Dear Dr. Zheng,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: No

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The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The manuscript entitled “Risk of Hepatitis B Virus Reactivation in the Treatment of HBsAg and HBV DNA Double-negative Lymphoma Patients” has been done for risk evaluation of HBV reactivation among patients who tested negative for both HBsAg and HBV DNA. The type of study and idea sounds interesting. The methodology used was standard for this type of study. Notably, HBV reactivation was detected in 3 patients who were negative for HBsAg, HBcAb and HBV DNA.

Reviewer #2: This was a well-designed study that analyzed the risk of HBV reactivation among HBsAg and HBV DNA double-negative lymphoma patients. The findings of this study contribute further evidence for the management, prevention, and control of HBV reactivation among lymphoma patients before treatment. While the study has merit, I note several omissions in the reporting of the study. The following comments need addressing:

1) A brief demographic information of participants can be added to the abstract.

2) Discussion section is well-written but should be more expanded. The author should better discuss the results from virological aspects by comparing with other similar studies.

3) HBV treatment options in China should be mentioned in the Discussion section.

4) The authors better mention some limitation of the study if there is any.

Reviewer #3: This study investigates the risk of HBV reactivation in lymphoma patients who are negative for both HBsAg and HBV DNA before undergoing treatment. The authors examine the influence of HBcAb status and age on the risk of reactivation, providing new insights into monitoring strategies for this seemingly low-risk population. The study is significant in identifying that reactivation can occur even in HBcAb-negative patients, thereby challenging existing assumptions. However, the manuscript is limited by its single-center, retrospective design, the lack of mechanistic understanding of HBV reactivation in HBcAb-negative patients, the absence of multivariate analysis, undefined monitoring intervals, and insufficient clinical recommendations. These limitations collectively affect the generalizability, analytical rigor, and practical applicability of the study. There are several comments regarding this article:

Major comments:

1. Introduction: The background focuses mostly on well-known facts (HBV reactivation risks in HBsAg+ and HBcAb+ patients), but does not thoroughly justify why studying HBsAg-/HBV DNA- patients is necessary. It mentions previous studies (refs [12–15]) showing reactivation in such patients yet fails to differentiate how this study adds novel value or addresses a specific gap.

2. Study population: The manuscript does not provide a thorough account of patient inclusion and exclusion criteria. For instance, it remains unclear whether individuals with prior HBV antiviral therapy were included, or if comorbidities and immunosuppressive treatments were considered. These omissions hinder reproducibility and raise potential concerns of bias. The authors should clearly delineate and quantify all inclusion and exclusion criteria, and ensure this information is appropriately reflected in Figure 1.

3. Detection methods for HBsAg, HBcAb and HBV DNA: Although the section notes the assessment of HBsAg, HBcAb, and HBV DNA, it fails to specify the frequency or timing of these tests during follow-up. This omission compromises the ability to accurately interpret the reported HBV reactivation rates and undermines the study’s methodological transparency.

4. The study’s analysis is restricted to only four variables—anti-HBc status, age, sex, and treatment duration—while omitting several well-established predictors of HBV reactivation in lymphoma patients, such as chemotherapy regimen and intensity (including rituximab and corticosteroid use), baseline liver function, and anti-HBs titers. This limited analytical scope compromises the comprehensiveness of risk assessment, increases the risk of omitted variable bias, and diminishes the clinical applicability and interpretive validity of the findings.

5. Statistical Analysis: The manuscript presents only univariate analyses, including chi-square and Fisher’s exact tests, without implementing multivariate approaches such as Cox regression to adjust for potential confounders. This methodological limitation hampers the ability to identify independent associations and undermines the validity and robustness of the findings, particularly in relation to critical variables such as age, sex, and treatment modalities.

6. Statistical Analysis: The treatment duration data are stratified into numerous narrow time intervals, some of which contain very small sample sizes (e.g., groups with only 3–5 patients). Such fragmentation undermines statistical power and renders intergroup comparisons unreliable and potentially misleading. To more appropriately assess the impact of treatment duration, the authors should consider applying time-dependent Cox regression, which accommodates continuous or time-varying effects and enhances analytical robustness.

7. The study exclusively reports serological HBV reactivation—defined by HBV DNA or HBsAg positivity—while omitting data on hepatitis flares, a clinically significant manifestation typically marked by elevated liver enzymes such as ALT. This limitation restricts the clinical interpretability of the findings, as serological reactivation does not always correlate with clinically meaningful hepatic events. Without information on liver function or flare severity, the study provides an incomplete assessment of HBV reactivation’s clinical impact, thereby reducing its practical utility for patient management.

8. The discussion section inadequately addresses several critical limitations of the study. Notably absent are acknowledgments of the retrospective, single-center design, the lack of multivariate analysis, and the omission of important clinical variables such as treatment type and hepatitis flare data. Failure to fully disclose these weaknesses undermines the study’s credibility, transparency, and scholarly rigor.

9. The discussion restates key findings without offering mechanistic insights into why reactivation is higher in HBcAb-positive and older individuals. This limits the depth and scientific value of the interpretation.

10.

Minor:

1. The statement, “HBsAg COI > 1.000 is considered positive. Conversely, an HBcAb COI < 1.000 is considered positive,” introduces ambiguity due to the misleading use of “conversely.” It is essential to clearly state that the cutoff values for positivity differ between antigens, with opposing threshold directions. Such clarification is necessary to ensure diagnostic clarity and preserve confidence in the study's validity.

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Reviewer #1: Yes:  Fatemeh Farshadpour

Reviewer #2: Yes:  Reza Taherkhani

Reviewer #3: No

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Dear Dr. Zheng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Aug 30 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Ashraf Elbahrawy

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: No

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #3: No

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: 1) In Table 1, please add the odds ratio.

2) Is it possible that the positive cases of infection observed during the treatment period could be the result of reinfection during hospital procedures or other factors rather than reactivation? If so, this concern can be mentioned in the manuscript.

Reviewer #3: The authors have made substantial improvements to the manuscript, particularly in expanding the introduction, clarifying laboratory methods, and elaborating on the virologic mechanisms underlying HBV reactivation in seronegative patients. The study contributes novel data to an underexplored patient group, and several of my prior concerns have been thoughtfully addressed. However, several critical limitations remain unresolved, particularly in the areas of analytical rigor, clinical data interpretation, and transparency. Below is a detailed evaluation of the responses to my original comments.

Major comments:

1. Introduction: The revised introduction better explains the rationale for studying HBsAg and HBV DNA double-negative patients. This population overlaps in part with previously studied HBsAg-/HBcAb+ individuals but also uniquely includes those who are HBsAg-/HBcAb-. The discussion of cccDNA persistence and latent HBV infection offers a plausible explanation for reactivation risk in HBcAb+ patients and supports the need for closer monitoring in this subgroup. However, the biological basis for HBV reactivation in HBsAg-/HBcAb- patients remains insufficiently addressed. The manuscript presents evidence of reactivation in a few HBcAb- individuals, but does not convincingly explain how latent infection could persist in the complete absence of both surface antigen and core antibody. Given that HBcAb is considered a marker of prior exposure and immune memory, its absence would typically suggest no past infection—and by extension, no residual cccDNA. The authors should clarify: (A) What mechanisms (e.g., seronegative occult HBV infection, transient HBcAb responses, immune escape variants) could explain cccDNA persistence in HBsAg-/HBcAb- patients? (B) Whether there is any literature support for HBV reactivation in truly seronegative individuals. (C) If the three HBcAb- cases in their cohort might reflect false-negative HBcAb results, delayed seroconversion, or low-level prior exposure not captured by standard serology.

Without further mechanistic explanation or discussion, the recommendation for routine monitoring of HBsAg-/HBcAb- patients may appear speculative. The authors are encouraged to address these biological uncertainties in the discussion section to support their conclusions.

2. Study population: While the authors have clarified basic inclusion criteria, important variables such as prior antiviral treatment, comorbidities, and immunosuppressive regimen details (e.g., rituximab or corticosteroid use) remain unreported. This omission limits reproducibility and raises concerns about potential selection bias. Furthermore, Figure 1 does not provide quantitative information about how many patients were excluded at each step based on specific criteria (e.g., HBsAg or HBV DNA positivity, lack of follow-up data, etc.). To enhance transparency and methodological rigor, the authors should revise Figure 1 to include the exact numbers excluded for each criterion.

3. The frequency and methodology for HBV marker testing are now more clearly described. The authors state that during each scheduled hospital visit, patients underwent simultaneous assessment of HBsAg, HBcAb, and HBV DNA. However, the manuscript still does not specify the actual frequency of these hospital visits—for example, whether testing was done every 1–2 weeks, monthly, or otherwise—and whether this schedule was consistent across all patients or individualized based on treatment plans. In addition, the authors do not describe how missing data were handled, such as whether all patients completed regular testing as planned or if certain timepoints were skipped or lost to follow-up. This information is crucial for assessing the reliability of the HBV reactivation detection timeline and the comparability of follow-up across subgroups.

4. The study omits several well-established risk factors for HBV reactivation, including chemotherapy regimen, corticosteroid use, baseline liver function, and anti-HBs titers. While the authors acknowledge data limitations, no descriptive statistics or stratified analyses were provided to mitigate this gap.

5. The authors limited their analysis to univariate statistics (chi-square and Fisher’s exact tests), citing incomplete data and retrospective design as the rationale. However, multivariate modeling is standard practice even in retrospective observational studies, particularly when identifying risk factors. Without such analysis, it is not possible to determine whether HBcAb positivity or advanced age are independent predictors of HBV reactivation, as potential confounders (e.g., treatment type, comorbidities) are not accounted for. The authors should either attempt a multivariate model using available covariates or explicitly acknowledge this as a major methodological limitation that affects the interpretability of their conclusions.

6. The study reports only serologic reactivation without ALT or clinical hepatitis data. For a retrospective study, liver function data (such as ALT) should be obtainable from the medical record system. Therefore, the authors should either include this information or clearly explain why it could not be retrieved.

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Reviewer #2: Yes:  Reza Taherkhani

Reviewer #3: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Risk of Hepatitis B Virus Reactivation in the Treatment of HBsAg and HBV DNA Double-negative Lymphoma Patients

PONE-D-25-21930R2

Dear Dr. Zheng,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Ashraf Elbahrawy

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewer #2:

Reviewer #3:

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Dear authors

The manuscript has been revised according to the comments. Thank you very much and good luck.

Reviewer #3: Thank you for the opportunity to provide a review of this well-designed study. All comments have been addressed and been responded well. The result is statistically confirmed from the clinical data in this paper. I suggest the acceptance of this manuscript for publication in the PLOS ONE.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #2: Yes:  Reza Taherkhani

Reviewer #3: No

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