In the last decade, there has been an upsurge of interest in developing malaria rapid diagnostic test (RDT) kits for the detection of Plasmodium species. Three antigens-Plasmodium falciparum histidine-rich protein 2 (PfHRP2), plasmodial aldolase and plasmodial lactate dehydrogenase (pLDH)-are currently used for RDTs. Tests targeting HRP2 contribute to more than 90 % of the malaria RDTs in current use. However, the specificities, sensitivities, numbers of false positives, numbers of false negatives and temperature tolerances of these tests vary considerably, illustrating the difficulties and challenges facing current RDTs. This paper describes recent developments in malaria RDTs, reviewing RDTs detecting PfHRP2, pLDH and plasmodial aldolase. The difficulties associated with RDTs, such as genetic variability in the Pfhrp2 gene and the persistence of antigens in the bloodstream following the elimination of parasites, are discussed. The prospect of overcoming the problems associated with current RDTs with a new generation of alternative malaria antigen targets is also described.

The molecular mechanisms regulating the sexual development of malaria parasites from gametocytes to oocysts in their mosquito vector are still largely unexplored. In other eukaryotes, NIMA-related kinases (Neks) regulate cell cycle progression and have been implicated in the regulation of meiosis. Here, we demonstrate that Nek-4, a new Plasmodium member of the Nek family, is essential for completion of the sexual cycle of the parasite. Recombinant Plasmodium falciparum Nek-4 possesses protein kinase activity and displays substrate preferences similar to those of other Neks. Nek-4 is highly expressed in gametocytes, yet disruption of the nek-4 gene in the rodent malaria parasite P. berghei has no effect on gamete formation and subsequent fertilization. However, further differentiation of zygotes into ookinetes is abolished. Measurements of nuclear DNA content indicate that zygotes lacking Nek-4 fail to undergo the genome replication to the tetraploid level that precedes meiosis. Cell cycle progression in the zygote is identified as a likely precondition for its morphological transition to the ookinete and for the successful establishment of a malaria infection in the mosquito.Malaria remains a devastating disease in most tropical and subtropical regions. The problem has been exacerbated over the last decades of the twentieth century by the emergence and spread of resistance of the causative agents, parasitic protists of the genus Plasmodium, to available antimalarials (Plasmodium falciparum is the species responsible for the vast majority of lethal cases) (1). Infection of the human host is initiated by the bite of an infected Anopheles mosquito, which delivers sporozoites into the bloodstream. The sporozoites rapidly gain the liver, where they invade hepatocytes and undergo a first round of schizogony. The merozoites produced in this process are released into the bloodstream and invade erythrocytes, where they undergo recurrent and synchronized schizogony. This is the phase of the parasite's life cycle that is responsible for malaria pathogenesis. A proportion of merozoites, upon invasion of a new red blood cell, do not enter schizogony, but arrest their cell cycle and develop into male or female gametocytes, the only forms capable of infecting the mosquito vector. Ingestion of gametocytes during a blood meal triggers their further development into gametes, a process, which for the male gametocyte, involves three rounds of genome replication and generation of eight flagellated male gametes. Fertilization is followed by nuclear fusion, one round of genome replication, and meiosis, which occurs within 3 h (2, 3). The nuclear envelope remains intact throughout this process, and meiosis is not followed by nuclear division. As a consequence the ookinete, a motile form that develops from the zygote and exits the mosquito midgut lumen, is tetraploid. The ookinete establishes an oocyst at the basal lamina, which produces several thousand sporozoites. These accumulate in the insect salivary glands and render the mosquito...

SummaryProcesses at the surface of Plasmodium falciparuminfected erythrocytes such as antigenic variation and cytoadhesion may be modulated by active signalling between host and parasite. Potential candidates for this role include the putative kinases of the FIKK family. The novel Apicomplexa-specific FIKK gene has expanded in P. falciparum to 20 sequence-related members distributed between 11 chromosomes. Specific antibodies raised against different members indicated that most FIKK proteins locate to punctate foci in the erythrocyte cytoplasm that colocalized with Maurer's clefts proteins. One FIKK member dissociates at the trophozoite stage from the Maurer's clefts and relocates with the erythrocyte cytoskeleton. Another FIKK protein, despite having a PEXEL motif, remains located within the parasite. FIKK proteins possess the essential residues for phosphotransferase activity. We show that protein kinase activity was detected in immunoprecipitates obtained with two anti-FIKK antibodies. Quantitative PCR analysis revealed differential gene transcription of the FIKK paralogues in asexual blood stages parasites. We observed significant changes in the transcription pattern between parasites with different adhesion phenotypes. Our data suggest a role of FIKK proteins in the remodelling of the erythrocyte surface and reveal the existence of an adaptable parasite system able to sense intra-and possibly extracellular changes.

The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission.

SummaryTwo members of the mitogen-activated protein kinase (MAPK) family have been previously characterized in Plasmodium falciparum , but in vitro attempts at identifying MAP kinase kinase (MAPKK) homologues have failed. Here we report the characterization of a novel plasmodial protein kinase, PfPK7, whose top scores in BLASTP analysis belong to the MAPKK3/6 subgroup of MAPKKs. However, homology to MAPKKs is restricted to regions of the C-terminal lobe of the kinase domain, whereas the N-terminal region is closer to fungal protein kinase A enzymes (PKA, members of the AGC group of protein kinases). Hence, PfPK7 is a 'composite' enzyme displaying regions of similarity to more than one protein kinase family, similar to a few other plasmodial protein kinases. PfPK7 is expressed in several developmental stages of the parasite, both in the mosquito vector and in the human host. Recombinant PfPK7 displayed kinase activity towards a variety of substrates, but was unable to phosphorylate the two P. falciparum MAPK homologues in vitro , and was insensitive to PKA and MEK inhibitors. Together with the absence of a typical MAPKK activation site in its T-loop, this suggests that PfPK7 is not a MAPKK orthologue, despite the fact that this enzyme is the most 'MAPKKlike' enzyme encoded in the P. falciparum genome. This is consistent with recent observations that the plasmodial MAPKs are not true orthologues of the ERK1/2, p38 or JNK MAPKs, and strengthens the evidence that classical three-component moduledependent MAPK signalling pathways do not operate in malaria parasites, a feature that has not been described in any other eukaryote.