Malaria vaccine research and testing in Africa.
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Journal Article↗
Biomedical subjects
D E Arnot
Publications and source records attributed to D E Arnot.
Seasonal changes in the Plasmodium falciparum population in individuals and their relationship to clinical malaria: a longitudinal study in a Sudanese village.
Residents of Daraweesh village in Sudan were monitored for Plasmodium falciparum infection and malaria morbidity in 3 malaria seasons from 1993 to 1996. Malaria parasites were detected microscopically and by polymerase chain reaction (PCR) in a series of cross-sectional surveys. PCR revealed submicroscopical infections during the dry season, particularly among individuals who had recovered from a malaria episode following successful drug treatment. Clinical and subclinical infections were contrasted by assaying for allelic polymorphism at 2 gene loci, MSP-1 and GLURP and 2 hypotheses examined with reference to these data: that clinical malaria is associated with infection with novel parasite genotypes not previously detected in that host, or alternatively, that clinical malaria episodes are associated with an increased number of clones in an infection. We detected more mixed infections among clinical isolates, but people carrying parasites during the dry season were not found to have an increased risk of disease in the following malaria season. There was a clear association of disease with the appearance of novel parasite genotypes.
Adolescent↗
The Plasmodium cell-cycle: facts and questions.
The Plasmodium life-cycle contains within it several discrete cell-cycles whose molecular controls are very poorly understood. Of the five (or more) periods of DNA synthesis within the P. falciparum life-cycle, that associated with erythrocytic schizogony has been studied in the greatest detail and even here, controversy exists as to the precise nature and timing of the major, cell-cycle events. Thus, while it is clear that DNA synthesis (S phase) starts around 28-31h after merozoite invasion (soon after the appearance of pigmented trophozoites), neither flow-cytometric nor precursor-incorporation studies have provided a definite answer as to whether rapid and successive G2 and then M (mitotic) phases occur during schizogony. These and other problems of interpretation of the P. falciparum cell-cycle are considered.
Animals↗
Seasonal variation in agglutination of Plasmodium falciparum-infected erythrocytes.
Agglutination and rosette formation are in vitro characteristics of Plasmodium falciparum-infected erythrocytes, which have been associated with host protective immune responses and also with parasite virulence. The present study was carried out in an area of seasonal and unstable malaria transmission in eastern Sudan. Plasma samples were obtained before, during, and after the transmission season from a volunteer cohort of 64 individuals seven years of age and older. These plasmas were assayed for their ability to agglutinate cultured parasitized erythrocytes originally obtained from acute malaria infection samples taken from five of the cohort members. Our data show that the capacity of donor plasma samples to agglutinate parasitized cells depended largely on the time of sampling relative to the transmission season, at least within this epidemiologic setting. Thus, although less than half of the pretransmission season samples could agglutinate any of the five lines of cultured parasites, all post-transmission season samples could agglutinate at least one of the parasite lines, with 74% agglutinating two or more lines. This increase in the agglutination capacity of individual plasma samples after the transmission season occurred essentially regardless of whether an individual had experienced a clinical malaria attack during the transmission season. The study thus confirms the acquisition of agglutinating antibodies following episodes of clinical malaria, but also demonstrates that such acquisition can take place in the absence of disease, presumably as a consequence of subclinical infection. This is the first demonstration of marked seasonal fluctuations in the capacity of individuals' sera to agglutinate parasitized red blood cells. Possible explanations for this effect include a decrease in the levels of agglutinating antibodies between seasons, or shifts in the antigens being recognized by such antibodies from one transmission season to the next. Finally, we showed the existence of marked seasonal fluctuation in the levels of agglutinating antibodies, either because levels of such antibodies are not sustained between seasons or because the antigens recognized change from one season to the next.
Adolescent↗
A Plasmodium falciparum homologue of the ATPase subunit of a multi-protein complex involved in chromatin remodelling for transcription.
A Plasmodium falciparum homologue of one of the components of a chromatin-remodelling complex which controls binding of transcription factors to nucleosome core particles has been cloned and characterised. The gene encodes 1422 amino acids with an estimated molecular mass of 167 kDa. The protein, SNF2L, shares 60% amino acid identity in its conserved DNA-dependent ATPase domain with yeast transcription factors originally identified by characterising mating type switch mutants. It also contains sequences related to the so-called SWI3, ADA2, N-CoR and TFIIIB B" or SANT DNA binding domains which are characteristic of these transcriptional activation factors. The SNF2L gene has two short introns in the 3' region of the coding sequence of the gene and is transcribed into a single approximately 6.5 kb messenger RNA species which is present throughout the asexual stages of the cell cycle. Southern blotting and pulsed field gel electrophoresis experiments show that SNF2L is a single copy gene. located on P. falciparum chromosome 11.
Adenosine Triphosphatases↗
Current status of the Plasmodium falciparum genome project.
The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures. Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes. Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases. A total of > 0.5 Mb of P. falciparum sequence tag data is available. The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome. Routes of access to project information and services are described.
Animals↗
Detection of very low level Plasmodium falciparum infections using the nested polymerase chain reaction and a reassessment of the epidemiology of unstable malaria in Sudan.
We have used the nested polymerase chain reaction (PCR) to assay for low level Plasmodium falciparum infections that were below the threshold of detection of blood film examination. This revealed a substantial group of asymptomatic, submicroscopically patent infections within the population of a Sudanese village present throughout the year although clinical malaria episodes were almost entirely confined to the transmission season. In our September, January, April, and June surveys, the PCR-detected prevalences were 13%, 19%, 24%, and 19%, respectively. These figures reveal a much higher prevalence of dry season infection than previous microscopic surveys have indicated. Furthermore, 20% of a cohort of 79 individuals were healthy throughout the September to November transmission season but were PCR-positive for P. falciparum in a least one of a series of samples taken in the ensuing months. Levels of exposure to P. falciparum infection were therefore higher than was previously believed in this region, highlighting the fact that many individuals were infected but healthy for most of the year. The reservoir parasite population was thus larger and more stable than previously thought, a finding that is consistent with the high levels of genetic variation at polymorphic loci reported from analysis of P. falciparum parasites in this area.
Adolescent↗
High proportion of subclinical Plasmodium falciparum infections in an area of seasonal and unstable malaria in Sudan.
In the present longitudinal study, a cohort (n = 98) of children and adults 5-30 years of age living in an area of highly seasonal and unstable malaria transmission were followed for malaria morbidity during several successive transmission seasons. Based on morbidity surveillance during 1993 and measurements of antibody titers to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), the cohort was divided into three groups: those who had at least one episode of clinical malaria (Group 1, n = 31), those who did not suffer from clinical malaria but had (Group 2, n = 63) or had not (Group 3, n = 4) a significant increase in antibody titers against the Pf155/RESA antigen. This increase was defined as equal to or greater than a four-fold increase in antibody titer in samples from same individuals taken at the beginning and the end of the malaria transmission season. Such increases in specific antibody levels suggested that the donors had been exposed to a P. falciparum blood-stage infection. Measurements of antibody titers to a peptide derived from the glutamate-rich protein exoantigen gave data parallel to those for Pf155/RESA. A surprisingly high fraction of individuals in the study cohort (approximately 66%) showed evidence of infection without ensuing clinical disease (Group 2).
Adolescent↗
Uptake and efflux of chloroquine by chloroquine-resistant Plasmodium falciparum clones recently isolated in Africa.
In recently isolated African Plasmodium falciparum clones, the intracellular chloroquine concentration at steady-state, under standard culture conditions, could not differentiate chloroquine-sensitive from resistant parasites. However, under an atmosphere of air the chloroquine-resistant P. falciparum clones released pre-accumulated [3H]chloroquine more rapidly than sensitive clones. The very fast efflux of the pre-accumulated drug from chloroquine-resistant (CQR) parasites resulted in a differential in the drug retained by resistant and sensitive parasites. The chloroquine-sensitive parasites retained 2-3 times more chloroquine than resistant parasites. The steady-state uptake of [3H]chloroquine appeared to be enhanced by verapamil and desipramine in the chloroquine-resistant clones, while the opposite was observed with sensitive clones. This confirmed the suggestion that verapamil inhibits the rapid efflux in CQR parasites resulting in a readily detectable increase in chloroquine accumulation. These observations indicate that the biochemical phenotypes of African chloroquine-resistant P. falciparum are similar to those reported from S.E. Asia and Latin America and are consistent with a common molecular basis for the phenomenon.
Africa↗
Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control.
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Amino Acid Sequence↗
Cloning and characterisation of a Plasmodium falciparum homologue of the Ran/TC4 signal transducing GTPase involved in cell cycle control.
On the basis of conserved sequences characteristic of the Ran/TC4 subfamily of the GTPase superfamily, a fragment of the gene encoding a Plasmodium falciparum Ran/TC4 homologue was amplified in the polymerase chain reaction. The fragment was used to screen a cDNA library to obtain clones which allowed determination of the complete gene sequence. The gene, designated pfran (Plasmodium falciparum ras-like nuclear protein), has around 70% amino acid identity with previously characterised Ran/TC4 proteins. Like other malarial mRNAs, the pfran mRNA contains a long (at least 679 bp) 5' untranslated region. Southern blotting experiments show that pfran is a single copy gene located on chromosome 11. RNA hybridisation experiments indicate that pfran mRNA is abundant in late trophozoite and schizont stages, but present at very low levels in gametocytes and early asexual stages.
Amino Acid Sequence↗
MVR-PCR analysis of hypervariable DNA sequence variation.
Techniques for accurate marking of infectious microbial agents circulating in populations would be very useful to epidemiologists. In this article, David Arnot, Cally Roper and Ali Sultan review recent progress in transferring MVR-PCR DNA finger-printing techniques from human forensic medicine to parasitology.
Journal Article↗
Plasmodium chabaudi: a short-term in vitro culture method and its application to chloroquine resistance testing.
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Animals↗
Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates.
DNA typing systems currently used in parasitology involve either hybridising Southern blots with repetitive sequence probes or amplifying genomic sequences using the polymerase chain reaction (PCR). Both such approaches assay allelic length variation, usually in unexpressed tandemly repeated DNA sequences. Where an appropriate target locus exists, an alternative PCR-based strategy which reveals allelic sequence variation in tandemly repeated DNA offers a more accurate and internally controlled assay. We describe such a strategy for the rapid extraction of information on tandem repeat sequence variation from hypervariable alleles, and apply it to the Plasmodium falciparum CS gene. The extreme variability of such DNA 'barcodes' can be used to identify parasite stocks and lineages. This system is also potentially useful for population genetic and epidemiological studies since it offers the possibility of following the spread of distinctively marked parasite genotypes in samples taken from infected individuals.
Animals↗
Possible mechanisms for the maintenance of polymorphisms in Plasmodium populations.
There are two views on the origin and maintenance of the high levels of polymorphism found in antigenic Plasmodium proteins. Immune selectionists consider that mutations which avoid stimulating a host response are frequent and advantageous. Proponents of the random genetic drift of selectively equivalent mutations hold that Plasmodium antigens are relatively unconstrained and can tolerate considerable structural diversity. Both sides agree that antigenic diversity is advantageous although selectionists see benefits in individual mutations whereas the proponents of random genetic drift see the advantage in the parasite's capacity to tolerate diversity per se.
Animals↗
Antigenic diversity in Thai Plasmodium vivax circumsporozoite proteins.
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Animals↗
Polymorphism in the circumsporozoite protein and anti-sporozoite malaria vaccines.
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Journal Article↗
Does biased gene conversion influence polymorphism in the circumsporozoite protein-encoding gene of Plasmodium vivax?
Variation between North Korean and Latin American isolates in the circumsporozoite (CS) protein encoding gene of the human malaria parasite Plasmodium vivax was studied. Polymorphic positions are confined to the central tandemly repeated sequences. Nucleotide substitutions in the tandem repeats produce variants; these substituted positions within the repeat array tend to be conserved between genes. The North Korean CS gene has a short insertion after the repeats encoding a 4-amino acid repeat (Ala-Gly-Gly-Asn) not found in the New World P. vivax genes. This sequence is found both flanking and within the tandem repeats of the CS genes of several strains of the Southeast Asian simian malaria parasite, Plasmodium cynomolgi. The intraspecific conservation of positions of variants within tandem repeat arrays and the interspecific conservation of probably ancestral repeat motifs at the end of these arrays are consistent with the occurrence of nonreciprocal genetic exchanges between the tandem repeats of these genes. However, a striking asymmetry in strand nucleotide composition within the tandem repeats of all CS genes leads us to suggest that biased correction of heteroduplexes formed during recombination plays a role in the evolution of these genes.
Amino Acid Sequence↗