title: 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.
authors.affiliation: Preventive Medicine and Biometrics, Uniformed Services University, Bethesda, Maryland, United States of America.
authors.forename: Robin H
authors.initials: RH
authors.lastname: Miller
authors.affiliation: Kondele Laboratory, U.S. Army Medical Research Unit-Kenya, Kisumu, Kenya.
authors.forename: Clifford O
authors.initials: CO
authors.lastname: Obuya
authors.affiliation: Kondele Laboratory, USAMRU-K, Kisumu, Kenya.
authors.forename: Elizabeth W
authors.initials: EW
authors.lastname: Wanja
authors.affiliation: Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
authors.forename: Bernhards
authors.initials: B
authors.lastname: Ogutu
authors.affiliation: Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya.
authors.forename: John
authors.initials: J
authors.lastname: Waitumbi
authors.affiliation: Department of Medical Microbiology and Immunology, University of California Davis School of Medicine, Davis, California, United States of America.
authors.forename: Shirley
authors.initials: S
authors.lastname: Luckhart
authors.affiliation: Preventive Medicine and Biometrics, Uniformed Services University, Bethesda, Maryland, United States of America.
authors.forename: V Ann
authors.initials: VA
authors.lastname: Stewart
mesh_terms: D001483:Base Sequence; D005838:Genotype; D006801:Humans; D007630:Kenya / Q000453:epidemiology; D008288:Malaria / Q000453:epidemiology; D008853:Microscopy; D041122:Plasmodium ovale / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013045:Species Specificity