A PCR-based assay for the sensitive and accurate determination of malaria parasite species that commonly infect humans, including discrimination of Plasmodium ovalecurtisi and Plasmodium ovalewallikeri

Abstract

There are six Plasmodium species that commonly infect humans including Plasmodium ovalecurtisi, and Plasmodium ovalewallikeri. To ensure successful malaria control, it is important to track their prevalence and understand their epidemiological and clinical characteristics. Current PCR-based malaria diagnostic techniques targeting the highly sensitive mitochondrial genome do not distinguish between the two Plasmodium ovale species. Here we present a PCR-based species typing method targeting the mitochondrial COXIII involving a sensitive, nested PCR with species-specific primers and a simple enzyme digestion to distinguish P. ovalecurtisi and P. ovalewallikeri. The assay is highly specific to each species and highly sensitive, detecting 0.125 and 0.25 parasites per 1 {micro}l of blood from in vitro cultured Plasmodium knowlesi and Plasmodium falciparum respectively. Furthermore, using 30 field samples, we validate its effectiveness in discriminating the two P. ovale spp., identifying 18 P. ovalecurtisi, 16 P. ovalewallikeri, 23 P. falciparum and 17 Plasmodium malariae infections. This novel assay allows the sensitive and accurate diagnosis of P. falciparum, Plasmodium vivax, P. malariae, P. knowlesi, P. ovalecurtisi, and P. ovalewallikeri and can be used to monitor human and zoonotic malaria infections for both diagnosis and epidemiological studies.