Plasmodium vivax and Plasmodium falciparum at the crossroads of exchange among islands in Vanuatu: implications for malaria elimination strategies.

Understanding the transmission and movement of Plasmodium parasites is crucial for malaria elimination and prevention of resurgence. Located at the limit of malaria transmission in the Pacific, Vanuatu is an ideal candidate for elimination programs due to low endemicity and the isolated nature of its island setting. We analyzed the variation in the merozoite surface protein 1 (msp1) and the circumsporozoite protein (csp) of P. falciparum and P. vivax populations to examine the patterns of gene flow and population structures among seven sites on five islands in Vanuatu. Genetic diversity was in general higher in P. vivax than P. falciparum from the same site. In P. vivax, high genetic diversity was likely maintained by greater extent of gene flow among sites and among islands. Consistent with the different patterns of gene flow, the proportion of genetic variance found among islands was substantially higher in P. falciparum (28.81-31.23%) than in P. vivax (-0.53-3.99%). Our data suggest that the current island-by-island malaria elimination strategy in Vanuatu, while adequate for P. falciparum elimination, might need to be complemented with more centrally integrated measures to control P. vivax movement across islands.

Plasmodium falciparum accompanied the human expansion out of Africa.

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

Phylogeny of Asian primate malaria parasites inferred from apicoplast genome-encoded genes with special emphasis on the positions of Plasmodium vivax and P. fragile.

Phylogenetic analyses of several marker genes have previously shown that Asian primate Plasmodium species (malaria parasites) were monophyletic including Plasmodium vivax, one of the four malaria parasites that infect humans. However, except for the presence of a few established groupings, phylogenetic relationships among the Asian primate Plasmodium species + P. vivax group have neither been clearly resolved with confident statistical supports, nor the closest relative to P. vivax was elucidated. Since comparative biological studies between P. vivax and its closest relative would provide valuable information on immunopathogenesity of vivax malaria, the phylogenetic positions of P. vivax in the clade comprised of Asian primate Plasmodium species are crucial. In order to clarify the phylogeny and evolution of Asian primate Plasmodium species including P. vivax, we obtained sequences of apicoplast genome-encoded genes for small subunit rRNA (SSUrRNA), large subunit rRNA (LSUrRNA), and caseinolytic protease C (ClpC) from 10 Plasmodium species: P. vivax, P. coatneyi, P. cynomolgi, P. fieldi, P. fragile, P. hylobati, P .inui, P. knowlesi, P. simiovale, and P. gonderi. Together with published sequences of apicoplast genome-encoded elongation factor Tu (EF-Tu) from these species, we performed phylogenetic analyses of a combined 4-gene data set using P. gonderi, an African old world monkey parasite, as an outgroup. The ML phylogeny based on a 'concatenate model' for combining information of the 4 genes clearly revealed close relationships between P. vivax and P. cynomolgi and monophyly of P. fragile with the P. coatneyi/P. knowlesi clade. When 'separate' models were assumed for combining phylogenetic information from the 4 genes that were independently analyzed, the support for the P. vivax/P. cynomolgi clade was substantially decreased, but the monophyly of P. fragile with the P. coatneyi/P. knowlesi clade was still robustly confirmed. The present analyses place P. fragile in a position that is incongruent with the early branching status of P. fragile amongst P-vivax-related primate Plasmodium species propose by Escalante et al. (Proc Natl Acad Sci USA 2005 102: 1980).

Recent independent evolution of msp1 polymorphism in Plasmodium vivax and related simian malaria parasites.

The Plasmodium MSP-1 is a promising malaria vaccine candidate. However, the highly polymorphic nature of the MSP-1 gene (msp1) presents a potential obstacle for effective vaccine development. To investigate the evolutionary history of msp1 polymorphism in P. vivax, we construct phylogenetic trees of msp1 from P. vivax and related monkey malaria parasite species. All P. vivax msp1 alleles cluster in the P. vivax lineage and are not distributed among other species. Similarly, all P. cynomolgi msp1 alleles cluster in the P. cynomolgi lineage. This suggests that, in contrast to presumed ancient origin of P. falciparum msp1 polymorphism, the origin of P. vivax msp1 polymorphism is relatively recent. We observed positive selection in the P. vivax lineage but not in P. cynomolgi. Also, positive selection acts on different regions of msp1 in P. vivax and P. falciparum. This study shows that the evolutionary history of msp1 differs greatly among parasite lineages.

Allelic dimorphism-associated restriction of recombination in Plasmodium falciparum msp1.

Allelic dimorphism is a characteristic feature of the Plasmodium falciparum msp1 gene encoding the merozoite surface protein 1, a strong malaria vaccine candidate. Meiotic recombination is a major mechanism for the generation of msp1 allelic diversity. Potential recombination sites have previously been mapped to specific regions within msp1 (a 5' 1-kb region and a 3' 0.4-kb region) with no evidence for recombination events in a central 3.5-kb region. However, evidence for the lack of recombination events is circumstantial and inconclusive because the number of msp1 sequences analysed is limited, and the frequency of recombination events has not been addressed previously in a high transmission area, where the frequency of meiotic recombination is expected to be high. In the present study, we have mapped potential allelic recombination sites in 34 full-length msp1 sequences, including 24 new sequences, from various geographic origins. We also investigated recombination events in blocks 6 to 16 by population genetic analysis of P. falciparum populations in Tanzania, where malaria transmission is intense. The results clearly provide no evidence of recombination events occurring between the two major msp1 allelic types, K1-type and Mad20-type, in the central region, but do show recombination events occurring throughout the entire gene within sequences of the Mad20-type. Thus, the present study indicates that allelic dimorphism of msp1 greatly affects inter-allelic recombination events, highlighting a unique feature of allelic diversity of P. falciparum msp1.

High frequency of recombination-driven allelic diversity and temporal variation of Plasmodium falciparum msp1 in Tanzania.

A major mechanism for the generation allelic diversity in the Plasmodium falciparum msp1 gene is meiotic recombination in the Anopheles mosquito. The frequency of recombination events is dependent on the intensity of transmission. Herein we investigate the frequency of recombination-driven allelic diversity and temporal variation of msp1 in Rufiji, eastern coastal Tanzania, where malaria transmission is intense. We identified 5' recombinant types, 3' sequence types, and msp1 haplotypes (unique associations of 5' recombinant types and 3' sequence types) to measure the extent and temporal variation of msp1 allelic diversity. The results show that msp1 haplotype diversity is higher in Tanzania as compared with areas with lower transmission rates. The frequencies of individual polymorphic regions/sites remained stable during the study period. However, the frequency distribution of msp1 haplotypes varied between 1993 and 1998. These results suggest that frequent recombination events between msp1 alleles intermittently generate novel alleles in high transmission areas.

Allelic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum on Palawan Island, the Philippines.

Allelic diversity of the Plasmodium falciparum merozoite surface protein 1 gene (msp1) is mainly generated by meiotic recombination at the mosquito stage. We investigated recombination-based allelic diversity of msp1 in a P. falciparum population from Palawan Island, the Philippines, where malaria transmission is moderate. We identified the 5' recombinant types, 3' sequence types and msp1 haplotypes (unique combinations of 5' recombinant type and 3' sequence type), and compared them with those of P. falciparum from the Solomon Islands, where malaria transmission is high. The mean number of 5' recombinant types per patient in Palawan was 1.44, which is comparable to the number for the Solomon Islands (1.41). The Palawan parasite population had 15 msp1 haplotypes, whereas the Solomon Islands population had only 8 haplotypes. The Palawan population showed strong linkage disequilibrium between polymorphic blocks/sites within msp1, which is comparable to the results for the Solomon Islands. These findings support our hypothesis that the extent of allelic diversity of msp1 is determined not only by the transmission intensity but also by the number of msp1 alleles prevalent in the local parasite population and the extent of mixed-allele infections. Contribution of a high prevalence of the chloroquine (CQ)-sensitive allele of P. falciparum CQ resistance transporter (pfcrt) to the relatively high msp1 diversity in the Palawan population is discussed.

Limited allelic diversity of Plasmodium falciparum merozoite surface protein 1 gene from populations in the Solomon Islands.

Meiotic recombination generates allelic diversity in the Plasmodium falciparum merozoite surface protein 1 (msp1) gene. In this study, we monitored recombination-based diversity of msp1 in Guadalcanal, the Solomon Islands, where malaria transmission is high. We identified 5' recombinant types, 3' sequence types, and msp1 haplotypes (unique associations of 5' recombinant types and 3' sequence types), and compared them with those from areas of low transmission in Thailand and Vanuatu. The mean number of 5' recombinant types per person (multiplicity) was lower in Guadalcanal than in Thailand. Guadalcanal populations had 6-8 msp1 haplotypes; the numbers are comparable to Vanuatu but much lower than in Thailand. There were marked geographic differences in distribution of msp1 haplo-types. Linkage disequilibrium in msp1 was stronger in Guadalcanal than in Thailand, suggesting limited recombination events in the Solomon Islands. We suggest that the frequency of recombination events in msp1 is determined not only by transmission intensity but by the number of msp1 alleles prevalent and multiplicity of infections.

Relative frequencies of polymorphisms of variation in Block 2 repeats and 5' recombinant types of Plasmodium falciparum msp1 alleles.

The mechanisms producing the genetic polymorphism at Plasmodium falciparum merozoite surface antigen-1 locus (pfmsp1) include the insertion and deletion of the different type of dimorphic Block 2 9-nucleotide repeat units as well as the intragenic recombination. To study relative occurrence frequencies of these two distinct mechanisms, we have developed a sensitive PCR strategy to identify both 5' recombinant types and the number of Block 2 repeats from the same sample. This method can specifically detect the target 5' recombinant type (Blocks 2-6) at the sensitivity of 1-4 copies of the pfmsp1. Applying the new method to field isolates from the Solomon Islands enabled us to identify six different 5' recombinant types and variation in Block 2 repeat number in three of them, thus distinguishing 10 different alleles. Distribution of these alleles in local three villages in the study area suggests that frequencies of variation in the number of Block 2 9-bp repeats and recombination events within Blocks 2-6 are mutually independent and the frequency of repeat variation is relatively high as compared to that of recombination events at the pfmsp1 locus in P. falciparum populations from the Solomon Islands.

Genetic distance in housekeeping genes between Plasmodium falciparum and Plasmodium reichenowi and within P. falciparum.

The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca2+ -ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672 +/- 0.0088 and 0.0011 +/- 0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30 +/- 5.40) x 10(4) and (11.62 +/- 7.56) x 10(4) years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively.

Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus.

The organization and allelic recombination of the merozoite surface protein-1 gene of Plasmodium vivax (PvMsp-1), the most widely prevalent human malaria parasite, were evaluated in complete nucleotide sequences of 40 isolates from various geographic areas. Alignment of 31 distinct alleles revealed the mosaic organization of PvMsp-1, consisting of seven interallele conserved blocks flanked by six variable blocks. The variable blocks showed extensive variation in repeats and nonrepeat unique sequences. Numerous recombination sites were distributed throughout PvMsp-1, in both conserved blocks and variable block unique sequences, and the distribution was not uniform. Heterozygosity of PvMsp-1 alleles was higher in Asia (0.953 +/- 0.009) than in Brazil (0.813 +/- 0.047). No identical alleles were shared between Asia and Brazil, whereas all but one variable block nonrepeat sequence found in Brazil occurred in Asia. These observations suggest that P. vivax populations in Asia are ancestral to Brazilian populations, and that PvMsp-1 has heterogeneity in frequency of allelic recombination events. Recurrent origins of new PvMsp-1 alleles by repeated recombination events were supported by a rapid decline in linkage disequilibrium between pairs of synonymous sites with increasing nucleotide distance, with little linkage disequilibrium at a distance of over 3 kb in a P. vivax population from Thailand, evidence for an effectively high recombination rate of the parasite. Meanwhile, highly reduced nucleotide diversity was noted in a region encoding the 19-kDa C-terminal epidermal growth factor-like domain of merozoite surface protein-1, a vaccine candidate.