The spectrum of clinical biomarkers in severe malaria and new avenues for exploration

Figures & data

Figure 1. Latest WHO guidelines on the clinical presentation of severe malaria [18].

Hb: hemoglobin, Hct: hematocrit, PD: parasite density, SBP: systolic blood pressure.

*These signs/symptoms are variably defined with respect to the Plasmodium species.

Definitions of Po and Pm severe malaria are not stated in the guidelines.

Figure 2A. Worldwide burden of severe malaria.

Figure 2A(1-4) In-hospital-attended patients due to Plasmodium spp species.

Figure 2B(1-4) P. falciparum mono-infections.

Figure 2C(1-4) P. vivax mono-infections in African and Eastern Mediterranean regions (A1, B1, C1), The Americas region (A2, B2, C2), South East Asia and West Pacific regions (A3, B3, C3), and India (A4, B4, C4), 2010—2020.

In Figure 2A(1-4), the total number of SM cases irrespective of the malaria species identified in studies was used to determine the proportion of SM in a country or an area.

Weighted proportions were calculated to depict hospital-admitted SM. For a same country or area, the weight of a study was determined by dividing the sample size of admitted patients in this study by the total number of admitted patients of all studies conducted in the country or area. The overall estimate of the SM proportion was then computed by summing up the product of SM prevalence in each study and the weight of the study.

LDR-FMA: ligase detection reaction-fluorescent microsphere assay, LM: light microscopy, PCR: polymerase chain reaction, QBC: quantitative buffy coat, RDT: rapid diagnostic test.

The maps were generated using the QGIS software.

The data are sourced from research articles retrieved from PubMed (https://pubmed.ncbi.nlm.nih.gov/), published in English and French languages We excluded studies with a small sample size, case—control design, published before 2010, imported malaria, studies with no country-wise data, and reviews/abstracts/book chapters.

Figure 2B. Continued.

Figure 2C. Continued.

Figure 3A. Continued.

Figure 3B. Importance (A) and categorization (B) of biomarkers in malaria infection.

Figure 4. Clinical biomarkers and severe malaria.

Figure 4A Origin of biomarkers.

Figure 4B Biological nature of biomarkers by malaria species.

Figure 4C Type of biomarkers evaluated.

Biomarkers for which reporting AUC/sensitivity/specificity values were included.

In 4B, the numbers above the histograms represent the total number of evaluated biomarkers for each malaria species.

In 4C, some biomarkers were studied in more than one malaria species (e.g. the platelet count was evaluated as biomarkers of severe malaria with Pf, Pv, or Pk).

AHSG: alpha-2-Heremans-Schmid glycoprotein, AUC: area under the curve, CAMs: cell adhesion molecules, CK-MB: cardiac disease creatine kinase muscle-brain type, CLI/CLU: circulatory complement‑lysis inhibitor or clusterin, DNA: deoxyribonucleic acid, eGRF: estimated glomerular filtration rate, HMGB1: high-mobility group box protein 1, HRP2: histidine-rich protein 2, IP-10: 10-kDa INF gamma-induced protein, Pf: Plasmodium falciparum, Pk: Plasmodium knowlesi, Pv: Plasmodium vivax, pLDH: parasite lactate dehydrogenase, RBC: red blood cell, sICAM-1: soluble intercellular cell adhesion molecule 1, SERPINA3: serpin peptidase inhibitor clade A member 3, sTie-2: soluble cognate receptor, sTREM 1: soluble triggering receptor expressed on myeloid cells 1, suPAR: soluble urokinase-type plasminogen activator, TNF: tumour necrosis factor, VEGF: vascular endothelial growth factor, vWF: von Willebrand factor.

Figure 5. Biomarkers evaluated for severe Pf-SM.

Biomarkers for AUC/sensitivity/specificity values were included.

Biomarkers for severe Pf malaria were categorized into three groups: i) suboptimal [the biomarker has AUC and/or sensitivity and specificity values below thresholds], ii) Good+ [the biomarker has AUC and/or sensitivity and specificity values above thresholds, but with limited evidence of its clinical utility due to several reasons, including a low number of studies (n = 1), no statistical significance was provided for AUC, evaluated in a specific population (i.e. nonimmune European travelers and evaluated by the same research team], and iii) Good++ [the biomarker has AUC and/or sensitivity and specificity values above thresholds, and strong evidence of its clinical utility due to several reasons including number of studies > 1, statistical significance provided for AUC, evaluated in different malaria endemic settings, evaluated in populations from malaria endemic areas, and evaluated by different research teams]. The Ang-2 and Ang-2/1 ratio (yellow star) showed good prognosis performances for most of the SM-related outcomes.

Ang: angiopoietin, CCL: chemokine ligand, cfDNA: cell-free deoxyribonucleic acid, CHI3L1: chitinase-3-like 1 protein, CRP: C-reactive protein, DNA: deoxyribonucleic acid, HMGB1: high-mobility group box protein 1, HRP2: histidine-rich protein 2, IFN: interferon, IL: interleukins, IP-10: 10-kDa INF gamma-induced protein, PC: platelet count, PCN: pigment-containing cells, PCT: procalcitonin, Pf: Plasmodium falciparum, PWD: platelet width distribution, RBCDW: red blood cell distribution width, RP: retinopathy positive, sICAM-1: soluble intercellular cell adhesion molecule 1, sTie-2: soluble cognate receptor, suPAR: soluble urokinase-type plasminogen activator, TNF: tumor necrosis factor, VCAM-1: vascular adhesion molecule 1, VEGF: vascular endothelial growth factor, vWF: von Willebrand factor.

Figure 6. Biomarkers evaluated for severe Pv and Pk malaria.

Biomarkers for AUC/sensitivity/specificity values were included.

Biomarkers for severe Pv and Pk malaria were categorized into three groups: i) suboptimal [the biomarker has AUC and/or sensitivity and specificity values below thresholds], ii) Good+ [the biomarker has AUC and/or sensitivity and specificity values above thresholds, but with limited evidence of its clinical utility due to several reasons including a low number of studies (n = 1), no statistical significance was provided for AUC, evaluated in a specific population (i.e. nonimmune European travelers, and evaluated by the same research team], iii) Good++ [the biomarker has AUC and/or sensitivity and specificity values above thresholds, and strong evidence of its clinical utility due to several reasons including number of studies > 1, statistical significance provided for AUC, evaluated in different malaria endemic settings, evaluated in populations from malaria endemic areas, and evaluated by different research teams].

Ang: angiopoietin, CRP: C-reactive protein, IFN: interferon, IL: Interleukin, Pk: Plasmodium knowlesi, Pv: Plasmodium vivax, sICAM-1: soluble intercellular cell adhesion molecule 1, SOD-1: superoxide dismutase 1, TNF-α: tumor necrosis factor alpha, VCAM-1: vascular adhesion molecule 1.

Table 1. Comparison of quantitative methods of SM biomarkers.

Techniques	Analysis time	Training	Cost	Workload	Reproducibility	Instrumentation required
Automated immunofluorescent assay^f	Minutes to hours	Low	Moderate	High	Moderate to high	Colorimeter/microscope
Bradford assay^a	Minutes	Low	Low	Low	Low to high	Spectrophotometer
Di-nitrophenyl hydrazine assay^a	Minutes	Low	Low	Low	Low	Spectrophotometer
ELISA^a	Hours	Low	Low to high	Low	High	Colorimeter/spectrophotometer
Flow cytometry^f	Minutes	High	High	Low	High	Flow cytometer
FRAP assay	Minutes	Low	Low	Low	High	Spectrophotometer
Genotyping/cloning	Several hours to days	High	High	High	High	Thermocycler
Hematological analyzer	Minutes	Low	High	Low	High	Robot
Immunocolorimetric assay^a	Minutes	Low	Moderate to high	Low	High	Spectrophotometer
Co-oximetry^f	Minutes	Low	Moderate	Low	High	Oximeter
Indirect potentiometry^f	Minutes	Low	Moderate	Low	High	Potential measuring device
Electrolyte analyzer	Minutes	Low	Moderate	Low	High	Robot
Light microscopy	Minutes to hours	High	Moderate	High	Low to high	Light microscope
Quantitative PCR	Minutes to hours	High	High	Low	High	Real-time thermocycler
Stable isotope dilution	Minutes to several days	High	Moderate to high	High	Low to high	Spectrophotometer
Thiobarbituric acid reaction assay^a	Minutes to hours	Low	Moderate to high	Low	Moderate to high	Spectrophotometer
Turbidimetric immunoassay^ae	Minutes	Low	Moderate to high	Low	High	Turbidimeter (turbidity meter)
B·R·A·H·M·S PCT-Q test^f	30 min	Low	Low	Low	High	-

aThese are commercial kits.

bThis method is used to measure the platelet count.

cThis method is used to measure blood sodium.

dThis method is used to measure blood carboxyhemoglobin.

eThis method is used to measure C-reactive protein.

fThis is a one-step immunochromatographic assay-based point-of-care detecting Procalcitonin using immunogold labeling.

Figure 7. Utility of omics technologies to accelerate the identification and validation of clinical biomarkers for SM.

(a) Blood samples are collected from a small number of individuals based on symptomatology of malaria (i.e. no malaria/healthy control, uncomplicated malaria, and severe malaria). These different clinical groups are defined based on the objectives of the study. (b) Samples are processed using omics technologies to identify RNA/protein/peptide expression patterns between these different clinical groups in order to identify differentially expressed molecules. (c) The most differentially expressed biomolecules are identified and characterized (d) Case–control studies may be implemented to evaluate the clinical value of these sets of selected biomolecules. Parameters such as sensitivity, specificity, and area under the curve can be evaluated. Other aspects such as reliability, simplicity, and practicality of the measurement method can be evaluated in parallel. In this scenario, three clinical groups were used as an example, but design could be made more complex by defining or adding supplementary clinical groups (e.g. i—severe Pf + Pv cases, ii—different severe malaria forms as cerebral malaria, severe malarial anemia, respiratory distress, iii—severe co-infections with Plasmodium and other pathogens – virus, bacteria, etc.) and then performing omics technologies as presented in the figure, and (e) the development of a rapid, reliable, and affordable assay system should be the final step for the utilization of clinical biomarkers.

Table 2. Some proposed severe malaria biomarkers for which clinical performances were not evaluated.

Biomarkers	Description	Author’s findings	References
Osteoprotegerin	Member of the TNF receptor family, but it is secreted and acts like a cytokine and plays a crucial role in osteoporosis.	The plasma level of this biomolecule was significantly higher in Pk-SM patients.	[54]
Cell-free Hb	Hb is released into the bloodstream during some RBC rupture inducing conditions as those seen in severe Pf malaria.	Cell-free Hb was significantly higher in Pk-SM as compared to Pk-UM and HC. Cell-free Hb was significantly correlated with parasitemia, lactate, creatinine, Ang-2, ICAM-1, and E-selectin.	[54]
Micro RNAs	MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs present in several cells (cellular miRNAs) and body fluids (circulating miRNAs).	MicroRNA 4497 is significantly more expressed in Pf-SM compared to Pf-UM patients. Other miRNAs (23a, 24, 27a, 125b, 181b, 150, and 210) are variably expressed, depending on the form of SM (i.e. CM, renal failure, pulmonary edema, ARDS, and metabolic acidosis).	[81–84]
Erythropoietin (Epo)	Epo is a protein mainly synthesized in the kidney in response to hypoxia and is crucial for the proliferation and differentiation of erythroid cell lines.	High levels of Epo (>200 units/L) were associated with a reduced risk of neurological sequelae in children with Pf-CM (OR = 0.18, 95% CI 0.01-0.93).	[85]
Galectin-9	Gal-9 is presumed to be involved in several physiological and pathological processes by binding molecules such as glycoproteins and glycolipids of immune cells and pathogens.	Gal‑9 levels were also higher in Pf-SM compared to Pf-UM.	[86]
Granzyme B (GrzB)	Member of a family of serine proteases expressed in the granules of natural killers and cytotoxic T cells.	Plasma GrzB levels were also higher in Pf-SM compared to Pf-UM in Ghanaian children.	[87]
Cell-specific microparticles (MP)	Fragments of the plasma membrane shed by various cell types under physiological stress conditions.	Platelet, erythrocyte, endothelial, and leukocyte MP levels were elevated in patients with cerebral dysfunctions and returned to normal by discharge. In Pf-CM patients, platelet MP were the most abundant and their levels significantly correlated with coma depth and thrombocytopenia.	[88]
Synapsin I	Member of the synapsin family produced in synaptic vesicles and involved in exocytosis transport mechanisms in synapses.	Significant increase in synapsin I in Pf-CM compared to non-CM and HC both in granule cells and glomerular synaptic complex.	[89]
Pantetheinase (PA)	Ubiquitous enzyme that hydrolyzes D-pantetheine into vitamin B5 (pantothenate) and cysteamine in the Coenzyme A pathway.	Patients with Pf-CM had lower PA than those with Pf-UM. Pf-CM patients had a lower serum PA than others with Pf-SMA or HC.	[90]
Extracellular vesicles (EVs)	EVs are lipid bilayer-delimited particles (30 nm to 4 µm) that are naturally released from a cell, and thus EVs can theoretically be released from any body cell type. EVs were thought to play only a structural function, but recent studies outline their role as mediators of crucial biological processes for several parasites including Plasmodium parasites (e.g. parasite virulence, cell invasion).	Some studies outlined decreased levels of some EVs in Pf-SM patients, especially in Pf-CM cases.	[91]
Hemopexin (Hpx)†	Hemopexin (Hpx) is a plasma protein belonging to the inflammation acute phase groups and mainly produced by the liver.	Hpx had good prognosis potential for discrimination between Pf-SM and Pf-UM patient cohorts. On admission, the Hemin-to-Hpx ratio was significantly higher in i) SMA patients vs non-SMA (0.124 vs 0.016, p < 0.0001) and ii) RD vs non-RD patients (0.063 vs 0.020, p < 0.01) in another study.	[78,92,93]
Hemin to hemopexin ratio	Hemin is an iron-containing porphyrin with chlorine that can be formed from a heme group such as heme B found in Hb.	On admission, the ratio was significantly higher in i) Pf-SMA vs Pf-non-SMA (0.124 vs 0.016, p < 0.0001)and ii) Pf-RD vs Pf-non-RD patients (0.063 vs 0.020, p < 0.01). This ratio was not associated with 48-h mortality (short-term) but was associated with 6-month mortality (long-term) (p = 0.012).	[93]
Ceruloplasmin†	Ceruloplasmin is a copper-containing glycoprotein found in the α2 globulin fraction of human serum. It is involved in the pathological process of several iron- and copper-related metabolic disorders such as Wilson’s disease.	Its plasma level was altered depending on the severity of malaria. Its performances for distinguishing Pf-SM and Pf-UM patients were lower than those reported for apolipoprotein E, retinol-binding protein 4 (RBP4), and Hpx.	[78,94]
Retinol-binding protein 4†	This protein is a protein of the lipocalin family and well known for its crucial role in the transport of retinol (vitamin A) in the bloodstream.	RBP 4 exhibited good prognosis potential for discrimination between Pf-SM and Pf-UM patients.	[78,95]
Plasminogen†	Protein playing a role in the coagulation process and other physiological and pathological processes (e.g. angiogenesis, inflammation, oncogenesis).	Its plasma level was altered depending on the severity of malaria. Its performances for distinguishing Pf-SM and Pf-UM patients were lower than those reported for apolipoprotein E, RBP4, and Hpx.	[78,96]
Apolipoproteins†	Group of proteins whose pathological increase in the body is related to the genesis of vascular diseases as arteriosclerosis and atherosclerosis.	Apolipoprotein E exhibited good prognosis potential for discrimination between Pf-SM and Pf-UM patients.	[64,78,97]
Endoglin	Transmembrane glycoprotein expressed on ECs operating as a co-receptor for several ligands of the TGF-β family. This protein is also a recognized marker of angiogenesis.	Endoglin levels were statistically higher in Pf-CM and Pf-SM as compared to Pf-UM and HC. No statistical difference between Pf-SM and Pf-CM.	[98]
B-cell-activating factor (BAFF)	Cytokine of the tumor necrosis family playing an important role in the differentiation and survival of B cells throughout their different developmental stages.	Significant progressive increase in BAFF concentration with disease severity (p = 0.0001). Children whose BAFF plasma levels were above the median BAFF plasma level were at a higher risk of being admitted to hospital either with Pf-UM or Pf-SM.	[99]
Complement fractions (C3, C4, Bb, C4d, iC3b, and SC5b-9)	Complement is a set of a small proteins produced by the liver and that are found in the bloodstream as inactive precursors. The proteins play a role in several immune processes such as inflammation,and phagocytosis.	On admission, SC5b-9, and C4d were significantly higher in Pf-SM compared to HC. C3 and iC3b were significantly lower in Pf-SM compared to HC. No difference for C4 between Pf-SM and HC.	[100]
BDCA3-positive dendritic cells	Dendritic cells are antigen-presenting cells that are crucial for the initiation of adaptive immune responses.	The frequency of BDCA3 dendritic cells was significantly increased in Pf-SM compared to HC but similar between SM and CP.	[101]
Vγ9 Vδ2 γδ T cells	Subtype of γδ T cells which acts as a bridge between the innate and adaptive immune response, and whose effector functions include the production of cytokines and direct cytotoxicity to pathogens/infected cells.	Vγ9 Vδ2 γδ T cells were lower in children presenting with Pf-UM and Pf-CM than in HC patients but did not vary between Pf-UM and Pf-CM patients (p = 0.224).	[102]
Asymmetric dimethylarginine (ADMA)	Biomolecules working as an endogenous inhibitor of nitric oxide synthase and thus contributing to endothelial dysfunction.	Pre-treatment ADMA levels were significantly reduced in Pf-SM and Pf-UM as compared HC. ADMA levels significantly increased in Pf-SM patients after commencement of antimalarial therapy.	[103]
Syndecan-1 and glycosaminoglycans	These are products of the degradation of glycocalyx and thus are considered as markers of its degradation.	These were significantly higher in Pk-SM as compared to Pk-UM and HC. These were significantly higher in Pv-SM as compared to Pv-UM and HC. They were significantly correlated with other putative SM biomarkers (e.g. Ang-2, ICAM-1, E-selectin, osteoprotegerin, ADMA).	[104]
pvcrt-oandpvmdr1	These two parasite genes are thought to be involved in the resistance of Pv parasites to antimalarial drugs such as chloroquine, as clearly established with their orthologues in Pf parasites.	The level of expression of these two genes was higher in Pv-SM compared to Pv-MM patients.	[105,106]

†Identified using proteomics analysis in some studies.

ADMA: asymmetric dimethylarginine, ARDS: acute respiratory distress syndrome, BAFF: B-cell-activating factor, CI: confidence interval, CM: cerebral malaria, ECs: Endothelial cells, CP: convalescent patients, crt-o: chloroquine resistance transporter-orthologue, Epo: erythropoietin, EVs: extracellular vesicles, Gal-9: galectin 9, GrzB: granzyme B, Hb : hemoglobin, HC: healthy control, Hpx: hemopexin, mdr1: multidrug resistance gene, MP: microparticles, OR: Odds ratio, PA: pantetheinase, Pf: Plasmodium falciparum, Pk: Plasmodium knowlesi, Pv: Plasmodium vivax, RBC: red blood cell, RBP4: retinol-binding protein 4, RD: respiratory distress, SM: severe malaria, SMA: severe malaria, anemia, TGF: transforming growth factor, TNF: tumor necrosis factor, UM: uncomplicated malaria, vWF: von Willebrand factor.

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Data availability statement

All data included in this study are fully available from the corresponding author on reasonable request.