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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

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          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.

          Author Summary

          Humans can be infected with five malaria parasite species: Plasmodium falciparum, P. vivax, P. malariae, P. knowlesi, and P. ovale. Although the vast majority of malaria morbidity and mortality worldwide can be attributed to P. falciparum, non-falciparum malaria parasites can also cause clinical disease. Researchers use nucleic acid based detection methods, such a polymerase chain reaction (PCR), to detect low-density malaria parasitemias that can evade microscopic detection. P. ovale was recently identified to exist as two subspecies, P. ovale curtisi and P. ovale wallikeri, that look identical but differ genetically. In this study, we developed a novel real-time PCR (qPCR) assay to detect all P. ovale parasites, based on a conserved gene between P. ovale curtisi and P. ovale wallikeri. We also used DNA sequencing to differentiate between P. ovale curtisi and P. ovale wallikeri from a small sample of P. ovale asymptomatic infections in Western Kenya. Through the use of our novel rbp2 qPCR assay, we aim to characterize the prevalence of P. ovale in future epidemiological studies in order to better understand this neglected malaria parasite species.

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          Most cited references47

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          High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction.

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            Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays.

            There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.
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              Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally.

              Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                January 2015
                15 January 2015
                : 9
                : 1
                Affiliations
                [1 ]Preventive Medicine and Biometrics, Uniformed Services University, Bethesda, Maryland, United States of America
                [2 ]Kondele Laboratory, U.S. Army Medical Research Unit-Kenya, Kisumu, Kenya
                [3 ]Kondele Laboratory, USAMRU-K, Kisumu, Kenya
                [4 ]Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya
                [5 ]Department of Medical Microbiology and Immunology, University of California Davis School of Medicine, Davis, California, United States of America
                US Food and Drug Administration, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: RHM SL VAS. Performed the experiments: RHM COO. Analyzed the data: RHM COO EWW VAS. Contributed reagents/materials/analysis tools: RHM COO EWW BO JW VAS. Wrote the paper: RHM COO SL VAS. Clinical Trial/Sample Collection: EWW BO JW VAS.

                Article
                PNTD-D-14-01487
                10.1371/journal.pntd.0003469
                4295880
                25590587
                8f94f869-46da-4692-b663-a8bfc8f5bdf4

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication

                Page count
                Figures: 5, Tables: 5, Pages: 19
                Product
                Funding
                The work described herein was funded partly by the Uniformed Services University of the Health Sciences Internal Grant RO87N7 (VAS) and partly by the National Institutes of Health 5R01AI104423-02 (VAS and SL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All sequence files are available from the NCBI GenBank database under accession numbers KM494978, KM494979, KM494980, KM494981, KM494982, KM494983, KM494984, KM494985, KM494986, KM494987, and KM494988. All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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