At the Principal Hospital of Dakar, venous blood sample collection took place in the Intensive Care Unit (ICU), infectious disease unit and the laboratory where all biological tests for the patients studied were performed, except for immunological tests. Immunological analyses, such as antibody quantification, were conducted at the Immunology and Genetics Unit of the Faculty of Medicine, Pharmacy, and Odontology at Cheikh Anta Diop University in Dakar.

We conducted a descriptive study with an analytical components, recruiting patients with severe forms of malaria (cerebral malaria) (CM) admitted to the hospital services. Similarly, there are also some patients consulted for uncomplicated malaria (UM) syndrome which was taking part in recruitment. In addition, non infected individuals were recruited and designated as the control patients group (CP).

The choice of period was based on the high transmission level of the parasite, particularly due to the abundance of vectors (Anopheles). This increased the probability of obtaining positive samples for P. falciparum. Recruitment took place from July to November in the following years: 2013, 2014, and 2015.

Patients were recruited based on World Health Organization (WHO) criteria and Saissy et al. studies for the different clinical forms of severe malaria requiring intensive care [12][13]. Life-threatening CM was defined following the WHO criteria as the presence of P. falciparum in blood smears accompanied by a coma with no other cause of cerebral symptoms. UM cases were defined as those with positive blood smear for P. falciparum and having fever, and no other symptoms of CM [12]. CP come from healthy volunteers living in Dakar.

Malaria diagnosis was screened using a rapid diagnostic test (RDT) for Plasmodium falciparum antigen detection and confirmed by microscopic examination of thick and thin blood smears for parasite identification and quantification. This study was approved by the Research Ethics Committee of Cheikh Anta Diop University in Dakar (protocol No. 001/2015/CER/UCAD).

Inclusion Criteria

During study periods, CM admitted to the ICU for severe malaria with a positive rapid diagnostic test (RDT) and confirmed by a positive thick blood smear and thin blood smear were included. These patients presented at least one severity criterion of malaria as defined by the WHO [12]. UM patients were recruited in the infectious disease unit during the same time-period. They were defined as having a positive RDT and blood smear for P. falciparum with fever and no symptoms of severe malaria. CP with negative thick blood smear; thin blood smear or a rapid diagnostic test (RDT) were also included.

Non-Inclusion Criteria

Patients were not included if they:

Did not have a thick blood smear, thin blood smear or a rapid diagnostic test (RDT) at admission.Were known to be infected with pathogens such as Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis.Did not provide consent.

Exclusion Criteria

CM patients were excluded from the study based on medical history, disease history, and potential co-morbidities that could interfere with the assays conducted in our investigations. Criteria includes: no stored sample, patients with gravid eclampsy in post pregnancy, septicemia (S. Pyogenes),pulmonary infection (E.coli, S. pneumoniae), gastro intestinal infection (Salmonella). UM patients were excluded because of: no consent from patient and no stored samples; pregnant patients; patients with acute infections associated with respiratory or intestinal infections, patients for whome data not available or insufficient. Samples with inadequate volume, hemolyzed blood, or those lost/damaged during long-distance transport and processing were systematically excluded from final analyses.

Out of 131 patients, only 107 considering exclusion criteria, were selected for the study. This includes 37 individuals consulting for a UM episode, 36 patients hospitalized for CM, and 34 CP.

Data collection was performed using a questionnaire based on the admission form and patient file. We considered different criteria such as sociological and demographic characteristics (age, sex, admission), biological data (blood count, blood group-rhesus factor, thick blood smear, and parasite density), serological and immunological characteristics (immunological markers of neutrophils, levels of IgE antibodies).

For each patient, a venous blood sample was taken in a tube containing Ethylenediaminetetraacetic acid (EDTA), and the preparation of a thick blood smear, thin blood smear, or a rapid diagnostic test (RDT) at the time of recruitment allowed for the detection and/or assessment of parasite density. The blood was then divided into two parts:

First Part:Used for the study of hematological data (complete blood count by an automated analyzer) and immunological data (by flow cytometry).Second Part:Centrifuged at 2000 rpm for 10 minutes, allowing for the isolation of plasma, which was then stored at −20˚C. These plasmas were diluted 1/100 at the time of IgE assays.

Two recombinant antigens were studied; both proteins expressed on the surface of the P. falciparum merozoite.

Merozoite Surface Protein-1p19 Antigen

Our study focuses on MSP-1p19, which may play a crucial role in food vacuole biogenesis and function throughout the intra-erythrocytic phase [14]. The protein used was genetically engineered using the Baculovirus/insect cell expression system, reproducing the parasite protein’s Epidermal Growth Factor-like (EGF-like) conformation developed by Longacre’s team at the Pasteur Institute in Paris.

Merozoite Surface Protein-4p20 Antigen

For this study, we used the MSP-4 construct: a full-length protein (MSP-4p40) from which we obtained the 20 kDa C-terminal fragment containing the EGF domain (MSP-4p20). These proteins were produced at the Parasite Vaccine Laboratory of the Pasteur Institute in Paris using the Baculovirus/insect cell expression system [15].

The determination of IgE levels directed against the studied antigens was conducted using Enzyme Linked Immunosorbent Assay (ELISA). Recombinant antigens were prepared in sterile phosphate‑buffered saline (PBS) and used to coat Immulon‑4 microplates at a final concentration of 0.5 µg/mL. Plasma samples were tested in duplicate at a 1:100 dilution, and IgE antibody levels were quantified by ELISA following procedures previously validated in similar immuno‑epidemiological studies [16]-[18]. Antibody detection relied on mouse monoclonal antibodies specific for human isotypes (Cappel^®, USA), followed by peroxidase‑conjugated goat anti‑mouse IgE (1:2000) (Sigma^®, St. Louis, MO, USA), each used at optimized working dilutions after preliminary titration [18][19]. To ensure inter‑plate consistency, standardized reference controls were included in every assay. The positive control consisted of a pooled serum sample from clinically immune adults residing in Dielmo, a holoendemic malaria village in Senegal. Two negative controls were used: pooled sera from non‑immune European donors and pooled sera from non‑immune African individuals from Dakar who tested negative for antibodies against P. falciparum schizont extract [15]-[18]. ELISA results were expressed as an optical density (OD) ratio, calculated as the OD of each sample divided by the OD of the naïve serum pool. Samples yielding an OD ratio greater than 2—which corresponds to the naïve control value plus three standard deviations—were classified as seropositive for prevalence analyses.

For flow cytometry, polymorphonuclear neutrophile cells (PMN) were isolated from peripheral blood (30 to 45 minutes after blood collection), counted, and then labeled with monoclonal antibodies anti-CD16/AF647, anti-CD49/FITC, anti-FcεRI/PE, anti-CD203/PerCP, and anti-IgE/PE in various combinations. Data acquisition by flow cytometry was performed using Cell Quest^® software before analysis with FlowJo^®10.0.6 according to the procedure shown in Figure 1. PMN were isolated via a sequential negative selection gating strategy within the polymorphonuclear cell gate. Briefly, polymorphonuclear basophils (CD203+) and polymorphonuclear eosinophils (CD49+) were systematically excluded by selecting CD203-CD49-polymorphonuclear, yielding a highly purified target PMN population for subsequent evaluation of surface FcεRI and IgE expression.

For statistical analysis, we utilized Statview 5.1 software (SAS Institute, Cary, NJ). Continuous variable distributions were verified to be non-Gaussian, thus non-parametric tests were chosen:

Mann-Whitney and Kruskal-Wallis tests for unpaired comparisons,Wilcoxon signed-rank test for paired comparisons,Spearman’s rank correlation tests to explore relationships between continuous variables.

A difference was considered statistically significant when the p-value was less than 0.05.

This study involved 107 patients divided into three groups:

36 Cerebral Malaria patients (CM)37 Uncomplicated Malaria cases (UM)34 people considered as Control Patients (CP) because they were free from malaria infection (Table 1).

Table 1. General characteristics of the patient cohorts.

n = count; M = male; F = female; d = days.

Demographic characteristics showed a predominance of male patients across all groups. The mean age was approximately 30 years, with a range of 15 to 80 years. Among CM patients, the length of hospital stay varied from 1 to 39 days, with an average duration that differed according to clinical outcome (9 days for survivors and 4 days for those who died). No statistically significant association was found between age, sex, clinical malaria form, or length of hospital stay.

Regarding clinical and biological parameters, patients were classified according to the clinical presentation of malaria. Parasitemia level in CM patients was significantly higher in patients with CM compared to those with UM (30,814.1 ±14,605.8 vs. 4812.6 ± 1452.7 trophozoites/µL) (Table 2).

In the hematological analysis, leukocyte count and polymorphonuclear neutrophil (PMN) levels were significantly elevated in CM patients compared to UM patients. Conversely, red blood cell count and hemoglobin levels were significantly lower in the CM group than in the UM group (Table 2). Finally, platelet counts exhibited a progressive decrease with clinical severity, being significantly lower in UM compared to CP, and further decreased in CM compared to UM patients (Table 2).

Table 2. Clinical and biological characteristics of the patient cohorts at the day of admission.

Data are expressed as Mean ±SD; Exact p-values indicate comparisons between CM vs. UM (first pcolumn) and UM vs. CP (second pcolumn); Abbreviations: CM = Cerebral Malaria; UM = Uncomplicated Malaria; CP = Control Patients.

With regard to PMN, their relative proportions differed markedly across groups: they accounted for 60.1% of circulating leukocytes in UM patients, 42.7% in CP, and only 10.3% in those with CM patients. This significantly reduced proportion of neutrophils in CM contrasts with the overall White Blood Cells (WBC) observed in these patients, which remained higher than in UM patients and in healthy controls.

Assessment of specific plasma IgE levels revealed distinct profiles according to malaria severity. Plasma anti-MSP4-p20 IgE levels were significantly higher in CM patients (median = 1.10 [IQR = 0.20]) compared to UM patients (median = 0.96 [IQR = 0.08]) (Mann-Whitney U = 173.5, p = 0.014). Similarly, plasma anti-MSP1-p19 IgE levels showed higher median values in CM cases (median = 1.86 [IQR = 1.15]) than in UM cases (median = 1.22 [IQR = 0.24]), representing a strong trend toward elevation in severe cases, although it did not reach formal statistical significance (Mann-Whitney U = 157.0, p = 0.070).

At the time of admission in ICU (DoA), we noted a high level of FcƐRI receptors on the surface of polymorphonuclear neutrophils (PMN-FcƐRI) in patients with CM (N = 36, including 27 survivors and 9 fatal cases). Comparison of these PMN-FcƐRI levels between patients suffering from UM (N = 37) and those with CM showed a statistically significant difference with stronger expression in cerebral forms (p= 0.017). However, at discharge from the ICU (DoD), the opposite was noted, i.e. a sharp decrease in the PMN-FcƐRI rate in patients with CM, follow-up was performed in surviving CM patients (N = 27; no discharge sample could be obtained for the 9 fatal cases) whereas there was a clear increase in these receptors in patients with UM patients. This latter variation was not statistically significant (Figure 2).

Figure 2. Comparison of polymorphonuclear neutrophils-FcƐRI receptors expression (PMN-FceRI positive events/all PMN events) (%) according to malaria form. DoA: Day of Admission to intensive care unit/DoD: Day of Discharge from ICU. Uncomplicated Malaria (UM): N = 37 at DoA and DoD, Cerebral Malaria (CM) at Admission (DoA): N = 36 total (27 survivors, 9 fatal cases). Cerebral Malaria (CM) at Discharge (DoD): N = 27 (surviving cases only).

The word At DoA, the level of IgE detected on the surface of neutrophils (PMN-IgE) was higher in CM than in UM. The gap narrowed at the discharge assessment; nevertheless, we observe a slightly elevated PMN-IgE level in cases of CM. (Figure 3). However, it should be noted that none of these variations were statistically significant.

The frequencies of positive PMN-FcƐRI and PMN-IgE events at DoA appear to be discriminating the outcome in patients suffering from CM. Indeed, in deceased patients with CM, higher levels of PMN-FcƐRI and PMN-IgE were found compared to patients that survived (p < 0.001). This analysis is illustrated in Figure 4(a) and Figure 4(b).

Among survivors, a significant decrease in the PMN-FcεRI/IgE levels studied was observed between the day of admission (DoA) and discharge (DoD) from the ICU (Figure 5).

Figure 3. Comparison of surface-bound IgE (Polymorphonuclear neutrophile-IgE positive events/ all PMN events) (%) distributed according to malaria form. PMN-IgE = Polymorphonuclear neutrophile-IgE; DoA = Day of Admission; DoD = Day of Discharge.

Figure 4. Comparison of polymorphonuclear neutrophils-FcƐRI (a) and polymorphonuclear neutrophils-IgE (b) positive events fequencies (%) according to the outcome of cerebral malaria.

Results show that overall, we found a positive correlation between PMN-FcεRI levels and PMN-IgE levels (rho = 0.29; p = 0.041) (Figure 6). Furthermore, a correlation was also observed between the PMN-IgE positive events and plasma IgE levels targeted against MSP-4p20 (p = 0.001; Rho = 0.374). However, we found no correlation between PMN-IgE and plasma anti-MSP1-19 IgE. Nor did we find any correlation between PMN-FcεRI and plasma IgE.

Figure 5. Comparison of PMN-FcεRI and PMN-IgE levels (%) in survivors of cerebral cases at admission and discharge.

Figure 6. Correlations between PMN-FcεRI and PMN-IgE levels in cerebral malaria cases.