African apes as reservoirs of Plasmodium falciparum and the origin and diversification of the Laverania subgenus

We investigated two mitochondrial genes (cytb and cox1), one plastid gene (tufA), and one nuclear gene (ldh) in blood samples from 12 chimpanzees and two gorillas from Cameroon and one lemur from Madagascar. One gorilla sample is related to Plasmodium falciparum, thus confirming the recently reported presence in gorillas of this parasite. The second gorilla sample is more similar to the recently defined Plasmodium gaboni than to the P. falciparum–Plasmodium reichenowi clade, but distinct from both. Two chimpanzee samples are P. falciparum. A third sample is P. reichenowi and two others are P. gaboni. The other chimpanzee samples are different from those in the ape clade: two are Plasmodium ovale, and one is Plasmodium malariae. That is, we have found three human Plasmodium parasites in chimpanzees. Four chimpanzee samples were mixed: one species was P. reichenowi; the other species was P. gaboni in three samples and P. ovale in the fourth sample. The lemur sample, provisionally named Plasmodium malagasi, is a sister lineage to the large cluster of primate parasites that does not include P. falciparum or ape parasites, suggesting that the falciparum + ape parasite cluster (Laverania clade) may have evolved from a parasite present in hosts not ancestral to the primates. If malignant malaria were eradicated from human populations, chimpanzees, in addition to gorillas, might serve as a reservoir for P. falciparum.     

African monkeys are infected by Plasmodium falciparum nonhuman primate-specific strains

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.     

RH5–Basigin interaction plays a major role in the host tropism of Plasmodium falciparum

Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175–GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175–GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)–Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5–BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.The most deadly of the malaria parasites, Plasmodium falciparum, is highly divergent from the other species of Plasmodium known to infect humans (1–3), with its closest relatives comprising a group of chimpanzee and gorilla parasites from the subgenus Laverania (3–7). Despite the origin of P. falciparum as a zoonosis, and the continuing coexistence of humans and apes in West and Central Africa, extensive field studies have failed to detect P. falciparum in wild-living chimpanzees and gorillas (5). Although there are reports of P. falciparum infecting chimpanzees either in certain captive settings (2) or following splenectomy and deliberate transfer of P. falciparum-infected human blood (8–10), the resulting infections have low parasitemia and are not known to result in malignant tertian malaria, suggesting a host-specific barrier for replete infection. The existence of host-specific barriers within the Laverania subgenus is supported further by the strict host specificity exhibited by ape Laverania parasites in the wild: Plasmodium reichenowi, Plasmodium billcollinsi, and Plasmodium gaboni infect only chimpanzees, and Plasmodium praefalciparum, Plasmodium blacklocki, and Plasmodium adleri are restricted to gorillas (3–6).The molecular basis for the host tropism of P. falciparum and other Laverania parasites is not currently understood and is difficult to investigate experimentally, because no ape Laverania parasites have been adapted to in vitro culture, and because ethical considerations clearly preclude the use of endangered African apes for in vivo experiments (3). In principle, the species specificity of known host–parasite interactions could be examined using recombinant proteins, but the technical challenges associated with expressing functional Plasmodium proteins in a recombinant form have made this approach difficult thus far (11). There also are multiple points in the complex Plasmodium life cycle that could represent restriction points, and some evidence suggests that transmission of Laverania to anthropophilic Anopheles species, for example, may be restricted (12). Although several restriction points are possible, the blood stage of Plasmodium infection, which is initiated when host erythrocytes are invaded by the parasite, is of particular interest. Erythrocyte invasion is an obligate stage in the parasite life cycle (13), and the inability of P. falciparum to produce infections of high parasitemia in chimpanzees by transfer of infected blood, despite contrived permissive experimental conditions, suggests that a host-specific barrier for infection exists at the blood stage. Moreover, erythrocyte invasion involves extracellular interactions between several different receptor–ligand pairs which are coevolving rapidly, driven by strong immune selection pressure and functional constraints (14, 15). This rapid coevolution means that interactions between parasite blood-stage ligands and erythrocyte receptors quickly could become host-specific, isolating a certain parasite species within a single host. Given these findings, erythrocyte invasion therefore is likely to represent a significant restriction point in determining host tropism.P. falciparum erythrocyte invasion depends on a partially redundant set of parasite ligands and erythrocyte receptors (14, 16–18), and there is experimental support for the suggestion that at least two of these interactions may be involved in P. falciparum host tropism. The interaction between Plasmodium falciparum Erythrocyte Binding Antigen-175 (PfEBA175) and the erythrocyte receptor Glycophorin-A (GYPA) is important for invasion and is known to require sialic acid residues displayed on GYPA (19, 20). The sialic acid content of human GYPA differs from that of other apes because humans lack a functional cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) enzyme. Consequently, human GYPA contains only N-Acetylneuraminic acid (Neu5Ac) sialic acids, whereas chimpanzees and gorillas, both of which have an active CMAH gene, contain a mixture of both Neu5Ac and its hydroxylated derivative, N-Glycolylneuraminic acid (Neu5Gc), with Neu5Gc being more abundant (21). This difference in GYPA sialic acid content led Martin et al. (10) to propose that the restriction of P. falciparum to humans results from the sialic acid-binding specificity in the PfEBA175–GYPA interaction. The cocrystal structure of the glycan-binding regions of P. falciparum EBA175 in complex with a Neu5Ac-containing glycan derivative (22) followed by modeling of the equivalent EBA175 sequence from the chimpanzee-restricted parasite, P. reichenowi (23), suggested a structural basis for this binding specificity. However, this hypothesis is inconsistent with a much earlier study by Orlandi et al. (24) in which the binding of native PfEBA175 to human erythrocytes was observed to be potently inhibited by soluble Neu5Gc and oligosaccharides containing Neu5Acα2–3Gal but not by Neu5Ac as a monosaccharide. This glycan-binding specificity was confirmed in a recent biochemical investigation using a recombinant protein consisting of the entire ectodomain of PfEBA175 (25). Furthermore, the sialic acid specificity of PfEBA175 orthologs cannot explain the restriction of ape Laverania parasites to their respective chimpanzee and gorilla hosts, both of which carry a functional CMAH gene (5). Therefore there is reasonable doubt about the importance of the EBA175–GYPA interaction in Laverania host tropism. A second and unrelated invasion ligand, Plasmodium falciparum Reticulocyte-binding protein homolog 5 (PfRH5) has also been implicated in host tropism by governing the ability of P. falciparum to infect New World Aotus monkeys (26, 27). The erythrocyte cell-surface receptor of PfRH5, Basigin (BSG), was identified only recently (28), and so the contribution of the PfRH5–BSG interaction toward determining host-species selectivity in P. falciparum is unknown.We recently have developed technology that enables the recombinant expression of P. falciparum cell-surface and secreted proteins in a functional form using a mammalian expression system (29). We have used this method to identify novel host–parasite interactions (28, 30), but here we apply it to investigate the relative roles of the EBA175–GYPA and PfRH5–BSG interactions as determinants of P. falciparum host tropism.     

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.     

Epidemiological aspects of vivax and falciparum malaria: global spectrum

Malaria, a mosquito-borne disease, is caused by the infection of apicomplexan parasites belonging to the genus Plasmodium, five species of which [Plasmodium vivax, Plasmodium falciparum (P. falciparum), Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi] account for all forms of human malaria. P. falciparum is responsible for the highest degree of complications (severe malarial anaemia and cerebral malaria) and mortality in the tropics and subtropics of the world. Despite the large burden of vivax malaria, it is overlooked and left in the shadow of severity of falciparum malaria in the globe, but current reports provide evidence of severe vivax malaria symptoms similar to P. falciparum infection. The major challenging factor is the emergence of multidrug resistant Plasmodium strains to the conventionally used antimalarials over the last two decades, and, more recently, to artemisinins. The WHO recommended artemisinin based combination therapies (ACTs). The non-ACT regimens are also found to be effective, safe, and affordable compared to ACTs. However, current successful antimalarial interventions are under threat from the ability of the parasite and its mosquito vector to develop resistance to medicines and insecticides, respectively. Hence, with widespread use of effective drugs and vector control with insecticide-treated bed nets and indoor residual spraying, an ideal malaria vaccine would be the actual means of malaria prevention. This review represents the current evidence, based upon the search of SCI-and non-SCI journal, on epidemiological aspects of two forms (vivax and falciparum) of human malaria, which is still a great global concern.     

From the Cover: Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe—where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum. Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.The genus Plasmodium contains two of the most significant human pathogenic organisms. One, Plasmodium vivax, is the most widely distributed human malaria parasite outside of Africa, with a range that extends well into temperate zones (1), whereas the other, Plasmodium falciparum, is the predominant malaria parasite of humans in subsaharan Africa and the one that causes >90% malarial deaths (2).Although an increasing number of studies have attempted to investigate the present-day distribution of Plasmodium parasites using DNA sourced from modern isolates of the pathogens, their origin and dispersal around the planet remain controversial. For example, the current global distribution of P. vivax includes Asia, the Middle East, South and Central America, and parts of Africa (3) and has likely resulted from complex dispersals involving intercontinental human population movements (4, 5). Although an African origin is likely (6), the current strains from this continent show little genetic polymorphism compared with the rest of the world (4), a fact that has been related to the emergence of Duffy-negative blood types—resistant to P. vivax—in human populations from this continent (7). Alternatively, it has been suggested that modern African parasites, at least in the Horn of Africa and Madagascar, could have been reintroduced via the import of East Asian/Indian P. vivax to the coastal areas of East Africa by early sea-going traders and that this could explain their lack of diversity (4). Intriguingly, the diversity of the American strains is comparable to those found in Oceania and Asia (8), raising questions regarding the origin of these strains. Some of the American strains are thought to have been brought there from Europe by Spanish sailors and colonists in the last few centuries (4, 9). An alternative possibility would imply a pre-Columbian entrance through the Bering Straits during the initial peopling of the Americas, although this seems unlikely due to the climatic conditions of this passage (9) and the long Beringian standstill that likely lasted up to several thousands of years (10). However, the existence of divergent mitochondrial lineages in the Americas seems to rule out the possibility of a single, recent introduction of P. vivax from Europe into this continent (8). At the nuclear level, the American isolates show geographic differentiation but also a common clustering regarding the Old World populations (5).With regard to the evolutionary history of P. falciparum, genetic analyses of Laveriana parasite species found in African chimpanzees and gorillas, considered to be precursors of the human parasites, prove an African origin (11), but the time by which the parasite dispersed out of Africa remains controversial. Although some suggest that the pathogen followed the original Homo sapiens dispersals around 60,000 y ago (12), others support a recent expansion within the last 5,000–10,000 y associated to the advent of agriculture (2). The recent analysis of 44 Indian field isolates showed, however, a higher than expected diversity and suggested that the Indian strains are also part of the ancestral distribution range of P. falciparum (13). Historical accounts seem to indicate that malaria spread from India into the Mediterranean at the time of Classical Greece; therefore, the now extinct European P. falciparum could be genetically related to some of these Indian parasites.The current controversies on the recent evolutionary history of P. vivax and P. falciparum partly relate to the lack of genetic evidence from the European parasite that was eradicated more than half century ago. In Spain, malaria had remained endemic until the early 20th century, in particular in Andalucia, Extremadura, and Ebro delta regions; it was declared oficially eradicated in 1964 (14). P. vivax and P. falciparum have been transmitted in southern Spain by two former malaria vectors: Anopheles atroparvus and Anopheles labranchiae. Both were present in the country but whereas An. atroparvus is still widely spread, An. labranchiae has not been found since the middle of the last century (15). An. atroparvus from different geographic origins are infected in laboratory with different world strains of P. vivax, whereas it is very difficult to do the same in the case of P. falciparum (16). Thus, the lack of genetic evidence of the former Plasmodium European parasites also hampers our understanding of their biology.To clarify the phylogenetic position of the extinct European Plasmodium strains, we report here the retrieval of the European P. vivax and P. falciparum genetic data from the analysis of three recently discovered old slides with blood drops from malaria-infected people living at the Ebro Delta in Spain in the first half of the 20th century that corresponds to local epidemics (Fig. 1).Open in a separate windowFig. 1.(A) Two of the Giemsa-stained slides analyzed in this study, labeled CM and CA (inferior stain). (B) Visualization of some Plasmodium parasites (arrows) in the CM slide under the microscope (400×).     

Evolutionary tree for apes and humans based on cleavage maps of mitochondrial DNA

The high rate of evolution of mitochondrial DNA makes this molecule suitable for genealogical research on such closely related species as humans and apes. Because previous approaches failed to establish the branching order of the lineages leading to humans, gorillas, and chimpanzees, we compared human mitochondrial DNA to mitochondrial DNA from five species of ape (common chimpanzee, pygmy chimpanzee, gorilla, orangutan, and gibbon). About 50 restriction endonuclease cleavage sites were mapped in each mitochondrial DNA, and the six maps were aligned with respect to 11 invariant positions. Differences among the maps were evident at 121 positions. Both conserved and variable sites are widely dispersed in the mitochondrial genome. Besides site differences, ascribed to point mutations, there is evidence for one rearrangement: the gorilla map is shorter than the other owing to the deletion of 95 base pairs near the origin of replication. The parsimony method of deriving all six maps from a common ancestor produced a genealogical tree in which the common and pygmy chimpanzee maps are the most closely related pair; the closest relative of this pair is the gorilla map; most closely related to this trio is the human map. This tree is only slightly more parsimonious than some alternative trees. Although this study has given a magnified view of the genetic differences among humans and apes, the possibility of a three-way split among the lineages leading to humans, gorillas, and chimpanzees still deserves serious consideration.     

Maurer's clefts,the enigma of Plasmodium falciparum

Plasmodium falciparum, the causative agent of malaria, completely remodels the infected human erythrocyte to acquire nutrients and to evade the immune system. For this process, the parasite exports more than 10% of all its proteins into the host cell cytosol, including the major virulence factor PfEMP1 (P. falciparum erythrocyte surface protein 1). This unusual protein trafficking system involves long-known parasite-derived membranous structures in the host cell cytosol, called Maurer’s clefts. However, the genesis, role, and function of Maurer’s clefts remain elusive. Similarly unclear is how proteins are sorted and how they are transported to and from these structures. Recent years have seen a large increase of knowledge but, as yet, no functional model has been established. In this perspective we review the most important findings and conclude with potential possibilities to shed light into the enigma of Maurer’s clefts. Understanding the mechanism and function of these structures, as well as their involvement in protein export in P. falciparum, might lead to innovative control strategies and might give us a handle with which to help to eliminate this deadly parasite.Since Charles Louis Alphonse Laveran discovered the malaria parasite in 1881 (1) in Algeria, while examining the blood of a patient who had died from marsh fever, research has been conducted on these deadly parasites. Laveran received the Nobel Prize in medicine for his discovery in 1907, which causally explained that malaria symptoms are caused by protozoan parasites, eventually described as Plasmodium species of the phylum Apicomplexa. Among the five species infecting humans, Plasmodium falciparum causes the most lethal forms of the disease, but zoonotic Plasmodium knowlesi infections can also be lethal.     

The malarial parasite Plasmodium falciparum imports the human protein peroxiredoxin 2 for peroxide detoxification

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer''s clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.     

Isolation and Characterization of the MSP1 Genes from Plasmodium malariae and Plasmodium ovale

The merozoite surface protein 1 (MSP1) is the principal surface antigen of the blood stage form of the Plasmodium parasite. Antibodies recognizing MSP1 are frequently detected following Plasmodium infection, making this protein a significant component of malaria vaccines and diagnostic tests. Although the MSP1 gene sequence has been reported for Plasmodium falciparum and Plasmodium vivax, this gene has not been identified for the other two major human-infectious species, Plasmodium malariae and Plasmodium ovale. MSP1 genes from these two species were isolated from Cameroon blood donor samples. The genes are similar in size to known MSP1 genes and encode proteins with interspecies conserved domains homologous to those identified in other Plasmodium species. Sequence and phylogenetic analysis of all available Plasmodium MSP1 amino acid sequences clearly shows that the Po and Pm MSP1 sequences are truly unique within the Plasmodium genus and not simply Pf or Pv variants.     

Plasmodium evasion of mosquito immunity and global malaria transmission: The lock-and-key theory

Plasmodium falciparum malaria originated in Africa and became global as humans migrated to other continents. During this journey, parasites encountered new mosquito species, some of them evolutionarily distant from African vectors. We have previously shown that the Pfs47 protein allows the parasite to evade the mosquito immune system of Anopheles gambiae mosquitoes. Here, we investigated the role of Pfs47-mediated immune evasion in the adaptation of P. falciparum to evolutionarily distant mosquito species. We found that P. falciparum isolates from Africa, Asia, or the Americas have low compatibility to malaria vectors from a different continent, an effect that is mediated by the mosquito immune system. We identified 42 different haplotypes of Pfs47 that have a strong geographic population structure and much lower haplotype diversity outside Africa. Replacement of the Pfs47 haplotypes in a P. falciparum isolate is sufficient to make it compatible to a different mosquito species. Those parasites that express a Pfs47 haplotype compatible with a given vector evade antiplasmodial immunity and survive. We propose that Pfs47-mediated immune evasion has been critical for the globalization of P. falciparum malaria as parasites adapted to new vector species. Our findings predict that this ongoing selective force by the mosquito immune system could influence the dispersal of Plasmodium genetic traits and point to Pfs47 as a potential target to block malaria transmission. A new model, the “lock-and-key theory” of P. falciparum globalization, is proposed, and its implications are discussed.The most deadly form of malaria in humans is caused by Plasmodium falciparum parasites. Malaria originated in Africa (1, 2) and is transmitted by anopheline mosquitoes. The disease became global as humans migrated to other continents and parasites encountered different mosquito species that were sometimes evolutionarily distant from African vectors (3). For example, anophelines of the subgenus Nyssorhynchus (malaria vectors in Central and South America, such as Anopheles albimanus) diverged from the subgenus Cellia (malaria vectors in Africa, India, and South Asia) about 100 Mya (4). P. falciparum parasites are transmitted by more than 70 different anopheline species worldwide (3), but compatibilities differ between specific vector–parasite combinations (5). For example, P. falciparum NF54 (Pf NF54), of putative African origin, effectively infects Anopheles gambiae, the main malaria vector in sub-Saharan Africa; but A. albimanus is highly refractory to this strain (6–8); whereas Asian P. falciparum isolates infect Anopheles stephensi (Nijmegen strain), a major vector in India, more effectively than A. gambiae (9). Similar differences in compatibility have been reported between Plasmodium vivax and different anopheline species (10, 11). The A. gambiae immune system can mount effective antiplasmodial responses mediated by the complement-like system that limit infection (12). We have previously shown that some P. falciparum lines can avoid detection by the A. gambiae immune system (13) and identified Pfs47 as the gene that mediated immune evasion (14). Here, we present direct evidence of selection of P. falciparum by the mosquito immune system and show that providing P. falciparum with a Pfs47 haplotype compatible for a given anopheline mosquito is sufficient for the parasite to evade mosquito immunity. The implications of P. falciparum selection by mosquitoes for global malaria transmission are discussed.     

Corticotropin-releasing hormone in chimpanzee and gorilla pregnancies.

In humans, the length of gestation and the onset of parturition have been linked to the exponential production of placental CRH and a late gestational decline in maternal plasma CRH-binding protein (CRH-BP). CRH has been shown to have direct effects on the myometrium and on the fetal adrenal, where it stimulates production of the estrogen precursor dihydroepiandrosterone sulfate. In vitro placental CRH production is stimulated by cortisol and inhibited by progesterone. To determine whether this mechanism might operate in other apes, we sampled eight chimpanzees and two gorillas through their pregnancies for CRH, CRH-BP, cortisol, estradiol, progesterone, and alpha-fetoprotein. We show that both chimpanzee and gorilla maternal plasma CRH concentrations rise exponentially as observed in the human. The gorillas exhibited a human-like antepartum fall in CRH-BP, whereas CRH-BP in the chimpanzee remained stable. Pregnancy-associated changes in cortisol, estradiol, progesterone, and alpha-fetoprotein were qualitatively similar to those observed in humans. Maternal plasma cortisol correlated with plasma CRH in both gorillas (r = 0.60; P < 0.05) and chimpanzees (r = 0.36; P < 0.02). Further, there was a strong correlation between plasma estradiol and the log of plasma CRH in the gorilla (r = 0.93; P < 0.0001) and in the chimpanzee (r = 0.72; P < 0.001), which is consistent with the hypothesis that placental CRH determines the placental production of estradiol by stimulating the production of fetal adrenal dehydroepiandrosterone sulfate. Plasma CRH and progesterone were positively correlated providing no in vivo support for progesterone inhibition of CRH release.     

The High Burden of Malaria in Primary School Children in Southern Malawi

Malaria among school children has received increased attention recently, yet there remain few detailed data on the health and educational burden of malaria, especially in southern Africa. This paper reports a survey among school children in 50 schools in Zomba District, Malawi. Children were assessed for Plasmodium infection, anemia, and nutritional status and took a battery of age-appropriate tests of attention, literacy, and numeracy. Overall, 60.0% of children were infected with Plasmodium falciparum, 32.4% were anemic and 32.4% reported sleeping under a mosquito net the previous night. Patterns of P. falciparum infection and anemia varied markedly by school. In multivariable analysis, higher odds of P. falciparum infection were associated with younger age and being stunted, whereas lower odds were associated with reported net use, higher parental education, and socioeconomic status. The odds of anemia were significantly associated with P. falciparum infection, with a dose–response relationship between density of infection and odds of anemia. No clear relationship was observed between health status and cognitive and educational outcomes. The high burden of malaria highlights the need to tackle malaria among school children.     

Origins and spread of pfdhfr mutant alleles in Plasmodium falciparum

The emergence and spread of Plasmodium falciparum parasite resistant to sulfadoxine and pyrimethamine (SP) poses a serious public health problem. Resistance is caused by point mutations in dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps), the two key enzymes in the folate biosynthetic pathway. The use of microsatellite markers flanking pfdhfr has recently shown that the invasion of limited resistant lineages may explain the widespread SP resistance in many endemic regions. In Africa, however, multiple indigenous origins of pfdhfr triple mutants have been demonstrated. More new independent lineages and routes of geographical spread of resistance may be found by further molecular evolutionary analyses using samples from various endemic regions. Here, I review recent studies about the history of SP usage and the evolution and spread of resistant lineages while addressing the technical issue of microsatellite analysis.     

A Cross-Sectional Survey of Plasmodium falciparum pfcrt Mutant Haplotypes in the Democratic Republic of Congo

Splenic infarction is a rare complication of malaria. We report two recent cases of splenic infarction after Plasmodium vivax infection. No systematic review of malaria-induced splenic infarction was available, therefore we conducted a systematic review of the English, French, and Spanish literature in PubMed and KoreaMed for reports of malaria-associated splenic infarction from 1960 to 2012. Of the 40 cases collected on splenic infarction by Plasmodium species, 23 involved P. vivax, 11 Plasmodium falciparum, one Plasmodium ovale, and five a mixed infection of P. vivax and P. falciparum. Of the 40 cases, 2 (5.0%) involved splenectomy and 5 (12.5%) were accompanied by splenic rupture. The median time from symptom onset to diagnosis was 8.5 days (range, 3–90 days). Improved findings after treatment were observed in 8 (88.9%) of 9 patients with splenic infarction on follow-up by computed tomography or ultrasonography. All patients survived after treatment with the exception of one patient with cerebral malaria. Clinicians should consider the possibility of splenic infarction when malaria-infected patients have left upper quadrant pain.     

Gene trees and hominoid phylogeny.

Here we present a DNA sequence study that incorporates intraspecific variation from all five genera of hominoids (apes and humans). Recently it has been claimed that using single individuals to analyze species' relationships might be misleading if within-species variation is great. Our results indicate that despite high intraspecific variation in mitochondrial cytochrome oxidase subunit II gene sequences of some hominoids, humans and chimpanzees are nonetheless significantly most closely related. We also report the observation that variation within the gorilla species exceeds that between common and pygmy chimpanzee species, a finding with implications for conservation. In contrast, humans are less mitochondrially diverse than lowland gorillas inhabiting western Africa.     

Evaluation of the NOW Malaria Immunochromatographic Test for Quantitative Diagnosis of Falciparum and Vivax Malaria Parasite Density

The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density.     

High Levels of Genetic Diversity of Plasmodium falciparum Populations in Papua New Guinea despite Variable Infection Prevalence

High levels of genetic diversity in Plasmodium falciparum populations are an obstacle to malaria control. Here, we investigate the relationship between local variation in malaria epidemiology and parasite genetic diversity in Papua New Guinea (PNG). Cross-sectional malaria surveys were performed in 14 villages spanning four distinct malaria-endemic areas on the north coast, including one area that was sampled during the dry season. High-resolution msp2 genotyping of 2,147 blood samples identified 761 P. falciparum infections containing a total of 1,392 clones whose genotypes were used to measure genetic diversity. Considerable variability in infection prevalence and mean multiplicity of infection was observed at all of the study sites, with the area sampled during the dry season showing particularly striking local variability. Genetic diversity was strongly associated with multiplicity of infection but not with infection prevalence. In highly endemic areas, differences in infection prevalence may not translate into a decrease in parasite population diversity.     

Marked Age-Dependent Prevalence of Symptomatic and Patent Infections and Complexity of Distribution of Human Plasmodium Species in Central Vietnam

In Vietnam, Plasmodium falciparum and P. vivax are responsible for most malaria infections, and P. malariae and P. ovale infections are rarely reported. Nevertheless, species-specific polymerase chain reaction analysis on 2,303 blood samples collected during a cross-sectional survey conducted in a forest area of central Vietnam identified 223 (9.7%) P. falciparum, 170 (7.4%) P. vivax, 95 (4.1%) P. malariae, and 19 (0.8%) P. ovale mono-infections and 164 (7.1%) mixed infections. Of the 671 Plasmodium-positive samples by polymerase chain reaction, only 331 were detected by microscopy. Microscopy poorly diagnosed P. malariae, P. ovale, and mixed infections. Clinical and sub-clinical infections occurred in all age groups. The risk for infection and disease decreased with age, probably because of acquired partial immunity. The common occurrence of sub-patent infections seems to indicate that the malaria burden is underestimated and that diagnostic and therapeutic policies should be adapted accordingly.