[Show abstract][Hide abstract] ABSTRACT: Some of the most valuable antimalarial compounds, including quinine and artemisinin, originated from plants. While these drugs have served important roles over many years for the treatment of malaria, drug resistance has become a widespread problem. Therefore, a critical need exists to identify new compounds that have efficacy against drug-resistant malaria strains. In the current study, extracts prepared from plants readily obtained from local sources were screened for activity against Plasmodium falciparum. Bioassay-guided fractionation was used to identify 18 compounds from five plant species. These compounds included eight lupane triterpenes (1-8), four kaempferol 3-O-rhamnosides (10-13), four kaempferol 3-O-glucosides (14-17), and the known compounds amentoflavone and knipholone. These compounds were tested for their efficacy against multi-drug-resistant malaria parasites and counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3) isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains can be identified from native and introduced plant species in the United States, which traditionally have received scant investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.
Full-text · Article · Dec 2015 · Journal of Natural Products
[Show abstract][Hide abstract] ABSTRACT: If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in 7 countries in Africa, SE Asia and S. America using a high density SNP/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500bp to 59kb, as well as 10,107 flanking, biallelic SNPs. Overall, CNVs were rare, small and skewed towards low frequency variants, consistent with the deleterious model. Relative to African and SE Asian populations, CNVs were significantly more common in S. America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g. DNA helicase, and 3 conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.
Full-text · Article · Nov 2015 · Molecular Biology and Evolution
[Show abstract][Hide abstract] ABSTRACT: Experimental genetic crosses in Plasmodium falciparum have played a pivotal role in the discovery of genes underlying several important phenotypic traits including drug resistance and host specificity. Previously, P. falciparum genetic crosses were carried out in splenectomized chimpanzees and in spite of the fact that three successful experimental crosses were generated, ethical and logistical concerns have now rendered this technology obsolete. Here we demonstrate a new model for P. falciparum experimental genetic crosses: a human hepatocyte-liver chimeric mouse (FRG huHep mouse) injected with human red blood cells (huRBCs) that allows for complete P. falciparum liver stage development and the transition of exo-erythrocytic merozoites to asexual blood stage development. Using this novel and versatile model, we have rapidly generated and analyzed three experimental crosses, including the identification of unique recombinant progeny from each cross. A chloroquine (CQ) sensitive transgenic strain, NF54HT-GFP[[unable to display character: –]]luc, was used as a parent in all three of our new experimental crosses and was crossed with three different strains: GB4 and 7G8, two strains used in a previous chimpanzee experimental cross, and NHP*, a recent field isolate from Southeast Asia. We characterized all crosses using microsatellite markers and further characterized progeny from the NF54HT-GFP[[unable to display character: –]]luc × NHP* cross using thousands of single nucleotide polymorphisms (SNPs) from next-generation sequence data and custom genotyping microarrays. These data were used to generate genetic maps and compute recombination rates across the genome. The high-density SNP-based linkage map will be used in conjunction with quantitative trait loci (QTL) mapping to assay a wide variety of quantifiable phenotypes such as drug responses, thus facilitating the study of complex genetic traits in P. falciparum.
[Show abstract][Hide abstract] ABSTRACT: There are enormous numbers of microsatellites repeats (~1 microsatellite every 650bp) in the genome of the malaria parasite, Plasmodium falciparum. These markers are multiallelic and therefore more informative than SNPs for many research questions, yet they are problematic to score using next generation sequence data, and remain an untapped source of genetic variation for population genomic analyses. We used three approaches to validate scoring of microsatellite loci from next generation sequence data: (1) we sequenced the progeny of a genetic cross (n=14), and examined microsatellite calling in genome regions inherited from each parent; (2) we sequenced (in duplicate) parasites from a mutation accumulation experiment (n=38), and measured reproducibility and accuracy of scoring in >36K microsatellite loci throughout the parasite genome. (3) We compared microsatellite genotypes in genome sequence data from 6 single clone patient samples that had previously been scored for 335 microsatellites using traditional capillary electrophoresis. These analyses define the robustness with which microsatellites bearing different sequence motifs can be scored, and provide a map of callable microsatellites for large scale analysis of malaria parasite populations. To examine the utility of these markers for population genomic analyses, we determined genome-wide patterns of microsatellite variation in sequence data from 50 isolates obtained from a focus of emerging drug resistance in South-East Asia. We identify loci under strong, recent, positive selection in these samples, including known drug resistance loci, and we recapture the kelch locus underpinning artemisinin resistance.
[Show abstract][Hide abstract] ABSTRACT: Genetic crosses of phenotypically distinct strains of the human malaria parasite Plasmodium falciparum are a powerful tool for identifying genes controlling drug resistance and other key phenotypes. Previous studies relied on the isolation of recombinant parasites from splenectomized chimpanzees, a research avenue that is no longer available. Here we demonstrate that human-liver chimeric mice support recovery of recombinant progeny for the identification of genetic determinants of parasite traits and adaptations.
[Show abstract][Hide abstract] ABSTRACT: Artemisinin-resistant falciparum malaria has emerged in Southeast Asia, posing a major threat to malaria control. It is characterised by delayed asexual-stage parasite clearance, which is the reference comparator for the molecular marker ‘Kelch 13’ and in vitro sensitivity tests. However, current cut-off values denoting slow clearance based on the proportion of individuals remaining parasitaemic on the third day of treatment ('day-3'), or on peripheral blood parasite half-life, are not well supported. We here explore the parasite clearance distributions in an area of artemisinin resistance with the aim refining the in vivo phenotypic definitions.
[Show abstract][Hide abstract] ABSTRACT: Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and whole-genome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) single-cell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections.
[Show abstract][Hide abstract] ABSTRACT: The analysis of in vitro anti-malarial drug susceptibility testing is vulnerable to the effects of different statistical approaches and selection biases. These confounding factors were assessed with respect to pfmdr1 gene mutation and amplification in 490 clinical isolates. Two statistical approaches for estimating the drug concentration associated with 50% effect (EC50 ) were compared: the commonly used standard two-stage (STS) method, and nonlinear mixed-effects modelling. The in vitro concentration-effect relationships for, chloroquine, mefloquine, lumefantrine and artesunate, were derived from clinical isolates obtained from patients on the western border of Thailand. All isolates were genotyped for polymorphisms in the pfmdr1 gene. The EC50 estimates were similar for the two statistical approaches but 15-28% of isolates in the STS method had a high coefficient of variation (>15%) for individual estimates of EC50 and these isolates had EC50 values that were 32 to 66% higher than isolates derived with more precision. In total 41% (202/490) of isolates had amplification of pfmdr1 and single nucleotide polymorphisms were found in 50 (10%). Pfmdr1 amplification was associated with an increase in EC50 for mefloquine (139% relative increase in EC50 for 2 copies, 188% for 3+ copies), lumefantrine (82% and 75% for 2 and 3+ copies respectively) and artesunate (63% and 127% for 2 and 3+ copies respectively). In contrast pfmdr1 mutation at codons 86 or 1042 were associated with an increase in chloroquine EC50 (44-48%). Sample size calculations showed that to demonstrate an EC50 shift of 50% or more with 80% power if the prevalence was 10% would require 430 isolates and 245 isolates if the prevalence was 20%. In conclusion, although nonlinear mixed-effects modelling did not demonstrate any major advantage for determining estimates of anti-malarial drug susceptibility, the method includes all isolates, thereby, potentially improving confirmation of candidate molecular markers of anti-malarial drug susceptibility.
[Show abstract][Hide abstract] ABSTRACT: Accurate measurement of malaria parasite clearance rates (CRs) following artemisinin (ART) treatment is critical for resistance
surveillance and research, and various CR metrics are currently used. We measured 13 CR metrics in 1472 ART-treated hyperparasitemia
infections for which 6-hour parasite counts and parasite genotypes (93 single nucleotide polymorphisms [SNPs]) were available.
We used heritability to evaluate the performance of each metric. Heritability ranged from 0.06 ± 0.06 (SD) for 50% parasite
clearance times to 0.67 ± 0.04 (SD) for clearance half-lives estimated from 6-hour parasite counts. These results identify
the measures that should be avoided and show that reliable clearance measures can be obtained with abbreviated monitoring
Full-text · Article · Apr 2013 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Background
Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies.
A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD). Most infections (81%) contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs), with 32% of MLGs recovered from multiple (2 – 28) independent subjects. We observed extremely low genotypic richness (R = 0.42) and long persistence of MLGs through time (median = 537 days, range = 1 – 2,997 days). There was a high probability (>5%) of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST = 0.084 - 0.279) were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD) decayed more rapidly (r2 = 0.17 for markers <10 kb apart) than observed previously in South American samples.
We conclude that Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies.
[Show abstract][Hide abstract] ABSTRACT: Pathogen control programs provide a valuable, but rarely exploited, opportunity to directly examine the relationship between population decline and population genetics. We investigated the impact of an ~12-fold decline in transmission on the population genetics of Plasmodium falciparum infections (n = 1731) sampled from four clinics on the Thai-Burma border over 10 years and genotyped using 96 genome-wide SNPs. The most striking associated genetic change was a reduction in the frequency of infections containing multiple parasite genotypes from 63% in 2001 to 14% in 2010 (P = 3 × 10(-15) ). Two measures of the clonal composition of populations (genotypic richness and the β-parameter of the Pareto distribution) declined over time as more people were infected by parasites with identical multilocus genotypes, consistent with increased selfing and a reduction in the rate at which multilocus genotypes are broken apart by recombination. We predicted that the reduction in transmission, multiple clone carriage and outbreeding would be mirrored by an increased influence of genetic drift. However, geographical differentiation and expected heterozygosity remained stable across the sampling period. Furthermore, N(e) estimates derived from allele frequencies fluctuation between years remained high (582 to ∞) and showed no downward trend. These results demonstrate how genetic data can compliment epidemiological assessments of infectious disease control programs. The temporal changes in a single declining population parallel to those seen in comparisons of parasite genetics in regions of differing endemicity, strongly supporting the notion that reduced opportunity for outbreeding is the key driver of these patterns.
Full-text · Article · Nov 2012 · Molecular Ecology
[Show abstract][Hide abstract] ABSTRACT: Evolving resistance to artemisinin-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia and has recently emerged in western Thailand, but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CRs) after treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6969 polymorphic single-nucleotide polymorphisms (SNPs) in 91 parasites from Cambodia, Thailand, and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chromosome 13 that shows strong association (P = 10(-6) to 10(-12)) with slow CRs, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.
[Show abstract][Hide abstract] ABSTRACT: Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand-Myanmar (Burma) border.
In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms.
3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5-2·7) in 2001, to 3·7 h (3·6-3·8) in 2010, compared with a mean of 5·5 h (5·2-5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010.
Genetically determined artemisinin resistance in P falciparum emerged along the Thailand-Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2-6 years.
The Wellcome Trust and National Institutes of Health.
[Show abstract][Hide abstract] ABSTRACT: Malaria infections containing multiple parasite genotypes are ubiquitous in nature, and play a central role in models of recombination, intra-host dynamics, virulence, sex ratio, immunity and drug resistance evolution in Plasmodium. While these multiple infections (MIs) are often assumed to result from superinfection (bites from multiple infected mosquitoes), we know remarkably little about their composition or generation. We isolated 336 parasite clones from eight patients from Malawi (high transmission) and six from Thailand (low transmission) by dilution cloning. These were genotyped using 384 single-nucleotide polymorphisms, revealing 22 independent haplotypes in Malawi (2-6 per MI) and 15 in Thailand (2-5 per MI). Surprisingly, all six patients from Thailand and six of eight from Malawi contained related haplotypes, and haplotypes were more similar within- than between-infections. These results argue against a simple superinfection model. Instead, the observed kinship patterns may be explained by inoculation of multiple related haploid sporozoites from single mosquito bites, by immune suppression of parasite subpopulations within infections, and serial transmission of related parasites between people. That relatedness is maintained in endemic areas in the face of repeated bites from infected mosquitoes has profound implications for understanding malaria transmission, immunity and intra-host dynamics of co-infecting parasite genotypes.
Full-text · Article · Mar 2012 · Proceedings of the Royal Society B: Biological Sciences
[Show abstract][Hide abstract] ABSTRACT: Malaria parasites vary in phenotypic traits of biomedical or biological interest such as growth rate, virulence, sex ratio and drug resistance, and there is considerable interest in identifying the genes that underlie this variation. An important first step is to determine trait heritability (H(2)). We evaluate two approaches to measuring H(2) in natural parasite populations using relatedness inferred from genetic marker data. We collected single-clone Plasmodium falciparum infections from 185 patients from the Thailand-Burma border, monitored parasite clearance following treatment with artemisinin combination therapy (ACT), measured resistance to six antimalarial drugs and genotyped parasites using 335 microsatellites. We found strong relatedness structure. There were 27 groups of two to eight clonally identical (CI) parasites, and 74 per cent of parasites showed significant relatedness to one or more other parasites. Initially, we used matrices of allele sharing and variance components (VC) methods to estimate H(2). Inhibitory concentrations (IC(50)) for six drugs showed significant H(2) (0.24 to 0.79, p = 0.06 to 2.85 x 10(-9)), demonstrating that this study design has adequate power. However, a phenotype of current interest--parasite clearance following ACT--showed no detectable heritability (H(2) = 0-0.09, ns) in this population. The existence of CI parasites allows the use of a simple ANOVA approach for quantifying H(2), analogous to that used in human twin studies. This gave similar results to the VC method and requires considerably less genotyping information. We conclude (i) that H(2) can be effectively measured in malaria parasite populations using minimal genotype data, allowing rational design of genome-wide association studies; and (ii) while drug response (IC(50)) shows significant H(2), parasite clearance following ACT was not heritable in the population studied.
Full-text · Article · Apr 2010 · Proceedings of the Royal Society B: Biological Sciences
[Show abstract][Hide abstract] ABSTRACT: Comparative genomic hybridization studies have revealed elevated copy number (CN) at the reticulocyte-binding protein 1 gene (PfRh1) in fast growing lab-adapted parasites, while genetic manipulation demonstrates a causal link between cell invasion and PfRh1 CN. We therefore examined PfRh1 copy number variation (CNV) in 202 single clone parasite isolates from four countries to quantify the extent of CNV within natural populations. Surprisingly, we found that no natural parasite infections showed elevated CN. In contrast, 4/28 independent laboratory reference strains show elevated CN. One possibility is that amplification of PfRh1 (or neighboring loci) is selected during laboratory culture. In the case of FCR3 group of parasites, clone trees show that PfRh1 amplification arose in laboratory lines following establishment in culture. These data show that CNV at PfRh1 is rare or non-existent in natural populations, but can arise during laboratory propagation. We conclude that PfRh1 CNV is not an important determinant of gene expression, cell invasion or growth rate in natural parasite populations.
[Show abstract][Hide abstract] ABSTRACT: In western Cambodia, malaria parasites clear slowly from the blood after treatment with artemisinin derivatives, but it is
unclear whether this results from parasite, host, or other factors specific to this population. We measured heritability of
clearance rate by evaluating patients infected with identical or nonidentical parasite genotypes, using methods analogous
to human twin studies. A substantial proportion (56%–58%) of the variation in clearance rate is explained by parasite genetics.
This has 2 important implications: (1) selection with artemisinin derivatives will tend to drive resistance spread and (2)
because heritability is high, the genes underlying parasite clearance rate may be identified by genome-wide association.
Trial Registration. ClinicalTrials.gov identifier: NCT00493363; Current Controlled Trials identifier: ISRCTN15351875.
Full-text · Article · Mar 2010 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: We genotyped 160 P. falciparum infections from Malawi for pfmdr-1 copy number changes and SNPs associated with in vivo tolerance and poor in vitro sensitivity to the component drugs of Coartem. We also measured in vitro susceptibility of 49 of these isolates to a variety of drugs in clinical use or with a potential for use in Africa. All 160 infections carried a single copy of pfmdr-1 but 34% exhibited sequence variation at 4 of the 5 polymorphic sites in pfmdr-1. Isolates carrying 86-Asn and 184-Tyr pfmdr-1 alleles were significantly less sensitive (p<0.001) to mefloquine, lumefantrine, artemether and dihydroartemisinin compared with those bearing 86-Tyr and 184-Phe polymorphisms. This study provides baseline measures prior to policy change: continued surveillance for changes in baseline drug susceptibility, pfmdr-1 copy number and SNPs, and other putative Coartem resistance loci will be necessary to provide an early warning of emerging Coartem resistance in this setting.
[Show abstract][Hide abstract] ABSTRACT: Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2-11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.