The efficacy of the analgesic GlyT2 inhibitor, ORG25543, is determined by two connected allosteric sites.

Glycine Transporter 2 (GlyT2) inhibitors have shown considerable potential as analgesics for the treatment of neuropathic pain but also display considerable side effects. One potential source of side effects is irreversible inhibition. In this study, we have characterized the mechanism of ORG25543 inhibition of GlyT2 by first considering three potential ligand binding sites on GlyT2-the substrate site, the vestibule allosteric site and the lipid allosteric site. The three sites were tested using a combination of molecular dynamics simulations and analysis of the inhibition of glycine transport of a series point mutated GlyT2 using electrophysiological methods. We demonstrate that the lipid allosteric site on GlyT2 is the most likely binding site for ORG25543. We also demonstrate that cholesterol derived from the cell membrane can form specific interactions with inhibitor-bound transporters to form an allosteric network of regulatory sites. These observations will guide the future design of GlyT2 inhibitors with the objective of minimising on-target side effects and improving the therapeutic window for the treatment of patients suffering from neuropathic pain.

The burden of submicroscopic and asymptomatic malaria in India revealed from epidemiology studies at three varied transmission sites in India.

Malaria in India, while decreasing, remains a serious public health problem, and the contribution of submicroscopic and asymptomatic infections to its persistence is poorly understood. We conducted community surveys and clinic studies at three sites in India differing in their eco-epidemiologies: Chennai (Tamil Nadu), Nadiad (Gujarat), and Rourkela (Odisha), during 2012-2015. A total of 6,645 subject blood samples were collected for Plasmodium diagnosis by microscopy and PCR, and an extensive clinical questionnaire completed. Malaria prevalence ranged from 3-8% by PCR in community surveys (24 infections in Chennai, 56 in Nadiad, 101 in Rourkela), with Plasmodium vivax dominating in Chennai (70.8%) and Nadiad (67.9%), and Plasmodium falciparum in Rourkela (77.3%). A proportional high burden of asymptomatic and submicroscopic infections was detected in community surveys in Chennai (71% and 71%, respectively, 17 infections for both) and Rourkela (64% and 31%, 65 and 31 infections, respectively). In clinic studies, a proportional high burden of infections was identified as submicroscopic in Rourkela (45%, 42 infections) and Chennai (19%, 42 infections). In the community surveys, anemia and fever were significantly more common among microscopic than submicroscopic infections. Exploratory spatial analysis identified a number of potential malaria hotspots at all three sites. There is a considerable burden of submicroscopic and asymptomatic malaria in malarious regions in India, which may act as a reservoir with implications for malaria elimination strategies.

Characterizing Antibody Responses to Plasmodium vivax and Plasmodium falciparum Antigens in India Using Genome-Scale Protein Microarrays.

Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.

The use of mosquito repellents at three sites in India with declining malaria transmission: surveys in the community and clinic.

BACKGROUND: Repellents such as coils, vaporizers, mats and creams can be used to reduce the risk of malaria and other infectious diseases. Although evidence for their effectiveness is limited, they are advertised as providing an additional approach to mosquito control in combination with other strategies, e.g. insecticide-treated nets. We examined the use of repellents in India in an urban setting in Chennai (mainly Plasmodium vivax malaria), a peri-urban setting in Nadiad (both P. vivax and P. falciparum malaria), and a more rural setting in Raurkela (mainly P. falciparum malaria). METHODS: The use of repellents was examined at the household level during a census, and at the individual level in cross-sectional surveys and among patients visiting a clinic with fever or other symptoms. Factors associated with their use were examined in a multivariate analysis, and the association between malaria and the use of repellents was assessed among survey- and clinic participants. RESULTS: Characteristics of participants differed by region, with more people of higher education present in Chennai. Use of repellents varied between 56-77 % at the household level and between 32-78 % at the individual level. Vaporizers were the main repellents used in Chennai, whereas coils were more common in Nadiad and Raurkela. In Chennai and Nadiad, vaporizers were more likely to be used in households with young male children. Vaporizer use was associated with higher socio-economic status (SES) in households in Chennai and Nadiad, whereas use of coils was greater in the lower SES strata. In Raurkela, there was a higher use of coils among the higher SES strata. Education was associated with the use of a repellent among survey participants in Chennai and clinic study participants in Chennai and Nadiad. Repellent use was associated with less malaria in the clinic study in Chennai and Raurkela, but not in the surveys, with the exception of the use of coils in Nadiad. CONCLUSIONS: Repellents are widely used in India. Their use is influenced by the level of education and SES. Information on effectiveness and guidance on choices may improve rational use.

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax.

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi.

Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical. We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes.

What is the value of reactive case detection in malaria control? A case-study in India and a systematic review.

BACKGROUND: Reactive case detection (RCD) for malaria is a strategy to identify additional malaria infections in areas of low malaria transmission and can complement passive surveillance. This study describes experiences with RCD in two Indian sites, and aimed to synthesize experiences with RCD across endemic countries. METHODS: RCD programmes were piloted in two urban areas of India with a low prevalence of mainly Plasmodium vivax malaria in 2014. Cases were identified in a clinic by microscopy and contacts were screened within 2 weeks; PCR, in addition to microscopy, was used to detect Plasmodium parasites. A systematic review was conducted to identify RCD experiences in the literature. RESULTS: In Chennai, 868 contacts were enrolled for 18 index cases of clinical malaria; in Nadiad, 131 contacts were enrolled for 20 index cases. No new malaria infections were detected in Nadiad among contacts, and four new infections were detected in Chennai (three P. vivax and one Plasmodium falciparum), of which two were among household members of index cases. An additional five studies describing results from an RCD strategy were identified in the literature: four in Africa and one in Thailand. Including the results from India, the average number of contacts screened per index case in a total of seven studies ranged from four to 50, and 126 in a case study in Thailand with one index case. Malaria was detected in 0-45 % of the contacted persons. The average number of index cases needed to be traced to find one new case of malaria ranged from one to five, and could not be assessed in one study in India (no contacts positive for 20 cases). Sharing the household with an index case was associated with a five-fold increased risk of malaria compared to contacts from households without an index case (pooled risk ratio 5.29, 95 % CI 3.31-8.47, I(2) 0 %, four studies). CONCLUSIONS: RCD in areas of low malaria transmission is a labour-intensive strategy, and its benefit is not clear. Studies are needed to assess how RCD can be optimized or into alternatives where interventions are targeted to family members or hotspots.

Malaria Diagnosis Across the International Centers of Excellence for Malaria Research: Platforms, Performance, and Standardization.

Diagnosis is "the act of identifying a disease, illness, or problem by examining someone or something." When an individual with acute fever presents for clinical attention, accurate diagnosis leading to specific, prompt treatment often saves lives. As applied to malaria, not only individual patient diagnosis is important but also assessing population-level malaria prevalence using appropriate diagnostic methods is essential for public health purposes. Similarly, identifying (diagnosing) fake antimalarial medications prevents the use of counterfeit drugs that can have disastrous effects. Therefore, accurate diagnosis in broad areas related to malaria is fundamental to improving health-care delivery, informing funding agencies of current malaria situations, and aiding in the prioritization of regional and national control efforts. The International Centers of Excellence for Malaria Research (ICEMR), supported by the U.S. National Institute of Allergy and Infectious Diseases, has collaborated on global efforts to improve malaria diagnostics by working to harmonize and systematize procedures across different regions where endemicity and financial resources vary. In this article, the different diagnostic methods used across each ICEMR are reviewed and challenges are discussed.

Sustainable in-well vapor stripping: A design, analytical model, and pilot study for groundwater remediation.

A sustainable in-well vapor stripping system is designed as a cost-effective alternative for remediation of shallow chlorinated solvent groundwater plumes. A solar-powered air compressor is used to inject air bubbles into a monitoring well to strip volatile organic compounds from a liquid to vapor phase while simultaneously inducing groundwater circulation around the well screen. An analytical model of the remediation process is developed to estimate contaminant mass flow and removal rates. The model was calibrated based on a one-day pilot study conducted in an existing monitoring well at a former dry cleaning site. According to the model, induced groundwater circulation at the study site increased the contaminant mass flow rate into the well by approximately two orders of magnitude relative to ambient conditions. Modeled estimates for 5h of pulsed air injection per day at the pilot study site indicated that the average effluent concentrations of dissolved tetrachloroethylene and trichloroethylene can be reduced by over 90% relative to the ambient concentrations. The results indicate that the system could be used cost-effectively as either a single- or multi-well point technology to substantially reduce the mass of dissolved chlorinated solvents in groundwater.

Chloroquine efficacy studies confirm drug susceptibility of Plasmodium vivax in Chennai, India.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (r(sadj): -0.00063, p = 0.3307) and the distribution of alleles among the Pvmdr1 SNPs and MS haplotypes showed no significant associations with IC50 values. CONCLUSIONS: 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 1076 L 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.

A call to arms: on refining Plasmodium vivax microsatellite marker panels for comparing global diversity.

BACKGROUND: Microsatellite (MS) markers have become an important tool for studying the population diversity, evolutionary history and multiplicity of infection (MOI) of malaria parasite infections. MS are typically selected on the basis of being highly polymorphic. However, it is known that the polymorphic potential (mutability) of each marker can vary as much as two orders of magnitude, which radically changes how diversity is represented in the genome from one marker to the next. Over the past decade, approximately 240 Plasmodium vivax MS have been published, comprising nine major panels of markers. Inconsistent usage of each panel has resulted in a surfeit of descriptive genetic diversity data that are largely incomparable between populations. The objective of this study was to statistically evaluate the quality of individual MS markers in order to validate a refined panel of markers that will provide a balanced picture of P. vivax population diversity. METHODS: All previously published data, including genetic diversity indices, MS parameters, and population parameters, were assembled from 18 different global studies into a flat file to facilitate statistical analysis and modelling using JMP® Genomics 6.0 (SAS Institute Inc, Cary, NC, USA). Statistical modeling was employed to down-select markers with extreme variation among the mean number of alleles, expected heterozygosity, maximum repeat length and/or chromosomal location of the repeat. Individual MS were analysed by step-down whole model linear regression and standard least squares fit models, both stratified by annual parasite incidence to identify MS markers with values significantly different from the mean. RESULTS: Of the 42 MS under evaluation in this study, 18 (nine high priority) were identified as ideal candidates for measuring population diversity between global regions, while five (two high priority) additional markers were identified as candidates for MOI studies. CONCLUSIONS: MS diversity was found to be a function of endemicity and motif structure. Evaluation of individual MS permitted the assembly of a refined panel of markers that can be reliably utilized in the field to compare population structures between global regions.

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.

The malaria parasite Plasmodium vivax exhibits greater genetic diversity than Plasmodium falciparum.

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.

Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade.

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.

The complexities of malaria disease manifestations with a focus on asymptomatic malaria.

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.

Quantification of Plasmodium falciparum malaria from complex infections in the Peruvian Amazon using quantitative PCR of the merozoite surface protein 1, block 2 (PfMSP1-B2): in vitro dynamics reveal density-dependent interactions.

The majority of Plasmodium falciparum field isolates are defined as complex infections because they contain multiple genetically distinct clones. Studying interactions between clones in complex infections in vivo and in vitro could elucidate important phenomena in malaria infection, transmission and treatment. Using quantitative PCR (qPCR) of the P. falciparum merozoite surface protein 1, block 2 (PfMSP1-B2), we provide a sensitive and efficient genotyping method. This is important for epidemiological studies because it makes it possible to study genotype-specific growth dynamics. We compared 3 PfMSP1-B2 genotyping methods by analysing 79 field isolates from the Peruvian Amazon. In vivo observations from other studies using these techniques led to the hypothesis that clones within complex infections interact. By co-culturing clones with different PfMSP1-B2 genotypes, and measuring parasitaemia using qPCR, we found that suppression of clonal expansion was a factor of the collective density of all clones present in a culture. PfMSP1-B2 qPCR enabled us to find in vitro evidence for parasite-parasite interactions and could facilitate future investigations of growth trends in naturally occurring complex infections.

Mutant pfcrt "SVMNT" haplotype and wild type pfmdr1 "N86" are endemic in Plasmodium vivax dominated areas of India under high chloroquine exposure.

BACKGROUND: Chloroquine resistance (CQR) phenotype in Plasmodium falciparum is associated with mutations in pfcrt and pfmdr-1 genes. Mutations at amino acid position 72-76 of pfcrt gene, here defined as pfcrt haplotype are associated with the geographic origin of chloroquine resistant parasite. Here, mutations at 72-76 and codon 220 of pfcrt gene and N86Y pfmdr-1 mutation were studied in blood samples collected across 11 field sites, inclusive of high and low P. falciparum prevalent areas in India. Any probable correlation between these mutations and clinical outcome of CQ treatment was also investigated. METHODS: Finger pricked blood spotted on Whatman No.3 papers were collected from falciparum malaria patients of high and low P. falciparum prevalent areas. For pfcrt haplotype investigation, the parasite DNA was extracted from blood samples and used for PCR amplification, followed by partial sequencing of the pfcrt gene. For pfmdr-1 N86Y mutation, the PCR product was subjected to restriction digestion with AflIII endonuclease enzyme. RESULTS: In 240 P. falciparum isolates with reported in vivo CQ therapeutic efficacy, the analysis of mutations in pfcrt gene shows that mutant SVMNT-S (67.50%) and CVIET-S (23.75%) occurred irrespective of clinical outcome and wild type CVMNK-A (7.91%) occurred only in adequate clinical and parasitological response samples. Of 287 P. falciparum isolates, SVMNTS 192 (66.89%) prevailed in all study sites and showed almost monomorphic existence (98.42% isolates) in low P. falciparum prevalent areas. However, CVIETS-S (19.51%) and CVMNK-A (11.84%) occurrence was limited to high P. falciparum prevalent areas. Investigation of pfmdr-1 N86Y mutation shows no correlation with clinical outcomes. The wild type N86 was prevalent in all the low P. falciparum prevalent areas (94.48%). However, mutant N86Y was comparably higher in numbers at the high P. falciparum prevalent areas (42.76%). CONCLUSIONS: The wild type pfcrt gene is linked to chloroquine sensitivity; however, presence of mutation cannot explain the therapeutic efficacy of CQ in the current scenario of chloroquine resistance. The monomorphic existence of mutant SVMNT haplotype, infer inbreeding and faster spread of CQR parasite in areas with higher P. vivax prevalance and chloroquine exposure, whereas, diversity is maintained in pfcrt gene at high P. falciparum prevalent areas.

Malaria in India: the center for the study of complex malaria in India.

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.

Sexual recombination is a signature of a persisting malaria epidemic in Peru.

BACKGROUND: The aim of this study was to consider the impact that multi-clone, complex infections have on a parasite population structure in a low transmission setting. In general, complexity of infection (minimum number of clones within an infection) and the overall population level diversity is expected to be minimal in low transmission settings. Additionally, the parasite population structure is predicted to be clonal, rather than sexual due to infrequent parasite inoculation and lack of recombination between genetically distinct clones. However, in this low transmission of the Peruvian Amazon, complex infections are becoming more frequent, in spite of decreasing infection prevalence. In this study, it was hypothesized that sexual recombination between distinct clonal lineages of Plasmodium falciparum parasites were altering the subpopulation structure and effectively maintaining the population-level diversity. METHODS: Fourteen microsatellite markers were chosen to describe the genetic diversity in 313 naturally occurring P. falciparum infections from Peruvian Amazon. The population and subpopulation structure was characterized by measuring: clusteredness, expected heterozygosity (He), allelic richness, private allelic richness, and linkage disequilibrium. Next, microsatellite haplotypes and alleles were correlated with P. falciparum merozoite surface protein 1 Block 2 (Pfmsp1-B2) to examine the presence of recombinant microsatellite haplotypes. RESULTS: The parasite population structure consists of six genetically diverse subpopulations of clones, called "clusters". Clusters 1, 3, 4, and 6 have unique haplotypes that exceed 70% of the total number of clones within each cluster, while Clusters 2 and 5 have a lower proportion of unique haplotypes, but still exceed 46%. By measuring the He, allelic richness, and private allelic richness within each of the six subpopulations, relatively low levels of genetic diversity within each subpopulation (except Cluster 4) are observed. This indicated that the number of alleles, and not the combination of alleles, are limited. Next, the standard index of association (IAS) was measured, which revealed a significant decay in linkage disequilibrium (LD) associated with Cluster 6, which is indicative of independent assortment of alleles. This decay in LD is a signature of this subpopulation approaching linkage equilibrium by undergoing sexual recombination. To trace possible recombination events, the two most frequent microsatellite haplotypes observed over time (defined by either a K1 or Mad20) were selected as the progenitors and then potential recombinants were identified in within the natural population. CONCLUSIONS: Contrary to conventional low transmission models, this study provides evidence of a parasite population structure that is superficially defined by a clonal backbone. Sexual recombination does occur and even arguably is responsible for maintaining the substructure of this population.

The Stark Law in retrospect.

Considering the ultimate goals of preventing the over-utilization of medical services and protecting the Medicare program, are the numerous phases of the Stark Law and their concomitant regulations effective; or, conversely, has the legislation served to impede entrepreneurialism among physicians to the detriment of innovations and better integration in the delivery of medical treatment? This article endeavors to answer the above question through an analysis of the policy goals behind the legislation; the evolution of its regulations; its effect on competitiveness in the field of medicine; and the ethical considerations implicated by the issue of physician self-referral. It further offers some proposals which attempt to address the problem of physician self-referral abuse while at the same time reducing the complexity and breadth of the Stark law and its regulations. The article concludes by noting that to truly change the practice of inappropriate self-referral as well as the culture of over-utilization, it is necessary not only to target specific relationships and practices prone to abuse, but to realign the financial incentives created by our current payment mechanisms as well.