Study design and sites
This was a cross-sectional analytical study that took place from December 2019 to February 2020 in Togo. Malaria transmission is stable throughout the country, with two predominant climates: the sub-equatorial with two rainy seasons in the southern part of the country and the tropical with a single rainy season in the northern part. Thus, three sentinel sites for monitoring the effectiveness of ACTs used for the treatment of uncomplicated malaria were used for this evaluation. The Social Medical Center (SMC) of Cacaveli, a public health facility in Lomé, the capital of Togo, was the first site, to which the SMC “UTB Circulaire” was added because of the relatively low patient attendance at the site. The SMC Ahépé, a public health facility in the Maritime region, was the second site, located 66 km from Lomé, to which the hospital “la Providence de Kouvé” was also added. The last site was the Sokodé polyclinic located in the central region approximatively 340 km north of Lomé. The first two sites were sub-equatorial, located respectively in urban and rural areas, and the third site was tropical, located in a semi-urban area.
Study population and sampling
The study population was symptomatic patients suspected of having malaria who were seen in consultation at the different sentinel sites and for whom TTBS was prescribed. Since the sensitivity of RDTs varies according to the parasite density [9, 10] and can reach 100 parasites/μl, which may vary from one product to another [11], blood smear-positive subjects were divided into two groups, a low parasite density group (patients with asexual parasitemia count per microliter between 50 and 1000) and a high parasite density group (those with asexual parasitemia per microliter between 2000 and 10,000). The control group comprised subjects negative for any species of Plasmodium.
Sample size
The sample size calculation methods of Buderer et al. [12] were used. For calculation of sensibility and specificity, we used the formula Np = \(\frac{{{Z}_{a/2}}^{2}\mathrm{se}(1-se)}{{E}^{2}}\) to estimate the number of cases (positives) to include and the formula Nn = \(\frac{{{Z}_{a/2}}^{2}\mathrm{sp}*(1-sp)}{{E}^{2}}\) to estimate the number of controls (negatives). A 90% sensitivity was estimated for low parasitemia and 95% for high parasitemia with a tolerated margin of error (E) of 5% and an accepted risk of error (α) of 5% (Zα/2 at 1.96); the size of positives with low parasitemia for inclusion was 139, and the number of positives with high parasitemia for inclusion was 73. For specificity estimated to 90%, the number of included controls was 139. Therefore, this study should include a total sample size of 351 participants.
Inclusion and non-inclusion criteria
Inclusion criteria were considered by group. Included in the low parasitemia group were patients with asexual parasitemia count per microliter between 50 and 1000; in the high parasitemia group, those with asexual parasitemia per microliter between 2000 and 10000; and in the control group, individuals with negative thick blood smear [13,14,15]. Signed written consent was obtained from each adult patient and the parent/guardian of the children before their enrollment in the study. Any person who did not meet the above criteria and who voluntarily declined to participate in the study was not included in the study population.
Data collection
A structured questionnaire was used to collect information on sociodemographic characteristics, clinical signs presented, history of the disease, and existence of other diseases if applicable.
Laboratory tests
Each patient had a capillary blood sampling for a TTBS. After the microscopy results were known (having parasitemia within a certain range or being malaria negative), a second sample was taken from the included subjects to test the RDTs evaluated for Plasmodium spp. infection, and dried blood spots (DBS) were performed on Wattman type III paper.
Thick and thin blood smear
The thick and thin smears were made on the same slide. Two slides were made. After fixing the thin blood smear with methanol for a few seconds, the first slide was stained by Giemsa 10%, 10 to 15 min, for initial screening (having parasitemia within a certain range or being malaria negative), and the second at 3%, 45 min for detailed examination to obtain definitive results. [16]. After drying, the slides were then read under an immersion microscope with an 100× objective to determine the positivity and identify the plasmodial species and estimate the parasitemia.
Rapid diagnostic tests
Two types of Advantage brand RDTs were evaluated: Advantage P.f. Malaria Card® (IR016025), which is specific for Plasmodium falciparum, and Advantage Malaria Pan + Pf Card® (IR231025), which can detect P….
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