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: 03 Feb 2019
Reviewer: Amit Goel
Reviewer's report:
The subjected manuscript describes he results of a prospective observational study which included  
patients with chronic hepatitis B virus (CHB) infection. Study included a total of 291 CHB patients  
who were in various phases of natural history of CHB: he immune-tolerant phase (IT, n = 35), HBeAg-
positive immune-active phase (EPIA, n = 121), inactive chronic hepatitis B(ICH, n = 58) and HBeAg-
negative immune reactive hepatitis (ENH, n = 77). They have studied and compared the serum HBV  
RNA among CHB patients in various phases. I have following comments
*Title and aim: The primary aim of the study was to evaluate the levels of HBV RNA in the  
consecutive phases of HBV infection in patients not receiving antiviral therapy; though the title  
is not indicative of it.
*Methods>Patients> Para 2: Patients with end-stage liver diseases were excluded though the text  
as well as table 1 clearly indicated that patients with liver disease severity upto CTP-class C  
were included. These things are contradictory to each other and shall be clarified.
*Study has included patients with chronic hepatitis B as well as those with liver cirrhosis. In a  
patient with HBV related cirrhosis, the parameters like HBeAg status, serum level of HBV  
DNA and serum ALT level carries no weight because all of them, with detectable HBV DNA,  
need treatment. Hence, the application of HBV RNA will be limited in this group; moreover the  
natural history of those with cirrhosis is very different than those without cirrhosis. Hence the  
study shall exclude them and restrict their analysis to those without cirrhosis. 
*Table 2: is a duplication of data as it is provided in text form
*The strength of correlation, at places in text, are expressed as strongly though the correlation  
co-efficient was ~ 0.5 or 06 which does not express a strong correlation
*Authors attempted to describe HBV RNA cut-off at 4.73 log 10 copies/ml for predicting HBeAg  
status but this analysis has no clinical relevance and can be omitted
*HBV RNA level were used `to differentiate between HBeAg negative CHB patients in either  
inactive carrier stage or aggressive phase. I will suggest them to use an uniform terminology for  
describing the various phases of CHB. Use of term aggressive phase instead of HBeAg negative  
hepatitis (ENH) will create confusion among readers. I will suggest that they shall use, in the  
entire manuscript, the terminology given by ASSLD 2016 guideline.
*Table 3: In an attempt to between HBeAg negative CHB patients in either inactive carrier or  
aggressive phases, ROC was drawn and cut-off for HBV RNA was found at 2.11 log 10  
copies/ml. In ROC, the performance of HBV RNA was compared with those of HBV DNA,  
qHBsAg,  and  their  combinations;  significance  of  this  comparison  is  not  clear  because  
comparison shall be done with gold standard which will be, in this case, liver biopsy; further,  
why the cut-off used for HBV DNA (>3.39 copies/ml) or qHBsAg (>2.74 log IU/mL) were  
used are not explained
*Figures (Box plots and scatter diagram) can be restricted to those showing the most important  
findings of the study.
*Conclusion: states that 'Serum HBV RNA levels can predict the natural course in chronic HBV-
infected individuals' which is not justifiable because only one time blood specimens were  
analyzed. Authors may say that serum HBV RNA can predict the stage of  CHB natural history  
for a given patient but not the natural course in HBV infected person.
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.
Yes
Are the conclusions 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  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:
Not suitable for publication unless extensively edited
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: 03 Feb 2019
Reviewer: Amit Goel
Reviewer's report:
The subjected manuscript describes he results of a prospective observational study which included  
patients with chronic hepatitis B virus (CHB) infection. Study included a total of 291 CHB patients  
who were in various phases of natural history of CHB: he immune-tolerant phase (IT, n = 35), HBeAg-
positive immune-active phase (EPIA, n = 121), inactive chronic hepatitis B(ICH, n = 58) and HBeAg-
negative immune reactive hepatitis (ENH, n = 77). They have studied and compared the serum HBV  
RNA among CHB patients in various phases. I have following comments
*Title and aim: The primary aim of the study was to evaluate the levels of HBV RNA in the  
consecutive phases of HBV infection in patients not receiving antiviral therapy; though the title  
is not indicative of it.
*Methods>Patients> Para 2: Patients with end-stage liver diseases were excluded though the text  
as well as table 1 clearly indicated that patients with liver disease severity upto CTP-class C  
were included. These things are contradictory to each other and shall be clarified.
*Study has included patients with chronic hepatitis B as well as those with liver cirrhosis. In a  
patient with HBV related cirrhosis, the parameters like HBeAg status, serum level of HBV  
DNA and serum ALT level carries no weight because all of them, with detectable HBV DNA,  
need treatment. Hence, the application of HBV RNA will be limited in this group; moreover the  
natural history of those with cirrhosis is very different than those without cirrhosis. Hence the  
study shall exclude them and restrict their analysis to those without cirrhosis. 
*Table 2: is a duplication of data as it is provided in text form
*The strength of correlation, at places in text, are expressed as strongly though the correlation  
co-efficient was ~ 0.5 or 06 which does not express a strong correlation
*Authors attempted to describe HBV RNA cut-off at 4.73 log 10 copies/ml for predicting HBeAg  
status but this analysis has no clinical relevance and can be omitted
*HBV RNA level were used `to differentiate between HBeAg negative CHB patients in either  
inactive carrier stage or aggressive phase. I will suggest them to use an uniform terminology for  
describing the various phases of CHB. Use of term aggressive phase instead of HBeAg negative  
hepatitis (ENH) will create confusion among readers. I will suggest that they shall use, in the  
entire manuscript, the terminology given by ASSLD 2016 guideline.
*Table 3: In an attempt to between HBeAg negative CHB patients in either inactive carrier or  
aggressive phases, ROC was drawn and cut-off for HBV RNA was found at 2.11 log 10  
copies/ml. In ROC, the performance of HBV RNA was compared with those of HBV DNA,  
qHBsAg,  and  their  combinations;  significance  of  this  comparison  is  not  clear  because  
comparison shall be done with gold standard which will be, in this case, liver biopsy; further,  
why the cut-off used for HBV DNA (>3.39 copies/ml) or qHBsAg (>2.74 log IU/mL) were  
used are not explained
*Figures (Box plots and scatter diagram) can be restricted to those showing the most important  
findings of the study.
*Conclusion: states that 'Serum HBV RNA levels can predict the natural course in chronic HBV-
infected individuals' which is not justifiable because only one time blood specimens were  
analyzed. Authors may say that serum HBV RNA can predict the stage of  CHB natural history  
for a given patient but not the natural course in HBV infected person.
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.
Yes
Are the conclusions 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  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:
Not suitable for publication unless extensively edited
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: 03 Feb 2019
Reviewer: Amit Goel
Reviewer's report:
The subjected manuscript describes he results of a prospective observational study which included  
patients with chronic hepatitis B virus (CHB) infection. Study included a total of 291 CHB patients  
who were in various phases of natural history of CHB: he immune-tolerant phase (IT, n = 35), HBeAg-
positive immune-active phase (EPIA, n = 121), inactive chronic hepatitis B(ICH, n = 58) and HBeAg-
negative immune reactive hepatitis (ENH, n = 77). They have studied and compared the serum HBV  
RNA among CHB patients in various phases. I have following comments
*Title and aim: The primary aim of the study was to evaluate the levels of HBV RNA in the  
consecutive phases of HBV infection in patients not receiving antiviral therapy; though the title  
is not indicative of it.
*Methods>Patients> Para 2: Patients with end-stage liver diseases were excluded though the text  
as well as table 1 clearly indicated that patients with liver disease severity upto CTP-class C  
were included. These things are contradictory to each other and shall be clarified.
*Study has included patients with chronic hepatitis B as well as those with liver cirrhosis. In a  
patient with HBV related cirrhosis, the parameters like HBeAg status, serum level of HBV  
DNA and serum ALT level carries no weight because all of them, with detectable HBV DNA,  
need treatment. Hence, the application of HBV RNA will be limited in this group; moreover the  
natural history of those with cirrhosis is very different than those without cirrhosis. Hence the  
study shall exclude them and restrict their analysis to those without cirrhosis. 
*Table 2: is a duplication of data as it is provided in text form
*The strength of correlation, at places in text, are expressed as strongly though the correlation  
co-efficient was ~ 0.5 or 06 which does not express a strong correlation
*Authors attempted to describe HBV RNA cut-off at 4.73 log 10 copies/ml for predicting HBeAg  
status but this analysis has no clinical relevance and can be omitted
*HBV RNA level were used `to differentiate between HBeAg negative CHB patients in either  
inactive carrier stage or aggressive phase. I will suggest them to use an uniform terminology for  
describing the various phases of CHB. Use of term aggressive phase instead of HBeAg negative  
hepatitis (ENH) will create confusion among readers. I will suggest that they shall use, in the  
entire manuscript, the terminology given by ASSLD 2016 guideline.
*Table 3: In an attempt to between HBeAg negative CHB patients in either inactive carrier or  
aggressive phases, ROC was drawn and cut-off for HBV RNA was found at 2.11 log 10  
copies/ml. In ROC, the performance of HBV RNA was compared with those of HBV DNA,  
qHBsAg,  and  their  combinations;  significance  of  this  comparison  is  not  clear  because  
comparison shall be done with gold standard which will be, in this case, liver biopsy; further,  
why the cut-off used for HBV DNA (>3.39 copies/ml) or qHBsAg (>2.74 log IU/mL) were  
used are not explained
*Figures (Box plots and scatter diagram) can be restricted to those showing the most important  
findings of the study.
*Conclusion: states that 'Serum HBV RNA levels can predict the natural course in chronic HBV-
infected individuals' which is not justifiable because only one time blood specimens were  
analyzed. Authors may say that serum HBV RNA can predict the stage of  CHB natural history  
for a given patient but not the natural course in HBV infected person.
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.
Yes
Are the conclusions 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  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:
Not suitable for publication unless extensively edited
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