Dyann F Wirth

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States

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Publications (205)1323.15 Total impact

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    ABSTRACT: Plasmodium vivax, one of the five species of Plasmodium parasites that cause human malaria, is responsible for 25-40% of malaria cases worldwide. Malaria global elimination efforts will benefit from accurate and effective genotyping tools that will provide insight into the population genetics and diversity of this parasite. The recent sequencing of P. vivax isolates from South America, Africa, and Asia presents a new opportunity by uncovering thousands of novel single nucleotide polymorphisms (SNPs). Genotyping a selection of these SNPs provides a robust, low-cost method of identifying parasite infections through their unique genetic signature or barcode. Based on our experience in generating a SNP barcode for P. falciparum using High Resolution Melting (HRM), we have developed a similar tool for P. vivax. We selected globally polymorphic SNPs from available P. vivax genome sequence data that were located in putatively selectively neutral sites (i.e., intergenic, intronic, or 4-fold degenerate coding). From these candidate SNPs we defined a barcode consisting of 42 SNPs. We analyzed the performance of the 42-SNP barcode on 87 P. vivax clinical samples from parasite populations in South America (Brazil, French Guiana), Africa (Ethiopia) and Asia (Sri Lanka). We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/μl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity. The markers are informative across all clinical samples evaluated (average minor allele frequency > 0.1). Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations. Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.
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    ABSTRACT: Host nutrition can affect the outcome of parasitic diseases through metabolic effects on host immunity and/or the parasite. Here we show that modulation of mouse immunometabolism through brief restriction of food intake (dietary restriction, DR) prevents neuropathology in experimental cerebral malaria (ECM). While no effects are detected on parasite growth, DR reduces parasite accumulation in peripheral tissues including the brain, and increases clearance in the spleen. Leptin, a host-derived adipokine linking appetite, energy balance and immune function, is required for ECM pathology and its levels are reduced upon DR. Recombinant leptin abrogates DR benefits, while pharmacological or genetic inhibition of leptin signalling protects against ECM. DR reduces mTORC1 activity in T cells, and this effect is abrogated upon leptin administration. Furthermore, mTORC1 inhibition with rapamycin prevents ECM pathology. Our results suggest that leptin and mTORC1 provide a novel mechanistic link between nutrition, immunometabolism and ECM pathology, with potential therapeutic implications for cerebral malaria.
    Nature Communications 01/2015; 6. DOI:10.1038/ncomms7050 · 10.74 Impact Factor
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    ABSTRACT: Complex malaria infections are defined as those containing more than one genetically distinct lineage of Plasmodium parasite. Complexity of infection (COI) is a useful parameter to estimate from patient blood samples because it is associated with clinical outcome, epidemiology and disease transmission rate. This manuscript describes a method for estimating COI using likelihood, called COIL, from a panel of bi-allelic genotyping assays. COIL assumes that distinct parasite lineages in complex infections are unrelated and that genotyped loci do not exhibit significant linkage disequilibrium. Using the population minor allele frequency (MAF) of the genotyped loci, COIL uses the binomial distribution to estimate the likelihood of a COI level given the prevalence of observed monomorphic or polymorphic genotypes within each sample. COIL reliably estimates COI up to a level of three or five with at least 24 or 96 unlinked genotyped loci, respectively, as determined by in silico simulation and empirical validation. Evaluation of COI levels greater than five in patient samples may require a very large collection of genotype data, making sequencing a more cost-effective approach for evaluating COI under conditions when disease transmission is extremely high. Performance of the method is positively correlated with the MAF of the genotyped loci. COI estimates from existing SNP genotype datasets create a more detailed portrait of disease than analyses based simply on the number of polymorphic genotypes observed within samples. The capacity to reliably estimate COI from a genome-wide panel of SNP genotypes provides a potentially more accurate alternative to methods relying on PCR amplification of a small number of loci for estimating COI. This approach will also increase the number of applications of SNP genotype data, providing additional motivation to employ SNP barcodes for studies of disease epidemiology or control measure efficacy. The COIL program is available for download from GitHub, and users may also upload their SNP genotype data to a web interface for simple and efficient determination of sample COI.
    Malaria Journal 01/2015; 14(1):4. DOI:10.1186/1475-2875-14-4 · 3.49 Impact Factor
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    ABSTRACT: This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)—immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3 % was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (p-value < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (p-value < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.
    American Journal of Hematology 01/2015; 90(1). DOI:10.1002/ajh.23860 · 3.48 Impact Factor
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    ABSTRACT: Background Drug resistance remains a major public health challenge for malaria treatment and eradication. Individual loci associated with drug resistance to many antimalarials have been identified, but their epistasis with other resistance mechanisms has not yet been elucidated.ResultsWe previously described two mutations in the cytoplasmic prolyl-tRNA synthetase (cPRS) that confer resistance to halofuginone. We describe here the evolutionary trajectory of halofuginone resistance of two independent drug resistance selections in Plasmodium falciparum. Using this novel methodology, we discover an unexpected non-genetic drug resistance mechanism that P. falciparum utilizes before genetic modification of the cPRS. P. falciparum first upregulates its proline amino acid homeostasis in response to halofuginone pressure. We show that this non-genetic adaptation to halofuginone is not likely mediated by differential RNA expression and precedes mutation or amplification of the cPRS gene. By tracking the evolution of the two drug resistance selections with whole genome sequencing, we further demonstrate that the cPRS locus accounts for the majority of genetic adaptation to halofuginone in P. falciparum. We further validate that copy-number variations at the cPRS locus also contribute to halofuginone resistance.Conclusions We provide a three-step model for multi-locus evolution of halofuginone drug resistance in P. falciparum. Informed by genomic approaches, our results provide the first comprehensive view of the evolutionary trajectory malaria parasites take to achieve drug resistance. Our understanding of the multiple genetic and non-genetic mechanisms of drug resistance informs how we will design and pair future anti-malarials for clinical use.
    Genome Biology 11/2014; 15(11):511. DOI:10.1186/PREACCEPT-1067113631444973 · 10.47 Impact Factor
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    ABSTRACT: As malaria transmission intensity has declined, Plasmodium falciparum parasite populations display decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006-2013 in Thiès, Senegal using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured:1) antibodies capable of inhibiting parasite growth in culture and 2) antibodies recognizing the surface of infected RBCs. IgGs obtained from volunteers in 2009 showed increased reactivity to the surface of CGS-parasitized erythrocytes over IgGs from 2008. As PfEMP-1 is a major variant surface antigen, we characterized the var genes expressed by CGS parasites after short term in vitro culture, by var Ups qRT-PCR and sequencing using degenerate DBL1α domain primers. CGS parasites show an upregulation of UpsA vars and 2-cysteine containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and there is year-to-year variation in immune recognition of surface antigens on CGS infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population.
    Infection and Immunity 11/2014; 83(1). DOI:10.1128/IAI.01979-14 · 4.16 Impact Factor
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    ABSTRACT: During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge. We developed a custom nCounter® gene expression platform and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy. We demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same samples measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections. Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.
    Genome Medicine 11/2014; 6(11):110. DOI:10.1186/s13073-014-0110-6 · 4.94 Impact Factor
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    ABSTRACT: Identifying the source of resurgent parasites is paramount to strategic and successful intervention for malaria elimination. Although malaria incidence in Panama is low, a recent outbreak resulted in a six-fold increase in reported cases. We hypothesized parasites sampled from this epidemic might be related and exhibit clonal population structure. We tested the genetic relatedness using informative single nucleotide polymorphisms and drug resistance loci. We found the parasites to be clustered into three clonal subpopulations and shared relatedness with parasites from Colombia. Two clusters of Panamanian parasites shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and likely resulted from epidemic expansion of imported or vestigial cases. Outbreak investigation using genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas of malaria elimination.
    The Journal of Infectious Diseases 10/2014; DOI:10.1093/infdis/jiu575 · 5.85 Impact Factor
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    The Journal of Infectious Diseases 10/2014; 211(7). DOI:10.1093/infdis/jiu565 · 5.78 Impact Factor
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    ABSTRACT: Here, we describe medicinal chemistry that was accelerated by a diversity-oriented synthesis (DOS) pathway, and in vivo studies of our previously reported macrocyclic antimalarial agent that derived from the synthetic pathway. Structure-activity relationships that focused on both appendage and skeletal features yielded a nanomolar inhibitor of P. falciparum asexual blood-stage growth with improved solubility and microsomal stability and reduced hERG binding. The build/couple/pair (B/C/P) synthetic strategy, used in the preparation of the original screening library, facilitated medicinal chemistry optimization of the antimalarial lead.
    Journal of Medicinal Chemistry 09/2014; 57(20). DOI:10.1021/jm500994n · 5.48 Impact Factor
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    ABSTRACT: Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure–activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg–1) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.
    Journal of Medicinal Chemistry 06/2014; DOI:10.1021/jm500535j · 5.48 Impact Factor
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    ABSTRACT: Malaria is a preventable and treatable disease, yet half of the world's population lives at risk of infection and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of PfDHODH inhibitors via in vitro resistance selections. We found eleven point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.
    Journal of Biological Chemistry 04/2014; 289(26). DOI:10.1074/jbc.M114.558353 · 4.60 Impact Factor
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    ABSTRACT: Malaria is receding in many endemic countries with intervention scale -up against the disease. However, this resilient scourge may persist in low-grade submicroscopic infections among semi-immune members of the population, and be poised for possible resurgence, creating challenges for detection and assessment of intervention impact. Parasite genotyping methods, such as the molecular barcode, can identify specific malaria parasite types being transmitted and allow tracking and evaluation of parasite population structure changes as interventions are applied. This current study demonstrates application of pre -amplification methods for successful detection and genotyping of residual Plasmodium falciparum infections during a dramatic malarial decline. The study was a prospective cross-sectional design and based on a 2,000 sq km vicinity of Macha Mission Hospital in southern Zambia. Willing and predominantly asymptomatic residents of all ages were screened for malaria by microscopy during the 2005 and 2008 transmission seasons, with simultaneous collection of dried blood spots (DBS) on filter paper, and extraction of Plasmodium falciparum DNA was performed. Plasmodium falciparum infections were genotyped using a 24 SNP-based molecular barcode assay using real-time PCR. Submicroscopic parasitaemia samples were subjected to pre-amplification using TaqMan PreAmp Master Mix following the manufacturer's instructions be fo re SNP barcode a na lysis. There was a dramatic decline of malaria between 2005 and 2008, and the geometric mean pa ra site density (95% CI) fell from 704/muL (390 -1,271) in 2005 to 39/muL (23 -68) in 2008, culminating in a large proportion of submicroscopic infections of which 90% failed to yield ample DNA for standard molecular characterization among 2008 samples. Pre -amplification enabled successful detection and genotyping of 74% of these low-grade reservoir infections, overall, compared to 54% that were detectable before pre -amplification (p <0.0005, n = 84). Furthermore, nine samples negative for parasites by microscopy and standard quantitative PCR amplification were positive after pre -amplification. Pre-amplification allows analysis of an otherwise undetectable parasite population and may be instrumental for parasites identification, tracking and assessing the impact of interventions on parasite populations during malaria control and elimination programmes when parasitaemia is expected to decline to submicroscopic levels.
    Malaria Journal 03/2014; 13(1):89. DOI:10.1186/1475-2875-13-89 · 3.49 Impact Factor
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    ABSTRACT: A challenge to conducting high-impact and reproducible studies of the mechanisms of P. falciparum drug resistance, invasion, virulence, and immunity is the lack of robust and sustainable in vitro culture in the field. While the technology exists and is routinely utilized in developed countries, various factors-from cost, to supply, to quality-make it hard to implement in malaria endemic countries. Here, we design and rigorously evaluate an adjustable gas-mixing device for the in vitro culture of P. falciparum parasites in the field to circumvent this challenge. The device accurately replicates the gas concentrations needed to culture laboratory isolates, short-term adapted field isolates, cryopreserved previously non-adapted isolates, as well as to adapt ex vivo isolates to in vitro culture in the field. We also show an advantage over existing alternatives both in cost and in supply. Furthermore, the adjustable nature of the device makes it an ideal tool for many applications in which varied gas concentrations could be critical to culture success. This adjustable gas-mixing device will dramatically improve the feasibility of in vitro culture of Plasmodium falciparum parasites in malaria endemic countries given its numerous advantages.
    PLoS ONE 03/2014; 9(3):e90928. DOI:10.1371/journal.pone.0090928 · 3.53 Impact Factor
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    ABSTRACT: Malaria is one of the most important tropical diseases that has caused devastation throughout the history of mankind. Malaria eradication programmes in the past have had many positive effects but failed to wipe out malaria from most tropical countries, including Sri Lanka. Encouraged by the impressive levels of reduction in malaria case numbers during the past decade, Sri Lanka has launched a programme to eliminate malaria by year 2014. This article reviews the historical milestones associated with the malaria eradication programme that failed subsequently and the events that led to the launch of the ongoing malaria elimination plans at national-level and its strategies that are operational across the entire country. The existing gaps in knowledge are also discussed together with the priority areas for research to fill in these gaps that are posing as challenges to the envisaged goal of wiping out malaria from this island nation.
    Malaria Journal 02/2014; 13(1):59. DOI:10.1186/1475-2875-13-59 · 3.49 Impact Factor
    This article is viewable in ResearchGate's enriched format
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    ABSTRACT: SUMMARY Here we examined whether the recent dramatic decline in malaria transmission in Sri Lanka led to a major bottleneck in the local Plasmodium vivax population, with a substantial decrease in the effective population size. To this end, we typed 14 highly polymorphic microsatellite markers in 185 P. vivax patient isolates collected from 13 districts in Sri Lanka over a period of 5 years (2003-2007). Overall, we found a high degree of polymorphism, with 184 unique haplotypes (12-46 alleles per locus) and average genetic diversity (expected heterozygosity) of 0·8744. Almost 69% (n = 127) isolates had multiple-clone infections (MCI). Significant spatial and temporal differentiation (F ST = 0·04-0·25; P⩽0·0009) between populations was observed. The effective population size was relatively high but showed a decline from 2003-4 to 2006-7 periods (estimated as 45 661 to 22 896 or 10 513 to 7057, depending on the underlying model used). We used three approaches - namely, mode-shift in allele frequency distribution, detection of heterozygote excess and the M-ratio statistics - to test for evidence of a recent population bottleneck but only the low values of M-ratio statistics (ranging between 0·15-0·33, mean 0·26) were suggestive of such a bottleneck. The persistence of high genetic diversity and high proportion of MCI, with little change in effective population size, despite the collapse in demographic population size of P. vivax in Sri Lanka indicates the importance of maintaining stringent control and surveillance measures to prevent resurgence.
    Parasitology 02/2014; 141(7):1-11. DOI:10.1017/S0031182013002278 · 2.36 Impact Factor
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    ABSTRACT: Elimination of malaria from Meso-America urges for the characterization of circulating field strains of Plasmodium parasites from the region. In this study we examined 39 DNA samples obtained from Plasmodium falciparum field isolates collected during an epidemic that occurred in Panama in 2003-2008, and characterized their genetic diversity and relatedness using a SNP-Based Molecular Barcode assay. Principal Component Analysis of the barcode data let us to characterize, map and group the isolates into three distinct clonal sub-populations (p < 0.0001), two from the pacific watershed of the isthmus identified as Madugandi and Darien that clustered together as one clonal group, while the other one belonging to the Guna Yala group clustered into two distinct clonal sub-populations. The identical barcodes observed in three subpopulations of P. falciparum field isolates indicates that these subpopulations are highly related and could be the result of clonal propagation, epidemic expansion, or both. These findings support the hypothesis that highly related parasite populations are evident in low transmission settings, such as that observed in Panama. The prevalence of antimalarial drug sensitivity associated SNPs in the Pfcrt, Pfmdr1, Pfdhps, Pfdhfr, Pfcytb and PfATPase6 gene loci was determined for 38 P. falciparum isolates using High Resolution Melting Analysis (HRM). HRM genotyping detected Pfcrt N75E, K76T, H97Q and A220S mutations in 100 % of the isolates. Pfmdr1 mutations N86Y, Y184F, N1042D and D1246Y were present in 26.0 %, 100 %, 26 % and 95 % of the isolates, respectively. Likewise, Pfdhps mutations A437G in 66 % and Pfdhfr C59R and S108N in 100%. But, no mutations were found in the Pfcytb and PfATPase6 genes. We anticipate that use of genomic tools such as the molecular barcode will detect changes in malaria parasite population structure that occur as malaria-reducing strategies are implemented regionally to lower transmission. Such tools allow tracking of specific parasite types and we anticipate that these data will help in the planning, design and implementation of malaria elimination programs tailored for the southern region of Meso-America.
    Keystone Symposia on Molecular and Cellular Biology, "The Science of Malaria Eradication", February 2-7, 2014. Fiesta Americana - Mérida, Yucatán, Mexico; 02/2014
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    ABSTRACT: The Senegalese National Malaria Control Programme has recommended use of rapid diagnostic tests (RDTs) that target the histidine-rich protein 2 (HRP2), specific to Plasmodium falciparum, to diagnose malaria cases. The target antigen has been shown to be polymorphic, which may explain the variability in HRP2-based RDT results reported in field studies. The genetic diversity of the pfhrp2 gene has not been investigated in depth in many African countries. The goal of this study is to determine the extent of polymorphism in pfhrp2 among Senegal, Mali and Uganda parasite populations, and discuss the implications of these findings on the utility of RDTs that are based on HRP2 detection. Sequencing data from the pfhrp2 locus were used to analyze the genetic diversity of this gene among three populations, with different transmission dynamics and malaria parasite ecologies. Nucleotide diversity (pi) and non-synonymous nucleotide diversity (piNS) were studied in the pfhrp2 gene from isolates obtained in Senegal. Amino acid repeat length polymorphisms in the PfHRP2 antigen were characterized and parameters of genetic diversity, such as frequency and correlation between repeats in these populations, were assessed. The diversity survey of the pfhrp2 gene identified 29 SNPs as well as insertion and deletion polymorphisms within a 918 bp region. The Senegal pfhrp2 exhibited a substantial level of diversity [pi = 0.00559 and piNS = 0.014111 (piS = 0.0291627)], similar to several polymorphic genes, such as msp1, involved in immune responses, and the gene encoding the SURFIN polymorphic antigen, which are surface exposed parasite proteins. Extensive repeat length polymorphisms in PfHRP2, as well as similar patterns in the number, organization and the type of predicted amino acid repeats were observed among the three populations, characterized by an occurrence of Type 2, Type 4 and Type 7 repeats. These results warrant deeper monitoring of the RDT target antigen diversity and emphasize that development of other essential genes as a target for diagnostic tools is critical.
    Malaria Journal 01/2014; 13(1):34. DOI:10.1186/1475-2875-13-34 · 3.49 Impact Factor
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    ABSTRACT: Drug resistance emerges in an ecological context where fitness costs restrict the diversity of escape pathways. These pathways are targets for drug discovery, and here we demonstrate that we can identify small-molecule inhibitors that differentially target resistant parasites. Combining wild-type and mutant-type inhibitors may prevent the emergence of competitively viable resistance. We tested this hypothesis with a clinically derived chloroquine-resistant (CQ(r)) malaria parasite and with parasites derived by in vitro selection with Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. We screened a chemical library against CQ(s) and CQ(r) lines and discovered a drug-like compound (IDI-3783) that was potent only in the CQ(r) line. Surprisingly, in vitro selection of Plasmodium falciparum resistant to IDI-3783 restored CQ sensitivity, thereby indicating that CQ might once again be useful as a malaria therapy. In parallel experiments, we selected P. falciparum lines resistant to structurally unrelated PfDHODH inhibitors (Genz-666136 and DSM74). Both selections yielded resistant lines with the same point mutation in PfDHODH:E182D. We discovered a compound (IDI-6273) more potent against E182D than wild-type parasites. Selection of the E182D mutant with IDI-6273 yielded a reversion to the wild-type protein sequence and phenotype although the nucleotide sequence was different. Importantly, selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites. These two examples demonstrate that the compromise between resistance and evolutionary fitness can be exploited to design therapies that prevent the emergence and spread of resistant organisms.
    Proceedings of the National Academy of Sciences 12/2013; 111(2). DOI:10.1073/pnas.1320886110 · 9.81 Impact Factor
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    ABSTRACT: Malaria treatment efforts are hindered by the rapid emergence and spread of drug resistant parasites. Simple assays to monitor parasite drug response in direct patient samples (ex vivo) can detect drug resistance before it becomes clinically apparent, and can inform changes in treatment policy to prevent the spread of resistance. Parasite drug responses to amodiaquine, artemisinin, chloroquine and mefloquine were tested in approximately 400 Plasmodium falciparum malaria infections in Thies, Senegal between 2008 and 2011 using a DAPI-based ex vivo drug resistance assay. Drug resistance-associated mutations were also genotyped in pfcrt and pfmdr1. Parasite drug responses changed between 2008 and 2011, as parasites became less sensitive to amodiaquine, artemisinin and chloroquine over time. The prevalence of known resistance-associated mutations also changed over time. Decreased amodiaquine sensitivity was associated with sustained, highly prevalent mutations in pfcrt, and one mutation in pfmdr1 -- Y184F -- was associated with decreased parasite sensitivity to artemisinin. Directly measuring ex vivo parasite drug response and resistance mutation genotyping over time are useful tools for monitoring parasite drug responses in field samples. Furthermore, these data suggest that the use of amodiaquine and artemisinin derivatives in combination therapies is selecting for increased drug tolerance within this population.
    Malaria Journal 12/2013; 12(1):441. DOI:10.1186/1475-2875-12-441 · 3.49 Impact Factor
    This article is viewable in ResearchGate's enriched format

Publication Stats

6k Citations
1,323.15 Total Impact Points

Institutions

  • 2010–2015
    • Broad Institute of MIT and Harvard
      • Therapeutics Platform
      Cambridge, Massachusetts, United States
  • 1985–2015
    • Harvard Medical School
      • Division of Medical Sciences
      Boston, Massachusetts, United States
  • 2014
    • Simmons College
      Boston, Massachusetts, United States
  • 1994–2014
    • Harvard University
      • Department of Immunology and Infectious Diseases
      Cambridge, Massachusetts, United States
    • Michigan State University
      Ист-Лансинг, Michigan, United States
  • 1987–2014
    • Massachusetts Department of Public Health
      Boston, Massachusetts, United States
  • 2007–2012
    • Cheikh Anta Diop University, Dakar
      Dakar, Dakar, Senegal
  • 2011
    • Beverly Hospital, Boston MA
      BVY, Massachusetts, United States
    • Loyola University Chicago
      • Department of Biology
      Chicago, IL, United States
  • 2009
    • State University of New York College at Geneseo
      • Department of Biology
      Geneseo, New York, United States
  • 2005
    • McLean Hospital
      Cambridge, Massachusetts, United States
    • University of California, San Francisco
      • Division of Hospital Medicine
      San Francisco, CA, United States
  • 2003–2004
    • University of Ibadan
      • College of Medicine
      Ibadan, Oyo State, Nigeria
  • 1998
    • Federal University of Rio de Janeiro
      • Instituto de Biofísica Carlos Chagas Filho (IBCCF)
      Rio de Janeiro, Rio de Janeiro, Brazil
  • 1993
    • Chulalongkorn University
      Krung Thep, Bangkok, Thailand
  • 1991
    • Armauer Hansen Research Institute
      Ādīs Ābeba, Ādīs Ābeba, Ethiopia
  • 1990
    • Armed Forces Institute of Pathology
      Ralalpindi, Punjab, Pakistan
  • 1989
    • U.S. Department of Health and Human Services
      Washington, Washington, D.C., United States