Selective sweeps and genetic lineages of Plasmodium falciparum drug -resistant alleles in Ghana.

BACKGROUND: In 2005, Ghana adopted artemisinin-based combination therapy (ACT) for primary treatment of falciparum malaria. A comprehensive study of the drug-resistance-associated mutations and their genetic lineages will lead to a better understanding of the evolution of antimalarial drug resistance in this region. METHODS: The pfcrt, pfmdr1, dhps, and dhfr mutations associated with chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) resistance and the microsatellite loci flanking these genes were genotyped in Plasmodium falciparum isolates from Ghana. RESULTS: The prevalence of mutations associated with both CQ and SP resistance was high in Ghana. However, we observed a decrease in prevalence of the pfcrt K76T mutation in northern Ghana after the change in drug policy from CQ to ACT. Analysis of genetic diversity and differentiation at microsatellite loci flanking all 4 genes indicated that they have been under strong selection, because of CQ and SP use. The triple-mutant pfcrt and dhfr alleles in Ghana were derived from Southeast Asia, whereas the double-mutant dhfr, dhps, and pfmdr1 alleles were of African lineage. CONCLUSION: Because of the possible role of pfmdr1 in amodiaquine and mefloquine resistance, demonstrating selection on pfmdr1 and defining lineages of resistant alleles in an African population holds great importance.

Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

Assessing how natural selection, negative or positive, operates on genes with low polymorphism is challenging. We investigated the genetic diversity of orthologous genes encoding the rhoptry-associated protein 1 (RAP-1), a low polymorphic protein of malarial parasites that is involved in erythrocyte invasion. We applied evolutionary genetic methods to study the polymorphism in RAP-1 from Plasmodium falciparum (n=32) and Plasmodium vivax (n=6), the two parasites responsible for most human malaria morbidity and mortality, as well as RAP-1 orthologous in closely related malarial species found in non-human primates (NHPs). Overall, genes encoding RAP-1 are highly conserved in all Plasmodium spp. included in this investigation. We found no evidence for natural selection, positive or negative, acting on the gene encoding RAP-1 in P. falciparum or P. vivax. However, we found evidence that the orthologous genes in non-human primate parasites (Plasmodium cynomolgi, Plasmodium inui, and Plasmodium knowlesi) are under purifying (negative) selection. We discuss the importance of considering negative selection while studying genes encoding proteins with low polymorphism and how selective pressures may differ among orthologous genes in closely related malarial parasites species.

A comparative study of the genetic diversity of the 42kDa fragment of the merozoite surface protein 1 in Plasmodium falciparum and P. vivax.

We investigated the genetic diversity of the 42kDa fragment of the merozoite surface protein 1 (MSP-1) antigen in Plasmodium falciparum and P. vivax, as well as in non-human primate malarial parasites. This fragment undergoes a proteolytic cleavage generating two fragments of 19kDa (MSP-1(19)) and 33kDa (MSP-1(33)) that are critical in erythrocyte invasion. We found that overall the MSP-1(33) fragment exhibits greater genetic diversity than the MSP-1(19) regardless of the species. We have found evidence for positive natural selection only in the human malaria parasites by comparing the rate of non-synonymous versus synonymous substitutions. In addition, we found clear differences between the two major human malaria parasites. In the case of P. falciparum, positive natural selection is acting on the MSP-1(19) region while the MSP-1(33) is neutral or under purifying selection. The opposite pattern was observed in P. vivax. Our results suggest different roles of this antigen in the host-parasite immune interaction in each of the major human malarial parasites.

Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum.

A pyrosequencing protocol was developed as a rapid and reliable method to identify the mutations of the dhfr and dhps genes of Plasmodium falciparum that are associated with antifolate resistance. The accuracy and specificity of this method were tested using six laboratory-cultured P. falciparum isolates harboring known single nucleotide polymorphisms (SNPs) in the genes dhfr (codons 50, 51, 59, 108, and 164) and dhps (codons 436, 437, 540, 581, and 613). The lowest threshold for detection of all the SNPs tested by pyrosequencing was the equivalent of two to four parasite genomes. Also, this method was highly specific for P. falciparum, as it did not amplify any DNA products from the other species of human malaria parasites. We also mixed wild-type and mutant-type parasite DNAs in various proportions to determine how pyrosequencing, restriction fragment length polymorphism (RFLP), and direct conventional sequencing (for dhfr) compared with each other in detecting different SNPs in the mixture. In general, pyrosequencing and RFLP showed comparable sensitivities in detecting most of the SNPs in dhfr except for the 164L mutation, which required at least twice the amount of DNA for pyroseqencing as for RFLP. For detecting SNPs in dhps, pyrosequencing was slightly more sensitive than RFLP and direct sequencing. Overall, pyrosequencing was faster and less expensive than either RFLP or direct sequencing. Thus, pyrosequencing is a practical alternative method that can be used in a high-throughput format for molecular surveillance of antimalarial-drug resistance.

Antifolate resistance in Plasmodium falciparum: multiple origins and identification of novel dhfr alleles.

BACKGROUND: Sulfadoxine-pyrimethamine has been widely used as first-line therapy for uncomplicated malaria throughout sub-Saharan Africa. Recent studies conducted in Asia and Africa suggest the triple-mutant dhfr genotype (51I/59R/108N) may have been generated as a single event in Southeast Asia, with subsequent spread of the single lineage to the African continent, but this hypothesis needs further validation. METHODS: Direct sequencing of polymerase chain reaction (PCR) products, pyrosequencing, and cloning of PCR products were utilized to identify mutations in dhfr. To investigate the evolutionary history of dhfr alleles, we assayed microsatellite loci flanking dhfr along chromosome 4. RESULTS: A total of 15 of 479 samples from western Kenya showed the presence of I164L, in 5 different genotypes. We document C50R in 2 of our samples. Using microsatellite markers, we show 2 haplotypes for both the 51I/108N/164L and 51I/59R/108N/164L genotypes. Our results also show multiple lineages for the triple-mutant dhfr genotype in Africa. CONCLUSIONS: These findings highlight the importance of local characterization of alleles before molecular surveillance of drug-resistant alleles is considered in different endemic settings and populations.

A monkey's tale: the origin of Plasmodium vivax as a human malaria parasite.

The high prevalence of Duffy negativity (lack of the Duffy blood group antigen) among human populations in sub-Saharan Africa has been used to argue that Plasmodium vivax originated on that continent. Here, we investigate the phylogenetic relationships among 10 species of Plasmodium that infect primates by using three genes, two nuclear (beta-tubulin and cell division cycle 2) and a gene from the plastid genome (the elongation factor Tu). We find compelling evidence that P. vivax is derived from a species that inhabited macaques in Southeast Asia. Specifically, those phylogenies that include P. vivax as an ancient lineage from which all of the macaque parasites could originate are significantly less likely to explain the data. We estimate the time to the most recent common ancestor at four neutral gene loci from Asian and South American isolates (a minimum sample of seven isolates per locus). Our analysis estimates that the extant populations of P. vivax originated between 45,680 and 81,607 years ago. The phylogeny and the estimated time frame for the origination of current P. vivax populations are consistent with an "out of Asia" origin for P. vivax as hominoid parasite. The current debate regarding how the Duffy negative trait became fixed in Africa needs to be revisited, taking into account not only human genetic data but also the genetic diversity observed in the extant P. vivax populations and the phylogeny of the genus Plasmodium.