Plasmodium vivax Diversity and Population Structure across Four Continents

Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11 microsatellite markers was either generated or compiled from 841 isolates from four continents collected in 1999–2008. Diversity was highest in South-East Asia (mean allelic richness 10.0–12.8), intermediate in the South Pacific (8.1–9.9) Madagascar and Sudan (7.9–8.4), and lowest in South America and Central Asia (5.5–7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60–80% in Latin American populations, suggesting that typing of 2–6 markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (FST>0.2). Outside Central Asia FST values were highest (0.11–0.16) between South American and all other populations, and lowest (0.04–0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations

Single-nucleotide polymorphism, linkage disequilibrium and geographic structure in the malaria parasite Plasmodium vivax: prospects for genome-wide association studies

<p>Abstract</p> <p>Background</p> <p>The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite <it>P. vivax </it>remain little characterized.</p> <p>Results</p> <p>We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of <it>P. vivax </it>in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of <it>P. vivax</it>. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the <it>pvmdr-1 </it>locus, putatively associated with drug resistance.</p> <p>Conclusion</p> <p>These findings support the feasibility of genome-wide association studies in carefully selected populations of <it>P. vivax</it>, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.</p> <p>See commentary: <url>http://www.biomedcentral.com/1741-7007/8/90</url></p

A lactate dehydrogenase ELISA-based assay for the in vitro determination of Plasmodium berghei sensitivity to anti-malarial drugs

BACKGROUND: Plasmodium berghei rodent malaria is a well-known model for the investigation of anti-malarial drug efficacy in vivo. However, the availability of drug in vitro assays in P. berghei is reduced when compared with the spectrum of techniques existing for Plasmodium falciparum. New alternatives to the current manual or automated methods described for P. berghei are attractive. The present study reports a new ELISA drug in vitro assay for P. berghei using two monoclonal antibodies against the parasite lactate dehydrogenase (pLDH). METHODS: This procedure includes a short-in vitro culture, the purification of schizonts and the further generation of synchronized mice infections. Early stages of the parasite are then incubated against different concentrations of anti-malarial drugs using micro-plates. The novelty of this procedure in P. berghei relies on the quantification of the drug activity derived from the amount of pLDH estimated by an ELISA assay using two monoclonal antibodies: 14C1 and 19G7. The IC(50)s obtained through the ELISA assay were compared with those from the micro-test. RESULTS: The initial parameters of the synchronized samples used in the in vitro assays were a parasitaemia of 0.5% and haematocrit of 1%, with an incubation period of 22 hours at 36.5°C. pLDH detection using a 14C1 coating at 10 μg/ml and 19G7 at 2.5 × 10(-3) μg/ml provided good readouts of optical densities with low background in negative controls and specific detection levels for all parasite stages. IC(50)s values derived from the ELISA assay for artesunate, chloroquine, amodiaquine and quinine were: 15, 7, 2, and 144 nM, respectively. When artesunate and chloroquine IC(50)s were evaluated using the micro-test similar values were obtained. CONCLUSION: This ELISA-based in vitro drug assay is easy to implement, fast, and avoids the use radioisotopes or expensive equipment. The utility of this simple assay for screening anti-malarial drug activity against P. berghei in vitro is demonstrated

Transdermal Glyceryl Trinitrate as an Effective Adjunctive Treatment with Artemether for Late-Stage Experimental Cerebral Malaria

Submitted by sandra infurna ([email protected]) on 2016-02-02T15:52:41Z No. of bitstreams: 1 leonardo3_carvalho_etal_IOC_2013.pdf: 1589877 bytes, checksum: 7e3c5586b089840035cd67a91ffe7045 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-02-02T16:06:26Z (GMT) No. of bitstreams: 1 leonardo3_carvalho_etal_IOC_2013.pdf: 1589877 bytes, checksum: 7e3c5586b089840035cd67a91ffe7045 (MD5)Made available in DSpace on 2016-02-02T16:06:26Z (GMT). No. of bitstreams: 1 leonardo3_carvalho_etal_IOC_2013.pdf: 1589877 bytes, checksum: 7e3c5586b089840035cd67a91ffe7045 (MD5) Previous issue date: 2013La Jolla Bioengineering Institute. San Diego, California, USA.La Jolla Bioengineering Institute. San Diego, California, USA.La Jolla Bioengineering Institute. San Diego, California, USA / Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.La Jolla Bioengineering Institute. San Diego, California, USA.La Jolla Bioengineering Institute. San Diego, California, USA.La Jolla Bioengineering Institute. San Diego, California, USA.La Jolla Bioengineering Institute. San Diego, California, USA.La Jolla Bioengineering Institute. San Diego, California, USA / Fundação Oswaldo cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Cerebral malaria (CM) is associated with low nitric oxide (NO) bioavailability, cerebrovascular constriction, occlusion, and hypoperfusion. Administration of exogenous NO partially prevents the neurological syndrome and associated vascular pathology in an experimental CM (ECM) mouse model. In this study, we evaluated the effects of transdermal glyceryl trinitrate in preventing ECM and, in combination with artemether, rescuing late-stage ECM mice from mortality. The glyceryl trinitrate and/or artemether effect on survival and clinical recovery was evaluated in C57BL/6 mice infected with P. berghei ANKA. NO synthase (NOS) expression in mouse brain was determined by Western blots. Mean arterial pressure (MAP) and pial arteriolar diameter were monitored using a tail-cuff blood pressure system and a cranial window preparation, respectively. Preventative administration of glyceryl trinitrate at 0.025 mg/h decreased ECM mortality from 67 to 11% and downregulated inducible NOS expression in the brain. When administered as adjunctive rescue therapy with artemether, glyceryl trinitrate increased survival from 47 to 79%. The adjunctive therapy caused a sustained reversal of pial arteriolar vasoconstriction in ECM mice, an effect not observed with artemether alone. Glyceryl trinitrate induced a 13% decrease in MAP in uninfected mice but did not further affect MAP in hypotensive ECM mice. Glyceryl trinitrate, when combined with artemether, was an effective adjunctive rescue treatment for ECM. This treatment ameliorated pial arteriolar vasospasm and did not significantly affect MAP. These results indicate that transdermal glyceryl trinitrate has potential to be considered as a candidate for adjunctive therapy for CM

Nitric Oxide Synthase Dysfunction Contributes to Impaired Cerebroarteriolar Reactivity in Experimental Cerebral Malaria

Made available in DSpace on 2015-10-07T18:53:03Z (GMT). No. of bitstreams: 3 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) leonardo_carvalho_etal_IOC_2013.pdf: 1076079 bytes, checksum: 74af087d55ef339c742479670722f02d (MD5) 1_leonardo_carvalho_etal_IOC_2013.pdf: 1076079 bytes, checksum: 74af087d55ef339c742479670722f02d (MD5) Previous issue date: 2013Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA.Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA.Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA.Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA.Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA.University of California. Department of Bioengineering. San Diego, CA, USA.Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA / Fundação Oswaldo Cruz. Instituto de pesquisa Clínica Evandro Chagas. Serviço de Parasitologia. Rio de Janeiro, RJ, Brasil.Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA.Center for Malaria Research. La Jolla Bioengineering Institute. San Diego, CA, USA / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre Malária. Rio de Janeiro, RJ, Brasil.Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment

Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n = 87) and P. falciparum (n = 164) parasites from Pursat\ud province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P = 0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P = 0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.National Institutes of Health (NIH) Grant RO1 AI 075416Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Grant 470570/2006-7Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Grant 07/51199-