New approaches for measuring fitness of Plasmodium falciparum mutations implicated in drug resistance

The repeated emergence of drug resistance in Plasmodium falciparum underscores the importance of understanding the genetic architecture of current resistance pathways, as well as any associated fitness costs. Why resistance emerges in particular regions of the world has been linked to particular genetic backgrounds that better tolerate resistance-associated polymorphisms; this is likely to play a key role in driving the epidemiology of drug resistance, however is infrequently studied at a large scale in a laboratory setting. The first results chapter establishes a barcoding approach for P. falciparum with the aim of tracking parasite growth in vitro. The strategy used was adapted for P. falciparum by using a pseudogene (PfRh3) as a safe harbour to insert unique molecular barcodes. These libraries of barcoded P. falciparum vectors were also used as a readout of transfection efficiency. The second chapter establishes a proof of principle for phenotyping by barcode sequencing, using a panel of barcoded parasites generated in different genetic backgrounds that comprise sufficient genetic diversity to pilot the method. These were grown in the presence and absence of antimalarial compounds, and growth phenotypes were measured in parallel using BarSeq. The third results chapter studies the contribution of mutations in Pfkelch13, a molecular marker of artemisinin resistance, to parasite fitness. Combining CRISPR/Cas9-based genome editing and high throughput sequencing, the impact of Pfkelch13 alleles on fitness in the context of particular strain backgrounds is revealed. In particular, the impact of genetic background in the emergence and spread of drug-resistant lineages (referred to as KEL1) in Southeast Asia carrying a Y580 Pfkelch13 allele. Overall, given the current pace of genome sequencing of pathogenic organisms such as P. falciparum, it will be important to increase the scale of experimental genetics, in order to tackle in real-time natural variation that might be under constant selection from drugs, thus anticipating the emergence of drug resistance in changing parasite populations. Through this work, tools were developed to facilitate parallel phenotyping by measuring in vitro growth using high-throughput sequencing. The work also develops novel approaches to address the importance of genetic background and a potential role for positive epistasis in a lineage responsible for the recent outbreak of drug-resistant malaria in Southeast Asia

Defining multiplicity of vector uptake in transfected Plasmodium parasites

Abstract: The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part because the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species

Dificultades de la adopción de niños en Colombia a partir de la jurisprudencia de la Corte Constitucional entre 2011 y 2016

This article aims to identify the administrative and judicial difficulties in the adoption process in Colombia, from the analysis of the Constitutional Court's judgments during the years 2011 to 2016. Taking as a basis the socio-legal type research and using an analytical descriptive method based on the study of judgments, defined from questions oriented to the difficulties in the adoption process. This research identified the adoption as a mechanism oriented towards satisfying the best interests of children when the family does not guarantee the conditions for the realization and exercise of their rights. Finally, it showed that there is still a lack of precision and effectiveness in applying the regulations and principles set forth in the Convention on the Rights of the Child.Este artículo pretende identificar las dificultades administrativas y judiciales en el proceso de adopción en Colombia a partir del análisis de las sentencias de la Corte Constitucional durante los años 2011 al 2016. Se parte de una investigación de tipo socio jurídica, utilizando el método analítico descriptivo basado en el estudio de las sentencias, identificadas desde preguntas orientadas a las dificultades en el proceso de adopción. Esta investigación permitió identificar la adopción como un mecanismo orientado primordialmente a satisfacer el interés superior de los niños cuando la familia no garantiza las condiciones para la realización y ejercicio de sus derechos; y evidenció que aún falta precisión y efectividad a la hora de aplicar la normatividad y los principios señalados en la Convención de los Derechos del Niño

Red blood cell tension protects against severe malaria in the Dantu blood group.

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.JCR, AM and DK were supported by the Wellcome Trust (206194/Z/17/Z). MPW is funded by a Wellcome Senior Fellowship (108070). TNW is funded through Fellowships awarded by the Wellcome Trust (091758 and 202800). SNK is supported by the Wellcome Trust-funded Initiative to Develop African Research Leaders (IDeAL) early-career postdoctoral fellowship (107769/Z/10/Z), supported through the DELTAS Africa Initiative (DEL-15-003). The Wellcome Trust provides core support to The KEMRI/Wellcome Trust Research Programme, Kilifi, Kenya (084535), Wellcome Sanger Institute, Cambridge, UK (206194/Z/17/Z) and the Wellcome Centre for Human Genetics, Oxford, UK (090532/Z/09/Z, 203141). PC is supported by the Engineering and Physical Sciences Research Council (EPSRC) (EP/R011443/1), and VI is supported by the EPSRC and the Sackler fellowship

Spatio-temporal dynamics of Plasmodium falciparum transmission within a spatial unit on the Colombian Pacific Coast

Funder: Newton-Caldas Fund Institutional Links, British Council, award G1854Funder: Newton-Caldas Fund Institutional Links, British Council, award G1854; Faculty of Medicine, Universidad Nacional de Colombia, awards HERMES 35988, 32309Abstract: As malaria control programmes concentrate their efforts towards malaria elimination a better understanding of malaria transmission patterns at fine spatial resolution units becomes necessary. Defining spatial units that consider transmission heterogeneity, human movement and migration will help to set up achievable malaria elimination milestones and guide the creation of efficient operational administrative control units. Using a combination of genetic and epidemiological data we defined a malaria transmission unit as the area contributing 95% of malaria cases diagnosed at the catchment facility located in the town of Guapi in the South Pacific Coast of Colombia. We provide data showing that P. falciparum malaria transmission is heterogeneous in time and space and analysed, using topological data analysis, the spatial connectivity, at the micro epidemiological level, between parasite populations circulating within the unit. To illustrate the necessity to evaluate the efficacy of malaria control measures within the transmission unit in order to increase the efficiency of the malaria control effort, we provide information on the size of the asymptomatic reservoir, the nature of parasite genotypes associated with drug resistance as well as the frequency of the Pfhrp2/3 deletion associated with false negatives when using Rapid Diagnostic Tests

Declines in prevalence alter the optimal level of sexual investment for the malaria parasite Plasmodium falciparum

International audienceSuccessful infectious disease interventions can result in large reductions in parasite prevalence. Such demographic change has fitness implications for individual parasites and may shift the parasite’s optimal life history strategy. Here, we explore whether declining infection rates can alter Plasmodium falciparum ’s investment in sexual versus asexual growth. Using a multiscale mathematical model, we demonstrate how the proportion of polyclonal infections, which decreases as parasite prevalence declines, affects the optimal sexual development strategy: Within-host competition in multiclone infections favors a greater investment in asexual growth whereas single-clone infections benefit from higher conversion to sexual forms. At the same time, drug treatment also imposes selection pressure on sexual development by shortening infection length and reducing within-host competition. We assess these models using 148 P. falciparum parasite genomes sampled in French Guiana over an 18-y period of intensive intervention (1998 to 2015). During this time frame, multiple public health measures, including the introduction of new drugs and expanded rapid diagnostic testing, were implemented, reducing P. falciparum malaria cases by an order of magnitude. Consistent with this prevalence decline, we see an increase in the relatedness among parasites, but no single clonal background grew to dominate the population. Analyzing individual allele frequency trajectories, we identify genes that likely experienced selective sweeps. Supporting our model predictions, genes showing the strongest signatures of selection include transcription factors involved in the development of P. falciparum ’s sexual gametocyte form. These results highlight how public health interventions impose wide-ranging selection pressures that affect basic parasite life history traits

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis.

Funder: Ministerio de Salud Pública del EcuadorThe human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring less genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to changes in drug regime. Using newly sequenced whole genomes, we measure relatedness between 166 parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than with other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore past selection events in the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in small populations, as will occur in any region nearing malaria elimination

Flexible and cost-effective genomic surveillance of P. falciparum malaria with targeted nanopore sequencing

Abstract Genomic surveillance of Plasmodium falciparum malaria can provide policy-relevant information about antimalarial drug resistance, diagnostic test failure, and the evolution of vaccine targets. Yet the large and low complexity genome of P. falciparum complicates the development of genomic methods, while resource constraints in malaria endemic regions can limit their deployment. Here, we demonstrate an approach for targeted nanopore sequencing of P. falciparum from dried blood spots (DBS) that enables cost-effective genomic surveillance of malaria in low-resource settings. We release software that facilitates flexible design of amplicon sequencing panels and use this software to design two target panels for P. falciparum. The panels generate 3–4 kbp reads for eight and sixteen targets respectively, covering key drug-resistance associated genes, diagnostic test antigens, polymorphic markers and the vaccine target csp. We validate our approach on mock and field samples, demonstrating robust sequencing coverage, accurate variant calls within coding sequences, the ability to explore P. falciparum within-sample diversity and to detect deletions underlying rapid diagnostic test failure

Spatio-temporal dynamics of Plasmodium falciparum transmission within a spatial unit on the Colombian Pacific Coast.

As malaria control programmes concentrate their efforts towards malaria elimination a better understanding of malaria transmission patterns at fine spatial resolution units becomes necessary. Defining spatial units that consider transmission heterogeneity, human movement and migration will help to set up achievable malaria elimination milestones and guide the creation of efficient operational administrative control units. Using a combination of genetic and epidemiological data we defined a malaria transmission unit as the area contributing 95% of malaria cases diagnosed at the catchment facility located in the town of Guapi in the South Pacific Coast of Colombia. We provide data showing that P. falciparum malaria transmission is heterogeneous in time and space and analysed, using topological data analysis, the spatial connectivity, at the micro epidemiological level, between parasite populations circulating within the unit. To illustrate the necessity to evaluate the efficacy of malaria control measures within the transmission unit in order to increase the efficiency of the malaria control effort, we provide information on the size of the asymptomatic reservoir, the nature of parasite genotypes associated with drug resistance as well as the frequency of the Pfhrp2/3 deletion associated with false negatives when using Rapid Diagnostic Tests

Spatio-temporal dynamics of Plasmodium falciparum transmission within a spatial unit on the Colombian Pacific Coast

As malaria control programmes concentrate their efforts towards malaria elimination a better understanding of malaria transmission patterns at fine spatial resolution units becomes necessary. Defining spatial units that consider transmission heterogeneity, human movement and migration will help to set up achievable malaria elimination milestones and guide the creation of efficient operational administrative control units. Using a combination of genetic and epidemiological data we defined a malaria transmission unit as the area contributing 95% of malaria cases diagnosed at the catchment facility located in the town of Guapi in the South Pacific Coast of Colombia. We provide data showing that P. falciparum malaria transmission is heterogeneous in time and space and analysed, using topological data analysis, the spatial connectivity, at the micro epidemiological level, between parasite populations circulating within the unit. To illustrate the necessity to evaluate the efficacy of malaria control measures within the transmission unit in order to increase the efficiency of the malaria control effort, we provide information on the size of the asymptomatic reservoir, the nature of parasite genotypes associated with drug resistance as well as the frequency of the Pfhrp2/3 deletion associated with false negatives when using Rapid Diagnostic Tests. © 2020, The Author(s)