Red blood cell indices and cytokine levels in complicated pediatric Malaria in unstable malaria transmission area of Vihiga highlands, Kenya

Study site and population

This study was a cross-sectional investigation in Vihiga County, Western Kenya, a highland location with unstable malaria transmission.^[11,12] After a written informed agreement, 309 children (males, n = 170 and females, n = 139) were recruited as healthy controls “presumptively healthy” (n = 82), uncomplicated malaria (n = 171), and severe malaria (n = 56). Centers for Disease Control and Prevention definitions of complicated Malaria included severe malaria anemia, cerebral Malaria, and parasitemia with metabolic acidosis in children.^[1] Cutoffs for anemia were 11.0 g/dL for 0.5–4.9 years, 11.5 g/dL for 5–11.9 years, and 12 g/dL for 12–14.9 years.^[13,14]

Sample and data collection

Enrolment questionnaires, checklists, and data capture forms collected sociodemographic, anthropometric, and clinical data. By venipuncture, 5 mL of venous blood was collected into heparinized green top BD vacutainer tubes (BD, UK). For hemoglobin and plasmodium species microscopy, finger-prick blood samples were taken.

Determination of parasitemia

Parasitemia was measured by counting asexual stage parasites per 200 white blood cells (WBCs) in blood smears stained with 3% Giemsa. A minimum of 200 high-power fields verified P. falciparum parasitemia microscopically. Malaria complications were diagnosed by erythrocyte morphology, cell size, shape, and staining pattern.

Hematological analysis

Beckman Coulter AcT diff2 hematology analyzers (GMI, US) performed complete blood counts(CBCs). The measurements included WBC, red blood cell (RBC) (×10⁶/mL), hemoglobin (g/dL), hematocrit (%), mean corpuscular volume (MCV) (fL), mean corpuscular hemoglobin (MCH) (pg), mean corpuscular hemoglobin concentration (MCHC) (g/dL), red cell distribution width (RDW) (%), reticulocyte count (%), WBC (×10⁹/L), lymphocytes (×10⁹/L), monocytes (×10⁹/L), platelets (×10⁹/L), and mean platelet volume (MPV). The parasitemia density per µL of blood was calculated using the formula: (parasites/300) × (WBCs/µL). Samples of plasma and serum were aliquoted into cryovials and stored at –70°C until use. May 2020, October 2020, December 2020, and April 2021 hemoglobin samples were taken at every time point. Photometry measured hemoglobin (hemo Control; EKF Diagnostics).

Isolation of peripheral mononuclear cells. Within 6 hours (h) of phlebotomy, density gradient centrifugation extracted peripheral blood mononuclear cells (PBMC) from whole blood. Ficoll-Hypaque density gradient centrifugation isolated the cells. For 30 min, anti-coagulated blood was carefully placed onto 5 mL of Ficoll-Hypaque in 1.5 mL tubes and centrifuged at 1500 rpm. From the buffy coat layer (containing mononuclear cells), a 10 mL pipette was used to collect PBMC into a 1.5 mL tube. The cells were rinsed with 12 mL of sterile phosphate-buffered saline (PBS) at PH 7.0 without calcium or magnesium. Next, they were centrifuged at 1300 rpm for 15 min at room temperature. Aspirate the supernatant, break the pellets with a light flick, and resuspend them in 2 mL complete DMEM with 1% FBS, HEPES (25 nmoL), L-glutamine (2 nmoL), and penicillin-streptomycin (100 U/mL) for 8 days. Using a hemocytometer, the cells were stained with 10 mL trypan blue 1:1 and counted. Microsoft Excel sheets were used to calculate cell counts and dilute them to 2 × 10⁵ cells/well for the test and cell culture. PBMCs were resuspended in full RPMI 1640 culture media. After stimulation with peptides, 2 × 10⁵ cells per well were cultivated in 96 well-round bottom plates at 37°C in 5% CO₂ humidity for 120 h. Harvested supernatants were kept at 80^oC for Bioplex assay cytokine testing.

Cytokine measurements

The cytokine 8-plex conjugated bead (25×) stock solution was vortexed as a working dilution of the anti-cytokine bead in a 96-well bioplex assay buffer. Millipore microtiter plates (Millipore Corporation, Billerica, MA) were prewetted with 100 µL Bioplex assay buffer per well and aspirated using a Millipore vacuum manifold. Next, 50 µL of the working bead solution was applied to the plate wells and aspirated using a Millipore vacuum funnel. To each well, 100 µL of bioplex wash buffer was added and aspirated. Add 50 µL of standard or sample to the wells, place the plate on a microplate shaker (Wilmington, NC) for 30 min at room temperature(RT), and gently mix. Plates were aspirated with a Millipore vacuum funnel to remove supernatant and washed 3 times with 100 µL of BioPlex wash buffer per well. In BioPlex detection antibody diluent, a 10× detection antibody stock solution was diluted. The detection antibody solution was vortexed, 25 µL added to each well, and incubated at RT for 30 min on a microplate shaker. Aspirate 100 µL of BioPlex wash buffer each well, wash the plate three times, and aspirate again to remove the wash buffer. Prepared a 100× working dilution of streptavidin-PE stock solution in bioplex assay buffer. Add 50 µL to each well and incubate for 10 min at RT on the microplate shaker. Aspiration was used to remove the buffer, followed by three washes with 100 µL of BioPlex wash buffer per well and another aspiration. To quantify cytokines, beads were resuspended in 125 µL of BioPlex assay solution per well, briefly mixed, and read on the BioPlex 200 system (Hercules, CA). By indirect enzyme-linked immunosorbent assay (ELISA) (Invitrogen, USA), plasma and culture supernatant cytokines were evaluated at days 0 and 14. To reduce test variability, batch analyses were performed for cytokine levels. ELISA was used to measure cytokine acquisition.^[15] Recombinant antigens (Wellcome genotype) were diluted to 5 µg/mL in PBS (50 µL/well) and incubated overnight at four °C on 96 well plates (maxisorp). After washing the plates four times with PBS and 0.05% Tween 20, they were blocked for 2 h with 200 µL of 1% (wt/vol) non-fat dry milk powder in PBS/T. In duplicate, each plate received positive controls (hyperimmune serum) and negative controls (non-immune resident serum). Plates were washed four times (Dako, Roskilde, Denmark), diluted 1:5,000 in PBS/T and applied to all wells with horseradish peroxidase (HRP)-conjugated streptavidin. Wells were emptied and rinsed four times with a wash buffer after incubation. Develop plates for 20 min with TMB substrate solution. Stopping reactions using two mol/L H₂SO₄. Plates were promptly examined at 450 nm, and optical density (OD) values recorded. Malaria-naïve and malaria-exposed individuals were used as negative and positive controls to determine the cutoff values for each antigen. Positive samples had OD greater than the mean + two standard deviations of the negative control plasma.

Ethical consideration

This protocol was approved by Masinde Muliro University of Science and Technology Institutional Ethical Review Committee (MMUST-IREC) protocol number MMUST/IERC/021/2021. The National Council on Science and Technology (NACOSTI) license number NACOSTI/P/21/14703 authorized the study. All participants signed consent forms.

Statistical analysis

Prism 7 statistical software (GraphPad Software, La Jolla, California, USA, www.graphpad.com) was used to analyze data. Medians and interquartile ranges show age, gender, red cell count, and cytokine levels. Comparisons were made between groups using the Kruskal–Wallis test, with Bonferroni correction at P < 0.017. Categorical variables were compared using Chi-square test. Statistical significance was defined as P < 0.05.

Demographic and clinical characteristics

Table 1 shows the study participants’ demographic and clinical features. There were 309 children, 82 (26.5%) malaria-free, and 227 (73.5%) malaria-infected. Malaria-infected subjects had 171 (75.3%) simple malaria and 56 (24.7%) severe malaria. Complicated malaria patients were younger (median, 8.5; interquartile range [IQR] 5 months; P = 0.014). Study groups likewise had similar gender distribution (P = 0.621). Clinical groups had similar axillary temperatures (P = 0.418). Fever was found in 92% and 96% of uncomplicated and complicated malarial children. Uncomplicated (74%) and complicated (73%) children had low feeding/appetite. Complicated Malaria caused 39% of children to have generalized pallor, compared to 9% of uncomplicated malaria patients. Jaundice rates were higher in children with complicated Malaria (13% vs. 2%). The complications malaria study group exhibited higher coughing and diarrhea than the simple malaria group. Convulsion was only documented in children with complicated Malaria.

Hematological profiles of the study participants

Table 2 shows the study participants’ hematological indicators. Complicated Malaria in children resulted in considerably lower hemoglobin levels (P < 0.0001) compared to uncomplicated Malaria (median, 9.9; IQR 3.6 g/dL) and healthy controls (median, 13.7; IQR 2.2 g/dL). Post hoc analyses showed decreased hematocrit, RBC count, MCH, MCHC, and platelets in complicated malaria patients compared to uncomplicated Malaria and healthy controls (median, 29.5; IQR, 7.3%; P < 0.017). Children with uncomplicated Malaria had a substantially higher MCV (median, 76.2; IQR 8.8 fL) than those with complicated Malaria (71.1; IQR 13.3fL). In addition, complicated malaria patients had lower MCV levels than healthy controls (median, 75.2; IQR 5.6fL vs. median, 71.1; IQR 13.3 fL). In contrast, the complicated malaria group had higher RDW, reticulocyte count, lymphocytes, monocytes, and MPV compared to other study groups (median, 24.0; IQR, 4.6%; P < 0.017). Three clinical groups had similar WBC counts (P = 0.068).

Serum cytokines levels

Figures 1 and 2 show study participants’ circulating cytokines. Figure 1 shows box plots for Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES), macrophage inflammatory protein 1 alpha (MIP1-α), macrophage inflammatory protein 1 beta (MIP1-β), IL-8, IL-10, and IL-12. Figure 2 shows IL-1β, IL-2, IL-4, IL-5, IL-6, and interferon gamma (IFN-ɤ) plots. Serum RANTES levels were substantially higher (P < 0.0001) in children with complicated Malaria (median, 20281.23; IQR 1019 pg/mL) compared to uncomplicated Malaria (median, 17267.73; IQR, 883 pg/mL) and healthy controls (median, 15801.31; IQR, 507 pg/mL IL-8 complicated Malaria (median, 32.06; IQR 11.5 pg/mL), uncomplicated Malaria (median, 16.05; IQR 8.7 pg/mL), healthy controls (median, 6.24; IQR 4.4 pg/mL), IL-10 complicated Malaria (median, 89.1; IQR 166.5 pg/mL), and uncomplicated Malaria. In contrast, children with uncomplicated Malaria had significantly higher levels of MIP1-α (median, 115.0; IQR 121.6 pg/mL) and MIP1-β (median, 286.0; IQR, 408.2 pg/mL) (P = 0.002 and P < 0.0001). IL-1β levels were higher in children with complicated Malaria (median, 3.47; IQR, 3.3 pg/mL) compared to uncomplicated Malaria (median, 2.56; IQR 2.1 pg/mL) and healthy controls (median, 1.93; IQR 0.9 pg/mL) (P < 0.0001). In complicated Malaria, IL-2, IL-5, IL-6, and IFN-ɤ levels were high (median, 1.71; IQR, 0.8 pg/mL; P < 0.0001; median, 1.82; IQR, 2.2 pg/mL; P < 0.0001; median, 171.7; IQR, 22.3 pg/mL; P < 0.0001; median, 102.0; IQR, 132.5 pg/mL; P < 0.0001). Healthy controls had significantly higher IL-4 levels (24.1; IQR 25.6 pg/mL) compared to uncomplicated and complicated malaria groups (median, 12.0; IQR 44.3 pg/mL and 12.6; IQR 16.8 pg/mL, respectively, P < 0.0001).

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Figure 1:

Serum levels of (a) Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES), (b)MIP1-α, (c) MIP1-β, (d)IL-12, (e)IL-10, and (f)IL-8. HC: Healthy controls, UM: Uncomplicated Malaria, CM: Complicated Malaria. Data was analyzed using the Kruskal–Wallis H-test. Post hoc analyses done using Bonferroni correction set at P < 0.017). IL: Interleukin, MIP1-α: Macrophage inflammatory protein 1 alpha, MIP1: Macrophage inflammatory protein 1 beta , ***Refers to P < 0.0001, **Refers to P < 0.001.

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Figure 2:

Serum levels of (a)IL-6, (b)IL-5, (c)IL-4, (d)IL-1β, (e) INF-ɤ, and (f)IL-2. HC: Healthy controls. UM: Uncomplicated malaria, CM: Complicated malaria. Data analyzed using Kruskal–Wallis H-test. Post hoc analyses done using Bonferroni correction set at P < 0.017). IL: Interleukin, INF-ɤ: Interferon gamma, ***Refers to P < 0.0001, *Refers to P < 0.05.

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Recommendations

• Enhance malaria diagnosis and management by incorporating hematological characteristics associated with complicated Malaria, such as lower hemoglobin levels and altered RBC parameters, highlighting the importance of early diagnosis and appropriate management. Health systems should ensure access to accurate and timely diagnostic tools and effective antimalarial treatments.

• Monitor and manage cytokine responses in complicated malaria patients. The elevated levels of proinflammatory and anti-inflammatory cytokines in complicated Malaria indicate dysregulated immune responses. Further research is needed to understand the role of cytokines in disease progression and to develop targeted interventions to modulate the immune response.