Research Article: Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya Utilizing a Novel Species-specific Real-time PCR Assay

Date Published: January 15, 2015

Publisher: Public Library of Science

Author(s): Robin H. Miller, Clifford O. Obuya, Elizabeth W. Wanja, Bernhards Ogutu, John Waitumbi, Shirley Luckhart, V. Ann Stewart, Alain Debrabant.

Abstract: BackgroundPlasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden.MethodsIn order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri.ResultsOur P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene.ConclusionsOur novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission.

Partial Text: Plasmodium ovale, the causative agent of benign tertian malaria, was identified as a distinct malaria parasite species in 1922 based on its characteristic oval morphology in infected erythrocytes [1]. P. ovale rarely causes severe disease in humans living in malaria endemic regions, but can cause serious clinical disease in naive travelers [2–9]. The actual prevalence and clinical relevance of P. ovale is likely underestimated for the following reasons. First, P. ovale is often found as a mixed infection with other malaria parasite species [10–12]. This can confound microscopic identification of P. ovale due to difficulties in differentiating P. ovale from other morphologically similar malaria parasites, such as P. vivax. Second, the characteristic low-level parasitemia of P. ovale infection further complicates microscopic detection due to the difficulty in finding and identifying low numbers of P. ovale parasites [13]. Finally, malaria Rapid Diagnostic Tests (RDTs) show a reduced ability to detect P. ovale compared to other human malaria parasites, resulting in false negative cases [14–16]. However, the use of extremely sensitive molecular detection methods, such as polymerase chain reaction (PCR), have revealed a higher prevalence of P. ovale and expanded the geographical distribution of this malaria parasite compared to what was previously identified based on microscopy [10, 17–20].

Based on multilocus genotyping using the rbp2, ssrRNA, and tra genes, we detected both P. ovale curtisi and P. ovale wallikeri in approximately equal frequencies in a small sample set from Western Kenya, a region in which P. ovale subspecies characterization had not been previously performed. The presence of both P. ovale subspecies in Western Kenya is in agreement with other studies in sub-Saharan Africa and P. ovale endemic regions that describe the sympatric distribution of P. ovale curtisi and P. ovale wallikeri [23, 27, 48]. We also identified additional allelic diversity within the tra gene in P. ovale samples from Kenya (Table 4) compared to what was previously identified in P. ovale samples from other malaria endemic regions [23]. This allelic diversity at the P. ovale tra gene is consistent with reports of other tra variants identified by DNA sequencing, however our tra sequences are unique from previously published tra gene sequences [30].



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