An observational study of malaria in British travellers: Plasmodium ovale wallikeri and Plasmodium ovale curtisi differ significantly in the duration of latency.
ABSTRACT: Ovale malaria is caused by two closely related species of protozoan parasite: Plasmodium ovale curtisi and Plasmodium ovale wallikeri Although clearly distinct genetically, there have been no studies comparing the morphology, life cycle or epidemiology of these parasites. We tested the hypothesis that the two species differ in the duration of latency prior to presentation with symptoms of blood-stage infection.PCR was used to identify P ovale curtisi and P ovale wallikeri infections among archived blood from UK malaria patients. Latency periods, estimated as the time between entry into the UK and diagnosis of malaria, were compared between the two groups.UK National Reference Laboratory.None. Archived parasite material and surveillance data for 74 P ovale curtisi and 60 P ovale wallikeri infections were analysed. Additional epidemiological data were taken from a database of 1045 imported cases.None.No differences between the two species were identified by a detailed comparison of parasite morphology (N=9, N=8, respectively) and sex ratio (N=5, N=4) in archived blood films. The geometric mean latency period in P ovale wallikeri was 40.6 days (95% CI 28.9 to 57.0), whereas that for P ovale curtisi was more than twice as long at 85.7 days (95% CI 66.1 to 111.1; p=0.002). Further, the proportion of ovale malaria sensu lato which occurred in patients reporting chemoprophylaxis use was higher than for Plasmodium falciparum (OR 7.56; p<0.0001) or P vivax (OR 1.82; p<0.0001).These findings provide the first difference of epidemiological significance observed between the two parasites which cause ovale malaria, and suggest that control measures aimed at P falciparum may not be adequate for reducing the burden of malaria caused by P ovale curtisi and P ovale wallikeri.
Project description: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.
Project description:It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasite, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. It was recently shown that these two parasite types are sympatric at the country level. However, it remains possible that localised geographic, temporal or ecological barriers exist within endemic countries which prevent recombination between the genomes of the two species. Here, using conventional and real-time quantitative PCR (qPCR) methods specifically designed to discriminate P. o. curtisi and P. o. wallikeri, it is shown that both species are present among clinic attendees in Congo-Brazzaville, and occur simultaneously both in lake-side and inland districts in Uganda and on Bioko Island, Equatorial Guinea. Thus P. o. curtisi and P. o. wallikeri in these localities are exactly sympatric in both time and space. These findings are consistent with the existence of a biological barrier, rather than geographical or ecological factors, preventing recombination between P. o. curtisi and P. o. wallikeri. In cross-sectional surveys carried out in Uganda and Bioko, our results show that infections with P. ovale spp. are more common than previously thought, occurring at a frequency of 1-6% in population samples, with both proposed species contributing to ovale malaria in six sites. Malaria elimination programmes in Africa need to include strategies for control of P. o. curtisi and P. o. wallikeri.
Project description:Malaria, due to Plasmodium ovale, can be challenging to diagnose due to clinically mild disease and low parasite burden. Two genetically distinct sub-species of P. ovale exist: Plasmodium ovale curtisi (classic) and Plasmodium ovale wallikeri (variant). It is presently unknown if the sub-species causing infection affects performance of malaria diagnostic tests. The aim of this work was to understand how the genetically distinct sub-species, P. o. curtisi and P. o. wallikeri, affect malaria diagnostic tests.Plasmodium ovale-positive whole blood specimens were sub-speciated by PCR and sequencing of 18S rRNA and dhfr-ts. Parasitaemia, morphology, pan-aldolase positivity, 18S copy number, and dhfr-ts sequences were compared between sub-species.From 2006 to 2015, 49 P. ovale isolates were identified, of which 22 were P. o. curtisi and 27 P. o. wallikeri; 80% were identified in the last five years, and 88% were acquired in West Africa. Sub-species did not differ by parasitaemia, 18S copy number, or pan-aldolase positivity. Lack of Schüffner's stippling was over-represented among P. o. wallikeri isolates (p = 0.02). Several nucleotide polymorphisms between the sub-species were observed, but they do not occur at sites believed to relate to antifolate binding.Plasmodium ovale is increasing among travellers to West Africa, although sub-species do not differ significantly by parasitologic features such as parasitaemia. Absence of Schüffner's stippling may be a feature specific to P. o. wallikeri and is a novel finding.
Project description:BACKGROUND:To assess the occurrence of Plasmodium ovale wallikeri and Plasmodium ovale curtisi species in travellers returning to Germany, two real-time PCR protocols for the detection and differentiation of the two P. ovale species were compared. Results of parasite differentiation were correlated with patient data. METHODS:Residual nucleic acid extractions from EDTA blood samples of patients with P. ovale spp. malaria, collected between 2010 and 2019 at the National Reference Centre for Tropical Pathogens in Germany, were subjected to further parasite discrimination in a retrospective assessment. All samples had been analysed by microscopy and by P. ovale spp.-specific real-time PCR without discrimination on species level. Two different real-time PCR protocols for species discrimination of P. o. curtisi and P. o. wallikeri were carried out. Results were correlated with patient data on gender, age, travel destination, thrombocyte count, and duration of parasite latency. RESULTS:Samples from 77 P. ovale spp. malaria patients were assessed, with a male:female ratio of about 2:1 and a median age of 30 years. Parasitaemia was low, ranging from few visible parasites up to 1% infected erythrocytes. Discriminative real-time PCRs revealed 41 cases of P. o. curtisi and 36 cases of P. o. wallikeri infections. Concordance of results by the two PCR approaches was 100%. Assessment of travel destinations confirmed co-existence of P. o. curtisi and P. o. wallikeri over a wide range of countries in sub-Saharan Africa. Latency periods for the two P. ovale species were similar, with median values of 56.0 days for P. o. curtisi and 58.0 days for P. o. wallikeri; likewise, there was no statistically significant difference in thrombocyte count with median values of 138.5/µL for patients with P. o. curtisi and 152.0/µL for P. o. wallikeri-infected patients. CONCLUSIONS:Two different real-time PCR protocols were found to be suitable for the discrimination of P. o. curtisi and P. o. wallikeri with only minor differences in sensitivity. Due to the overall low parasitaemia and the lack of differences in severity-related aspects like parasite latency periods or thrombocyte counts, this study supports the use of P. ovale spp. PCR without discrimination on species level to confirm the diagnosis and to inform clinical management of malaria in these patients.
Project description:Plasmodium ovale curtisi and Plasmodium ovale wallikeri are distinct species of malaria parasite which are sympatric throughout the tropics, except for the Americas. Despite this complete overlap in geographic range, these two species do not recombine. Although morphologically very similar, the two taxa must possess distinct characters which prevent recombination between them. We hypothesised that proteins required for sexual reproduction have sufficiently diverged between the two species to prevent recombination in any mosquito blood meal in which gametocytes of both species are ingested. In order to investigate possible barriers to inter-species mating between P. ovale curtisi and P. ovale wallikeri, homologues of genes encoding sexual stage proteins in other plasmodia were identified and compared between the two species. Database searches with motifs for 6-cysteine, Limulus Coagulation factor C domain-containing proteins and other relevant sexual stage proteins in the genus Plasmodium were performed in the available P. ovale curtisi partial genome database (Wellcome Trust Sanger Institute, UK). Sequence fragments obtained were used as the basis for PCR walking along each gene of interest in reference isolates of both P. ovale curtisi and P. ovale wallikeri. Sequence alignment of the homologues of each gene in each species showed complete dimorphism across all isolates. In conclusion, substantial divergence between sexual stage proteins in the two P. ovale spp. was observed, providing further evidence that these do not recombine in nature. Incompatibility of proteins involved in sexual development and fertilisation thus remains a plausible explanation for the observed lack of natural recombination between P. ovale curtisi and P. ovale wallikeri.
Project description:As indigenous malaria has decreased over recent decades, the increasing number of imported malaria cases has provided a new challenge for China. The proportion of imported cases due to Plasmodium ovale has increased during this time, and the difference between P. ovale curtisi and P. ovale wallikeri is of importance. To better understand P. ovale epidemiology and the differences between the two subspecies, information on imported malaria in Henan Province was collected during 2010-2017. We carried out a descriptive study to analyze the prevalence, proportion, distribution, and origin of P. o. curtisi and P. o. wallikeri. It showed that imported P. ovale spp. accounts for a large proportion of total malaria cases in Henan Province, even more than that of P. vivax. This suggests that the proportion of P. ovale cases is underestimated in Africa. Among these cases, the latency period of P. o. curtisi was significantly longer than that of P. o. wallikeri. More attention should be paid to imported ovale malaria to avoid the reintroduction of these two subspecies into China.
Project description:Plasmodium ovale is rare and not exactly known to be autochthonous in Malaysia. There are two distinct forms of the parasite, namely P. ovale curtisi (classic form) and P. ovale wallikeri (variant form). Here, the first sequence confirmed case of an imported P. ovale wallikeri infection in Malaysia is presented. Microscopy found Plasmodium parasites with morphology similar to P. ovale or Plasmodium vivax in the blood films. Further confirmation using polymerase chain reaction (PCR) targeting the small-subunit rRNA gene of the parasite was unsuccessful. Genus-specific PCR was then performed and the product was sequenced and analysed. Sequence analyses confirmed the aetiological agent as P. ovale wallikeri. New species-specific primers (rOVA1v and rOVA2v) were employed and P. ovale wallikeri was finally confirmed. The findings highlight the need to look out for imported malaria infections in Malaysia and the importance of a constantly updated and validated diagnostic technique.
Project description:Plasmodium ovale curtisi and Plasmodium ovale wallikeri are two sympatric human malaria species prevalent in Africa, Asia and Oceania. The reported prevalence of both P. ovale spp. was relatively low compared to other malaria species, but more sensitive molecular detection techniques have shown that asymptomatic low-density infections are more common than previously thought. Whole genome sequencing of both P. ovale spp. revealed genetic dissociation between P. ovale curtisi and P. ovale wallikeri suggesting a species barrier. In this study we further evaluate such a barrier by assessing polymorphisms in the genes of three vaccine candidate surface protein: circumsporozoite protein/ thrombospondin-related anonymous-related protein (ctrp), circumsporozoite surface protein (csp) and merozoite surface protein 1 (msp1). The complete coding sequence of ctrp and csp, and a partial fragment of msp1 were isolated from 25 P. ovale isolates and compared to previously reported reference sequences. A low level of nucleotide diversity (Pi = 0.02-0.10) was observed in all three genes. Various sizes of tandem repeats were observed in all ctrp, csp and msp1 genes. Both tandem repeat unit and nucleotide polymorphism in all three genes exhibited clear dimorphism between P. ovale curtisi and P. ovale wallikeri, supporting evidence of non-recombination between these two species.
Project description:BACKGROUND: In 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. METHODS: A 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. RESULTS: Out 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. CONCLUSION: This 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:Plasmodium ovale, considered the rarest of the malaria parasites of humans, consists of two morphologically identical but genetically distinct sympatric species, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. These parasites resemble morphologically to Plasmodium vivax with which they also share a tertian periodicity and the ability to cause relapses, making them easily misidentified as P. vivax. Plasmodium ovale infections are rarely reported, but given the likelihood of misidentification, their prevalence might be underestimated.Morphological and molecular analysis of confirmed malaria cases admitted in Singapore in 2012-2014 detected nine imported P. ovale cases that had been misidentified as P. vivax. Since P. ovale had not been previously officially reported in Singapore, a retrospective analysis of available, frozen, archival blood samples was performed and returned two additional misidentified P. ovale cases in 2003 and 2006. These eleven P. ovale samples were characterized with respect to seven molecular markers (ssrRNA, Potra, Porbp2, Pog3p, dhfr-ts, cytb, cox1) used in recent studies to distinguish between the two sympatric species, and to a further three genes (tufa, clpC and asl).The morphological features of P. ovale and the differential diagnosis with P. vivax were reviewed and illustrated by microphotographs. The genetic dimorphism between P. ovale curtisi and P. ovale wallikeri was assessed by ten molecular markers distributed across the three genomes of the parasite (Genbank KP050361-KP050470). The data obtained for seven of these markers were compared with those published and confirmed that both P. ovale species were present. This dimorphism was also confirmed for the first time on: (1) two genes from the apicoplast genome (tufA and clpC genes); and, (2) the asl gene that was used for phylogenetic analyses of other Plasmodium species, and that was found to harbour the highest number of dimorphic loci between the two P. ovale species.Misidentified P. ovale infections are reported for the first time among imported malaria cases in Singapore. Genetic dimorphism between P. ovale curtisi and P. ovale wallikeri was confirmed using markers from the parasites' three genomes. The apparent increase of imported P. ovale since 2012 (with yearly detection of cases) is puzzling. Given decrease in the overall number of malaria cases recorded in Singapore since 2010 the 'resurgence' of this neglected species raises public health concerns.