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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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Related in: MedlinePlus

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.
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pntd.0003469.g002: 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.

Mentions: Plasmid standard curve analysis of rbp2 qPCR assay. Since all 22 P. ovale microscopy positive samples were successfully amplified and sequenced using the rbp2 primers, we developed an rbp2 based qPCR assay to detect all P. ovale subspecies simultaneously in a single assay. Efficiency of the rbp2 qPCR assay was analyzed using the non-linearized rbp2 plasmid 10-fold serial dilution standard curve. Efficiency ranged from 90%–99% for six consecutive qPCR experiments with a coefficient of correlation (R2) greater than 0.99. A representative qPCR amplification plot and standard curve are shown in Fig. 2 and 3, respectively. All 22 P. ovale samples identified as P. ovale positive by expert microscopy were detected using our rbp2 qPCR assay. There was no difference in PCR efficiency or R2 value based on the conformation (linearized vs. non-linearized) of the rbp2 plasmid standard curve (Pearson product-moment correlation = 0.998, P<0.001).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0003469.g002: 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.
Mentions: Plasmid standard curve analysis of rbp2 qPCR assay. Since all 22 P. ovale microscopy positive samples were successfully amplified and sequenced using the rbp2 primers, we developed an rbp2 based qPCR assay to detect all P. ovale subspecies simultaneously in a single assay. Efficiency of the rbp2 qPCR assay was analyzed using the non-linearized rbp2 plasmid 10-fold serial dilution standard curve. Efficiency ranged from 90%–99% for six consecutive qPCR experiments with a coefficient of correlation (R2) greater than 0.99. A representative qPCR amplification plot and standard curve are shown in Fig. 2 and 3, respectively. All 22 P. ovale samples identified as P. ovale positive by expert microscopy were detected using our rbp2 qPCR assay. There was no difference in PCR efficiency or R2 value based on the conformation (linearized vs. non-linearized) of the rbp2 plasmid standard curve (Pearson product-moment correlation = 0.998, P<0.001).

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