PONE-D-21-20229

Genotypic glucose-6-phosphate dehydrogenase (G6PD) deficiency protects against Plasmodium falciparum infections in individuals living in Ghana

PLOS ONE

Dear Dr. Kumi,

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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: Partly

Reviewer #2: Partly

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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?

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Reviewer #1: Yes

Reviewer #2: Yes

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

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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: Dear Authors,

I find your finding to be really interesting and important if done and analyzed properly. However, I found a lot of redundancy in the data presented and also few very confusing data/figures/tables.

Please clarify the following:

1. How do you enrolled randomly your samples from the main study of 19869 participants?

2. G6PD B genotype is actually wild type, as the authors have mentioned that this genotype has ‘complete’ G6PD activity – meaning, normal G6PD activity. So when this is considered as part of the G6PD genotype categorization, therefore, one must have in mind that this is the normal genotype. The G6PD A variant although a variant but has normal G6PD activity as well. Thus, it safe to say, BB, BA, AA, are normal. G6PD A- is considered as the variant that has less G6PD activity than A or B variant and thus can hemolyze upon oxidative trigger. Thus, please work around this facts when discussing about your results. so when we are discussing G6PD A- variant, then we are talking about G6PD deficiency.

3. No need to say percentage prevalence as normally prevalence is already in % as it is the proportion of the population having malaria or having G6PD deficiency.

4. Table 1 there is no data on mean of Age (SEM). To be more informative, the malaria data should be divided between male and female as perhaps this can explain why A-A- is more resistant to malaria infection compared to A-.

5. Figure 2A, the y-axis is not prevalence but number of subjects, while your x-axis should be malaria+ NOT microscopy +

6. Figure 2B, what does mean prevalence mean in this instance? I actually found the figure to be very confusing. Malaria+ within G6PD deficient subject OR malaria+ in 6108 total participants of the study? Again, y-axis should be number of participants.

7. Figure 2C, this is better to describe all those participants who have certain G6PD genotypes and malaria+ as well (Delete Figure 2A and B as these are redundant). Also please make stacked bars instead of the present bargraph, this way is easier to get your point across.

8. Figure 3D, y-axis can be Sensitivity (no need to add 100%) and y-axis as 1-Specificity.

9. Table 2A, why is there a - in front of the %? I think this number represents the percentage and if so, please be consistent with the rest of the table. Also please divide this by gender for easier reading: males (B,A or A-) and females (BB, BA, BA-, AA, AA-, and A-A-).

10. Table 2B: the number in bracket is the percentage?

11. Lines 203-206: “The odds of detecting microscopic malaria among the individuals' G6PDd genotypic variants showed B (p<0.0006), BB (p<0.0001) and BA (p<0.0001) genotypic variants influence malaria infections while A-A- genotypic variant protect individuals from malaria infection with OR (95%CI) of 0.0784 (0.0265-0.2319), p<0.0001 (Table 3).” The genotypes B, BB and BA do not influence malaria infection but rather do not have any protection against malaria infection, in comparison to A-A- that showed some degree of protection. Please change the wording here. However, how do you explain the male hemizygous A- (same low G6PD activity as female homozygous A-A-) that has higher OR than A-A- in detecting malaria in this variant? From this Table, it seems that hemizygous G6PD deficient male (A-) does not offer protection as it has the same OR as A, B, AA, and even has higher OR than BB, BA, and BA-?

12. Same question as before, in Line 212, "The mean (SEM) prevalence.." what does it mean?

13. Figure 3A - delete since this information is shown already in Fig 2C and Table 2B. Delete Fig 3B.

14. Line 568 “D) the plot of the adjusted p value…” please change to C) however, this figure should be deleted as again very redundant information and confusing to see.

15. Lines 219-222 “the mean difference (95% CI) for BA vs B (-546.7(-997.3-116.1), p=0.0064), B vs A-A- (527.3 (96.75-957.9), p=0.009), B vs AA- (468.3 (37.75-898.9), p=0.026; for males were B vs A- (435.7 (5.081-807.9), p=0.046 and BB vs A-A- (395.3 (-35.25-825.9), p=0.0896.” This observation cannot be made as there is no logic or order as to why you compare B to AA- or B to A-A- as this is normal male to heterozygous and homozygous G6PD deficient females. Also line 221, it is for females not males in comparing BB to A-A-.

16. Figure 4A - delete as this is shown already in Figure 4C.

17. Figure 4B is confusing, what does mean 1 and 2 mean?

18. Figure 5A, please use 3D histogram rather than you current graph for better grasp of which region has highest G6PD genotype of which kind.

19. Figure 5B, delete as this is shown in Table 2A already.

20. Supp Fig 1 A is pretty difficult to distinguish, perhaps different colour scheme? As for your x-axis, it's better to put actual age range rather than age category unless you state this in the legend.

21. Supp. Fig 1C, please explain why there are 2 sets of genotypes, are tehse based on different age categories? If so, please indicate in your graph.

22. Please explain why only homozygous deficient females have highest protection to malaria infection compared to hemizygous deficient males?

Reviewer #2: The manuscript reported a cross-sectional survey of G6PDd genotypic variants in Ghana. The objective of this study was to identify the prevalence and distribution of G6PD genotypic variants in Ghana as well as determine whether G6PDd genotypic variants protect against symptomatic malaria infection. A total of 6108 subjects were enrolled in the. The authors concluded that G6PDd genotypic variants, A-A-, AA- and A- protect against P. falciparum, P. ovale and P. malariae infection in Ghana.

Comment:

1. It will be useful to have some ideas about the G6PD ranges across the region? Is there any difference in the G6PD activities according to tribes?

2. While there is selective protection of G6PDd against severe malaria that may also extend to asymptomatic infection. A molecular test would be a useful addition as it offers better sensitivity over microscopy. The authors could have done a diagnostic PCR for Plasmodium species as they had DNA samples from DBS.

3. Do the authors have serological evidence to support their claim? Severely deficient or heterozygous females may not be sero naïve.

4. Studies conducted in other parts of the world could not document the protective effect of G6PD deficiency. I would suggest the authors discuss this aspect (e.g.

1. https://doi.org/10.1371/journal.pmed.1003576 ) in their discussion section.

5. Although provided separately, a title is missing for Table1, Table 3, and all figures.

6. I think the sample size is not sufficient to draw a conclusion that G6PDd genotypic variants, A-A-, AA- and A- protect against P. ovale and P. malariae infection in Ghana.

7. In some places scientific names were not italicized.

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: Review PONE-D-21-20229.docx

Genotypic glucose-6-phosphate dehydrogenase (G6PD) deficiency protects against Plasmodium falciparum infection in individuals living in Ghana

PONE-D-21-20229R1

Dear Dr. Kumi,

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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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 #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

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

Reviewer #1: Yes

Reviewer #2: 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

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: Yes

Reviewer #2: 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: Dear Authors,

I am pleased to see that the revised manuscript showed very good improvement in terms of clarity and statistical analysis.

I have spotted only 1 very minor mistake:

Line 116, delete "then" in ...the then 10 regions of Ghana...

I also would like to comment whether the reason you've shown very good protection against homozygous deficient females with A-A- genotype was because the number of subjects with this genotype is very small almost ten fold less than those with hemizygous deficient males A-?

Also, whether there are more chances of males being outside than females that you see this protection?

As I said before, your study is very interesting. It would be nice if you can pair this genotype with phenotype results as well.

Thank you.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No