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PONE-D-23-03162

Genetic characteristics of P. falciparum parasites collected from 2012 to 2016 and anti-malaria resistance along the China-Myanmar border

PLOS ONE

Dear Dr. Li,

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.

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PLOS ONE

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   " WHO Mekong Malaria Programme (WP/10/MVP/005837) from a Consolidated Grant from USAID-PMI to WHO; the Round 6 (CHN-607-G09-M) and 10 (CHN-011-G15-M) Global Fund grant to fight AIDS, Tuberculosis and Malaria (GFATM) in China. It was also supported by the National Science Foundation of China (No. 81673113)" 

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: No

Reviewer #2: Yes

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4. 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 #1: No

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: Overview:

Li et al’s manuscript titled Genetic characteristics of P. falciparum parasites collected from 2012 to 2016 and anti-malaria resistance along the China-Myanmar border examined the frequencies of several molecular markers associated with delayed artemisinin clearance and measured clearance times of P. falciparum strains collected along the China-Myanmar border. This study is interesting, as it takes place in the decade immediately preceding China’s malaria-free certification in 2021.

The primary purpose of the study was to assess the level of delayed artemisinin clearance at the China-Myanmar border. To do this, they assessed SNPs in the K13-propellar domain microsatellites flanking the K13 gene on chromosome 13, and copy number variation of pfmdr1 and pfpm2. Clearance times following DHA-PIP treatment were also measured.

Overall, the actual results appear to be sound, and I recommend publication. The observation of different genetic structure in the NW and SW collection sites is interesting and appears to be consistent with the fragmented parasite populations seen in SE Asia.

However, the language style will need to be revised before final acceptance. The manuscript is understandable but awkward in too many areas to list. I have, however, left some recommendations specific recommendations to improve the presentation of the paper.

Major comments

Introduction

1) The introduction should be refocused to provide greater epidemiological context to the importance of the China-Myanmar border. It is obvious that a major motivation for the study was to study the drug resistance profiles of individuals moving between China and Myanmar, which was important because China was actively applying for malaria-free certification in this time period

a. The introduction can be improved by explaining the status of malaria transmission in Yunnan province between 2012-2016, and the drug resistance profile in Myanmar (or possibly Laos), as these are likely where the parasites seen at the China-Myanmar border are coming from.

2) Too much time is spent describing CQ resistance and its spread. --CQ is not relevant to this study and I would advise focusing specifically on describing what is known about ACTs and DHA-PIP. For example, much of lines 68-75 can be cut.

3) Line 61 – “In response to widespread resistance….” is technically correct but skips a lot of steps. ACTs were used after many drugs, including CQ began to fail due to the emergence of drug resistance.

Methods

1) The map used in Fig 1 should be rescaled so that it is zoomed into the China-Myanmar border. The actual study sites are too small to see.

2) Line 179 – “all data were collected in Excel 2016” – I don’t think this is relevant detail

3) Line 181 – “Column statistics Method in Column Analysis” is uninformative and the name/description of the exact test being used should be used instead

Results

1) The description of the sampling design could be improved by including a flow chart or some other visualization to help the reader understand and remember

2) Line 195 – The term “K13 genotype” is misleading—I think it can be clarified to state “Samples that were successfully genotyped at K13”. Same at Line 200 – It should be rephrased as “Among the 175 parasites that were successfully genotyped at K13…”

3) Table 1 is confusing because it is unclear which regions correspond to which year. Example: was Ruili collected in 2012 or 2013? Some shading or coloring would help clarify

4) Were any parasites observed with multiple K13 mutations?

5) Line 262 replace with “We did not observe any amplification of the pfpm2 gene in our samples”.

6) Line 263 – It is strange that the estimated CNV for pfpm2 is less than 1, as this would imply a deletion in some of the samples. I am guessing this is because the equations used to estimate CNV are not completely accurate—but if there truly are deletions of pfpm2 in the data that would be useful to report

a. Line 263 – “NW region significantly lower…” – this might be technically true, but I doubt that this is due to actual biology… I think it is more likely to be because the equation used is not completely accurate

7) Line 278: the name of the test should be used (I think it is an ANOVA?)

Discussion

1) Line 325-326 – “Following the spread of chloroquine….”, Technically true, but ultimately irrelevant. Would suggest this first section focus more on the specific implications of drug resistance of at the China-Myanmar border. Lines 329-331 should be expanded upon

2) The most interesting finding to me is that PCTs in the W group are much lower than the PCT values in the ML group. Both groups are wild type at K13, which would imply that the ML group has other mutations contributing to delayed clearance—I think this is an important finding and should be discussed more

3) Lines 347-350 – I think the phrasing can be improved. The low He observed in F466I could be due to strong, recent drug-mediated positive selection for the mutation due to ACT use in the region. It could also indicate that F466I arrived at in Yunnan border from a recent importation event that rapidly expanded in NW Yunnan province – both could result in extremely low He.

4) Lines 353-357 – This is also interesting and should be expanded. Given that the samples in SW Yunnan were collected during an outbreak, it is entirely possible that the parasites in the ML group are all clonal or extremely clonal to one another and derived from a recent importation event.

5) It is curious that C580Y was not found at high frequencies in this study. Some discussion about why this might be would be useful. Additional discussion for why F446I is so prevalent would also be interesting

Minor Comments

1) In general, the name of the statistical test used to estimate p-values should be included when reporting p-values

2) Line 160 – Pf3D7 is misspelled (Pf37D)

3) lines 167 and 169-Equation is misspelled (“equitation”) in

4) Line 176-177: The equations used for He and Fst could be useful

5) The results from SW region should also be included in Table 1, even though there were no K13 mutants observed. Alternatively, Table 1 should be renamed “Distribution of K13 genotypes in NW Yunnan”

6) Line 268 – the section starting with “The prevalence of isolates with multiple pfmdr1 copies…” should be made into its own paragraph

7) The language style of the manuscript should be reviewed by a dedicated editor

Reviewer #2: 1. It would be worth mentioning about the drug policy in the different study area. Although author mentioned the drug of choice in line no. 98-101 but if possible then mentioned the specific drug used in the study area.

2. Since the study follows the standard WHO protocol for efficacy monitoring, the sample size as per WHO protocol was not adequate to conclude for each site vs each year which need to explain.

3. The study was not planned for PCT (6 hr smear preparation and examine) so association between PCT and mutation may not provide the actual data. Therefore, author needs to clarify in starting while discussing the associations.

4. Author did analyse only the copy number variation in MDR1 gene and explained on the association of SW region vs NW region however, author did not perform the mdr1 mutation analysis so that the statement may be more conclusive as mutation along with CNV play a role in artemisinin resistance. If not possible then it should come into the study limitation.

5. F446I mutation are found particularly one study sites (Yingjiang) with lower frequency in 2012 (35%) and subsequently it raised to 90% in 2013 and 2014. Please discuss the possible cause of the spread particularly in one site. If possible, the corelate the epidemiological data of the study site for recombination point of view.

6. What were the QA/QC procedures for K13 sequencing? Were the sequences of the F446I containing samples confirmed; either by re-sequencing, or any other method?

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Reviewer #1: No

Reviewer #2: Yes: Praveen K Bharti

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PONE-D-23-03162R1Genetic characteristics of P. falciparum parasites collected from 2012 to 2016 and anti-malaria resistance along the China-Myanmar borderPLOS ONE

Dear Dr. Li,

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 Sep 29 2023 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.

Please include the following items when submitting your revised manuscript:

• 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,

Himanshu Gupta

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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 #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?

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: 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 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: (No Response)

Reviewer #2: 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 #1: (No Response)

Reviewer #2: Yes

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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 #1: Overall, I find the paper much improved and am satisfied with the changes. I have a few minor points though:

Introduction & Discussion – The wording needs to be checked by a dedicated editor for grammar and phrasing. I have included grammatical and phrasing suggestions where I could but I probably did not catch all them all.

Line 206-225: There is no reason to have all the statistics (the 25% per = 2.74, per = 5.02, etc etc) reported in the main text—They are all already Table 2. I suggest using the main text to highlight only the key results.

Minor comments

• Line 39: Missing a the: In (the) NW (north-west Yunnan…)

• Line 47: Change phrasing: no difference in PCT50s were present between…

• Line 47-50: having the actual values of He for each of the groups could be useful

• Line 52: would change the word inferred to hypothesized—I am more used to being used in the statistical sense (ie for model selection or statistical parameter inference)

• Line 63: backward economic development – would advise a rephrase to avoid offending people

• Line 65: Malaria is one of <the> infectious diseases – missing a the

• Line 67: “last reported local case” instead of last report oflocal <sic> case

• Line 68: Most of them (78.02, 4099/5254) were missing –missing a were

• Line 70: The emergence and spread

• Line 74: suggest replacing “because” with “following”

• Line 77-80: Rephrase as: In China, ACTs were used as the official first-line drugs to treat uncomplicated P. falciparum malaria starting in 2006.

• Line 84: Rephrase: Treatment with DHA-PIP initially demonstrated prolonged parasite clearance time( PCT), but began to fail as the partner drugs began failing in the GMS around 2010.

• Line 171: replace treated with examined

• Line 306: Rephrase “ so surveillalnce sites are usually set in these Prefectures in those days.” The phrasing is too casual for a scientific paper in my opinion

• Line 401: oppression is not the right word—advise selective pressure</sic></the>

Reviewer #2: Author have addressed all the concern and revised the manuscript accordingly. The revised version of the manuscript may accept for the publications.

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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.

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

**********

[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.

Genetic characteristics of P. falciparum parasites collected from 2012 to 2016 and anti-malaria resistance along the China-Myanmar border

PONE-D-23-03162R2

Dear Dr. Li,

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, as well as improves the English throughout the manuscript, as pointed out by the reviewer.

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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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Himanshu Gupta

Academic Editor

PLOS ONE

Additional Editor Comments (optional): There is a requirement to improve the English throughout the manuscript.

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 #1: All comments have been addressed

**********

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 #1: Yes

**********

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

Reviewer #1: Yes

**********

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 #1: (No Response)

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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 #1: (No Response)

**********

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 #1: Line 323: “following with [the] civil war in Kachin” – remove “the”

Line 324: “We [preferred] the F446I…” -- “preferred” is the wrong word

Line 351: the high prevalence of mutant K13 [which] was …” – remove “which”

Line 356: “[Besides], this study was not planned…” replace besides with However

Line 363 “resistance to [multiply]” – replace multiply with multiple

Line 386 multiply should be replaced with multiple

Line 389 “which perhaps were interfered by PIP…” is awkward, suggest a rephrase

Line 391 remove “ayway”

Line 429-431 – “Otherwise the values of copy number…” is awkward, suggest a rephrase

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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.

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

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