Origin and evolution of sulfadoxine resistant Plasmodium falciparum.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

Sequence analysis of Plasmodium falciparum cytochrome b in multiple geographic sites.

BACKGROUND: The antimalarial drug atovaquone specifically targets Plasmodium falciparum cytochrome b (Pfcytb), a mitochondrial gene with uniparental inheritance. Cases of resistance to atovaquone associated with mutant Pfcytb have been reported, justifying efforts to better document the natural polymorphism of this gene. To this end, a large molecular survey was conducted in several malaria endemic areas where atovaquone was not yet in regular use. METHODS: The polymorphism of the Pfcytb was analysed by direct sequencing of PCR products corresponding to the full length coding region. Sequence was generated for 671 isolates originating from three continents: Africa (Senegal, Ivory Coast, Central African Republic and Madagascar), Asia (Cambodia) and South America (French Guiana). RESULTS: Overall, 11 polymorphic sites were observed, of which eight were novel mutations. There was a large disparity in the geographic distribution of the mutants. All isolates from Senegal, Central African Republic and Madagascar displayed a Camp/3D7 wild type Pfcytb sequence, as did most samples originating from Cambodia and Ivory Coast. One synonymous (t759a at codon V253V) and two non-synonymous (t553g and a581g at codons F185V and H194R, respectively) singletons were detected in Ivory Coast. Likewise, two synonymous (a126t and c793t at codons -T42T and L265L, respectively) singletons were observed in Cambodia. In contrast, seven mutated sites, affecting seven codons and defining four mutant haplotypes were observed in French Guiana. The wild type allele was observed in only 14% of the French Guiana isolates. The synonymous c688t mutation at position L230L was highly prevalent; the most frequent allele was the c688t single mutant, observed in 84% of the isolates. The other alleles were singletons (a126t/a165c, a4g/a20t/a1024c and a20t/t341c/c688t corresponding to T42T/S55S, N2D/N71I/I342L, N71I/L114S/L230L, respectively" please replace with ' corresponding to T42T/S55S, N2D/N71I/I342L and N71I/L114S/L230L, respectively). The codon 268 polymorphisms associated with atovaquone resistance were not observed in the panel the isolates studied. Overall, the wild type PfCYTb protein isoform was highly predominant in all study areas, including French Guiana, suggesting stringent functional constraints. CONCLUSION: These data along with previously identified Pfcytb field polymorphisms indicate a clustering of molecular signatures, suggesting different ancestral types in South America and other continents. The absence of mutations associated with most atovaquone-proguanil clinical failures indicates that the atovaquone-proguanil association is an interesting treatment option in the study areas.

World Antimalarial Resistance Network (WARN) II: in vitro antimalarial drug susceptibility.

Intrinsic resistance of Plasmodium falciparum is clearly a major determinant of the clinical failure of antimalarial drugs. However, complex interactions between the host, the parasite and the drug obscure the ability to define parasite drug resistance in vivo. The in vitro antimalarial drug susceptibility assay determines ex-vivo growth of parasite in the presence of serial drug concentrations and, thus, eliminates host effects, such as drug metabolism and immunity. Although the sensitivity of the parasite to various antimalarials provided by such a test provides an important indicator of intrinsic parasite susceptibility, there are fundamental methodological issues that undermine comparison of in vitro susceptibility both between laboratories and within a single laboratory over time. A network of laboratories is proposed that will agree on the basic parameters of the in vitro test and associated measures of quality control. The aim of the network would be to establish baseline values of sensitivity to commonly used antimalarial agents from key regions of the world, and create a global database, linked to clinical, molecular and pharmacology databases, to support active surveillance to monitor temporal trends in parasite susceptibility. Such a network would facilitate the rapid detection of strains with novel antimalarial resistance profiles and investigate suitable alternative treatments with retained efficacy.

A sero-epidemiological study of malaria in human and monkey populations in French Guiana.

This paper describes a sero-epidemiological study of malaria prevalence in French Guiana. An immunofluorescence assay and an enzyme-linked immunosorbent assay were used to detect antibodies against blood-stage antigens and synthetic peptides mimicking the repetitive epitope of the sporozoites of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae/brasilianum, in 218 human sera and 113 non-human primate sera collected in French Guiana. Almost all the monkey sera tested had antibodies against malaria blood-stages (98%) and a large majority (73%) also tested positive with the P. malariae/brasilianum circumsporozoite peptide. A number of primate samples also reacted positively with P. falciparum NANP repeats in a very specific manner, suggesting that monkeys in the rainforest are bitten by mosquitoes infected with human malaria parasites. Seroprevalences were lower in the humans tested but Indian tribes on the borders with Suriname and Brazil were clearly more exposed to malaria than other ethnic groups, with a prevalence of nearly 70% seropositivity. P. vivax infections accounted for much of the observed pattern of reactivity, but there was also a high frequency of positive reactions to the P. brasilianum/malariae peptide. Similarly, a large proportion of the sera obtained from Bush Negro populations tested positive for P. malariae/brasilianum repeats. These data add to the emerging evidence that non-human primates might constitute a natural reservoir, not only for simian, but also for human malaria, and therefore suggest that they might be responsible for the maintenance of foci of P. malariae, and possibly of other malaria species, in isolated areas of the Amazonian rainforest.