PONE-D-22-05138Gut microbiota diversity in patients with strongyloidiasis differs little in two different regions in endemic areas of ThailandPLOS ONE

Dear Dr. Maleewong,

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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?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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 requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Summary

The manuscriot “Gut microbiota diversity in patients with strongylodiasis differs little in two different regions in endemic area of Thailand” by Maleewong et. al. surveyed gut microbiome from 5 individuals from two different regions and either infected or uninfected with intestinal nematode S. stercoralis. Authors report no significant difference as a function on infection, but observed differences in microbiomes of uninfected individual from different regions.

Major Concerns----------

I feel that the authors’ utilization of statistical analysis needs to be reconsidered to make the most of the data they have collected. A t-test was used for comparing sample types. The author’s experimental design however is highly appropriate to a two-factor Analysis of Variance 2-ANOVA (factors = NE/ST and infected/uninfected). I strongly suggest authors perform this or similar analysis, and perhaps additional statistical significance in the data may be uncovered.

Throughout manuscript, but particularly apparent for example in Venn-diagram (Fig 2), it is unclear how Authors combined experimental data from individuals. For example, in Fig 2 there are 1403 OTUs unique to SsST. Are those OTUs present in at least one individual from SsST? All SsST individuals? This should be made clear in analysis. I would additionally suggest, if authors have not already done so, remove OTUs found only in one or in very few individuals to remove very low prevalence/abundance OTUs that are on the edge of detectability or may even be sequencing artifacts.

Throughout the manuscript, but for example L39-41, Authors report changes in abundance that do not reach the level of statistical significance. I would state that there is no such thing as a ‘non-statistically significant change in abundance’; a change is statistically significant or else there is no change. I strongly recommend removing all reporting of non-significant results.

Two sequencing methods were reported as used in this study: Illumina and IonS5. These sequencing methods each have their own specific sequencing biases. While I do not think that mixing data from different sequencing technologies is inherently incompatible, Authors should, at the very least, identify which samples derive from which method, and ideally should present some analysis indicating that sequencer technology did not contribute to biases in OTU reporting.

Infection positive individuals are referred to in the manuscript as “patients”. What can Authors report about selection of infected and non-infected individuals? Are non-infected individuals also ‘pateints’? If possible, Authors should present some minimal aggregate demographic information for experimental groups (e.g. range of ages, percent male/female, or presence of co-morbidities for hospitalized patients).

Authors did not find significant differences in alpha or beta diversity as a function of strongylodiasis infection. The experimental sample size per experimental group however was relatively modest (N=5). The effects of sample size on analysis should be considered in Discussion section.

In the Introduction, Authors report “diverse and contrasting results” from prior investigations into the effect of parasites on host microbiomes. This requires elaboration. One significant frustration for me in reading this paper was lack of context. A clear description of what is known or suspected about the interactions between parasite infections and microbiomes, what specific question this study seeks to address, where the results of this study fall into the spectrum of previously reported 'diverse and contrasting results', and what makes this study unique relative to previous studies is needed.

Minor Concerns----------

In my opinion, the Abstract is too detailed. It would serve better by simply stating results in prose and saving details like p-values for Results section.

I cannot see where the number of samples analyzed is explicitly stated in manuscript. (N=5 for each condition, but only inferred that from supplemental data table). Number of samples should be in Methods (and if I missed it somehow, then perhaps it should be stated more prominently).

Discussion includes a number of Author-picked examples of taxa from analysis with a brief discussion of its possible role in community. The discussion may be stronger if some of the more tenuous links to possible functions are omitted and say nothing about a particular taxon rather than speculate too freely.

Figure 4: I don’t think that Figure 4 contributes much to description of data. Maybe an alternative version of this figure or just prose description of data in main text.

In Discussion (L235-236), “It has been suggested that the gut microbiota influences human health”. I suggest that it is safe to call the link between microbiome and human health well-established at this time.

Recommendation----------

Major Revision

Reviewer #2: 6. Review Comments to the Author (minimum 200 characters)

The manuscript entitled “Gut microbiota diversity in patients with strongyloidiasis differs little in two different regions in endemic areas of Thailand” by Rodpai et al, showed that S. stercoralis infection leads to only minor alterations in the relative abundance of individual bacterial species in the human gut, and no detectable effect was observed on community structure and diversity. Some differences in species abundance were noted between geographical isolates. There are several points concerning clarity in the manuscript.

- line 72-74: S. stercoralis infection prevalence in 4 regions of Thailand. Explain more why the study area focused only in the southern and the northeastern areas and only one province from each area was selected.

- Please give more information about the sample size. Only 10 stool samples (5 SS positive and 5 SS negative) from each province were used for microbial composition analysis.

- line 85- amount of fecal samples and number of volunteers should be added

- Diet, age and health condition play an important role on microbial gut composition, therefore basic information of the volunteers, for examples age, health condition, diet, should be added.

-line 89- A portion means how many grams of each fecal sample

-line 91 The stool samples were categorized in 4 groups. Give more detail about DNA preparation of stool samples from each area. Is it prepared separately, or are pool samples from each area together and then extracted for the DNA?

Discussion

Line 243- please discuss more about the Ruminococcaceae abundance in uninfected groups and low abundance in infected groups.

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Reviewer #1: No

Reviewer #2: Yes: Wilawan Pumidonming

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Attachments

Attachment

Submitted filename: Review-update.docx

Gut microbiota diversity in human strongyloidiasis differs little in two different regions in endemic areas of Thailand

PONE-D-22-05138R1

Dear Dr. Maleewong,

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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Kind regards,

Marcello Otake Sato, Ph.D., D.V.M.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have adequately addressed to comments mentioned in a previous round of review.

The data support the conclusions. The statistical analysis been performed appropriately. This manuscript is now acceptable for publication.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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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

Reviewer #3: Yes: Ana Julia Pinto Fonseca Sieuve Afonso

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