PNTD-D-26-00567

Identification of two genomic cryptotypes of Plasmodium malariae  in Africa

PLOS Neglected Tropical Diseases

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PLOS Neglected Tropical Diseases

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Reviewers' Comments:

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The joining of the NHP and human data doesn’t make clear sense. There is a very solid analysis of P. malariae population structure in humans in Africa and a novel observation of P. brasilianum in NHPs in South America. Aside from the observation that there is P. brasilianum in NHPs there seems to be no real value from including the P. brasilianum data here. The major finding (and title) of the paper is not reliant on this data and it is confusing to include here. I would suggest the authors consider whether the article would be better served by separating these two datasets and only focusing on the malariae data here.

Reviewer #2: There are no concerns with respect to the methodology.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Analysis of the population structure defining cryptotypes

Definition – can the authors clarify what they mean by cryptotype in this context? The P. falciparum observation was a small number of loci around the genome where LD was maintained despite being on different chromosomes. In P. ovale the observation is that there are two “sibling” species which are sympatric but rarely recombine. The identification of segregation across the first few PCs seems closer to the ovale scenario, though latter data less so. A very clear definition of what is expected for each scenario would be valuable.

Driving factors – it is stated that no biological or geographical criteria allow these clusters to be distinguished. What factors were assessed, in addition what technical factors were assessed (i.e. sequencing depth, sequencing batch, instrument, etc). Do the clusters related to transmission intensity, mosquito species prevalence or historical use of antimalarials?

Figure 4: Was K=2 the best performing value of K?

Line 444-453: Did you examine if the selection of samples results in a geographical separation. There is higher yellow ancestry in East Africa than West Africa. Could the samples used for this analysis be marked on Fig. 3?

You state in the discussion that haplotype based signatures of selection are powerful for detecting recent events – why were they not used here?

Line 129: densify should be replaced with an alternative word.

Figure 3: If the best K is 6, why are the pie charts shown from K=3?

Figure 4: Using shades of red and yellow to define countries and red and yellow to define groups which are not related to geography is confusing. Could the country colors come from a different palate?

Reviewer #2: The analyses match the analytical plans, and the results clearly and fully presented, as are the figures. (minor suggestions are made below)

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions and Discussion cover all these areas

Reviewer #2: The conclusions are supported by the data, and the limitations indicated. (See "Summary and General Comments")

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: See "Summary and General Comments"

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: It is becoming increasingly clear that infections by P. malariae are by no means rare (at least in Africa). It is also known that these parasites cause infections that can persist for years. Thus, it is becoming important to study this species in the context of efforts to eliminate malaria. Yet, this parasite species has been relatively poorly studied, and molecular investigations focusing on this parasite are few.

The authors present a very interesting set of analyses of P. malariae populations, based on whole genome sequences, that substantially extends and improves on a recent analysis by Ibrahim et al. (2024). In addition to previous sequenced genomes, those of 45 new isolates from Africa were added, and of particular novelty the authors provide the genomes of two additional P. brasilianum isolates one from Colombia and another from French Guiana.

Their analyses confirmed previous observations concerning the differences between the populations of P. malariae across the continents and interestingly revealed the occurrence of two sympatric genetically distinct groups within the African isolates, which the authors consider as cryptotypes that nonetheless recombine.

The manuscript is well written, the data is clearly and convincingly presented, and fully supports the conclusions drawn by the authors. The salient critique I have is that the discussion presented in the manuscript deserves to be expanded to confront the data with some of the parasitological parameters of P. malariae. As it stands, the discussion generally reiterates the results presented, with little substantive speculation as to the nature or origin of the two cryptotypes.

General comments

- Why were the two P. malariae-like parasites (Rutledge et al. 2017), as well as the P. brasilianum genome that was previously obtained from a Bolivian strain (Bajic et al. 2022), not included with the other 179 isolates in the main analysis? These omissions are regrettable and somewhat weaken the manuscript, and I suggest that they should be added. This is because it would have helped resolve the phylogeny of the P. malariae-like parasite from chimpanzees and to provide a comparison of the 3 P. brasilianum isolates that were collected in distant geographical regions.

- The speculations concerning the cause of divergence between the two cryptotypes are based on the identifications of 315 genes that appear to have been differentially selected in the two populations (112 Y and 203 for R). For the discussion the authors selected only 22 of these genes, as they are known to be, or extrapolated as, implicated in host-parasite interactions. Speculation as to the nature of the selective forces were limited to a few lines (587-596). This is a major weakness of this manuscript, since this differential selection is what is the most interesting aspect of the manuscript. It is clear that the authors cannot speculate on the vast majority of the genes they identified since their function is unknown or only ascribed putatively. However, there are many avenues that would have been important to discuss.

First, there are numerous antigens that are considered to be important in the acquisition/evasion of immunity with some displaying significant diversity. It is of interest that none were identified in the analysis presented in S2 Table. It would have been of distinct interest to compare the diversity of such genes between the isolates from the two clusters (presumably these could be easily identified from the whole genome sequences).

Second, what the evidence that the two cryptotypes recombine. Is it possible that recombination is rare? Are mixed infections (Y+R) common? If the two types recombine frequently, one would expect them to merge into a single type eventually. If they do not, might this be a vase of speciation? This deserves speculation.

Third, there are some data on the transmission of P. malariae (and P. brasilianum) by different mosquito species. Could the authors speculate as to the differences in the anopheline species that occur between the different regions of the African continent, and whether this could have contributed to the generation of two cryptotypes (admixture of the two parasite types could have been recent, as large population movement between West and East Africa is likely to be recent).

Fourth, the genetic make-up of the human hosts might have been another contributory factor.

Of note the distribution of haemoglobinopathies differ across the African continent. This is also the case for Duffy negativity. Indeed, there was recent speculation (Culleton et al. 2023 Trends in Parasitology, in which one of the co-authors of this manuscript is also a co-author) that P. malariae might preferentially invade reticulocytes. Although infection of Duffy negative individuals by P. malariae occurs, the course of the infection might differ between Duffy negative and Duffy positive persons. It would be of interest for the authors to indicate the ethnic origin of the patients from whom the samples were collected. Since nearly all the African samples collected were from persons returning to the UK or France, it is possible that some could have been of African descent while others not (I am aware that this might be difficult for the samples collected in the UK).

Minor comments

Line 89 It is now recognised that the population of P. ovale is actually composed of two distinct species. In this case the use of the trinomial leads to confusion (as this is restricted to sub-species in the ICZN). I suggest that the denominations P. ovalecurtisi and P. ovalewallikeri should be used.

Line 113-115 References numbering is incorrect.

Figure 2. It is very difficult to distinguish between the various colours. Could I suggest that you restrict these to one colour for each geographic set: North Africa, West Africa, Central Africa, etc…. since there is no discussion as to variations between the parasites within each of these geographical groupings.

Line 249 Define IBD.

Line 481 Only 20 of the 22 genes are shown in bold in S2 Table.

Line 581-582 One cannot state that the cluster-specific adaptation signals are “dominated” by host-interaction genes (22 out of 315 is hardly a dominant proportion) The word to use should be “comprise”.

Line 584 There is no Figure 4C.

Line 617 It will be more correct to replace “challenge” by “add to”.

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Reviewer #1: No

Reviewer #2: No

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Dear Dr. Lefebvre,

We are pleased to inform you that your manuscript 'Identification of two genomic cryptotypes of Plasmodium malariae  in Africa' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Sanjai Kumar

Guest Editor

PLOS Neglected Tropical Diseases

Hira Nakhasi

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

***********************************************************

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: The authors adequately addressed all the comments

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: The authors adequately addressed all the comments

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The authors adequately addressed all the comments

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have appropriately addressed all comments in the revised manuscript

Reviewer #2: The authors adequately addressed all the comments. The manuscript is suitable for publication

**********

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Reviewer #1: No

Reviewer #2: No