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1.  Genetic diversity of Trichomonas vaginalis reinfection in HIV-positive women 
Sexually transmitted infections  2013;89(6):473-478.
Recently developed genotyping tools allow better understanding of Trichomonas vaginalis population genetics and epidemiology. These tools have yet to be applied to T vaginalis collected from HIV+ populations, where understanding the interaction between the pathogens is of great importance due to the correlation between T vaginalis infection and HIV transmission. The objectives of the study were twofold: first, to compare the genetic diversity and population structure of T vaginalis collected from HIV+ women with parasites from reference populations; second, to use the genetic markers to perform a case study demonstrating the usefulness of these techniques in investigating the mechanisms of repeat infections.
Repository T vaginalis samples from a previously described treatment trial were genotyped at 11 microsatellite loci. Estimates of genetic diversity and population structure were determined using standard techniques and compared with previously reported estimates of global populations. Genotyping data were used in conjunction with behavioural data to evaluate mechanisms of repeat infections.
T vaginalis from HIV+ women maintain many of the population genetic characteristics of parasites from global reference populations. Although there is evidence of reduced diversity and bias towards type 1 parasites in the HIV+ population, the populations share a two-type population structure and parasite haplotypes. Genotyping/behavioural data suggest that 36% (12/33) of repeat infections in HIV+ women can be attributed to treatment failure.
T vaginalis infecting HIV+ women is not genetically distinct from T vaginalis infecting reference populations. Information from genotyping can be valuable for understanding mechanisms of repeat infections.
PMCID: PMC3755760  PMID: 23694936
2.  Microsatellite analysis of chloroquine resistance associated alleles and neutral loci reveal genetic structure of Indian Plasmodium falciparum 
Efforts to control malignant malaria caused by Plasmodium falciparum are hampered by the parasite’s acquisition of resistance to antimalarial drugs, e.g., chloroquine. This necessitates evaluating the spread of chloroquine resistance in any malaria-endemic area. India displays highly variable malaria epidemiology and also shares porous international borders with malaria-endemic Southeast Asian countries having multi-drug resistant malaria. Malaria epidemiology in India is believed to be affected by two major factors: high genetic diversity and evolving drug resistance in P. falciparum. How transmission intensity of malaria can influence the genetic structure of chloroquine-resistant P. falciparum population in India is unknown. Here, genetic diversity within and among P. falciparum populations is analyzed with respect to their prevalence and chloroquine resistance observed in 13 different locations in India. Microsatellites developed for P. falciparum, including three putatively neutral and seven microsatellites thought to be under a hitchhiking effect due to chloroquine selection were used. Genetic hitchhiking is observed in five of seven microsatellites flanking the gene responsible for chloroquine resistance. Genetic admixture analysis and F-statistics detected genetically distinct groups in accordance with transmission intensity of different locations and the probable use of chloroquine. A large genetic break between the chloroquine-resistant parasite of the Northeast-East-Island group and Southwest group (FST = 0.253, P<0.001) suggests a long period of isolation or a possibility of different origin between them. A pattern of significant isolation by distance was observed in low transmission areas (r = 0.49, P=0.003, N = 83, Mantel test). An unanticipated pattern of spread of hitchhiking suggests genetic structure for Indian P. falciparum population. Overall, the study suggests that transmission intensity can be an efficient driver for genetic differentiation at both neutral and adaptive loci across India.
PMCID: PMC3799960  PMID: 23871774
Plasmodium falciparum; India; Chloroquine resistance; Hitchhiking; Transmission area; Gene-flow
3.  Ahead of the curve: Next generation estimators of drug resistance in malaria infections 
Trends in parasitology  2013;29(7):321-328.
Drug resistance is a major obstacle for controlling infectious diseases. A key challenge is detecting the early signs of drug resistance when little is known about its genetic basis. Focusing on malaria parasites, we propose a way to do this. Newly developing or low level resistance at low frequency in patients can be detected through a phenotypic signature: individual parasite variants clearing more slowly following drug treatment. Harnessing the abundance and resolution of deep sequencing data, our ‘selection differential’ approach addresses some limitations of extant methods of resistance detection, should allow for the earliest detection of resistance in malaria or other multi-clone infections, and has the power to uncover the true scale of the drug resistance problem.
PMCID: PMC3694767  PMID: 23746748
Plasmodium; deep sequencing; parasite clearance curves; mixed infections; selection differential; artemisinin
4.  A review of malaria transmission dynamics in forest ecosystems 
Parasites & Vectors  2014;7:265.
Malaria continues to be a major health problem in more than 100 endemic countries located primarily in tropical and sub-tropical regions around the world. Malaria transmission is a dynamic process and involves many interlinked factors, from uncontrollable natural environmental conditions to man-made disturbances to nature. Almost half of the population at risk of malaria lives in forest areas. Forests are hot beds of malaria transmission as they provide conditions such as vegetation cover, temperature, rainfall and humidity conditions that are conducive to distribution and survival of malaria vectors. Forests often lack infrastructure and harbor tribes with distinct genetic traits, socio-cultural beliefs and practices that greatly influence malaria transmission dynamics. Here we summarize the various topographical, entomological, parasitological, human ecological and socio-economic factors, which are crucial and shape malaria transmission in forested areas. An in-depth understanding and synthesis of the intricate relationship of these parameters in achieving better malaria control in various types of forest ecosystems is emphasized.
PMCID: PMC4057614  PMID: 24912923
Forest malaria; Transmission dynamics; Deforestation; Vector behavior; Socio-economic factors; Tribal communities
5.  The Tc1/mariner transposable element family shapes genetic variation and gene expression in the protist Trichomonas vaginalis 
Mobile DNA  2014;5:12.
Trichomonas vaginalis is the most prevalent non-viral sexually transmitted parasite. Although the protist is presumed to reproduce asexually, 60% of its haploid genome contains transposable elements (TEs), known contributors to genome variability. The availability of a draft genome sequence and our collection of >200 global isolates of T. vaginalis facilitate the study and analysis of TE population dynamics and their contribution to genomic variability in this protist.
We present here a pilot study of a subset of class II Tc1/mariner TEs that belong to the T. vaginalis Tvmar1 family. We report the genetic structure of 19 Tvmar1 loci, their ability to encode a full-length transposase protein, and their insertion frequencies in 94 global isolates from seven regions of the world. While most of the Tvmar1 elements studied exhibited low insertion frequencies, two of the 19 loci (locus 1 and locus 9) show high insertion frequencies of 1.00 and 0.96, respectively. The genetic structuring of the global populations identified by principal component analysis (PCA) of the Tvmar1 loci is in general agreement with published data based on genotyping, showing that Tvmar1 polymorphisms are a robust indicator of T. vaginalis genetic history. Analysis of expression of 22 genes flanking 13 Tvmar1 loci indicated significantly altered expression of six of the genes next to five Tvmar1 insertions, suggesting that the insertions have functional implications for T. vaginalis gene expression.
Our study is the first in T. vaginalis to describe Tvmar1 population dynamics and its contribution to genetic variability of the parasite. We show that a majority of our studied Tvmar1 insertion loci exist at very low frequencies in the global population, and insertions are variable between geographical isolates. In addition, we observe that low frequency insertion is related to reduced or abolished expression of flanking genes. While low insertion frequencies might be expected, we identified two Tvmar1 insertion loci that are fixed across global populations. This observation indicates that Tvmar1 insertion may have differing impacts and fitness costs in the host genome and may play varying roles in the adaptive evolution of T. vaginalis.
PMCID: PMC4021607  PMID: 24834134
DNA transposable element; Mariner transposase; Trichomonas vaginalis; Population genetics; Gene expression
6.  Chloroquine efficacy studies confirm drug susceptibility of Plasmodium vivax in Chennai, India 
Malaria Journal  2014;13:129.
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.
CQ efficacy was evaluated with a 28-day in vivo therapeutic study, while CQ sensitivity was measured with an in vitro drug susceptibility assay. In both studies, isolates also underwent molecular genotyping to investigate correlations between parasite diversity and drug susceptibility to CQ. Molecular genotyping included sequencing a 604 base pair (bp) fragment of the P. vivax multidrug resistant gene-1 (Pvmdr1) for single nucleotide polymorphisms (SNPs) and also the amplification of eight microsatellite (MS) loci located across the genome on eight different chromosomes.
In the 28-day in vivo study (N=125), all subjects were aparasitaemic by Day 14. Passive case surveillance continuing beyond Day 28 in 22 subjects exposed 17 recurrent infections, which ranged from 44 to 148 days post-enrollment. Pvmdr1 sequencing of these recurrent infections revealed that 93.3% had identical mutant haplotypes (958M/Y976/1076L) to their baseline Day 0 infection. MS genotyping further revealed that nine infection pairs were related with ≥75% haplotype similarity (same allele at six or more loci). To test the impact of this mutation on CQ efficacy, an in vitro drug assay (N=68) was performed. No correlation between IC50 values and the percentage of ring-stage parasites prior to culture was observed (rsadj: -0.00063, p = 0.3307) and the distribution of alleles among the Pvmdr1 SNPs and MS haplotypes showed no significant associations with IC50 values.
Plasmodium vivax was found to be susceptible to CQ drug treatment in both the in vivo therapeutic drug study and the in vitro drug assay. Though the mutant 1076L of Pvmdr1 was found in a majority of isolates tested, this single mutation did not associate with CQ resistance. MS haplotypes revealed strong heterogeneity in this population, indicating a low probability of reinfection with highly related haplotypes.
PMCID: PMC4021252  PMID: 24685286
Plasmodium vivax; Chloroquine; In vitro; In vivo; Genetic diversity; Chennai
7.  Getting trichy: tools and approaches to interrogating Trichomonas vaginalis in a post-genome world 
Trends in parasitology  2012;29(1):17-25.
Trichomonas vaginalis is a parasite of the urogenital tract in men and women, with a worldwide presence and significant implications for global public health. T. vaginalis research entered the age of genomics with the publication of the first genome sequence in 2007, yet subsequent utilization of other ‘omics’ technologies and methods has been slow. Here, we review some of the tools and approaches available to interrogate T. vaginalis biology, with an emphasis on recent advances and current limitations, and draw attention to areas where further efforts are needed to effectively examine the complex and intriguing biology of the parasite.
PMCID: PMC3534864  PMID: 23219217
Trichomonas vaginalis; population genetics; -omics; molecular tools; evolution
8.  Genome sequence of the human malaria parasite Plasmodium falciparum 
Nature  2002;419(6906):10.1038/nature01097.
The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host–parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.
PMCID: PMC3836256  PMID: 12368864
9.  Association between Trichomonas vaginalis and vaginal bacterial community composition among reproductive-age women 
Sexually transmitted diseases  2012;39(10):807-812.
Some vaginal bacterial communities are thought to prevent infection by sexually transmitted organisms. Prior work demonstrated that the vaginal microbiota of reproductive-age women cluster into five types of bacterial communities; 4 dominated by Lactobacillus species (L. iners, L. crispatus, L. gasseri, L. jensenii), and one (termed community state type (CST) IV) lacking significant numbers of lactobacilli and characterized by higher proportions of Atopobium, Prevotella, Parvimonas, Sneathia, Gardnerella, Mobiluncus, and other taxa. We sought to evaluate the relationship between vaginal bacterial composition and Trichomonas vaginalis.
Self-collected vaginal swabs were obtained cross-sectionally from 394 women equally representing four ethnic/racial groups. T. vaginalis screening was performed using PCR targeting the 18S rRNA and β-tubulin genes. Vaginal bacterial composition was characterized by pyrosequencing of barcoded 16S rRNA genes. A panel of eleven microsatellite markers was used to genotype T. vaginalis. The association between vaginal microbiota and T. vaginalis was evaluated by exact logistic regression.
T. vaginalis was detected in 2.8% of participants (11/394). Of the eleven T. vaginalis-positive cases, eight (72%) were categorized as CST-IV, two (18%) as communities dominated by L. iners and one (9%) as L. crispatus-dominated (p-value:0.05). CST-IV microbiota were associated with an 8-fold increased odds of detecting T. vaginalis compared to women in the L. crispatus-dominated state (OR:8.26, 95% CI:1.07–372.65). Seven of the 11 T. vaginalis isolates were assigned to two genotypes.
T. vaginalis was associated with vaginal microbiota consisting of low proportions of lactobacilli and high proportions of Mycoplasma, Parvimonas, Sneathia, and other anaerobes.
PMCID: PMC3458234  PMID: 23007708
Trichomonas vaginalis; vaginal microbiota
10.  Neutral Polymorphisms in Putative Housekeeping Genes and Tandem Repeats Unravels the Population Genetics and Evolutionary History of Plasmodium vivax in India 
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.
Author Summary
Plasmodium vivax is the most prevalent human malaria parasite outside Africa, responsible for significant morbidity, although it is commonly referred to as a benign malaria agent with respect to P. falciparum. Inferring the evolutionary history of an infectious agent provides important information for designing control measures. In this study, we developed a panel of novel molecular markers including housekeeping genes, minisatellites and microsatellites to infer the evolutionary history of P. vivax on the Indian subcontinent. The panel of markers uncovered an ancient and long evolutionary history and a stable population structure of P. vivax in India.
PMCID: PMC3777877  PMID: 24069480
11.  Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade 
Nature genetics  2012;44(9):1051-1055.
Plasmodium cynomolgi, a malaria parasite of Asian Old World monkeys, is the sister taxon of Plasmodium vivax, the most prevalent human malaria species outside Africa. Since P. cynomolgi shares many phenotypic, biologic and genetic characteristics of P. vivax, we generated draft genome sequences of three P. cynomolgi strains and performed comparative genomic analysis between them and P. vivax, as well as a third previously sequenced simian parasite, Plasmodium knowlesi. Here we show that genomes of the monkey malaria clade can be characterized by 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 for mapping parasite traits and studying parasite populations. The P. cynomolgi genome is a critical step in developing a model system for P. vivax research, and to counteract the neglect of P. vivax.
PMCID: PMC3759362  PMID: 22863735
Microsatellite markers derived from simple sequence repeats have been useful in studying a number of human pathogens, including the human malaria parasite Plasmodium falciparum. Genetic markers for P. vivax would likewise help elucidate the genetics and population characteristics of this other important human malaria parasite. We have identified a locus in a P. vivax telomeric clone that contains simple sequence repeats. Primers were designed to amplify this region using a two-step semi-nested polymerase chain reaction protocol. The primers did not amplify template obtained from non-infected individuals, nor DNA from primates infected with the other human malaria parasites (P. ovale, P. malariae, or P. falciparum). The marker was polymorphic in P. vivax-infected field isolates obtained from Papua New Guinea, Indonesia and Guyana. This microsatellite marker may be useful in genetic and epidemiologic studies of P. vivax malaria.
PMCID: PMC3728893  PMID: 14640496
13.  The evolution of infectious agents in relation to sex in animals and humans: brief discussions of some individual organisms 
The following series of concise summaries addresses the evolution of infectious agents in relation to sex in animals and humans from the perspective of three specific questions: (1) what have we learned about the likely origin and phylogeny, up to the establishment of the infectious agent in the genital econiche, including the relative frequency of its sexual transmission; (2) what further research is needed to provide additional knowledge on some of these evolutionary aspects; and (3) what evolutionary considerations might aid in providing novel approaches to the more practical clinical and public health issues facing us currently and in the future?
PMCID: PMC3694397  PMID: 21824167
evolution; infectious agents; sexual transmission; econiche
14.  The malaria parasite Plasmodium vivax exhibits greater genetic diversity than Plasmodium falciparum 
Nature genetics  2012;44(9):1046-1050.
We sequenced and annotated the genomes of four Plasmodium vivax strains collected from disparate geographical locations, tripling the number of genome sequences available for this understudied parasite and providing the first genome-wide perspective of global variability within this species. We observe approximately twice as much SNP diversity among these isolates as we do among a comparable collection of isolates of Plasmodium falciparum, a malaria parasite that causes higher mortality. This indicates a distinct history of global colonization and/or a more stable demographic history for P. vivax than 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 the capacity for greater functional variation within 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.
PMCID: PMC3432710  PMID: 22863733
15.  Malaria in India: The Center for the Study of Complex Malaria in India 
Acta Tropica  2011;121(3):267-273.
Malaria is a major public health problem in India and one which contributes significantly to the overall malaria burden in Southeast Asia. The National Vector Borne Disease Control Program of India reported ~1.6 million cases and ~1100 malaria deaths in 2009. Some experts argue that this is a serious underestimation and that the actual number of malaria cases per year is likely between 9 and 50 times greater, with an approximate 13-fold underestimation of malaria-related mortality. The difficulty in making these estimations is further exacerbated by (i) highly variable malaria eco-epidemiological profiles, (ii) the transmission and overlap of multiple Plasmodium species and Anopheles vectors, (iii) increasing antimalarial drug resistance and insecticide resistance, and (iv) the impact of climate change on each of these variables. Simply stated, the burden of malaria in India is complex. Here we describe plans for a Center for the Study of Complex Malaria in India (CSCMi), one of ten International Centers of Excellence in Malaria Research (ICEMRs) located in malarious regions of the world recently funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The CSCMi is a close partnership between Indian and United States scientists, and aims to address major gaps in our understanding of the complexity of malaria in India, including changing patterns of epidemiology, vector biology and control, drug resistance, and parasite genomics. We hope that such a multidisciplinary approach that integrates clinical and field studies with laboratory, molecular, and genomic methods will provide a powerful combination for malaria control and prevention in India.
PMCID: PMC3294179  PMID: 22142788
Malaria; Plasmodium; Anopheles; India; Genomics; Epidemiology
17.  Extensive Genetic Diversity, Unique Population Structure and Evidence of Genetic Exchange in the Sexually Transmitted Parasite Trichomonas vaginalis 
Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite's genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes.
Methodology/Principal Findings
Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages.
Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease.
Author Summary
The human parasite Trichomonas vaginalis causes trichomoniasis, the world's most common non-viral sexually transmitted infection. Research on T. vaginalis genetic diversity has been limited by a lack of appropriate genotyping tools. To address this problem, we recently published a panel of T. vaginalis-specific genetic markers; here we use these markers to genotype isolates collected from ten regions around the globe. We detect high levels of genetic diversity, infer a two-type population structure, identify clinically relevant differences between the two types, and uncover evidence of genetic exchange in what was believed to be a clonal organism. Together, these results greatly improve our understanding of the population genetics of T. vaginalis and provide insights into the possibility of genetic exchange in the parasite, with implications for the epidemiology and control of the disease. By taking into account the existence of different types and their unique characteristics, we can improve understanding of the wide range of symptoms that patients manifest and better implement appropriate drug treatment. In addition, by recognizing the possibility of genetic exchange, we are more equipped to address the growing concern of drug resistance and the mechanisms by which it may spread within parasite populations.
PMCID: PMC3313929  PMID: 22479659
18.  The complexities of malaria disease manifestations with a focus on asymptomatic malaria 
Malaria Journal  2012;11:29.
Malaria is a serious parasitic disease in the developing world, causing high morbidity and mortality. The pathogenesis of malaria is complex, and the clinical presentation of disease ranges from severe and complicated, to mild and uncomplicated, to asymptomatic malaria. Despite a wealth of studies on the clinical severity of disease, asymptomatic malaria infections are still poorly understood. Asymptomatic malaria remains a challenge for malaria control programs as it significantly influences transmission dynamics. A thorough understanding of the interaction between hosts and parasites in the development of different clinical outcomes is required. In this review, the problems and obstacles to the study and control of asymptomatic malaria are discussed. The human and parasite factors associated with differential clinical outcomes are described and the management and treatment strategies for the control of the disease are outlined. Further, the crucial gaps in the knowledge of asymptomatic malaria that should be the focus of future research towards development of more effective malaria control strategies are highlighted.
PMCID: PMC3342920  PMID: 22289302
Asymptomatic malaria; Host factors; Parasite factors; Transmission dynamics
19.  Microsatellite polymorphism in the sexually transmitted human pathogen Trichomonas vaginalis indicates a genetically diverse parasite 
Given the growing appreciation of serious health sequelae from widespread Trichomonas vaginalis infection, new tools are needed to study the parasite's genetic diversity. To this end we have identified and characterized a panel of 21 microsatellites and six single-copy genes from the T. vaginalis genome, using seven laboratory strains of diverse origin. We have (1) adapted our microsatellite typing method to incorporate affordable fluorescent labeling, (2) determined that the microsatellite loci remain stable in parasites continuously cultured up to 17 months, and (3) evaluated microsatellite marker coverage of the six chromosomes that comprise the T. vaginalis genome using fluorescent in situ hybridization (FISH). We have used the markers to show that T. vaginalis is a genetically diverse parasite in a population of commonly used laboratory strains. In addition, we have used phylogenetic methods to infer evolutionary relationships from our markers in order to validate their utility in future population analyses. Our panel is the first series of robust polymorphic genetic markers for T. vaginalis that can be used to classify and monitor lab strains, as well as provide a means to measure the genetic diversity and population structure of extant and future T. vaginalis isolates.
PMCID: PMC2974001  PMID: 20813140
Population genetics; Trichomonas vaginalis; sexually transmitted infection; microsatellite; FISH; genetic markers
20.  Genome sequences reveal divergence times of malaria parasite lineages 
Parasitology  2010;138(13):1737-1749.
The evolutionary history of human malaria parasites (genus Plasmodium) has long been a subject of speculation and controversy. The complete genome sequences of the two most widespread human malaria parasites, P. falciparum and P. vivax, and of the monkey parasite P. knowlesi are now available, together with the draft genomes of the chimpanzee parasite P. reichenowi, three rodent parasites, P. yoelii yoelli, P. berghei and P. chabaudi chabaudi, and one avian parasite, P. gallinaceum.
We present here an analysis of 45 orthologous gene sequences across the eight species that resolves the relationships of major Plasmodium lineages, and provides the first comprehensive dating of the age of those groups.
Our analyses support the hypothesis that the last common ancestor of P. falciparum and the chimpanzee parasite P. reichenowi occurred around the time of the human-chimpanzee divergence. P. falciparum infections of African apes are most likely derived from humans and not the other way around. On the other hand, P. vivax, split from the monkey parasite P. knowlesi in the much more distant past, during the time that encompasses the separation of the Great Apes and Old World Monkeys.
The results support an ancient association between malaria parasites and their primate hosts, including humans.
PMCID: PMC3081533  PMID: 21118608
21.  Why Is Plasmodium vivax a Neglected Tropical Disease? 
PMCID: PMC3125139  PMID: 21738804
22.  Phylogeny of Parasitic Parabasalia and Free-Living Relatives Inferred from Conventional Markers vs. Rpb1, a Single-Copy Gene 
PLoS ONE  2011;6(6):e20774.
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.
Principal Findings
Comparing parabasalid EF1α, α-tubulin, enolase and MDH protein-coding genes with information from the Trichomonas vaginalis genome reveals difficulty in resolving the history of species or isolates apart from duplicated genes. A conserved single-copy gene encodes the largest subunit of RNA polymerase II (Rpb1) in T. vaginalis and other eukaryotes. Here we directly sequenced Rpb1 degenerate PCR products from 10 parabasalid genera, including several T. vaginalis isolates and avian isolates, and compared these data by phylogenetic analyses. Rpb1 genes from parabasalids, diplomonads, Parabodo, Diplonema and Percolomonas were all intronless, unlike intron-rich homologs in Naegleria, Jakoba and Malawimonas.
The phylogeny of Rpb1 from parasitic and free-living parabasalids, and conserved Rpb1 insertions, support Trichomonadea, Tritrichomonadea, and Hypotrichomonadea as monophyletic groups. These results are consistent with prior analyses of rDNA and GAPDH sequences and ultrastructural data. The Rpb1 phylogenetic tree also resolves species- and isolate-level relationships. These findings, together with the relative ease of Rpb1 isolation, make it an attractive tool for evaluating more extensive relationships within Parabasalia.
PMCID: PMC3111441  PMID: 21695260
23.  Hundreds of microsatellites for genotyping Plasmodium yoelii parasites 
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 (MS) 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 microsatellite (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.
PMCID: PMC2787103  PMID: 19450732
Rodent malaria parasite; Simple sequence repeat (SSR); Genetic markers; Genotyping; MS
24.  A Systems-Based Analysis of Plasmodium vivax Lifecycle Transcription from Human to Mosquito 
Up to 40% of the world's population is at risk for Plasmodium vivax malaria, a disease that imposes a major public health and economic burden on endemic countries. Because P. vivax produces latent liver forms, eradication of P. vivax malaria is more challenging than it is for P. falciparum. Genetic analysis of P. vivax is exceptionally difficult due to limitations of in vitro culture. To overcome the barriers to traditional molecular biology in P. vivax, we examined parasite transcriptional changes in samples from infected patients and mosquitoes in order to characterize gene function, define regulatory sequences and reveal new potential vaccine candidate genes.
Principal Findings
We observed dramatic changes in transcript levels for various genes at different lifecycle stages, indicating that development is partially regulated through modulation of mRNA levels. Our data show that genes involved in common biological processes or molecular machinery are co-expressed. We identified DNA sequence motifs upstream of co-expressed genes that are conserved across Plasmodium species that are likely binding sites of proteins that regulate stage-specific transcription. Despite their capacity to form hypnozoites we found that P. vivax sporozoites show stage-specific expression of the same genes needed for hepatocyte invasion and liver stage development in other Plasmodium species. We show that many of the predicted exported proteins and members of multigene families show highly coordinated transcription as well.
We conclude that high-quality gene expression data can be readily obtained directly from patient samples and that many of the same uncharacterized genes that are upregulated in different P. vivax lifecycle stages are also upregulated in similar stages in other Plasmodium species. We also provide numerous examples of how systems biology is a powerful method for determining the likely function of genes in pathogens that are neglected due to experimental intractability.
Author Summary
Most of the 250 million malaria cases outside of Africa are caused by the parasite Plasmodium vivax. Although drugs can be used to treat P. vivax malaria, drug resistance is spreading and there is no available vaccine. Because this species cannot be readily grown in the laboratory there are added challenges to understanding the function of the many hypothetical genes in the genome. We isolated transcriptional messages from parasites growing in human blood and in mosquitoes, labeled the messages and measured how their levels for different parasite growth conditions. The data for 5,419 parasite genes shows extensive changes as the parasite moves between human and mosquito and reveals highly expressed genes whose proteins might represent new therapeutic targets for experimental vaccines. We discover sets of genes that are likely to play a role in the earliest stages of hepatocyte infection. We find intriguing differences in the expression patterns of different blood stage parasites that may be related to host-response status.
PMCID: PMC2850316  PMID: 20386602
25.  Comparative genomics of the neglected human malaria parasite Plasmodium vivax 
Nature  2008;455(7214):757-763.
The human malaria parasite Plasmodium vivax is responsible for 25-40% of the ~515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated in the laboratory except in non-human primates. We determined the genome sequence of P. vivax in order to shed light on its distinctive biologic features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternate invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance scientific investigation into this neglected species.
PMCID: PMC2651158  PMID: 18843361

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