Molecular surveillance of malaria in the Central Highlands,Vietnam

Vietnam achieved outstanding success against malaria in the last few decades. The mortality and morbidity of malaria in Vietnam have decreased remarkably in recent years, but malaria is still a major public health concern in the country, particularly in the Central Highlands region. In this study, molecular analyses of malaria parasites in the Central Highlands were performed to understand the population structure and genetic diversity of the parasites circulating in the region. Plasmodium falciparum (68.7%) and P. vivax (27.4%) along with mixed infections with P. falciparum/P. vivax (3.9%) were detected in 230 blood samples from patients with malaria. Allele-specific nested-polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of pfmsp-1, pfama-1, pvcsp, and pvmsp-1 revealed complex genetic makeup in P. falciparum and P. vivax populations of Vietnam. Substantial multiplicity of infection (MOI) was also identified, suggesting significant genetic diversity and polymorphism of P. falciparum and P. vivax populations in the Central Highlands of Vietnam. These results provide fundamental insight into the current patterns of dispersion and genetic nature of malaria parasites as well as for the development of malaria elimination strategies in the endemic region.     

Mating Patterns of Plasmodium falciparum

Recent empirical data have enabled a more informed debate over the extent of clonality in Plasmodium falciparum populations. Oocyst heterozygosity data reveal that the mating structure of malaria populations varies according to the transmission intensity. This finding provides a more detailed picture of the malaria mating structure than previous conclusions, which were based on indirect measures of population mating structure, ie. linkage disequilibrium analyses. In this article, Ric Paul and Karen Day discuss aspects of the genetic structure of malaria populations as evidenced by oocyst heterozygosity and linkage disequilibrium data. They address the difficulties of performing genetic analyses of malaria parasite population structure inherent in parasite sampling, why two identical parasites are rarely observed in the field and how features of the epidemiology determine parasite population structure.     

Evolutionary genetics of Trypanosoma and Leishmania

We review recent advances in the study of population structure and phylogenetic diversity of parasites belonging to the genera Trypanosoma and Leishmania. In all species properly analyzed, these parasites exhibit a basically clonal population structure, with occasional bouts of genetic exchange or hybridization, and a strong structuration of their populations into discrete evolutionary lineages. On an evolutionary scale, the impact of sex appears to be greater in African than in American trypanosomes. The taxonomic status of some Leishmania 'species' is questionable.     

Genetic diversity of Plasmodium falciparum and its implications in the epidemiology of malaria

Genetic diversity provides Plasmodium falciparum with the potential capacity of avoiding the immune response, and possibly supporting the selection of drug or vaccine resistant parasites. These genetic characters play key roles in the selection of appropriate malaria control measures. Diverse clones of Plasmodium falciparum, often denoted as strains, has been documented, and the degree of genetic diversity supported by several kinds of PCR (polymerase chain reaction) assays. Many studies in different endemic regions with differences in their level of disease transmission have clarified the interactions between the parasite populations and malaria epidemiology. This paper describes recombination events of the malaria parasite life cycle that originate such genetic diversity in P. falciparum, reviewing different studies on this aspect and its implications in the immunity and development of control measures in regions with different degrees of endemicity.     

Distribution,prevalence and host specificity of avian malaria parasites across the breeding range of the migratory lark sparrow (Chondestes grammacus)

The lark sparrow (Chondestes grammacus) is a ground-nesting passerine that breeds across much of the central North American steppe and sand barrens. Through genotyping and sequencing of avian malaria parasites we examined levels of malaria prevalence and determined the distribution of Haemoproteus and Plasmodium lineages across the breeding range of the lark sparrow. Analysis of 365 birds collected from five breeding locations revealed relatively high levels of malaria prevalence in adults (80 %) and juveniles (46 %), with infections being primarily of Haemoproteus (91 % of sequenced samples). Levels of genetic diversity and genetic structure of malaria parasites with respect to the avian host populations revealed distinct patterns for Haemoproteus and Plasmodium, most likely as a result of their distinct life histories, host specificity, and transmission vectors. With the exception of one common Haemoproteus haplotype detected in all populations, all other haplotypes were either population-specific or shared by two to three populations. A hierarchical analysis of molecular variance of Haemoproteus sequences revealed that 15–18 % of the genetic variation can be explained by differences among host populations/locations (p < 0.001). In contrast to the regional patterns of genetic differentiation detected for the lark sparrow populations, Haemoproteus parasites showed high levels of population-specific variation and no significant differences among regions, which suggests that the population dynamics of the parasites may be driven by evolutionary processes operating at small spatial scales (e.g., at the level of host populations). These results highlight the potential effects of host population structure on the demographic and evolutionary dynamics of parasites.     

Plasmodium falciparum: differential selection of drug resistance alleles in contiguous urban and peri-urban areas of Brazzaville, Republic of Congo

The African continent is currently experiencing rapid population growth, with rising urbanization increasing the percentage of the population living in large towns and cities. We studied the impact of the degree of urbanization on the population genetics of Plasmodium falciparum in urban and peri-urban areas in and around the city of Brazzaville, Republic of Congo. This field setting, which incorporates local health centers situated in areas of varying urbanization, is of interest as it allows the characterization of malaria parasites from areas where the human, parasite, and mosquito populations are shared, but where differences in the degree of urbanization (leading to dramatic differences in transmission intensity) cause the pattern of malaria transmission to differ greatly. We have investigated how these differences in transmission intensity affect parasite genetic diversity, including the amount of genetic polymorphism in each area, the degree of linkage disequilibrium within the populations, and the prevalence and frequency of drug resistance markers. To determine parasite population structure, heterozygosity and linkage disequilibrium, we typed eight microsatellite markers and performed haplotype analysis of the msp1 gene by PCR. Mutations known to be associated with resistance to the antimalarial drugs chloroquine and pyrimethamine were determined by sequencing the relevant portions of the crt and dhfr genes, respectively. We found that parasite genetic diversity was comparable between the two sites, with high levels of polymorphism being maintained in both areas despite dramatic differences in transmission intensity. Crucially, we found that the frequencies of genetic markers of drug resistance against pyrimethamine and chloroquine differed significantly between the sites, indicative of differing selection pressures in the two areas.     

Historical Shifts in Brazilian P. falciparum Population Structure and Drug Resistance Alleles

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.     

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

A comparison of Anopheles gambiae and Plasmodium falciparum genetic structure over space and time

Population genetic structure and subdivision are key factors affecting the evolution of organisms. In this study, we analysed and compared the population genetic structure of the malaria parasite Plasmodium falciparum and its mosquito vector Anopheles gambiae over space and time in the Nianza Province, near Victoria Lake in Kenya. The parasites were collected from mosquitoes caught in six villages separated by up to 68 km in 2002 and 2003. A total of 545 oocysts were dissected from 122 infected mosquitoes and genotyped at seven microsatellite markers. Five hundred and forty-seven mosquitoes, both infected and uninfected, were genotyped at eight microsatellites. For the parasite and the vector, the analysis revealed no (or very little) genetic differentiation among villages. This may be explained by high local population sizes for the parasite and the mosquito. The small level of genetic differentiation observed between populations may explain the speed at which antimalarial drug resistance and insecticide resistance spread into the African continent.     

Within-population variation in prevalence and lineage distribution of avian malaria in blue tits, Cyanistes caeruleus

The development of molecular genetic screening techniques for avian blood parasites has revealed many novel aspects of their ecology, including greatly elevated diversity and complex host-parasite relationships. Many previous studies of malaria in birds have treated single study populations as spatially homogeneous with respect to the likelihood of transmission of malaria to hosts, and we have very little idea whether any spatial heterogeneity influences different malaria lineages similarly. Here, we report an analysis of variation in the prevalence and cytochrome b lineage distribution of avian malaria infection with respect to environmental and host factors, and their interactions, in a single blue tit (Cyanistes caeruleus) population. Of 11 Plasmodium and Haemoproteus cytochrome b lineages found in 997 breeding individuals, the three most numerous (pSGS1, pTURDUS1 and pBT7) were considered separately, in addition to analyses of all avian malaria lineages pooled. Our analyses revealed marked spatial differences in the prevalence and distribution of these lineages, with local prevalence of malaria within the population ranging from over 60% to less than 10%. In addition, we found several more complex patterns of prevalence with respect to local landscape features, host state, parasite genotype, and their interactions. We discuss the implications of such heterogeneity in parasite infection at a local scale for the study of the ecology and evolution of infectious diseases in natural populations. The increased resolution afforded by the combination of molecular genetic and geographical information systems (GIS) tools has the potential to provide many insights into the epidemiology, evolution and ecology of these parasites in the future.     

Plasmodium relictum infection and MHC diversity in the house sparrow (Passer domesticus)

Antagonistic coevolution between hosts and parasites has been proposed as a mechanism maintaining genetic diversity in both host and parasite populations. In particular, the high level of genetic diversity usually observed at the major histocompatibility complex (MHC) is generally thought to be maintained by parasite-driven selection. Among the possible ways through which parasites can maintain MHC diversity, diversifying selection has received relatively less attention. This hypothesis is based on the idea that parasites exert spatially variable selection pressures because of heterogeneity in parasite genetic structure, abundance or virulence. Variable selection pressures should select for different host allelic lineages resulting in population-specific associations between MHC alleles and risk of infection. In this study, we took advantage of a large survey of avian malaria in 13 populations of the house sparrow (Passer domesticus) to test this hypothesis. We found that (i) several MHC alleles were either associated with increased or decreased risk to be infected with Plasmodium relictum, (ii) the effects were population specific, and (iii) some alleles had antagonistic effects across populations. Overall, these results support the hypothesis that diversifying selection in space can maintain MHC variation and suggest a pattern of local adaptation where MHC alleles are selected at the local host population level.     

Genetic linkage and association analyses for trait mapping in Plasmodium falciparum

Genetic studies of Plasmodium falciparum laboratory crosses and field isolates have produced valuable insights into determinants of drug responses, antigenic variation, disease virulence, cellular development and population structures of these virulent human malaria parasites. Full-genome sequences and high-resolution haplotype maps of SNPs and microsatellites are now available for all 14 parasite chromosomes. Rapidly increasing genetic and genomic information on Plasmodium parasites, mosquitoes and humans will combine as a rich resource for new advances in our understanding of malaria, its transmission and its manifestations of disease.     

Genetic fingerprints of parasites causing severe malaria in a setting of low transmission in Sudan

In this study we intended to examine the extent of genetic diversity of Plasmodium falciparum parasites causing severe malaria (SM). For this purpose, 100 parasite isolates were obtained from patients with SM and uncomplicated malaria, from an area of low and unstable malaria transmission in Sudan. The diversity of infection (DOI) was estimated by relating the number of the different parasite genotypes that were detected to the total number of parasites that were genotyped (parasite population/subpopulation). We used different molecular markers individually (pfcrt-76, pfmr1-86, GLURP size and MSP2 family and size) and as a group to set a multilocus genetic profile for each parasite isolate. The DOI as estimated by MSP2 and GLURP was 0.553 and 0.435, respectively. However, combination of all four molecular markers (multilocus genetic profile) revealed a fingerprint pattern of genetic diversity with a DOI of 0.936, indicating that in SM infection, diversity is the rule and homogeny is the exception. Furthermore, our clinical data suggest that the virulence markers might also be more diverse than expected. In conclusion, the results are unexpected and overturn the assumption that parasites causing SM are a limited subpopulation of virulent parasites or of a clonal nature. However, it was more likely that there was a genetically unique parasite in each infection.     

Mitochondrial genome sequences support ancient population expansion in Plasmodium vivax

Examination of nucleotide diversity in 106 mitochondrial genomes of the most geographically widespread human malaria parasite, Plasmodium vivax, revealed a level of diversity similar to, but slightly higher than, that seen in the virulent human malaria parasite Plasmodium falciparum. The pairwise distribution of nucleotide differences among mitochondrial genome sequences supported the hypothesis that both these parasites underwent ancient population expansions. We estimated the age of the most recent common ancestor (MRCA) of the mitochondrial genomes of both P. vivax and P. falciparum at around 200,000-300,000 years ago. This is close to the previous estimates of the time of the human mitochondrial MRCA and the origin of modern Homo sapiens, consistent with the hypothesis that both these Plasmodium species were parasites of the hominid lineage before the origin of modern H. sapiens and that their population expansion coincided with the population expansion of their host.     

Faster clonal turnover in high‐infection habitats provides evidence for parasite‐mediated selection

According to the Red Queen hypothesis for sex, parasite‐mediated selection against common clones counterbalances the reproductive advantage of asexual lineages, which would otherwise outcompete sexual conspecifics. Such selection on the clonal population is expected to lead to a faster clonal turnover in habitats where selection by parasites is stronger. We tested this prediction by comparing the genetic structure of clonal and sexual populations of freshwater snail Potamopyrgus antipodarum between years 2003 and 2007 in three depth‐specific habitats in Lake Alexandrina (South Island, New Zealand). These habitats differ in the risk of infection by castrating trematodes and in the relative proportion of sexual individuals. As predicted, we found that the clonal structure changed significantly in shallow and mid‐water habitats, where prevalence of infection was high, but not in the deep habitat, where parasite prevalence was low. Additionally, we found that both clonal diversity and evenness of the asexual population declined in the shallow habitat. In contrast, the genetic structure (based on F–statistics) of the coexisting sexual population did not change, which suggests that the change in the clonal structure cannot be related to genetic changes in the sexual population. Finally, the frequency of sexuals had no effect on the diversity of the sympatric clonal population. Taken together, our results show a more rapid clonal turnover in high‐infection habitats, which gives support for the Red Queen hypothesis for sex.     

Genetic hypervariability of telomere-related sequences is associated with meiosis in Plasmodium falciparum.

Sequences related to those near chromosome telomeres in the human malaria parasite, Plasmodium falciparum, were extremely unstable during a genetic cross between two different clonal genotypes. Many progeny of the heterologous cross displayed telomere-homologous restriction fragments found in neither parent. A significant number of the new fragments resulted from rearrangements at chromosome-internal locations which were bounded by more complex tracts of DNA sequence. The same instability was not seen to arise during an inbreeding cross, nor during mitotic replication of parasites. Thus, a form of genetic hypervariability results from molecular events which occur during meiotic reduction and is apparent only in a cross between heterologous strains of parasite. Since other sequences were entirely stable under the same conditions, it appears that chromosome-internal blocks of telomeric sequences in the P. falciparum genome may designate conditionally unstable chromosomal domains. We discuss some potential implications of these findings for the population biology of P. falciparum.     

Genetic mapping in the human malaria parasite Plasmodium falciparum

The Plasmodium falciparum genome sequence has boosted hopes for a new era of malaria research and for the application of comprehensive molecular knowledge to disease control, but formidable obstacles remain: approximately 60% of the predicted P. falciparum proteins have no known functions or homologues, and most life cycle stages of this haploid eukaryotic parasite are relatively intractable to cultivation and biochemical manipulation. Genetic mapping based on high-resolution maps saturated with single-nucleotide polymorphisms or microsatellites is now providing effective strategies for discovering candidate genes determining important parasite phenotypes. Here we review classical linkage studies using laboratory crosses and population associations that are now amenable to genome-wide approaches and are revealing multiple candidate genes involved in complex drug responses. Moreover, mapping by linkage disequilibrium is practicable in cases where chromosomal segments flanking drug-selected genes have been preserved in populations during relatively recent P. falciparum evolution. We discuss the advantages and limitations of these various genetic mapping strategies, results from which offer complementary insights to those emerging from gene knockout experiments and/or high-throughput genomic technologies.     

The paradoxical population genetics of Plasmodium falciparum

Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies.     

Plasmodium falciparum: population genetic analysis by multilocus enzyme electrophoresis and other molecular markers.

Abderrazak, S. B., Oury, B, Lal, A. A., Bosseno, M.-F., Force-Barge, P., Dujardin, J.-P., Fandeur, T., Molez, J.-F., Kjellberg, F., Ayala, F. J., and Tibayrenc, M. 1999. Plasmodium falciparum: Population genetic analysis by multilocus enzyme electrophoresis and other molecular markers. Experimental Parasitology 92, 232-238. The population structure of Plasmodium falciparum, the agent of malignant malaria, is uncertain. We have analyzed multilocus enzyme electrophoresis (MLEE) polymorphisms at 7-12 gene loci in each of four populations (two populations in Burkina Faso, one in Sudan, one in Congo), plus one "cosmopolitan" sample consisting of parasite cultures from 15 distant localities in four different continents. We have also performed random amplified polymorphic DNA analysis (RAPD) and restriction fragment length polymorphism (RFLP) and characterized gene varia tion at four antigen genes in the Congo population. All genetic assays show abundant genetic variability in all populations analyzed. With the isoenzyme assays, strong linkage disequilibrium is apparent in at least two local populations, the Congo population and one population from Burkina Faso, as well as in the cosmopolitan sample, and less definitely in the other Burkina Faso population. However, no linkage disequilibrium is detected in the Congo population with the molecular assays. We failed to detect any nonrandom association between the different kinds of genetic markers; that is, MLEE with RAPD or RFLP, RAPD with RFLP, and so on. Although isoenzyme data show statistical departures from panmictic expectations, these results suggest that in the areas under survey, P. falciparum populations do not undergo predominant clonal evolution and show no clear-cut subdivisions, un like Trypanosoma cruzi, Leishmania sp., and other major parasitic species. We discuss the epidemiological and taxonomical significance of these results.