Malaria continues to be the most prevalent infection in the world, causing great mortality in children up to 5 years of age living in endemic areas. In Brazil, the most prevalent malaria is caused by Plasmodium vivax, followed by P. falciparum, associated with higher morbidity and mortality. P. malariae is the least prevalent (0.5-1%), is frequently asymptomatic and presents low morbidity and mortality. In the thick smear test P. malariae can be easily confused with P. vivax due to common morphological characteristics, often passing unnoticed in mixed infections. Few parasites may not be detected even by nested-PCR. In this way, it is possible to assume that cases of malaria caused by P. malariae may be underreported. The present pilot study aims to design primers in the sequence encoding the mitochondrial cytochrome b (cyt b) gene because it is a gene with more copies/ parasite than the 18S rRNA, preferably used in the diagnosis and speciation of Plasmodium. We intend to develop a faster, more sensitive and specific quantitative molecular tool than nested-PCR. For the primers selection we will use the technique known as PCR-ASO (Allele Specific Oligonucleotide), in quantitative amplifications (qPCR-ASO). This is a considerable technical challenge as the cyt b sequence has more than 90% similarity between species. After the standardization step, we will validate qPCR-ASO by testing at least 30 samples with defined diagnosis and species, and the MacNemar test will determine the agreement or disagreement between the results obtained by the reference tests (thick smear and nested-PCR) in relation to the results of determined by qPCR-ASO.
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