Comparative Analysis of Oral Prevotella intermedia, Tannerella forsythia, Streptococcus sanguinis, and Streptococcus mutans in Patients with Esophageal Squamous Cell Carcinoma and Healthy Controls in Mthatha, South Africa
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe topic of the study is intersting and fits the journal's aims&scope. The possible effect of oral bacteria on carcinoma development is investigated. The authors detected the disbalance in oral microbiota in patients with esophagial cancer. Unfortunately, the sample size is reltiveley small and the risk of bias is high as the authors did not consider dental and periodontal status of study participants. To make the study design more transparent several corrections in the manuscript may be provided:
1. The abstract should be structured according to the article’s main parts.
2. In the Introduction it is mentioned that oral bacteria such as Streptococcus mitis, Streptococcus anginosus, and Treponema denticola are frequently found in esophageal cancer patients (lines 46-48). Nevertheless, study aim was to analyze and compare the presence and abundance of specific oral bacteria (Streptococcus mutans, Streptococcus sanguinis, Prevotella intermedia, and Tannerella forsythia) in the oral microbiome of ESCC patients and healthy controls main parts (Lines 53-54).
Please, add more information to Introduction section explaining your selection of bacteria evaluated within the study.
3. Materials and methods:
- Please add inclusion and exclusion (non-inclusion) criteria
- Please add information about sample size calculation
- Please add more information related to possible bias and bias prevention
4. Results
- Please add to Table 1 information about the mean age in both study groups. Were the study groups matched by age?
- The potential difference in the number of bacteria in saliva may be associated with different levels of oral hygiene in patients. Did you measure any Plaque index? Did you consider multiple caries or periodontal disease?
5. Discussion
- Lines 155-156: Ignored for a long time, until 1994 when the role of H. pylori bacteria was established 155 in the etiology of gastric cancer [23]. - Please, double-check the citation and edit the whole sentence. It seems, a part of the sentence is absent.
6. Conclusion
- Lines 217-220: This work thus confirms that an imbalance in the selected bacteria plays a crucial and indicative role in the development or complications of esophageal tumors in South African population. – This statement is not supported by the results. The results just demonstrate the difference in oral microbiota in gastroesophagela cancer patients and healthy controls, but do not estimate the causation.
Author Response
All the corrections are visible in color in the document.
comments 1- The abstract should be structured according to the article’s main parts.
Answer 1: The abstract has been corrected according to the author's guidelines.
- In the Introduction it is mentioned that oral bacteria such as Streptococcus mitis, Streptococcus anginosus, and Treponema denticola are frequently found in esophageal cancer patients (lines 46-48). Nevertheless, study aim was to analyze and compare the presence and abundance of specific oral bacteria (Streptococcus mutans, Streptococcus sanguinis, Prevotella intermedia, and Tannerella forsythia) in the oral microbiome of ESCC patients and healthy controls main parts (Lines 53-54).
comments 2- Please, add more information to Introduction section explaining your selection of bacteria evaluated within the study.
Answer 2: The justification for the selection of bacteria has been added in the introduction, lines 47-54, as recommended by the reviewer.
- Materials and methods:
comments 3- Please add inclusion and exclusion (non-inclusion) criteria
Answer 3: The inclusion and exclusion criteria have been added to the document on page 2, section 2.2, as recommended by the reviewer.
comments 4- Please add information about sample size calculation
Answer 4: The sample size was calculated using the formula for a case-control study [Bland, 2000], considering a significance level (α) of 0.05 and a power (1-β) of 0.70. With expected exposure proportions of 0.2 for controls and 0.3 for cases, the required sample size was determined to be 48 (24 cases and 24 controls). See section data collection, page 3, lines 92-95.
comments 5- Please add more information related to possible bias and bias prevention
Answer 5: In this study to reduce selection bias we recruited every participant that met the inclusion criteria, all patients diagnosed with OSCC at Nelson Mandela Academic Hospital during the study period and healthy controls were matched to OSCC patients based on age, geographic location (Mthatha) and gender. See section data collection, page 3, lines 95-99.
- Results
comments 6- Please add to Table 1 information about the mean age in both study groups. Were the study groups matched by age?
Answer 6: The average age has been added to Table 2. Although they are not exactly matched, the average ages are not very different.
comments 7- The potential difference in the number of bacteria in saliva may be associated with different levels of oral hygiene in patients. Did you measure any Plaque index? Did you consider multiple caries or periodontal disease?
Answer 7: We did not measure any Plaque Index or any of the above-mentioned parameters; we measured bacterial expression in whole unstimulated saliva. Thank you for the suggestion; we will consider it in future studies.
- Discussion
comments 8- Lines 155-156: Ignored for a long time, until 1994 when the role of H. pylori bacteria was established 155 in the etiology of gastric cancer [23]. - Please, double-check the citation and edit the whole sentence. It seems, a part of the sentence is absent.
Answer 8: This sentence has been rephrased as suggested by the reviewer as follows: "Many years ago, the role played by microorganisms in cancer was ignored, but the discovery that the bacterium Helicobacter pylori (H. pylori) promotes gastric cancer brought the issue back into focus in 1994 (Hou et al., 2022)." The reference has been reviewed.
- Conclusion
comments 9- Lines 217-220: This work thus confirms that an imbalance in the selected bacteria plays a crucial and indicative role in the development or complications of esophageal tumors in South African population. – This statement is not supported by the results. The results just demonstrate the difference in oral microbiota in gastroesophagela cancer patients and healthy controls, but do not estimate the causation.
Answer 9: This section has been rephrased, see the conclusion section, page 8, lines 252–256.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Sirs,
I carefully read your paper and I congrats with you for the nice work you did.
As Introdution and study design are appropriate, same for the result, I fond discuccion adequately supported from literature.
However let me suggest you some papers that could improve your paper by means of introducing the concept of oral health management
Yu Y, Xia L, Wang Z, Zhu T, Zhao L, Fan S. A cross-cohort study identifies potential oral microbial markers for esophageal squamous cell carcinoma. iScience. 2024 Nov 22;27(12):111453. doi: 10.1016/j.isci.2024.111453. PMID: 39758985; PMCID: PMC11699290.
https://pubmed.ncbi.nlm.nih.gov/36141674/
https://pubmed.ncbi.nlm.nih.gov/38668014/
Author Response
comment :
However, let me suggest you some papers that could improve your paper by means of introducing the concept of oral health management: Yu Y, Xia L, Wang Z, Zhu T, Zhao L, Fan S. A cross-cohort study identifies potential oral microbial markers for esophageal squamous cell carcinoma. iScience. 2024 Nov 22;27(12):111453. doi: 10.1016/j.isci.2024.111453. PMID: 39758985; PMCID: PMC11699290. https://pubmed.ncbi.nlm.nih.gov/36141674/.; https://pubmed.ncbi.nlm.nih.gov/38668014/.
Answers
All the corrections are visible in color in the document.
The references mentioned by the reviewer have been utilized and cited in the introduction (reference 11,12,14).
Reviewer 3 Report
Comments and Suggestions for AuthorsPlease see the attached comments
Comments for author File: 
Comments.pdf
Author Response
All the corrections are visible in color in the document.
Comment 1: Line 39. Please use italics in bacterial names
Answer 1: The names of the bacteria have been italicized throughout the document.
Comment 2: Table 1. How the primers and probes were selected? The sequences among Streptococcus species and the probe design are challenging; there are easily sone crossreactivity. How you tested that only selected Streptococcus not others were measured?
Answer 2: We used primers and probes specific for the selected bacteria for one full reaction.
Comment 3: Table 2. Please add the BMI values.
Answer 3: On this study we were not looking at the weight and height of the participants so on our questionnaires there was no space for weight and height.
Comment 4: Table2 Before the experiment controls and cases should be selected carefully or explaned why they are randomly selected. There are significant differences on many parameters. The p values should be added on the table. In the discussion it should be explaned how these so different groups cannot be compared. Please add also the statistical methods in m&m section.
Answer 4: Regarding the selection of controls and cases, we ensured that participants were selected based on strict inclusion and exclusion criteria to minimize selection bias. The groups were matched based on key characteristics such as age, gender, and geographical location to improve comparability. Significant differences observed in several parameters are noted, and the corresponding p-values have been added to Table 2 for clarity.
Comment 5: Fig1. Please add the y axis value (theoretical cell count or what ?). Please add the total amounts also. Are the values mean or medians? Add 95%CI or quarter ranges (depending on previous presentation). Add The p values. Why The same results are presented twice in Fig1 and table 3. There are no unit values in the table 3 either.
Answer 5: Figure 1 has been corrected according to the reviewer's recommendations, see page 5, section 3.2. Standard deviations have been added, along with p-values, and the unit of the (y) axis has been included as recommended by the reviewer.
Comment 6: Table3. Results should be presented as adjusted values i.e corrected with Socio-demographic characteristics age, sex, education, missing of teeth, alcohol consumption and smoking.
Answer 5: Table 3 has been corrected according to the reviewer's recommendations (see page 7, section 3.3).
Reviewer 4 Report
Comments and Suggestions for AuthorsDear Aurhors,
I carefully read your manuscript entitled: " Comparative analysis of oral Prevotella intermedia, Tannerella forsythia, Streptococcus sanguinis, and Streptococcus mutans in esophageal squamous cell cancer patients and healthy controls in Mthatha-South Africa ".
Here my comments.
I think the introduction will benefit from the addition of α-diversity which is expressed through several indices, such as Shannon index; and β-diversity and its several indexes. For this reason I suggest to read and cite the introduction of the following article: https://pubmed.ncbi.nlm.nih.gov/36141674/.
Author Response
Comment : For this reason I suggest to read and cite the introduction of the following article: https://pubmed.ncbi.nlm.nih.gov/36141674/.
All the corrections are visible in color in the document.
Answer: The reference mentioned by the reviewer has been utilized and cited in the introduction (reference 11).
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors provided the required revisions and the manuscript may be published.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear sirs
Unfortunately the authors have not understand /corrected the manucript according the suggested comments as I asked. The mist importantly, table 3 &4 do not contain the informaation that was required: cases vs controls do those bacteria effect on cancer when the results are adjusted with significant sosio-demograpic variables.
Reviewer 4 Report
Comments and Suggestions for AuthorsDear Authors, I apprecciated your efforts to improve the manuscript.
I found a better overall quality. For this reaspn I recommand the acceptance
