Cytoadherence and virulence - the case of Plasmodium knowlesi malaria.

BACKGROUND: Cytoadherence of infected red blood cells to brain endothelium is causally implicated in malarial coma, one of the severe manifestations of falciparum malaria. Cytoadherence is mediated by specific binding of variant parasite antigens, expressed on the surface of infected erythrocytes, to endothelial receptors including, ICAM-1, VCAM and CD36. In fatal cases of severe falciparum malaria with coma, blood vessels in the brain are characteristically congested with infected erythrocytes. Brain sections from a fatal case of knowlesi malaria, but without coma, were similarly congested with infected erythrocytes. The objective of this study was to determine the binding phenotype of Plasmodium knowlesi infected human erythrocytes to recombinant human ICAM-1, VCAM and CD36. METHODS: Five patients with PCR-confirmed P. knowlesi malaria were recruited into the study with consent between April and August 2010. Pre-treatment venous blood was washed and cultured ex vivo to increase the proportion of schizont-infected erythrocytes. Cultured blood was seeded into Petri dishes with triplicate areas coated with ICAM-1, VCAM and CD36. Following incubation at 37°C for one hour the dishes were washed and the number of infected erythrocytes bound/mm2 to PBS control areas and to recombinant human ICAM-1 VCAM and CD36 coated areas were recorded. Each assay was performed in duplicate. Assay performance was monitored with the Plasmodium falciparum clone HB3. RESULTS: Blood samples were cultured ex vivo for up to 14.5 h (mean 11.3 ± 1.9 h) to increase the relative proportion of mature trophozoite and schizont-infected red blood cells to at least 50% (mean 65.8 ± 17.51%). Three (60%) isolates bound significantly to ICAM-1 and VCAM, one (20%) isolate bound to VCAM and none of the five bound significantly to CD36. CONCLUSIONS: Plasmodium knowlesi infected erythrocytes from human subjects bind in a specific but variable manner to the inducible endothelial receptors ICAM-1 and VCAM. Binding to the constitutively-expressed endothelial receptor CD36 was not detected. Further work will be required to define the pathological consequences of these interactions

Comparison of the airway microbiota in children with chronic suppurative lung disease

Rationale: The airway microbiota is important in chronic suppurative lung diseases (CSLD), such as primary ciliary dyskinesia (PCD) and cystic fibrosis (CF). This comparison has not previously been described but is important because difference between the two diseases may relate to the differing prognoses and lead to pathological insights and potentially, new treatments. Objectives: To compare the longitudinal development of the airway microbiota in children with PCD to that of CF and relate this to age and clinical status. Methods: Sixty-two age-matched children (age range 0.5–17 years) with PCD or CF (n=31 in each group) were recruited prospectively and followed for 1.1 years. Throat swabs or sputum as well as clinical information were collected at routine clinical appointments. 16S rRNA gene sequencing was performed. Measurements and Main Results: The microbiota was highly individual and more diverse in PCD and differed in community composition when compared with CF. Whilst Streptococcus was the most abundant genus in both conditions, Pseudomonas was more abundant in CF with Haemophilus more abundant in PCD (Padj=0.0005). In PCD only, an inverse relationship was seen in the relative abundance of Streptococcus and Haemophilus with age. Conclusions: Bacterial community composition differs between children with PCD and those with CF. Pseudomonas is more prevalent in CF and Haemophilus in PCD, at least until infection with Pseudomonas supervenes. Interactions between organisms, particularly members of Haemophilus, Streptococcus, and Pseudomonas genera appear important. Study of the interactions between these organisms may lead to new therapies or risk stratification

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

Spatial and temporal distribution of the malaria mosquito Anopheles arabiensis in northern Sudan: influence of environmental factors and implications for vector control

BACKGROUND: Malaria is an important public health problem in northern Sudan, but little is known about the dynamics of its transmission. Given the characteristic low densities of Anopheles arabiensis and the difficult terrain in this area, future vector control strategies are likely to be based on area-wide integrated pest management (AW-IPM) that may include the sterile insect technique (SIT). To support the planning and implementation of future AW-IPM activities, larval surveys were carried out to provide key data on spatial and seasonal dynamics of local vector populations. METHODS: Monthly cross-sectional larval surveys were carried out between March 2005 and May 2007 in two localities (Dongola and Merowe) adjacent to the river Nile. A stratified random sampling strategy based on the use of Remote Sensing (RS), Geographical Information Systems (GIS) and the Global Positioning System (GPS) was used to select survey locations. Breeding sites were mapped using GPS and data on larval density and breeding site characteristics were recorded using handheld computers. Bivariate and multivariate logistic regression models were used to identify breeding site characteristics associated with increased risk of presence of larvae. Seasonal patterns in the proportion of breeding sites positive for larvae were compared visually to contemporaneous data on climate and river height. RESULTS: Of a total of 3,349 aquatic habitats sampled, 321 (9.6%) contained An. arabiensis larvae. The frequency with which larvae were found varied markedly by habitat type. Although most positive sites were associated with temporary standing water around the margins of the main Nile channel, larvae were also found at brickworks and in areas of leaking pipes and canals - often far from the river. Close to the Nile channel, a distinct seasonal pattern in larval populations was evident and appeared to be linked to the rise and fall of the river level. These patterns were not evident in vector populations breeding in artificial water sources away from the river. CONCLUSION: The GIS-based survey strategy developed in this study provides key data on the population dynamics of An. arabiensis in Northern State. Quantitative estimates of the contributions of various habitat types and their proximity to settlements provide a basis for planning a strategy for reducing malaria risk by elimination of the vector population

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

Ethnic differences in Glycaemic control in people with type 2 diabetes mellitus living in Scotland

Background and Aims: Previous studies have investigated the association between ethnicity and processes of care and intermediate outcomes of diabetes, but there are limited population-based studies available. The aim of this study was to use population-based data to investigate the relationships between ethnicity and glycaemic control in men and women with diabetes mellitus living in Scotland.&lt;p&gt;&lt;/p&gt; Methods: We used a 2008 extract from the population-based national electronic diabetes database of Scotland. The association between ethnicity with mean glycaemic control in type 2 diabetes mellitus was examined in a retrospective cohort study, including adjustment for a number of variables including age, sex, socioeconomic status, body mass index (BMI), prescribed treatment and duration of diabetes.&lt;p&gt;&lt;/p&gt; Results: Complete data for analyses were available for 56,333 White Scottish adults, 2,535 Pakistanis, 857 Indians, 427 Chinese and 223 African-Caribbeans. All other ethnic groups had significantly (p&#60;0.05) greater proportions of people with suboptimal glycaemic control (HbA1c &#62;58 mmol/mol, 7.5%) compared to the White Scottish group, despite generally younger mean age and lower BMI. Fully adjusted odds ratios for suboptimal glycaemic control were significantly higher among Pakistanis and Indians (1.85, 95% CI: 1.68–2.04, and 1.62,95% CI: 1.38–1.89) respectively.&lt;p&gt;&lt;/p&gt; Conclusions: Pakistanis and Indians with type 2 diabetes mellitus were more likely to have suboptimal glycaemic control than the white Scottish population. Further research on health services and self-management are needed to understand the association between ethnicity and glycaemic control to address ethnic disparities in glycaemic control.&lt;p&gt;&lt;/p&gt

In vivo [64Cu]CuCl2 PET imaging reveals activity of Dextran-Catechin on tumor copper homeostasis

Given the strong clinical evidence that copper levels are significantly elevated in a wide spectrum of tumors, copper homeostasis is considered as an emerging target for anticancer drug design. Monitoring copper levels in vivo is therefore of paramount importance when assessing the efficacy of copper-targeting drugs. Herein, we investigated the activity of the copper-targeting compound Dextran-Catechin by developing a [64Cu]CuCl2 PET imaging protocol to monitor its effect on copper homeostasis in tumors. Methods: Protein expression of copper transporter 1 (CTR1) in tissue microarrays representing 90 neuroblastoma patient tumors was assessed by immunohistochemistry. Western blotting analysis was used to study the effect of Dextran-Catechin on the expression of CTR1 in neuroblastoma cell lines and in tumors. A preclinical human neuroblastoma xenograft model was used to study anticancer activity of Dextran-Catechin in vivo and its effect on tumor copper homeostasis. PET imaging with [64Cu]CuCl2 was performed in such preclinical neuroblastoma model to monitor alteration of copper levels in tumors during treatment. Results: CTR1 protein was found to be highly expressed in patient neuroblastoma tumors by immunohistochemistry. Treatment of neuroblastoma cell lines with Dextran-Catechin resulted in decreased levels of glutathione and in downregulation of CTR1 expression, which caused a significant decrease of intracellular copper. No changes in CTR1 expression was observed in normal human astrocytes after Dextran-Catechin treatment. In vivo studies and PET imaging analysis using the neuroblastoma preclinical model revealed elevated [64Cu]CuCl2 retention in the tumor mass. Following treatment with Dextran-Catechin, there was a significant reduction in radioactive uptake, as well as reduced tumor growth. Ex vivo analysis of tumors collected from Dextran-Catechin treated mice confirmed the reduced levels of CTR1. Interestingly, copper levels in blood were not affected by treatment, demonstrating potential tumor specificity of Dextran-Catechin activity. Conclusion: Dextran-Catechin mediates its activity by lowering CTR1 and intracellular copper levels in tumors. This finding further reveals a potential therapeutic strategy for targeting copper-dependent cancers and presents a novel PET imaging method to assess patient response to copper-targeting anticancer treatments

High prevalence of trypanosomes in European badgers detected using ITS-PCR.

BACKGROUND: Wildlife can be important sources and reservoirs for pathogens. Trypanosome infections are common in many mammalian species, and are pathogenic in some. Molecular detection tools were used to measure trypanosome prevalence in a well-studied population of wild European badgers (Meles meles). FINDINGS: A nested ITS-PCR system, that targeted the ribosomal RNA gene locus, has been widely used to detect pathogenic human and animal trypanosomes in domestic animals in Africa and some wildlife hosts. Samples from a long-term DEFRA funded capture-mark-recapture study of wild badgers at Woodchester Park (Gloucestershire, SW England) were investigated for trypanosome prevalence. A total of 82 badger blood samples were examined by nested ITS-PCR. Twenty-nine of the samples were found to be positive for trypanosomes giving a prevalence of 35.4 % (25.9 % - 46.2 %; 95 % CI). Infection was not found to be linked to badger condition, sex or age. Analysis of DNA sequence data showed the badgers to be infected with Trypanosoma (Megatrypanum) pestanai and phylogenetic analysis showed the Woodchester badger trypanosomes and T. pestanai to cluster in the Megatrypanum clade. CONCLUSIONS: The results show that the ITS Nested PCR is an effective tool for diagnosing trypanosome infection in badgers and suggests that it could be widely used in wildlife species with unknown trypanosomes or mixed infections. The relatively high prevalence observed in these badgers raises the possibility that a significant proportion of UK badgers are naturally infected with trypanosomes

A randomized, controlled, double-blind crossover study on the effects of isoeffective and isovolumetric intravenous crystalloid and gelatin on blood volume, and renal and cardiac hemodynamics

Background & aimsBlood volume expanding properties of colloids are superior to crystalloids. In addition to oncotic/osmotic properties, the electrolyte composition of infusions may have important effects on visceral perfusion, with infusions containing supraphysiological chloride causing hyperchloremic acidosis and decreased renal blood flow. In this non-inferiority study, a validated healthy human subject model was used to compare effects of colloid (4% succinylated gelatin) and crystalloid fluid regimens on blood volume, renal function, and cardiac output.MethodsHealthy male participants were given infusions over 60 min > 7 days apart in a randomized, crossover manner. Reference arm (A): 1.5 L of Sterofundin ISO, isoeffective arm (B): 0.5 L of 4% Gelaspan®, isovolumetric arm (C): 0.5 L of 4% Gelaspan® and 1 L of Sterofundin ISO (all B. Braun, Melsungen, Germany). Participants were studied over 240 min. Changes in blood volume were calculated from changes in weight and hematocrit. Renal volume, renal artery blood flow (RABF), renal cortex perfusion and diffusion, and cardiac index were measured with magnetic resonance imaging.ResultsTen of 12 males [mean (SE) age 23.9 (0.8) years] recruited, completed the study. Increase in body weight and extracellular fluid volume were significantly less after infusion B than infusions A and C, but changes in blood volume did not significantly differ between infusions. All infusions increased renal volume, with no significant differences between infusions. There was no significant difference in RABF across the infusion time course or between infusion types. Renal cortex perfusion decreased during the infusion (mean 18% decrease from baseline), with no significant difference between infusions. There was a trend for increased renal cortex diffusion (4.2% increase from baseline) for the crystalloid infusion. All infusions led to significant increases in cardiac index.ConclusionsA smaller volume of colloid (4% succinylated gelatin) was as effective as a larger volume of crystalloid at expanding blood volume, increasing cardiac output and changing renal function. Significantly less interstitial space expansion occurred with the colloid

Adaptation of in vitro cytoadherence assay to Plasmodium knowlesi field isolates

P. knowlesi was the first Plasmodium species in which antigenic variation was observed. Variation was due to schizont infected cell agglutination (SICAvar) antigens expressed by the parasite and transported to the exposed surface of the host erythrocyte [1]. PfEMP1 is P. falciparum’s orthologue of P. knowlesi’s SICA proteins [2]. In P. falciparum PfEMP1 is associated with infected erythrocytes binding to receptors such as ICAM-1 expressed on the endothelial cells of the host microvasculature. Here, we use a static protein assay [3] to determine if naturally occurring human P. knowlesi infections can cause erythrocytes to bind to ICAM-1, VCAM-1 and CD36