Mind the gap between non-activated (non-aggressive) and activated (aggressive) indoor fungal testing: impact of pre-sampling environmental settings on indoor air readings

Article title: Mind the gap between non-activated (non-aggressive) and activated (aggressive) indoor fungal testing: impact of pre-sampling environmental settings on indoor air readings.

Nice that the authors have added a case study. However I have some comments to that and other minor suggestions to the manuscript. In the file there are also small grammatical suggestions.

I would recommend that the authors change the headings throughout the manuscript:

1. Introduction

1.1 Non-activated (non-aggressive) and activated (aggressive) protocols

1.2 The effect of activities and environmental context to the sampling readings

2. Materials and Methods

2.1. Laboratory experiments

2.2. Case study

3. Results

3.1. Laboratory experiments

3.2. Case study

4. Discussion

5. Conclusion

Other suggestions

Abstract: delete “enormous”. The diversity is not that big when you consider how many fungal species there exits (see Andersen B., et al. (2021) in your reference list).

Introduction: Use “diversity" or "biota" instead of “flora”. Flora is restricted for plants. For fungi you can use mycobiota or fungal biodiversity or fungal diversity.

Sec. 2.1 (5 lines under fig. 3): Should probably be “floor” instead of “wall”.

Sec. 2.2: When was the samples taken. Give data or at least month. Since the outdoor fungal species change during the year, it is important to know which species that can come in through the windows.

Move the paragraph “air sampling…” up (see file).

How big was the room and for how long did you run the blower?

How did you get hold of the equipment and filters for both analyses? Which blower was used for activation? Which pump was used for sampling?

Did you sample both NAHA and DNA (all 3 filters) at the same time?

Where was the samples taken? In the middle of the room. And in what hight?

Fungal names have to be in italics and abbreviated when it has been written out once in each "chapter".

Sec. 3.2: Spell out RFU first time in a new “chapter”.

Would it be possible to make the figure larger/taller. The bars are difficult to distinguish from each other.

I take it that there were no Chaetomium or Trichoderma found at al.

For your discussion you could point out that different fungal species have different spore sizes and shapes. Aspergillus versicolor spores are 2,5-3,0 my and globose, while Stachybotrys chartarum have ellipsoid spores that are 8-12 x 4-5 µ. Furthermore, Stachybotrys and Trichoderma spores are produced in slime heads and stick to the mycelium. Ulocladium have the biggest spores (up to 20 x 16 µ).

I suggest you get a copy of the textbook "Food and Indoor fungi" (Samson et al., 2019). There you can see the different fungi you are analysing for and if they can grow indoors or not.

In your case study it does not make sense to make these types of analyses. You are only analysing for 16 species and if there is no fungal growth, you should not find any Stachybotrys , Chaetomium or Trichoderma in the indoor environment. However, the level of A. versicolor is a bit high suggesting perhaps minor humidity problem.

So in this case, where HouseTest analysis show low species richness, it is a good thing (= no fungal growth indoors). Most of the fungal species in the assay are those that grow on building materials and NOT the ones that grow on food items or live in the soil. The HouseTest is biased toward indoor fungi and limited in the number of fungal species, which is excellent for the purpose of analysing for fungal growth in indoor environments, but not for biodiversity studies. I suggest you delete the whole paragraph.

4. Discussion: You results just show - very nicely - the effect of "airing the house". Opening windows and doors is a very good way of getting rid of fungal spores and humid air. That is probably also where A. fumigatus comes from. This fungus lives in compost and garbage outside (Samson et al., (2019)).

5. Conclusions : Since your manuscript is about protocols, many readers would like to know what you suggest/recommend based on your results. Please state:

1) Blow duration:

2) Waiting time before sampling:

3) Sampling volume:

4) Sampling hight:

5) Type of analysis (NAHA or DNA)?

I suggest that the authors send a copy of the manuscript to Mycometer and HouseTest for method check and correct conclusions.

Kind regards

Birgitte

I'd like to thank the authors for their detailed responses to my comment, and updating the paper.

Some minor comments:

You have a 'Results and discussion' section and a 'Discussion' section. This would be better as a Results section and a Discussion section

In the paper, you refer to sections, but might be best to refer to section names (e.g. Methods) or even subsection names.

In the first paragraph, you state that ‘…indoor fungal testing has not yet been standardised’, and then reference a standard (BS EN ISO 16000-19:2014). This could be clarified.

The term activated vs non-activated is a bit confusing, given active vs passive air sampling methods.

I imagine the methods also reflect the research questions. ie. what are all the types of fungi in the building (activated) vs what are people exposed to under normal operating conditions (non-activated). This is covered somewhat in section 2.1, but could be expanded on.

section 2.2 – air speed can also lead to the air pressure differentials which could draw spores out of spaces where they would not normally come from. It may not only be a question of the duration of aerosolising spores, but also the pressure differential created.

The experimental section is interesting, but particle sensors are used, not bioaerosol samplers. So this would show the effects of air speed on all particles not just fungal spores.

UCL Press 13-05-2022: Mind the gap between non-activated (non-aggressive) and activated (aggressive) indoor fungal testing: a short paper .

Interesting paper, but for a short paper it is quit long, especially the introduction. Perhaps sections 2.1 and 2.2 could be condensed into 10-15 lines together with table 2 since your results do not involve fungal counts as such.

It is important that the authors distinguish between the state (of thr air) of the room prior to air sampling and the air sampling method itself. Air sampling itself that can be either passive (or non-volumetric (e.g. open Petri dishes)) or active (or volumetric (e.g. Andersen sampler, Slit-to-agar sampler etc.)).

I think that it would make more sense to the reader to use the terms “passive”, “active” and “aggressive” for the preparation/set-up prior to air sampling , instead of non-active, non-aggressive, and then define in the text what you mean and non-volumetric and volumetric for the sampling. Intuitively the reader will know what the terms involve. Table 1 could be used for the definitions. It also makes the headline 2. Non-activated (non-aggressive) and activated (aggressive) protocols is difficult to understand.

Hence a protocol could be either “passive and non-volumetric” (a person sneaks into an empty room, place two Petri dishes on the table, removes the lids and sneaks out again), “active and volumetric” (several persons move freely about in the room and sample several times with a MAS100 air sampler) or “aggressive and non-volumetric” (a person use a leaf blower in the room for 5 min, place two Petri dishes on the table, removes the lids and walks out).

The authors should also state something about safety measures for the person, who operates the leaf blower – such as dust mask, respirator etc.

Layout:

It would be easier for the reader if the authors could follow the “normal” layout:

1. Introduction

1.1. subsection etc.

2. Materials and Methods

3. Results and Discussion

4. Conclusion

5. References

Furthermore, Table headings have to be “on top” of the table as opposed to figure legends, which have to be below the figures and please insert page numbers.

Language and wording:

Though the manuscript is well written, changing some words might help the reader.

Change reserve(s) to reservoir (s) throughout the text (e.g. reservoir of plant pathogenic propagules in soil)

Use particles counted and particle counts instead of particle readings or particles measured.

Change blowing duration(s) to blowing time .

In the figures the unit is minutes, but it is difficult to relate to 4000 min. or 10000 min. Please change to hours . You of course keep the blowing time in minutes.

Specific questions:

How high/low was the blower placed?

Did the experiment run continuously for 10000 min. (167 hours or 7 days)?

Was the room sealed of for all 7 days?

The dust you used in your experiment was that just what was in the chamber, or did you add more?

Usually electric equipment, like the blower, produce particles during operation and thereby increase the number of particles in the sealed chamber. Small particles may also stick together and decrease in numbers. Have you seen that in your study?

Conclusion:

You do not state best practice for air sampling after aggressive preparation prior to air sampling according to your results. What would you recommend for blowing time and for sampling hight?

Have the authors tested if particle counts and volumetric air sampling show the same results?