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Journal of Molecular and Genetic Medicine
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Malaria epidemiology: Insights from the genome of the malaria parasite

Alyssa E Barry*

Department of Medical Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010, USA

*Corresponding Author:
Alyssa Barry
Tel: +1 212 263 6765
Fax: +1 212 263 8116
Email: [email protected]

Received date: 23 November 2005; Revised date: 21 December 2005; Accepted date: 23 December 2005; Published date: 30 December 2005

Citation: Journal of Molecular and Genetic Medicine (2005), 1(2), 76-86

Copyright: © 2005 Alyssa E Barry, This is an open access article, published under the terms of the Licence for Users available at http://www.libpubmedia.co.uk/ MedJ/LicenceForUsers.pdf. This licence permits noncommercial use, distribution and reproduction of the article, provided the original work is appropriately acknowledged with correct citation details.

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Keywords

Malaria, Plasmodium, epidemiology, genomics, diversity, evolution, population genetics

Introduction

Malaria continues to be one of the most devastating infec-tious diseases of our time, rivaling HIV and tuberculosis as a killer disease in tropical and subtropical regions ((WHO, 2005), Figure 1). Around 3.2 billion people are at risk of malaria each year (WHO, 2005), with around 500 million people proceeding to clinical disease, and 2-3 million deaths occurring (Snow et al, 2005). Over 90% of these deaths occur in sub-Saharan Africa (WHO, 2005). The burden of morbidity and mortality is biased towards young children, not yet immune to clinical symptoms (Snow et al, 2005), and pregnant women where parasites are seques-tered in the placenta (Rowe and Kyes, 2004). Despite much suffering and many years of research there is no effective vaccine and drugs are either too expensive for the majority of people that are at risk of disease, or no longer effective due to extensive drug resistance of the malaria parasite. Hence, there is a shortage of effective interven-tions for malaria.

molecular-genetic-medicine-global-distribution-malaria

Figure 1. The global distribution of malaria in 2004 (WHO 2005). Reproduced from WHO website (WHO 2005; http://www.who.int/malaria/malariaendemiccountries.html); reprinted with permission.

The parasites that cause malarial disease are protozoan organisms that also infect many animal species including primates, lizards and birds. Four Plasmodium species are responsible for human malaria: P. falciparum, P. vivax, P. ovale and P. malariae. P. falciparum is the most virulent parasite, and is responsible for the majority of malaria-related mortality. It is found in all malaria endemic regions of the world and is the most common human malaria para-site in Africa (WHO, 2005). P. vivax is rarely found in Africa, but is the most common species outside Africa (Mendis et al, 2001; Carter and Mendis, 2002).

The malaria parasite has a complex lifecycle involving both asexual and sexual stages with obligatory phases in both humans and the female Anopheles mosquito (Box 1). For most of its lifecycle the parasite has a haploid genome, but following sexual reproduction in the mosquito it un-dergoes a brief period of diploidy. Here, recombination occurs with a high crossover rate (Su et al, 1999) resulting in the re-assortment of alleles. Recombination allows the generation of novel parasite genomes if the blood meal con-tains two or more genetically distinct clones. Out-crossing occurs commonly in regions of high Plasmodium transmis-sion (high rates of infection) such as Africa and Papua New Guinea (PNG) because there are many multiple infections (Babiker et al, 1994; Paul et al, 1995). This creates genetic diversity and provides opportunities for the dissemination of advantageous alleles among parasites of a population.

Epidemiology can be described as the study of disease patterns. Such studies can be applied to the control of that disease. The epidemiology of malaria is described as en-demic (stable) when a consistent pattern of Plasmodium transmission is found in humans over a number of years. Malaria epidemiology in areas of stable transmission is typified by an age dependent pattern of non-sterilizing immunity in the human host (Cattani et al, 1986; Molineaux, 1988). This is thought to be due to the gradual acquisition of antibodies to diverse parasite surface anti-gens (Bull et al, 1998). Stable malaria is generally charac-terized by a genetically diverse parasite population (or many novel genomes). At the other extreme, epidemic (unstable) malaria occurs when there is a large increase in the prevalence of cases at any period in time. In unstable malaria, parasites are generally less genetically diverse. This is due to clonal expansion of single genomes in the absence of immune pressure (Arez et al, 1999; Laserson et al, 1999; Hoffmann et al, 2003). Unstable malaria may also be associated with diverse parasites where infections have been able to persist sub-patently in the host between trans-mission seasons (Babiker, 1998). Globally, a spectrum of epidemiologies of malaria showing varying transmission intensities can be found (Molineaux, 1988; Gilles, 1993; Snow et al, 2005). Transmission differences were shown to occur within continents and within countries (Snow et al., 2005; Guerra et al., 2006, in press). Variations can also oc-cur locally, for example, in PNG, varying malaria epidemi-ology was observed between villages and even between households within the same village (Cattani et al, 1986).

A complex interplay between history, environment, vector, host and parasite has resulted in the varying patterns of malaria epidemiology across the globe. We are only just beginning to understand how parasite genetic diversity relates to these patterns. The advent of molecular genetic technologies combined with a complete genome sequence for P. falciparum (Gardner et al, 2002) has allowed the measurement of genetic diversity using a variety of mark-ers throughout the genome. In this article I will review the post-genomic epidemiology of malaria by discussing the Malaria Genome Project, and studies of genome wide di-versity, evolution and population genetics of the most studied human malaria parasites, P. falciparum and P. vivax. The impact of the Malaria Genome project on inves-tigations into two parasite survival characteristics that are issues for disease control, namely drug resistance and variant antigens will also be discussed. (For a review on malaria epidemiology, see Greenwood et al, 2005)

The Malaria Genome Project

The Malaria Genome Project began as an initiative in 1996 by a consortium of genome centers including the Sanger Institute, The Institute for Genomic Research (TIGR)/Naval Medical Research Centre and Stanford Uni-versity. The aim was to sequence the genome of the P. falciparum (clone 3D7). The majority of the genome was finished and annotated in 2002 (Gardner et al, 2002) and is now complete, with the exception of a small number of repetitive regions that have been difficult to sequence. The 14 chromosome nuclear genome was found to consist of 23 Mb of DNA sequence containing 5268 predicted genes (Gardner et al, 2002). P. falciparum also contains a mito-chondrial genome of approximately 6 kb, and a plastid ge-nome of 35 kb, as do the other malaria species. Approxi-mately 60% of the predicted genes did not match any se-quence in the public databases and were thus annotated as “hypothetical” proteins. This shows how much more we have to learn about the biology of this parasite, and the large potential for identifying new drug and vaccine targets.

All genome information is readily accessible online via the databases PlasmoDB (www.plasmodb.org) or GeneDB (www.genedb.org) (reviewed in Aslett et al, 2005). For each predicted gene a number of parameters were deter-mined using bioinformatic tools. These include homology-based functional annotations, protein motifs and domains. Queries can be made using keywords, regular expressions (a string of characters eg: a sequence motif) or by DNA or protein sequence homology using the Basic Local Align-ment Search Tool (BLAST) (Altschul et al, 1990). Ex-pressed sequence tags (ESTs), microarray (Bozdech et al, 2003; Le Roch et al, 2003) and proteomic (Florens et al, 2002; Lasonder et al, 2002) data have also been aligned to the P. falciparum genome to reveal the stage-specificity of each gene. Gene ontology (biological processes), paralogs (homologs within the same species), orthologs (homologs within different species), and syntenic regions with other species (conserved order of genes) can be viewed. Plas-moDB also shows the locations of the identified single nucleotide polymorphisms (SNPs) (Mu et al, 2002; Mu et al, 2005a). Researchers are able to contribute their own data to the databases to enhance these features.

More recently, the Sanger Institute, the Institute for Ge-nomic Research (TIGR) and the Broad Institute have been sequencing the genomes of a number of additional Plas-modium species including rodent and primate malaria parasites (Table 1). These include four additional P. falci-parum genomes: a fresh clinical isolate originating in Ghana; a well-studied clone, known as IT, from Brazil; and HB3 and Dd2, the parents of a genetic cross to map chloroquine resistance (Su et al, 1997). Clone 3D7 is as-sumed to be of African origin, although its precise origin was unknown because it was isolated from Amsterdam airport (Walliker et al, 1987). The additional genomes will complement the completed 3D7 genome, and reveal any loss of genetic material which is known to occur after long periods of parasite culture (Day et al, 1993). Sequencing of the P. vivax (clone SalI), genome is almost complete and the manuscript is due to be submitted in early 2006 (J. Carlton, personal communication), while the P. ovale and P. malariae genome projects are in the planning stages (M. Berriman, personal communication). Sequencing of additional non-human parasites will aid genetic studies in model organism parasites as well as to complete synteny maps revealing insights into genome structure and evolu-tion (Hall and Carlton, 2005). Comparative genomics of non-human primate species that are closely related to hu-man malaria species may reveal genetic traits associated with host switch and co-evolution.

Species Strain Host Genome sequence Institution Reference
P. berghei ANKA rodent African thicket 8X Sanger http://www.sanger.ac.uk/Projects/Protozoa
P. chabaudi AS rats African thicket 8X Sanger http://www.sanger.ac.uk/Projects/Protozoa
P. yoelii yoelii 17XNL rats 5X TIGR, NMRCSanger, TIGR, Stan- Carlton et al, 2002
P. falciparum 3D7 Ghana human 18X ford Gardner et al, 2002
P. falciparum clinical human 8X Sanger http://www.sanger.ac.uk/Projects/Protozoa
P. falciparum IT human 1X Sanger http://www.sanger.ac.uk/Projects/Protozoa
P. falciparum HB3 human 8X Broad Institute http://www.broad.mit.edu/seq/msc
P. falciparum Dd2 human in progress Broad Institute http://www.broad.mit.edu/seq/msc Carlton 2003;
P. vivax SalI human 10X TIGR http://www.tigr.org/tdb/e2k1/pva1/
P. malariae   human planned Sanger M. Berriman, personal communication
P. ovale   human planned Sanger M. Berriman, personal communication
P. knowlesi   Monkey 8X Sanger http://www.sanger.ac.uk/Projects/Protozoa
P. reichenowi Dennis Chimpanzee in progress Sanger http://www.sanger.ac.uk/Projects/Protozoa
P. gallinaceum   Bird 3X Sanger http://www.sanger.ac.uk/Projects/Protozoa

Table 1. Summary of Plasmodium genome projects

Genomic Diversity of Malaria Parasites

Genetic polymorphisms have been used as molecular markers to determine the evolution and the population genetics of the malaria parasite. Knowledge of the ge-nomic diversity of P. vivax is not as broad as that for P. falciparum. This is mostly due to the lack of a complete genome sequence, and an existing method for its in vitro culture. But as the sequencing of the P. vivax genome pro-ceeds, more studies will be possible. An investigation of the genetic diversity in P. ovale showed the existence of much variation with two major lineages (Win et al, 2004; reviewed in Collins and Jeffrey, 2005), however the analy-sis of polymorphisms in large stretches of genomic DNA are yet to be done for this species, and for P. malariae, so they will not be discussed further.

Genetic polymorphisms that have been used to study di-versity in the P. falciparum and P. vivax genomes fall into three major categories: (1) single nucleotide polymor-phisms (SNPs); (2) microsatellites and other repeats; and (3) indels.

SNPs

SNPs confer point mutations in the nucleotide sequence that may or may not encode amino acid polymorphisms. These are known as nonsynonymous (or amino acid) and synonymous polymorphisms respectively. In the genomes of both P. falciparum and P. vivax the pattern of SNPs is described as a mosaic, consisting of long stretches of monomorphic DNA interspersed with islands of high den-sity SNPs (Volkman et al, 2001; Mu et al, 2002; Volkman et al, 2002; Feng et al, 2003). The majority of genes are relatively monomorphic (few differences among ge-nomes), whilst only a few are highly polymorphic (many differences among genomes). Predominantly nonsynony-mous polymorphisms are found in P. falciparum genes (Rich et al, 1998). A study of chromosome 2 showed that the genes with many SNPs (both nonsynonymous and synonymous) contained transmembrane domains. This suggests that they are exported to the surface and that their abundance of SNPs is due to exposure to selection by the host immune system (Volkman et al, 2002). By mapping SNPs throughout the genome, novel antigens, that may be vaccine candidates, or other functionally important genes (under selection), may be discovered.

Microsatellites and other repeats

Microsatellites are small (1-4 base pair (bp)) tandem re-peats, while minisatellites can be classified as consisting of repeat units of more than 5 bp. Repeats diversify through the expansion and contraction of repeat arrays. They change at a faster rate than SNPs due to replication slippage (Su et al, 1999; Anderson et al, 2000a). Amongst even the most monomorphic P. falciparum sequences lay polymorphic microsatellites and minisatellites. Microsatel-lite polymorphisms are widespread throughout non-coding sequences of P. falciparum (Volkman et al, 2001). The majority of microsatellites consist of 1-3 bp TA repeats (e.g., T, TA or TAA), although some more complex re-peats have been found (Anderson et al, 2000b; Volkman et al, 2001; Mu et al, 2002). This may in part be a conse-quence of the ~ 80% AT rich genome of P. falciparum (Gardner et al, 2002). In contrast, the P. vivax genome contains around 55% AT nucleotides (Carlton, 2003) and polymorphic microsatellites appear to be less diverse (Leclerc et al, 2004) and less common (Feng et al, 2003; Leclerc et al, 2004). On the other hand, polymorphic minisatellites were found to be more abundant in the P. vivax genome than in P. falciparum which may also be a consequence of their different base compositions (Feng et al, 2003).

Indels

Indels are inserts or deletions of 1 or more base pairs in non-repetitive DNA sequence. A 2 nucleotide deletion, and 1 nucleotide insert were found over 4106 bp of se-quence in a study of introns of P. falciparum (Volkman et al, 2001). Only 3 single nucleotide indels were found in over 100 kb of P. vivax DNA, but these were observed within repetitive sequences (Feng et al, 2003).

Evolution of Malaria Parasites

Investigations into the origins and history of malaria para-sites have given insights into what conditions may have led to the origin and spread of disease. This was done through comparison with the natural population history of the Anophelene vector, humans (and our ancestors) and non-human primates. This knowledge may show how we could avoid similar situations in the future, and explain the complex epidemiology of present day malaria. Further-more, a parasite of ancient origin will be more genetically diverse and perhaps pose a greater challenge for control, whereas homogeneous parasite populations may be more equally susceptible to antimalarial interventions. Both P. falciparum and P. vivax have been successful at adapting to new environmental challenges. They contain polymor-phic surface antigen and drug resistance genes (see sec-tions below), and as discussed above P. falciparum has an abundance of polymorphic microsatellites (Su et al, 1999; Anderson et al, 2000a). This led to the assumption that malaria parasites were of ancient origin.

To determine the origin of an organism, the time to the most recent common ancestor (MRCA) can be calculated using SNPs among a few isolates with distinct global origins. However, the time to MRCA should be calculated using only synonymous polymorphisms because they are consid-ered to accumulate randomly, and are neutrally evolving (i.e. a type of molecular clock), whereas nonsynonymous polymorphisms are more likely to have accumulated under selection. Alternatively, other putatively neutral SNPs such as those within non-coding sequences or neutrally selected genes (eg: housekeeping genes) can be used.

P. falciparum

An examination of diversity in a collection of genes from P. falciparum revealed a high ratio of nonsynonymous to syn-onymous polymorphism (Rich et al, 1998). Using the syn-onymous polymorphisms, the time to MRCA was calculated to approximately 6,000 years ago. Similar studies suggested the MRCA was much more ancient at around 150-200,000 years (reviewed in Hughes and Verra, 2002). The authors examined mostly antigen and drug resistance encoding loci, known to be under selection, but that were available from the GenBank database prior to the genome project. It was there-fore possible that there were biased ratios of nonsynonymous to synonymous polymorphisms, and errors in the GenBank data (Barry et al, 2003). Further studies were carried out to clarify this paradox using genes under putatively neutral selection and careful sequencing procedures.

A study of the introns of putative housekeeping genes de-scribed a time to MRCA of 10,000-25,000 years (Volkman et al, 2001). In contrast, a further examination of coding and non-coding regions in selected and neutral genes across chromosome 3, predicted a much older MRCA of 100,000-180,000 years ago (Mu et al, 2002). The difference in MRCA estimates was partially resolved by analysis of the 6 kb mitochondrial genome. This confirmed P. falciparum was likely to be widespread in the human population for more than 50,000 years but had undergone a major recent expansion around 10,000 years ago (Joy et al, 2003).

A recent expansion is supported by human migration pat-terns and behavior (reviewed in Hume et al, 2003) and a time to MRCA of around 10,000 years, is coincident with the advent of agriculture and humans living in large popu-lations (Coluzzi, 1999; reviewed in Hartl et al, 2003). P. falciparum appears to have experienced this rapid expan-sion and spread around the world in parallel with its hu-man host and with the speciation of Anopheles mosquitoes (Coluzzi, 1999). Behavior changes of the human host may have contributed to an increase in parasite population size. For example, the development of large sedentary agricul-tural settlements could have allowed the expansion of host, vector, and consequently Plasmodium populations. A number of major bottlenecks that would reduce genetic diversity may have also occurred when the parasite adapted to the different species of Anopheles worldwide (Hartl et al, 2003; Hume et al, 2003; Joy et al, 2003).

P. vivax

P. vivax appears to have undergone a different mode of evolution to that of P. falciparum. The fact that P. vivax can relapse may have resulted in the wider distribution (outside Africa). The higher frequency of SNPs (Feng et al, 2003; Escalante et al, 2005) indicated that P. vivax would have an older MRCA (Escalante et al, 2005). It was previously suggested that the emergence of Duffy antigen negativity in humans resulted in the near-eradication of P. vivax from Africa (Miller et al, 1976). Two studies exam-ined SNPs in the mitochondrial DNA in P. vivax and ref-erence strains for primate malarias to investigate evolution in P. vivax (Jongwutiwes et al, 2005; Mu et al, 2005b). Both described a history of P. vivax consistent with an origin in Asia and horizontal transfer from Old World monkeys (Asian macaques) between 45,000-300,000 years ago. These data support an argument where P. vivax mi-grated into, not out-of Africa (Jongwutiwes et al, 2005; Mu et al, 2005b). An investigation of the circumsporozoite protein (CSP) gene suggested that host transfer occurred very recently between humans and New World monkeys (South American platyrrhines) and that it involved at least two independent events (Lim et al, 2005).

The P. vivax story still needs some resolution, through analysis of larger regions of genomic sequence as was done for P. falciparum, plus sequencing of more isolates from both human (including P. vivax isolates from Africa) and monkeys. Although, results from these studies show that changes in human behavior thousands of years ago contributed at least to the widespread expansion and dis-tribution of P. falciparum.

Population Genetics of P. Falciparum

Examining parasite populations in depth (many isolates) and comparing them to distant populations permits the analysis of the geographic structure (i.e., how populations from different parts of the world relate to each other). Population genetics can be used to predict and monitor the effects of disease interventions, especially if specific loci are monitored. Measuring the diversity gives an indication of the range (e.g., number and characteristics of alleles), organization (e.g., allele prevalences), outbreeding (e.g., random or self mating within the mosquito midgut) and natural history, since high levels of polymorphism would suggest a long history of the population. A reduction in these parameters following interventions would be indica-tors that the intervention was successful. Whereas, the increased prevalence of an allele in association with resis-tance (eg: drug resistance, see section below) would indi-cate that a new intervention should be implemented. Com-parisons among populations such as measuring gene flow (differentiation) could predict how quickly resistance may be spread to other populations, as well as its natural his-tory, such as whether it is the origin or a subpopulation of other populations. Of course, the calculation of population genetic parameters is much more complicated than out-lined here (Hartl and Clark, 1997).

To examine the worldwide population structure of P. fal-ciparum a limited number of studies have examined P. falciparum population genetics on a global or local level using haplotypes of putatively neutral microsatellites, pre-viously developed for defining recombination parameters and genetic crosses (Su et al, 1999), or they have used SNPs in nuclear genes (Mu et al, 2005a).

Global population structure

The first study to examine the global population structure of P. falciparum was performed using microsatellites (Anderson et al, 2000a), while a second used SNPs from chromosome 3 and transporter genes (Mu et al, 2005a). Parasite populations sampled included Africa, Asia, PNG, and Latin America. P. falciparum was found to have a com-plex global population structure. Diversity was correlated with the endemicity and transmission intensity of each popu-lation in both studies (Anderson et al, 2000a; Mu et al, 2005a) as were levels of linkage equilbrium (Anderson et al., 2000a) and recombination rates (Mu et al, 2005a). African parasites were the most diverse followed by PNG parasites. Asian parasites had a moderate level of diversity and Latin American parasites showed very low levels of diversity and high levels of linkage disequilibrium. Populations were found to be structured according to their continental origins (Anderson et al, 2000a; Mu et al, 2005a). There was some gene flow between Asian and PNG parasites when microsa-tellites were analyzed (Anderson et al, 2000a), but it was not possible to distinguish between them using the chromosome 3 SNPs (Mu et al, 2005a), demonstrating the importance of using a variety of genetic markers. Within Latin America, populations were significantly differentiated in the microsa-tellite study. Such differences may be attributed to multiple independent introductions of the parasite into Latin America with the African slave trade around 500 years ago (Anderson et al, 2000a).

Local population structure

The above-described studies generalize the levels of global population structure into trends observed in low (e.g., Asia and Latin America) and high transmission regions (eg: Africa and PNG). However, there was not extensive sam-pling within regions to examine populations in relatively close proximity to each other. It is not unusual to find mi-cro-variation with mosaic patterns of malaria epidemiol-ogy within regional or local areas, as measured by parasite prevalence and density (Cattani et al, 1986). This pre-sumably also affects the genetic structure of parasite popu-lations. Variation among different parasite populations in the Congo and Kenya reflect spatial population genetic heterogeneity and inbreeding in stable malarious areas (Durand et al, 2003, Razakanrainibe et al, 2005). Further studies are required to examine local population structure in endemic regions.

Local population genetic investigations have also been performed within countries with unstable malaria. Malaria control efforts resulted in a ten-fold decrease in the inci-dence of P. falciparum malaria in Malaysian Borneo from 1995-1999 (Singh and Cox-Singh, 2001). Population ge-netic parameters were measured in eight P. falciparum foci in two regional areas (three and five populations each) separated by a geographic barrier (Anthony et al., 2005). Seven populations showed evidence of clonal expansion with high levels of linkage disequilibrium. The remaining population showed high levels of diversity and no linkage disequilibrium. However, the time of sample collection may have biased the results for this latter population. Ma-laria control efforts in this region appear to have affected parasite diversity and the population genetic data suggests that it may be easier to maintain low prevalence (Anthony et al, 2005). A study in Brazil, described low levels of di-versity and significant linkage disequilibrium for three of five populations studied, and indicated a reduced recombi-nation rate due to inbreeding rather than epidemic expan-sion. The remaining two populations showed no linkage disequilibrium suggesting that, because resistance alleles are common throughout the region, there may be an in-creased spread of multilocus drug resistance phenotypes (Machado et al, 2004).

Levels of differentiation in Malaysian Borneo were corre-lated with distance between populations (Anthony et al, 2005). However, in the Brazilian Amazon, population ge-netic analysis of five parasite populations in three states showed no isolation by distance (Machado et al, 2004). The multiple origins of Latin American parasite popula-tions, described above, may be the reason for this disparity (Anderson et al, 2000a).

Drug Resistance

Monitoring the emergence and spread of drug resistance will influence drug policies so that public health bodies can take appropriate action. This may involve switching to a second line drug when resistance arises or the use of combination therapies (White, 1999). Drug resistance genes can be examined for the prevalence of mutations conferring resistance before, during and following the use of a drug in the community.

The potential for differential levels of drug resistance phe-notypes to be encoded by additional genes is well recog-nized. But at present major drug resistance markers have only been identified for chloroquine (P. falciparum chrloroquine resistance transporter (pfcrt)) and antifolates (dihyrofolate reductase (dhfr) and dihydropteroate syn-thase (dhps)) discussed below. Amplification of the mul-tidrug resistance transporter (Pfmdr1) has been implicated in the increased transport of drug from the parasite (re-viewed in Duraisingh and Cowman, 2005).

Chloroquine resistance (CQR)

Chloroquine resistant (CQR) parasites were originally dis-covered in Asia and Latin America in the late 1950’s and subsequently spread to all malaria endemic areas (Payne, 1987). This was disastrous for the control of malaria as chloroquine was the most effective, safe and cheap treat-ment available at the time. The mode of action of chloro-quine is not known and therefore an obvious target was not available to study the molecular mechanism for CQR. The range of CQR levels suggested that resistance was encoded by multiple genes, but genetic cross and association studies revealed the gene pfcrt to be a significant contributor (Su et al, 1997; Fidock et al, 2000). There are two known allelic variants of pfcrt, African/Asian and Latin American (Fidock et al, 2000). The Latin American pfcrt allele was also found in PNG which suggested an independent origin of CQR in PNG to that in Asia (Mehlotra et al, 2001). This was sur-prising as it was expected that the Asian allele would be found, because that there is gene flow between Asian and PNG parasites (Anderson et al, 2000).

The availability of the P. falciparum genome sequence al-lowed the CQR selective sweep to be studied in more detail. The genomic region surrounding pfcrt was examined by genotyping 15 flanking microsatellite loci spanning 200 kb, in 87 worldwide isolates from each of the major malaria endemic regions (Wootton et al, 2002). Microsatellite hap-lotypes were different for PNG and Latin American para-sites. This confirmed independent origins of CQR in these two locations. A third pfcrt allele was also found in other parts of Latin America suggesting an additional origin of CQR in the Americas. Furthermore, Asia and Africa shared a common microsatellite haplotype, but the Asian CQR haplotype was more diverse. This suggested that it spread from an origin in Asia to Africa. Together this data revealed that there were at least four origins of CQR world-wide (Wootton et al, 2002). The Asian haplotype has also been identified in the Amazon basin further highlighting the impact of travel in the spread of drug resistance (Vieira et al, 2004). A genome wide study also reported reduced allelic diversity on chromosomes 1, 5, 6, 7, 10 and 12 that were not associated with CQR, suggesting other selective sweeps in the global population of P. falciparum. The mo-lecular mechanism behind these putative sweeps is yet to be determined (Wootton et al, 2002).

Antifolate resistance

The antifolate drug combination sulfadoxine-pyrimethamine (SP) was introduced in the late 1970s-80s following the widespread failure of chloroquine. This drug disrupts the folate synthesis pathway of P. falciparum by targeting DHPS (sulfadoxine), and DHFR (pyrimethamine). Previously these two drugs had been used independently, with the use of sufonamides during World War II, and pyrimethamine in the 1950s. Conse-quently, point mutations arose in the genes encoding these enzymes and allowed the rapid development of resistance to the combination of the two drugs (Plowe et al, 1997; Wang et al, 1997; Sims et al, 1999). Single (S108N), dou-ble (N51I:S108N or C59R:S108N), triple (N51I:C59R:S108N or C59R:S108N:I164L) or quadruple (N51I:C59R:S108N:I164L) point mutations in the dhfr gene are responsible for increasing levels of resistance respectively (Plowe et al, 1997). Single (A437G) or double (S436F:A437G or A437G:K534E) mutations in the dhps gene result in a similar phenotype (Wu et al., 1996; Triglia et al., 1997; Triglia et al., 1998). By examining flaking microsatellite haplotypes in African parasites, three inde-pendent origins of dhfr double mutants (two for the N51I:S108N allele and one for the C59R:S108N allele), and one independent origin of the dhfr triple mutant (N51I:C59R:S108N) were found. The other dhfr triple mutant allele (C59R:S108N:I164L) was not found in Af-rica. For dhps, multiple origins of the single mutant allele were found in Africa, while only one origin of one of the double mutant alleles (A437G:534E) was identified (Roper et al, 2003). The dhfr triple mutant alleles found in Africa were shown to have originated in Asia where both triple mutant alleles and the quadruple mutant allele are common (Nair et al, 2003; Roper et al, 2004). The distri-bution of dhps and dhfr mutant alleles in South America indicated that SP resistance had a single origin (Cortese et al, 2002). Further examination of flanking microsatellites in the South American isolates is needed to ascertain whether these alleles also originated in Asia.

Genetic studies on the genes encoding both CQR and anti-folate resistance and the flanking genomic regions show that gene flow as a consequence of human migration rather than the emergence of new mutations are a major factor in spreading anti-malarial drug resistance. This has implica-tions for the implementation of drug policies. For example, introducing combination therapies may slow the evolution of resistance because multiple mutations do not occur as often as single mutations do (White, 1999; Mackinnon, 2005), thus appearing to be mainly spread by human mi-gration (Nair et al, 2003; Roper et al, 2004).

Drug Resistance

Monitoring the emergence and spread of drug resistance will influence drug policies so that public health bodies can take appropriate action. This may involve switching to a second line drug when resistance arises or the use of combination therapies (White, 1999). Drug resistance genes can be examined for the prevalence of mutations conferring resistance before, during and following the use of a drug in the community.

The potential for differential levels of drug resistance phe-notypes to be encoded by additional genes is well recog-nized. But at present major drug resistance markers have only been identified for chloroquine (P. falciparum chrloroquine resistance transporter (pfcrt)) and antifolates (dihyrofolate reductase (dhfr) and dihydropteroate syn-thase (dhps)) discussed below. Amplification of the mul-tidrug resistance transporter (Pfmdr1) has been implicated in the increased transport of drug from the parasite (re-viewed in Duraisingh and Cowman, 2005).

Chloroquine resistance (CQR)

Chloroquine resistant (CQR) parasites were originally dis-covered in Asia and Latin America in the late 1950’s and subsequently spread to all malaria endemic areas (Payne, 1987). This was disastrous for the control of malaria as chloroquine was the most effective, safe and cheap treat-ment available at the time. The mode of action of chloro-quine is not known and therefore an obvious target was not available to study the molecular mechanism for CQR. The range of CQR levels suggested that resistance was encoded by multiple genes, but genetic cross and association studies revealed the gene pfcrt to be a significant contributor (Su et al, 1997; Fidock et al, 2000). There are two known allelic variants of pfcrt, African/Asian and Latin American (Fidock et al, 2000). The Latin American pfcrt allele was also found in PNG which suggested an independent origin of CQR in PNG to that in Asia (Mehlotra et al, 2001). This was sur-prising as it was expected that the Asian allele would be found, because that there is gene flow between Asian and PNG parasites (Anderson et al, 2000).

The availability of the P. falciparum genome sequence al-lowed the CQR selective sweep to be studied in more detail. The genomic region surrounding pfcrt was examined by genotyping 15 flanking microsatellite loci spanning 200 kb, in 87 worldwide isolates from each of the major malaria endemic regions (Wootton et al, 2002). Microsatellite hap-lotypes were different for PNG and Latin American para-sites. This confirmed independent origins of CQR in these two locations. A third pfcrt allele was also found in other parts of Latin America suggesting an additional origin of CQR in the Americas. Furthermore, Asia and Africa shared a common microsatellite haplotype, but the Asian CQR haplotype was more diverse. This suggested that it spread from an origin in Asia to Africa. Together this data revealed that there were at least four origins of CQR world-wide (Wootton et al, 2002). The Asian haplotype has also been identified in the Amazon basin further highlighting the impact of travel in the spread of drug resistance (Vieira et al, 2004). A genome wide study also reported reduced allelic diversity on chromosomes 1, 5, 6, 7, 10 and 12 that were not associated with CQR, suggesting other selective sweeps in the global population of P. falciparum. The mo-lecular mechanism behind these putative sweeps is yet to be determined (Wootton et al, 2002).

Antifolate resistance

The antifolate drug combination sulfadoxine-pyrimethamine (SP) was introduced in the late 1970s-80s following the widespread failure of chloroquine. This drug disrupts the folate synthesis pathway of P. falciparum by targeting DHPS (sulfadoxine), and DHFR (pyrimethamine). Previously these two drugs had been used independently, with the use of sufonamides during World War II, and pyrimethamine in the 1950s. Conse-quently, point mutations arose in the genes encoding these enzymes and allowed the rapid development of resistance to the combination of the two drugs (Plowe et al, 1997; Wang et al, 1997; Sims et al, 1999). Single (S108N), dou-ble (N51I:S108N or C59R:S108N), triple (N51I:C59R:S108N or C59R:S108N:I164L) or quadruple (N51I:C59R:S108N:I164L) point mutations in the dhfr gene are responsible for increasing levels of resistance respectively (Plowe et al, 1997). Single (A437G) or double (S436F:A437G or A437G:K534E) mutations in the dhps gene result in a similar phenotype (Wu et al., 1996; Triglia et al., 1997; Triglia et al., 1998). By examining flaking microsatellite haplotypes in African parasites, three inde-pendent origins of dhfr double mutants (two for the N51I:S108N allele and one for the C59R:S108N allele), and one independent origin of the dhfr triple mutant (N51I:C59R:S108N) were found. The other dhfr triple mutant allele (C59R:S108N:I164L) was not found in Af-rica. For dhps, multiple origins of the single mutant allele were found in Africa, while only one origin of one of the double mutant alleles (A437G:534E) was identified (Roper et al, 2003). The dhfr triple mutant alleles found in Africa were shown to have originated in Asia where both triple mutant alleles and the quadruple mutant allele are common (Nair et al, 2003; Roper et al, 2004). The distri-bution of dhps and dhfr mutant alleles in South America indicated that SP resistance had a single origin (Cortese et al, 2002). Further examination of flanking microsatellites in the South American isolates is needed to ascertain whether these alleles also originated in Asia.

Genetic studies on the genes encoding both CQR and anti-folate resistance and the flanking genomic regions show that gene flow as a consequence of human migration rather than the emergence of new mutations are a major factor in spreading anti-malarial drug resistance. This has implica-tions for the implementation of drug policies. For example, introducing combination therapies may slow the evolution of resistance because multiple mutations do not occur as often as single mutations do (White, 1999; Mackinnon, 2005), thus appearing to be mainly spread by human mi-gration (Nair et al, 2003; Roper et al, 2004).

Variant Antigens

Parasites express antigens on their surface during the ex-tracellular stages (sporozoite, merozoite) and on the sur-face of the host cell during the intracellular stages (liver, intraerythrocytic and gametocyte stages). Such antigens are being investigated as vaccine candidates (reviewed in Ballou et al, 2004), but they are highly polymorphic due to diversifying selection by the host immune system. This has hampered efforts to develop an effective vaccine, but identifying all the variants in a population through popula-tion genetics (see section above) and including them in the vaccine preparation may show promise (Genton et al, 2002). Because they are so diverse these variant antigens can be used to determine the molecular epidemiology of malaria. For example they have been used to monitor mul-tiple infection rates, and to follow individual genomes throughout the course of an infection (reviewed in Greenwood, 2002).

Newly discovered antigen genes within the parasite ge-nome offers hope in the search for an effective malaria vaccine (reviewed in Doolan et al, 2003). Parallel ge-nomic technologies have proven successful in the search for novel vaccine candidates. For example, proteomic analysis of proteins cleaved from the surface of infected erythrocytes resulted in the discovery of novel surface proteins. This was achieved by mapping the protein se-quences obtained back to the genome sequence, followed by the examination of expression patterns using im-munofluorescence (Florens et al, 2004; Winter et al, 2005). Furthermore, the analysis of polymorphisms across whole chromosomes using oligonucleotide arrays pro-vided SNP maps across the genome (Volkman et al, 2002; Carret et al, 2005). Regions of high SNP density corre-lated with well known antigen genes in both P. falcipa-rum (Volkman et al, 2002; Carret et al, 2005) and P. vivax (Feng et al, 2003). Therefore, novel antigen genes may be discovered when this technology is expanded. In other studies, a motif was discovered that predicts export from the parasite during the asexual stages. Genome wide searches revealed around 400 genes within the P. falcipa-rum genome involved in remodeling of the host erythro-cyte, including 160 proteins not previously known to be exported (Hiller et al, 2004; Marti et al, 2004). Some of these may also be vaccine candidates if also found to be on the parasite or infected erythrocyte surface. Accumu-lating credentials from post-genomic technologies such as those described here have great utility in the search for new vaccine candidates.

The majority of well known P. falciparum surface anti-gens are encoded by single copy genes, with the exception of PfEMP1 and RIFIN, encoded by up to around 60 and around 177 genes respectively (Gardner et al., 2002). The PfEMP1 coat switches by differential expression of var genes, allowing antigenic variation and sequestration within the host to avoid detection by the host immune system (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). The Malaria Genome Project allowed an entire var gene repertoire to be described for the first time. Three major var gene groups (A, B and C) were defined, each having a specific structural and genomic organization (Kraemer & Smith, 2003; Lavstsen et al., 2003). Analysis of a small (~100 kb) region of the P. vivax genome re-vealed a multigene family (approximately 600 genes) that encode diverse immunogenic parasite surface antigens known as VIR (reviewed in del Portillo, 2004). The pub-lication of the P. vivax genome will reveal an entire vir repertoire that will uncover the structure and organization of this multigene family similar to that for the P. falcipa-rum var genes.

Concluding Remarks

Global populations of P. falciparum and P. vivax show signatures of their natural history in their genomes. Their genomes are composed of relatively monomorphic se-quences with highly polymorphic “islands” that are con-centrated at the telomeres (Volkman et al, 2002; Carret et al, 2005). Overlaying the genomes of the four additional P. falciparum genome sequences will further contribute to a genome wide map of genetic diversity within this species. Such genome wide polymorphism analyses have been carried out using comparative genome hybridization (CGH) microarrays (Volkman et al, 2002; Carret et al, 2005). Ex-amining the polymorphic regions of the genome in many isolates from distinct populations has already begun to un-cover the relationships between parasite genetic diversity and the epidemiology of malaria across the globe (Anderson et al., 2000; Mu et al., 2005a). These studies have shown that parasite populations display varying degrees of diver-sity and differentiation that reflect the evolutionary history of the parasite and its human host, and the epidemiology of malaria. Within localized regions, micro-variation was ob-served (Durand et al., 2003, Razakanrainibe et al. 2005), but the extent of this needs further investigation. Equally impor-tant to study, are unusually monomorphic regions. These indicate selective sweeps due to linkage disequilibrium, such as the CQR locus discussed above. A large-scale SNP mapping project known as HapMap is also currently under-way to identify regions of linkage disequilibrium across the genome (http://www.broad.mit.edu/infect). Combining this information with genome wide functional annotations may thereby enhance possibilities of finding novel drug and vac-cine candidates, and the genes associated with specific phe-notypes such as drug resistance and virulence.

Descriptions of the genomic diversity, evolution and popu-lation genetics of P. falciparum using SNPs could not have been done without the large regions of annotated genomic sequence provided by the Malaria Genome Project. It will now be necessary to carefully monitor each distinct parasite population as disease interventions (eg: drugs and vaccines) are being applied, to monitor and predict outcomes. We now have a wide-range of polymorphic markers for P. falcipa-rum with which to do this, and genome wide markers for P. vivax are imminent. Further investigations into the genomic diversity, evolution and population genetics of the other human malaria parasites will be possible when their genome projects are complete.

In addition to the parasite genomes described, the entire human (HGPConsortium, 2004) and the African mosquito vector, Anopheles gambiae (Holt et al, 2002) genomes have been sequenced. There is now great opportunity for investigations into host-parasite interactions and co-evolution (Hoffman et al, 2002). The epidemiology of ma-laria has been well defined using traditional tools such as the microscope, but now we have the opportunity of exam-ining each species on an entirely new level. The comple-tion of the P. falciparum genome has undoubtedly resulted in a new approach to malaria epidemiology by producing the molecular tools to map, characterize and monitor its genes in natural populations of parasites. Monitoring genes that are important to parasite survival as well as neutral markers will assist in implementing the appropriate dis-ease control measures.

Acknowledgements

I thank colleagues at New York University School of Medicine for helpful comments in the preparation of the manuscript: Karen Day, Freya Fowkes and Rebecca Staf-ford-Allen. I would also like to thank two anonymous refe-rees for their suggestions to improve the manuscript.

Statement of Competing Interests

The author declared no competing interests.

List of Abbreviations

PNG; Papua New Guinea

BLAST; Basic Local Alignment Search Tool

SNP; single nucleotide polymorphism

MRCA; most recent common ancestor

CQR; chloroquine resistance

Pfcrt; Plasmodium falciparum chloroquine resistance transporter

SP; Sulfadoxine-pyrimethamine

dhfr; Dihydrofolate reductase

dhps; Dihydropterate synthase

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Clinical Journals

Datta A

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Engineering Journals

James Franklin

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Food & Nutrition Journals

Katie Wilson

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General Science

Andrea Jason

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Genetics & Molecular Biology Journals

Anna Melissa

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Immunology & Microbiology Journals

David Gorantl

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Materials Science Journals

Rachle Green

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1-702-714-7001Extn: 9039

Nursing & Health Care Journals

Stephanie Skinner

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1-702-714-7001Extn: 9039

Medical Journals

Nimmi Anna

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1-702-714-7001Extn: 9038

Neuroscience & Psychology Journals

Nathan T

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Pharmaceutical Sciences Journals

Ann Jose

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Social & Political Science Journals

Steve Harry

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