Qualitative Comparison of Hydrogen Peroxide Decontamination Systems: Vapor vs. Aerosol
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsValidated decontamination methods are vital to maintaining safety of laboratories working with high consequence pathogens, and fumigation is necessary for airborne transmissible pathogens. Testing the validity of these methods provides assurance. This paper therefore has merit in addressing this issue, including advantages and limitations of different delivery systems, however I believe it has missed an opportunity to provide more valuable data.
By placing the biological indicator discs into nutrient broth and incubating, all this provides is a yes/no answer and the averaging of the results for various locations does not tell the reader enough about performance in the different locations., and therefore enough about penetrability and dispersion. Conventionally decontamination performance is measured as log kill, and such data would have been achievable if the authors had instead eluted the spores from discs into isotonic solution, serially diluted and inoculated spread plates, this achieving quantification of the surviving spores at any point, resulting in a log kill value. In my opinion I would like this experiment to be repeated using this method to add value to the limited data in the manuscript at present. It would not be a major undertaking to repeat this experiment as I assume this is the authors' laboratory, As it stands, the results are not providing anything more than the validation data for the authors' laboratory which is probably a statutory requirement anyway. However, the comparison of the two hydrogen peroxide delivery methods is interesting.
Some other observations. The BSL3 is quite small (I'm guessing about 4m x 4m) so doesn't greatly challenge fumigant dispersion. A larger laboratory would be better. I'm guessing as the Class II biosafety cabinet was left on during the experiments it was a recirculating rather than ducted to atmosphere version otherwise the fumigant would be quickly depleted. However it does not specify this. Recirculating cabinets in BSL3 laboratories are unusual. The laboratory door was 'closed' but was it also sealed to prevent fumigant leakage? This needs to be with plastic tape (not duct tape) to prevent penetration. I assume the lids were on the petri dishes? It does not say. The materials and methods states biological safety cabinets plural but I guess from the rest of the text that was a typo and there was just one. A relatively small laboratory in any case would not accommodate more than one for them to perform safely.
Comments on the Quality of English LanguageJust minor word corrections
Author Response
Reviewer 1:
I would like to express my sincere gratitude for your thorough and insightful evaluation of our manuscript. Your thoughtful comments and constructive suggestions have greatly contributed to the improvement of our work. I appreciate the time and effort you dedicated to reviewing our article, as well as the valuable recommendations that have enhanced the quality of our research.
Comment 1: Repeating Experiments for a Quantitative Study
Response 1 : The aim of this study is to compare the two methods, relying primarily on commercial biological indicators, which are frequently used for the qualification of DSVA devices. Your comment is very pertinent. However, unfortunately, we are unable to repeat the experiments.
Comment 2: BSL3 Dimensions
Response 2 :The BSL3 laboratory is relatively small (approximately 4 m x 4 m), which limits potential issues with the dispersion of the fumigant. However, it is true that a larger laboratory would allow for a better evaluation of the distribution and effectiveness of the treatment over a larger area. We will consider your suggestion for future studies or configurations.
Comment 3: Class II biosafety cabinet operation
Response 3 : Thank you for your observation. The purpose of operating the Class II biosafety cabinet during the decontamination cycle is not to recirculate the air, but to verify whether the decontamination agent reaches all internal surfaces of the cabinet, even when the Class II biosafety cabinet is in operation. This approach allows us to assess the effectiveness of the decontaminant distribution within the cabinet, although we cannot guarantee that this distribution is entirely homogeneous. Air recirculation occurs at the end of the contact cycle. For the HPV system, the device automatically catalyzes the Hâ‚‚Oâ‚‚ vapor into water and oxygen. For the aPH system, recirculation is achieved by manually activating the air treatment unit from outside the BSL3.
Comment 4: Laboratory Door Status
Response 4 :The BSL3 laboratory door was closed and sealed during the decontamination cycle. We used the integrated pressure-sealing system within the laboratory, which ensures proper airtightness. We did not use adhesive tape for sealing, but the pressure system ensures that leaks of the decontaminating agent are adequately controlled.
Comment 5: Petri dish in position 19
Response 5 :No, the Petri dish in 19 and 20 positions were closed.
Comment 6: Number of BCCII
Response 6 : Yes, there was only one microbiological safety cabinet.
Reviewer 2 Report
Comments and Suggestions for AuthorsReview of “Qualitative Comparison of Hydrogen Peroxide Decontamination Systems: Vapor vs. Aerosol”
The manuscript provides a nice direct comparison of hydrogen peroxide aerosols (aHP) versus vapourized hydrogen peroxide (VHP) systems for room disinfection. It uses a carefully controlled set up and set of conditions that allow for a better and direct comparison of vapour versus aerosols, which is lacking in the literature. Even if the disinfection efficacy is somewhat similar in the two methods, this information is a useful contribution and other observations are interesting. However, there are a number of clarifications and corrections that should be considered before the manuscript would be acceptable. These are listed below in rough order of appearance in the manuscript.
1. Section 2.1 first line: “area” should be “volume”.
2. Section 2.1: presumably each device was located at the same position, but it might be useful to explicitly state this.
3. Section 2.1: just to be completely clear, is the concentration based on 4.5 g of 35% solution, or of pure H2O2? Some literature can be confusing on this type of information.
4. Table 1: this nicely summarizes the sampling locations, but a diagram might be more effective for visualization, with the table data provided in Supplementary materials?
5. Section 2.3: “Mesalabs” should be “MesaLabs”, I believe.
6. Section 2.3: It seems from the supplementary data spreadsheet that there were 7 biological indicator discs exposed at each position? Is this correct, and if so it would be useful to mention this in the methods as it elevates the statistical confidence.
7. Section 2.4: details on the supplier of the HPV-CI indicators should be given.
8. Section 3.1: it is not quite clear why there was a one hour difference in cycle length between the two methods, and some discussion or explanation would be useful. Earlier, the decontamination cycle times seemed to be stated to be the same for both methods in Section 2.1, where “60 or 90 minutes” is mentioned. However, this is only a ½ hour difference and therefore a bit confusing.
9. Section 3.1, the last line has “161” which seems like a typo?
10. Figure 1 caption “Analyze” should be “Analysis of”
11. Figure 2: the devices were apparently at location 0 cm, but at what height?
12. Section 3.3, first paragraph: This paragraph refers to Figure 3, but I think that Figure 2 is the intention. Also, the roles and significance of positions 19 and 20 are somewhat unclear. In Section 2.1 they are mentioned in Table 1 but without comment, and so in Section 3.3 it is somewhat confusing. Some more discussion would be useful around what purpose these serve and whatever hypothesis they are testing.
13. Section 3.3, 2nd paragraph and Figure 3: although there are small differences, it seems that they might be statistically insignificant. Were any statistical tests done to examine this? One complicating factor is that the bioindicator method used here is binomial, with either a positive or negative growth observation. Was the standard deviation calculation for binomial distributions used?
14. Figure 3: I'm not sure that Figure 3 is very useful or informative. The fact that some samples required up to 3 days to show visible turbidity (growth) is not unusual and it is more of a limitation of the method rather than an observation on the efficacy of aHP or HPV. Use of plate counts might have been more quantitative for assessing the extent of disinfection. These disc growth methods are strictly only binomial, and although you might infer some level of disinfection from the time required to show growth it is not very quantitative.
15. Figure 3: the label on the y-axis is confusing (% positive). At first glance it appears that over 90% of the samples were positive for growth, which would be a poor disinfection result. It seems that the data is based on % positive for negative growth, which is a bit awkward.
16. In the submitted spreadsheet, the "stdev.s" function seems to be used for standard deviation estimates. However, since the data is binomial, this function is not really correct since it's based on normal distributions. I don't think that there is enough binomial data to be considered approximately normal, but this should be looked into.
17. Section 4.: “aPH” should be “aHP”.
18. Section 4.: the comparison with literature results is useful, but could possibly be done in a table format for easier communication, with some discussion around important observations.
19. Section 4., 2nd paragraph: “Moreover, there was a decline in efficacy…”. This discussion is a bit misleading. There isn't really a decline in disinfection efficacy after 3 days of incubation. It's just that the low initial levels of viable bacteria on the discs took a while to grow to a visible concentration. One might conclude that the initial efficacy was lower, since there was growth eventually but to quantify this is difficult. It also illustrates that a 5 or 7 day incubation is really required for proper evaluation using this method.
20. Section 4, 3rd paragraph: regarding the discussion of diffusion and Petri dishes. Possibly there may also be some differences in the individual Petri dishes, with respect to how well the lids fit, and this may affect the results? Were these plastic disposable Petri dishes? It is not unusual to find some small variability between these plastic items.
Author Response
Reviewer 2:
Thank you very much for your detailed and insightful review of our manuscript. Your careful analysis and constructive feedback have been instrumental in refining our work. I deeply appreciate the time and effort you invested in reviewing our article and the valuable suggestions you provided, which have significantly improved the quality of our research.
Comment 1 : Section 2.1 first line: “area” should be “volume”.
Response 1 : It's modified in the text.
Comment 2 : Section 2.1: presumably each device was located at the same position, but it might be useful to explicitly state this.
Response 2 : It's modified in the text.
Comment 3 : Section 2.1: just to be completely clear, is the concentration based on 4.5 g of 35% solution, or of pure H2O2? Some literature can be confusing on this type of information.
Response 3 : It's modified in the text.
Comment 4 : Table 1: this nicely summarizes the sampling locations, but a diagram might be more effective for visualization, with the table data provided in Supplementary materials?
Response 4 : I have added a figure showing the positions.
Comment 5 : Section 2.3: “Mesalabs” should be “MesaLabs”, I believe.
Response 5 : It's modified in the text.
Comment 6 : Section 2.3: It seems from the supplementary data spreadsheet that there were 7 biological indicator discs exposed at each position? Is this correct, and if so it would be useful to mention this in the methods as it elevates the statistical confidence.
Response 6 : No, only one biological indicator disk was exposed at each position. The number 7 actually refers to the number of days during which the cultures were monitored to see if there was any germination of bacterial spores after the decontamination cycles.
Comment 7 : Section 2.4: details on the supplier of the HPV-CI indicators should be given.
Response 7 : It's modified in the text.
Comment 8 : Section 3.1: it is not quite clear why there was a one hour difference in cycle length between the two methods, and some discussion or explanation would be useful. Earlier, the decontamination cycle times seemed to be stated to be the same for both methods in Section 2.1, where “60 or 90 minutes” is mentioned. However, this is only a ½ hour difference and therefore a bit confusing.
Response 8 : Decontamination cycles using vaporized hydrogen peroxide (HPV) are typically shorter than those using aerosolized hydrogen peroxide (aHP) due to the faster and more uniform distribution of the vapor and quicker drying. Additionally, the HPV system is a closed-loop system that recovers Hâ‚‚Oâ‚‚ from the atmosphere by catalyzing it into water and oxygen, which reduces the aeration phase compared to the aHP system.
Comment 9: Section 3.1, the last line has “161” which seems like a typo?
Response 9 : It's modified in the text.
Comment 10 : Figure 1 caption “Analyze” should be “Analysis of”
Response 10 : It's modified in the text.
Comment 11 : Figure 2: the devices were apparently at location 0 cm, but at what height?
Response 11 : For aPH system 52 cm and for 71 cm.
Comment 12: Section 3.3, first paragraph: This paragraph refers to Figure 3, but I think that Figure 2 is the intention. Also, the roles and significance of positions 19 and 20 are somewhat unclear. In Section 2.1 they are mentioned in Table 1 but without comment, and so in Section 3.3 it is somewhat confusing. Some more discussion would be useful around what purpose these serve and whatever hypothesis they are testing.
Response 12 : No, it is indeed Figure 3. This first paragraph outlines the expected results of the biological indicators: the absence of germination in positions 1 to 18 and the presence of germination in positions 19 and 20 (with samples placed in Petri dishes with lids). This is why Figure 4 is also included.
Response 12 (part 2) : Positions 19 and 20 involve spore discs placed in sealed Petri dishes. Under normal conditions, we anticipated that positions 19 and 20 would show germination for both methods. However, surprisingly, some trials in position 19 for both methods displayed delays or reductions. These observations led us to hypothesize that the laminar flow within the BCCII improves the diffusibility of Hâ‚‚Oâ‚‚ within the BCCII.
Comment 13 : Section 3.3, 2nd paragraph and Figure 3: although there are small differences, it seems that they might be statistically insignificant. Were any statistical tests done to examine this? One complicating factor is that the bioindicator method used here is binomial, with either a positive or negative growth observation. Was the standard deviation calculation for binomial distributions used?
Response 13 : Since this study is qualitative in nature, statistical analyses to determine the significance of the observed differences could not be conducted.
Comment 14 : Figure 3: I'm not sure that Figure 3 is very useful or informative. The fact that some samples required up to 3 days to show visible turbidity (growth) is not unusual and it is more of a limitation of the method rather than an observation on the efficacy of aHP or HPV. Use of plate counts might have been more quantitative for assessing the extent of disinfection. These disc growth methods are strictly only binomial, and although you might infer some level of disinfection from the time required to show growth it is not very quantitative.
Response 14 : Figure 3 presents the assessment of bacterial growth over a period of 7 days. Although this study does not rely on classical solid media culture methods, the observed delay in germination can also occur with liquid media culture methods, which may be attributed to oxidative stress that slows down growth. Whether using solid or liquid media, exposure of bacteria to hydrogen peroxide induces a similar metabolic stress, resulting in delayed growth. Therefore, it is pertinent to retain this figure in the article. Thank you for your feedback; it is important to note that a study based on a binary response (positive or negative) should not exhibit significant variability.
Comment 15 : Figure 3: the label on the y-axis is confusing (% positive). At first glance it appears that over 90% of the samples were positive for growth, which would be a poor disinfection result. It seems that the data is based on % positive for negative growth, which is a bit awkward.
Response 15 : It's modified in the graph.
Comment 16 : In the submitted spreadsheet, the "stdev.s" function seems to be used for standard deviation estimates. However, since the data is binomial, this function is not really correct since it's based on normal distributions. I don't think that there is enough binomial data to be considered approximately normal, but this should be looked into.
Response 16 : It's modified in the graph.
Comment 17 : Section 4.: “aPH” should be “aHP”.
Response 17 : It's modified in the text.
Comment 18 : Section 4.: the comparison with literature results is useful, but could possibly be done in a table format for easier communication, with some discussion around important observations.
Response 18 : Thank you for your comment, but I only have two studies comparing aHP and HPV. I don't find it relevant to include a table with such a small number of studies.
Comment 19 : Section 4., 2nd paragraph: “Moreover, there was a decline in efficacy…”. This discussion is a bit misleading. There isn't really a decline in disinfection efficacy after 3 days of incubation. It's just that the low initial levels of viable bacteria on the discs took a while to grow to a visible concentration. One might conclude that the initial efficacy was lower, since there was growth eventually but to quantify this is difficult. It also illustrates that a 5 or 7 day incubation is really required for proper evaluation using this method.
Response 19 : It's modified in the text.
Comment 20 : Section 4, 3rd paragraph: regarding the discussion of diffusion and Petri dishes. Possibly there may also be some differences in the individual Petri dishes, with respect to how well the lids fit, and this may affect the results? Were these plastic disposable Petri dishes? It is not unusual to find some small variability between these plastic items.
Response 20 : Thank you for your observation. The Petri dishes used were made of plastic, which can introduce some variability, particularly concerning the fit of the lids. However, within the context of this qualitative study, this variability remains significant. It is important to note that the aHP method is based on droplet dispersion, while the HPV method relies on vapor, which partly explains the observed differences.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for the amendments made and explanation of what can or cannot be done to make further amendments. I can see that within those limitations, and with the changes made, the paper now has greater merit and provides useful data for the reader to make choices over fumigation methods applied in laboratories.
I would just like to suggest a further change. I was not just asking about whether the BCC is recirculating for purposes of fumigation, but this is important for the reader to know, to differentiate it from BCCs that vent directly to atmosphere as a fundamental of their mode of operation. Therefore I suggest rewording line 73 to "was equipped with a Biosafety Cabinet Class II (BCCII) whose mode of operation is to recirculate air into the laboratory through a double HEPA filter".
Comments on the Quality of English Language
A few minor typographical errors that will be picked up at final edit
Author Response
Comment 1 : rewording line 73 to "was equipped with a Biosafety Cabinet Class II (BCCII) whose mode of operation is to recirculate air into the laboratory through a double HEPA filter".
Response 1 : It's modified in the text.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have made some significant clarifications to the manuscript and methods in particular. Although the results are somewhat qualitative, the manuscript is a useful contribution and comparison of two different room disinfection methods under controlled conditions.
Author Response
Dear Reviewer,
Thank you for your valuable feedback. We appreciate your recognition of the clarifications we made to the manuscript and methods. We are pleased that you find the manuscript to be a useful contribution.
While we would have liked to provide further clarifications, this is unfortunately not possible under the current conditions. We hope that the revisions we have made will be clear enough for the readers.
Best regards,
Courti Ibtissam
