Metagenomic Shotgun Sequencing Analysis of Canalicular Concretions in Lacrimal Canaliculitis Cases

Round 1

Reviewer 1 Report

I read with great interest the article entitled “Metagenomic shotgun sequencing analysis of canalicular con-2 cretions in lacrimal canaliculitis cases” attempting to describe the results of using Next-Generation Sequencing (NGS) for metagenomic shotgun sequencing analysis of canalicular concretion samples from primary lacrimal canaliculitis.

However, several flaws and questions are raised in this manuscript that need to be addressed and clarified.

MAJOR CONCERNS

1. The article reports: “We reviewed the medical records of patients with primary lacrimal canaliculitis who were examined in Toho University Omori Medical Center Hospital, Amagasaki Medical Center Hospital, Jikei University Hospital, Saitama Medical Center Hospital from February 2015 through July 2017.” Please clarify whether the work is retrospective or prospective through the text and in the title.
2. The Methods section reports: “After each sample was collected, it was placed in an Eppendorf tube and stored in a freezer at -80℃ until DNA extraction. All specimens were frozen and sent to the Department of Microbiology and Infectious Diseases at the Faculty of Medicine, Toho University School of Medicine.” The Authors are invited to clarify the concept of “sample”. Did the Authors analyze tears or canalicular concretions? This is a fundamental point!
3. The introduction and discussion sections are too short and incomplete. Clearly, several issues should be deepened. Please note that the presence of canalicular concretions (or related debris) may alter the physiological tear behavior and the overall tear turnover as described in the following articles:                                                                         (A) Fourier-domain OCT imaging of the ocular surface and tear film dynamics: A review of the state of the art and an integrative model of the tear behavior during the inter-blink period and visual fixation. Journal of clinical medicine, 2020, 9.3: 668.                                       (B)The bull’s eye pattern of the tear film in humans during visual fixation on en-face optical coherence tomography. Scientific reports, 2019, 9.1: 1-9.

Please cite the aforementioned articles and discuss this point. This reinforces the article.

4. Another important concern is as follows: What is the usefulness of this article? What are the original findings of this research article compared to the others? Please clarify.

MINOR CONCERNS

5. The methods section in the abstract is insufficient.It should be rewritten.
6. Did you perform sample size calculation? Please specify.

In sum, the article is quite interesting, but discussion should be improved as suggested. At the moment, the paper is not ready for publication. I have some concerns about some areas noted above, particularly in bibliographic research, and I would like to see the revised version again before allowing it to be published.

Author Response

Dear: Reviewer 1 

Please see attachment, thank you.

Best regard

Yukinobu Okajima

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript retrospectively reviewed the medical records of patients with primary lacrimal canaliculitis by using metagenomic shotgun sequencing analysis using next- generation sequencing of pathogens . These results may provide new information in clinical treatment of lacrimal canaliculitis .

Major concerns:

1. In this retrospective results, though  shortgun sequencing and NGS may reveal genome of pathogens, authors should compare this results with their own traditional culture results in these 17 patients. Is these data available ?
2. Are the results of shortgun sequencing can contribute the choices of antibiotics in the treatment of patients with lacrimal canaliculitis? How of this examination can impact the treatment choices?
3. For clinical applications, the treatment results of these 17 patients based on the shortgun sequencing should be provided in another table.

Minor concerns:

1. Line179-191. Comparing the current results with the culture results, 
anaerobic bacteria such as Actinomyces spp. were detected in 15 eyes, while in the culture results, Actinomyces spp. was detected in 5% to 44.4%. ---- 1. with other culture results. 2. comparing with percentage. And all the following sentences.

Author Response

Dear :Reviewer2

Please see the attachment. Thank you.

Best  Regards

Yukinobu Okajima

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I would like to commend the authors for such an interesting work. The article is ready for publication. 

Reviewer 2 Report

No further comments