Reviewer’s report
Title:  Serum HBV RNA shows superiority in differentiating  ‘aggressive HBeAg-negative chronic  
hepatitis B’ in treatment-naïve chronic HBV-infected individuals
Version: 1 Date: 04 Feb 2019
Reviewer: Ahmed Mohamed Elsharkawy
Reviewer's report:
In this paper Liu et al aim to study levels of HBV RNA as a marker of cccDNA activity in patients with  
chronic HBV. Their premise is that this marker may act as a better differentiator of those patients with e  
antigen negative disease and a more aggressive biological course. The authors have used the modern  
nomenclature recommended in the 2017 EASL clinical practice guidelines for e antigen negative  
patients and are to be congratulated for this. The fact the HBV RNA levels are higher in e antigen  
positive patients compared to e antigen negative patients makes biological sense and is reassuring. The  
authors then show data to support the hypothesis that serum HBV RNA is better at differentiating  
chronic hepatitis in e antigen negative patients compared to other parameters. This is a good sized  
patient cohort with good numbers in the four chronic categories as defined. I think that the authors  
should address the following issues - 
1.There are a number of grammatical and spelling mistakes in the document that need to be  
corrected - e.g the word efficacy is misspelt in the first paragraph of the introduction.
2.The phrase 'liver invasion' is not appropriate in the first paragraph of the introduction and  
should be modified.
3. It is confusing that the authors use copies/ml for HBV DNA whilst they use IU/ml for qHBsAg.  
It would be better to use IU/ml for HBV DNA (as is used in AASLD and EASL guidelines)  
throughout the manuscript.
4.For the sake of consistency the authors should refer to e antigen positive chronic infection  
(rather than immunotolerant) and e antigen positive chronic hepatitis (rather than EPIA).
5.It is arguable that 3-6 months follow up is sufficient to characterise an e antigen negative patient  
into a chronic infection vs a chronic hepatitis phenotype. The authors should provide more  
justification for why they used such a short follow up period.
6.It is not clear how long the patients had been diagnosed with HBV and were under active follow  
up prior to enrolment in the study - this information would be helpful to the reader.
7.Incomplete data on 77 patients seems quite a large number - could the authors please explain  
why this came about?
8.The observation that the median level of HBV RNA in the EPIA group is lower than in the IT  
group is an intriguing one. The authors should speculate as to why this is.
9.In table 1, it is not clear what the p values relate to.
10.The  authors  claim  in  the  results  section  that  HBV  RNA is  better  than  HBV  DNA  at  
differentiating the two phases of e antigen negative disease and yet the predictive accuracy are  
exactly the same in table 3. This needs to be reconciled.
11.One major weakness of the study is that there were no repeated samples from the same patient  
over a period of time. In normal clinical practise patients are often not categorised into one of  
the 4 categories until they have had more than one reading. This limitation is highlighted by the  
authors but should be given more prominence.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Unable to assess
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you recommend an additional  
statistical review?
If  an  additional  statistical  review  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:
Needs some language corrections before being published
Declaration 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  
your 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:  Serum HBV RNA shows superiority in differentiating  ‘aggressive HBeAg-negative chronic  
hepatitis B’ in treatment-naïve chronic HBV-infected individuals
Version: 1 Date: 04 Feb 2019
Reviewer: Ahmed Mohamed Elsharkawy
Reviewer's report:
In this paper Liu et al aim to study levels of HBV RNA as a marker of cccDNA activity in patients with  
chronic HBV. Their premise is that this marker may act as a better differentiator of those patients with e  
antigen negative disease and a more aggressive biological course. The authors have used the modern  
nomenclature recommended in the 2017 EASL clinical practice guidelines for e antigen negative  
patients and are to be congratulated for this. The fact the HBV RNA levels are higher in e antigen  
positive patients compared to e antigen negative patients makes biological sense and is reassuring. The  
authors then show data to support the hypothesis that serum HBV RNA is better at differentiating  
chronic hepatitis in e antigen negative patients compared to other parameters. This is a good sized  
patient cohort with good numbers in the four chronic categories as defined. I think that the authors  
should address the following issues - 
1.There are a number of grammatical and spelling mistakes in the document that need to be  
corrected - e.g the word efficacy is misspelt in the first paragraph of the introduction.
2.The phrase 'liver invasion' is not appropriate in the first paragraph of the introduction and  
should be modified.
3. It is confusing that the authors use copies/ml for HBV DNA whilst they use IU/ml for qHBsAg.  
It would be better to use IU/ml for HBV DNA (as is used in AASLD and EASL guidelines)  
throughout the manuscript.
4.For the sake of consistency the authors should refer to e antigen positive chronic infection  
(rather than immunotolerant) and e antigen positive chronic hepatitis (rather than EPIA).
5.It is arguable that 3-6 months follow up is sufficient to characterise an e antigen negative patient  
into a chronic infection vs a chronic hepatitis phenotype. The authors should provide more  
justification for why they used such a short follow up period.
6.It is not clear how long the patients had been diagnosed with HBV and were under active follow  
up prior to enrolment in the study - this information would be helpful to the reader.
7.Incomplete data on 77 patients seems quite a large number - could the authors please explain  
why this came about?
8.The observation that the median level of HBV RNA in the EPIA group is lower than in the IT  
group is an intriguing one. The authors should speculate as to why this is.
9.In table 1, it is not clear what the p values relate to.
10.The  authors  claim  in  the  results  section  that  HBV  RNA is  better  than  HBV  DNA  at  
differentiating the two phases of e antigen negative disease and yet the predictive accuracy are  
exactly the same in table 3. This needs to be reconciled.
11.One major weakness of the study is that there were no repeated samples from the same patient  
over a period of time. In normal clinical practise patients are often not categorised into one of  
the 4 categories until they have had more than one reading. This limitation is highlighted by the  
authors but should be given more prominence.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Unable to assess
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you recommend an additional  
statistical review?
If  an  additional  statistical  review  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:
Needs some language corrections before being published
Declaration 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  
your 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:  Serum HBV RNA shows superiority in differentiating  ‘aggressive HBeAg-negative chronic  
hepatitis B’ in treatment-naïve chronic HBV-infected individuals
Version: 1 Date: 04 Feb 2019
Reviewer: Ahmed Mohamed Elsharkawy
Reviewer's report:
In this paper Liu et al aim to study levels of HBV RNA as a marker of cccDNA activity in patients with  
chronic HBV. Their premise is that this marker may act as a better differentiator of those patients with e  
antigen negative disease and a more aggressive biological course. The authors have used the modern  
nomenclature recommended in the 2017 EASL clinical practice guidelines for e antigen negative  
patients and are to be congratulated for this. The fact the HBV RNA levels are higher in e antigen  
positive patients compared to e antigen negative patients makes biological sense and is reassuring. The  
authors then show data to support the hypothesis that serum HBV RNA is better at differentiating  
chronic hepatitis in e antigen negative patients compared to other parameters. This is a good sized  
patient cohort with good numbers in the four chronic categories as defined. I think that the authors  
should address the following issues - 
1.There are a number of grammatical and spelling mistakes in the document that need to be  
corrected - e.g the word efficacy is misspelt in the first paragraph of the introduction.
2.The phrase 'liver invasion' is not appropriate in the first paragraph of the introduction and  
should be modified.
3. It is confusing that the authors use copies/ml for HBV DNA whilst they use IU/ml for qHBsAg.  
It would be better to use IU/ml for HBV DNA (as is used in AASLD and EASL guidelines)  
throughout the manuscript.
4.For the sake of consistency the authors should refer to e antigen positive chronic infection  
(rather than immunotolerant) and e antigen positive chronic hepatitis (rather than EPIA).
5.It is arguable that 3-6 months follow up is sufficient to characterise an e antigen negative patient  
into a chronic infection vs a chronic hepatitis phenotype. The authors should provide more  
justification for why they used such a short follow up period.
6.It is not clear how long the patients had been diagnosed with HBV and were under active follow  
up prior to enrolment in the study - this information would be helpful to the reader.
7.Incomplete data on 77 patients seems quite a large number - could the authors please explain  
why this came about?
8.The observation that the median level of HBV RNA in the EPIA group is lower than in the IT  
group is an intriguing one. The authors should speculate as to why this is.
9.In table 1, it is not clear what the p values relate to.
10.The  authors  claim  in  the  results  section  that  HBV  RNA is  better  than  HBV  DNA  at  
differentiating the two phases of e antigen negative disease and yet the predictive accuracy are  
exactly the same in table 3. This needs to be reconciled.
11.One major weakness of the study is that there were no repeated samples from the same patient  
over a period of time. In normal clinical practise patients are often not categorised into one of  
the 4 categories until they have had more than one reading. This limitation is highlighted by the  
authors but should be given more prominence.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Unable to assess
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you recommend an additional  
statistical review?
If  an  additional  statistical  review  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:
Needs some language corrections before being published
Declaration 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  
your 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