PONE-D-24-27945Low prevalence of Plasmodium falciparum histidine-rich protein 2 and 3 gene deletions in malaria-endemic states of IndiaPLOS ONE

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Comments to the Author

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

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: No

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: N/A

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

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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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: Yes

Reviewer #2: Yes

Reviewer #3: 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: This manuscript describes the results of surveillance of pfhrp2 and pfhrp3 deletions in P. falciparum samples from malaria-endemic states in India. The outcomes are highly relevant for the Indian malaria control programme and show that HRP2 RDTs can still be used reliably for diagnosing malaria. The manuscript itself would benefit from a number of adaptations and additions, mainly in the methods and discussion.

Importantly, Table 1 and 2 were not part of the submission and could thus not be reviewed. As such, the data were not fully available at this point.

Major comments

- Methods: please include more information on the patients from whom the samples were collected. How were these patients identified? Were they symptomatic cases presenting at hospitals? Were there certain inclusion/exclusion criteria?

- How sensitive is the nested PCR + gel readout method? Is it possible that the samples marked with a deletion had low parasite DNA and that there was insufficient hrp product to visualize this on gel?

- Results: apart from percentages, it would be good to also present the absolute numbers of samples and deletions per year and state.

- The start of the discussion (until line 142) is more of a review of the malaria situation in India and the used diagnostics, with the content partly overlapping with the introduction. I suggest the authors rewrite this section, so that it reflects the main outcomes of their study

- Can the authors think of possible reasons why the deletion rate in this study was (much) lower than in some previous ones?

- Lines 155-161: This information is not very relevant for the interpretation of this study, especially when considering that deletion rates are still far below the threshold of 5%.

- A critical reflection on the limitations of the study design should be added to the discussion, e.g. the used methodology for detecting deletions, representativeness of sample, etc.

Minor comments

- Line 22: please rephrase this sentence, as the deletions are not found in RDTs (but in the parasite) and have not been widely reported in India so far.

- Line 42: please rephrase this sentence

- Lines 55-58: the issue of deletions is mentioned twice

- Lines 70-73: this part feels a bit redundant in the introduction and would fit better in the discussion

- Line 88: please explain why the exon 2 segment was chosen for detection of gene deletions

- Line 88-98: this section would be easier to understand if the use of nested PCR is mentioned at the start

- Line 90-91: please clarify what is confirmed exactly by testing for the additional markers

- DNA samples had been stored at -20C for up to 9 years. This is quite long, and at -20C there is a risk of DNA degradation. DNA integrity was checked by detection of msp1 and 2 markers, but these are located on different chromosomes (9 and 2). How do the authors assess the risk of DNA degradation of the subtelomeric regions on which pfhrp2 and 3 are found?

- Line 108: contrary to what is stated here, deletions were reported for Assam, Meghalaya and Mizoram in Figure 2

- Line 110: idem as above, Figure 2 reports deletions in 2017. Please check.

- Line 110-111 and 121-122: were these samples also positive for 18s rRNA?

- Line 151-152: I would omit the numbers from Africa, they are less relevant for this study.

- Line 169: please clarify how the study data will aid to comprehend these evolutionary mechanisms

Layout and editing:

- Please italicize Latin organism names, e.g. Plasmodium falciparum (line 21) and P. falciparum (line 24) (please also check the rest of the manuscript)

- Line 79: add “detection of” before “Pfhrp2”

- Line 113: avoid the use of contractions (didn’t)

- Throughout the manuscript, please be consistent in the notation of the HRP2 gene (hrp2/pfhrp2/P. falciparum hrp2)

Reviewer #2: The manuscript is technically sound and the data of the manuscript support the conclusion.

Statistical analysis performed propely

All the data is available in the manuscript,

the manuscript is written in standard English and intelligible fasion

The abstract is written in the clear language to show the manuscript

the conclusion clear and precise, but a bit sronger recommendation is needed.

Reviewer #3: Low prevalence of Plasmodium falciparum histidine-rich protein 2 and 3 gene deletions in malaria-endemic states of India

The authors present a short report on hrp deletions in India across multiple malaria endemic states spanning several years. The sample number analysed is large and the authors find a few suspected deletions. It would have been great to have some more details on the size of the study sites and distances between the sampling locations. The map (figure 1) has no scale, and if I interpret this correctly, the samples were collected from different locations in different years. As there is no details provided in what malaria transmission is like in the different states and whether it is homogeneous across states, not too many conclusions can be drawn.

No RDT data is presented for the samples here – all samples were microscopy positive but in the absence of direct comparison to RDT performance, no testing recommendations can be made.

It would have been beneficial to see the microcopy parasite density data - overall but also for the suspected deletions.

The report would have benefited from more details in the experimental design and methods used especially in hrp testing where essentially, the inability to amplify a gene suggests that the gene is deleted. The authors need to show the data e.g. add an agarose gel, positive and negative controls and number of repetitions, and the LOD of all assays.

Both tables are missing from the copy of the manuscript that I received.

In the discussion there is no mention of multiclonal infections that potentially contain hrp-deleted minority clones.

The discussion could also include a paragraph on methodological difficulties of hrp testing e.g. When is a deletion a “real” deletion and when is it a PCR failure (only mentioning 18s, msp testing in a couple of sentences is not enough)?

In summary, experiments are not described in sufficient detail and conclusions are not presented in an appropriate fashion and therefore it is very difficult to judge whether the data is supported.

Some more detailed comments:

Line 21 + – Plasmodium falciparum and pfhrp2– please use italics when referring to organisms and gene names throughout the manuscript. Also Genus = capital & species = lower case. Gene names all in lower case.

Line 29 – Please keep nomenclature the same e.g. refer to hrp2 gene as either pfhrp2 or hrp2 but use the same nomenclature throughout the manuscript.

Lins 41-42 – “Despite the significant reduction in malaria cases, in recent years, the total of 0.22 million cases reported in 2023” – the sentence lacks meaning, please revise.

Line 76 – Could you please indicate exactly how the whole blood was collected and stored? Seeing as the hrp deletion analysis depends on PCRs where the absence of product means the presence of a deletion, it would be great to know what state the samples were in when they were tested.

Line 85 - Please give your elution volume.

Line 87 and paragraph – It would be great to include more details on the confirmation PCRs as well as the hrp PCRs. It is important to understand the limit of detection of all PCRs, what positive and negative controls were used, and whether the samples were run multiple times etc. You give some more information in the results, but I believe this should already be clearly explained in the methods as part of the experimental design.

Line 106 + - please always give % with associated numbers e.g. line 105!

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Reviewer #1: Yes: Norbert van Dijk

Reviewer #2: Yes: ABEBECH TESFAYE TOLESSA

Reviewer #3: No

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Low prevalence of Plasmodium falciparum histidine-rich protein 2 and 3 gene deletions in malaria-endemic states of India

PONE-D-24-27945R1

Dear Dr. Bharti,

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,

Henk Schallig, Ph.D

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 #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 #3: Yes

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

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 #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 #3: 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 #3: (No Response)

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Reviewer #3: No

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