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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

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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: Yes

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: Manuscript Title: Seroprevalence of Diphtheria and Tetanus Antibodies among Children and Adolescents in High- and Low-Immunization Coverage Areas in the Lao People's Democratic Republic

Manuscript Number: PONE-D-25-45990

Recommendation: Minor Revision

________________________________________

Summary Evaluation

This manuscript presents a well-designed cross-sectional sero-epidemiological study assessing immunity to Diphtheria and Tetanus among children and adolescents aged 1–19 years in Lao People's Democratic Republic, in Oudomxay (high coverage) and Xaisomboun (low coverage). Using multistage cluster sampling and validated ELISA assays, it provides robust data on age- and location-specific seroprevalence, revealing important immunity gaps in school-aged children and among ethnic minority groups, notably the Hmong.

The study is timely and policy-relevant. It highlights the risk posed by waning immunity in the absence of booster doses beyond infancy, supporting the introduction of World Health Organization-recommended boosters in Lao PDR.

However, several methodological clarifications are needed to strengthen transparency and interpretation. Most importantly, vaccination history should be more rigorously reported: the proportion of participants with vaccination cards (by age and province), recall–card concordance, and how discrepancies were handled. Missing data handling should be described, and 95% confidence intervals added to adjusted odds ratios (Table 2). The unexpected 100% Diphtheria seropositivity among 1–4 year-olds with “no vaccination history” likely reflects undocumented doses and should be re-examined with denominators and CIs provided. The sample size calculation and selection process should be clarified, and the representativeness of the two provinces briefly discussed. Finally, the discussion should frame lower Tetanus seropositivity among Hmong participants as likely driven by structural or geographic factors rather than ethnicity per se.

Overall, this is a timely and valuable study that would benefit from these clarifications.

Major comment :

1. Vaccination history: card availability and recall accuracy

Vaccination history was partly based on caregiver recall, but the frequency of card availability and the accuracy of recall are not reported.

- Please report the proportion of participants with vaccination cards (overall, by age and province) and assess concordance between recall and cards (e.g. agreement or κ).

- Clarify how discrepancies were handled and discuss potential misclassification bias in the Limitations.

- Consider presenting the results for three-level vaccine history (documented vaccinated / documented unvaccinated / recall-only or unknown).

2. Table 2: add 95% CIs

Table 2 shows adjusted ORs without confidence intervals.

- Please add the 95% CIs for all adjusted ORs and note in the footnote how clustering and covariates were handled.

3. Figure 4: 100% diphtheria seropositivity in 1–4 y with no vaccination history

It is unexpected that 1–4 y children with “no vaccination history” show 100% diphtheria seropositivity, as maternal antibodies wane by 12 months.

- Provide denominators (n) and 95% CIs for each bar.

- Recheck vaccination classification—acknowledge that very likely reflect undocumented doses.

- Consider separating “no doses” from “unknown/no card” and note that maternal IgG cannot explain this pattern.

4. Handling of Missing Data

Table 1 indicates missing values for key variables (e.g., Penta3 history, ethnicity, income). Please clarify how these were handled in the regression models (e.g., listwise deletion vs. imputation) as it can meaningfully affect regression estimates and interpretation of associations.

Minor Comments

1. Clarify Ethnicity Terminology

For clarity, explicitly state early in the Methods that Hmong is an ethnic group, to ensure international readers understand this categorization.

2. Interpretation of Ethnicity vs. Socioeconomic and Geographical Factors

The discussion attributes lower tetanus seropositivity to “Hmong ethnicity.” However, ethnicity may act as a proxy for geographic remoteness, structural exclusion, or unmeasured socioeconomic barriers (e.g., lower access to health services, transportation, or education). The authors note that differences in caregiver education, occupation, and income were not statistically significant, yet these contextual factors likely influence vaccination uptake. Consider reframing to emphasize that “ethnicity” may reflect underlying structural determinants and avoid implying a direct causal effect.

3. Association Between Vaccination and Seropositivity

Clarify that the lack of a significant association between vaccination history and diphtheria seropositivity likely reflects the persistent circulation of Diphtheria and frequent outbreaks affecting both vaccinated and unvaccinated individuals. This context is key to understanding why vaccination status was only associated with Tetanus seropositivity.

4. Sample Size Description

There is some ambiguity in the description of the sample size calculation and selection. The Methods state that “192 participants per group were selected, totaling 800,” yet also mention that “to support multistage cluster sampling, a target of approximately 960 participants was set.” This appears inconsistent and could confuse readers about how the final sample size was determined.

Please clarify:

- Precisely how the figure of 192 participants per age group was calculated (e.g., based on assumed prevalence, confidence level, design effect, expected response rate);

- why this initial total (192 × 4 age groups = 768 or “800” as written) differs from the final target of 960;

- and how this target was operationalized within the multistage cluster design (e.g., number of clusters/villages, participants per cluster, and any oversampling to account for non-response).

Providing a concise flow (calculation → adjustment for cluster design → final enrolled number) would help readers understand the sampling framework and ensure transparency of the study design.

5. Representativeness of Selected Provinces

Please clarify how the two selected provinces (Oudomxay and Xaisomboun) compare to national averages in terms of immunization coverage, demographics, and health service access. This would help readers assess the external validity and generalizability of the findings, given that the provinces were purposively chosen to contrast coverage levels.

6. Typographical Errors

- In the Statistical Analysis section, correct “seropositivity rare” to “seropositivity rate.”

- Consider

Reviewer #2: The study addresses an important public health issue by evaluating vaccination coverage and seroprevalence of diphtheria and tetanus among subjects aged 1-19 years, in Oudomxay and Xaisomboun provinces of Lao PDR. The use of serological data in combination with vaccination history provides valuable insight into population-level immunity and possible gaps in immunization coverage.

The manuscript is concise and presents significant results that could have meaningful implications for immunization strategies. However, several areas require clarification and expansion before the manuscript is acceptable for publication.

1. Please provide the response rate of the study in both the abstract and results sections. In addition, please discuss the potential implications of selection bias arising from non-response.

2. In Table 2, please provide 95% confidence intervals (CIs) alongside the adjusted odds ratios (aORs).

3. I recommend adding a graphical representation of the U-shaped seropositivity pattern by age group for each antigen (diphtheria and tetanus) stratified by province.

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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: No

Reviewer #2: No

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Seroprevalence of Diphtheria and Tetanus Antibodies among Children and Adolescents in High- and Low-Immunization Coverage Areas in the Lao People’s Democratic Republic

PONE-D-25-45990R1

Dear Dr. Yokobori,

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,

Ray Borrow, Ph.D., FRCPath

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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 #2: Yes

**********

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

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

Reviewer #2: All my concerns have been properly responded. I now suggest the editorial acceptance of the manuscript, in principle.

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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 #2: No

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