Detection of SARS-CoV-2 in the Indoor and Outdoor Areas of Urban Public Transport Systems of Three Major Cities of Portugal in 2021

Round 1

Reviewer 1 Report

The authors perform a novel study evaluating air samples for SARS-CoV-2 in indoor and outdoor spaces of public transport systems in Portugal. 2 positive air samples were detected at a train station, one at the front of station and the other at the entrance hall, with an interval of approximately 10 minutes between each other.

Major points:

1. It was noted that the 2 air samples were taken on the same day, 10 minutes between each other, at different locations of the train station of City A -authors to clarify in the methods the following:

• Authors to elaborate on whether the cyclonic microbial air sampler was sterilized after each air sampling measurement, if the air sampler was cleaned/sterilized between each air sampling measurement and also between change of location of air sampling. What is the manufacturer’s recommendation for cleaning/sterilization between each measurement?

• Given that there were 2 consecutive air samples testing positive for SARS-CoV-2, what is the likelihood of residual SARS-CoV-2 RNA within the air sampler from the first air sampling that was positive, contaminating the second air sampling?

• For the research assistant/researcher deploying the air sampler, was the research assistant/researcher wearing a mask and sterilizing their hands when setting up the air sampler?

2. Authors should consider further discussion of their findings in comparison with references 7, 15,16,23 and 24. What do they think is the major contributing factor to discrepant results in air sampling across the studies, as well as their study?

3. What was the prevailing public health policy in Portugal on mask wearing in public spaces?

• What quality masks are acceptable by law? Is wearing cloth masks in open public areas acceptable
• Authors commented that there could be a possibility of improper mask wearing. Are masks mandatory and what are the existing penalties for not mask wearing?

The manuscript generally reads well and requires only minor revisions, with significant findings that could have public health implications. Authors to address the above concerns.  

Author Response

Dear Editor,

Thank you so much for your letter. I am resubmitting a revised and reworked paper.

A detailed document was attached with the authors’ response to the reviewers’ comments.

Modifications were incorporated in the manuscript in highlighted in yellow for responses to the reviewer’s comments.

Best regards,

Priscilla Gomes da Silva

Reviewer 1

The authors perform a novel study evaluating air samples for SARS-CoV-2 in indoor and outdoor spaces of public transport systems in Portugal. 2 positive air samples were detected at a train station, one at the front of station and the other at the entrance hall, with an interval of approximately 10 minutes between each other.

Major points:

1. It was noted that the 2 air samples were taken on the same day, 10 minutes between each other, at different locations of the train station of City A -authors to clarify in the methods the following:

• Authors to elaborate on whether the cyclonic microbial air sampler was sterilized after each air sampling measurement, if the air sampler was cleaned/sterilized between each air sampling measurement and also between change of location of air sampling. What is the manufacturer’s recommendation for cleaning/sterilization between each measurement?

Answer: Thank you for the important remark. Cleaning of the sampler was performed between each air sampling measurement and/or change of location according to the manufacturer’s recommendation. We have clarified in the methods section the cleaning procedure of the equipment. Please refer to lines 106-110.

• Given that there were 2 consecutive air samples testing positive for SARS-CoV-2, what is the likelihood of residual SARS-CoV-2 RNA within the air sampler from the first air sampling that was positive, contaminating the second air sampling?

Answer: Thank you for this valuable question. Some things were interpreted in order to consider that there was no contamination of the second air sample. First, we did the disinfection of the air sampler between samplings. This was performed according to the manufacturer’s instructions. Additionally, only sterile cones were used. Second, the first air sample had lower viral load than the second. Finally, both samples presented positivity to different genomic regions (i.e. 1^st sample was positive to N2 and 2^nd sample to N1) which shows that viruses from sample 1 were different from sample 2.

For the research assistant/researcher deploying the air sampler, was the research assistant/researcher wearing a mask and sterilizing their hands when setting up the air sampler?

Answer: Thank you for this remark. Yes, the researcher in charge of the air sampling was wearing KN95 masks and gloves at all times. Information about this was added to the methodology section so it is clear in the manuscript that all prevention methods were followed. Please refer to lines 116-118.

1. Authors should consider further discussion of their findings in comparison with references 7, 15,16,23 and 24. What do they think is the major contributing factor to discrepant results in air sampling across the studies, as well as their study?

Answer: Thank you for the important remark. We have added a paragraph on the discussion for that. Please refer to lines 179-189..

1. What was the prevailing public health policy in Portugal on mask wearing in public spaces?

Answer: Thank you for the question. The prevailing public health policy in Portugal at the time required that it was mandatory to wear masks inside all indoor and outdoor public spaces. Please refer to lines 199-201.

• What quality masks are acceptable by law? Is wearing cloth masks in open public areas acceptable

Answer: Surgical masks or higher were acceptable by law at that time. Cloth masks are not considered acceptable by law. We have specified that in the manuscript. Please refer to line 200.

• Authors commented that there could be a possibility of improper mask wearing. Are masks mandatory and what are the existing penalties for not mask wearing?

Answer: Thank you for your comment. The existing penalties in Portugal at the time were a fee that varied between 100-500€ for people not following the rule of wearing proper masks in indoor and outdoor environments. We have added that information to the manuscript. Please refer to line 201.

The manuscript generally reads well and requires only minor revisions, with significant findings that could have public health implications. Authors to address the above concerns.  

Answer: Thank you for your very kind comment.

Reviewer 2 Report

In the submitted manuscript “Detection of SARS-CoV-2 in indoor and outdoor areas of urban public transport systems of three major cities of Portugal, 2021” (Manuscript Number: ijerph-1668247), the authors conducted a cross-sectional study between April 5^th to April 30^th 2020 (at that time, low incidence of COVID-19 was reported). As data on the presence of SARS CoV-2 RNA in public transport system is scarce, the authors aimed to assess the presence of SARS CoV-2 RNA in public transport system in indoor and outdoor spaces in 3 major cities in Portugal. Air (n=31) and surface (n=70) samples were collected and analyzed through RT-qPCR. Only 2 air samples from an outdoor and a partially open space were positive for SARS-CoV-2 RNA. The author conclude that the risk of transmission in outdoor environments is higher than in indoor when the first are crowded and the majority of people are not wearing protective masks. However, more studies are needed to fully elucidate the real risk of infection in outdoor spaces.

A rational of the study is interesting. However, in my point-of-view, there are some major points that need to be clarified:

1. In “Introduction” section,
   1. In Line 40, please reformat the reference (Dyani, 2021).
   2. In Line 60, please reformat the reference (Luo, 2020).
   3. In Line 62, please reformat the reference (Harris, 2020).
   4. In Line 66, replace “The first one by [15] …” with “The first one by Di Carlo et.al. [15] …”. Line 68 and 70 as well.

2. In “Methods” section,
   1. How the authors ensure that viral RNA in samples was not degraded during storage in PBS at 4 oC before arrival to the laboratory?

3. In “Results” section,
   1. As the authors compared SARS-CoV-2 detection between indoor and outdoor areas, Table 1 should clearly indicate which sample location is considered as indoor area or as outdoor area

4. In “Discussion” section,
   1. The authors mentioned that “No positive surface sample was detected”. That may result from the inappropriate of the sampling method used in this study. Please clarify.
   2. The authors mentioned that that the risk of transmission in outdoor environments is higher than in indoor. They should provide the percentages of positive samples in outdoor environments versus that in indoor environments. What are the denominators of sample in outdoor/indoor environments? As the small number of sample used in this study, it is difficult to demonstrate a significant difference between the rates of SARS-CoV-2 positive samples in outdoor environments versus that in indoor environments. Therefore, the conclusion may not be true.

5. In the Reference part, some references are not relevant.
   1. The format of some references are not correct, for example #3, #19, #20, etc.

Author Response

Dear Editor,

Thank you so much for your letter. I am resubmitting a revised and reworked paper.

A detailed document was attached with the authors’ response to the reviewers’ comments.

Modifications were incorporated in the manuscript in highlighted in yellow for responses to the reviewer’s comments.

Best regards,

Priscilla Gomes da Silva

Reviewer 2

In the submitted manuscript “Detection of SARS-CoV-2 in indoor and outdoor areas of urban public transport systems of three major cities of Portugal, 2021” (Manuscript Number: ijerph-1668247), the authors conducted a cross-sectional study between April 5^th to April 30^th 2020 (at that time, low incidence of COVID-19 was reported). As data on the presence of SARS CoV-2 RNA in public transport system is scarce, the authors aimed to assess the presence of SARS CoV-2 RNA in public transport system in indoor and outdoor spaces in 3 major cities in Portugal. Air (n=31) and surface (n=70) samples were collected and analyzed through RT-qPCR. Only 2 air samples from an outdoor and a partially open space were positive for SARS-CoV-2 RNA. The author conclude that the risk of transmission in outdoor environments is higher than in indoor when the first are crowded and the majority of people are not wearing protective masks. However, more studies are needed to fully elucidate the real risk of infection in outdoor spaces.

A rational of the study is interesting. However, in my point-of-view, there are some major points that need to be clarified:

1. In “Introduction” section,

• In Line 40, please reformat the reference (Dyani, 2021).

Answer: Thank you for the comment. The reference was reformatted accordingly.

• In Line 60, please reformat the reference (Luo, 2020).

Answer: Thank you for the comment. The reference was reformatted accordingly.

• In Line 62, please reformat the reference (Harris, 2020).

Answer: Thank you for the comment. The reference was reformatted accordingly.

• In Line 66, replace “The first one by [15] …” with “The first one by Di Carlo et.al. [15] …”. Line 68 and 70 as well.

Answer: Thank you for the comment. The references were corrected accordingly.

2. In “Methods” section,

• How the authors ensure that viral RNA in samples was not degraded during storage in PBS at 4 oC before arrival to the laboratory?

Answer: Thank you for the important question. Sterile cones and swabs, as well as sterile RNAse-free 1.5 mL eppendorfs were used to store the samples throughout the whole experiment. All samples were kept at 4ºC and RNA extraction was performed within 12 hours. We have added that information to the manuscript. Please refer to lines 113-115.

3. In “Results” section,

• As the authors compared SARS-CoV-2 detection between indoor and outdoor areas, Table 1 should clearly indicate which sample location is considered as indoor area or as outdoor area

Answer: Thank you for the remark. We have updated the table accordingly.

4. In “Discussion” section,

• The authors mentioned that “No positive surface sample was detected”. That may result from the inappropriate of the sampling method used in this study. Please clarify.

Answer: Thank you for this important remark. We have added a clarification about this in the discussion. Please refer to lines 161-165.

• The authors mentioned that that the risk of transmission in outdoor environments is higher than in indoor. They should provide the percentages of positive samples in outdoor environments versus that in indoor environments. What are the denominators of sample in outdoor/indoor environments? As the small number of sample used in this study, it is difficult to demonstrate a significant difference between the rates of SARS-CoV-2 positive samples in outdoor environments versus that in indoor environments. Therefore, the conclusion may not be true.

Answer: Thank you for this remark. You are right, therefore we have toned down the conclusion accordingly. Please refer to lines 215-216.

5. In the Reference part, some references are not relevant.

• The format of some references are not correct, for example #3, #19, #20, etc.

Answer: Thank you for the commented. The citations and references were all reviewed and corrected accordingly.