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Context and Aim: Given the challenges of microscopy, we compared its performance with SD-Bioline malaria rapid diagnostic test (MRDT) and polymerase chain reaction (PCR) and evaluated the time it took for positive results to become negative after treatment of children with acute uncomplicated malaria. Subjects and Methods: We present the report of 485 participants with complete MRDT, microscopy, and PCR data out of 511 febrile children aged 3-59 months who participated in a cohort study over a 12-month period in rural and urban areas of Ibadan, Nigeria. MRDT-positive children received antimalaria and tested at every visit over 28 days. Speciation was also carried out by PCR. Results: With microscopy as the gold standard, SD-Bioline™ had 95.2% sensitivity, 66.4% specificity, 67.5% positive predictive value (PPV), and 94.9 negative predictive value (NPV), while with PCR the findings were 84.3% sensitivity, 66.5% specificity, 72.7% PPV, and 80.1% NPV. PCR speciation of malaria parasites revealed 91.6% Plasmodium falciparum, 18.9% Plasmodium malariae, and 4.4% Plasmodium ovale. Among the 47 children with P. malariae infections, 66.0% were coinfected with P. falciparum, while 54.6% cases of P. ovale occurred as coinfections with P. falciparum. The median time to a negative MRDT was 23.2 days, while the median time to a negative malaria microscopy was 3.8 days. The two survival curves were significantly different. Conclusions: The SD-BiolineTM MRDT performed well, with remarkable persistence of rapid test-positive for an average of 23 days post treatment. The prevalence of P. malaria is somewhat greater than expected. [ABSTRACT FROM AUTHOR]