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All issues pointed by the reviewers were addressed and the revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Gwyn Gould, a PeerJ Section Editor covering this Section #]
no comment
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Please address the concerns of both reviewers and amend the manuscript accordingly.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The manuscript is generally well written and professionally presented. The introduction provides sufficient background on the clinical significance of HBV RNA and outlines the rationale for investigating its predictive value in chronic hepatitis B (CHB) patients undergoing NUC therapy.
However, the following improvements are recommended:
1. Figure 1A: Please consider adding asterisks to indicate statistically significant differences between the SR and NSR groups at relevant time points. This will enhance visual interpretation and highlight meaningful comparisons.
2. Figure 3: The AUROC values are mentioned in the text but not shown in the figure panels. I suggest adding the AUROC values directly on each graph (baseline, week 12, and week 24) for clarity and to aid the reader in understanding the differences in predictive performance over time.
3. HBeAg detection method: Although HBeAg is a primary endpoint used for group stratification and regression analysis, the methodology for measuring HBeAg is not described. Please specify the assay type (e.g., ELISA or CLIA), the manufacturer, and whether the assay was qualitative or quantitative.
Several aspects of the methodology require clarification:
1. The assays used for HBV RNA and HBV DNA quantification appear to differ from the commonly used standardized methods. It is unclear whether these methods were validated in-house or externally for analytical performance (e.g., sensitivity, specificity, reproducibility, and limit of detection). The authors should clarify this and cite relevant validation studies or manufacturer data, particularly since the study’s conclusions depend on these measurements.
2. The assignment of patients to ETV, TDF, or TAF treatment groups is not described. Was treatment allocation randomized, physician-determined, or based on patient preference or clinical characteristics? This has implications for potential selection bias, especially since the TAF group showed a significantly higher HBeAg seroconversion rate despite having the smallest sample size (n=28).
The core findings are well presented, and the use of multivariate regression and ROC curve analysis supports the conclusion that HBV RNA at week 12 may serve as a valuable early predictor of HBeAg seroconversion.
1. In Table 1, while the distribution of patients receiving different antiviral treatments (ETV, TDF, TAF) between the SR and NSR groups is clearly shown, no statistical test is provided to assess whether this distribution is significantly different. A chi-square test or similar analysis should be added to confirm whether treatment types were evenly distributed or if a significant imbalance exists that could affect outcomes.
2. As noted above, AUROC values should be added to Figure 3 for easier visualization, and significance markers should be added to Figure 1A where appropriate.
This is a clinically relevant and well-designed study that investigates the predictive value of HBV RNA for HBeAg seroconversion in CHB patients undergoing NUC therapy. The identification of week-12 HBV RNA level as an early predictor is novel and of potential clinical utility.
Addressing the above methodological and statistical issues would significantly enhance the clarity, reproducibility, and interpretability of the study findings.
Title and Abstract:
Title
• The title captures the core content of the manuscript and includes keywords: chronic hepatitis B, HBV RNA, HBeAg, and antiviral therapy. So, it is highly informative.
Abstract:
• The abstract is comprehensive and well-organized, clearly defining the background, objective, methods, results, and conclusions.
• It effectively highlights the clinical significance of the HBV RNA as a predictor for HBeAg seroconversion in chronic hepatitis B patients receiving antiviral therapy.
• The abstract helps the reader to understand the study’s objective and main findings. To make it more remarkable, the author should indicate the time point with a significant finding in the first sentence of the results section.
• Please state the specific time point (12th week) in the opening sentence of the results section to highlight the most novel finding (Page 6, Line 34).
Introduction:
The information provided in the background is adequate for understanding the research.
• The author outlines the worldwide burden of chronic hepatitis B (CHB) and the significance of antiviral treatment (Page 7, Lines 52-70).
• The author explains the limitations of existing markers, such as HBV DNA, explaining that their undetectable levels do not indicate the presence of covalently closed circular DNA (cccDNA) activity in hepatocytes. This supports the study of HBV RNA that provides more direct evidence for viral replication status and the efficacy of treatment in CHB patients (Pages 7 and 8, Lines 71-84).
• The literature used is appropriately referenced and relevant to the topic.
• The introduction provides a solid foundation by highlighting the relevance of studying serum HBV RNA as a predictor for virological response to antiviral therapy and seroconversion of HBsAg or HBeAg in HBV patients. However, it can be strengthened by providing a detailed overview of the current knowledge regarding HBV RNA as a predictor, before mentioning that the “clinical value of HBV RNA for virological responses and drug discontinuance is still unknown” (Page 8, Lines 86-87). The author should briefly outline the findings of previous studies (e.g., Mak et al., 2023, Luo et al.), which mentioned the pgRNA to specifically identify the gap that this research paper aims to fill.
Figures & Tables
• The figures (1, 2, 3, 4) and tables (1 and 2) are clear and legible, presenting the study’s data. They accurately represent the findings and appear to be free from unnecessary modifications.
• The author uses line charts, boxplots, and scatter plots to visualize the data, which is appropriate for visualizing the data trends and correlations.
Material and Methods
• The materials and methods section provides sufficient information to understand the study design, clinical data collection, and laboratory assessment.
• It clearly states the patient enrollment criteria, drug regimens, exclusion criteria, and the statistical methods appropriate for the study’s objectives, design, and data types.
• Please improve the method section by providing specific experimental details that would enable the replication of the experiments conducted.
o For HBV real-time fluorescent quantitative PCR, please indicate the amount of DNA sample and the DNA sequence of the primers (page 9, Lines 128-129).
o For HBV ribonucleic acid quantitative PCR, please indicate the amount of RNA sample and the sequence of the gene-specific primers and probes (page 9, Lines 130-137).
• The study duration is from January 2023 to December 2023; however, the follow-up periods were described as up to 60 weeks and up to 120 weeks. Please clarify the timeline of the study and explain that patient enrollment was staggered over the year, allowing for some to reach 60 weeks of follow-up within the study timeframe (Page 8, Lines 112-114).
• The author acknowledged that the data after 60 weeks were not included due to the high rate of follow-up loss (Page 8, Line 115), which is a significant limitation of the study. Please provide details on the number of patients lost at each follow-up point and discuss how this loss impacts the results, especially the non-significant differences in HBV RNA after 36 weeks. Consider the limitation regarding follow-up duration on the overall interpretation.
• The author mentions that patients receiving TAF had a higher SR rate than others. However, this study does not appear to be a randomized controlled trial. Please acknowledge this limitation. I suggest performing a head-to-head comparison of these antiviral drugs to strengthen the manuscript.
Results
• The study presents novel findings regarding the clinical value of HBV RNA, especially as an early predictor of HbeAg seroconversion at the 12-week time point.
• The finding that patients treated with the antiviral drug tenofovir alafenamide (TAF) showed a higher HBeAg seroconversion rate compared to entecavir (ETV) and disoproxil fumarate (TDF) is also significant.
• The presented data (including HBeAg seroconversion rates, dynamic changes of HBV RNA and DNA, correlations between markers, and ROC curve analyses) appear plausible and credible.
• The study provides robust evidence supporting the use of HBV RNA as an early predictor that could refine the antiviral treatment monitoring for CHB patients, advancing clinical practice in hepatology.
• The results showed statistically significant differences in the baseline HBeAg, ALT, and GGT levels between the SR and NSR groups (Page 10, Lines 177-182). This significant finding would suggest that these variables can serve as baseline predictors. Although the authors stated these findings, they did not explore this further in the multivariate analysis presented in Table 2 (Page 20). Please include these significant baseline characteristics in the multivariate model to assess their independent predictive value in addition to HBV RNA.
• The results state that “The HBV RNA levels in the RS and NSR group showed a similar trend of change (Figure 1B)”. This seems to contradict the following sentence, which notes a statistical difference at several time points (Page 10, Lines 190- 195). For clarification, the terminology should be changed. The author should note that although the trend (reduction over time) is similar between the two groups, the magnitude of the decrease and the absolute values are significantly different.
Discussion
• The discussion highlights that HBV RNA level declines more slowly than HBV DNA, supporting the idea that HBV DNA negativity alone is insufficient to indicate the actual viral replication status.
• The importance of HBV RNA in CHB patients as an early predictor for HBeAg seroconversion is discussed well, aligning with the study's goals.
• The author discusses the positive correlation between the baseline level of HBV RNA in chronic hepatitis B patients and HBV DNA and HBsAg levels. This correlation was reduced following TAF treatment for the HBeAg SR group compared to the other antiviral drugs used.
• The author highlights the importance of HBV RNA negativity for drug discontinuation but cites a prospective discontinuation study (Page 12, Line 251-260). Since the current study did not investigate drug discontinuation, this is an area of extrapolation. Please revise this section to position this as a need for future research proposed by the current study, rather than a direct conclusion drawn from their findings.
• The author attributes the declining correlation between HBV RNA, HBV DNA, and HBsAg after antiviral treatment to the active status of cccDNA, which is a reasonable hypothesis. It would be better for the author to state this as a hypothesis and suggest further research to evaluate this theory (Page 13, Lines 290-299).
• The author acknowledges that this study is a prospective research study with a limitation of a short follow-up period; therefore, its results require long-term validation studies with large sample sizes.
Overall, the discussion is practical in the interpretation of the study findings, and it highlights the clinical implications of using HBV RNA as an optimal marker for CHB management and treatment.
Conclusions
• The conclusions align well with the findings of the study and indicate that:
1. HBV RNA level correlates positively with HBV DNA and HBsAg.
2. HBV RNA (at 12 weeks) is an early predictor for HBeAg.
3. TAF shows a higher HBeAg seroconversion rate compared to other treatments used.
The conclusions are strong and directly supported by the presented data.
The author stated that TAF had a higher HBeAg SR rate compared to other antiviral drugs tested. However, the author needs to clarify that this result was based on an observational finding in a non-randomized cohort. The author should emphasize the need for a prospective study to validate these findings.
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