Molecular analysis of HBV pre-core gene mutations in patients co-infected with HIV at a tertiary care hospital in North India
This article has been Reviewed by the following groups
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Objective: Hepatitis B virus (HBV) spontaneous mutations may impact the severity of liver disease. This study aimed to assess the mutations in the PC region in HBV-HIV co-infected patients. Additionally, we explored its association with genotypes and examined the clinical implications. Methods: A total of 100 HBV-HIV co-infected patients and 50 HBV mono-infected patients were included in the study. We focused on the pre-core region of the HBV genome, sequencing it to identify PC mutant variants. PCR products were purified using the QIAquick Gel Extraction Kit, quantified via spectrophotometry, and sequenced using the Sanger method. The resulting sequences were assembled, annotated, and aligned in a single reading frame. Subsequent mutational and phylogenetic analyses were performed using UGENE software to determine the genotypes of the isolates. Results: The pre-core region was successfully amplified and sequenced in 27 samples, comprising 16 from HBV-HIV co-infected patients and 11 from HBV mono-infected patients. Phylogenetic analysis identified two HBV genotypes: genotype D, which was predominant and found in 24 samples (88.9%), and genotype A, present in 3 samples (11.1%). A T-to-C mutation at nucleotide position 1912 was detected in 48.1% of the patients. Furthermore, several additional pre-core mutations were observed, including A1850T, C1858T, G1899A, G1862T, G1951T, T1812C, and T1809G, along with novel mutations such as C1936T, A2011G, T2020A, and C2044T. Notably, the prevalence of these pre-core mutations did not significantly differ between the HBV mono-infected and HBV-HIV co-infected groups. Conclusion: This study underscored the prevalence of pre-core mutations in HBV-HIV co-infected patients. Although several of these mutations have been previously reported, our findings also revealed novel variants. Further research is needed to elucidate the clinical significance of these new mutations.
Article activity feed
-
Thank you for submitting your paper to Access Microbiology. It has now been reviewed and I would like you to revise the paper in line with the reviewers' reports and any Editorial Office requirements below. The reviewer reports can be found at the bottom of the email.
-
Comments to Author
I herein append my observations and suggestions: Line 41: Gene should always to italicised Line 70-71:This section is not clear. What did the author want to. Communicate to the reader in response to HBV antigen presentation in relation to PC region. Especially reference 14 was not correctly interpretated. Line 74-76: Not clear the relationship between ECORI (a restriction enzyme) and HCC and HBV. Let the author be very careful when making comparison so as not to lose meaning of thought Line 79-80: Not clear about the mechanisms that guide/inform this conclusion Line 87-88: .. imply that there were 200 patients and not 100 as presented. There are 100 HIV/HBV co-infection; HIV positive and HBV positive. So why 100 samples. Where is the control group? What of the pre-core gene for HIV positive but are …
Comments to Author
I herein append my observations and suggestions: Line 41: Gene should always to italicised Line 70-71:This section is not clear. What did the author want to. Communicate to the reader in response to HBV antigen presentation in relation to PC region. Especially reference 14 was not correctly interpretated. Line 74-76: Not clear the relationship between ECORI (a restriction enzyme) and HCC and HBV. Let the author be very careful when making comparison so as not to lose meaning of thought Line 79-80: Not clear about the mechanisms that guide/inform this conclusion Line 87-88: .. imply that there were 200 patients and not 100 as presented. There are 100 HIV/HBV co-infection; HIV positive and HBV positive. So why 100 samples. Where is the control group? What of the pre-core gene for HIV positive but are HBV negative. What of HBV positive but HIV negative. On the same note, did the author determine the HBV status of the participants before the start of the study (determine the presence of HBVsAg) Line 138: Include contry of origin of manufacturers of the kits. Give a brief description of the procedure on how the DNA was extracted from collected blood/serum; amplification conditions according to the kit information for replication. Line 140- 142:Provide the quantities of the PCR reagents used from the master mix. How much of the primer did the author use Line 145: Provide reference (literature) from where the PCR conditions were obtained … according to XX et al., (Year), the amount of current (Millivolts was not given) Line 146: How did the author arrive at 320bp before electrophoresis (speculation) Line 147: How did the author obtain the DNA from the gel to gel extraction kit? Line 150: No! the purified DNA must fist be amplified using a dye either SYBR Gold or SYBR Green or Florescent dye before sequencing. Line 157: Provide the reference/literature used as standard/control Line 186: what was the gender of the 45% severity in HIV/HBV Mild/ Moderate/ Severe, how did the author arrive at this categorization? Is there a baseline cut /limit to consider established by liver cancer organizations Line 192- 93: There is result for ALP but there is no method on how this was determined in the method section Line 195: ALT - what is the mean value as implied in the text? Line 196: What does the author mean by an upward trend was observed? (this was valued from what to what- range/rate?) and what are the baseline limits Line 192-201: What did the author want to convey in this paragraph? What inference is the author making out of these data? Part of these section could be taken to appendix to avoid confusion to the reader Line 209: .. the prevalence of HBV antigen increased progressively … from what to what? Line 217: Where is the data for the HIV positive and HIV negative in relation to respective mutations. What type of mutations were these? Was this mutation permanent or it was caused by medication hence not adaptive or mal adaptive to the genome, can it be inherited? Is this a form of gene plasticity? Is there a viral evolution being observed and by what mechanism? Figure 4 should be cross-linked and not be part of the data set in the text Discussion Line 8 from above …. Identify 30 mutation… this statement is not correct because no data on HBV negative was presented for comparison Paragraph 3 from the top is not correct since comparison made with a different section of the pre-core region B and not D. Remember this data is to be used for patient management hence need to be correct statistically. Paragraph on --- Ninety percent---.. (This part needs to be properly substantiated so that it is accepted scientifically as the new normal since it is different from what is known. Conclusion Based on the above observations, it is not easy to make conclusive conclusion on of the study. Thank You.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Not at all
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: I didn’t see how they were handled after they had signed the consent form to be involved in the study (were they treated and if yes what were the drug regime?)
-
Comments to Author
The manuscript addresses an important aspect of virology with potential relevance to clinical outcomes and/or basic virus-host interactions. The study appears to be well-conceived and methodologically sound in many aspects. However, a few areas could benefit from clarification, more rigorous interpretation, and enhanced contextualization within existing literature. The novelty of the work isn't coming through clearly—please spell out what gap this fills and how it builds on or goes beyond earlier studies. Some key experimental details are missing—please flesh out things like viral strain info, infection quantification, and how you ensured data quality. The stats section is pretty thin—please add a dedicated part explaining your test choices and how you handled things like multiple comparisons. Some …
Comments to Author
The manuscript addresses an important aspect of virology with potential relevance to clinical outcomes and/or basic virus-host interactions. The study appears to be well-conceived and methodologically sound in many aspects. However, a few areas could benefit from clarification, more rigorous interpretation, and enhanced contextualization within existing literature. The novelty of the work isn't coming through clearly—please spell out what gap this fills and how it builds on or goes beyond earlier studies. Some key experimental details are missing—please flesh out things like viral strain info, infection quantification, and how you ensured data quality. The stats section is pretty thin—please add a dedicated part explaining your test choices and how you handled things like multiple comparisons. Some conclusions feel a bit overconfident—please tone down causal language and acknowledge where the data might have limits. A few figures are hard to read or missing labels—please improve clarity and consider adding raw data to the supplement for transparency. Line 89-90: Consider rephrasing to improve clarity; the current sentence structure is awkward. Line 120: Abbreviation used here (e.g., MOI) should be defined at first use. Table 1: Ensure consistent formatting, particularly regarding units and decimal alignment. Discussion section: The connection to broader virological implications (e.g., implications for vaccine design or therapeutic targeting) could be better emphasized. Supplementary data: If used, ensure it is referenced properly in the main text.If there's A simple schematic of your workflow would really help readers follow the design. Please add more on how these results might matter clinically, especially if you used human samples or animal models. The limitations section could use more detail—think sample size, strain generalizability, or any possible batch effects.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
The authors have satisfied my concerns, the authors should provide a figure legend or proper color guide for the various color sections in the phylogenetic tree in Figure 3
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner …
Comments to Author
The authors have satisfied my concerns, the authors should provide a figure legend or proper color guide for the various color sections in the phylogenetic tree in Figure 3
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
-
Thank you for submitting your manuscript for publication in Access Microbiology. It has been examined by expert reviewers who have concluded that the work is of potential interest to the readership of Access Microbiology. However, based on the comments received, it is clear that a major revision of this manuscript will be required before a decision can be made on its publication. I will be pleased to consider a revised manuscript along with a document including a point by point response to each of the reviewers comments. Your revised manuscript may be returned to one or more of the original reviewers, along with your itemised response to the reviewers’ comments.
-
Comments to Author
MAJOR REVISIONS Abstract: The abstract seems very vague and confusing, particularly the results section. The methods section needs to be properly re-written to summarize what methods were used to generate results. Authors should delete names of kits or reagents used for analysis in the abstract. The authors also do not need to mention primers used. The results section should be improved upon firstly the authors should organize the result summary in a meaningful order e.g, prevalent of positive samples, followed by distribution of genotypes, results of phylogeny and finally mutation results. The conclusion of the abstract should also just reflect the contribution and recommendation of the study, not discussing the results further. Methods: The Flow chart in Figure 1 needs to be improved upon in visual …
Comments to Author
MAJOR REVISIONS Abstract: The abstract seems very vague and confusing, particularly the results section. The methods section needs to be properly re-written to summarize what methods were used to generate results. Authors should delete names of kits or reagents used for analysis in the abstract. The authors also do not need to mention primers used. The results section should be improved upon firstly the authors should organize the result summary in a meaningful order e.g, prevalent of positive samples, followed by distribution of genotypes, results of phylogeny and finally mutation results. The conclusion of the abstract should also just reflect the contribution and recommendation of the study, not discussing the results further. Methods: The Flow chart in Figure 1 needs to be improved upon in visual quality and clarity a similar flow chart can be seen in Faneye et al, 2024, Access Microbiol. 6. 0000821. Authors are advised to either delete the first paragraph of section2.4, page 6, lines 153-155, it does not describe any known bioinformatic process or recast to reflect the actual method used including software utilized for alignment and mutational analysis. The accession numbers section should be moved to the data summary section in line with the journal requirement. The phylogenetic analysis description in Page 7, line 165-66, should be moved upward to the alignment section. Also, more details to the type pf phylogenetic analysis done, software that was used, including the models utilized and tree rooting selection should be included in the revised phylogenetic analysis description. The sub-heading of the Alignment sub-section should also be changed into Sequence alignment and Phylogenetic analysis Results: The authors have reported results of the PCR as well as phylogenetic analysis, however there are some major revisions that still need to be done to the result section. Firstly, in the mutation analysis, only the nucleotide sequence was analyzed, the amino acid sequence mutations which have actual phenotypic effect (Sobajo et al, 2023, Viruses. 15, 2188; Faneye et al, 2024. Access Microbiol. 6. 000821.). The data in Table 1 should be subjected to statistical testing to determine significance and the results discussed. The phylogenetic tree in Figure 3 seems confusing, authors are advised to use a better graphical display of the tree such as Figtree or IToL to properly display the tree topology with appropriate bootstrap values and highlight genotypes and sub-genotypes. Discussion; The authors should include discussion on the synonymous amino acid mutations and their possible effect on the clinical course of HBV infection or their effect on HBV transmissibility or pathogenesis. MINOR REVISIONS Figure 2 can be moved to the supplementary materials section. The authors should include an alignment file for the mutation analysis done for the study sequences as supplementary material. Figure 4, the authors should please highlight the reference sequence used to compare study sequences in the figure. Authors are advised if table 4 can be converted to a visual chart such as heatmap, it seems confusing in its current form, also the number of isolates analyzed in genotype A for each mutation is too small to justify any statistical testing, so authors are advised to remove P values from the table.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
The manuscript titled "Molecular analysis of HBV pre-core gene mutations in patients co-infected 2 with HIV at a tertiary care hospital in North India" is an important study mainly focussing on Mutations in the HBV gene in context of HBV. In the present study, the authors aim to analyze the mutations in the PC region of HBV in patients co-infected with HIV (100 patients) and those mono-infected with HBV (50 patients) in North India. The authors amplified the HBV PC region of 27 patients and identified multiple mutations, particularly the A1912C mutation, present in 48.1% (13/27) of patients. Additionally, 3/27 (11.1%) of the HBV positive samples were identified as genotype A, while 88.9% (24/27) were genotype D, with no significant difference in the incidence of PC mutations. The manuscript is within …
Comments to Author
The manuscript titled "Molecular analysis of HBV pre-core gene mutations in patients co-infected 2 with HIV at a tertiary care hospital in North India" is an important study mainly focussing on Mutations in the HBV gene in context of HBV. In the present study, the authors aim to analyze the mutations in the PC region of HBV in patients co-infected with HIV (100 patients) and those mono-infected with HBV (50 patients) in North India. The authors amplified the HBV PC region of 27 patients and identified multiple mutations, particularly the A1912C mutation, present in 48.1% (13/27) of patients. Additionally, 3/27 (11.1%) of the HBV positive samples were identified as genotype A, while 88.9% (24/27) were genotype D, with no significant difference in the incidence of PC mutations. The manuscript is within the journal's scope. It's well-written and includes a current overview of the literature. The abstract is written in a clear and concise manner. The materials and processes have been meticulously presented. The findings have been thoroughly explored, and suitable figures and tables have been included including the phylogenetic analysis and mutation detection. I have the following comments for the authors to consider. 1. The authors should standardize and define the abbreviations throughout the manuscript. For instance, the abbreviation for Hepatitis B virus appears multiple times without proper notation. 2. Figure 3: The phylogenetic tree is apparently incorrect. If you investigate the tree, HBV genotype E is positioned in the middle of genotype D sequence clades. Moreover, HSP 17 did not clustered with genotype A but with other genotypes. Lastly, please remove the "0" on the tree for improved readability.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-