Study area and population

This cross-sectional study was conducted from August to October 2020 at the Oke-Mapo Primary Health Center in Owo, Ondo State, Southwestern Nigeria. The town is located 305 m above sea level and has a population of about 222,000. The clinic is generally focused on the treatment of common childhood infections with referrals for more serious conditions to the district and federal hospitals in Owo. Malaria treatment guidelines in Nigeria recommend screening individuals presenting with a history of fever in the past 48 h or having a measured temperature of >37.5°C for malaria using a Plasmodium falciparum histidine-rich protein 2 (HRP-2)-based mRDT kit or microscopy, followed by treatment with artemisinin-based combination therapy if the test returns positive [13]. The mRDT kit available at the clinic at the time of the study was the Standard Diagnostics (SD Bioline®), malaria Ag P.f RDT kit.

Informed consent were provided by the caregivers for children aged less than 15 years where the decision to screen for malaria was made by the attending healthcare worker. Once permission was given, we documented the child’s age, gender, and complaints on presentation. In addition to the blood sample collected for the mRDT test, we collected three additional drops of blood on a slide for later malaria screening via microscopy. The slides were labeled with the participant’s initials and screening number.

The blood smear was dried at room temperature and stained with 3% Giemsa stain for 30 min and was thereafter rinsed and dried. The stained slides were independently examined under the microscope by two laboratory scientists, and up to 100 high-power fields were viewed before the sample was declared negative. When a parasite was seen, quantification was done by counting parasites in relation to 200 white blood cells and assuming an average white cell count of 8000 cells/μL, following published protocols [14]. For the hematocrit (PCV) determination, microhematocrit tubes were filled to the predefined mark and centrifuged for 5 min at 11,000 rpm in a microhematocrit rotor (Hawksley and Sons Ltd, Sussex, UK), and the PCV value was measured with a hematocrit reader.

To determine the sample size for this study, we assumed a 5% discordance in results between microscopy [15] and mRDT. We estimated that in 250 paired samples, we would be able to detect this level of discordance with a 90% power and a 5% significance level. This number included an additional 10% increase in the estimate to account for samples that were destroyed during processing.

Data collected on paper forms were entered in an access database and were verified against the source document. We presented a description of the study population and the association between malaria covariates, including age and gender, assessed using a regression model. We also determined the sensitivity, specificity, and predictive values of mRDT compared to microscopy.

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