Reviewer ’s report  
 
Title: Contributions  of HLA haplotypes, IL8 level and Toxoplasma gondii infection in defining 
new phenotypes in celiac disease  
 
Version: 0 Date: 24 Jan 2018  
 
Reviewer: Fabiana Zingone  
 
Reviewer's report:  
 
The authors in this study analysed the presence of antibodies specific against Toxoplasma gondii, 
the level of IL8 and HLA -DQ2 and HLA -DQ8 alleles in 150 healthy subjects and 150 patients 
diagnosed with CD, looking for the correlation between these paramet ers and the damages in 
intestinal mucosal. The authors found that past infection with Toxoplasma gondii was more 
frequent in controls than in celiac patients. However, in this latter group the authors found a 
significant relationship between the severity o f mucosal damages and anti -TG IgG level. Instead, 
mean serum levels of IL8 was significantly higher in CD patients compared with control. The 
authors decided to look for Toxoplasma gondii infection because it results in a vigorous 
inflammatory with histolo gic characteristics similar to celiac disease. The reviewer believes that 
the work needs some major revisions and clarifications.  
- It is not clear how the patients were enrolled ("Randomly enrolled?). Please clarify.  
 
- How and where were the controls sel ected?. Please clarify.  
 
- The authors say "We considered a diagnosis of CD if the patients had high levels of IgA 
anti-tTG > 3 times of upper normal limit and confirmed by appropriate histology and HLA 
typing. For instance in patients with a phenotype of M arsh I -II, the diagnosis was confirmed 
as potential celiac disease (PCD) by HLA typing and high titers of IgA anti -tTG, 3 times 
higher than cut off or positive at any titer, but confirmed by IgA EmA positivity". Is the IgA 
EmA performed in any case or only  according to the Marsh results?  
 
- Despite the prevalence of anti -TG IgG positivity is lower in CD than controls the authors 
found a significant relationship between the severity of mucosal damages and a positive 
anti-Toxoplasma gondii IgG (P=0.02). It would be interesting to analyse the  duodenal 
mucosa aspects in non -CD subjects with high anti -TG IgG level. This should be done in 
other class of patients needed routine gastroscopy. The authors could discuss this possibility.  
 
- It is not specified in the introduction session why the autho rs decide to analyse also HLA 
and IL8 to reach their aim. The results on IL8 and HLA create some confusions to the 
reader. I suggest rewriting the session regarding these points (HLA and IL8) to better clarify 
the background, the results and the clinical i mplication of what the authors find.  
 
- I would avoid discussing about NCGS. It is out of the authors purpose.   
 
- In the conclusion the authors write: "our results indicated that T. gondii infection in 
combination with high level of IL8, increases the ris k of severity of histology in celiac 
disease patients" and in the result session the authors report that "no significant relationship 
were observed between the level of mucosal damage and IL8 levels in CD patients". Please 
clarify this point.  
I believe th at to better show a possible relationship between Toxoplasma gondii and duodenal 
damage in celiac patients, an epidemiological study with a larger sample size is needed.  What 
about all patients without history of Toxoplasma gondii?  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the concl usions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical re view is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
  
Declarat ion of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose  financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If you r reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses.  I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/ licenses/by/4.0/ ). I understand that any comments 
which I do  not wish to be included in my named report can be included as confidential comments 
to the  editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Contributions  of HLA haplotypes, IL8 level and Toxoplasma gondii infection in defining 
new phenotypes in celiac disease  
 
Version: 0 Date: 24 Jan 2018  
 
Reviewer: Fabiana Zingone  
 
Reviewer's report:  
 
The authors in this study analysed the presence of antibodies specific against Toxoplasma gondii, 
the level of IL8 and HLA -DQ2 and HLA -DQ8 alleles in 150 healthy subjects and 150 patients 
diagnosed with CD, looking for the correlation between these paramet ers and the damages in 
intestinal mucosal. The authors found that past infection with Toxoplasma gondii was more 
frequent in controls than in celiac patients. However, in this latter group the authors found a 
significant relationship between the severity o f mucosal damages and anti -TG IgG level. Instead, 
mean serum levels of IL8 was significantly higher in CD patients compared with control. The 
authors decided to look for Toxoplasma gondii infection because it results in a vigorous 
inflammatory with histolo gic characteristics similar to celiac disease. The reviewer believes that 
the work needs some major revisions and clarifications.  
- It is not clear how the patients were enrolled ("Randomly enrolled?). Please clarify.  
 
- How and where were the controls sel ected?. Please clarify.  
 
- The authors say "We considered a diagnosis of CD if the patients had high levels of IgA 
anti-tTG > 3 times of upper normal limit and confirmed by appropriate histology and HLA 
typing. For instance in patients with a phenotype of M arsh I -II, the diagnosis was confirmed 
as potential celiac disease (PCD) by HLA typing and high titers of IgA anti -tTG, 3 times 
higher than cut off or positive at any titer, but confirmed by IgA EmA positivity". Is the IgA 
EmA performed in any case or only  according to the Marsh results?  
 
- Despite the prevalence of anti -TG IgG positivity is lower in CD than controls the authors 
found a significant relationship between the severity of mucosal damages and a positive 
anti-Toxoplasma gondii IgG (P=0.02). It would be interesting to analyse the  duodenal 
mucosa aspects in non -CD subjects with high anti -TG IgG level. This should be done in 
other class of patients needed routine gastroscopy. The authors could discuss this possibility.  
 
- It is not specified in the introduction session why the autho rs decide to analyse also HLA 
and IL8 to reach their aim. The results on IL8 and HLA create some confusions to the 
reader. I suggest rewriting the session regarding these points (HLA and IL8) to better clarify 
the background, the results and the clinical i mplication of what the authors find.  
 
- I would avoid discussing about NCGS. It is out of the authors purpose.   
 
- In the conclusion the authors write: "our results indicated that T. gondii infection in 
combination with high level of IL8, increases the ris k of severity of histology in celiac 
disease patients" and in the result session the authors report that "no significant relationship 
were observed between the level of mucosal damage and IL8 levels in CD patients". Please 
clarify this point.  
I believe th at to better show a possible relationship between Toxoplasma gondii and duodenal 
damage in celiac patients, an epidemiological study with a larger sample size is needed.  What 
about all patients without history of Toxoplasma gondii?  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the concl usions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical re view is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
  
Declarat ion of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose  financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If you r reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses.  I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/ licenses/by/4.0/ ). I understand that any comments 
which I do  not wish to be included in my named report can be included as confidential comments 
to the  editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Contributions  of HLA haplotypes, IL8 level and Toxoplasma gondii infection in defining 
new phenotypes in celiac disease  
 
Version: 0 Date: 24 Jan 2018  
 
Reviewer: Fabiana Zingone  
 
Reviewer's report:  
 
The authors in this study analysed the presence of antibodies specific against Toxoplasma gondii, 
the level of IL8 and HLA -DQ2 and HLA -DQ8 alleles in 150 healthy subjects and 150 patients 
diagnosed with CD, looking for the correlation between these paramet ers and the damages in 
intestinal mucosal. The authors found that past infection with Toxoplasma gondii was more 
frequent in controls than in celiac patients. However, in this latter group the authors found a 
significant relationship between the severity o f mucosal damages and anti -TG IgG level. Instead, 
mean serum levels of IL8 was significantly higher in CD patients compared with control. The 
authors decided to look for Toxoplasma gondii infection because it results in a vigorous 
inflammatory with histolo gic characteristics similar to celiac disease. The reviewer believes that 
the work needs some major revisions and clarifications.  
- It is not clear how the patients were enrolled ("Randomly enrolled?). Please clarify.  
 
- How and where were the controls sel ected?. Please clarify.  
 
- The authors say "We considered a diagnosis of CD if the patients had high levels of IgA 
anti-tTG > 3 times of upper normal limit and confirmed by appropriate histology and HLA 
typing. For instance in patients with a phenotype of M arsh I -II, the diagnosis was confirmed 
as potential celiac disease (PCD) by HLA typing and high titers of IgA anti -tTG, 3 times 
higher than cut off or positive at any titer, but confirmed by IgA EmA positivity". Is the IgA 
EmA performed in any case or only  according to the Marsh results?  
 
- Despite the prevalence of anti -TG IgG positivity is lower in CD than controls the authors 
found a significant relationship between the severity of mucosal damages and a positive 
anti-Toxoplasma gondii IgG (P=0.02). It would be interesting to analyse the  duodenal 
mucosa aspects in non -CD subjects with high anti -TG IgG level. This should be done in 
other class of patients needed routine gastroscopy. The authors could discuss this possibility.  
 
- It is not specified in the introduction session why the autho rs decide to analyse also HLA 
and IL8 to reach their aim. The results on IL8 and HLA create some confusions to the 
reader. I suggest rewriting the session regarding these points (HLA and IL8) to better clarify 
the background, the results and the clinical i mplication of what the authors find.  
 
- I would avoid discussing about NCGS. It is out of the authors purpose.   
 
- In the conclusion the authors write: "our results indicated that T. gondii infection in 
combination with high level of IL8, increases the ris k of severity of histology in celiac 
disease patients" and in the result session the authors report that "no significant relationship 
were observed between the level of mucosal damage and IL8 levels in CD patients". Please 
clarify this point.  
I believe th at to better show a possible relationship between Toxoplasma gondii and duodenal 
damage in celiac patients, an epidemiological study with a larger sample size is needed.  What 
about all patients without history of Toxoplasma gondii?  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the concl usions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical re view is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
  
Declarat ion of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose  financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If you r reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses.  I agree for my report to be made available under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/ licenses/by/4.0/ ). I understand that any comments 
which I do  not wish to be included in my named report can be included as confidential comments 
to the  editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant