Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin resistance.

Malaria is a protozoan disease transmitted by the bite of the Anopheles mosquito. Among five species that can infect humans, Plasmodium falciparum is responsible for the most severe human malaria. Resistance of P. falciparum to chloroquine and pyrimethamine/sulfadoxine, conventionally used antimalarial drugs, is already widely distributed in many endemic areas. As a result, artemisinin-based combination therapies have been rapidly and widely adopted as first-line antimalarial treatments since the mid-2000s. Recent population and evolutionary genetic analyses have proven that the geographic origins of parasite lineages resistant to the conventional drugs are considerably limited. Almost all resistance emerged from either Southeast Asia or South America. The Greater Mekong subregion in Southeast Asia is probably the most alarming source of resistance, from which P. falciparum resistant to chloroquine and pyrimethamine/sulfadoxine dispersed to Africa. The emergence of artemisinin resistance has also recently been confirmed in the Greater Mekong. The WHO Global Malaria Programme has recently launched a "Global Plan for Artemisinin Resistance Containment," which aims to prevent the spread of artemisinin resistance while also stopping the emergence of novel resistance. However, an inadequate understanding of a mechanism of artemisinin resistance and the lack of reliable genetic markers to monitor artemisinin resistance make it difficult to survey the spread of resistance. Elucidation of such markers would substantially contribute to the design of an effective policy for the containment of artemisinin resistance.

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5.

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS=0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (Fst) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants.

Limited geographical origin and global spread of sulfadoxine-resistant dhps alleles in Plasmodium falciparum populations.

BACKGROUND: Plasmodium falciparum malaria resistant to chloroquine and pyrimethamine originated in limited foci and migrated to Africa. It remains unresolved whether P. falciparum resistance to sulfadoxine, which is conferred by mutations in dihydropteroate synthase (DHPS), evolved following a similar pattern. METHODS: The dhps locus of 893 P. falciparum isolates from 12 countries in Asia, the Pacific Islands, Africa, and South America was sequenced. Haplotypes of 6 microsatellite loci flanking the dhps locus were determined to define the genetic relationships among sulfadoxine-resistant lineages. RESULTS: Six distinct sulfadoxine-resistant lineages were identified. Highly resistant lineages appear to have originated only in Southeast Asia and South America. Two resistant lineages found throughout Southeast Asia have been introduced to East Africa, where they appear to have spread. CONCLUSIONS: The infrequent selection of parasites highly resistant to sulfadoxine and the subsequent migration of resistant lineages from Asia to Africa are similar to the patterns observed in chloroquine and pyrimethamine resistance. These findings strongly suggest that the global migration of resistant parasites has played a decisive role in the establishment of drug-resistant P. falciparum parasites, and that similar patterns may be anticipated for the spread of artemisinin resistance.

Spread and evolution of Plasmodium falciparum drug resistance.

Worldwide spread of Plasmodium falciparum drug resistance to conventional antimalarials, chloroquine and sulfadoxine/pyrimethamine, has been imposing a serious public health problem in many endemic regions. Recent discovery of drug resistance-associated genes, pfcrt, pfmdr1, dhfr, and dhps, and applications of microsatellite markers flanking the genes have revealed the evolution of parasite resistance to these antimalarials and the geographical spread of drug resistance. Here, we review our recent knowledge of the evolution and spread of parasite resistance to chloroquine and sulfadoxine/pyrimethamine. In both antimalarials, resistance appears to be largely explained by the invasion of limited resistant lineages to many endemic regions. However, multiple, indigenous evolutionary origins of resistant lineages have also been demonstrated. Further molecular evolutionary and population genetic approaches will greatly facilitate our understanding of the evolution and spread of parasite drug resistance, and will contribute to developing strategies for better control of malaria.

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.

Allelic diversity at the Merozoite surface protein-1 (MSP-1) locus in natural Plasmodium falciparum populations: a brief overview

The merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum codes for a major asexual blood-stage antigen currently proposed as a major malaria vaccine candidate. The protein, however, shows extensive polymorphism, which may compromise its use in sub-unit vaccines. Here we compare the patterns of allelic diversity at the MSP-1 locus in wild isolates from three epidemiologically distinct malaria-endemic areas: the hypoendemic southwestern Brazilian Amazon (n=54), the mesoendemic southern Vietnam (n=238) and the holoendemic northern Tanzania (n=79). Fragments of the variable blocks 2, 4a, 4b and 6 or 10 of this single-copy gene were amplified by the polymerase chain reaction, and 24 MSP-1 gene types were defined as unique combinations of allelic types in each variable block. Ten different MSP-1 types were identified in Brazil, 23 in Vietnam and 13 in Tanzania. The proportion of genetically mixed infections (isolates with carrying more one MSP-1 version) ranged from 39 per cent in Brazil to 44 per cent in Vietnam and 60 per cent in Tanzania. The vast majority (90 per cent) of the typed parasite populations from Brazil and Tanzania belonged to the same seven most frequent MSP-1 gene types. In contrast, these seven types corresponded to only 61 per cent of the typed parasite populations from Vietnam. Non-random associations were found between allelic types in blocks 4a and 6 among Vietnamese isolates, the same pattern being observed in independent studies performed in 1994, 1995 and 1996. These results suggest that MSP-1 is under selective pressure in the local parasite population. Nevertheless, the finding that similar MSP-1 type frequencies were found in 1994 and 1996 argues against the prominence of short-term frequency-dependent immune selection of MSP-1 polymorphisms. Non-random associations between MSP-1 allelic types, however, were not detected among isolates from Brazil and Tanzania. A preliminary analysis of the distribution od MSP-1 gene types per host among isolates from Tanzania, but not among those from Brazil and Vietnam, shows significant deviation from that expected under the null hypothesis of independent distribution of parasites carrying different gene types in the human hosts. Some epidemiological consequences of these findings are discussed.