A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues

A novel method for the isolation of schistosome eggs and miracidia from livers of mice infected with Schistosoma japonicum or S. mansoni is described. The method employed collagenase B to degrade the interstitial matrix of mouse liver tissue, after which the schistosome eggs were separated from the liver cells by 2 single-step density centrifugations through Percoll. Using this procedure sufficient quantities of miracidia were obtained to generate a cDNA library. Southern blot analysis demonstrated that miracidia isolated by this method were free from contaminating host DNA

A review of mixed malaria species infections in anopheline mosquitoes

BACKGROUND: In patients with malaria mixed species infections are common and under reported. In PCR studies conducted in Asia mixed infection rates often exceed 20%. In South-East Asia, approximately one third of patients treated for falciparum malaria experience a subsequent Plasmodium vivax infection with a time interval suggesting relapse. It is uncertain whether the two infections are acquired simultaneously or separately. To determine whether mixed species infections in humans are derived from mainly from simultaneous or separate mosquito inoculations the literature on malaria species infection in wild captured anopheline mosquitoes was reviewed. METHODS: The biomedical literature was searched for studies of malaria infection and species identification in trapped wild mosquitoes and artificially infected mosquitoes. The study location and year, collection methods, mosquito species, number of specimens, parasite stage examined (oocysts or sporozoites), and the methods of parasite detection and speciation were tabulated. The entomological results in South East Asia were compared with mixed infection rates documented in patients in clinical studies. RESULTS: In total 63 studies were identified. Individual anopheline mosquitoes were examined for different malaria species in 28 of these. There were 14 studies from Africa; four with species evaluations in individual captured mosquitoes (SEICM). One study, from Ghana, identified a single mixed infection. No mixed infections were identified in Central and South America (seven studies, two SEICM). 42 studies were conducted in Asia and Oceania (11 from Thailand; 27 SEICM). The proportion of anophelines infected with Plasmodium falciparum parasites only was 0.51% (95% CI: 0.44 to 0.57%), for P. vivax only was 0.26% (95% CI: 0.21 to 0.30%), and for mixed P. falciparum and P. vivax infections was 0.036% (95% CI: 0.016 to 0.056%). The proportion of mixed infections in mosquitoes was significantly higher than expected by chance (P < 0.001), but was one fifth of that sufficient to explain the high rates of clinical mixed infections by simultaneous inoculation. CONCLUSIONS: There are relatively few data on mixed infection rates in mosquitoes from Africa. Mixed species malaria infections may be acquired by simultaneous inoculation of sporozoites from multiply infected anopheline mosquitoes but this is relatively unusual. In South East Asia, where P. vivax infection follows P. falciparum malaria in one third of cases, the available entomological information suggests that the majority of these mixed species malaria infections are acquired from separate inoculations

Overestimating resistance in field testing of malaria parasites: simple methods for estimating high EC(50 )values using a Bayesian approach

Conventional methods of assessing in-vitro antimalarial drug-concentration effect relationships in field testing of fresh isolates assess each parasite isolate individually. This leads to systematic overestimation of EC(50 )values for the most resistant isolates, and thus overestimation of the degree of resistance. In antimalarial drug-susceptibility studies conducted on the north-western border of Thailand the overestimation of EC(50 )for the most resistant isolate ranged from 15% for artesunate to 43% for mefloquine. If isolates cannot be stored for re-testing, more accurate estimations of the degree of resistance can be obtained using a Bayesian approach to data analysis which is described here

Modelling the cost-effectiveness of pulse oximetry in primary care management of acute respiratory infection in rural northern Thailand

Objectives We aimed to determine the cost-effectiveness of supplementing standard care with pulse oximetry among children <5 years with acute respiratory infection (ARI) presenting to 32 primary care units in a rural district (total population 241,436) of Chiang Rai province, Thailand, and to assess the economic effects of extending pulse oximetry to older patients with ARI in this setting. Methods We performed a model-based cost-effectiveness analysis from a health systems perspective. Decision trees were constructed for three patient categories (children <5 years, children 5–14 years, and adults), with a 1-year time horizon. Model parameters were based on data from 49,958 patients included in a review of acute infection management in the 32 primary care units, published studies, and procurement price lists. Parameters were varied in deterministic sensitivity analyses. Costs were expressed in 2021 US dollars with a willingness-to-pay threshold per DALY averted of 8624 US dollars. Results The annual direct cost of pulse oximetry, associated staff, training, and monitoring was 24,243 US dollars. It reduced deaths from severe lower respiratory tract infections in children <5 years by 0.19 per 100,000 patients annually. In our population of 14,075 children <5 years, this was equivalent to 2.0 DALYs averted per year. When downstream costs such as those related to hospitalisation and inappropriate antibiotic prescription were considered, pulse oximetry dominated standard care, saving 12,757 US dollars annually. This intervention yielded smaller mortality gains in older patients but resulted in further cost savings, primarily by reducing inappropriate antibiotic prescriptions in these age groups. The dominance of the intervention was also demonstrated in all sensitivity analyses. Conclusions Pulse oximetry is a life-saving, cost-effective adjunct in ARI primary care management in rural northern Thailand. This finding is likely to be generalisable to neighbouring countries with similar disease epidemiology and health systems

Shadows and traces in bicategories

Traces in symmetric monoidal categories are well-known and have many applications; for instance, their functoriality directly implies the Lefschetz fixed point theorem. However, for some applications, such as generalizations of the Lefschetz theorem, one needs "noncommutative" traces, such as the Hattori-Stallings trace for modules over noncommutative rings. In this paper we study a generalization of the symmetric monoidal trace which applies to noncommutative situations; its context is a bicategory equipped with an extra structure called a "shadow." In particular, we prove its functoriality and 2-functoriality, which are essential to its applications in fixed-point theory. Throughout we make use of an appropriate "cylindrical" type of string diagram, which we justify formally in an appendix.Comment: 46 pages; v2: reorganized and shortened, added proof for cylindrical string diagrams; v3: final version, to appear in JHR

Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro.

BACKGROUND: Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays. RESULTS: Morphotype was associated with survival in the presence of H2O2 and antimicrobial peptide LL-37, but not with susceptibility to acid, acidified sodium nitrite, or resistance to lysozyme, lactoferrin, human neutrophil peptide-1 or human beta defensin-2. Incubation under anaerobic conditions was a strong driver for switching of type III to an alternative morphotype. Differences were noted in the survival and replication of the three types following uptake by human macrophages, but marked strain-to strain-variability was observed. Uptake of type III alone was associated with colony morphology switching. CONCLUSIONS: Morphotype is associated with phenotypes that alter the ability of B. pseudomallei to survive in adverse environmental conditions.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The Association of Ambient Air Pollution With Cataract Surgery in UK Biobank Participants: Prospective Cohort Study

Purpose: Air pollution is associated with chronic diseases of later life. Cataract is the most common cause of blindess globally. It is biologically plausible that cataract risk is increased by pollution exposure. Therefore, the relationship between air pollution and incident cataract surgery was examined. Methods: This was a prospective, observational study involving 433,727 UK Biobank participants. Ambient air pollution measures included particulates, nitrogen dioxide (NO2) and nitrogen oxides (NOx). Outdoor air pollution was estimated based on land use regression models. Participants undergoing cataract surgery in either eye were ascertained via data linkage to the National Health Service procedure statistics. Those undergoing cataract surgery within 1 year of baseline assessment and those reporting cataract at baseline were excluded. Cox proportional hazards models were used to examine the associations between air pollutants and incident cataract surgery, adjusting for sociodemographic and lifestyle factors. Results: There were 16,307 incident cases of cataract surgery. Higher exposure to PM2.5 was associated with a 5% increased risk of incident cataract surgery (per interquartile range [IQR] increase). Compared to the lowest quartile, participants with exposures to PM2.5, NO2, and NOx in the highest quartile were 14%, 11%, and 9% more likely to undergo cataract surgery, respectively. A continuous exposure-response relationship was observed, with the likelihood of undergoing cataract surgery being progressively higher with greater levels of PM2.5, NO2, and NOx (P for trend P < 0.001). Conclusions: Although the results of our study showed a 5% increased risk of future cataract surgery following an exposure to PM2.5, NO2, and NOx, the effect estimates were relatively small. Further research is required to determine if the associations identified are causal

Spread of anti-malarial drug resistance: Mathematical model with implications for ACT drug policies

BACKGROUND: Most malaria-endemic countries are implementing a change in anti-malarial drug policy to artemisinin-based combination therapy (ACT). The impact of different drug choices and implementation strategies is uncertain. Data from many epidemiological studies in different levels of malaria endemicity and in areas with the highest prevalence of drug resistance like borders of Thailand are certainly valuable. Formulating an appropriate dynamic data-driven model is a powerful predictive tool for exploring the impact of these strategies quantitatively. METHODS: A comprehensive model was constructed incorporating important epidemiological and biological factors of human, mosquito, parasite and treatment. The iterative process of developing the model, identifying data needed, and parameterization has been taken to strongly link the model to the empirical evidence. The model provides quantitative measures of outcomes, such as malaria prevalence/incidence and treatment failure, and illustrates the spread of resistance in low and high transmission settings. The model was used to evaluate different anti-malarial policy options focusing on ACT deployment. RESULTS: The model predicts robustly that in low transmission settings drug resistance spreads faster than in high transmission settings, and treatment failure is the main force driving the spread of drug resistance. In low transmission settings, ACT slows the spread of drug resistance to a partner drug, especially at high coverage rates. This effect decreases exponentially with increasing delay in deploying the ACT and decreasing rates of coverage. In the high transmission settings, however, drug resistance is driven by the proportion of the human population with a residual drug level, which gives resistant parasites some survival advantage. The spread of drug resistance could be slowed down by controlling presumptive drug use and avoiding the use of combination therapies containing drugs with mismatched half-lives, together with reducing malaria transmission through vector control measures. CONCLUSION: This paper has demonstrated the use of a comprehensive mathematical model to describe malaria transmission and the spread of drug resistance. The model is strongly linked to the empirical evidence obtained from extensive data available from various sources. This model can be a useful tool to inform the design of treatment policies, particularly at a time when ACT has been endorsed by WHO as first-line treatment for falciparum malaria worldwide

Computational analysis of binding between malarial dihydrofolate reductases and anti-folates

BACKGROUND: Plasmodium falciparum readily develops resistance to the anti-folates pyrimethamine and proguanil via a characteristic set of mutations in the dihydrofolate reductase (PfDHFR) gene that leads to reduced competitive drug binding at the enzyme's active site. Analogous mutations can be found in the DHFR gene in isolates of Plasmodium vivax (PvDHFR) although anti-folates have not been widely used for the treatment of this infection. Here the interactions between DHFR inhibitors and modelled structures of the DHFR enzymes of Plasmodium malariae (PmDHFR) and Plasmodium ovale (PoDHFR) are described, along with an investigation of the effect of recently reported mutations within PmDHFR. METHODS: DHFR models for PmDHFR and PoDHFR were constructed using the solved PfDHFR-TS and PvDHFR structures respectively as templates. The modelled structures were docked with three DHFR inhibitors as ligands and more detailed interactions were explored via simulation of molecular dynamics. RESULTS: Highly accurate models were obtained containing sets of residues that mediate ligand binding which are highly comparable to those mediating binding in known crystal structures. Within this set, there were differences in the relative contribution of individual residues to inhibitor binding. Modelling of PmDHFR mutant sequences revealed that PmDHFR I170M was associated with a significant reduction in binding energy to all DHFR inhibitors studied, while the other predicted resistance mutations had lesser or no effects on ligand binding. CONCLUSIONS: Binding of DHFR inhibitors to the active sites of all four Plasmodium enzymes is broadly similar, being determined by an analogous set of seven residues. PmDHFR mutations found in field isolates influenced inhibitor interactions to a varying extent. In the case of the isolated I170M mutation, the loss of interaction with pyrimethamine suggests that DHFR-inhibitor interactions in P. malariae are different to those seen for DHFRs from P. falciparum and P. vivax

Protein-based signatures of functional evolution in Plasmodium falciparum

Abstract. Background: It has been known for over a decade that Plasmodium falciparum proteins are enriched in non-globular domains of unknown function. The potential for these regions of protein sequence to undergo high levels of genetic drift provides a fundamental challenge to attempts to identify the molecular basis of adaptive change in malaria parasites. Results: Evolutionary comparisons were undertaken using a set of forty P. falciparum metabolic enzyme genes, both within the hominid malaria clade (P. reichenowi) and across the genus (P. chabaudi). All genes contained coding elements highly conserved across the genus, but there were also a large number of regions of weakly or non-aligning coding sequence. These displayed remarkable levels of non-synonymous fixed differences within the hominid malaria clade indicating near complete release from purifying selection (dN/dS ratio at residues non-aligning across genus: 0.64, dN/dS ratio at residues identical across genus: 0.03). Regions of low conservation also possessed high levels of hydrophilicity, a marker of non-globularity. The propensity for such regions to act as potent sources of non-synonymous genetic drift within extant P. falciparum isolates was confirmed at chromosomal regions containing genes known to mediate drug resistance in field isolates, where 150 of 153 amino acid variants were located in poorly conserved regions. In contrast, all 22 amino acid variants associated with drug resistance were restricted to highly conserved regions. Additional mutations associated with laboratory-selected drug resistance, such as those in PfATPase4 selected by spiroindolone, were similarly restricted while mutations in another calcium ATPase (PfSERCA, a gene proposed to mediate artemisinin resistance) that reach significant frequencies in field isolates were located exclusively in poorly conserved regions consistent with genetic drift. Conclusion: Coding sequences of malaria parasites contain prospectively definable domains subject to neutral or nearly neutral evolution on a scale that appears unrivalled in biology. This distinct evolutionary landscape has potential to confound analytical methods developed for other genera. Against this tide of genetic drift, polymorphisms mediating functional change stand out to such an extent that evolutionary context provides a useful signal for identifying the molecular basis of drug resistance in malaria parasites, a finding that is of relevance to both genome-wide and candidate gene studies in this genus. © 2011 Gardner et al; licensee BioMed Central Ltd