PONE-D-23-20941Detection through the use of RT-MqPCR of asymptomatic reservoirs of malaria in samples of patients from the indigenous Comarca of Guna Yala, Panama: Essential method to achieve the elimination of malaria.PLOS ONE

Dear Dr. Cáceres Carrera, Thank you for submitting your manuscript to PLoS ONE. After careful consideration, we felt that your manuscript requires substantial revision, following which it can possibly be reconsidered, thus governing the decision of a “major revision”. As requested by the reviewers, the structure and content of the manuscript need to be deeply revised. The authors need to revise the text making it more concise and (considerably) shorter, eliminating unnecessary repetitions and removing far-fetched conclusions. In addition, the authors need to address several concerns, particularly related to the data available (raw data), statistical analysis, methods and results. Finally, we strongly suggest a professional copy-editing service. For your guidance, a copy of the reviewers' comments was included below.

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Luzia H Carvalho, Ph.D.

Academic Editor

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?

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: No

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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: No

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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: The work presents a topic of extreme relevance for understanding the epidemiology of malaria.

However, it is necessary to make major changes to the text to better understand the work.

I would like to see the raw population data and the RT-MqPCR results, with the sample amplification curves. To better analyze the results.

I suggest passing the article through an English reviewer.

59: Confirm the number of positives in the study. In the text this value is 13.4%. reconfirm this value.

60: Confirm the number of positives in Irgandi. In the text this value is 1.8%. reconfirm this value.

65: To better characterize the age of your population, it would be extremely important to write the age in months of children under one year old. I suggest changing the minus sign to x months until 21 years.

101: cite a reference for this sentence.

111: Wouldn't that be a strategy for eradicating and eliminating malaria?

117: Change the parasites/ml for parasites/ul. parasites/ul is the most used unit.

128: add Polymerase chain reaction (PCR).

135: change for parasites/μl.

137: I couldn't understand what an nPCR is and what its target is.

163: It would be interesting to reference studies that demonstrate the role of asymptomatic people in the transmission of malaria.

194: Throughout the text the degree sign Celsius is written with a subscript. Change to °.

203: The sentence is incomplete, please add "malaria parasites".

211: The peak number of malaria cases was based on which study or census?

216: Was there any statistical calculation to establish the ideal sample number? If yes, please describe clearly.

223: change the celsius degree sign.

251: Describe the age of minor participants more clearly. It's a little vague, just write smaller ones. The more detailed the better the work.

259: What is the average amount of blood collected in ul?

281: The RT-MqPCR part needs to be rewritten or more information needs to be added.

282: DNA or cDNA? Describe how the DNA was extracted. What kit is used? and What is the final extraction yield in uL?

282: What is the target of this PCR?

345: Clearly describe the age of children under one year in months.

353: Clearly describe the age of children under one year in months.

359: Clearly describe the age of children under one year in months.

378: add uL.

383: describe the age of children under one year in months.

386: I would like to see the RT-MqPCR results, with the sample amplification curves. To better analyze the results. I suggest adding these graphs in the supplementary results.

394: The frequencies in the text are not the same as those in table 3. Choose whether to use the number of samples or inhabitants.

457: Have epidemiological studies been carried out with anopheles in these regions?

1251: Improve the description of the legend in figure 1. Add what the green color on the map and the red balls mean.

1225: Define what is a title and what is a legend in figure 2.

1237: improve the description of the legend for figure 3. Describe what the color red means.

1261: Clearly describe the age of children under one year in months, in the group from < 1 to 10 years.

1277: It would be interesting to demonstrate the age and location of each positive sample.

1279: Rewrite the table in English.

Reviewer #2: Major Concerns

Overall, the manuscript presents us interesting data on the detection of asymptomatic cases of P. vivax on three distinct locations in a Comarca in Panama. And, according to the authors, this is the first ever report of asymptomatic cases in the Country. Therefore, the manuscript does hold scientific relevance.

However, apart from the Introduction, in the following sections, the text is increasingly repetitive and lacks a linear. objectivity. For example, the argument that asymptomatic infections have epidemiological consequences is repeated and rephrased in almost all sections of the manuscript. These repetitive arguments make the text unnecessarily long.

The data presented is based on the results of light microscopy and RT-MqPCR diagnostic tests from 551 asymptomatic people in three different localities. The take home message is that PCR over performs LM in the detection of asymptomatic cases - which was expected. The manuscript then tries to explore some population variables, such as sex and age, but the messages are not clear, specially since no statistical analysis is shown (not in the text and not even on the tables). Although the Methods section cites the Chi-square test, no clear indication of its use is presented.

The proper use of statistical analyses could help improve the manuscript as it would reveal whether any of the variables are indeed distinct among localities. If the authors have already performed the proper analyses, they should report them appropriately in the manuscript, specially in the tables.

The tables should be reformatted as well. Table 2 has terms to be translated into English. And Table 3 has too much information on display and without the results from the statistical analyses, some of these data are simply confusing, as for example, the many percentages.

There are several conclusions/inferences in the manuscript that are not backed by the data (specially without the statistical support) that the authors should consider removing. For example, this study is not suitable for inferences on sensitivity specificity of diagnostic tests, or for policy recommendations that children are more prone to asymptomatic infections.

In summary, my suggestion is that the authors revise the text making it more concise and (considerably) shorter, eliminating unnecessary repetitions and removing far fetched conclusions. Also, perform and report the statistical analysis on population variables.

Minor concerns

Pg11 L233 → positive LM OR PCR (not and)

Section Results, Analysis of the study population → The numbers and percentages are not adding correctly

Paragraph at line 351 → Why are CI values informed for a known exact number of people?

The title of table 2 says 3 communities, but the data is not presented by community

According to FIG4. Irgandi had the most cases in 2021, but had fewer asymptomatic cases. How is this interpreted?

Suggestion → reorganize TABLE 3 to have ML and RT-MqPCR as lines not columns

Discussion

"one of the most important variables that determines the intensity and prevalence of malaria in this region is the internal migration in the region of native people and the arrival of immigrants from South America who are symptomatic and asymptomatic carriers"

What is the migrant status of the participants? Are there more migrants in any of the communities?

Line 525 → These percentages are relative to which total numbers?

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Reviewer #1: Yes: Dra. Gabriela Maíra Pereira de Assis

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-23-20941_reviewer_GMPA.pdf

PONE-D-23-20941R1Detection through the use of RT-MqPCR of asymptomatic reservoirs of malaria in samples of patients from the indigenous Comarca of Guna Yala, Panama: Essential method to achieve the elimination of malaria.PLOS ONE

Dear Dr. Carrera,After careful consideration, we have concluded that your manuscript has the potential to be published although some aspects of the manuscript will need to be changed prior to formal acceptance. Specifically, the language needs to be properly adjusted otherwise it might compromise the MS.

Please submit your revised manuscript by May 01 2024 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,

Luzia H Carvalho, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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

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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

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

Reviewer #1: 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 #1: 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: Yes

**********

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: The manuscript is well written and includes all the requested corrections. The scientific findings are extremely relevant to the scientific community and health organizations.

Two additional corrections need to be made:

Table 1 - correct "Femen" to female

Table 2 - correct "porcentaje" to percentage

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Reviewer #1: No

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Detection through the use of RT-MqPCR of asymptomatic reservoirs of malaria in samples of patients from the indigenous Comarca of Guna Yala, Panama: Essential method to achieve the elimination of malaria.

PONE-D-23-20941R2

Dear Dr. Carrera,

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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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,

Luzia H Carvalho, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: