Coamplification of Leucocytozoon by PCR diagnostic tests for avian malaria: a cautionary note.

A number of PCR assays have now been described for detecting species of the avian malaria parasites Plasmodium and Haemoproteus from blood samples. The published protocols amplify both genera simultaneously, owing to the high degree of sequence similarity between them in target genes. However, the potential for coamplification in these assays of a third, closely related hematozoan parasite, Leucocytozoon spp. has been largely overlooked. In this paper, we highlight the importance of this issue, showing that coamplification of Leucocytozoon spp. occurs in several of the protocols designed to amplify avian malaria parasites. This leads not only to scoring of false positives but, in cases of mixed Leucocytozoon/malaria infections, may also lead to scoring of false negatives. We, therefore, advocate the use of a post-PCR diagnostic step, such as RFLP analysis or sequencing, to assess the contribution of Leucocytozoon spp. to overall prevalence.

Light and electron microscopical observations of the effects of high-density lipoprotein on growth of Plasmodium falciparum in vitro.

Human serum high-density lipoprotein (HDL) is necessary and sufficient for the short-term maintenance of Plasmodium falciparum in in vitro culture. However, at high concentrations it is toxic to the parasite. A heat-labile component is apparently responsible for the stage-specific toxicity to parasites within infected erythrocytes 12-42 h after invasion, i.e. during trophozoite maturation. The effects of HDL on parasite metabolism (as determined by nucleic acid synthesis) are evident at about 30 h after invasion. Parasites treated with HDL show gross abnormalities by light and electron microscopy.

Recent origin of Plasmodium falciparum from a single progenitor.

Genetic variability of Plasmodium falciparum underlies its transmission success and thwarts efforts to control disease caused by this parasite. Genetic variation in antigenic, drug resistance, and pathogenesis determinants is abundant, consistent with an ancient origin of P. falciparum, whereas DNA variation at silent (synonymous) sites in coding sequences appears virtually absent, consistent with a recent origin of the parasite. To resolve this paradox, we analyzed introns and demonstrated that these are deficient in single-nucleotide polymorphisms, as are synonymous sites in coding regions. These data establish the recent origin of P. falciparum and further provide an explanation for the abundant diversity observed in antigen and other selected genes.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

Age- and species-specific duration of infection in asymptomatic malaria infections in Papua New Guinea.

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.

Genetic diversity and dynamics of plasmodium falciparum and P. vivax populations in multiply infected children with asymptomatic malaria infections in Papua New Guinea.

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.

Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum.

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.

Complex mutations in a high proportion of microsatellite loci from the protozoan parasite Plasmodium falciparum.

Microsatellite loci are generally assumed to evolve via a stepwise mutational process and a battery of statistical techniques has been developed in recent years based on this or related mutation models. It is therefore important to investigate the appropriateness of these models in a wide variety of taxa. We used two approaches to examine mutation patterns in the malaria parasite Plasmodium falciparum: (i) we examined sequence variation at 12 tri-nucleotide repeat loci; and (ii) we analysed patterns of repeat structure and heterozygosity at 114 loci using data from 12 laboratory parasite lines. The sequencing study revealed complex patterns of mutation in five of the 12 loci studied. Alleles at two loci contain indels of 24 bp and 57 bp in flanking regions, while in the other three loci, blocks of imperfect microsatellites appear to be duplicated or inserted; these loci essentially consist of minisatellite repeats, with each repeat unit containing four to eight microsatellites. The survey of heterozygosity revealed a positive relationship between repeat number and microsatellite variability for both di- and trinucleotides, indicating a higher mutation rate in loci with longer repeat arrays. Comparisons of levels of variation in different repeat types indicate that the mutation rate of dinucleotide-bearing loci is 1.6-2.1 times faster than trinucleotides, consistent with the lower mean number of repeats in trinucleotide-bearing loci. However, despite the evidence that microsatellite arrays themselves are evolving in a manner consistent with stepwise mutation model in P. falciparum, the high frequency of complex mutations precludes the use of analytical tools based on this mutation model for many microsatellite-bearing loci in this protozoan. The results call into question the generality of models based on stepwise mutation for analysing microsatellite data, but also demonstrate the ease with which loci that violate model assumptions can be detected using minimal sequencing effort.

Do malaria parasites mate non-randomly in the mosquito midgut?

Polymerase chain reaction (PCR)-based genotyping of oocysts dissected from mosquito midguts has previously been used to investigate overall levels of inbreeding within malaria parasite populations. We present a re-analysis of the population structure of Plasmodium falciparum malaria using diploid genotypes at three antigen-encoding loci in 118 oocysts dissected from 34 mosquitoes. We use these data to ask whether mating is occurring at random within the mosquito midgut, as is generally assumed. We observe a highly significant deficit of heterozygous oocysts within mosquitoes at all three loci, suggesting that fusion of gametes occurs non-randomly in the mosquito gut. A variety of biological explanations, such as interrupted feeding of mosquitoes, positive assortative mating and outcrossing depression, could account for this observation. However, an alternative artefactual explanation--the presence of non-amplifying or null alleles--can account for the observed data equally well, without the need to invoke non-random mating. To evaluate this explanation further, we estimate the frequencies of null alleles within the oocyst population using maximum likelihood, by making the assumption that non-amplifying oocysts at any of the three loci are homozygous for null alleles. Observed levels of visible heterozygotes fit closely with those expected under random mating when non-amplifying oocysts are accounted for. Other lines of evidence also support the artefactual explanation. Overall inbreeding coefficients have been recalculated in the light of this analysis, and may be considerably lower than those estimated previously. In conclusion, we suggest that the deficit of heterozygotes observed is unlikely to indicate non-random mating within the mosquito gut and is better explained by misscoring of heterozygotes as homozygotes.

Commitment to gametocytogenesis in Plasmodium falciparum.

To achieve transmission, a subpopulation of asexually dividing bloodstream forms of the human malaria parasite Plasmodium falciparum withdraws from the cell cycle to develop into gametocytes - cells specialized for sexual reproduction and invasion of the mosquito vector. For natural selection to maximize transmission to new hosts, a balance must have evolved between asexual replication and sexual differentiation. Here, Mike Dyer and Karen Day consider observations on the process of commitment to gametocytogenesis and use this information as the framework for a model that begins to explain the control of the dynamics between asexual and sexual development.

Cross-species interactions between malaria parasites in humans.

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.

Plasmodium falciparum: histidine-rich protein II is expressed during gametocyte development.

Both early gametocytes (I-II) and asexual trophozoite stages of Plasmodium falciparum digest hemoglobin and detoxify haem by polymerizing it into parasite pigment called hemozoin. The mechanism of polymerization is unclear but it has been proposed that histidine-rich protein II may facilitate transport of hemoglobin to the food vacuole and catalyze the polymerization in asexual stages. We describe the transcription of histidine-rich protein II in gametocytes by Northern blot analysis and the expression of the protein in these stages by immunoprecipitation and Western blotting. Localization of histidine-rich protein II within the gametocyte by immunofluorescence assay and immunoelectron microscopy clearly illustrated the presence of this molecule in the infected red cell cytosol in the early stages of gametocyte development and internalization in the later gametocyte as it matures. There is a strong correlation between the stage-specific trafficking of histidine-rich protein II in gametocytes and the susceptibility of early but not late gametocytes to the antimalarial drug chloroquine.

Malaria transmission and naturally acquired immunity to PfEMP-1.

Why there are so few gametocytes (the transmission stage of malaria) in the blood of humans infected with Plasmodium spp. is intriguing. This may be due either to reproductive restraint by the parasite or to unidentified gametocyte-specific immune-mediated clearance mechanisms. We propose another mechanism, a cross-stage immunity to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1). This molecule is expressed on the surface of the erythrocyte infected with either trophozoite or early gametocyte parasites. Immunoglobulin G antibodies to PfEMP-1, expressed on both life cycle stages, were measured in residents from an area where malaria is endemic, Papua New Guinea. Anti-PfEMP-1 prevalence increased with age, mirroring the decline in both the prevalence and the density of asexual and transmission stages in erythrocytes. These data led us to propose that immunity to PfEMP-1 may influence malaria transmission by regulation of the production of gametocytes. This regulation may be achieved in two ways: (i) by controlling asexual proliferation and density and (ii) by affecting gametocyte maturation.

Polymorphism at the merozoite surface protein-3alpha locus of Plasmodium vivax: global and local diversity.

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.

Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples.

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.

Virulence and transmission success of the malarial parasite Plasmodium falciparum.

Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host-parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages.

Application of genetic markers to the identification of recrudescent Plasmodium falciparum infections on the northwestern border of Thailand.

Parasite genotyping by the polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in a Karen population resident on the northwestern border of Thailand where malaria transmission is low (one infection/person/year). Plasmodium falciparum infections were genotyped for allelic variation in three polymorphic antigen loci, merozoite surface proteins-1 and -2 (MSP-1 and -2) and glutamaterich protein (GLURP), before and after antimalarial drug treatment. Population genotype frequencies were measured to provide the baseline information to calculate the probability of a new infection with a different or the same genotype to the initial pretreatment isolate. Overall, 38% of the infections detected following treatment had an identical genotype before and up to 121 days after treatment. These post-treatment genotypes were considered recrudescent because of the low (< 5%) probability of repeated occurrence by chance in the same patient. This approach allows studies of antimalarial drug treatment to be conducted in areas of low transmission since recrudescences can be distinguished confidently from newly acquired infections.

Plasmodium falciparum: analysis of the antibody specificity to the surface of the trophozoite-infected erythrocyte.

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.