Detecting Closer to Care: Combining Phage and LAMP to Detect Tuberculosis, Bovine TB and Johne’s Disease

Round 1

Reviewer 1 Report

Comments from a Reviewer

Please refer to the advice below and rewrite the target disease to focus on the problem.

Title: The meaning of "Detecting closer to care" is not clear. In the first place, isn't "Care" inappropriate for bovine tuberculosis and bovine Johne's disease? The correct response to these mycobacterial infections is to kill or isolate cattle that are suspected of being infected.

Abstract: In the introduction, the authors state, ”There is an unmet need for new diagnostics that are effective, near-curable, and quick and easy to perform during the early stages of infection.'' The meaning of "Detecting closer to care" is not clear. In the first place, isn't "Care" inappropriate for bovine tuberculosis and bovine Johne's disease? The correct response to cholera mycobacterial infections is to kill or isolate cattle that are suspected of being infected.

Introduction
L26-27: First, bovine Johne's disease, tuberculosis, and human tuberculosis should not be discussed together. For bovine Johne's disease, infected or suspected cattle can be identified by repeated absorption ELISA and fecal PCR methods. By repeating this inspection and culling at regular intervals, the herd can be purified. Bovine tuberculosis can be diagnosed with a high probability using conventional intradermal tests and interferon-gamma assays. Cattle that test positive for these tests can be purified by being promptly isolated and culled. Some countries have achieved a high level of cleanliness. Countries that do not take these preventive measures continue to leave potentially infected cows on farms and continue producing positive cows.

 As the author states, tuberculosis and non-tuberculous mycobacterial diseases in humans are still difficult to diagnose and treat. Since the same measures as for livestock cannot be taken, it is not desirable to focus solely on the difficulty in the common biological nature of the Mycobacterium.

L27-32: Rather than explaining the structure and resistance of mycobacteria, the author should describe the cutting-edge limitations of mycobacterial diagnosis to date. Why did your country not achieve the eradication of bovine paratuberculosis and tuberculosis, although another country achieved realized the eradication of the diseases. This will be the premise for the solution of old problems and the need for new diagnostic methods.

L33-38: I think the author should start the introduction from this part. As mentioned later, quantitative PCR and interferon-gamma assays are expensive. But they are reasonable cost for developed countries.

L46-47: Because antibody-positive cows have not been properly culled, subclinically infected cows will continue to be on the farm forever, incurring epidemic prevention costs forever. Only the companies that manufacture and sell testing reagents will continue to make profits, and the miserable situation of farmers will continue. This situation has not changed since 50 years ago, when this reviewer began researching Johne's disease. Isn't this because the naive approach of the department in charge of national epidemic prevention to protect dairy farmers and consumers has not been corrected? I hope the authors think about these problems logically as scientists.

L49: Some countries have achieved cleanliness with known diagnostic methods. However, I think that developing countries need cheaper and more practical diagnostic technology. However, no matter what kind of diagnostic method is developed, if the state is unable to pay the compensation for infected cattle after diagnosis, infected cattle will continue to exist on farms, and epidemic prevention efforts will become endless.

L49-56: Once again, it is essential that animals that test positive using current diagnostic methods be compensated and culled by the state. If this is not the case, it may be possible to suppress the spread of infection by keeping the animal isolated after diagnosis, but it may not be effective enough to clean up the farm.

L57-58: The difficulties in diagnosing tuberculosis in bovines and humans and Johne's disease in bovines cannot be discussed together. Isn't Johne's disease more difficult to diagnose than tuberculosis?

L58-60: Host immune reaction against MAP is very complicated, but the infected host appears specific immunoreaction in antibody and CMI depending on the stage of the infection. The author emphasizes the thickness of the Mycobacterial cell wall in the prevention of host immunity, but the author should know that MAP cell wall antigen, called ethanol-extracted antigen induces host antibody production in an earlier stage than cytoplasmic antigens.

L62-63: References required.

L63-67: In the first place, is it necessary to detect "live MAP" when diagnosing paratuberculosis using blood samples?

L84~: OK. But why did you use ATCC 19698 instead of K10 which is the current standard stain?

L186~: OK. But Need to find out more Mycobacteria species?

L344-345: The authors say that 15% of the cows on the farm are ELISA positive, but considering the sensitivity of the ELISA infection stage, isn't this a severely contaminated farm? Cows that test positive for ELISA are at an advanced stage of paratuberculosis, so don't the infected cows before that make up the majority of the farm?

Why are you keeping ELISA-positive cows alive on such heavily contaminated farms? This is unthinkable based on the reviewer's common sense. I'm sure the milk should not get from the infected cattle in the contaminated farms, or transporting culled cows to slaughterhouses on the public health. Please answer. Think livestock health and public health. The correct administrative action would be to immediately kill ELISA-positive cows before leisurely considering new diagnostic methods.

L352-355: The clinical samples mentioned here are not described in Materials and Methods. For tuberculosis patients, information such as whether they are HIV positive or not and their CD4 cell count is required. In particular, it would be unusual for bacteremia to be detected in 7/10 blood samples of non-HIV-infected tuberculosis patients. I don't think you should write what should be written on M&M in the Discussion.

L363-367: Although we do not deny the development and evaluation of new testing methods, at the current stage, it is possible to diagnose infected cattle by at least regularly testing a combination of absorption ELISA and fecal PCR. First, repeat the diagnosis and make full use of it to establish a paratuberculosis-free farm.

L379-382: It seems that this method should be studied specifically for the diagnosis of human tuberculosis and non-tuberculous mycobacterial infections and the determination of bacteremia.

There is no need at this stage for the application of paratuberculosis in cattle. This is because cows that test positive with the current absorption ELISA method suggest highly infected status, so it is meaningless whether or not M. paratuberculosis is detected in the cow's blood. The path to cleanliness is to test all cattle using the absorption ELISA method and fecal PCR method, and then repeat the test until at least zero positive cattle remain.

L395-398: Although the research in this paper is meaningful for understanding the infection status of human tuberculosis patients, diagnosis and culling of Johne's disease and tuberculosis in cattle in the field should be carried out by making full use of current diagnostic methods. At that time, if the government is reluctant to provide the necessary security funds for epidemic prevention, researchers and researchers should stand up and advocate for the safety of farmers and consumers. Please do your best.

Author Response

Reviewer 1 response

We would like to thank the Reviewer for their comments on improving our manuscript. Below are responses to each point (in bold) and where applicable the manuscript has been changed and highlighted for convenience.

Title: The meaning of "Detecting closer to care" is not clear. In the first place, isn't "Care" inappropriate for bovine tuberculosis and bovine Johne's disease? The correct response to these mycobacterial infections is to kill or isolate cattle that are suspected of being infected.

 The ‘care’ element is taken from point of care as diagnostic test, although you are correct the animals are not ‘cared’ for this is the literal meaning and we are referring only developing on diagnostic tests that can be used closer to the patient or animal. The same terminology has been described in articles describing point of care tests for bovine tuberculosis – for example:

Kelley, H., Waibel, S.M., Sidiki, S. et al. Accuracy of Two Point-of-Care Tests for Rapid Diagnosis of Bovine Tuberculosis at Animal Level using Non-Invasive Specimens. Sci Rep 10, 5441 (2020). https://doi.org/10.1038/s41598-020-62314-2.

It should also be pointed out we are talking about human as well and bovine disease, however we do take your point and will clarify in the introduction.

Abstract: In the introduction, the authors state, ”There is an unmet need for new diagnostics that are effective, near-curable, and quick and easy to perform during the early stages of infection.'' The meaning of "Detecting closer to care" is not clear. In the first place, isn't "Care" inappropriate for bovine tuberculosis and bovine Johne's disease? The correct response to cholera mycobacterial infections is to kill or isolate cattle that are suspected of being infected.

See above, however the statement closer to care as been removed to enable further explanation in the introduction. 

Introduction
L26-27: First, bovine Johne's disease, tuberculosis, and human tuberculosis should not be discussed together. For bovine Johne's disease, infected or suspected cattle can be identified by repeated absorption ELISA and fecal PCR methods. By repeating this inspection and culling at regular intervals, the herd can be purified. Bovine tuberculosis can be diagnosed with a high probability using conventional intradermal tests and interferon-gamma assays. Cattle that test positive for these tests can be purified by being promptly isolated and culled. Some countries have achieved a high level of cleanliness. Countries that do not take these preventive measures continue to leave potentially infected cows on farms and continue producing positive cows.

We are describing the biology of mycobacterium and feel that they can be put together as we are not describing the individual diseases caused, rather talking about the infective agent. Here we have clarified the difficulties associated with thick cell wall and slow growth rate but adding ‘by culture’ to line 31 to be explicit about the challenges the biology of mycobacteria can cause.

We are further not sure where the comments regarding ELISA and faecal PCR are relevant here as the UK has a major problem and early detection coupled with ease of detection has not been possible with these diseases.

 As the author states, tuberculosis and non-tuberculous mycobacterial diseases in humans are still difficult to diagnose and treat. Since the same measures as for livestock cannot be taken, it is not desirable to focus solely on the difficulty in the common biological nature of the Mycobacterium.

Again we believe the shared biology is important but agree the same control measures are carried out. We have added ‘and in humans treatment’ to line 49 to distinguish between human and animal disease control. 

L27-32: Rather than explaining the structure and resistance of mycobacteria, the author should describe the cutting-edge limitations of mycobacterial diagnosis to date. Why did your country not achieve the eradication of bovine paratuberculosis and tuberculosis, although another country achieved realized the eradication of the diseases. This will be the premise for the solution of old problems and the need for new diagnostic methods.

 We have focused on the structure and resistance as this is the most relevant elements for our paper as we are describing a bacteriophage approach to detecting mycobacteria. The phage approach circumvents the issues of thick cell wall and slow growth rate allowing rapid detection. I am not sure the history of each disease is relevant to our manuscript as we are touching on three separate diseases, bovine TB which is endemic to regions of the UK, Johne’s disease which is considered endemic worldwide and human tuberculosis which is endemic primarily in the global south in LMICs.

L33-38: I think the author should start the introduction from this part. As mentioned later, quantitative PCR and interferon-gamma assays are expensive. But they are reasonable cost for developed countries.

These technologies are very expensive even in high income countries for the detection of bovine TB and Johne’s disease. For TB where in the UK it is less burdensome these costs are indeed reasonable, however we are keen to show that the technology has the a potential worldwide impact and therefore being as low cost and usable as possible. 

L46-47: Because antibody-positive cows have not been properly culled, subclinically infected cows will continue to be on the farm forever, incurring epidemic prevention costs forever. Only the companies that manufacture and sell testing reagents will continue to make profits, and the miserable situation of farmers will continue. This situation has not changed since 50 years ago, when this reviewer began researching Johne's disease. Isn't this because the naive approach of the department in charge of national epidemic prevention to protect dairy farmers and consumers has not been corrected? I hope the authors think about these problems logically as scientists.

 There may well be issues politically with regards to these diseases that have exacerbated the problem, however as the focus on this paper is on novel technology, we feel it would be out of scope to comment on failings in the manuscript and would most probably be more appropriate in a review on Johne’s disease.

L49: Some countries have achieved cleanliness with known diagnostic methods. However, I think that developing countries need cheaper and more practical diagnostic technology. However, no matter what kind of diagnostic method is developed, if the state is unable to pay the compensation for infected cattle after diagnosis, infected cattle will continue to exist on farms, and epidemic prevention efforts will become endless.

 We do agree with this entirely but may be out of scope of the manuscript.

L49-56: Once again, it is essential that animals that test positive using current diagnostic methods be compensated and culled by the state. If this is not the case, it may be possible to suppress the spread of infection by keeping the animal isolated after diagnosis, but it may not be effective enough to clean up the farm.

 We do agree with this entirely but may be out of scope of the manuscript.

L57-58: The difficulties in diagnosing tuberculosis in bovines and humans and Johne's disease in bovines cannot be discussed together. Isn't Johne's disease more difficult to diagnose than tuberculosis?

The difficulties are the same especially during early infection where no good diagnostic test exists. Again the idea of the manuscript is to describe the develop of a novel tool that may be used for diagnostic purposes for mycobacteria in general and can be applied to different disease states. 

L58-60: Host immune reaction against MAP is very complicated, but the infected host appears specific immunoreaction in antibody and CMI depending on the stage of the infection. The author emphasizes the thickness of the Mycobacterial cell wall in the prevention of host immunity, but the author should know that MAP cell wall antigen, called ethanol-extracted antigen induces host antibody production in an earlier stage than cytoplasmic antigens.

 Here we are not talking about host immunity as we clearly have stated molecular biomarkers – reference DNA/RNA which is difficult to access inside the cell.

L62-63: References required.

 These have been added

L63-67: In the first place, is it necessary to detect "live MAP" when diagnosing paratuberculosis using blood samples?

The power of bacteriophages are that they are akin to culture where only viable cells are detected. This is important for when we detect MAP (or any mycobatceria) as we can determine that there is live viable mycobacteria within their host and therefore the host is infected.

L84~: OK. But why did you use ATCC 19698 instead of K10 which is the current standard stain?

There are many standard strains of MAP and ATCC19698 is a readily available cattle type strain of MAP.

L186~: OK. But Need to find out more Mycobacteria species?

Not sure of the point you are making here in the ethics section? Could you please clarify?

L344-345: The authors say that 15% of the cows on the farm are ELISA positive, but considering the sensitivity of the ELISA infection stage, isn't this a severely contaminated farm? Cows that test positive for ELISA are at an advanced stage of paratuberculosis, so don't the infected cows before that make up the majority of the farm?

We would consider this to be a high prevalence herd, however there are no animals with clinical signs of Johne’s from this herd. Our data presented here is a pilot to determine which animals had viable MAP compared to ELISA and that this will hopefully inform larger follow up studies to understand the relationship between ELISA and phage positivity rate. We do agree with the reviewer that there is a high chance of Johne’s infection on these herds.

Why are you keeping ELISA-positive cows alive on such heavily contaminated farms? This is unthinkable based on the reviewer's common sense. I'm sure the milk should not get from the infected cattle in the contaminated farms, or transporting culled cows to slaughterhouses on the public health. Please answer. Think livestock health and public health. The correct administrative action would be to immediately kill ELISA-positive cows before leisurely considering new diagnostic methods.

 This is not our farm and so we cannot comment on individual farmers management practices. We are merely testing animals based on ELISA tests. As stated however there is no clinical signs of JD on these farms and there is no legal obligation to cull or remove infected animals with JD.

L352-355: The clinical samples mentioned here are not described in Materials and Methods. For tuberculosis patients, information such as whether they are HIV positive or not and their CD4 cell count is required. In particular, it would be unusual for bacteremia to be detected in 7/10 blood samples of non-HIV-infected tuberculosis patients. I don't think you should write what should be written on M&M in the Discussion.

 As mentioned the samples were opportunistically obtained and none with HIV positive. This has been added to the materials and methods. We have reference our previous studies that have demonstrated viable mycobacteria can be found in the blood of TB patients (Verma et al 2019) and this finding is not unexpected.

L363-367: Although we do not deny the development and evaluation of new testing methods, at the current stage, it is possible to diagnose infected cattle by at least regularly testing a combination of absorption ELISA and fecal PCR. First, repeat the diagnosis and make full use of it to establish a paratuberculosis-free farm.

 This is stated in the paragraph were our data supports further testing and evaluation of the technology.

L379-382: It seems that this method should be studied specifically for the diagnosis of human tuberculosis and non-tuberculous mycobacterial infections and the determination of bacteremia.

There is no need at this stage for the application of paratuberculosis in cattle. This is because cows that test positive with the current absorption ELISA method suggest highly infected status, so it is meaningless whether or not M. paratuberculosis is detected in the cow's blood. The path to cleanliness is to test all cattle using the absorption ELISA method and fecal PCR method, and then repeat the test until at least zero positive cattle remain.

 We would like to disagree with the reviewer here as ELISA testing is not 100% sensitive especially during early stages of infection were transmission can occur. Here we describe a new potential tool that may be used to tackle JD and other mycobacterial infections.

L395-398: Although the research in this paper is meaningful for understanding the infection status of human tuberculosis patients, diagnosis and culling of Johne's disease and tuberculosis in cattle in the field should be carried out by making full use of current diagnostic methods. At that time, if the government is reluctant to provide the necessary security funds for epidemic prevention, researchers and researchers should stand up and advocate for the safety of farmers and consumers. Please do your best.

We are not entirely sure of the meaningfulness of this comment with regards to our manuscript as we are describing a novel tool and not commenting on the politics or countries in how they control their diseases.

Reviewer 2 Report

The manuscript titled “Detecting closer to care: combining phage and LAMP to detect tuberculosis, bovine TB and Johne’s disease”, is an interesting study on a phage-based approach that diagnoses the viability of certain pathogenic mycobacteria in blood samples. According to the results, the phage-based lysis coupled with LAMP could reduce the time needed for sample processing (centrifuging, cleaning,...) when compared to Actiphage coupled with qPCR, although both approaches showed correlated endpoints. I believe that the reproducibility of the assay should be tested in a larger number of clinical samples. In addition, incorporating a complementary assay like ELISA along with phage-LAMP may offer a comprehensive insight into the state of diseases alongside the viability of pathogenic mycobacteria. 

To improve the manuscript, please find my comments as follows:

Line 49-51:This sentence has been revised for clarity: The welfare and economic importance of TB, bTB, and JD underline the need for affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free diagnostics that can be easily delivered to the end-users. 

Line 53: Could you describe what are the other options?

Line 57: This sentence is repeated before (check a few lines ago), please remove it: The thick and waxy cell wall common to Mycobacterium makes these bacteria difficult to lyse [11].

Line 58: Remove the dot before preventing.

Line 59: Do you have any references for this statement? The mycobacterial cell wall is difficult to lyse and it creates a defensive barrier against immune cells, however, this difficulty doesn't impede the identification of biomarkers.

Line 61: Lysing mycobacterial cell walls was not the primary application of mycobacteriophages. In addition, mycobacteriophages might be lytic or lysogenic which should be mentioned. To enhance the understanding of mycobacteriophages, I suggest first describing a background about the mycobacteriophages and outlining their applications. For instance: Mycobacteriophages were primarily used as molecular tools useful in the study of mycobacteria, especially their genetics". https://doi.org/10.1016/B978-012373944-5.00020-1

or they could be used for therapeutic purposes. https://doi.org/10.1159/000519870

Line 64: You may cite the following article referring to a recent work about the detection of viability of MAP in milk samples via a novel phage assay:

https://doi.org/10.1038/s41598-021-04451-w 

Line 65-67: Please cite your statement.

Line 71-75: the sentence should be revised for clarity and improved flow. It has some missing parts, and the flow should be improved.  

Line 90-91: Is there any reason that you did not agitate the culture? Because, if the mycobacterial culture doesn't get agitated, clumps form. 

Line 92: Approximately 1x105 cfu/ml of each bacterium was used for what purpose? it should be mentioned!

Line 97: what was the composition of the media you used for serial dilution and also for conveying the phage?

Line 101: Did you add any supplements or chemicals to the media containing phages such as OADC or CaCl2?

Line 134-135: Change the dots with coma before optimizing, before the effect of primer.

Line 169: I suggest changing the order of paragraphs: I think the Phage-LAMP assay should be located before 2.4. NAAT optimization and testing. Lines 140-146 should be discussed after 2.5 and 2.6 sections. 

Line 188: I think the first two lines (188-189)  should be replaced with the following sentence: The performance of the assay is influenced by some factors such as temperature and time of exposure, loop primer sequence, and the composition of the NAAT master mix. Then you can explain how each factor affects the performance of the assay.

Line 223-229: Please clarify whether you are discussing qPCR or LAMP results. This ambiguity could be seen in the figure caption. "Line 231: Effect of clean and concentration on crude and concentrated DNA lysate". I think it should be revised like this: The efficiency of LAMP on clean-concentrated DNA compared to crude-concentrated DNA.  

Line 242-243: Please define TOD and LOD at their first use

Line 355: remove one of "in"

Minor editing of English language required. I suggest revising the sentences indicated in the comments for further clarity and flow. 

Author Response

Reviewer 2 response

We would like to thank the Reviewer for their comments on improving our manuscript. Below are responses to each point (in bold) and where applicable the manuscript has been changed and highlighted for convenience.

The manuscript titled “Detecting closer to care: combining phage and LAMP to detect tuberculosis, bovine TB and Johne’s disease”, is an interesting study on a phage-based approach that diagnoses the viability of certain pathogenic mycobacteria in blood samples. According to the results, the phage-based lysis coupled with LAMP could reduce the time needed for sample processing (centrifuging, cleaning,...) when compared to Actiphage coupled with qPCR, although both approaches showed correlated endpoints. I believe that the reproducibility of the assay should be tested in a larger number of clinical samples. In addition, incorporating a complementary assay like ELISA along with phage-LAMP may offer a comprehensive insight into the state of diseases alongside the viability of pathogenic mycobacteria. 

Thank you for your overview and we agree further evaluation would be required including comparison with immunological based tests are far larger cohorts of patients/animals.

To improve the manuscript, please find my comments as follows:

Line 49-51:This sentence has been revised for clarity: The welfare and economic importance of TB, bTB, and JD underline the need for affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free diagnostics that can be easily delivered to the end-users. 

Thank you this has been modified.

Line 53: Could you describe what are the other options?

We have removed this sentence as the next sentence essential repeats this with the added ‘options’

Line 57: This sentence is repeated before (check a few lines ago), please remove it: The thick and waxy cell wall common to Mycobacterium makes these bacteria difficult to lyse [11].

This has been removed from lines 27-28

Line 58: Remove the dot before preventing.

This has been modified.

Line 59: Do you have any references for this statement? The mycobacterial cell wall is difficult to lyse and it creates a defensive barrier against immune cells, however, this difficulty doesn't impede the identification of biomarkers.

This has been modified focussing on DNA and molecular diagnostics

Line 61: Lysing mycobacterial cell walls was not the primary application of mycobacteriophages. In addition, mycobacteriophages might be lytic or lysogenic which should be mentioned. To enhance the understanding of mycobacteriophages, I suggest first describing a background about the mycobacteriophages and outlining their applications. For instance: Mycobacteriophages were primarily used as molecular tools useful in the study of mycobacteria, especially their genetics". https://doi.org/10.1016/B978-012373944-5.00020-1

or they could be used for therapeutic purposes. https://doi.org/10.1159/000519870

Line 64: You may cite the following article referring to a recent work about the detection of viability of MAP in milk samples via a novel phage assay:

https://doi.org/10.1038/s41598-021-04451-w 

Thank you for these citation and comment – thy have been added.

Line 65-67: Please cite your statement.

This has been modified

Line 71-75: the sentence should be revised for clarity and improved flow. It has some missing parts, and the flow should be improved.  

This has been modified.

Line 90-91: Is there any reason that you did not agitate the culture? Because, if the mycobacterial culture doesn't get agitated, clumps form. 

For MAP especially we have found the opposite is true and more clumps form if agitated.

Line 92: Approximately 1x105 cfu/ml of each bacterium was used for what purpose? it should be mentioned!

This has been modified. To ensure lack of detection was not due to low levels of bacteria creating a false negative signal.

Line 97: what was the composition of the media you used for serial dilution and also for conveying the phage?

This has been modified – this is supplemented media as mentioned earlier.

Line 101: Did you add any supplements or chemicals to the media containing phages such as OADC or CaCl2?

This has been modified – as we did.

Line 134-135: Change the dots with coma before optimizing, before the effect of primer.

This has been modified.

Line 169: I suggest changing the order of paragraphs: I think the Phage-LAMP assay should be located before 2.4. NAAT optimization and testing. Lines 140-146 should be discussed after 2.5 and 2.6 sections. 

This has been modified.

Line 188: I think the first two lines (188-189)  should be replaced with the following sentence: The performance of the assay is influenced by some factors such as temperature and time of exposure, loop primer sequence, and the composition of the NAAT master mix. Then you can explain how each factor affects the performance of the assay.

This has been modified.

Line 223-229: Please clarify whether you are discussing qPCR or LAMP results. This ambiguity could be seen in the figure caption. "Line 231: Effect of clean and concentration on crude and concentrated DNA lysate". I think it should be revised like this: The efficiency of LAMP on clean-concentrated DNA compared to crude-concentrated DNA.  

This has been modified.

Line 242-243: Please define TOD and LOD at their first use

This has been modified in Lines 201 and 202

Line 355: remove one of "in

This has been modified

Round 2

Reviewer 1 Report

I have attached a comment sheet.

Comments for author File: Comments.pdf

Author Response

Reviewer 1 second response.

We thank you for your rigorous review of our manuscript. In general your points are well received and we have added a comment in the discussion regarding using current testing and the need for strong government action plans to help reduce the burden of Johne’s disease on farms. Lines 53-56 and Lines 402-404.

We hope this satisfies your comments.