Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary:

The author conducted a systematic evaluation of the process limit of detection (PLOD) for four pathogenic microorganisms in surface water and wastewater by using the Nanotrap particles with the KingFisher™ Apex robotic platform, qPCR, and dPCR. Through seeding known concentration targets into uncontaminated samples, the recovery, PLOD, and positive ratio were reported and compared. This work is designed well and is significant to further strengthen current environmental sample-based disease surveillance. However, the discussion and the comparison between the present and previous studies are weak. 

Comments:

1. Discussion could be added to discuss if there is a correlation between seeding level and recovery.
2. In most cases, at the lowest seeding level, wastewater usually generated more positive results than surface water, which is in contrast to the common sense that wastewater contains more PCR inhibitors that could negatively affect detection performance. The author can add more discussion to give some explanation. For example, the wastewater might have a higher background of the targets. Although it was tested as negative before seeding, it just means the target concentration is below the detection method. The sample might still contain targets, and these targets will be added to the overall amount after seeding, hence inducing a higher positive ratio in wastewater samples.

3. The recoveries are pretty low for most targets. Could the author compare them with previous studies in detail and give a suggestion about whether the workflow used in the present study is suitable for future applications?

Author Response

Reviewer 1 Comments:

The author conducted a systematic evaluation of the process limit of detection (PLOD) for four pathogenic microorganisms in surface water and wastewater by using the Nanotrap particles with the KingFisher™ Apex robotic platform, qPCR, and dPCR. Through seeding known concentration targets into uncontaminated samples, the recovery, PLOD, and positive ratio were reported and compared. This work is designed well and is significant to further strengthen current environmental sample-based disease surveillance. However, the discussion and the comparison between the present and previous studies are weak. 

1. Discussion could be added to discuss if there is a correlation between seeding level and recovery.

 

Response: We analyzed that the seeding levels are not correlated with recovery rates. This was added to the Discussion part.

 

1. In most cases, at the lowest seeding level, wastewater usually generated more positive results than surface water, which is in contrast to the common sense that wastewater contains more PCR inhibitors that could negatively affect detection performance. The author can add more discussion to give some explanation. For example, the wastewater might have a higher background of the targets. Although it was tested as negative before seeding, it just means the target concentration is below the detection method. The sample might still contain targets, and these targets will be added to the overall amount after seeding, hence inducing a higher positive ratio in wastewater samples.

 

Response: The reviewer raised an interesting and important question about the background(residue) of target pathogen and PCR inhibition in wastewater. I have seen that many seeding experiments testing the limit of detection of pathogens in wastewater use wastewater knowing residue amount of pathogen in wastewater. In my opinion, this type of research design is not appropriate since the wastewater may contain trace amount of the target pathogen that is under the limit of detection as what the review pointed out. In this study, we collected a septic tank from a resident whose family had no previous history of infection with the four pathogens. Therefore, there is no issue about residue pathogens in wastewater. For the PCR inhibition question, it is not an issue in this study since we use one wastewater sample for all the seeding experiments. Whether or not the sample has PCR inhibition, the quantification and recovery efficiencies are comparable among different seeding levels and detection methods.

3. The recoveries are pretty low for most targets. Could the author compare them with previous studies in detail and give a suggestion about whether the workflow used in the present study is suitable for future applications?

Response: The low recovery efficiencies of the four target pathogens in this study are in the range of the recovery efficiency of bacteria and viruses in wastewater reported in the literature. Typically, the pathogen recovery efficiency in wastewater is low because pathogen was lost or underestimated in each step of the concentration, nucleic acid extraction, and PCR detection.  I added more detailed information in the Discussion. In addition, the workflow used in this study can be applicable in future studies except that manual nucleic acid extraction can replace the automatic extraction method, which our previous study showed no difference between the manual and automatic extraction methods (Liu et al, 2023, Frontiers in Microbiology). This suggestion was also added in the Discussion part.

Reviewer 2 Report

Comments and Suggestions for Authors

The introduction is short, but it introduces the topic very well. The methodology is described in detail. It allows for the experiment to be recreated.

The quantitative assessment of the number of bacteria being determined is intriguing in the methodological part. Were any attempts made to verify the results obtained on the spectrophotometer with those obtained using culture methods? Does this match? The adopted method of quantitative assessment of bacteria is simplified, the result may be disturbed by the culture medium itself and the products of bacterial metabolism. Please add this to the article in the discussion section - one paragraph is enough.
The results are presented very well and properly discussed.
The discussion is correct, it results from the presented material.

Please add a short summary. A few sentences of conclusions from the conducted research.

Relevant literature, most of the literature is from 2020.

Author Response

Reviewer 2 Comments

1. The introduction is short, but it introduces the topic very well. The methodology is described in detail. It allows for the experiment to be recreated. The quantitative assessment of the number of bacteria being determined is intriguing in the methodological part. Were any attempts made to verify the results obtained on the spectrophotometer with those obtained using culture methods? Does this match? The adopted method of quantitative assessment of bacteria is simplified, the result may be disturbed by the culture medium itself and the products of bacterial metabolism. Please add this to the article in the discussion section - one paragraph is enough. The results are presented very well and properly discussed. The discussion is correct, it results from the presented material.

Response: The reviewer raised a great comment. We did not try to validate the molecular results by culture methods since we know that pathogen culture from wastewater samples is challenging because the wastewater contains matrix that significantly interfere with the growth of pathogens. We added a few sentences in the Discussion part.

 

Please add a short summary. A few sentences of conclusions from the conducted research.

Response: A short summary was added to the Discussion.

 

Relevant literature, most of the literature is from 2020.

Response: I tried to add a few latest papers.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All comments have been added. I suggested that the final summary should be a separate chapter, but since it is very short it can stay that way.