Conceived and designed the experiments: FEL NT ROO SK SL CCJ WCL KCK. Performed the experiments: FEL EIL NR. Analyzed the data: FEL MH CCJ. Contributed reagents/materials/analysis tools: NT ROO SK SL CCJ. Wrote the paper: FEL MH CCJ WCL KCK.
The University Health Network holds intellectual property related to the role of angiogenic factors in the pathogenesis of infectious disease. The authors report no conflict of interest with respect to this manuscript.
Limited tools exist to identify which individuals infected with
Based on the hypothesis that endothelial activation and blood-brain-barrier dysfunction contribute to CM pathogenesis, we examined the endothelial regulators, angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), in serum samples from
ANG-1 and the ANG-2/1 ratio are promising clinically informative biomarkers for CM. Additional studies should address their utility as prognostic biomarkers and potential therapeutic targets in severe malaria.
Although greater than 500 million
The discovery of a reliable laboratory test that accurately identifies individuals with, or at risk of, CM would be valuable. The capacity for early detection and intervention in cases of severe malaria and CM would have clinical and economic impact, particularly in resource-poor settings where effective allocation of limited health resources is essential. Several studies have examined the correlation of serum markers, such as cytokines, with severe and complicated malaria. Elevated levels of TNF have been associated with severe malaria
Endothelial cell activation and dysfunction have been implicated in the pathogenesis of CM, in which the endothelium responds to parasite-induced inflammation and mediates parasitized erythrocyte sequestration, especially in vital organs such as the brain
In addition to systemic endothelial activation, recent work has focused on mechanisms by which malaria may compromise the structural and functional integrity of the blood-brain-barrier (BBB), leading to leakage of plasma proteins, perivascular edema and neuronal injury
Based on the hypothesis that dysregulation of angiopoietins may be associated with endothelial and BBB dysfunction during malaria infection, we examined whether both ANG-1 and ANG-2 were clinically informative biomarkers for cerebral malaria. We report that angiopoietin levels were accurate biomarkers of CM and predicted mortality in African children.
Individuals (≥13 years of age) admitted to the Hospital for Tropical Disease (Mahidol University, Thailand) for ongoing studies of anti-malarial drug efficacy were eligible for enrolment. The institutional review board of Mahidol University approved the study, and written informed consent was obtained from all patients or their legal guardians. Venous blood samples were collected from 50 patients with
Group | Adult (Thailand) | Pediatric (Uganda) | ||||
N | Age | Parasites/µl | N | Age | Parasites/µl | |
10 | 32 (25–48) | 0 | 28 | 7 (3.2–12) | 0 | |
25 | 22 (14–63)* | 2.2×104 (170-1.9×105)* | 67 | 7 (3–12) | 3.3×104 (48-2.4×105)* | |
25 | 25 (17–50) | 3.1×105 (500-2.1×106)* |
69 | 5.4 (3.2–12) * |
4.0×105 (32-9.3×105)* |
Age and parasitemia are presented as median (range). *p<0.05 vs. HC and †p<0.05 vs. UM (Kruskal-Wallis test with Dunn's multiple comparison post-test).
The Ugandan study population has been previously described
Sample handling and quantification of serum biomarker levels: Serum derived from patient blood was immediately frozen, shipped on dry ice, and maintained at −80°C until use. The serum used was thawed (on ice) and re-frozen a maximum of 3 times. Serum concentrations of ANG-1, ANG-2 and TNF were measured by ELISA (R&D Systems, Minneapolis MN; TNF: eBioscience, San Diego CA). Concentrations were interpolated from 4-parameter-fit standard curves generated using a standard curve of recombinant human proteins. The upper and lower limits of detection for each assay were as follows: ANG-1 (10,000–156.25 pg/ml), ANG-2 (3,500–54.69 pg/ml) and TNF (500–7.8 pg/ml). Samples were diluted between 1∶2 to 1∶50 in assay diluent to fall within the range of the standard curves, as per the manufacturers' instructions. TNF levels in Ugandan children were measured as described
Statistical analysis was performed using GraphPad Prism v4.03 (San Diego, CA). Serum protein levels were analyzed using a Kruskal-Wallis test, followed by Dunn's multiple comparison tests. Receiver operating characteristic (ROC) curves and area under the ROC curves were generated using (SPSS 16.0. Cutoff values were derived mathematically from the ROC curves, using the point on the ROC curve with the lowest value for the formula: (1-sensitivity)2+(1-specificity)2. Angiopoietin levels and survival outcomes were analyzed using the Wilcoxon rank-sum test. Multivariable logistic regression modeling was used to examine the independent predictive value of biomarkers on outcome (CM vs.UM) in order to account for potential confounding effects of multiple covariates (SPSS 16.0). Hosmer Lemeshow test was used to verify model goodness of fit.
In Thailand, serum ANG-1 levels were significantly lower in adults with CM compared to either adults with UM or healthy controls, and in adults with UM compared to healthy controls (
A. Serum concentrations of angiopoietin-1 (ANG-1), angiopoietin-2 (ANG-2), the ratio of ANG-2∶ANG-1 (RATIO, expressed as log base 10) and tumour necrosis factor (TNF) were measured in 10 healthy controls (HC), 25 consecutive uncomplicated malaria (UM) patients, and in consecutive 25 cerebral malaria (CM) patients. B. Receiver operating characteristic curves (blue line) were generated for each test to compare CM with UM patients, with the null hypothesis (green line) that area under the curve equals 0.5.
The manifestations and outcomes of severe and CM may differ between adults and children and between varying genetic backgrounds of patient and parasite populations
A. Serum concentrations of angiopoietin-1 (ANG-1), angiopoietin-2 (ANG-2), the ratio of ANG-2∶ANG-1 (RATIO, expressed as log base 10), and tumour necrosis factor (TNF) were measured in 28 healthy controls (HC), 67 uncomplicated malaria (UM) patients, and in 69 cerebral malaria (CM) patients. B. Receiver operating characteristic curves (blue line) were generated for each test to compare CM with UM patients, with the null hypothesis (green line) that area under the curve equals 0.5.
Comparisons of the median and range of each serum biomarker concentration (
Marker | Adult (Thailand) | Pediatric (Uganda) | ||||
HC | UM | CM | HC | UM | CM | |
378 (151–946) | 82.25 (27.3–379) | 3.51 (0.001–15.3) | 64.4 (23.5–101) | 25.0 (0.39–64.9) | 9.0 (0.39–37.5) | |
0.0089 (0.005–0.847) | 1.84 (0.25–5.44) | 6.19 (0.78–35) | 0.068 (0.068–1.33) | 0.28 (0.068–10.0) | 0.83 (0.068–33.5) | |
0.00003 (0.000013–0.0021) | 0.017 (0.03–0.11) | 3.47 (0.15–204) | 0.0015 (0.00071–0.014) | 0.013 (0.0011–13.0) | 0.14 (0.0024–81.5) | |
0 (0–0) | 0 (0–44.8) | 6.51 (0–73.8) | 0 (0–31.3) | 70.6 (0–658) | 76 (0–559) |
Values are presented as median (range).
ROC curves for the biomarkers, examining CM patients as “cases” and uncomplicated malaria patients as “controls”, were plotted and compared to assess the ability of each marker to discriminate between patients with and without cerebral complications (
Marker | Adult (Thailand) | Pediatric (Uganda) | ||
AUC (95% CI) | P | AUC (95% CI) | P | |
1 (1–1) | <0.001 | 0.785 (0.709–0.861) | <0.001 | |
0.835 (0.719–0.951) | <0.001 | 0.688 (0.595–0.780) | <0.001 | |
1 (1–1) | <0.001 | 0.779 (0.702–0.856) | <0.001 | |
0.834 (0.713–0.955) | <0.001 | 0.557 (0.453–0.661) | 0.268 |
P values are based on the null hypothesis that AUC = 0.5.
Compared to ANG-1 and ANG-2 as biomarkers of CM, previously studied markers of severe and CM such as TNF (
The diagnostic accuracy (sensitivity, specificity, positive and negative likelihood ratios) for each biomarker, stratified by patient population, are reported in
Marker | Adult (Thailand) | Pediatric (Uganda) | ||||||||
Cut-off | SEN | SPEC | LR(+) | LR(−) | Cut-off | SEN | SPEC | LR(+) | LR(−) | |
21.26 ng/ml | 1 (0.87–1) | 1 (0.87–1) | ∞ |
0 |
15.05 ng/ml | 0.70 (0.58–0.79) | 0.75 (0.63–0.83) | 2.7 (1.8–4.3) |
0.40 (0.28–0.60) |
|
3.04 ng/ml | 0.72 (0.52–0.86) | 0.84 (0.65–0.94) | 4.5 (1.8–11) |
0.33 (0.17–0.64) |
0.39 ng/ml | 0.83 (0.72–0.90) | 0.60 (0.48–0.71) | 2.1 (1.5–2.8) |
0.29 (0.17–0.51) |
|
0.131 | 1 (0.87–1) | 1 (0.87–1) | ∞ |
0 |
0.052 | 0.73 (0.61–0.82) | 0.70 (0.58–0.79) | 2.4 (1.6–3.6) |
0.39 (0.26–0.59) |
|
1.46 pg/ml | 0.76 (0.57–0.89) | 0.88 (0.70–0.96) | 6.3 (2.1–19) |
0.27 (0.13–0.56) |
81.1 pg/ml | 0.48 (0.36–0.61) | 0.62 (0.49–0.74) | 1.3 (0.84–2.0) | 0.82 (0.60–1.1) |
significantly different from 1 (p<0.05).
Although higher parasitemia is generally associated with an increased risk of severe malaria or CM, severe disease can occur in individuals with relatively low peripheral parasitemias. In the Thai population, patients with CM had significantly higher parasitemias than in uncomplicated malaria patients (p<0.001); however, this was not the case in Ugandan children (
Predictor | Adjusted OR (95%CI) | p |
Group: | ||
Thailand | 1.0 |
|
Uganda | 0.36 (0.029–4.7) | 0.44 |
Age | 0.96 (0.86–1.1) | 0.53 |
Parasitemia (parasites/µL) | 1.00 (1.00–1.00) | 0.20 |
ANG-1 (ng/mL) | 0.899 (0.864–0.934) |
<0.001 |
ANG-2 (ng/mL) | 1.10 (0.944–1.28) | 0.22 |
Ratio (ANG-2/ANG-1) | 1.01 (0.932–1.09) | 0.82 |
TNF (pg/mL) | 1.00 (0.994–1.003) | 0.91 |
baseline comparator group.
Adjusted odds ratio represents the incremental odds of CM for every unit increase (1 ng/mL) in the ANG-1 level.
We examined angiopoietin levels at presentation and subsequent survival in children with CM and observed that ANG-1 levels and the ratio of ANG-2∶ANG-1 were related to mortality. Higher ANG-1 levels at presentation were associated with protection from fatal CM (median (range): non-fatal CM 9.1 (0.39 to 38) versus fatal CM 0.39 (0.39 to 4.6), p = 0.027;
Serum concentrations of angiopoietin-1 (ANG-1) were measured in 69 cerebral malaria (CM) patients at presentation and compared to outcome. Higher ANG-1 levels at presentation were associated with protection from fatal cerebral malaria. *p = 0.027, non-fatal CM versus fatal CM (Wilcoxon rank-sum test).
This study provides evidence implicating dysregulation of angiopoietins in the pathogenesis of CM and suggests that they may be clinically informative biomarkers of this syndrome. Since the manifestations of severe malaria may differ between children and adults and in varying backgrounds, we measured serum ANG-1 and ANG-2 levels in two geographically and genetically diverse patient and parasite populations and demonstrate that these endothelial regulators were accurate discriminators of CM vs. UM in both settings. In both adults from Thailand (
No laboratory tests are currently available to definitively confirm a diagnosis of CM, and misdiagnosis may result in increased adverse outcomes
An ideal biomarker for CM might be expected to possess a number of logistical, diagnostic/prognostic and therapeutic attributes, including 1) capacity to be easily measured in a readily available specimen such as serum or whole blood by a standardized assay that requires limited specialized equipment and performed with minimal training, 2) reliable detection, with high sensitivity and specificity of individuals with either established CM or at risk of progression to CM, and 3) detection of determinants likely to be involved in the underlying pathogenesis of the disorder (rather than bystander reactions/epiphenomena), thereby providing a metric of the underlying disease process, as well as representing potential therapeutic targets for intervention.
Despite the growing realization that CM is a complex multisystem disorder, our data suggest that angiopoietins meet several of these criteria and may represent clinically useful biomarkers for this syndrome. Angiopoietins appear to be robust and accessible targets, readily detectable by standard immunoassays in serum or whole blood. ROC curve analysis in both Ugandan pediatric and Thai adult populations indicated that ANG-1 and ANG-2 were highly accurate tests for the detection of CM and its discrimination from uncomplicated disease (
Our observations that ANG-1 and ANG-2 are dysregulated in patients with CM, supports the hypothesis that they may be involved in the pathogenesis of this syndrome. As key regulators of endothelial integrity, there are several mechanisms by which angiopoietins may contribute to the pathophysiology of CM. Although the role of BBB disruption in CM remains controversial
It will be important to dissect the putative mechanisms by which angiopoietins may contribute to malaria pathogenesis in animal models where endothelial and BBB dysfunction and vascular leak are central features of disease
One limitation of our study is the relatively small sample sizes, particularly in the Thai population. The sensitivity and specificity of ANG-1 levels and the ANG-2/ANG-1 ratio for the diagnosis of CM was 100% in the Thai population and somewhat lower in the Ugandan pediatric cohort. It will be important to confirm and extend our observations and further assess performance and specificity in larger prospective clinical trials, especially those assessing malarial retinopathy and autopsy studies with histopathologically confirmed cases of CM
In summary, these data suggest that the dysregulation of angiogenic factors may be involved in the pathogenesis of cerebral malaria and that serum ANG-1 and ANG-2 levels are accurate biomarkers to discriminate CM from uncomplicated disease and predict survival in African children with cerebral involvement.
We thank our study teams of medical officers, nurses, data entry clerks and office staff members for their efforts and the patients and their families for their participation in the study.