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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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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).
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pntd.0003469.g005: 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).

Mentions: Quantification comparison: Microscopy versus rbp2 qPCR. Quantitative parasitemia determined by expert microscopy (parasites per microliter) was compared to the rbp2 copy number per microliter based on the rbp2 plasmid standard curve (Fig. 5). A modest correlation was determined (R2 = 0.6595). This lack of a strong correlation is not surprising, as all P. ovale parasitemias were low, ranging from 16–3800 parasites/μl, and such low-level parasitemias are notoriously difficult to quantify accurately by microscopy [40, 45–47]. Additionally, the samples utilized for comparison were mixed malaria species infections, mainly with P. falciparum. Mixed species infections create further difficulties for the accurate quantification of P. ovale-specific parasitemia based on light microscopy, but single-species P. ovale infected samples were not available.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003469.g005: 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).
Mentions: Quantification comparison: Microscopy versus rbp2 qPCR. Quantitative parasitemia determined by expert microscopy (parasites per microliter) was compared to the rbp2 copy number per microliter based on the rbp2 plasmid standard curve (Fig. 5). A modest correlation was determined (R2 = 0.6595). This lack of a strong correlation is not surprising, as all P. ovale parasitemias were low, ranging from 16–3800 parasites/μl, and such low-level parasitemias are notoriously difficult to quantify accurately by microscopy [40, 45–47]. Additionally, the samples utilized for comparison were mixed malaria species infections, mainly with P. falciparum. Mixed species infections create further difficulties for the accurate quantification of P. ovale-specific parasitemia based on light microscopy, but single-species P. ovale infected samples were not available.

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