Malarial merozoites use an array of ligands, including members of the Reticulocyte Binding Like (RBL) super-family of invasion proteins, to identify and invade erythrocytes. RBL family members are large Type I membrane anchored proteins expressed at the invasive end of merozoites that share homology with the Reticulocyte Binding Proteins 1 and 2 (PvRBP1 and 2) of Plasmodium vivax. Plasmodium species vary widely both in the number and sequence of their RBL genes, with the recently completed Plasmodium falciparum genome containing five RBL genes. Of these, three encode proteins shown to be involved in erythrocyte invasion, a fourth is a pseudogene, and the role of the fifth is as yet unclear. In order to identify sequence similarities and differences that may have functional implications for erythrocyte invasion as well as to gain insights into the recent evolutionary history of the P. falciparum RBL genes, we have sequenced all five corresponding RBL genes from the chimpanzee parasite Plasmodium reichenowi, which is the closest phylogenetic relative of P. falciparum, yet is unable to invade human erythrocytes. Two of the five P. falciparum RBL genes have highly conserved complete open reading frames in both species, while the other three genes show evidence of gene conversion and rapid evolution. The RBL super-family, therefore, appears to be surprisingly dynamic and divergent, implying that it is involved in species-specific aspects of erythrocyte recognition and invasion.

Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii.

The availability of complete genomic sequences for hundreds of organisms promises to make obtaining genome-wide estimates of substitution rates, selective constraints and other molecular evolution variables of interest an increasingly important approach to addressing broad evolutionary questions. Two of the programs most widely used for this purpose are codeml and baseml, parts of the PAML (Phylogenetic Analysis by Maximum Likelihood) suite. A significant drawback of these programs is their lack of a graphical user interface, which can limit their user base and considerably reduce their efficiency.

Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated.

Assessing the Plasmodium vivax burden in India is complicated by the potential threat of an emerging chloroquine (CQ) resistant parasite population from neighbouring countries in Southeast Asia. Chennai, the capital of Tamil Nadu and an urban setting for P. vivax in southern India, was selected as a sentinel site for investigating CQ efficacy and sensitivity in vivax malaria.

In densely populated urban environments, the distribution of microbes and the drivers of microbial community assemblages are not well understood. In sprawling metropolitan habitats, the "urban microbiome" may represent a mix of human-associated and environmental taxa. Here we carried out a baseline study of automated teller machine (ATM) keypads in New York City (NYC). Our goal was to describe the biodiversity and biogeography of both prokaryotic and eukaryotic microbes in an urban setting while assessing the potential source of microbial assemblages on ATM keypads. Microbial swab samples were collected from three boroughs (Manhattan, Queens, and Brooklyn) during June and July 2014, followed by generation of Illumina MiSeq datasets for bacterial (16S rRNA) and eukaryotic (18S rRNA) marker genes. Downstream analysis was carried out in the QIIME pipeline, in conjunction with neighborhood metadata (ethnicity, population, age groups) from the NYC Open Data portal. Neither the 16S nor 18S rRNA datasets showed any clustering patterns related to geography or neighborhood demographics. Bacterial assemblages on ATM keypads were dominated by taxonomic groups known to be associated with human skin communities (Actinobacteria, Bacteroides, Firmicutes, and Proteobacteria), although SourceTracker analysis was unable to identify the source habitat for the majority of taxa. Eukaryotic assemblages were dominated by fungal taxa as well as by a low-diversity protist community containing both free-living and potentially pathogenic taxa (Toxoplasma, Trichomonas). Our results suggest that ATM keypads amalgamate microbial assemblages from different sources, including the human microbiome, eukaryotic food species, and potentially novel extremophilic taxa adapted to air or surfaces in the built environment. DNA obtained from ATM keypads may thus provide a record of both human behavior and environmental sources of microbes. IMPORTANCE Automated teller machine (ATM) keypads represent a specific and unexplored microhabitat for microbial communities. Although the number of built environment and urban microbial ecology studies has expanded greatly in recent years, the majority of research to date has focused on mass transit systems, city soils, and plumbing and ventilation systems in buildings. ATM surfaces, potentially retaining microbial signatures of human inhabitants, including both commensal taxa and pathogens, are interesting from both a biodiversity perspective and a public health perspective. By focusing on ATM keypads in different geographic areas of New York City with distinct population demographics, we aimed to characterize the diversity and distribution of both prokaryotic and eukaryotic microbes, thus making a unique contribution to the growing body of work focused on the "urban microbiome." In New York City, the surface area of urban surfaces in Manhattan far exceeds the geographic area of the island itself. We have only just begun to describe the vast array of microbial taxa that are likely to be present across diverse types of urban habitats.

We sequenced and annotated the genomes of four P. vivax strains collected from disparate geographic locations, tripling the number of genome sequences available for this understudied parasite and providing the first genome-wide perspective of global variability in this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of P. falciparum, a malaria-causing parasite that results in higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax relative to P. falciparum, which is thought to have undergone a recent population bottleneck. The SNP diversity, as well as additional microsatellite and gene family variability, suggests a capacity for greater functional variation in the global population of P. vivax. These findings warrant a deeper survey of variation in P. vivax to equip disease interventions targeting the distinctive biology of this neglected but major pathogen.

The evolutionary history and age of Plasmodium vivax has been inferred as both recent and ancient by several studies, mainly using mitochondrial genome diversity. Here we address the age of P. vivax on the Indian subcontinent using selectively neutral housekeeping genes and tandem repeat loci. Analysis of ten housekeeping genes revealed a substantial number of SNPs (n = 75) from 100 P. vivax isolates collected from five geographical regions of India. Neutrality tests showed a majority of the housekeeping genes were selectively neutral, confirming the suitability of housekeeping genes for inferring the evolutionary history of P. vivax. In addition, a genetic differentiation test using housekeeping gene polymorphism data showed a lack of geographical structuring between the five regions of India. The coalescence analysis of the time to the most recent common ancestor estimate yielded an ancient TMRCA (232,228 to 303,030 years) and long-term population history (79,235 to 104,008) of extant P. vivax on the Indian subcontinent. Analysis of 18 tandem repeat loci polymorphisms showed substantial allelic diversity and heterozygosity per locus, and analysis of potential bottlenecks revealed the signature of a stable P. vivax population, further corroborating our ancient age estimates. For the first time we report a comparable evolutionary history of P. vivax inferred by nuclear genetic markers (putative housekeeping genes) to that inferred from mitochondrial genome diversity.

Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.

The study of the three protagonists in malaria-the Plasmodium parasite, the Anopheles mosquito, and the human host-is key to developing methods to control and eventually eliminate the disease. Genomic technologies, including the recent development of next-generation sequencing, enable interrogation of this triangle to an unprecedented level of scrutiny, and promise exciting progress toward real-time epidemiology studies and the study of evolutionary adaptation. We discuss the use of genomics by the International Centers of Excellence for Malaria Research, a network of field sites and laboratories in malaria-endemic countries that undertake cutting-edge research, training, and technology transfer in malarious countries of the world.

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.

Whole-genome comparisons are highly informative regarding genome evolution and can reveal the conservation of genome organization and gene content, gene regulatory elements, and presence of species-specific genes. Initial comparative genome analyses of the human malaria parasite Plasmodium falciparum and rodent malaria parasites (RMPs) revealed a core set of 4,500 Plasmodium orthologs located in the highly syntenic central regions of the chromosomes that sharply defined the boundaries of the variable subtelomeric regions. We used composite RMP contigs, based on partial DNA sequences of three RMPs, to generate a whole-genome synteny map of P. falciparum and the RMPs. The core regions of the 14 chromosomes of P. falciparum and the RMPs are organized in 36 synteny blocks, representing groups of genes that have been stably inherited since these malaria species diverged, but whose relative organization has altered as a result of a predicted minimum of 15 recombination events. P. falciparum-specific genes and gene families are found in the variable subtelomeric regions (575 genes), at synteny breakpoints (42 genes), and as intrasyntenic indels (126 genes). Of the 168 non-subtelomeric P. falciparum genes, including two newly discovered gene families, 68% are predicted to be exported to the surface of the blood stage parasite or infected erythrocyte. Chromosomal rearrangements are implicated in the generation and dispersal of P. falciparum-specific gene families, including one encoding receptor-associated protein kinases. The data show that both synteny breakpoints and intrasyntenic indels can be foci for species-specific genes with a predicted role in host-parasite interactions and suggest that, besides rearrangements in the subtelomeric regions, chromosomal rearrangements may also be involved in the generation of species-specific gene families. A majority of these genes are expressed in blood stages, suggesting that the vertebrate host exerts a greater selective pressure than the mosquito vector, resulting in the acquisition of diversity.

Plasmodium vivax in southern Mexico exhibits different infectivities to 2 local mosquito vectors, Anopheles pseudopunctipennis and Anopheles albimanus. Previous work has tied these differences in mosquito infectivity to variation in the central repeat motif of the malaria parasite's circumsporozoite (csp) gene, but subsequent studies have questioned this view. Here we present evidence that P. vivax in southern Mexico comprised 3 genetic populations whose distributions largely mirror those of the 2 mosquito vectors. Additionally, laboratory colony feeding experiments indicate that parasite populations are most compatible with sympatric mosquito species. Our results suggest that reciprocal selection between malaria parasites and mosquito vectors has led to local adaptation of the parasite. Adaptation to local vectors may play an important role in generating population structure in Plasmodium. A better understanding of coevolutionary dynamics between sympatric mosquitoes and parasites will facilitate the identification of molecular mechanisms relevant to disease transmission in nature and provide crucial information for malaria control.

Trichomonas vaginalis, the most common nonviral sexually transmitted parasite, causes ∼283 million trichomoniasis infections annually and is associated with pregnancy complications and increased risk of HIV-1 acquisition. The antimicrobial drug metronidazole is used for treatment, but in a fraction of clinical cases, the parasites can become resistant to this drug. We undertook sequencing of multiple clinical isolates and lab derived lines to identify genetic markers and mechanisms of metronidazole resistance. Reduced representation genome sequencing of ∼100 T. vaginalis clinical isolates identified 3,923 SNP markers and presence of a bipartite population structure. Linkage disequilibrium was found to decay rapidly, suggesting genome-wide recombination and the feasibility of genetic association studies in the parasite. We identified 72 SNPs associated with metronidazole resistance, and a comparison of SNPs within several lab-derived resistant lines revealed an overlap with the clinically resistant isolates. We identified SNPs in genes for which no function has yet been assigned, as well as in functionally-characterized genes relevant to drug resistance (e.g., pyruvate:ferredoxin oxidoreductase). Transcription profiles of resistant strains showed common changes in genes involved in drug activation (e.g., flavin reductase), accumulation (e.g., multidrug resistance pump), and detoxification (e.g., nitroreductase). Finally, we identified convergent genetic changes in lab-derived resistant lines of Tritrichomonas foetus, a distantly related species that causes venereal disease in cattle. Shared genetic changes within and between T. vaginalis and Tr. foetus parasites suggest conservation of the pathways through which adaptation has occurred. These findings extend our knowledge of drug resistance in the parasite, providing a panel of markers that can be used as a diagnostic tool.

The small subunit ribosomal RNA (SSU rRNA) gene is a widely used molecular marker to study the diversity of life. Sequencing of SSU rRNA gene amplicons has become a standard approach for the investigation of the ecology and diversity of microbes. However, a well-curated database is necessary for correct classification of these data. While available for many groups of Bacteria and Archaea, such reference databases are absent for most eukaryotes. The primary goal of the EukRef project (eukref.org) is to close this gap and generate well-curated reference databases for major groups of eukaryotes, especially protists. Here we present a set of EukRef-curated databases for the excavate protists-a large assemblage that includes numerous taxa with divergent SSU rRNA gene sequences, which are prone to misclassification. We identified 6121 sequences, 625 of which were obtained from cultures, 3053 from cell isolations or enrichments and 2419 from environmental samples. We have corrected the classification for the majority of these curated sequences. The resulting publicly available databases will provide phylogenetically based standards for the improved identification of excavates in ecological and microbiome studies, as well as resources to classify new discoveries in excavate diversity.

Durgama Anchalare Malaria Nirakaran (DAMaN) is a multi-component malaria intervention for hard-to-reach villages in Odisha, India. The main component, malaria camps (MCs), consists of mass screening, treatment, education, and intensified vector control. We evaluated MC effectiveness using a quasi-experimental cluster-assigned stepped-wedge study with a pretest-posttest control group in 15 villages: six immediate (Arm A), six delayed (Arm B), and three previous interventions (Arm C). The primary outcome was PCR + Plasmodium infection prevalence. The time (i.e., baseline vs. follow-up 3) x study arm interaction term shows that there were statistically significant lower odds of PCR + Plasmodium infection in Arm A (AOR = 0.36, 95% CI = 0.17, 0.74) but not Arm C as compared to Arm B at the third follow-up. The cost per person ranged between US$3-8, the cost per tested US$4-9, and the cost per treated US$82-1,614, per camp round. These results suggest that the DAMaN intervention is a promising and financially feasible approach for malaria control.

Protists are ubiquitous components of terrestrial and aquatic environments, as well as animal and human microbiomes. Despite this, little is known about protists in urban environments. The ~7400-mile sewer system of New York City (NYC) collects human waste from ~8 million human inhabitants as well as from animals, street runoff, and groundwater, providing an ideal system to study these microbes. We used 18S rRNA amplicon sequencing and shotgun metagenomic sequencing to profile raw sewage microbial communities. Raw sewage samples were collected over a 12-month period from 14 treatment plants of the five NYC boroughs, and compared with samples from other environments including soil, stormwater, and sediment. Sewage contained a diverse protist community dominated by free-living clades, and communities were highly differentiated across environments. Seasonal differences in protist composition were observed; however, network analysis and functional profiling demonstrated that sewage communities were robust and functionally consistent. Protists typically associated with human and animal guts or feces were frequently detected. Abundance of these parasites varied significantly both spatially and temporally, suggesting that spikes could reflect trends in the source population. This underscores sewage as a valuable model system for monitoring patterns in urban microbes and provides a baseline protist metagenome of NYC.

P. cynomolgi, a malaria-causing parasite of Asian Old World monkeys, is the sister taxon of P. vivax, the most prevalent malaria-causing species in humans outside of Africa. Because P. cynomolgi shares many phenotypic, biological and genetic characteristics with P. vivax, we generated draft genome sequences for three P. cynomolgi strains and performed genomic analysis comparing them with the P. vivax genome, as well as with the genome of a third previously sequenced simian parasite, Plasmodium knowlesi. Here, we show that genomes of the monkey malaria clade can be characterized by copy-number variants (CNVs) in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites and CNVs in the P. cynomolgi genome, providing a map of genetic variation that can be used to map parasite traits and study parasite populations. The sequencing of the P. cynomolgi genome is a critical step in developing a model system for P. vivax research and in counteracting the neglect of P. vivax.

Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb).

Parabasalia are single-celled eukaryotes (protists) that are mainly comprised of endosymbionts of termites and wood roaches, intestinal commensals, human or veterinary parasites, and free-living species. Phylogenetic comparisons of parabasalids are typically based upon morphological characters and 18S ribosomal RNA gene sequence data (rDNA), while biochemical or molecular studies of parabasalids are limited to a few axenically cultivable parasites. These previous analyses and other studies based on PCR amplification of duplicated protein-coding genes are unable to fully resolve the evolutionary relationships of parabasalids. As a result, genetic studies of Parabasalia lag behind other organisms.