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This study will be conducted at Chulaimbo Sub-District Hospital in Kisumu County, Western Kenya Latitude: 002 0 North, Longitude: 34037' East. The Hospital is located about 18 km North West of Kisumu City at an altitude of 1300 meters above the sea level. The climatic condition of this place is equatorial sub-humid Savannah type influenced by continental rainfall regime of Lake Victoria Basin. This is a malaria holoendemic region and despite reports of a decline in malaria incidences in a number of malaria endemic countries, this region according to latest studies has recorded resurgence in clinical malaria (Okiro et al. 2010).
3.2 Study Population
3.2.1 Inclusion Criteria
This is a longitudinal study that would enroll 100 children between the ages of 6 months to 5 years presenting at Chulaimbo Sub-District Hospital with acute uncomplicated malaria diagnosis. These children will be recruited into the study after informed written consent from their parents or guardians for blood samples to be drawn, and admission with a diagnosis of acute malaria morbidity as defined by an axillary temperature ≥37.5 and Plasmodium falciparum parasitaemia ≥ 5000/µL. Enrolled children should not present with anemia and upper respiratory tract infections (URTIs) and they should also be HIV negative. The children would be followed up at 7 days and 30 days after the acute infection. At follow up time points, the children will be examined clinically and treated if necessary. In addition, 30 healthy age-matched children who are parasite negative and with normal body temperature of 37°C would be enrolled as controls.
3.3 Sample Size Calculation
The sample size for the study has been calculated using the Gpower 3.1software. To determine the sample size, an effect size of 0.5, alpha error of 0.05, and power of 0.80 and allocation ratio of 0.3 were used. With these, the sample size was calculated to be 130 (100 for cases and 30 for healthy controls).
3.4 Blood Sample Collection
3ml of blood would be drawn from children presenting with acute malaria at Chulaimbo Sub-district Hospital. Additional 5ml blood samples would be drawn after 7 days and 30 days post-treatment. The blood will be transferred into a 15 ml Falcon tube containing 50 units of heparin. A small aliquot of this blood will be used for thick and thin smear for the detection of the parasite by microscopy. Another small aliquot will be collected in ethylene-diamine-tetra-acetic acid (EDTA) for whole blood differential counts. These would be done in accordance with approved guidelines established by the Research Ethics Boards of SUNY Upstate Medical University, the Kenya Medical Research Institute and declaration of Helsinki.
3.5 Peripheral Blood Mononuclear Cells (PBMCs) Isolation
Isolation of peripheral blood mononuclear cells (PBMCs) will be done using Ficoll-hypaque density gradient centrifugation. After collection in heparinized tubes, whole blood will be carefully layered on 5ml of Ficoll-hypaque in sterile 15ml tubes under sterile conditions in laminar flow hood. Then the tubes will be spun at 1500 rpm for 30 minutes (Sorvall RT6000D, ThermoFischer Scientific, MA, USA) and this will allow separation of whole blood into various constituent parts. The plasma on top would be sucked with sterile 5ml pipette tubes and aliquoted into sterile Sarsdedt (SarsdedtTM, Newton, Germany) tubes. This will then be frozen at -800C for later use for antibody and cytokine detection. The (buffy coat) layer containing PBMCs would then be carefully transferred into a new sterile 15ml tube then topped with sterile 1- PBS (pH 7.0) up to 12ml mark and mixed thoroughly, followed by another spinning at 1000 rpm for 15 minutes (Sorvall RT6000D, ThermoFischer Scientific, MA, USA). After spinning, the supernatant will be carefully aspirated off and the pellet broken by gently tapping on the tube with the knuckles. The cells would then be re-suspended in 12ml of sterile 1- PBS (pH 7.0) and again centrifuged for 10 minutes at 1000rpm (Sorvall RT6000D, ThermoFischer Scientific, MA, USA) for 10 minutes. The supernatant will be carefully aspirated and then the pellet broken by gently tapping with the knuckles before finally re-suspending in 1ml of culture media (RPMI 1640; GIBCO, Invitrogen, Paisley, Scotland, UK) supplemented with L-glutamine, human serum type AB, HEPES and gentamycin (cRPMI 1640). The viability of the re-suspended PBMCs will be performed by mixing the re-suspended PBMCs with Turk's solution in 1:1 ratio followed by dispensing 10µL of the mixture into a haemocytometer for PMBC enumeration using a light microscope.
3.6 Measurement of anti-schizont IgG and IgM antibodies by ELISA
To determine the total immunoglobulin M (IgM) and immunoglobulin G (IgG) in plasma, plates would be coated with 50µL/well of polyclonal rabbit anti-human IgM or IgG in 0.1 carbonate buffer, pH 9.6. After coating, the plates will be incubated overnight at 4°C. Then the coating solution will be poured off from the plate. After washing twice with PBS, the wells will be blocked with 150µL of blocking buffer, and then incubated at 37°C for 1 hour. Washing will be done 4 times with 200µL wash buffer after which 100µL/well of samples and/ or standards in desired dilutions (usually 1:100; 1:400) in sample dilution buffer will be added. Then it will be incubated at 37°C for 1 hour. This will be followed with washing 4 times with 200µL of PBST. After washing, 100µL/well of conjugate (Rabbit anti-human IgG or IgM Horse radish peroxidase (HRP) (Dako, (PO214) Glostrup, Denmark) diluted 1:4000 in sample/conjugate dilution buffer will be added. Then it will be incubated at 37°C for 1 hour followed with washing 4 times with 200µL of PBST. After washing, 100µL/well of substrate solution will be added and then incubated in the dark (or covered with foil paper) for 15-30 minutes at RT. The development of colour reaction will be checked after which 100µL/well of stop solution will be added. The measurement will be done at 450nm after 20- 30 minutes on an ELISA reader; Opsys MRTM (DYNEX Technologies, Inc; (Sully field Circle, Chantilly, VA) or West Sussex UK) using RevelationTM QuickLink Software (Version 4.25).
3.7 Assaying of the TNF cytokines (BAFF, TWEAK and APRIL) by ELISA
Plasma levels of BAFF, APRIL and TWEAK will be determined using Enzyme-Linked Immunosorbent Assay (ELISA). Plates would be coated with 50µL/well of mouse IgG1 anti-human BAFF and APRIL and also anti-TWEAK monoclonal antibodies at 1µg/m L in PBS. After coating, the plates will be incubated overnight at 4°C after which the coating solution will be poured off from the plate. After washing twice with PBS, the wells will be blocked with 150µL of 0.5% Bovine Serum Albumin (BSA) and then incubated at 37°C for 1 hour. Washing will be done 4 times with 200µL wash buffer after which 100µL/well of samples and/ or standards in desired dilutions (usually 1:100; 1:400) in sample dilution buffer will be added. Then it will be incubated at 37°C for 1 hour. This will be followed with washing 4 times with 200µL of PBST. After washing, 100µL/well of conjugate (Biotinylated goat anti-human BAFF, APRIL and TWEAK) diluted 1:4000 in sample/conjugate dilution buffer will be added. Then it will be incubated at 37°C for 1 hour followed with washing 4 times with 200µL of PBST. After washing, 100µL/well of substrate solution will be added and then incubated in the dark (or covered with foil paper) for 15-30 minutes at room temperature. The substrate reaction will then be stopped by addition of 50 µL of the stop solution and optical density read at 450nm after 20-30 minutes on an ELISA reader (Opsys MRTM (DYNEX Technologies, Inc; (Sully field Circle, Chantilly, VA) or West Sussex UK) using RevelationTM QuickLink Software Version 4.25). Data obtained will be exported to Excel Microsoft and then transferred to GraphPad Prism version 4 for graphical representation and statistical analysis.
3.8 Flow Cytometry
Eight-colour flow cytometry will be used to characterize the BAFF, APRIL and TWEAK receptors as well as to define B-cell sub-populations. PBMCs would be thawed and viable cells counted. For analysis of BAFF, APRIL and TWEAK receptors as well as B-cell sub-populations PBMCs will be stained with the anti-BAFF-R (anti-CD237), polyclonal anti-human BCMA (anti-CD267), anti-FN14 (anti-CD266), anti -CD19-PE, anti-IgD-FITC, anti-CD27-PE and anti-CD10-PE, monoclonal antibodies for 30 minutes in the dark. Followed by washing in flow buffer and fixing in 1% paraformaldehyde for 15 minutes, washed and re-suspended in 300ul of flow buffer. Data obtained will be collected using a FACsCalibur or LSRII flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, USA) and analysed using FlowJo software (Tree Star Inc. San Carlos, CA, USA).
3.8 Statistical Analysis.
The analysis of data will be done using GraphPadPrism version 5. Statistical differences between continuous variables between defined groups like children suffering acute uncomplicated clinical malaria will be determined using unpaired t test while within group comparisons will be done using repeated measures analysis of variances with a Bonferroni post. Correlations among different parameters will be analyzed by the Pearson's correlation.