Dear Dr. Fang,

The manuscript has been evaluated by two reviewers, and their comments are available below. The reviewers note that a strength of the manuscript is the large sample size; however, they each raise concerns regarding the statistical analyses that must be fully addressed as a prerequisite to further consideration. In addition, the identity of the health examination database must be described in more detail.

Could you please carefully revise the manuscript to address all comments raised?

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PLOS One

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“This work was supported in part by the 2022 National Key Research and Development Program of China, Key Special Project on “Active Health and Science and Technology Response to Population Aging” (No. 2022YFC3600903), entitled “Research on the Construction and Output Technology of Active Health Knowledge System.” The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. “

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Additional Editor Comments (if provided):

1) As you revise your manuscript to address the reviewers' comments, please include in the Methods a description of the health examination database used in this research. If, for example, the database is composed of patient records from Shanghai Health and Medical Center (or another hospital or clinic), please state this in the Methods. If the study used third-party data (for example, the National Health and Nutrition Examination Survey (NHANES)) this also needs to be clearly indicated in the Methods.

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Thank you for the opportunity to review this manuscript. This is a well-conducted study with a large sample size and appropriate statistical analyses. The manuscript is generally well written, and the conclusions are supported by the results. I have a few suggestions that may improve the manuscript.

1- Please expand the discussion on residual confounding, especially because information on smoking, alcohol use, diet, physical activity, and medications was not available.

2- The association found after adjustment is statistically significant but very small. Please emphasize that the clinical significance of this finding may be limited.

3- Please provide a reference for the definition of hyperuricemia used in the study.

4- Please check the manuscript for small inconsistencies in percentages and table formatting.

5- Please ensure that the manuscript includes a clear Data Availability Statement in accordance with PLOS ONE requirements.

Overall, I believe the manuscript is scientifically sound and would be suitable for publication after these minor revisions.

Reviewer #2: Overall assessment

This manuscript evaluates the cross-sectional association between Helicobacter pylori infection, hyperuricemia, and serum uric acid levels in a large health examination population. The large sample size, use of the ^13C-urea breath test, sequential regression models, sex-stratified analyses, and sensitivity analyses are important strengths.

The main finding is that the crude association between H. pylori positivity and hyperuricemia becomes non-significant after adjustment for metabolic, renal, hepatic, and inflammation-related variables. A small association with continuous serum uric acid remains, but its magnitude is unlikely to be clinically relevant.

The study is potentially valuable, but several methodological and interpretative issues should be addressed. I recommend major revision.

Major comments

1. Interpretation of sequential adjustment

The authors frequently state that metabolic, renal, hepatic, and inflammatory factors “explain” the association. However, attenuation after adjustment in a cross-sectional analysis does not establish mediation or causality. Some adjusted variables may be confounders, whereas others could potentially lie on the pathway between infection and uric acid metabolism.

The authors should clarify the purpose of each model and replace causal wording such as “explained by” with more cautious language, for example:

“The association was substantially attenuated after adjustment for measured metabolic, renal, hepatic, and inflammation-related variables.”

The proposed “shared metabolic-inflammatory phenotype” should be presented as a possible interpretation rather than a demonstrated mechanism.

2. Participant selection and missing data

The cohort decreased from 97,379 unique participants to 72,392 participants with valid H. pylori and uric acid data and then to 67,827 participants in the regression analyses.

The authors should provide a participant flow diagram and clarify:

whether the ^13C-urea breath test was routine or optional;

why such a large number of participants lacked relevant measurements;

whether included and excluded participants differed;

and whether selection for H. pylori testing could have introduced bias.

The statement that missingness was below 1% for core covariates appears inconsistent with the exclusion of 4,565 participants after the baseline analysis. A table reporting missing data for each variable is required. Sequential models should also be repeated in the same complete-case population to ensure that attenuation is not partly caused by changing sample sizes.

3. Description of the ^13C-urea breath test

The test protocol is insufficiently described. The Methods should include:

manufacturer and analytical system;

administered ^13C-urea dose;

timing of breath samples;

positivity threshold;

fasting requirements;

quality-control procedures;

and required withdrawal periods for proton pump inhibitors, antibiotics, and bismuth compounds.

The potential influence of recent treatment on false-negative results should be discussed.

4. Laboratory methods

The manuscript should provide details regarding the measurement of serum uric acid, creatinine, glucose, lipids, liver enzymes, WBC, and hs-CRP, including analytical methods, equipment, reagents, fasting conditions, and quality control.

The sex-specific hyperuricemia thresholds of >420 μmol/L in men and >360 μmol/L in women should be supported by an appropriate reference.

5. Covariate selection

The rationale for including some variables and excluding others is unclear. For example, the model includes BMI but not waist circumference, systolic but not diastolic blood pressure, fasting glucose but not HbA1c, and ALT and GGT but not AST.

The authors should explain whether covariates were selected based on prior evidence, a causal framework, data availability, or statistical criteria. They should also clarify whether the analysis plan was established before inspecting the results.

6. Statistical analysis

Because this is a cross-sectional study, the manuscript should refer to prevalence or prevalence odds rather than “risk.”

Since hyperuricemia prevalence is relatively high, adjusted prevalence ratios obtained using Poisson regression with robust variance may be easier to interpret than odds ratios.

The authors should also:

evaluate non-linear relationships of continuous covariates;

assess residuals and heteroscedasticity in linear regression;

consider transformations for skewed variables;

and report the exact software packages and functions used.

The statement that regression estimates were generated using “matrix-based” routines is not sufficiently reproducible. The analyses should preferably be validated using established statistical software, and the code should be made available.

7. Clinical relevance and hs-CRP analysis

The fully adjusted increase in serum uric acid was only 2.14 μmol/L, indicating very limited clinical relevance. This should remain central to the interpretation.

Moreover, after additional adjustment for hs-CRP, the association with continuous serum uric acid was no longer statistically significant. This result should be discussed more clearly. It may reflect additional inflammatory adjustment, reduced statistical power, selection bias in the hs-CRP subset, or a combination of these factors.

8. Conclusions

The conclusion should be more cautious. The study supports substantial attenuation after adjustment but does not establish that H. pylori and hyperuricemia share a specific biological phenotype.

A more appropriate conclusion would be:

In this cross-sectional health examination population, the crude association between H. pylori positivity and hyperuricemia was substantially attenuated after adjustment for measured metabolic, renal, hepatic, and inflammation-related variables. A small adjusted difference in continuous serum uric acid remained, but its clinical relevance was limited. Longitudinal and eradication-based studies are required to clarify temporality and potential mechanisms.

Minor comments

Replace “risk” with “prevalence,” “prevalence odds,” or “cross-sectional association.”

Use eGFR, not EGFR, consistently.

Add a participant flow diagram.

Report standardized differences in Table 1, as very small differences may produce P<0.001 in such a large cohort.

Clarify the sample size and denominator for each analysis.

Report the number and percentage of participants with available hs-CRP data.

Clarify whether participants using urate-lowering medication, diuretics, antibiotics, proton pump inhibitors, or bismuth were included.

Shorten the Discussion, particularly the repetitive interpretation of model attenuation.

Expand the literature concerning previous studies of H. pylori, hyperuricemia, and gout.

The manuscript would benefit from careful English-language editing.

Recommendation

The study addresses an interesting question and benefits from a large sample size. However, participant selection, missing data, laboratory methods, statistical reproducibility, and interpretation of adjusted associations require clarification.

Recommendation: Major Revision

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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 #1: Yes: Zean Zefenkey

Reviewer #2: No

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Helicobacter pylori infection, hyperuricemia, and serum uric acid levels: the role of metabolic, renal, and inflammatory factors in a large health examination population

PONE-D-26-26394R1

Dear Dr. Fang,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Ahmed E. Abdel Moneim

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: The authors have carefully addressed the comments raised during the previous review, and the revised manuscript has been substantially improved, particularly with regard to the statistical analyses, methodological description, and interpretation of the findings. The major concerns have been adequately resolved.

I noted only a few very minor editorial inconsistencies in the revised/marked-up version, mainly related to sample-size notation, table numbering, and wording in table notes. These should be corrected during final proofreading.

Overall, I consider the manuscript suitable for publication in PLOS ONE after these minor editorial corrections.

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

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

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