The authors have declared that no competing interests exist.
Collected clinical data: SD DK MD JMA MD. Conceived and designed the experiments: NKMM DLvdH TMLM JMA L SA AR MD CAL RMF. Performed the experiments: NKMM DLvdH TMLM. Analyzed the data: NKMM WG MPF RMF. Wrote the paper: NKMM MPF RMF.
Inflammatory cytokinemia and systemic activation of the microvascular endothelium are central to the pathogenesis of
We measured levels of UA and soluble forms of intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-Selectin), thrombomodulin (sTM), tissue factor (sTF) and vascular endothelial growth factor (VEGF) in the plasma of Malian children aged 0.5–17 years with uncomplicated malaria (UM, n = 487) and non-cerebral severe malaria (NCSM, n = 68). In 69 of these children, we measured these same factors once when they experienced a malaria episode and twice when they were healthy (i.e., before and after the malaria transmission season). We found that levels of UA, sICAM-1, sVCAM-1, sE-Selectin and sTM increase during a malaria episode and return to basal levels at the end of the transmission season (p<0.0001). Plasma levels of UA and these four endothelial biomarkers correlate with parasite density and disease severity. In children with UM, UA levels correlate with parasite density (r = 0.092, p = 0.043), sICAM-1 (r = 0.255, p<0.0001) and sTM (r = 0.175, p = 0.0001) levels. After adjusting for parasite density, UA levels predict sTM levels.
Elevated UA levels may contribute to malaria pathogenesis by damaging endothelium and promoting a procoagulant state. The correlation between UA levels and parasite densities suggests that parasitized erythrocytes are one possible source of excess UA. UA-induced shedding of endothelial TM may represent a novel mechanism of malaria pathogenesis, in which activated thrombin induces fibrin deposition and platelet aggregation in microvessels. This protocol is registered at clinicaltrials.gov (NCT00669084).
Although the mechanisms of endothelial activation, damage and dysfunction in malaria have not been fully elucidated, the systemic microvascular sequestration of
UA is a weak organic acid present mainly as monosodium urate at physiological pH, and is found as microscopic crystals in diseases (e.g., gout) associated with elevated UA levels
In Malian children, we recently found that plasma UA levels increase during an acute episode of
All protocol activities were approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Odontostomatology at the University of Bamako, Mali, and the Institutional Review Board of the U.S. National Institute of Allergy and Infectious Diseases. Written informed consent was obtained from the parent or guardian of all children. The protocol is registered at clinicaltrials.gov (NCT00669084).
This study was conducted within a 4-year (2008–2012) prospective cohort study of malaria incidence in three rural Malian villages (Kenieroba, Fourda and Bozokin) (Lopera-Mesa
UM was defined by (i) fever (axillary temperature ≥ 37.5°C, or history of fever in the previous 24 h) with or without other symptoms of malaria (headache, body aches and malaise), (ii) the presence of any asexual
Venous blood (2–10 ml) was obtained from a sub-cohort of 69 healthy children aged 3–11 years before and after the 2009 transmission season, at which times they had undetectable parasitemia. Venous blood was also obtained from all children who presented with malaria. Blood samples were collected by venipuncture in sodium heparin Vacutainers® (Becton Dickinson, Franklin Lakes, NJ) and transported for 2 h on ice to the University of Bamako. Plasma was separated from whole blood by centrifugation at 2500 rpm for 10 min at ambient temperature, aliquotted, immediately stored at −80°C, shipped in a liquid nitrogen dry shipper to NIAID, and maintained at −80°C until use.
Plasma samples were thawed at room temperature and centrifuged at 14000 rpm for 10 min at 4°C before measuring UA and endothelial biomarker levels. UA levels were quantified in triplicate by a colorimetric method with a linear detection range of 0.22–30 mg/dl using a QuantiChrom™ Uric Acid Assay Kit (Bioassay Systems, Hayward, CA). Levels of sICAM-1, sVCAM-1 and sE-Selectin were quantified using a Human Adhesion Molecule MultiAnalyte Profiling Base kit (R&D Systems, Minneapolis, MN); samples were diluted 30-fold according to the manufacturer’s instructions and analyzed using a Luminex200™ flow-based sorting and detection platform (Invitrogen, Carlsbad, CA). sTM levels were measured using a Human Thrombomodulin Quantikine ELISA (R&D Systems); samples were diluted 10-fold according to the manufacturer’s instructions and optical densities measured using a microplate reader set to 450 nm with 750 nm wavelength correction. sTF levels were quantified using the Human Coagulation Factor III/Tissue Factor Quantikine ELISA (R&D Systems); samples were diluted 2-fold and assayed according to the manufacturer’s instructions. VEGF levels were measured using the Human VEGF single bead Luminex Kit (Invitrogen); samples were diluted 3-fold, assayed according to the manufacturer’s instructions, and analyzed using a Luminex200™ flow-based sorting and detection platform (Invitrogen).
Due to technical issues, there is a small number of children with missing UA or endothelial biomarker levels [UM (UA, 2; sICAM1, 4; sVCAM1, 4; sE-selectin, 4; sTM, 5; sTF, 5; VEGF, 15) and NCSM (VEGF, 6)]. For clarity of presentation, we refer to ‘487’ children with UM and ‘68’ children with NCSM throughout this report. To compare groups of children, Fisher’s exact test was used for categorical variables (sex) and the Mann-Whitney test for continuous variables (age, hemoglobin level, parasite density, UA and biomarker levels). The Wilcoxon signed rank test was used to compare the changes in levels of UA and biomarkers in children either from before the transmission season to a malaria episode, or from a malaria episode to after the transmission season. Confidence intervals on the fold-change over time used t-tests on the log-transformed responses. Spearman’s correlation test was used to measure correlations between UA levels, biomarker levels and parasite densities. Spearman’s correlation is based on ranks, so it measures the correlation for the bulk of the data and is not overly influenced by points at the extremes. We sometimes additionally check for continued significance after adjusting p-values with Holm’s multiple comparison correction test because we were testing six biomarkers
We studied 555 Malian children aged 0.5–17 years who presented with UM (n = 487) or NCSM (n = 68) in the 2009 transmission season. Most children with NCSM had prostration, repetitive vomiting, cessation of eating and drinking or some combination of these criteria. Only 2 children with NCSM developed severe anemia. None of the children died. The groups of children with UM and NCSM did not differ significantly in sex (female: 52.4% for UM
Parameter |
Uncomplicated malaria (n = 487) | Non-cerebral severe malaria (n = 68) | p-value |
|
255/232 | 36/32 | 1.0 |
|
6 | 5 | 0.23 |
(3–9) | (3–8) | ||
|
10.3 | 9.9 | 0.04 |
(9.1–11.4) | (9.0–10.7) | ||
|
12300 | 35363 | <0.0001 |
(2250–28950) | (14250–61556) |
The median (IQR) of each variable is shown, except for sex ratio.
p-values were calculated using the Mann-Whitney test, unless otherwise specified.
p-value was calculated using the Fisher’s exact test.
To determine whether plasma UA levels increase during a malaria episode and return to basal levels thereafter, we measured UA levels in paired plasma samples from 69 children before and after the 2009 transmission season, when they were healthy and without parasitemia, and at their first episode of malaria in the interim. We found that geometric mean basal UA levels increase 1.23-fold (95% CI 1.12–1.35, p<0.0001) during a malaria episode and then decrease by a factor of 1/1.23 = 0.82 (95% CI 0.75–0.89, p<0.0001) to basal levels after the transmission season (
A. UA levels were quantified in paired plasma samples from 69 Malian children before and after the 2009 malaria transmission season, and at their first episode of malaria in the interim. B. UA levels were quantified in plasma samples from Malian children with uncomplicated (UM, n = 487) or non-cerebral severe malaria (NCSM, n = 68). Boxplots show the median, interquartile range, with outliers shown as open circles beyond the range. Data points are displayed by density
Parameter |
Before | Episode | p-value |
After | p-value |
(n = 69) | (n = 69) | (Before |
(n = 69) | (Episode |
|
|
3.31 | 3.95 | <0.0001 | 3.31 | <0.0001 |
(2.93–3.86) | (3.46–4.59) | (2.93–3.66) | |||
|
369 | 530 | <0.0001 | 388 | <0.0001 |
(325–455) | (440–607) | (343–475) | |||
|
712 | 1151 | <0.0001 | 774 | <0.0001 |
(621–816) | (971–1518) | (674–988) | |||
|
53.5 | 92.7 | <0.0001 | 55.5 | <0.0001 |
(42.4–72.5) | (73.7–124) | (43.3–67.9) | |||
|
3.66 | 4.90 | <0.0001 | 3.62 | <0.0001 |
(2.89–4.24) | (3.71–5.96) | (2.83–4.07) | |||
|
35.5 | 33.0 | 0.0194 | 31.9 | 0.1691 |
(27.0–45.6) | (21.9–43.7) | (24.03–39.4) | |||
|
4.54 | 4.71 | 0.1740 | 6.50 | 0.1319 |
(2.30–7.38) | (2.63–9.15) | (3.29–9.50) |
The median (IQR) of each variable is shown.
p-values were calculated using the Wilcoxon signed rank test.
To determine whether plasma levels of UA and endothelial biomarkers increase with disease severity, we compared these levels in 487 and 68 children with UM and NCSM, respectively. We found that levels of UA, sICAM-1, sVCAM-1, sE-Selectin and sTM are higher in children with NCSM compared to those with UM (
Parameter |
Uncomplicated malaria | Non-cerebral severe malaria | p-value |
(n = 487) | (n = 68) | (UM |
|
|
4.81 | 5.11 | 0.014 |
(3.98–5.65) | (4.41–6.23) | ||
|
711 | 1374 | <0.0001 |
(555–886) | (773–1584) | ||
|
1036 | 1178 | 0.0001 |
(847–1243) | (1006–1418) | ||
|
87.3 | 109 | <0.0001 |
(60.5–123) | (81.7–155) | ||
|
5.52 | 6.68 | <0.0001 |
(4.42–6.65) | (5.48–7.98) | ||
|
35.7 | 31.5 | 0.031 |
(26.6–48.9) | (22.1–43.8) | ||
|
4.04 | 4.05 | 0.698 |
(2.29–7.62) | (1.99–7.13) |
The median (IQR) of each variable is shown.
p-values were calculated using the Mann-Whitney test.
We explored whether plasma UA during a malaria episode is associated with
To explore whether parasite-derived UA (in addition to other parasite-derived factors) contribute to the endothelial pathology of malaria, we analyzed correlations between UA and endothelial biomarker levels in 487 children with UM. Interestingly, UA levels correlate only with sICAM-1 (r = 0.255, p<0.0001) and sTM (r = 0.175, p = 0.0001) levels; these correlations remain significant after using Holm’s correction to adjust for testing six biomarkers. We used Spearman correlations, which are driven by the bulk of the data, located in the middle range of UA levels (
The levels of UA, sICAM-1 and sTM were quantified in plasma samples from 487 Malian children at their first episode of uncomplicated
To further investigate a specific role for UA in the endothelial pathology of malaria, we investigated whether UA levels predict those of six endothelial biomarkers – independent of the effects of parasite density. Using a series of simple linear models with each log10 biomarker as a response, we found that, for a fixed log10 parasite density, there is a 1.29-fold (95% CI 1.07–1.56, p = 0.008) increase in sTM for each log10 increase in UA. Fold-changes in the other five biomarkers are not significant or the linear model assumptions were not met.
We hypothesized that UA contributes to the pathogenesis of
Studying the role of UA in the pathogenesis of endovascular diseases such as cardiovascular disease, hypertension and diabetes has been challenging
To our knowledge, we report the first data implicating UA in the endothelial pathology of human malaria. Our study has several strengths. The major finding of an association between UA and sTM levels is based on data from a large number (n = 487) of Malian children with UM and is adjusted for the effects of parasite density – a surrogate for UA precipitates and other parasite-derived virulence factors that we have not measured. Also, we show that sTM levels increase in UM and further increase in NCSM, thus implicating the shedding of TM in the pathogenesis of malaria in our study population. Finally, our study suggests that parasitized RBCs contribute only modestly to the excess UA in plasma; however, regardless of the source of increased UA levels, their association with increased sTM levels justifies a variety of experiments to evaluate the potential role of soluble UA in modulating the shedding of TM from MVECs. One potential source of excess soluble UA in plasma is the cytosol of schizont-infected RBCs, which lyse during parasite development in microvessels. Whether this parasite-derived UA mediates the shedding of TM from the surface of MVECs can now be tested
One significant limitation of our study is that it does not identify the sources of excess UA in our cohort of Malian children with malaria. Possible sources include fasting and the release of free iron, which may upregulate xanthine oxidase activity. Parasite-derived UA precipitates, which have been directly observed
We thank Ababacar Diouf, Kazutoyo Miura, Sam Moretz for providing the samples from the sub-cohort study; Greg Tullo for organizing the epidemiologic data; Jennifer Kirk for the initial statistical data analysis; Karim Traoré, Seidina Diakité and Dick Sakai for logistical support in Mali; Robert Gwadz and Thomas Wellems for their support of this study.