Detecting Detection: International Perspectives on the Uses of a Plot

One of the problems with books which are relatively general in nature is that many of the individual contributions tend to be so narrow and specialised that only the author has any knowledge of (or interest in) the issues under discussion. At first sight this appears to be the case with Detecting Detection. Fortunately, however, first impressions are deceiving. Although the essays in the volume deal with writers as diverse and disparate as the Catalano-Spanish writer Juan Marse, the Bulgarian-French philosopher Julia Kristeva and the once-vaunted giant of English literature,Graham Greene, among numerous others, there is much to be enjoyed and learnt, even if some of the works under discussion are unfamiliar to the crime fiction reader and/or scholar to whom the book initially appears to be directed.

Lanthanide Chelates as Donors in Fluorescence Resonance Energy Transfer: Exciting Prospects for Bioaffinity Assay Detection

Fluorescence resonance energy transfer (FRET) is a non-radiative energy transfer from a fluorescent donor molecule to an appropriate acceptor molecule and a commonly used technique to develop homogeneous assays. If the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, FRET might occur. As a consequence, the emission of the donor is decreased and the emission of the acceptor (if fluorescent) increased. Furthermore, the distance between the donor and the acceptor needs to be short enough, commonly 10-100 Å. Typically, the close proximity between the donor and the acceptor is achieved via bioaffinity interactions e.g. antibody binding antigen. Large variety of donors and acceptors exist. The selection of the donor/acceptor pair should be done not only based on the requirements of FRET but also the performance expectancies and the objectives of the application should be considered. In this study, the exceptional fluorescence properties of the lanthanide chelates were employed to develop two novel homogeneous immunoassays: a non-competitive hapten (estradiol) assay based on a single binder and a dual-parametric total and free PSA assay. In addition, the quenching efficiencies and energy transfer properties of various donor/acceptor pairs were studied. The applied donors were either europium(III) or terbium(III) chelates; whereas several organic dyes (both fluorescent and quenchers) acted as acceptors. First, it was shown that if the interaction between the donor/acceptor complexes is of high quality (e.g. biotin-streptavidin) the fluorescence of the europium(III) chelate could be quenched rather efficiently. Furthermore, the quenching based homogeneous non-competitive assay for estradiol had significantly better sensitivity (~67 times) than a corresponding homogeneous competitive assay using the same assay components. Second, if the acceptors were chosen to emit at the emission minima of the terbium(III) chelate, several acceptor emissions could be measured simultaneously without significant cross-talk from other acceptors. Based on these results, the appropriate acceptors were chosen for the dual-parameter assay. The developed homogeneous dual-parameter assay was able to measure both total and free PSA simultaneously using a simple mix and measure protocol. Correlation of this assay to a heterogeneous single parameter assay was excellent (above 0.99 for both) when spiked human plasma samples were used. However, due to the interference of the sample material, the obtained concentrations were slightly lower with the homogeneous than the heterogeneous assay, especially for the free PSA. To conclude, in this work two novel immunoassay principles were developed, which both are adaptable to other analytes. However, the hapten assay requires a rather good antibody with low dissociation rate and high affinity; whereas the dual-parameter assay principle is applicable whenever two immunometric complexes can form simultaneously, provided that the requirements of FRET are fulfilled.

Inorganic Nanoparticles and the Immune System: Detection, Selective Activation and Tolerance

The immune system is the responsible for body integrity and prevention of external invasion. On one side, nanoparticles are no triggers that the immune system is prepared to detect, on the other side it is known that foreign bodies, not only bacteria, viruses and parasites, but also inorganic matter, can cause various pathologies such as silicosis, asbestosis or inflammatory reactions. Therefore, nanoparticles entering the body, after interaction with proteins, will be either recognized as self-agents or detected by the immune system, encompassing immunostimulation or immunosuppression responses. The nature of these interactions seems to be dictated not specially by the composition of the material but by modifications of NP coating (composition, surface charge and structure). Herein, we explore the use of gold nanoparticles as substrates to carry multifunctional ligands to manipulate the immune system in a controlled manner, from undetection to immunostimulation. Murine bone marrow macrophages can be activated with artificial nanometric objects consisting of a gold nanoparticle functionalized with peptides. In the presence of some conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines were induced. The biochemical type of response depended on the type of conjugated peptide and was correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical NP conjugate design to either activate the immune system or hide from it, in order to reach their targets before being removed by phagocytes. Additionally, it opens up the possibility to modulate the immune response in order to suppress unwanted responses resulting from autoimmunity, or allergy or to stimulate protective responses against pathogens.

Comparison of Potassium and Sodium Content in Diet and Non-Diet Soft Drinks by Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

Capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) was used for determination of sodium and potassium concentrations in diet and non-diet soft drinks. Higher sodium concentrations were found in the diet samples due to the utilization of sodium salts of cyclamate and saccharine as sweeteners. The CE-C4D method can be used by food industries and health regulatory agencies for monitoring sodium and potassium content, not only in soft drink but in many others food products.

Eye Fundus Image Analysis for Automatic Detection of Diabetic Retinopathy

Diabetes is a rapidly increasing worldwide problem which is characterised by defective metabolism of glucose that causes long-term dysfunction and failure of various organs. The most common complication of diabetes is diabetic retinopathy (DR), which is one of the primary causes of blindness and visual impairment in adults. The rapid increase of diabetes pushes the limits of the current DR screening capabilities for which the digital imaging of the eye fundus (retinal imaging), and automatic or semi-automatic image analysis algorithms provide a potential solution. In this work, the use of colour in the detection of diabetic retinopathy is statistically studied using a supervised algorithm based on one-class classification and Gaussian mixture model estimation. The presented algorithm distinguishes a certain diabetic lesion type from all other possible objects in eye fundus images by only estimating the probability density function of that certain lesion type. For the training and ground truth estimation, the algorithm combines manual annotations of several experts for which the best practices were experimentally selected. By assessing the algorithm’s performance while conducting experiments with the colour space selection, both illuminance and colour correction, and background class information, the use of colour in the detection of diabetic retinopathy was quantitatively evaluated. Another contribution of this work is the benchmarking framework for eye fundus image analysis algorithms needed for the development of the automatic DR detection algorithms. The benchmarking framework provides guidelines on how to construct a benchmarking database that comprises true patient images, ground truth, and an evaluation protocol. The evaluation is based on the standard receiver operating characteristics analysis and it follows the medical practice in the decision making providing protocols for image- and pixel-based evaluations. During the work, two public medical image databases with ground truth were published: DIARETDB0 and DIARETDB1. The framework, DR databases and the final algorithm, are made public in the web to set the baseline results for automatic detection of diabetic retinopathy. Although deviating from the general context of the thesis, a simple and effective optic disc localisation method is presented. The optic disc localisation is discussed, since normal eye fundus structures are fundamental in the characterisation of DR.

Mild Cognitive Impairment and Early Detection of Alzheimer`s Disease. A Positron Emission Tomography Study

Alzheimer`s disease (AD) is characterised neuropathologically by the presence of extracellular amyloid plaques, intraneuronal neurofibrillary tangles, and cerebral neuronal loss. The pathological changes in AD are believed to start even decades before clinical symptoms are detectable. AD gradually affects episodic memory, cognition, behaviour and the ability to perform everyday activities. Mild cognitive impairment (MCI) represents a transitional state between normal aging and dementia disorders, especially AD. The predictive accuracy of the current and commonly used MCI criteria devide this disorder into amnestic (aMCI) and non-amnestic (naMCI) MCI. It seems that many individuals with aMCI tend to convert to AD. However many MCI individuals will remain stable and some may even recover. At present, the principal drugs for the treatment of AD provide only symptomatic and palliative benefits. Safe and effective mechanism-based therapies are needed for this devastating neurodegenerative disease of later life. In conjunction with the development of new therapeutic drugs, tools for early detection of AD would be important. In future one of the challenges will be to detect at an early stage these MCI individuals who will convert to AD. Methods which can predict which MCI subjects will convert to AD will be much more important if the new drug candidates prove to have disease-arresting or even disease–slowing effects. These types of drugs are likely to have the best efficacy if administered in the early or even in the presymptomatic phase of the disease when the synaptic and neuronal loss has not become too widespread. There is no clinical method to determine with certainly which MCI individuals will progress to AD. However there are several methods which have been suggested as predictors of conversion to AD, e.g. increased [11C] PIB uptake, hippocampal atrophy in MRI, low CSF A beta 42 level, high CSF tau-protein level, apolipoprotein E (APOE) ε4 allele and impairment in episodic memory and executive functions. In the present study subjects with MCI appear to have significantly higher [¹¹C] PIB uptake vs healthy elderly in several brain areas including frontal cortex, the posterior cingulate, the parietal and lateral temporal cortices, putamen and caudate. Also results from this PET study indicate that over time, MCI subjects who display increased [¹¹C] PIB uptake appear to be significantly more likely to convert to AD than MCI subjects with negative [¹¹C] PIB retention. Also hippocampal atrophy seems to increase in MCI individuals clearly during the conversion to AD. In this study [¹¹C] PIB uptake increases early and changes relatively little during the AD process whereas there is progressive hippocampal atrophy during the disease. In addition to increased [¹¹C] PIB retention and hippocampal atrophy, the status of APOE ε4 allele might contribute to the conversion from MCI to AD.

Fraud detection in the banking sector : a multi-agent approach

Fraud is an increasing phenomenon as shown in many surveys carried out by leading international consulting companies in the last years. Despite the evolution of electronic payments and hacking techniques there is still a strong human component in fraud schemes. Conflict of interest in particular is the main contributing factor to the success of internal fraud. In such cases anomaly detection tools are not always the best instruments, since the fraud schemes are based on faking documents in a context dominated by lack of controls, and the perpetrators are those ones who should control possible irregularities. In the banking sector audit team experts can count only on their experience, whistle blowing and the reports sent by their inspectors. The Fraud Interactive Decision Expert System (FIDES), which is the core of this research, is a multi-agent system built to support auditors in evaluating suspicious behaviours and to speed up the evaluation process in order to detect or prevent fraud schemes. The system combines Think-map, Delphi method and Attack trees and it has been built around audit team experts and their needs. The output of FIDES is an attack tree, a tree-based diagram to ”systematically categorize the different ways in which a system can be attacked”. Once the attack tree is built, auditors can choose the path they perceive as more suitable and decide whether or not to start the investigation. The system is meant for use in the future to retrieve old cases in order to match them with new ones and find similarities. The retrieving features of the system will be useful to simplify the risk management phase, since similar countermeasures adopted for past cases might be useful for present ones. Even though FIDES has been built with the banking sector in mind, it can be applied in all those organisations, like insurance companies or public organizations, where anti-fraud activity is based on a central anti-fraud unit and a reporting system.

Immunohistochemical detection of Clostridia species in paraffin-embedded tissues of experimentally inoculated guinea pigs

Blackleg is caused by Clostridium chauvoei, whereas malignant oedema is caused by C. chauvoei, C. septicum, C. sordellii, C. perfringens type A, and/or C. novyi type A. Anti-C. chauvoei, anti-C. septicum, anti-C. sordellii and anti-C. novyi type A polyclonal antibodies were produced in rabbits and purified in a column of DEAE-cellulose. Aliquots of the antisera were conjugated with fluorescein isothiocyanate and the remaining was used for the streptavidin biotin peroxidase technique (SBPT). SBPT was standardized to detect C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs. SBPT was compared to a fluorescent antibody technique (FAT). Sections and smears of muscle from inoculation area (MIA), heart, liver, spleen and kidney, were obtained for both SBPT and FAT. Cross-reactions between the different Clostridial species were not observed. C. chauvoei and C. septicum were detected in all specimens from the animals inoculated with these microorganisms, while only sections of muscle obtained from all the animals inoculated with C. sordellii and C. novyi type A were positive. The same results observed by the SBPT, were obtained on tissue smears of these microorganisms stained by the FAT. The results indicate that SBPT is suitable for detection of C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs.

Ultrasound call detection in capybara

The vocal repertoire of some animal species has been considered a non-invasive tool to predict distress reactivity. In rats ultrasound emissions were reported as distress indicator. Capybaras[ vocal repertoire was reported recently and seems to have ultrasound calls, but this has not yet been confirmed. Thus, in order to check if a poor state of welfare was linked to ultrasound calls in the capybara vocal repertoire, the aim of this study was to track the presence of ultrasound emissions in 11 animals under three conditions: 1) unrestrained; 2) intermediately restrained, and 3) highly restrained. The ultrasound track identified frequencies in the range of 31.8±3.5 kHz in adults and 33.2±8.5 kHz in juveniles. These ultrasound frequencies occurred only when animals were highly restrained, physically restrained or injured during handling. We concluded that these calls with ultrasound components are related to pain and restraint because they did not occur when animals were free of restraint. Thus we suggest that this vocalization may be used as an additional tool to assess capybaras[ welfare.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC). The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

Detection and characterization of fibropapilloma associated herpesvirus of marine turtles in Rio Grande do Sul, Brazil

Fibropapillomatosis (FP) is a benign tumoral disease that affects sea turtles, hampering movement, sight and feeding, ultimately leading to death. In Brazil, the disease was described for the first time in 1986. Research suggests the involvement of a herpesvirus in association with environmental and genetic factors as causal agents of FP. The objective of the present study was to detect and characterize this herpesvirus in sea turtles living in the coast of state Rio Grande do Sul (RS), Brazil. From October 2008 to July 2010, 14 turtles were observed between the beaches of Torres and Tavares, of which 11 were green turtles (Chelonia mydas) and 3 were loggerhead turtles (Caretta caretta). All turtles were young and mean curved carapace length was 37.71±7.82cm, and varied from 31 to 55cm. Only one green turtle presented a 1cm, papillary, pigmented fibropapilloma. Skin and fibropapilloma samples were analyzed by conventional and real time PCR assays to detect and quantify herpesvirus. All skin samples were negative, though the fibropapilloma specimen was positive in both tests. Viral load was 9,917.04 copies of viral genome per milligram of tissue. The DNA fragment amplified from the fibropapilloma sample was sequenced and allocated in the Atlantic phylogeographic group. This study reports the first molecular characterization of herpesvirus associated with fibropapilloma in turtles from the coast of RS.

Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil

Salmonella spp. are considered the main agents of foodborne disease and Salmonella Enteritidis is one of the most frequently isolated serovars worldwide. The virulence of Salmonella spp. and their interaction with the host are complex processes involving virulence factors to overcome host defenses. The purpose of this study was to detect virulence genes in S. Enteritidis isolates from poultry in the South of Brazil. PCR-based assays were developed in order to detect nine genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH and spvC) associated with the virulence in eighty-four isolates of S. Enteritidis isolated from poultry. The invA, hilA, sivH, sefA and avrA genes were present in 100% of the isolates; lpfA and sopE were present in 99%; agfA was present in 96%; and the spvC gene was present in 92%. It was possible to characterize the isolates with four different genetic profiles (P1, P2, P3 and P4), as it follows: P1, positive for all genes; P2, negative only for spvC; P3, negative for agfA; and P4, negative for lpfA, spvC and sopE. The most prevalent profile was P1, which was present in 88% of the isolates. Although all isolates belong to the same serovar, it was possible to observe variations in the presence of these virulence-associated genes between different isolates. The characterization of the mechanisms of virulence circulating in the population of Salmonella Enteritidis is important for a better understanding of its biology and pathogenicity. The frequency of these genes and the establishment of genetic profiles can be used to determine patterns of virulence. These patterns, associated with in vivo studies, may help develop tools to predict the ability of virulence of different strains.