PREreview of The Impact of Malaria-Induced Neutrophil Subset Shift and a Link to Burkitt Lymphoma

Does the introduction explain the objective of the research presented in the preprint?

Yes. 

The introduction provides background information behind chronic Plasmodium falciparum (Pf) malaria and explains how it is strongly linked to endemic Burkitt lymphoma (BL) that is known to impair neutrophil effector functions and neutrophil activation in children. 

The introduction then includes existing literature on neutrophil subset dynamics based on consistent inflammatory exposure, which includes Pf malaria. 

The introduction could maybe add a little more information on other past studies related to the study’s aim in more detail. The introduction could also add testable hypotheses for future functional studies.

It is also necessary to address that different cancer types showing distinct neutrophil profiles suggest tumor-specific effects rather than malaria-driven predisposition.

The aim of this study was clearly stated: it was to ascertain the effects of chronic Pf-malaria exposure on neutrophil subset composition and evaluate their association with BL. Neutrophil subsets from healthy malaria-naive adults, children with different Pf-malaria exposure histories, and children with BL from Western Kenya were profiled and then characterized by expression of CD15, CD16, CD10, CD11b, CD182, CD184, and CD62L. Researchers used a combination of these specific biomarkers to define subsets.

Are there any methodological issues or are the methods well-suited for this research?

Somewhat Appropriate with Relevant Techniques

Strengths of Methods:

1. The grouping of the study design is well-suited for correlating neutrophil profiles with malaria exposure history while strengthening the specificity of any BL associations. Study design includes malaria-exposed healthy children, malaria-naive healthy children, children with BL, children with non-BL type cancers, and healthy adults as a control. 

2. Rigorous neutrophil immunophenotyping allows for multi-parametric subset identification by using a nine-color flow panel and including FMOs and compensation controls that ensure reliability by reducing false positives. 

3. Strong serological and molecular evidence provided robust confirmation of malaria exposure by utilizing AMA1 IgG levels and the ultrasensitive varATS qPCR for low-density parasitemia detection, which are highly effective because long-term exposure trends can be assessed. The varATS qPCR also targets multicopy genomic sequences, increasing sensitivity.

4. No discredited methods were used as all techniques are standard and validated.

Potential Major Methodological Issues:

1. There is a small sample size for each group of participants. I would suggest increasing each group’s sample size with about 10-20 more participants. Sample sizes: 19 healthy children malaria-exposed, 11 children malaria-naive, 11 children diagnosed with BL, and 5 children diagnosed with non-BL cancers. There were only 3 Kenyan adult healthy controls.

2. Neutrophil phenotypes could have been affected by sample handling time. Neutrophils are highly sensitive to marker shedding or activation ex vivo. Thus, it is important to confirm marker stability under fixation conditions and that next-day acquisition maintains unbiased subset frequencies.

3. Potential confounders are not explicitly addressed like nutritional status, other treatments participants may have taken prior to the study, or concurrent infections they experience. These confounders can affect subset frequencies. 

4. Flow cytometry detail and qPCR detection: While there is strong information on straining, gating, and controls for flow cytometry, there is no information on the thresholds for debris exclusion, number of events acquired, or doublet discrimination parameters that allow for transparency. qPCR detection did not include limitations in the detection and quantification (LOD, LOQ). 

5. Acknowledge Possible Eosinophil Contamination: If eosinophil exclusion cannot be retrospectively validated, I suggest performing sensitivity analysis showing the subset frequencies remain consistent when analyzing only high side-scatter cells. 

Overall, the methods are generally well designed with some major issues that need to be addressed. There are some minor issues like clarifications on the interpretation of AMA1 vs. qPCR exposure differences and few spelling errors (“spnning”, “polymeraase”). Also, neutrophil subset distributions can vary with age during childhood development. If groups are not age-matched, observed differences could partially reflect age effects rather than malaria or disease effects.

Are the data presentations and collections well-suited to represent the data?

Highly suitable

The results are clearly stated and well aligned with the study’s objectives. With the high-quality immunophenotyping and appropriation use of multiple comparison groups, there are clear and consistent biological trends that yield highly significant p-values. However, there are a few major and minor issues that are necessary to be addressed.

Major Issues:

1. Some of the results rely on low median values, making findings sensitive to measurement variability. 

2. Some tables or figures are mentioned but not shown as a visual (e.g.: S2 and S3 tables). Presentation of these tables/figures may need more detail. 

Minor Issues:

1. The referencing of p-values could be clearer with multiple comparisons. 

2. Occasional typos and spelling errors like “Mann-Whithney” in Figure 3. 

Are the conclusions supported by the data?

Mostly supported by the data with few inconsistencies. 

Frequencies of five neutrophil subsets in malaria-exposed (ME) and malaria-naive (MN) children were compared: aged, atypical aged, immature, mature, and mature inactive.

• Malaria-exposed (ME) children showed a marked reduction in mature and mature-inactive neutrophils and a significant increase in aged and atypical aged neutrophils compared to malaria-naive (MN) children, indicating that malaria drives neutrophil aging. 

• Chronic malaria exposure shifts neutrophil profiles in children to resemble adult patterns. Children with Burkitt lymphoma (BL) had higher frequencies of immature neutrophils than ME children, suggesting cancer-driven expansion of this subset for immune evasion. 

• A strong positive correlation in immature neutrophils between ME and BL children indicates a similar expansion pattern in both groups.

These conclusions were directly supported by flow cytometry data with clear statistical significance.  Although much of the data appears to come from in vitro and small-scale clinical cohorts, which are informative but not yet definitive enough to change medical practice or public-health strategies. Linking a specific immune cell phenotype to one regional cancer type limits its global immediacy.

Despite the established link between cancer and immature neutrophils, the data suggest that chronic or repeated malaria exposure may also be contributing to the elevated presence of immature neutrophils, pointing to a possible immunological bridge between malaria and BL pathogenesis.

Major Issues: 

1. Conclusions about causality between malaria or BL and neutrophil subset shifts are suggestive but there is no mechanistic evidence behind the associations. 

2. Small sample sizes and endemic regions of Western Kenya limit generalizability of the results.

How clearly do the authors discuss and interpret their findings and potential next steps for the research?

Somewhat clear with few issues

The researchers generally discussed and interpreted their findings, supporting their conclusions by citing previous studies published in recent years. The discussion section also includes potential implications for understanding immune modulation in malaria-endemic regions. However, future steps for research are limited and not fully detailed. There is also limited discussion of mechanistic pathways that explain neutrophil shifts.

Does the paper maintain professional integrity and ethics?

Professional integrity and ethics are maintained.

• Ethical approval was obtained from multiple recognized review boards.

• Informed consent and assent were obtained for minors, adolescents, and adults.

Would you recommend this preprint to others?

Yes, after major and minor issues are fixed.

I would recommend it after further improvements because of its high quality. Findings are interesting and the study provides clinically relevant insights that could lead to promising advancements in future research.

Competing interests

The authors declare that they have no competing interests.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they used generative AI to come up with new ideas for their review.