Dielectric characterization of Plasmodium falciparum infected red blood cells using microfluidic impedance cytometry

Although malaria is the world’s most life-threatening parasitic disease, there is no clear understanding of how certain biophysical properties of infected cells change during the malaria infection cycle. In this article, we use microfluidic impedance cytometry to measure the dielectric properties of Plasmodium falciparum-infected red blood cells (i-RBCs) at specific time-points during the infection cycle. Individual parasites were identified within i-RBCs using Green Fluorescent Protein (GFP) emission. The dielectric properties of cell sub-populations were determined using the multi-shell model. Analysis showed that the membrane capacitance and cytoplasmic conductivity of i-RBCs increased along the infection time-course, due to membrane alterations caused by parasite infection. The volume ratio occupied by the parasite was estimated to vary from <10% at earlier stages, to ~90% at later stages. This knowledge could be used to develop new label-free cell sorting techniques for sample pre-enrichment, improving diagnosis

The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression

Anopheles gambiae mosquitoes are primary human malaria vectors, but we know very little about their mechanisms of transcriptional regulation. We profiled chromatin accessibility by the assay for transposase-accessible chromatin by sequencing (ATAC-seq) in laboratory-reared A. gambiae mosquitoes experimentally infected with the human malaria parasite Plasmodium falciparum. By integrating ATAC-seq, RNA-seq and ChIP-seq data, we showed a positive correlation between accessibility at promoters and introns, gene expression and active histone marks. By comparing expression and chromatin structure patterns in different tissues, we were able to infer cis-regulatory elements controlling tissue-specific gene expression and to predict the in vivo binding sites of relevant transcription factors. The ATAC-seq assay also allowed the precise mapping of active regulatory regions, including novel transcription start sites and enhancers that were annotated to mosquito immune-related genes. Not only is this study important for advancing our understanding of mechanisms of transcriptional regulation in the mosquito vector of human malaria, but the information we produced also has great potential for developing new mosquito-control and anti-malaria strategies

The impact of storage conditions on human stool 16S rRNA microbiome composition and diversity

Background: Multiple factors can influence stool sample integrity upon sample collection. Preservation of faecal samples for microbiome studies is therefore an important step, particularly in tropical regions where resources are limited and high temperatures may significantly influence microbiota profiles. Freezing is the accepted standard to preserve faecal samples however, cold chain methods are often unfeasible in fieldwork scenarios particularly in low and middle-income countries and alternatives are required. This study therefore aimed to address the impact of different preservative methods, time-to-freezing at ambient tropical temperatures, and stool heterogeneity on stool microbiome diversity and composition under real-life physical environments found in resource-limited fieldwork conditions. Methods: Inner and outer stool samples collected from one specimen obtained from three children were stored using different storage preservation methods (raw, ethanol and RNAlater) in a Ugandan field setting. Mixed stool was also stored using these techniques and frozen at different time-to-freezing intervals post-collection from 0–32 h. Metataxonomic profiling was used to profile samples, targeting the V1–V2 regions of 16S rRNA with samples run on a MiSeq platform. Reads were trimmed, combined and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology and R software. Results: Child donor was the greatest predictor of microbiome variation between the stool samples, with all samples remaining identifiable to their child of origin despite the stool being stored under a variety of conditions. However, significant differences were observed in composition and diversity between preservation techniques, but intra-preservation technique variation was minimal for all preservation methods, and across the time-to-freezing range (0–32 h) used. Stool heterogeneity yielded no apparent microbiome differences. Conclusions: Stool collected in a fieldwork setting for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout

Quantification of female and male Plasmodium falciparum gametocytes by reverse transcriptase quantitative PCR

The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite fitness. This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female (Pfs25) and male (Pfs230p) gametocyte specific RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy. Accurate quantification of the ratio and densities of female and male gametocytes will increase understanding of P. falciparum transmission and improve the evaluation of transmission blocking interventions

Impact of repeated four-monthly anthelmintic treatment on Plasmodium infection in preschool children: a double-blind placebo-controlled randomized trial

<p>Abstract</p> <p>Background</p> <p>Helminth infections can alter susceptibility to malaria. Studies need to determine whether or not deworming programs can impact on <it>Plasmodium </it>infections in preschool children.</p> <p>Methods</p> <p>A double-blind placebo-controlled randomised trial was conducted to investigate the impact of anthelmintic treatment on <it>Plasmodium </it>infection in children aged 12-59 months. Children were randomly assigned to receive either albendazole or placebo every four months for 12 months with a follow-up at 14 months.</p> <p>Results</p> <p>320 children (out of 1228, 26.1%) complied with all the follow-up assessments. <it>Plasmodium </it>prevalence and mean <it>Plasmodium </it>parasite density was significantly higher in the treatment group (44.9% and 2319 ± SE 511) compared to the placebo group (33.3% and 1471 ± 341) at baseline. The odds of having <it>Plasmodium </it>infection increased over time for children in both the placebo and treatment groups, however this increase was significantly slower for children in the treatment group (P = 0.002). By month 14, mean <it>Plasmodium </it>density had increased by 156% in the placebo group and 98% in the treatment group but the rate of change in <it>Plasmodium </it>density was not significantly different between the groups. The change from baseline in haemoglobin had a steeper increase among children in the treatment group when compared to the placebo group but this was not statistically significant.</p> <p>Conclusions</p> <p>Repeated four-monthly anthelminthic treatments for 14 months resulted in a significantly lower increase in the prevalence of <it>Plasmodium </it>infection in preschool children which coincided with a reduction in both the prevalence and intensity of <it>A. lumbricoides </it>infections.</p> <p>Trial Registration</p> <p>Current controlled trials ISRCTN44215995</p

Alternative splicing of the Anopheles gambiae Dscam gene in diverse Plasmodium falciparum infections

Background: In insects, including Anopheles mosquitoes, Dscam (Down syndrome cell adhesion molecule) appears to be involved in phagocytosis of pathogens, and shows pathogen-specific splice-form expression between divergent pathogen (or parasite) types (e.g. between bacteria and Plasmodium or between Plasmodium berghei and Plasmodium falciparum). Here, data are presented from the first study of Dscam expression in response to genetic diversity within a parasite species. Methods: In independent field and laboratory studies, a measure of Dscam splice-form diversity was compared between mosquitoes fed on blood that was free of P. falciparum to mosquitoes exposed to either single or mixed genotype infections of P. falciparum. Results: Significant increases in Anopheles gambiae Dscam (AgDscam) receptor diversity were observed in parasite-exposed mosquitoes, but only weak evidence that AgDscam diversity rises further upon exposure to mixed genotype parasite infections was found. Finally, a cluster of AgDscam exon 4 variants that become especially common during Plasmodium invasion was identified. Conclusions: While the data clearly indicate that AgDscam diversity increases with P. falciparum exposure, they do not suggest that AgDscam diversity rises further in response to increased parasite diversit

Host candidate gene polymorphisms and clearance of drug-resistant Plasmodium falciparum parasites

Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches

N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60

Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1)-Pro(223)) and N2 (Gly(224)-Gly(411)), and the ADH region contains C1 (Gly(412)-Val(648)) and C2 (Pro(649)-Val(866)). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.The N2 subdomain exhibited the greatest affinity for Hsp60 with a K(D) of 9.50±2.6 nM. The K(D) of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies

Information use and plasticity in the reproductive decisions of malaria parasites

BACKGROUND: Investment in the production of transmissible stages (gametocytes) and their sex ratio are malaria parasite traits that underpin mosquito infectivity and are therefore central to epidemiology. Malaria parasites adjust their levels of investment into gametocytes and sex ratio in response to changes in the in-host environment (including red blood cell resource availability, host immune responses, competition from con-specific genotypes in mixed infections, and drug treatment). This plasticity appears to be adaptive (strategic) because parasites prioritize investment (in sexual versus asexual stages and male versus female stages) in manners predicted to maximize fitness. However, the information, or ‘cues’ that parasites use to detect environmental changes and make appropriate decisions about investment into gametocytes and their sex ratio are unknown. METHODS: Single genotype Plasmodium chabaudi infections were exposed to ‘cue’ treatments consisting of intact or lysed uninfected red blood cells, lysed parasitized RBCs of the same clone or an unrelated clone, and an unmanipulated control. Infection dynamics (proportion of reticulocytes, red blood cell and asexual stage parasite densities) were monitored, and changes in gametocyte investment and sex ratio in response to cue treatments, applied either pre- or post-peak of infection were examined. RESULTS AND CONCLUSIONS: A significant reduction in gametocyte density was observed in response to the presence of lysed parasite material and a borderline significant increase in sex ratio (proportion of male gametocytes) upon exposure to lysed red blood cells (both uninfected and infected) was observed. Furthermore, the changes in gametocyte density and sex ratio in response to these cues depend on the age of infection. Demonstrating that variation in gametocyte investment and sex ratio observed during infections are a result of parasite strategies (rather than the footprint of host physiology), provides a foundation to investigate the fitness consequences of plasticity and explore whether drugs could be developed to trick parasites into making suboptimal decisions