Expression of specific var gene subtypes is differentially associated with severe malaria syndromes
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eLife Assessment
This valuable study uses a large cohort of clinical malaria cases collected over 18 years to address a critical knowledge gap regarding the role of PfEMP1 variants across distinct severe malaria syndromes. The conclusions are potentially of importance and interest to those who study malaria severity, but the evidence is incomplete, largely due to a lack of clarity on data inclusion and the correct use of statistical tests. More up-to-date data analysis methods would further strengthen the conclusions.
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Abstract
The virulence of Plasmodium falciparum is closely linked to P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by a diverse var gene family. PfEMP1 mediates parasite immune evasion and vascular adhesion of infected red blood cells, contributing to severe disease. While expression of group A or domain cassettes (DC8 and DC13)-containing var genes have been associated with severe malaria, comparisons between the severe malaria syndromes remain limited. Furthermore, interactions between specific var gene expression and the rosetting phenotype, a known marker of severe malaria, are incompletely understood across severe malaria syndromes.
We analyzed parasites and clinical data from 712 Kenyan children with non-severe and severe malaria syndromes collected over 18 years in Kilifi, Kenya. We used RT-qPCR and DBLα-tag sequencing to quantify var gene expression. We show that parasites expressing var genes containing the cys2 MFK+REY−motif or encoding DC8 domains were associated with impaired consciousness (IC), while rosetting was associated with respiratory distress (RD) and severe malarial anaemia (SMA), but not IC. These findings demonstrate that distinct PfEMP1 variants are preferentially expressed in specific severe malaria syndromes, highlighting potential targets for variant-specific future therapeutic and diagnostic strategies.
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eLife Assessment
This valuable study uses a large cohort of clinical malaria cases collected over 18 years to address a critical knowledge gap regarding the role of PfEMP1 variants across distinct severe malaria syndromes. The conclusions are potentially of importance and interest to those who study malaria severity, but the evidence is incomplete, largely due to a lack of clarity on data inclusion and the correct use of statistical tests. More up-to-date data analysis methods would further strengthen the conclusions.
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Reviewer #1 (Public review):
Summary:
Severe childhood malaria is associated with three main overlapping syndromes: impaired consciousness (IC), respiratory distress (RD), and severe malaria anaemia (SMA). One central feature of severe malaria, driven by host and parasite factors, is the sequestration of parasitized red blood cells in vascular beds, leading to impaired tissue perfusion and lactic acidosis. The causing agent, the parasite ligand PfEMP1, is expressed on the surface of infected red blood cells, where it binds to a broad range of different endothelial receptors. Accumulation of parasite-infected erythrocytes in the host's microvasculature has been repeatedly confirmed for cerebral malaria, but there are scarce data on the extent of sequestration in the other severe malaria syndromes. However, the absence of effective …
Reviewer #1 (Public review):
Summary:
Severe childhood malaria is associated with three main overlapping syndromes: impaired consciousness (IC), respiratory distress (RD), and severe malaria anaemia (SMA). One central feature of severe malaria, driven by host and parasite factors, is the sequestration of parasitized red blood cells in vascular beds, leading to impaired tissue perfusion and lactic acidosis. The causing agent, the parasite ligand PfEMP1, is expressed on the surface of infected red blood cells, where it binds to a broad range of different endothelial receptors. Accumulation of parasite-infected erythrocytes in the host's microvasculature has been repeatedly confirmed for cerebral malaria, but there are scarce data on the extent of sequestration in the other severe malaria syndromes. However, the absence of effective adjunctive therapies for severe malaria implies that our understanding of its pathogenesis remains incomplete. Thus, by comparing var gene expression from a large Kenyan cohort (n=372 severe cases; n=340 non-severe cases), this study addresses a critical knowledge gap regarding the role of PfEMP1 across distinct severe malaria syndromes. The substantial sample size, phenotypic stratification, and use of two complementary methods (DBLa-tag sequencing and RT-qPCR), along with data about the parasite's ability to form rosettes and antibody level assessments, provide a strong setup. Var gene expression data - either proportions of different DBLa-tags classified by the number of cysteine residues and presence of particular motifs or relative expression RT-qPCR data from a set of primer pairs targeting conserved regions of var groups or particular domains - is associated with (a) severe malaria syndromes, (b) variant expression homogeneity, (c) rosetting ability, and (d) mortality using independent linear regression models, spearman ranks correlations, or logistic regression models. In summary, the study confirms that A-type and DC8-containing gene expression correlate with IC, that RD is associated with rosetting, and that SMA is linked to a high variant expression homogeneity (VEH) of var-A expression, which may indicate a longer infection duration. However, some findings remain inconclusive. For example, when analyzing pure syndromes, several associations changed: DC8 expression was also found to be significantly enriched in SMA (with multiple primer pairs) and RD, not exclusively with IC. Additionally, rosetting was associated with DC8 expression but not with IC, even though IC itself is linked to DC8 expression. Overall, the findings are significant and supported by a large dataset, though the reported evidence remains largely associative rather than mechanistic.
Strengths:
As the authors stated themselves, one of the key unresolved questions is whether severity-causing parasites are biologically different from parasites responsible for asymptomatic infections. This study is among the first to address this question using data from a large, phenotypically stratified cohort. The use of two complementary methods (DBLa-tag sequencing and RT-qPCR), together with data on the parasites' ability to form rosettes and assessments of antibody levels, provides an excellent experimental framework.
Weaknesses:
Even when assessing var gene expression using two different approaches - DBLα-tag sequencing and RT-qPCR targeting pre-defined variants - only a glimpse of the parasites' actual biology is captured. Moreover, a well-known confounder in gene expression studies of P. falciparum field isolates is variation in parasite age (hours post-invasion) or synchronicity, both of which significantly influence var gene expression. The methods employed in this study, unfortunately, do not allow for controlling or correcting for these factors. Then, the old classification system of DBLa-tag data developed by Bull et al is certainly still valid; however, more recent advances in bioinformatic tool development now allow for a more in-depth exploration of DBLa-tag datasets. Tools such as Varia (doi: 10.1186/s12859-022-04573-6), cUPS (https://doi.org/10.1371/journal.ppat.1012813), and upsML (doi: https://doi.org/10.1101/2025.05.19.654848) enable the prediction of DBLa-tag-connected PfEMP1 domains and the var group affiliations.
As A-type var gene expression has already been associated with severity, most expression studies (including this one) have a selection bias towards A- and B/A-type var genes. Here, A- and B/A-types are covered by 8 primer pairs (gpA1, gpA2, 4x DC8, DC13, DC4), whereas high polymorphic B-types are targeted by only 2 primer pairs (b1, DC9) and C-types only by a single primer (c2). Thus, any association with A-type expression is more likely to be observed, although evidence is accumulating that parasites are preferably expressing B-type var genes at the onset of blood stage infection in naïve/less immune individuals; this is also consistent with the observation of the authors that VEH is positively associated with immunity (measured as anti-IE) and negatively associated with temperature.
I am not an expert in biostatistics, but to my understanding, independently performed regressions should be corrected for multiple testing.Overall, the authors largely achieved their aims, identifying specific var groups associated with different severity syndromes. However, due to the complexity of var gene data and the interdependence of parameters, the resulting picture is not entirely clear. Some opposite results between different analyses may also be difficult for the reader to interpret. Nevertheless, this study can be considered a pioneering effort, providing valuable insights into the complex interplay of var gene expression across different severity syndromes and offering useful data for the field. Follow-up studies will be important to validate these findings and further dissect the mechanisms linking parasites gene expression to clinical outcomes.
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Reviewer #2 (Public review):
Summary:
The manuscript presents results of a study using two complementary approaches (RT-qPCR and DBL) to analyze the putative relationship between var gene transcription (and hence, PfEMP1 expression) and clinical presentation among Kenyan children with Plasmodium falciparum malaria. Binary rosetting (yes/no) data are used in a similar way. The study includes samples collected over a period of almost 20 years from about 700 children presenting with either severe (impaired consciousness [IC], respiratory distress [RD], severe anemia [SA]) or non-severe malaria. During the study period, the study area experienced a remarkable drop in P. falciparum transmission intensity.
Strengths:
The study stands on the shoulders of many similar studies of this kind, both by the authors and by other research teams, and …
Reviewer #2 (Public review):
Summary:
The manuscript presents results of a study using two complementary approaches (RT-qPCR and DBL) to analyze the putative relationship between var gene transcription (and hence, PfEMP1 expression) and clinical presentation among Kenyan children with Plasmodium falciparum malaria. Binary rosetting (yes/no) data are used in a similar way. The study includes samples collected over a period of almost 20 years from about 700 children presenting with either severe (impaired consciousness [IC], respiratory distress [RD], severe anemia [SA]) or non-severe malaria. During the study period, the study area experienced a remarkable drop in P. falciparum transmission intensity.
Strengths:
The study stands on the shoulders of many similar studies of this kind, both by the authors and by other research teams, and the inferences made largely confirm those made previously. The current study has analytical rigor and a large sample size. Disentangling the multiple parameters of the above-mentioned relationship is of obvious and crucial importance to an improved understanding of P. falciparum malaria pathogenesis and of the targets and mechanisms of protective immunity to the disease. The present study is a valuable effort towards that. The study is well-structured, and the figures are clear.
Weaknesses:
It is somewhat unclear to this reviewer to what extent the samples and data analyzed and reported here are new (i.e., not used/analyzed in previous studies). If there is substantial overlap with earlier studies, this is a weakness because of the risk of circular inferences. The Discussion section would benefit from less repetition of the results section and a more in-depth discussion of the findings obtained relative to the existing literature. Better inclusion of key primary references is recommended.
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Reviewer #3 (Public review):
Summary:
In this manuscript, Ndugwa et al. attempt to link specific severe malaria manifestations with particular var gene expression patterns. This is an important question, and the dataset the authors have assembled over decades is impressive. However, greater clarity in the descriptions and statistics would, in my view, help this reviewers, and readers in general develop a more precise understanding of the significance of the findings.
Strengths:
The study addresses a critically important question in malaria pathogenesis, and the dataset is extensive and represents a significant long-term effort by the authors.
Weaknesses:
The Results section often lacks clarity: clinical group definitions (NS, non-IC, non-SMA, mild vs. moderate) are sometimes ambiguous, and key methodological details, including the VEH …
Reviewer #3 (Public review):
Summary:
In this manuscript, Ndugwa et al. attempt to link specific severe malaria manifestations with particular var gene expression patterns. This is an important question, and the dataset the authors have assembled over decades is impressive. However, greater clarity in the descriptions and statistics would, in my view, help this reviewers, and readers in general develop a more precise understanding of the significance of the findings.
Strengths:
The study addresses a critically important question in malaria pathogenesis, and the dataset is extensive and represents a significant long-term effort by the authors.
Weaknesses:
The Results section often lacks clarity: clinical group definitions (NS, non-IC, non-SMA, mild vs. moderate) are sometimes ambiguous, and key methodological details, including the VEH index calculation, RT-qPCR quantification, antibody detection methods, and rosetting assays, are either missing from the results text or poorly explained in the figure legends. Additionally, figure presentation requires improvement, with inconsistent reporting of sample sizes, undefined colors, and p-values that overlap with data points rather than being clearly displayed above them.
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