Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

Background: The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. Methods: DNA was extracted from two RDT devices (Paracheck-PfW and SD Bioline Malaria Pf/Pan (R)), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman (R) 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. Results: The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. Conclusions: The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.ACT Consortium through Bill and Melinda Gates Foundation; Swedish International Development Agency (SIDA) [SWE 2009-193]; Swedish Civil Contingencies Agency (MSB) [2010-7991]; Swedish Medical Research Council (VR) [2009-3785]; Goljes Foundationinfo:eu-repo/semantics/publishedVersio

Direct 3D Tomographic Reconstruction and Phase-Retrieval of Far-Field Coherent Diffraction Patterns

We present an alternative numerical reconstruction algorithm for direct tomographic reconstruction of a sample refractive indices from the measured intensities of its far-field coherent diffraction patterns. We formulate the well-known phase-retrieval problem in ptychography in a tomographic framework which allows for simultaneous reconstruction of the illumination function and the sample refractive indices in three dimensions. Our iterative reconstruction algorithm is based on the Levenberg-Marquardt algorithm. We demonstrate the performance of our proposed method with simulation studies

Connecting and dating with tephras: principles, functioning, and application of tephrochronology in Quaternary research

Tephrochronology, the characterisation and use of volcanic-ash layers as a unique chronostratigraphic linking, synchronizing, and dating tool, has become a globally-practised discipline of immense practical value in a wide range of subjects including Quaternary stratigraphy, palaeoclimatology, palaeoecology, palaeolimnology, physical geography, geomorphology, volcanology, geochronology, archaeology, human evolution, anthropology, and human disease and medicine. The advent of systematic studies of cryptotephras – the identification, correlation, and dating of sparse, fine-grained glass-shard concentrations ‘hidden’ within sediments or soils – over the past ~20 years has been revolutionary. New cryptotephra techniques developed in northwestern Europe and Scandinavia in particular and in North America most recently adapted or improved to help solve problems as they arose, have now been applied to sedimentary sequences (including ice) on all the continents. The result has been the extension of tephra isochrons over wide areas hundreds to several thousands of kilometres from source volcanoes. Taphonomic and other issues, such as quantifying uncertainties in correlation, provide scope for future work. Developments in dating and analytical methods have led to important advances in the application of tephrochronology in recent times. In particular: (i) the ITPFT (glass fission-track) method has enabled landscapes and sequences to be dated where previously no dates were obtainable or where dating was problematic; (ii) new EMPA protocols enabling narrow-beam analyses (<5 um) of glass shards, or small melt inclusions, have been developed, meaning that small (typically distal) glass shards or melt inclusions <~10 um in diameter can now be analysed more efficaciously than previously (and with reduced risk of accidentally including microlites in the analysis as could occur with wide-beam analyses); (iii) LA-ICPMS method for trace element analysis of individual shards <~10 um in diameter is generating more detailed ‘fingerprints’ for enhancing tephra-correlation efficacy (Pearce et al., 2011, 2014; Pearce, 2014); and (iv) the revolutionary rise of Bayesian probability age modelling has helped to improve age frameworks for tephras of the late-glacial to Holocene period especially

Connecting with tephras: principles, functioning, and applications of tephrochronology in Quaternary science

Tephrochronology is a unique method for linking and dating geological, palaeoecological, palaeoclimatic, or archaeological sequences or events. The method relies firstly on stratigraphy and the law of superposition, which apply in any study that connects or correlates deposits from one place to another. Secondly, it relies on characterising and hence identifying or ‘fingerprinting’ tephra layers using either physical properties evident in the field or those obtained from laboratory analysis, including mineralogical examination by optical microscopy or geochemical analysis of glass shards or crystals (e.g., Fe-Ti oxides, ferromagnesian minerals) using the electron microprobe and other tools. Thirdly, the method is enhanced when a numerical age is obtained for a tephra layer by (1) radiometric methods such as radiocarbon, fission-track, U-series, or Ar/Ar dating, (2) incremental dating methods including dendrochronology or varved sediments or layering in ice cores, or (3) age-equivalent methods such as palaeomagnetism or correlation with marine oxygen isotope stages or palynostratigraphy. Once known, that age can be transferred from one site to the next using stratigraphic methods and by matching compositional characteristics, i.e., comparing ‘fingerprints’ from each layer. Used this way, tephrochronology is an age-equivalent dating method

Arrangement of Annexin A2 tetramer and its impact on the structure and diffusivity of supported lipid bilayers

Annexins are a family of proteins that bind to anionic phospholipid membranes in a Ca2+-dependent manner. Annexin A2 forms heterotetramers (Anx A2t) with the S100A10 (p11) protein dimer. The tetramer is capable of bridging phospholipid membranes and it has been suggested to play a role in Ca2+-dependent exocytosis and cell-cell adhesion of metastatic cells. Here, we employ x-ray reflectivity measurements to resolve the conformation of Anx A2t upon Ca2+-dependent binding to single supported lipid bilayers (SLBs) composed of different mixtures of anionic (POPS) and neutral (POPC) phospholipids. Based on our results we propose that Anx A2t binds in a side-by-side configuration, i.e., both Anx A2 monomers bind to the bilayer with the p11 dimer positioned on top. Furthermore, we observe a strong decrease of lipid mobility upon binding of Anx A2t to SLBs with varying POPS content. X-ray reflectivity measurements indicate that binding of Anx A2t also increases the density of the SLB. Interestingly, in the protein-facing leaflet of the SLB the lipid density is higher than in the substrate-facing leaflet. This asymmetric densification of the lipid bilayer by Anx A2t and Ca2+ might have important implications for the biochemical mechanism of Anx A2t-induced endo- and exocytosis.Comment: 27 pages, 7 figures; supplementary material available upon request from the author