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

Miller RH, Obuya CO, Wanja EW, Ogutu B, Waitumbi J, Luckhart S, Stewart VA - PLoS Negl Trop Dis (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Preventive Medicine and Biometrics, Uniformed Services University, Bethesda, Maryland, United States of America.

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.

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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).
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pntd.0003469.g004: 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).

Mentions: Spiking experiments, in which P. falciparum DNA or P. vivax DNA was added to the rbp2 plasmid standard curve samples and subsequently utilized as template for the rbp2 qPCR did not significantly alter the Cq values compared to when the standard curve plasmid samples were run alone (ANOVA, P = 0.9993, Fig. 4).


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

Miller RH, Obuya CO, Wanja EW, Ogutu B, Waitumbi J, Luckhart S, Stewart VA - PLoS Negl Trop Dis (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295880&req=5

pntd.0003469.g004: 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).
Mentions: Spiking experiments, in which P. falciparum DNA or P. vivax DNA was added to the rbp2 plasmid standard curve samples and subsequently utilized as template for the rbp2 qPCR did not significantly alter the Cq values compared to when the standard curve plasmid samples were run alone (ANOVA, P = 0.9993, Fig. 4).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Preventive Medicine and Biometrics, Uniformed Services University, Bethesda, Maryland, United States of America.

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.

Show MeSH
Related in: MedlinePlus