Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

<p>Abstract</p> <p>Background</p> <p>Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.</p> <p>Results</p> <p>EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10²to 1.3 × 10⁸copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³to 2.0 × 10⁵copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴to 1.1 × 10⁵copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10²to 5.6 × 10³copies/ml in HIV-infected patients (n = 12), and 2.0 × 10²to 9.1 × 10⁴copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).</p> <p>Conclusion</p> <p>Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.</p

Scenes, saliency maps and scanpaths

The aim of this chapter is to review some of the key research investigating how people look at pictures. In particular, my goal is to provide theoretical background for those that are new to the field, while also explaining some of the relevant methods and analyses. I begin by introducing eye movements in the context of natural scene perception. As in other complex tasks, eye movements provide a measure of attention and information processing over time, and they tell us about how the foveated visual system determines what to prioritise. I then describe some of the many measures which have been derived to summarize where people look in complex images. These include global measures, analyses based on regions of interest and comparisons based on heat maps. A particularly popular approach for trying to explain fixation locations is the saliency map approach, and the first half of the chapter is mostly devoted to this topic. A large number of papers and models are built on this approach, but it is also worth spending time on this topic because the methods involved have been used across a wide range of applications. The saliency map approach is based on the fact that the visual system has topographic maps of visual features, that contrast within these features seems to be represented and prioritized, and that a central representation can be used to control attention and eye movements. This approach, and the underlying principles, has led to an increase in the number of researchers using complex natural scenes as stimuli. It is therefore important that those new to the field are familiar with saliency maps, their usage, and their pitfalls. I describe the original implementation of this approach (Itti & Koch, 2000), which uses spatial filtering at different levels of coarseness and combines them in an attempt to identify the regions which stand out from their background. Evaluating this model requires comparing fixation locations to model predictions. Several different experimental and comparison methods have been used, but most recent research shows that bottom-up guidance is rather limited in terms of predicting real eye movements. The second part of the chapter is largely concerned with measuring eye movement scanpaths. Scanpaths are the sequential patterns of fixations and saccades made when looking at something for a period of time. They show regularities which may reflect top-down attention, and some have attempted to link these to memory and an individual’s mental model of what they are looking at. While not all researchers will be testing hypotheses about scanpaths, an understanding of the underlying methods and theory will be of benefit to all. I describe the theories behind analyzing eye movements in this way, and various methods which have been used to represent and compare them. These methods allow one to quantify the similarity between two viewing patterns, and this similarity is linked to both the image and the observer. The last part of the chapter describes some applications of eye movements in image viewing. The methods discussed can be applied to complex images, and therefore these experiments can tell us about perception in art and marketing, as well as about machine vision