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Multilocus genotyping of microbial pathogens has revealed a range of population structures, with some bacteria showing extensive recombination and others showing almost complete clonality. The population structure of the protozoan parasite Plasmodium falciparum has been harder to evaluate, since most studies have used a limited number of antigen-encoding loci that are known to be under strong selection. We describe length variation at 12 microsatellite loci in 465 infections collected from 9 locations worldwide. These data reveal dramatic differences in parasite population structure in different locations. Strong linkage disequilibrium (LD) was observed in six of nine populations. Significant LD occurred in all locations with prevalence <1% and in only two of five of the populations from regions with higher transmission intensities. Where present, LD results largely from the presence of identical multilocus genotypes within populations, suggesting high levels of self-fertilization in populations with low levels of transmission. We also observed dramatic variation in diversity and geographical differentiation in different regions. Mean heterozygosities in South American countries (0.3-0.4) were less than half those observed in African locations (0. 76-0.8), with intermediate heterozygosities in the Southeast Asia/Pacific samples (0.51-0.65). Furthermore, variation was distributed among locations in South America (F:(ST) = 0.364) and within locations in Africa (F:(ST) = 0.007). The intraspecific patterns of diversity and genetic differentiation observed in P. falciparum are strikingly similar to those seen in interspecific comparisons of plants and animals with differing levels of outcrossing, suggesting that similar processes may be involved. The differences observed may also reflect the recent colonization of non-African populations from an African source, and the relative influences of epidemiology and population history are difficult to disentangle. These data reveal a range of population structures within a single pathogen species and suggest intimate links between patterns of epidemiology and genetic structure in this organism.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3alpha, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6.6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co-infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5-14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

The burden and duration of asymptomatic malaria infections were measured in residents of the malaria endemic village of Gonoa, Madang Province, Papua New Guinea. Plasmodium falciparum, P. vivax and P. malariae infections in people aged 4 years to adulthood were compared. Frequent sampling at 3-day intervals for up to 61 days allowed assessment of individual episodes of infection. Statistical assessment of P. falciparum detection revealed a periodicity consistent with synchronous replication of this species over periods up to 27 days. The duration of P. falciparum episodes was longer across all age groups than that of P. vivax and P. malariae. A trend for decreasing duration with age was also noted in data from each species. This was most prominent in P. falciparum infections: median duration in 4-year-olds was > 48 days compared with a median between 9 and 15 days in older children and adults. The results are consistent with the slow acquisition of immunity to antigenically diverse Plasmodium populations and suggest a faster rate of acquisition to P. vivax and P. malariae than to P. falciparum.

The dynamics of multiple Plasmodium infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.

Allelic diversity at the Plasmodium vivax merozoite surface protein-3alpha (PvMsp-3alpha) locus was investigated using a combined polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) protocol. Symptomatic patient isolates from global geographic origins showed a high level of polymorphism at the nucleotide level. These samples were used to validate the sensitivity, specificity, and reproducibility of the PCR/RFLP method. It was then used to investigate PvMsp3alpha diversity in field samples from children living in a single village in a malaria-endemic region of Papua New Guinea, with the aim of assessing the usefulness of this locus as an epidemiologic marker of P. vivax infections. Eleven PvMsp-3alpha alleles were distinguishable in 16 samples with single infections, revealing extensive parasite polymorphism within this restricted area. Multiple infections were easily detected and accounted for 5 (23%) of 22 positive samples. Pairs of samples from individual children provided preliminary evidence for high turnover of P. vivax populations.

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0.21 to 0.87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

Recent research has shown that many parasite populations are made up of a number of epidemiologically distinct strains or genotypes. The implications of strain structure or genetic diversity for parasite population dynamics are still uncertain, partly because there is no coherent framework for the interpretation of field data. Here, we present an analysis of four published data sets for vector-borne microparasite infections where strains or genotypes have been distinguished: serotypes of African horse sickness (AHS) in zebra; types of Nannomonas trypanosomes in tsetse flies; parasite-induced erythrocyte surface antigen (PIESA) based isolates of Plasmodium falciparum malaria in humans, and the merozoite surface protein 2 gene (MSP-2) alleles of P. falciparum in humans and in anopheline mosquitoes. For each data set we consider the distribution of strains or types among hosts and any pairwise associations between strains or types. Where host age data are available we also compare age-prevalence relationships and estimates of the force of infection. Multiple infections of hosts are common and for most data sets infections have an aggregated distribution among hosts with a tendency towards positive associations between certain strains or types. These patterns could result from interactions (facilitation) between strains or types, or they could reflect patterns of contact between hosts and vectors. We use a mathematical model to illustrate the impact of host-vector contact patterns, finding that even if contact is random there may still be significant aggregation in parasite distributions. This effect is enhanced if there is non-random contact or other heterogeneities between hosts, vectors or parasites. In practice, different strains or types also have different forces of infection. We anticipate that aggregated distributions and positive associations between microparasite strains or types will be extremely common.

Current opinion supports the view that immunity to the surface of the trophozoite-infected erythrocyte (IE) is to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1). Here we provide further evidence using the mutant cell line 1776/C10 which no longer expresses PfEMP-1 at the IE surface, due to a subtelomeric deletion in chromosome 9. We have measured antibody reactivity to this mutant in comparison to it's intact isogenic parent line 1776, which does express PfEMP-1, using the sensitive technique of flow cytometry. IgG-specific antibodies (subclass IgG1) in the plasma of hyperimmune adults, reacted to 1776 but never to the 1776/C10 mutant. Antibody subclasses were also measured in individual plasma samples to the surface of trophozoite-IE. Predominantly IgG1 antibodies were detected, with a few individual plasma having additional IgG3 antibodies. Previous studies have used the agglutination assay to measure sero-conversion to PfEMP-1. Here we show that both agglutination and flow cytometric methods are comparable, suggesting that agglutination of trophozoite-IE is mediated by IgG antibodies. Comparison of the isogenic cell lines 1776 and 1776/C10 differing in expression of PfEMP-1 provides further evidence that IgG antibodies, in particular of the cytophilic subclasses, mediate recognition of PfEMP-1.

Description of the genetic structure of malaria parasite populations is central to an understanding of the spread of multiple-locus drug and vaccine resistance. The Plasmodium falciparum mating patterns from madang, Papua New Guinea, where intense transmission of malaria occurs, are described here. A high degree of inbreeding occurs in the absence of detectable linkage disequilibrium. This contrasts with other studies, indicating that the genetic structure of malaria parasite populations is neither clonal nor panmictic but will vary according to the transmission characteristics of the region.

Molecular genetic analyses of P. falciparum have led to the cloning and sequencing of a number of antigens that are potential candidates for vaccination against malaria. Seroepidemiological studies in endemic areas have attempted to assess the relative importance of these antigens in protection against malaria. In this paper, we attempt to evaluate the relative contributions of conserved and strain-specific immune responses by modelling their influence of age-specific patterns of infection and disease. The modelling exercises in this paper clearly demonstrate that the observed patterns of age-prevalence are best explained by proposing that the accumulation to a threshold of an immune response against a conserved determinant is required for protection against infection, while 'anti-disease' immunity develops more linearly with exposure. This is compatible with the conjecture that the parasite population is structured into several independently transmitted strains, that each confers some degree of 'anti-disease' immunity, but does not protect against further infection by the same strain. Within this framework, the average duration of parasitaemia increases with age, as previously encountered strains endure for longer periods at a subclinical level. Indirect evidence for the increase in duration of parasitaemia with age may be obtained from a comparison of age-prevalence curves between dry and rainy seasons. By using mathematical methods to structure epidemiological and immunological information, we provide a coherent theoretical framework for the dissection of the important components of naturally acquired immunity to malaria.

Active community and self-reporting surveillance techniques have been used to describe the dynamics of febrile illness and associated malaria infection in children aged 2 to 15 years from a rural area of Madang Province, Papua New Guinea (PNG). Both history of fever and fever in association with parasitaemia appeared to be reliable indicators of malaria morbidity in this endemic area. Parasite density was observed to be a major determinant of mild malarial disease at both the population level and within an individual. Age-specific prevalence of febrile illness correlated with age-specific patterns of parasite density but not of parasite prevalence. Seasonal changes in fever incidence correlated with parasite density. The transition from afebrile to febrile state within an individual was generally associated with an increase in parasite density. Surveillance and self-reported febrile cases (which differ in severity on the basis of the perceived need for treatment) could be distinguished on the basis of parasite density. Thus surveillance techniques divide clinical malaria in rural PNG into 'mild' and 'very mild' forms. The age-specific pattern of decline of prevalence of malaria-associated febrile illness and parasite density is best explained by induction of strain-specific anti-disease immunity upon infection with a given strain of Plasmodium falciparum. The fever threshold in self-reporting febrile cases was seen to decrease with age and can be explained by an age-specific decline in anti-toxic immunity.

The average age of humans at their first infection with Plasmodium falciparum is typically less than 1 year in most endemic areas. This has been interpreted as evidence of the high transmissibility of the parasite, with the implication that control of malaria will require high levels of coverage with a potential vaccine. This interpretation is challenged by mathematical models that demonstrate that the long period required to develop immunity to malaria permits a high risk (or low average age) of infection even when parasite transmissibility is low. Patterns of seroconversion to five antigenically distinct isolates of P. falciparum in a highly malarious area of Papua New Guinea indicate that each is only mildly transmissible and that malaria, as a construct of several such independently transmitted strains, has a basic reproductive rate (or transmissibility) that is an order of magnitude lower than other estimates.

Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.

The frequency of recombination between unlike genotypes is central to understanding the generation of genetic diversity in natural populations of malaria. Here we suggest a way of investigating the problem which could complement conventional biochemical approaches to the population genetics of malaria. Sex allocation theory is one of the most successful areas of evolutionary biology. A well-supported prediction is that progressively less female-biased sex ratios are favoured with more outcrossing; equal numbers of males and females being evolutionarily stable in randomly mating outbred populations. We present a simple game theory model to support the idea that outcrossing rates in malaria will be correlated with the sex ratio of gametocytes in the peripheral blood of vertebrate hosts. Blood films from epidemiological surveys and culture-adapted isolates from Madang Province, Papua New Guinea, were used to estimate average gametocyte sex ratio of Plasmodium falciparum in the area. The geometric mean proportion of males in the population was 0.18 (95% confidence limits: 0.15-0.22). From our model, we estimate that, on average, 36% of zygotes are the result of outcrossing. This estimate assumes that most microgametes released following exflagellation are capable of fertilization. If, on average, fewer than about 70% of microgametes are capable of fertilization (as is the case in at least one other species of Plasmodium), the observed sex ratio would be consistent with between zero and 36% of zygotes being the result of outcrossing. These estimates suggest that there is usually a numerically dominant genotype in the gametocyte population in a blood meal, and that a considerable amount of selfing is occurring in P. falciparum populations in the Madang region, even though it is an area of intense year-round transmission.

The development of antibodies to infective stages of the filarial parasite, Wuchereria bancrofti, with age of the host human population was studied by immunofluorescence, immunoprecipitation and immunoblotting assays. Among individuals under 20 years of age, few had detectable antibodies to the infective (L3) larval surface by IFA: only 2 out of 10 scored positive. However, all adults (over 20 years) were positive in this assay although the utilization of isotypes varied between different individuals. Whilst antibodies to the L3 surface are therefore acquired after prolonged exposure to infection (greater than 20 years), recognition patterns of L3 surface labelled antigens, measured by immunoprecipitation analysis iodinated proteins on SDS-PAGE, and of somatic L3 proteins on immunoblots, were equivalent in the two age groups. Thus, a critical surface antigen, recognised in an age-dependent manner, is present on the L6 cuticle but cannot be resolved as a conventional protein or glycoprotein constituent.