Non-woven masks comprising filters with 99% viral filtration efficacy with either aluminium or cotton gauzes attached to plastic collection cases on each half of the mask. A small cohort of hospitalized patients (n=29) was enrolled in the study, who wore the experimental masks for 3 hours. The Ct values of the nucleocapsid gene and envelope gene of SARS-CoV-2 were significantly higher in the aluminium gauze than in the cotton gauze. SARS-CoV-2 RNA was detected in the masks of eight out of 12 vaccinated patients but not in breath condensates collected behind either aluminium or cotton gauzes. This interesting proof of concept study indicating that non-woven masks with an aluminium gauze may be effective against SARS-CoV-2 transmission better than non-woven masks with cotton gauzes in clinical settings.

There are the following major comments on the study:

1. How did the experimenters control the collection of breath condensate?  The amount of breath condensate collected in the study would directly affect the RNA amount and therefore the PCR product and Ct values of SARS-CoV gene expression. The method suggests that three hundred microliters of the specimen was transferred to the sample chamber for PCR testing but the total collected condensate was 178±95ul, and it is therefore not clear how it was possible.

2. The CT values in most of the patients are either near 40 or higher. In this window range, it may not be appropriate to correlate the CT values with other variables quantitatively. Chances of false-positive also might be there.

In the main text, it is mentioned that the patient parameters are mentioned to be given in the table, which is missing in the text and must provide basic demography, comorbidity, symptoms, and WHO classification of disease severity.

In figure 3C, the add-on aluminium gauze experiment was done only in 3 subjects, which is too-small sample size for clinical study-oriented results. 

Author Response

Reviewer’s comment 1:

How did the experimenters control the collection of breath condensate?  The amount of breath condensate collected in the study would directly affect the RNA amount and therefore the PCR product and Ct values of SARS-CoV gene expression. The method suggests that three hundred microliters of the specimen was transferred to the sample chamber for PCR testing but the total collected condensate was 178±95ul, and it is therefore not clear how it was possible.

Answer 1:

Thank you for your comments. We revised a part of method section.

“Briefly, the contents of the specimen collection tube and gauze or filter were mixed by rapidly inverting the tube five times. Three hundred microliters of the mixed specimen was transferred to the sample chamber of the assay cartridge. For breath condensates, all volume of breath condensate was transferred to the sample chamber of the assay cartridge. They were loaded onto the GeneXpert® platform.”

Reviewer’s comment 2:

The CT values in most of the patients are either near 40 or higher. In this window range, it may not be appropriate to correlate the CT values with other variables quantitatively. Chances of false-positive also might be there.

Answer 2:

Thank you for your instructive comments. The upper limits of Ct values were different for the amplification reactions of Applied Biosystems (usually Ct < 40) and Xpert Xpress SARS-CoV-2, according to the manufacturer’s datasheet. As you advised, we added a new sentence on the limitation section.

“Second, the Ct values were above 40 for most patients. Although the Xpert Xpress assay detects Ct values less than 45, high Ct values may result in false positives. Therefore, the present data should be confirmed in COVID-19 patients with Ct values less than 40.”

Reviewer’s comment 3:

In the main text, it is mentioned that the patient parameters are mentioned to be given in the table, which is missing in the text and must provide basic demography, comorbidity, symptoms, and WHO classification of disease severity.

Answer 3:

Thank you for your comments. We added a table 1 as you suggested.

Reviewer’s comment 4:

In figure 3C, the add-on aluminium gauze experiment was done only in 3 subjects, which is too-small sample size for clinical study-oriented results.

Answer 4:

Thank you for your instructive comments. We could perform the add-on aluminium gauze experiment only in 3 patients because there were so few samples with Ct values less than 40. The limitation was the same as in answer 2.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I had reviewed the paper and all comments except one were appropriately responded. Nevertheless, the remaining comment is extremely important as it questioned the concept behind correlating very high CT values with other variables. Authors need to show the specificity at more than 40 is appropriate. The CT values indicating very low viral load, then it’s noteworthy that how the barrier capacity of testes masks could be compared. Writing a limitation doesn’t help much.

Either authors should consider adding more data points with small subset with lower than 35 CT values as generally accepted for positive PCR products or provide the QCdata of melting curves to show the specificity of the PCR product to consider these results.

Author Response

Thank you for your instructive comments. We performed a subgroup analysis with a Ct value of less than 35 for either gauze or filter. All eight patients analyzed had Ct values less than 35 of E gene in the cotton gauze. The trend was the same as for the group as a whole. We added new sentences and figures as follows.

“Since many of the Ct values were greater than 35, we analyzed the Ct values in the data where the Ct value of either gauze or filter was less than 35. All eight patients analyzed had Ct values less than 35 of E gene in the cotton gauze. In this subgroup analysis, the Ct values of the N2 and E genes of SARS-CoV-2 were significantly higher in the aluminium gauze than in the cotton gauze (Figure 3C).”

Author Response File: Author Response.pdf