Molecular analysis of HBV pre-core gene mutations in patients co-infected with HIV at a tertiary care hospital in North India
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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.
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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.
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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
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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
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