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A new real-time PCR for the detection of Plasmodium ovale wallikeri.

ABSTRACT: It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite's small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 11 to P. ovale wallikeri. Thus, this assay provides a useful tool that can be applied to epidemiological investigations of the two newly recognized distinct P. ovale species, that might reveal if these species also differ in their clinical manifestation, drugs susceptibility and relapse periodicity. The results presented confirm that P. ovale wallikeri is not confined to Southeast Asia, since the majority of the patients analyzed in this study had acquired their P. ovale infection in African countries, mostly situated in West Africa.

SUBMITTER: Calderaro A

PROVIDER: S-EPMC3480495 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A new real-time PCR for the detection of Plasmodium ovale wallikeri.

Calderaro Adriana A Piccolo Giovanna G Gorrini Chiara C Montecchini Sara S Rossi Sabina S Medici Maria Cristina MC Chezzi Carlo C Snounou Georges G

PloS one 20121024 10

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasites: P. ovale curtisi and P. ovale wallikeri. We have extended and optimized a Real-time PCR assay targeting the parasite's small subunit ribosomal RNA (ssrRNA) gene to detect both these species. When the assay was applied to 31 archival blood samples from patients diagnosed with P. ovale, it was found that the infection in 20 was due to P. ovale curtisi and in the remaining 1 ...[more]

PMID: 23110165

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Project description:BackgroundPlasmodium 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.MethodsIn 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.ResultsOur 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.ConclusionsOur 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.

Project description:BackgroundIn Ethiopia Plasmodium falciparum and Plasmodium vivax are the dominant species accounting for roughly 60 and 40% of malaria cases, respectively. Recently a major shift from P. falciparum to P. vivax has been observed in various parts of the country but the epidemiology of the other human malaria species, Plasmodium ovale spp. and Plasmodium malariae remains poorly understood. The aim of this study was to assess P. ovale curtisi and wallikeri infection in north-west Ethiopia by using microscopy and nested PCR.MethodsA health institution-based survey using non-probability sampling techniques was conducted at Maksegnet, Enfranze and Kola Diba health centres and Metema hospital in North Gondar. Three-hundred patients with signs and symptoms consistent with malaria were included in this study and capillary blood was collected for microscopic examination and molecular analysis of Plasmodium species. Samples were collected on Whatman 903 filter papers, stored in small plastic bags with desiccant and transported to Vienna (Austria) for molecular analysis. Data from study participants were entered and analysed by SPSS 20 software.ResultsOut of 300 study participants (167 males and 133 females), 184 samples were classified positive for malaria (133 P. falciparum and 51 P. vivax) by microscopy. By species-specific PCR 233 Plasmodium spp (95% CI: 72.6-82) were detected and the majority 155 (66.5%, 95% CI: 60.2-72.3) were P. falciparum followed by P. vivax 69 (29.6%, 95% CI; 24.1-35.8) and 9 (3.9%, 95% CI: 2-7.2) samples were positive for P. ovale. Seven of P. ovale parasites were confirmed as P. ovale wallikeri and two were confirmed as P. ovale curtisi. None of the samples tested positive for P. malariae. During microscopic examination there were high (16.3%) false negative reports and all mixed infections and P. ovale cases were missed or misclassified.ConclusionThis study indicates that P. ovale malaria is under-reported in Ethiopia and provides the first known evidence of the sympatric distribution of indigenous P. ovale wallikeri and P. ovale curtisi in Ethiopia. Therefore, further studies assessing the prevalence of the rare species P. ovale and P. malariae are urgently needed to better understand the species distribution and to adapt malaria control strategies.

Project description:BackgroundPlasmodium ovale is relatively unfamiliar to Chinese staff engaged in malaria diagnosis. In 2013, dried blood spots of four unidentified but suspected ovale malaria samples were sent to the National Malaria Reference Laboratory (NMRL) for reconfirmation.MethodsPartial and complete, small, subunit ribosomal DNA (SSU rDNA) sequences of four samples were obtained with PCR-cloning-sequencing method. Obtained sequences were analyzed by aligning with each other and with nine SSU rDNA sequences of six known Plasmodium parasites. A phylogenetic tree was constructed based on complete SSU rDNA sequences and 12 same gene sequences derived from six known Plasmodium parasites and three Babesia parasites. Primary structure of conservative and variable regions of variant sequences was determined also by comparing them with those of six known Plasmodium parasites. To confirm their existence in genome, they were redetected with primers matching their variable regions. PCR systems aimed to roughly detect any eukaryotes and prokaryotes respectively were also applied to search for other pathogens in one of four patients.ResultsTotally, 19 partial and 23 complete SSU rDNA sequences obtained from four samples. Except eight variant sequences, similarities among sequences from same DNA sample were in general high (more than 98%). The phylogenetic analysis revealed that three cases were infected by P. ovale wallikeri and one by P. ovale curtisi. Four of the variant sequences which obtained from four samples relatively showed high similarities with each other (98.5%-100%). Identical variant sequences actually could be re-obtained from each DNA sample. Their primary structure of conservative and variable regions showed quite fit with that of six known Plasmodium parasites. The test for prokaryote pathogens showed negative and the tests for eukaryotes only found DNA sequences of Human and P. ovale parasites.ConclusionBoth P. ovale wallikeri and P. ovale curtisi infections are present in imported malaria cases of China. New type of partial SSU rDNA sequence which assumed to express in a certain life stage of P. ovale was obtained from both P. ovale wallikeri and P. ovale curtisi samples. This discovery would supply information and clues to identify and understand P. ovale parasites more accurately.

Dataset Information

A new real-time PCR for the detection of Plasmodium ovale wallikeri.

Publications

A new real-time PCR for the detection of Plasmodium ovale wallikeri.

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