Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

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

The 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.

Genomic epidemiological models describe pathogen evolution across fitness valleys.

Genomics is fundamentally changing epidemiological research. However, systematically exploring hypotheses in pathogen evolution requires new modeling tools. Models intertwining pathogen epidemiology and genomic evolution can help understand processes such as the emergence of novel pathogen genotypes with higher transmissibility or resistance to treatment. In this work, we present Opqua, a flexible simulation framework that explicitly links epidemiology to sequence evolution and selection. We use Opqua to study determinants of evolution across fitness valleys. We confirm that competition can limit evolution in high-transmission environments and find that low transmission, host mobility, and complex pathogen life cycles facilitate reaching new adaptive peaks through population bottlenecks and decoupling of selective pressures. The results show the potential of genomic epidemiological modeling as a tool in infectious disease research.

Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study.

Multiplexed PCR amplicon sequencing (AmpSeq) is an increasingly popular application for cost-effective monitoring of threatened species and managed wildlife populations, and shows strong potential for the genomic epidemiology of infectious disease. AmpSeq data from infectious microbes can inform disease control in multiple ways, such as by measuring drug resistance marker prevalence, distinguishing imported from local cases, and determining the effectiveness of therapeutics. We describe the design and comparative evaluation of two new AmpSeq assays for Plasmodium falciparum malaria parasites: a four-locus panel ("4CAST") composed of highly diverse antigens, and a 129-locus panel ("AMPLseq") composed of drug resistance markers, highly diverse loci for inferring relatedness, and a locus to detect Plasmodium vivax co-infection. We explore the performance of each panel in various public health use cases with in silico simulations as well as empirical experiments. The 4CAST panel appears highly suitable for evaluating the number of distinct parasite strains within samples (complexity of infection), showing strong performance across a wide range of parasitaemia levels without a DNA pre-amplification step. For relatedness inference, the larger AMPLseq panel performs similarly to two existing panels of comparable size, despite differences in the data and approach used for designing each panel. Finally, we describe an R package (paneljudge) that facilitates the design and comparative evaluation of genetic panels for relatedness estimation, and we provide general guidance on the design and implementation of AmpSeq panels for the genomic epidemiology of infectious disease.

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.

Spleen-dependent regulation of antigenic variation in malaria parasites: Plasmodium knowlesi SICAvar expression profiles in splenic and asplenic hosts.

BACKGROUND: Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera. PRINCIPAL FINDINGS: We have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry. SIGNIFICANCE: SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying antigenic variation in the context of the host environment.

[Differentiation of Entamoeba histolytica from Entamoeba dispar using Gal/GalNAc-lectin and polymerase chain reaction]. / Diferenciación del complejo Entamoeba histolytica/Entamoeba dispar mediante Gal/GalNAc-lectina y PCR en aislamientos colombianos.

BACKGROUND: Entamoeba histolytica and Entamoeba dispar are morphologically identical. However, the former is highly pathogenic and the latter is not. AIM: To differentiate Entamoeba histolytica from Entamoeba dispar through ELISA and PCR techniques in Colombian isolates from feces. MATERIAL AND METHODS: Descriptive study of Colombian fecal samples from 53 males and 47 women, that were positive for the complex E. histolytica/E. dispar on light microscopy. Positive samples were cultured on Robinson medium to isolate trophozoites. The presence of specific Gal/ GalNAc-lectin was determined by ELISA and polymerase chain reaction in genomic DNA, using the combination of three nucleotides that recognize a variable region of 16S small subunit ribosomal RNA, generating a 166 base pair (bp) product for E. histolytica and 752 pb product for E. dispar. RESULTS: After verification, only eight of the 100 samples were positive for the complex E. histolytica/E. dispar and were cultivated. Isolates were obtained in six cultures, one corresponded to E. histolytica and six to E. dispar. CONCLUSIONS: The presence of E. histolytica/E. dispar complex was largely overestimated with light microscopy. In the few samples where isolates were obtained, the technique described differentiated between both strains.

Diferenciación del complejo Entamoeba histolytica/Entamoeba dispar mediante Gal/GalNAc-lectina y PCR en aislamientos colombianos / Differentiation of entamoeba histolytica from entamoeba dispar using Gal/GalNAc-lectin and polymerase chain reaction

Background: Entamoeba histolytica and Entamoeba dispar are morphologically identical. However, the former is highly pathogenic and the latter is not. Aim: To differentiate Entamoeba histolytica from Entamoeba dispar through ELISA and PCR techniques in Colombian isolates from feces. Material and Methods: Descriptive study of Colombian fecal samples from 53 males and 47 women, that were positive for the complex E. histolytica/E. dispar on light microscopy. Positive samples were cultured on Robinson medium to isolate trophozoites. The presence of specific Gal/ GalNAc-lectin was determined by ELISA and polymerase chain reaction in genomic DNA, using the combination of three nucleotides that recognize a variable region of 16S small subunit ribosomal RNA, generating a 166 base pair (bp) product for E. histolytica and 752 pb product for E. dispar. Results: After verification, only eight of the 100 samples were positive for the complex E. histolytica/E. dispar and were cultivated. Isolates were obtained in six cultures, one corresponded to E. histolytica and six to E. dispar. Conclusions: The presence of E. histolytica/E. dispar complex was largely overestimated with light microscopy. In the few samples where isolates were obtained, the technique described differentiated between both strains.

Prospects for malaria elimination in non-Amazonian regions of Latin America.

Latin America contributes 1-1.2 million clinical malaria cases to the global malaria burden of about 300 million per year. In 21 malaria endemic countries, the population at risk in this region represents less than 10% of the total population exposed worldwide. Factors such as rapid deforestation, inadequate agricultural practices, climate change, political instability, and both increasing parasite drug resistance and vector resistance to insecticides contribute to malaria transmission. Recently, several malaria endemic countries have experienced a significant reduction in numbers of malaria cases. This is most likely due to actions taken by National Malaria Control Programs (NMCP) with the support from international funding agencies. We describe here the research strategies and activities to be undertaken by the Centro Latino Americano de Investigación en Malaria (CLAIM), a new research center established for the non-Amazonian region of Latin America by the National Institute of Allergy and Infectious Diseases (NIAID). Throughout a network of countries in the region, initially including Colombia, Guatemala, Panama, and Peru, CLAIM will address major gaps in our understanding of changing malaria epidemiology, vector biology and control, and clinical malaria mainly due to Plasmodium vivax. In close partnership with NMCPs, CLAIM seeks to conduct research on how and why malaria is decreasing in many countries of the region as a basis for developing and implementing new strategies that will accelerate malaria elimination.

Origin and dissemination across the Colombian Andes mountain range of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum.

The therapeutic efficacy of sulfadoxine-pyrimethamine (SP) in treating uncomplicated Plasmodium falciparum malaria is unevenly distributed in Colombia. The Andes mountain range separates regions in the west where malaria is endemic from those in the east and constitutes a barrier against gene flow and the dispersal of parasite populations. The distribution of dhfr and dhps genotypes of 146 P. falciparum samples from the eastern Amazon and Orinoco basins and Northwest and Southwest Pacific regions of Colombia was consistent with the documented levels of therapeutic efficacy of SP. The diversity of four dhfr- and dhps-linked microsatellites indicated that double- and triple-mutant alleles for both resistance loci have a single origin. Likewise, multilocus association genotypes, including two unlinked microsatellite loci, suggested that genetic exchanges between the eastern Orinoco and Northwest Pacific populations has taken place across the Andes, most probably via migration of infected people.

Phylogenetic and structural information on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in Plasmodium provides functional insights.

Plasmodium is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PDH) plays an important role in glycolysis and is, therefore, a potential target for antimalarial drug development. The g3pdh gene of nine Plasmodium species was sequenced from genomic DNA and the type and origin determined by phylogenetic analysis. Substitutions were analyzed over a wide phylogenetic spectrum in relation to the known three-dimensional structures of the P. falciparum and human proteins. Substitutions were found within the functional domains (Rossman NAD+-binding and catalytic domains). A number of replacements within the adenosyl-binding surfaces were found to be conserved within the Chromoalveolates, others in the Apicomplexa, and still others within the genus Plasmodium, all of which were different from the human sequence. These sites may prove to be of functional importance and provide insights for drug-targeting studies, as have other regions examined in Leishmania and Toxoplasma G3PDH research.

Dramatic difference in diversity between Plasmodium falciparum and Plasmodium vivax reticulocyte binding-like genes.

Malaria parasite proteins involved in erythrocyte invasion are considered important vaccine targets. Members of the reticulocyte binding-like (RBL) family of Plasmodium merozoite proteins are found in human, simian, and rodent malaria parasites and function in the initial steps of erythrocyte selection and invasion. The RBL genes are large, ranging in size from 7.7 to 10 kb, and the extent of any sequence diversity in parasite populations is unknown. We present the first assessment of sequence diversity within RBL genes from the two major human malaria parasites: Plasmodium falciparum and P. vivax. Polymorphism within the RBL genes is generally limited, except for P. vivax reticulocyte binding protein 2 (PvRBP2), which has nucleotide diversity levels 25-fold higher than the other RBL genes. The PvRBP2 haplotypes appear to fall into two distinct classes of alleles, suggesting large-scale dimorphism in this gene. Polymorphisms were frequently clustered, suggesting that different RBL domains may be evolving under different selection and functional pressures.

A SICAvar switching event in Plasmodium knowlesi is associated with the DNA rearrangement of conserved 3' non-coding sequences.

Plasmodium knowlesi variant antigens are expressed at the surface of infected erythrocytes and are encoded by the Schizont Infected Cell Agglutination variant antigen (SICAvar) multigene family. The 3' region of the SICAvar gene locus encoding the 205 kDa variant antigen expressed in the Pk1(B+)1+ parasites was found to be altered compared to the Pk1(A+) parental clone. Here we report that this alteration is the result of a DNA rearrangement and that the original and altered 205 SICAvar alleles appear to encode bona fide variant antigens. Importantly, 205A and 205B SICAvar RNA sequences are detectable in similar apparent quantities as determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) amplification experiments. However, expression of the 205 kDa SICA protein at the surface of the infected erythrocyte is not characteristic of the Pk1(A+) parasites and the 205 SICAvar transcript has not been detected in Pk1(A+) parasites by northern blot analysis. Furthermore, we report that many distinct SICAvar transcripts were detected in P. knowlesi Pk1(B+)1+ cDNA library hybridization screens. Of special interest, in light of these data, distinctive differences at the 3' end of the 205A and 205B alleles are observed, which may be of functional importance. An analysis of the 3' untranslated region (UTR) of SICAvar genes in more than 100 sequences revealed a surprising common sequence pattern characterized by blocks of imperfect, GT-rich, heptad repeated motifs (Block I), followed by A and T rich homopolymers (Block II) and in a large number of genes, GC-rich segments (Block III). We show that this region undergoes extensive recombination and that the preferential stability of the 205 SICAvar transcript in Pk1(B+)1+ parasites may be associated with the presence of its specific Block III sequences. We speculate that the conserved yet polymorphic SICAvar 3'UTR sequences, and comparable regions in P. falciparum var genes, function in the stage-specific and developmentally regulated post-transcriptional gene silencing (PTGS) of variant antigen transcripts.

Phylogenetic analysis of CSP and MSP-9 gene sequences demonstrates the close relationship of Plasmodium coatneyi to Plasmodium knowlesi.

Plasmodium coatneyi is a simian malaria parasite with various biological features similar to the human malaria P. falciparum and potential as a model for severe cases of malaria. We have characterized two single-copy genes from P. coatneyi, the circumsporozoite protein and merozoite surface protein-9 genes, and determined its phylogenetic relationship among Plasmodium species. This study demonstrates that while it has similarities to P. falciparum, P. coatneyi belongs to a distant clade including several simian malaria parasites and the human malaria P. vivax. P. coatneyi forms a monophyletic group with P. knowlesi, demonstrating their close relationship despite some very distinctive biological characteristics.

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys.

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.

Histoplasmosis epidemica: II. Hallazgos en la Cueva del eden (Cunday, Tolima) / Epidemic hystoplasmosis: II. Findings at the Eden Cave (Cunday, Tolima, Col.)

Se informa el estudio realizado en la Cueva del Eden (Cunday, Tolima) para aislar, de las tierras de la cueva y de murcielagos alli capturados, al Histoplasma Capsulatum, como agente causal de una anterior epidemia de histoplasmosis ocurrida entre visitantes de la cueva. El agente se aislo en 8 de las 27 muestras de tierra y guano de murcielago. Se capturaron 233 ejemplares pertenecientes a 4 generos de murcielagos y en los cultivos practicados en higado, pulmon y bazo de cien de ellos, no fue posible aislar el H. Capsulatum