Study design, setting and participants

The study was a cross-sectional, descriptive, and analytical study, carried out at the Mother and Child Centre (MCC), Chantal Biya Foundation, Yaoundé, Cameroon, over a period of 7 months (November 2018 to June 2019). A total of 126 children (aged 6 months to 15 years) were initially selected based on presenting complaints. The children to take part in the study were selected by a single paediatrician who thoroughly examined the children for a concomitant clinical focus of bacterial infection. Those with overt evidence of clinical bacterial infections were excluded. These included anomalies to various organ systems such as bulging tympanic membranes, hyperemic conjunctiva, erythematous oropharynx, lung crackles, lymphadenopathy and present costovertebral and supra-pubic tenderness. A complete blood count was also performed to exclude all cases with hyper leukocytosis. A total of 83 children were retained for the study. Figure 1 shows the flow diagram of how the study participants were enrolled into the different phases of the study.

Fig. 1

Flow diagram of participant recruitment in the study

Assessment of malaria using a rapid diagnostic test for malaria

This was done using rapid diagnostic test (RDT) kits for malaria (Standard Diagnostics BIOLINE®, Malaria Ag. P.f/Pan) with peripheral venous blood which had been collected into a 3-5 ml EDTA tube. Whole blood (0.5 mL) was pipetted into the EDTA tube and dropped in the sample hole quickly enough to avoid blood clotting. Two drops of the buffer were put into the appropriate hole. After waiting for 15–20 minutes, the results were read. The RDT was considered valid with the presence of the control line.

Peripheral blood smear for malaria and assessment of severity

Parasite density was evaluated by May-Grunwald and Giemsa stains of thin and thick smears, respectively, using blood from the EDTA tubes. The parasite density was calculated per microlitre of blood using each patient’s white blood cell count from a full blood count analysis. (©2017 Shenzhen Mindray BC 3D Bio-medical Electronics Co. Ltd). Thin films were examined only for malaria parasite species. Severe malaria was defined using the WHO malaria severity criteria [6]: impaired consciousness, prostration, convulsions, acidosis, hypoglycaemia, severe anemia, renal impairment, jaundice, pulmonary edema, significant bleeding, shock, hyperthemia at 40o c and hyperparasitemia. Severe malaria was diagnosed if one or more criteria for severity were present.

Assessment of plasma procalcitonin levels

Blood collected in the EDTA tubes was centrifuged, plasma was obtained and stored in a − 80 °C freezer in cryotubes pending batch analysis. Analysis was completed within 2 months of storage. The samples did not undergo any freeze-thaw process before analysis. Plasma PCT was then measured using Human PCT Enzyme-Linked Immunosorbent Assay (ELISA) Kit. Catalog No: E-EL-H1492 96 Test Elabscience® following the manufacturer’s instructions at the Clinical Diagnosis Laboratory of the National Obesity Centre, Yaoundé Central Hospital. The sensitivity of the assay was 18.75 pg/ml. The kit was able to recognise natural and some recombinant human PCT. No significant cross-reactivity or interference between human PCT and analogues was observed. The coefficient of variation (precision within an assay and between assays) was < 10%.

Study outcome variables

The primary outcome was the difference in serum PCT levels between the cases with uncomplicated and severe malaria. Other secondary outcome variables included presenting complaints at inclusion into the study and malaria parasite density.

Statistical analysis

Data was entered with Epi info version 7.2.2.6 and analysed with SPSS (Statistical Package for Social Sciences) version 22.0. Student t-test was used to compare means while Mann-Whitney U test was used to compare medians. Correlation between continuous variables was examined using Spearman correlation coefficient. P values less than 0.05 were considered statistically significant.

Ethical consideration and confidentiality

Ethical clearance was obtained from the Institutional Review Board (IRB) of the Faculty of Medicine and Biomedical Sciences (FMBS) – University of Yaoundé I (No: 117/UY1/FMSB/VDRC/CSD), Centre Regional Ethics Committee for Human Research (No: 1582/AP/MINSANTE/SG/DRSPC/CRERSH) and the parents/guardians of the participants provided a signed written informed consent before participating in the study.