Studies of hepatic latency in human ovale malaria

Malaria in humans is caused by several species of parasite belonging to the Plasmodium group, which pass from infected mosquitoes into the person's liver, where after a period of incubation the infection emerges into the blood, causing the fevers and other symptoms we associate with malaria. One of the species causing malaria in humans is Plasmodium ovale, which was first discovered in 1922. It was not until 2010, however, that we were able to provide convincing evidence that these were actually two different parasites which could not be distinguished from each other just by looking down a microscope. We developed some simple DNA testing methods which allow health workers to tell which of these two species is present; using these it is now clear that this type of malaria is actually more widespread than had been thought. Although ovale malaria does not commonly cause a life-threatening form of malaria, two features make it hard to control, and a serious challenge for efforts to eliminate malaria altogether in some countries. Firstly, it is often present in people's blood in relatively small numbers at the same time as other malaria parasites which are more abundant, and so is under-recognised. Secondly, it shares with one other species, Plasmodium vivax, the ability to "hibernate" at the liver stage for months or years, being resistant to almost all known malaria drugs when it is in this resting phase. Efforts to study these "sleeping" forms in the liver for P. vivax malaria have not been very successful, and although we have some ideas about them, these "sleeping" stages remain largely a mystery of modern biology. As host institution for the UK Malaria Reference Laboratory, we receive blood samples from malaria cases across the UK, and each year there are more than fifty ovale cases among them. We thus expect approximately one per week, and therefore have sufficient numbers coming through to plan a new study of these parasites. We have developed a way to purify the parasites in patient blood away from human blood cells, and we will seek to feed these to mosquitoes, and then infect liver cells obtained from the ongoing research work of colleagues at UCL, nearby. If this is successful, we will have a new approach for studying liver-stage malaria, and in particular a new window into the mysterious world of the "sleepy" liver-stage parasites. This information should also help us to better understand these stages in P. vivax malaria, also.

Ovale malaria is caused by two closely related parasite species, Plasmodium ovale curtisi (Poc) and P. o. wallikeri (Pow). These have recently been shown to differ in the duration of the latent period prior to development of a symptomatic blood-stage infection, implying differences in liver stage biology. The literature contains a single study on liver-stage schizonts (1954) and none on hypnozoites of ovale malaria, yet recent data suggest that ovale parasites can evade chemotherapy in some individuals, presumably through the emergence of hypnozoites into the blood after drug levels have fallen. Thus P. ovale spp. liver-stage biology has important public health consequences for malaria control and elimination efforts in sub-Saharan Africa, where the parasite is widespread. We have established a transient 48 - 72 hour culture method with ex vivo ovale parasites from blood samples submitted to the UK Malaria Reference Laboratory that sustains schizont and gametocyte maturation, but not-erythrocyte invasion. This will permit studies of mRNA expression, sex ratio and ex-flagellation in ovale sexual stage parasites for the first time. We will attempt experimental mosquito infections and initiation of in vitro liver stage cultures by inoculation of primary human hepatocytes and cell lines with sporozoites from both Poc and Pow. The ex vivo blood stage material will also be used to for next-generation sequencing on the LSHTM in-house Illumina platform. Bioinformatic approaches will be used to identify genes shared with the relapsing parasite P. vivax, but not with P. knowlesi, as a means to generate a list of candidate genes important in the formation of hypnozoites. Patterns of ovale relapse in Malian children treated with ACT for falciparum malaria will be deduced from screening of follow-up blood samples in large clinical trials currently underway; this will add to our knowledge of the role of hypnozoites in maintaining ovale transmission in Africa.

This project will have impact on the following stake-holders:
-travel medicine specialists, as we hope to produce better understanding of relapse patterns with ovale malaria in Africa
-drug development groups beyond academia as new pathways to the identification of targets in Plasmodium spp. hypnozoites may be found
-malaria control programmes in endemic countries, as a better understanding of ovale malaria relapse (whether symptomatic or asymptomatic) following ACT treatment of falciparum malaria cases is likely to suggest changes to treatment policy and a need for more specific diagnostics
-immunologists and vaccine developers, as the Poc and Pow sporozoite-infected mosquitoes could be used for irradiation experiments and elicitation of ovale-specific antibodies in mice.