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.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.

Graphical abstractResearch highlights► We propose that two related species of malaria parasite cause ovale malaria. ► Discriminatory PCR tests identified both species in African surveys. ► Plasmodium ovale curtisi and Plasmodium ovale wallikeri were found together in time and space. ► Lack of recombination between them is not due to geographic or temporal separation. ► Therefore, the species barrier may be maintained by a biological mechanism(s).

Two hundred and seventy four asymptomatic Ghanaian school-children aged 5 to 17 years were screened for malaria parasites by examination of blood films. One hundred and fifty five microscopically-positive individuals were treated with dihydroartemisinin-piperaquine and followed for 3 weeks. Retrospective species-specific PCR of all 274 screened samples identified an additional 60 children with sub-patent parasitaemia, and a substantial proportion of co-infections with Plasmodium malariae, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. One hundred individuals harboured at least one non-falciparum parasite species. Using standard double-read microscopy, the 21-day efficacy of treatment against Plasmodium falciparum was 91.4% among the 117 children seen at all 5 visits. Using nested PCR to test 152 visit 5 blood samples, 22 were found to be parasite-positive. Twenty individuals harboured P. falciparum, four harboured P. ovale spp. and two P. malariae, with four of these 22 isolates being mixed species infections. The persistent detection of low density Plasmodium sp. infections following antimalarial treatment suggests these may be a hitherto unrecognised obstacle to malaria elimination.

ObjectivesOvale 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.DesignPCR 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.SettingUK National Reference Laboratory.ParticipantsNone. 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.OutcomesNone.ResultsNo 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).ConclusionsThese 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.

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

Malaria in humans is caused by six species of Plasmodium parasites, of which the nuclear genome sequences for the two Plasmodium ovale spp., P. ovale curtisi and P. ovale wallikeri, and Plasmodium malariae have not yet been analyzed. Here we present an analysis of the nuclear genome sequences of these three parasites, and describe gene family expansions therein. Plasmodium ovale curtisi and P. ovale wallikeri are genetically distinct but morphologically indistinguishable and have sympatric ranges through the tropics of Africa, Asia and Oceania. Both P. ovale spp. show expansion of the surfin variant gene family, and an amplification of the Plasmodium interspersed repeat (pir) superfamily which results in an approximately 30% increase in genome size. For comparison, we have also analyzed the draft nuclear genome of P. malariae, a malaria parasite causing mild malaria symptoms with a quartan life cycle, long-term chronic infections, and wide geographic distribution. Plasmodium malariae shows only a moderate level of expansion of pir genes, and unique expansions of a highly diverged transmembrane protein family with over 550 members and the gamete P25/27 gene family. The observed diversity in the P. ovale wallikeri and P. ovale curtisi surface antigens, combined with their phylogenetic separation, supports consideration that the two parasites be given species status.

Molecular markers for surveillance of Plasmodium falciparum resistance to current antimalarials are sorely needed. A 28-day efficacy study of artemether-lumefantrine in eastern Sudan identified 5 treatment failures among 100 evaluable patients; 9 further individuals were parasite positive by PCR during follow-up. Polymorphisms in pfatpase6 and pfmdr1 were evaluated by DNA sequencing. One individual carried parasites with a novel pfmdr1 polymorphism (F1044L). pfmdr1 gene amplification in parasites prior to treatment occurred in three individuals who had recurrent infection during follow-up.