Review of '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