Lisa C. Ranford-Cartwright

Random mating in a natural population of the malaria parasite Plasmodium falciparum

The genetic structure of a population of the malaria parasite Plasmodium falciparum has been examined in a village in Tanzania. Seventeen alleles of the merozoite surface protein MSP-1 and 23 of MSP-2 were detected by the polymerase chain reaction (PCR) among the blood parasites of the inhabitants. Most infections contained mixtures of genetically distinct parasite clones. PCR was then used to examine individual P. falciparum oocysts, the products of fertilization events, in wild-caught mosquitoes. Forty-five out of 71 oocysts were heterozygous for one or both genes, showing that crossing between clones was taking place frequently, following uptake of mixtures of gametocytes by the mosquitoes. The frequency of heterozygous forms showed that random mating events probably occurred within mosquito bloodmeals between gametes belonging to different parasite clones.

Real-time quantitative PCR in parasitology

Standard techniques for counting parasites are often time-consuming, difficult and inaccurate, and occasionally unpleasant. Real-time quantitative polymerase chain reaction has recently been applied to parasitology, specifically Plasmodium, Toxoplasma, Leishmania and Neospora. These techniques are truly quantitative, give results over a range of 6-7 orders of magnitude, are quick to perform and require no manipulations post-amplification. They can be used to count genome numbers and to study levels of gene expression. The advantages and limitations of existing thermocyclers and applicable detection systems are discussed here, and promising new developments are highlighted.

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.

The Plasmodium eukaryotic initiation factor-2α kinase IK2 controls the latency of sporozoites in the mosquito salivary glands

Sporozoites, the invasive form of malaria parasites transmitted by mosquitoes, are quiescent while in the insect salivary glands. Sporozoites only differentiate inside of the hepatocytes of the mammalian host. We show that sporozoite latency is an active process controlled by a eukaryotic initiation factor-2α (eIF2α) kinase (IK2) and a phosphatase. IK2 activity is dominant in salivary gland sporozoites, leading to an inhibition of translation and accumulation of stalled mRNAs into granules. When sporozoites are injected into the mammalian host, an eIF2α phosphatase removes the PO4 from eIF2α-P, and the repression of translation is alleviated to permit their transformation into liver stages. In IK2 knockout sporozoites, eIF2α is not phosphorylated and the parasites transform prematurely into liver stages and lose their infectivity. Thus, to complete their life cycle, Plasmodium sporozoites exploit the mechanism that regulates stress responses in eukaryotic cells.

Plasmodium falciparum Accompanied the Human Expansion out of Africa

Plasmodium falciparum is distributed throughout the tropics and is responsible for an estimated 230 million cases of malaria every year, with a further 1.4 billion people at risk of infection. Little is known about the genetic makeup of P. falciparum populations, despite variation in genetic diversity being a key factor in morbidity, mortality, and the success of malaria control initiatives. Here we analyze a worldwide sample of 519 P. falciparum isolates sequenced for two housekeeping genes (63 single nucleotide polymorphisms from around 5000 nucleotides per isolate). We observe a strong negative correlation between within-population genetic diversity and geographic distance from sub-Saharan Africa (R(2) = 0.95) over Africa, Asia, and Oceania. In contrast, regional variation in transmission intensity seems to have had a negligible impact on the distribution of genetic diversity. The striking geographic patterns of isolation by distance observed in P. falciparum mirror the ones previously documented in humans and point to a joint sub-Saharan African origin between the parasite and its host. Age estimates for the expansion of P. falciparum further support that anatomically modern humans were infected prior to their exit out of Africa and carried the parasite along during their colonization of the world.

Estimation of inbreeding coefficients from genotypic data on multiple alleles, and application to estimation of clonality in malaria parasites

Methods for estimating probability of identity by descent (f) are derived for data on numbers of genotypes at single loci and at pairs of loci with many alleles at each locus. The methods are general, but are specifically applied to data on genotype frequencies in zygotes of the malaria parasite sampled from its mosquito host in order to find the extent of outcrossing in the parasite and the degree of clonality in populations. It is assumed that zygotes are the outcome either of gametes of the same clone, in which they are identical at all loci, or are products of two random, unrelated clones. From the estimate of f an effective number of clones per human host can also be derived. For Plasmodium falciparum from a Tanzanian village, estimates of f are 0.33 from data on zygote frequencies at two multiallelic loci, indicating that two-thirds of zygotes produce recombinant type.

Molecular analysis of recrudescent parasites in a Plasmodium falciparum drug efficacy trial in Gabon.

Recrudescent Plasmodium falciparum parasites were sampled from 108 children taking part in a drug efficacy trial in Gabon. A finger-prick blood sample was taken from each child before treatment, and a post-treatment sample taken of the recrudescent parasites. Sample deoxyribonucleic acid was amplified by the polymerase chain reaction using primers specific to the P. falciparum antigen genes MSP-1, MSP-2 and GLURP. Seventy-seven children had identical parasites in their pre- and post-treatment samples, indicating genuine recrudescences of resistant parasites. Fourteen children had completely different parasites in their pre- and post-treatment samples, indicating either a fresh infection from a mosquito or growth of a population of parasites not detected in the pre-treatment sample, perhaps due to sequestration. The remaining 17 children had a mixture of pre-treatment and new parasites in their post-treatment samples. This study demonstrated the use of polymorphic markers to confirm whether parasites in patients with clinical recrudescences after drug treatment are genuinely resistant.

3. Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania

Plasmodium falciparum parasites exist as genetically distinct haploid clones in infected people. In the Kilombero valley in south-east Tanzania, at least 85% of the inhabitants of Michenga village harbour more than one clone. Using 2 highly polymorphic unlinked markers, it has been estimated that each infected person harbours between one and 6 P. falciparum clones at any one time, with a mean of 3.5 clones. When mosquitoes acquire gametocytes of 2 different clones in a blood meal, crossing generates recombinant clones differing from their parental genotypes. The inbreeding coefficient of the parasite population has been estimated as 0.33.

Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics

The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen‐activated protein kinase (MAPK) homologues, pfmap‐1 and pfmap‐2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map‐2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map‐2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non‐essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap‐2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap‐1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap‐2 protein levels are elevated in pfmap‐1‐ parasites, suggesting that Pfmap‐1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.

Commitment to sexual differentiation in the human malaria parasite, Plasmodium falciparum

The differentiation of the two sexes in the gametocytogenesis of Plasmodium falciparum was investigated using a plaque assay and antibodies specific for various stages and sexes of gametocytes. Immunofluorescence assays on plaques of cultured parasites grown in monolayers of erythrocytes revealed that the merozoites released from a single sexually-committed schizont became either all male or all female gametocytes. Thus, the commitment of this species to differentiate into one sex or the other is likely to occur prior to the nuclear division of the sexually-committed schizont. The characteristic female-biased gametocyte sex ratio observed for many Plasmodium species is manifested in P. falciparum by a greater percentage of schizonts that produce female gametocytes (67-71%) than those that yield males. From the plaque assay, it was determined that the number of gametocytes produced per sexually-committed schizont was similar for both sexes, indicating that allocation of parasite resources was equal for each sex of gametocyte. The timing of sexual differentiation and features of the gametocyte sex ratio is discussed in relation to previous observations on P. falciparum and related malaria parasites.