Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya utilizing a novel species-specific real-time PCR assay

Abstract

Background: Plasmodium 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.

Methods: In 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.

Results: Our 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.

Conclusions: Our 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.

Figure 1. P. ovale reticulocyte binding protein 2 (rbp2) sequence alignment.

The P. ovale curtisi (GU813971) and P. ovale wallikeri (GU813972) rbp2 sequences were aligned using EMBL-EBI Clustal Omega program and visualized in Jalview with the default Jalview nucleotide color scheme (green for adenine, orange for cytosine, red for guanine, and blue for thymine). Primers and probe were designed based on conserved regions between the two P. ovale subspecies. The forward (PoRBP2fwd1) and reverse (PoRBP2rev1) primers are indicated by arrows and the hydrolysis probe (PoRBP2p) binding site (boxed) is located in between the forward and reverse primer.

Figure 2. P. ovale rbp2 qPCR dynamic range.

A ten-fold serial dilution of rbp2 plasmid (1 to 100,000 copies/μl) is shown in the amplification plot. The cycle threshold was determined automatically by the ABI 7500 fast system software program. The negative control sample (red line) shows no amplification over the cycle threshold for 60 cycles.

Figure 3. P. ovale rbp2 plasmid standard curve.

A representative standard curve demonstrates linearity based on 10-fold serial dilutions (1 to 100,000 copies/μl) of rbp2 plasmid.

Figure 4. P. ovale rbp2 qPCR specificity.

Serial dilutions of rbp2 plasmid were spiked with P. falciparum DNA (10,000 parasites/μl) or P. vivax DNA (517 parasites/μl). Cq values were unchanged in the presence of DNA from additional malaria parasite species (P = 0.9993).

Figure 5. Comparison of microscopy and P. ovale rbp2 qPCR results.

P. ovale parasitemias based on microscopy (log parasites/μl) were compared to rbp2 qPCR results (log rbp2 copy number/μl). A limited correlation was found between parasitemia and rbp2 plasmid copy number (R2 = 0.6595).