Abstract
Introduction:
The association between thrombocytopenia and parasite density or disease severity are described in numerous studies. In recent years, several studies described the protective role of platelets in directly killing Plasmodium parasites, mediated by platelet factor 4 binding to Duffy antigen.
Objective:
To evaluate the protective role of platelets in young children that are Duffy antigen-negative, such as those in sub-Saharan Africa.
Methods:
A zero-inflated negative binomial (ZINB) model was used to relate platelet count and parasite density data collected in a longitudinal birth cohort. Platelet factors were measured by ELISA in samples collected from malaria-infected children participated in a cross-sectional study.
Results:
We described that an increase of 10,000 platelets/μl was associated with a 2.76% reduction in parasite count. Increasing levels of platelet factor 4 and CXCL7 levels were also significantly associated with reduction in parasite count.
Conclusions:
Platelets play a protective role in reducing parasite burden in Duffy-negative children, possibly mediated through activation of the innate immune system.
Introduction
Infection with Plasmodium falciparum is a major public health problem. Clinical presentation varies from asymptomatic infection to severe disease. In areas of high malaria transmission, young children bear most of the burden of severe disease and mortality [1].
In addition to their role in hemostasis, platelets contribute to both innate and adaptive immune responses [2]. In the context of Plasmodium infections in humans, reduction in platelet counts during malaria infection is one of the common hematological changes observed, in both non-immune young children and semi-immune adults living in malaria-endemic areas. Among symptomatic adults in Ghana, 47% presented with thrombocytopenia, and platelet counts were significantly lower with increasing parasite density [3]. In a study conducted near the Thai-Myanmar border that included both children and adults, 84.9% of individuals with clinical malaria presented with thrombocytopenia [4]. Asymptomatic P. falciparum infection among children aged 5–11 years (but not adolescents) in Mozambique and children aged 2–9 in Nigeria were associated with a significant reduction in platelet counts compared to counts in uninfected children [5, 6]. The proportion of malaria-infected children with thrombocytopenia was almost 50%, and thrombocytopenia was associated with increased parasite density [7].
In model studies of in vitro cultured P. falciparum parasites, platelets inhibited parasite growth that occurred when platelets were allowed to directly interact with infected erythrocytes and parasite killing by platelets was mediated by platelet factor 4 (PF4, CXCL4). PF4 is taken up by infected erythrocytes following PF4 binding to Duffy antigen expressed on the surface of erythrocytes (reviewed in [8]). PF4 is taken up by infected erythrocytes following PF4 binding to Duffy antigen expressed on the surface of erythrocytes. A study conducted in patients infected with P. falciparum, P. vivax or P. knowlesi in Indonesia and Malaysia confirmed that platelets play a role in killing parasites in vivo that is mediated by PF4 [9].
Most people living in Sub-Saharan Africa are Duffy-negative [10]. This raises the question of whether platelets contribute to parasite control in the absence of PF4-Duffy antigen mediated killing of parasites. Here, we examine the relationship between platelet counts and parasite density in young children that participated in a longitudinal birth cohort. In addition, we related plasma levels of soluble platelet chemokines PF4 and CXCL7 with parasite density in samples collected from children that participated in a cross-sectional study.
Methods
Study population and clinical procedures
The study was conducted in Ouélessébougou, Mali located 80 Km south of Bamako. Children were enrolled between January 2011 and December 2014 into a longitudinal birth cohort, and between August 2022 and December 2022 into a cross-sectional study of hospitalized children and community control. A parent or guardian provided informed consent for their child’s participation in the study. The protocol and study procedures were approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (ClinicalTrials.gov ID NCT01168271), and the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Bamako, Mali.
Children in the longitudinal cohort were seen monthly during the malaria transmission season (July-December) and every two months during the dry season (January-June), and any time the child was sick. Scheduled and unscheduled visits included clinical examination and blood smear microscopy for detection of malaria parasites and complete blood count.
Children in the cross-sectional study were enrolled at the hospital (n=40) and in the community (n=120). Children enrolled in the community were matched for age to children enrolled at the hospital, and were malaria-positive at enrollment based on rapid diagnostic test (RDT). Assessments included clinical examination, blood smear microscopy and complete blood count. P. falciparum parasite biomass was also measured by P. falciparum histidine rich protein 2 (PfHRP2) ELISA as previously described [11].
Iron status was based on zinc protoporphyrin (ZPP)/heme (H) ratio in the longitudinal cohort, defined as ZPP/H ≥80 μmol/mol. In the cross-sectional study, iron status was defined based on ferritin concentration corrected for inflammation. Iron deficiency (ID) was defined as ferritin concentration of <30 ng/ml in the absence of inflammation (CRP <8.2 μg /ml), or ferritin concentration of <70 ng/ml in the presence of inflammation (CRP >8.2 μg/ml) [12]. Ferritin and CRP levels were measured using DuoSet ELISA (R&D systems) according to the manufacturer’s instructions.
Platelet factors
To measure platelet factor, blood was collected in acid citrate dextrose (ACD) tubes and platelet poor plasma was prepared as previously described [13]. Briefly, blood samples were centrifuge for 10 minutes at 1,000 g. Plasma was transferred to a new tube and centrifuge at 10,000 g for 10 minutes and the plasma supernatant was transferred to a new tube. Plasma samples were stored at −80°C until use. PF4 and CXCL7 levels were measured using DuoSet ELISA (R&D systems) according to the manufacturer’s instructions.
Statistical analysis
To evaluate the association between platelet counts and parasite density in the longitudinal cohort, a zero-inflated negative binomial (ZINB) model with random effects to account for multiple data points per child was used. This model was employed as children were not infected with malaria during most visits, resulting in 89.5% of blood smears showing zero parasite counts.
The associations between platelet factors and parasite density in the cross-sectional study were also analyzed using a ZINB model, as 47 of 120 children showed parasite counts of zero. The associations between PfHRP2 levels and platelet factors were evaluated using a zero-inflated gamma model. PfHRP2 concentration was below the level of detection in 21 samples, which were imputed as zero.
Significance level was set at a p value of less than 0.05.
Results
Study population
Longitudinal birth cohort
1462 children participated in the mother-infant longitudinal cohort (Table 1). Children were enrolled at birth and followed for a median of 28.2 months [interquartile range (IQR), 18.5–39.6]. In this cohort, iron status was based on zinc protoporphyrin (ZPP)/heme (H) ratio. Elevated ZPP is an indicator of iron-deficient erythropoiesis and can be used as an indicator of iron status [14]. 1045 children were iron-deficient during follow up, and 1029 children experienced at least one malaria infection during the study. Platelet counts were significantly lower (p<0.0001) during blood smear-positive visits. Median (IQR) platelet count in visits with a positive blood smear was 243×103/μl (160 ×103/μl −353 ×103/μl), and with a negative blood smear, 442 ×103/μl (359×103/μl −540 ×103/μl).
Table 1.
Study population
| Longitudinal birth cohort | |
|---|---|
| Number of children | 1462 |
| Median follow up duration | 28.3 (18.5–39.6) months |
| Hemoglobin genotype | |
| AA | 1173 |
| AC1 | 149 |
| AS2 | 140 |
| Hospitalized and community control | |
| Number of children | 160 |
| Median age at enrollment | 36 months |
| Hemoglobin genotype | |
| AA | 134 |
| AC | 16 |
| AS3 | 10 |
including 2 children with HbCC
including 3 children with HbSS and 6 children with HbSC
including 1 child with HbSC
Hospitalized and community control study
Since platelet-poor plasma samples were not available from children that participated in the longitudinal birth cohort, platelet soluble factors were measured in platelet-poor plasma samples collected from 160 children that participated in a cross-sectional study. This cohort included malaria-infected children admitted to the hospital (n=40) and malaria-infected children in the community matched for age (n=120). Median age of children enrolled at the hospital and in the community was 36 months (Table 1).
All 160 children were malaria-positive at enrollment based on rapid diagnostic test. Blood smears were negative for 42 children enrolled in the community as well as for 5 hospitalized children. 33.1% of children were iron-deficient defined by ferritin levels corrected for inflammation status. Platelet counts were significantly lower in children with febrile or severe malaria than children with non-febrile malaria infections. Median (IQR) platelet count in children with febrile or severe malaria was 123 ×103/μl (73×103/μl −193 ×103/μl), and in children with non-febrile infection, 243 ×103/μl (139 ×103/μl −345×103/μl).
Increase in platelet count is associated with reduced parasite density
Within the longitudinal cohort, the blood smear results were negative in 89.5% of the visits. Therefore, a zero-inflated negative binomial regression model with random effect (ZINBRE) was fitted to account for multiple visits per child. The main goal of this analysis was to evaluate the association between platelet count and parasite density, rather than modeling the probability of excess zeros; below we describe the negative binomial component of the model (count model), and the zero-inflated component is described in Supplementary Table 1. The model was adjusted for age, as well as host factors known to reduce parasite density including hemoglobin type and ID. An increase of 10,000 platelets/μl was associated with a 2.76% decrease in parasite count (Table 2, model 1). Hemoglobin AS was also associated with a significant reduction in parasite density, as previously reported for this cohort [15]. ID was negatively associated with parasite count, but the effect was not significant, unlike previous studies describing ID associated with reduction in malaria infection and parasite density [12, 16]. One possible explanation for the non-significant association between ID and parasite density is collider bias. An earlier study reported that thrombocytosis in young children is commonly associated with non-malaria infections and ID [17]. Although platelets have been associated with reduction in parasite density, malaria infection is associated with a reduction in platelet count as described above. Because platelet count is potentially affected by both ID and parasite count, platelet count measured during infection is a collider for the association between ID and parasite density. This collider bias may influence the magnitude and direction of the coefficient, making it difficult to interpret the degree of ID effect on reduction in parasite burden. Therefore, we separately evaluated the association between ID and parasite density. In a ZINBRE model adjusted for hemoglobin type and age, ID was associated with a significant 20.5% reduction in parasite density (Table 2, model 2).
Table 2.
Zero-inflated negative binomial model with random effects for the association between platelets and parasite density
| Variable | Model 1 Coefficient (95% CI) |
Model 1 P value |
Model 2 Coefficient (95% CI) |
Model 2 P value |
|---|---|---|---|---|
| Platelet count | −0.0028 (−0.0033, −0.0024) | <0.0001 | ||
| Iron deficiency | −0.128 (−0.285, 0.030) | 0.1 | −0.229 (0.387, −0.071) | 0.005 |
| HGB AC | −0.099 (−0.382, −0.184) | 0.5 | −0.213 (−0.495, 0.069) | 0.1 |
| HGB AS | −1.028 (−1.334, −0.722) | <0.0001 | −1.179 (−1.487, −0.870) | <0.0001 |
| Age | 0.033 (0.027, 0.040) | <0.0001 | 0.040 (0.0328, 0.047) | <0.0001 |
Increased PF4 and CXCL7 levels are associated with a reduction in parasite density
To further evaluate platelet contribution to the reduction in parasite density, we measured activation markers platelet factor 4 (PF4, CXCL4) and CXCL7, and related these levels with parasite density in our cross-sectional study. The models were adjusted for age, platelet count, hemoglobin type and ID. PF4 levels were negatively associated with parasite density (Table 3A). Parasite density was reduced by 16.7% for an increase of 100 ng/ml of PF4. Similarly, an increase of 100 ng/ml in CXCL7 concentration was associated with a 10.3% reduction in parasite density (Table 3B).
Table 3.
Zero-inflated negative binomial analysis for the association between platelet factors and parasite density measured by blood smear microscopy
| A. Platelet factor 4 (PF4) | ||
|---|---|---|
| Variable | Coefficient (95% CI) | P value |
| PF4 | −0.0018 (−0.003- −0.0004) | 0.01 |
| Platelet count | −0.004 (−0.008- −0.0009) | 0.01 |
| Age | 0.005 (−0.009–0.019) | 0.5 |
| HGB AS | −3.226 (−4.630- −1.822) | <0.0001 |
| Iron deficiency | 0.35 (−0.475–1.180) | 0.4 |
| B. CXCL7 | ||
|---|---|---|
| Variable | Coefficient (95% CI) | P value |
| CXCL7 | −0.0011 (−0.002, −0.0006) | <0.0001 |
| Platelet count | −0.005 (−0.008, −0.001) | 0.006 |
| Age | 0.005 (−0.009, 0.019) | 0.5 |
| HGB AS | −3.10 (−4.483, −1.718) | <0.0001 |
| Iron deficiency | 0.39 (−0.445, 1.231) | 0.4 |
We also evaluated the relationships between P. falciparum histidine rich protein 2 (PfHRP2) levels, another measure of parasite biomass and PF4 and CXCL7 levels (Tables 4A, B). The models were adjusted for age, platelet count, hemoglobin type and ID. CXCL7 levels were negatively associated with PfHRP2 concentration. An increase of 100 ng/ml of CXCL7 was associated with a 6.7% reduction in PfHRP2 concentration, but this relationship did not achieve significance (p=0.06).
Table 4.
Zero-inflated gamma analysis for the association between platelet factors and parasite density measured by PfHRP2 concentration
| A. Platelet factor 4 (PF4)a | ||
|---|---|---|
| Variable | Coefficient (95% CI) | P value |
| PF4 | −0.0004 (−0.001, 0.0006) | 0.4 |
| Platelet count | −0.005 (−0.008, −0.001) | 0.006 |
| Age | −0.003 (−0.016, 0.010) | 0.7 |
| HGB AS | −2.07 (−3.29, −0.85) | 0.0009 |
| Iron deficiency | −2.51 (−3.36, −1.66) | <0.0001 |
| B. CXCL7 | ||
|---|---|---|
| Variable | Coefficient (95% CI) | P value |
| CXCL7 | −0.0007 (−0.001, 0.00004) | 0.06 |
| Platelet count | −0.005 (−0.008, −0.001) | 0.007 |
| Age | −0.004 (−0.02, 0.009) | 0.6 |
| HGB AS | −2.10 (−3.32, −0.89) | 0.0007 |
| Iron deficiency | −2.41 (−3.29, −1.54) | <0.0001 |
Discussion
In malaria-endemic regions, immunity to severe malaria develops at a young age, but anti-parasite immunity that can control parasite density is observed at an older age [1, 18, 19]. In addition to naturally acquired immunity, host factors also contribute to control of parasite density [1]. In the current study, we examined the associations between platelet count and parasite density in the context of a longitudinal birth cohort as well as a cross-sectional study. Results from the longitudinal birth cohort show that increased platelet count was associated with decreasing parasite density. The cross-sectional study of malaria-infected children showed increasing platelet factors PF4 and CXCL7 levels were associated with reduction in parasite density (p=0.01, and p<0.0001 respectively), further supporting the role of platelets in reducing parasite burden.
The association between platelets and P. falciparum parasites is complex. Surface-expressed platelet proteins like CD36, PECAM1/CD31 and gC1qR mediate adhesion of infected erythrocytes (IE) [20–22], resulting in platelet-mediated clumping [23]. Platelet-mediated clumping was higher in parasite samples collected from children with hyperparasitemia, but similar between children with severe disease or uncomplicated malaria [24]. The association between platelets and malaria pathogenesis was also reported in histopathological studies describing the accumulation of platelets and monocytes together with IEs in brain vasculature of children that died from cerebral malaria, compared to children that died of severe malarial anemia or nonmalarial encephalopathy [25]. Among children with cerebral malaria, those that were retinopathy-negative (an indicator of a less severe form of cerebral malaria) had higher PF4 plasma levels and platelet-derived growth factor compared to retinopathy-positive children [26]. The authors suggested increased platelet activation in retinopathy-negative children may indicate platelet activation at an earlier stage of the disease, resulting in a less severe form of cerebral malaria [26].
Here, we described that PF4 is associated with a reduction in parasite density, consistent with the protective role of platelets during malaria infection described by McMorran et al [8]. Further, the associations between increased platelet counts and reduced parasitemia is similar to a study conducted in Indonesia and Malaysia, in which parasitemia was negatively associated with platelet counts and IE-platelet complexes, with higher platelet-IE complexes in individuals with low compared to high parasitemia [9]. The proportion of IE killed by platelets, indicated by PF4 and TUNEL staining, negatively correlated with parasite density [9]. Platelet activation in response to malaria infection, platelet binding to IE, shorter life span during malaria infection, and peripheral destruction [27] could be the major reasons for thrombocytopenia associated with malaria infection. Overall, platelets may contribute to reducing parasite density as described here, and disease severity, but are also part of disease pathogenesis through IE adhesion and clumping.
The current study expands on the protective role of platelets during malaria infection that is mediated by reducing parasite density in a population that is Duffy antigen-negative. Therefore, parasite killing is not directly mediated by intraerythrocytic accumulation of PF4 but by a different, currently unknown mechanism. Platelet-soluble mediators like PF4 and CXCL7 are involved in recruiting immune cells like monocytes and neutrophils to the site of inflammation [28]. We speculate that factors secreted by platelets in response to infection mediate reduction in parasite density by recruiting immune cells to the site of inflammation.
PfHRP2 concentrations have been used to estimate total parasite biomass [29]. A limitation of the study is that parasite biomass was not measured in the longitudinal cohort, thus platelet counts were only related to parasite density based on blood smear microscopy count. PfHRP2 levels were measured in the cross-sectional study that was used to relate platelet factors with parasite density. Although PF4 and CXCL7 levels were associated with decreased parasite density measured by blood smear microscopy, the negative association between CXCL7 and PfHRP2 approached significance, but not PF4. We speculate that the lack of significant associations between PfHRP2 and platelet factors PF4 and CXCL7 could be partly due to the fact that PfHRP2 can be detected in blood several weeks after infection is resolved [30] that may explain in part that some children in the current study were infected by PfHRP2-based RDT but not by blood smear microscopy; at this stage platelet activation is similar to pre-infection state.
In our previous birth cohort study conducted in Tanzania, we described that ID was associated with reduction in malaria infection, severe malaria and parasite density [12]. We proposed that ID is associated with reduction in parasite density by limiting parasite access to iron and by increasing inducible nitric oxide synthase expression, resulting in higher nitric oxide levels that mediate macrophage activity against the parasites [12]. In the longitudinal birth cohort study described here, ID as a covariate in the model that included platelet count was not significantly associated with reduction in parasite density. We proposed that our estimate of the association between ID and parasite density is subject to collider bias when adjusted for platelet count measured during infection. Alternatively, because ID is associated with thrombocytosis, it is possible that reduction in parasite density in iron-deficient children is mediated in part by increasing platelet counts; in this case, adjustment for platelet count could mask some of the association between ID and parasite count.
In summary, we describe that platelets and platelet factors are associated with reduction in parasite density in young children residing in sub-Saharan Africa. Because our cohort was from west Africa, and therefore overwhelmingly Duffy-negative, our results suggest platelet protective activity is mediated by non-PF4/Duffy interactions. In the population described here, parasite killing by platelet cannot be directly mediated by PF4/Duffy, and the effect of CXCL7 on parasite density is similar to PF4, suggesting platelets contribute to controlling parasitemia by activating other immune cells such as monocytes and neutrophils.
Supplementary Material
Highlights.
Platelet count is negatively associated with parasitemia in Malian children
Increased PF4 and CXCL7 levels are associated with reduction in parasite count
Platelets play a role in controlling parasite density in Malian children
Acknowledgements
This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Footnotes
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Competing Interests
The authors declare no competing interests are associated with this manuscript.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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