Population genetic structure and genomic divergence in Plasmodium knowlesi

Plasmodium knowlesi infections in humans have been increasingly seen in many countries across Southeast Asia, with cases mainly concentrated in Malaysia, since a major focus of infections was first described in Malaysian Borneo over 10 years ago. Clinical presentations show a wide spectrum of illness from mild to fatal, with the possible occurrence of asymptomatic infections. Two monkey species have been identified as the chief reservoir hosts; long-­‐tailed macaque (Macaca fascicularis) and pig-­‐tailed macaque (M. nemestrina). In order to explore the transmission of P. knowlesi infections, it is important to study the population genetic structure of this parasite. To address this, a microsatellite genotyping toolkit consisting of 10 loci specific for P. knowlesi was developed and validated. Using these highly polymorphic markers, analysis of more than 500 P. knowlesi infections from human and wild macaque hosts across Malaysian Borneo and humans of peninsular Malaysia showed remarkable population genetic structure. Human clinical isolates were shown to comprise highly divergent subpopulations, respectively associated with forest-­‐dwelling long-­‐tailed macaque (Cluster 1) and pig-­‐tailed macaque (Cluster 2) reservoir hosts. After analysis of initial whole genome sequence data, re-­‐assessment of population genetic structure was undertaken by microsatellite analysis of more samples from wild macaques and humans from peninsular Malaysia, showing profound geographical divergence between Borneo (sympatric Cluster 1 and Cluster 2) and mainland peninsular Malaysia (Cluster 3). The overall three major subpopulations demonstrated by microsatellite 4 analyses matched the analysis inferred by the genome-­‐wide sequence analysis of clinical isolates. To allow further investigation of variation in genome-­‐wide divergence between the sympatric subpopulations in Borneo, a simple laboratory kit consisting of allele-­‐specific PCR assays was developed to distinguish the two subpopulations. This eased in identifying to which subpopulations P. knowlesi infections belonged, and subsequently generating more genome-­‐wide sequences for comparative study. Further analyses revealed remarkable heterogeneity in the level of divergence between the sympatric subpopulation across the genome. Genomic architectures showed 20 high-­‐divergence regions scattered in different chromosomes. These findings suggest independent adaption of parasites in different macaque hosts that persist sympatrically

Severe malaria - a case of fatal Plasmodium knowlesi infection with post-mortem findings: a case report.

BACKGROUND: Zoonotic malaria caused by Plasmodium knowlesi is an important, but newly recognized, human pathogen. For the first time, post-mortem findings from a fatal case of knowlesi malaria are reported here. CASE PRESENTATION: A formerly healthy 40 year-old male became symptomatic 10 days after spending time in the jungle of North Borneo. Four days later, he presented to hospital in a state of collapse and died within two hours. He was hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values; he was also thrombocytopenic and eosinophilic. Dengue haemorrhagic shock was suspected and a post-mortem examination performed. Investigations for dengue virus were negative. Blood for malaria parasites indicated hyperparasitaemia and single species P. knowlesi infection was confirmed by nested-PCR. Macroscopic pathology of the brain and endocardium showed multiple petechial haemorrhages, the liver and spleen were enlarged and lungs had features consistent with ARDS. Microscopic pathology showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. Brain sections were negative for intracellular adhesion molecule-1. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis and endothelial cells in heart sections were prominent. CONCLUSIONS: The overall picture in this case was one of systemic malaria infection that fit the WHO classification for severe malaria. Post-mortem findings in this case were unexpectedly similar to those that define fatal falciparum malaria, including cerebral pathology. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain. These results suggest that further study of knowlesi malaria will aid the interpretation of, often conflicting, information on malaria pathophysiology in humans

Contribution of Plasmodium knowlesi to Multispecies Human Malaria Infections in North Sumatera, Indonesia.

Background: As Indonesia works toward the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in 3 communities in North Sumatera Province. Methods: A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi. Results: Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%). Conclusions: Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities

Magnetism, Spin-Orbit Coupling, and Superconducting Pairing in UGe2_2

A consistent picture on the mean-field level of the magnetic properties and electronic structure of the superconducting itinerant ferromagnet UGe$_2$ is shown to require inclusion of correlation effects beyond the local density approximation (LDA). The "LDA+U" approach reproduces both the magnitude of the observed moment, composed of strongly opposing spin and orbital parts, and the magnetocrystalline anisotropy. The largest Fermi surface sheet is comprised primarily of spin majority states with orbital projection $m_{\ell}$=0, suggesting a much simpler picture of the pairing than is possible for general strong spin-orbit coupled materials. This occurrence, and the quasi-two-dimensional geometry of the Fermi surface, support the likelihood of magnetically mediated p-wave triplet pairing.Comment: accepted for publication in Phys. Rev. Lett; URL for better quality image of Fig.3 (2MB) at http://yammer.ucdavis.edu/public/UGe2/fig3.ep

Análise da acurácia dos métodos cinemáticos de posicionamento GPS em aplicações costeiras

O NAVSTAR GPS vem sendo aplicado nas mais diversas áreas entre elas encontram-se o ambiente costeiro. Os levantamentos geodésicos podem ser utilizados para estudar as variações posicionais que ocorrem em sistemas costeiros dinâmicos. O presente trabalho tem por objetivo avaliar a acurácia dos métodos cinemáticos de posicionamento GPS em um esporão arenoso localizado na Ilha do Mel, município de Paranaguá, Estado do Paraná, Brasil. Os testes foram realizados com os seguintes métodos de posicionamento geodésico: Absoluto Cinemático (MAC), Relativo Cinemático (MRC) e Diferencial (RTK e DGPS). Uma estação móvel composta por dois receptores GPS foi construída para efetuar simultaneamente a captação de dados e assim analisar os métodos propostos em um único levantamento geodésico. Os resultados mais satisfatórios foram oriundos da solução RTK, apresentando os menores valores para o Erro Médio Quadrático (EMQ), 0,003 m, 0,007 m e 0,149 m nas componentes Norte (N), Este (E) e Altura Geométrica h, respectivamente. Por fim, recomendações da aplicabilidade dos métodos estudados para a área costeira são apresentadas

Contribution of Plasmodium knowlesi to multi-species human malaria infections in North Sumatera, Indonesia

Background As Indonesia works towards the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in three communities in North Sumatera Province. Methods A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified by nested PCR of rRNA genes, and a novel assay which amplifies a conserved sequence specific for the sicavar gene family of P. knowlesi. Results 614 of 3,731 participants (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1,169 individuals (31.3%). In total, 377 participants (11.8%) harboured P. knowlesi. Also present were P. vivax (14.3%), P. falciparum (10.5%) and P. malariae (3.4%). Conclusions Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multi-species parasitaemia is relatively common in North Sumatera, and so PCR-based surveillance is required to support control and elimination activities

Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism.

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology

Disease progression in Plasmodium knowlesi malaria is linked to variation in invasion gene family members.

Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥ 0.34, p =  <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region

Impaired Brain Dopamine and Serotonin Release and Uptake in Wistar Rats Following Treatment with Carbotplatin

Chemotherapy-induced cognitive impairment, known also as “chemobrain”, is a medical complication of cancer treatment that is characterized by a general decline in cognition affecting visual and verbal memory, attention, complex problem solving skills, and motor function. It is estimated that one-third of patients who undergo chemotherapy treatment will experience cognitive impairment. Alterations in the release and uptake of dopamine and serotonin, central nervous system neurotransmitters that play important roles in cognition, could potentially contribute to impaired intellectual performance in those impacted by chemobrain. To investigate how chemotherapy treatment affects these systems, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to measure dopamine and serotonin release and uptake in coronal brain slices containing the striatum and dorsal raphe nucleus, respectively. Measurements were taken from rats treated weekly with selected doses of carboplatin and from control rats treated with saline. Modeling the stimulated dopamine release plots revealed an impairment of dopamine release per stimulus pulse (80% of saline control at 5 mg/kg and 58% at 20 mg/kg) after 4 weeks of carboplatin treatment. Moreover, Vmax, the maximum uptake rate of dopamine, was also decreased (55% of saline control at 5 mg/kg and 57% at 20 mg/kg). Nevertheless, overall dopamine content, measured in striatal brain lysates by high performance liquid chromatography, and reserve pool dopamine, measured by FSCV after pharmacological manipulation, did not significantly change, suggesting that chemotherapy treatment selectively impairs the dopamine release and uptake processes. Similarly, serotonin release upon electrical stimulation was impaired (45% of saline control at 20 mg/kg). Measurements of spatial learning discrimination were taken throughout the treatment period and carboplatin was found to alter cognition. These studies support the need for additional neurochemical and behavioral analyses to identify the underlying mechanisms of chemotherapy-induced cognitive disorders