Assessment by doppler ultrasound of entheseal lesions in spondyloarthritis : a longitudial study to determine structural damage and disease activity lesions

RESUMO: Enthesitis is the hallmark of spondyloarthritis (SpA), and is observed in all subtypes. Wide information on SpA abnormalities, including synovitis, tendinitis and enthesitis, can be efficiently perceived by Doppler ultrasound. Furthermore, several studies on imaging of enthesis showed that imaging techniques are better than clinical examination to detect enthesis alterations; and vascularized enthesitis detected by Doppler ultrasound appears to be a valuable diagnostic tool to confirm SpA diagnosis. However, data published until now concerning entheseal elementary alterations that characterize SpA enthesitis (enthesis inflammatory activity) or enthesopathy (permanent structural changes) reflect rather the authors’ empiric opinion than a methodological validation process. In this sense it seems crucial to identify elementary entheseal lesions associated with activity or damage, in order to improve monitoring and treatment response in SpA patients. The development of better assessment tools is today a challenge and a need in SpA. The first study of this thesis focused on the analysis of the reliability of inter-lector and inter-ultrasonography equipment of Madrid sonography enthesitis index (MASEI). Fundamental data for the remaining unrolling project validity. In the second and third studies we concerned about two entheseal elemental lesions: erosions and bursa. In literature erosions represent a permanent structural damage, being useful for monitoring joint injury, disease activity and therapeutic response in many rheumatic diseases; and to date, this concept has been mostly applied in rheumatoid arthritis (RA). Unquestionably, erosion is a tissue-related damage and a structural change. However, the hypothesis that we decided to test was if erosions represent a permanent structural change that can only grow and worsen over time, as occurs in RA, or a transitory alteration. A longitudinal study of early SpA patients was undertaken, and the Achilles enthesis was used as a model. Our results strongly suggested that previously detected erosions could disappear during the course of the disease, being consistent with the dynamic behavior of erosion over time. Based on these striking results it seems reasonable to suggest that the new-bone formation process in SpA could be associated with the resolution of cortical entheseal erosion over time. These results could also be in agreement with the apparent failure of anti-tumor necrosis factor (TNF) therapies to control bone proliferation in SpA; and with the relation of TNF-α, Dickkopf-related protein 1 (Dkk-1) and the regulatory molecule of the Wnt signaling pathway in the bone proliferation in SpA. In the same model, we then proceeded to study the enthesis bursa. Interestingly, the Outcome Measures in Rheumatology Clinical Trials (OMERACT) enthesopathy definition does not include bursa as an elementary entheseal lesion. Nonetheless, bursa was included in 46% of the enthesis studies in a recently systematic literature review, being in agreement with the concept of “synovio-entheseal complex” that includes the link between enthesitis and osteitis in SpA. It has been clarified in recent data that there is not only a close functional integration of the enthesis with the neighboring bone, but also a connection between enthesitis and synovitis. Therefore, we tried to assess the prevalence and relevance of the bursa-synovial lesion in SpA. Our findings showed a significant increase of Achilles bursa presence and thickness in SpA patients compared to controls (healthy/mechanical controls and RA controls). These results raise awareness to the need to improve the enthesopathy ultrasonographic definition. In the final work of this thesis, we have explored new perspectives, not previously reported, about construct validity of enthesis ultrasound as a possible activity outcome in SpA. We performed a longitudinal Achilles enthesis ultrasound study in patients with early SpA. Achilles ultrasound examinations were performed at baseline, six- and twelve-month time periods and compared with clinical outcome measures collected at basal visit. Our results showed that basal erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are higher in patients with Doppler signal in enthesis, and even that higher basal ESR, CRP and Ankylosing Spondylitis Disease Activity Score (ASDAS) predicted a higher Doppler signal (an ultrasound alteration accepted as representative of inflammation) six months later. Patients with very high disease activity assessed by ASDAS (>3.5) at baseline had significantly higher Achilles total ultrasound score verified at the same time; and ASDAS <1.3 predicted no Doppler signal at six and twelve months. This seems to represent a connection between classical biomarkers and clinical outcomes associated with SpA activity and Doppler signal, not only at the same time, but also for the following months. Remarkably, patients with inactive disease (ASDAS < 1.3) at baseline had no Doppler signal at six and twelve months. These findings reinforce the potential use of ultrasound related techniques for disease progression assessment and prognosis purposes. Intriguingly, Ankylosing Spondylitis Disease Activity Index (BASDAI) didn’t show significant differences between different cut-offs concerning ultrasound lesions or Doppler signal, while verified with ASDAS. These results seem to indicate that ASDAS reflects better than BASDAI what happens in the enthesis. The work herein discussed clearly shows the potential utility of ultrasound in enthesis assessment in SpA patients, and can be important for the development of ultrasound activity and structural damage scores for diagnosis and monitoring purposes. Therefore, local promotion of this technique constitutes a medical intervention that is worth being tested in SpA patients for diagnosis, monitoring and prognosis purposes.

Performance of a rapid diagnostic test for the detection of visceral leishmaniasis in a large urban setting

Introduction Rapid diagnostic tests (RDTs) may improve the early detection of visceral leishmaniasis (VL), but their real-world performance requires additional study. Therefore, we evaluated the performance of an rK39-based RDT (Kalazar Detect™) for the detection of VL in an endemic, large urban area. Methods Data were collected from a registry of rK39 RDT performed at 11 emergency care units in Belo Horizonte, Brazil, and from a national database of reportable communicable diseases of the Sistema de Informação de Agravos de Notificação (SINAN). Results The rapid rK39 test was performed in 476 patients, with 114 (23.9%) positive results. The analysis of rK39 RDT performance was based on 381 (80%) cases reported to the SINAN database, of which 145 (38.1%) were confirmed cases. Estimates for sensitivity and specificity were 72.4% (95% CI: 64.6-79%) and 99.6% (95%CI: 97.6-99.9%), respectively. Positive and negative predictive values were estimated at 99.1% (95%CI: 94.9-99.8%) and 85.5% (95%CI: 80.8-89.1%), respectively. In addition, close agreement between the rK39 RDT and indirect immunofluorescence was observed. Conclusions In summary, the rK39 RDT showed a high specificity but only moderate sensitivity. In endemic areas for VL, treatment may be considered in cases with clinical manifestations and a positive rK39 RDT, but those with a negative test should be subjected to further investigation.

Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil

Introduction A sero-epidemiological survey was undertaken to detect the circulation of arboviruses in free-living non-human primates. Methods Blood samples were obtained from 16 non-human primates (13 Sapajus spp. and three Alouatta caraya) that were captured using terrestrial traps and anesthetic darts in woodland regions in the municipalities of Campo Grande, Aquidauana, Jardim, Miranda and Corumbá in the State of Mato Grosso do Sul, Brazil. The samples were sent to the Instituto Evandro Chagas (IEC) in Ananindeua, Pará, Brazil, to detect antibodies against 19 species of arboviruses using a hemagglutination inhibition test (HI). Results Of the 16 primates investigated in the present study, five (31.2%) were serologically positive for an arbovirus. Of these five, two (12.5%) exhibited antibodies to the Flavivirus genus, one (6.2%) exhibited a monotypic reaction to Cacipacoré virus, one (6.2%) was associated with Mayaro virus, and one (6.2%) was positive for Oropouche virus. Conclusions Based on the positive serology observed in the present study, it was possible to conclude that arboviruses circulate among free-living primates. The viruses in the areas studied might have been introduced by infected humans or by primates from endemic or enzootic areas. Studies of this nature, as well as efficient and continuous surveillance programs, are needed to monitor viral activities in endemic and enzootic regions.

Characterization of an OPCPA laser amplifier based on the nonlinear crystal YCOB

The work described in this thesis was performed at the Laboratory for Intense Lasers (L2I) of Instituto Superior Técnico, University of Lisbon (IST-UL). Its main contribution consists in the feasibility study of the broadband dispersive stages for an optical parametric chirped pulse amplifier based on the nonlinear crystal yttrium calcium oxi-borate (YCOB). In particular, the main goal of this work consisted in the characterization and implementation of the several optical devices involved in pulse expansion and compression of the amplified pulses to durations of the order of a few optical cycles (20 fs). This type of laser systems find application in fields such as medicine, telecommunications and machining, which require high energy, ultrashort (sub-100 fs) pulses. The main challenges consisted in the preliminary study of the performance of the broadband amplifier, which is essential for successfully handling pulses with bandwidths exceeding 100 nm when amplified from the μJ to 20 mJ per pulse. In general, the control, manipulation and characterization of optical phenomena on the scale of a few tens of fs and powers that can reach the PW level are extremely difficult and challenging due to the complexity of the phenomena of radiation-matter interaction and their nonlinearities, observed at this time scale and power level. For this purpose the main dispersive components were characterized in detail, specifically addressing the demonstration of pulse expansion and compression. The tested bandwidths are narrower than the final ones, in order to confirm the parameters of these elements and predict the performance for the broadband pulses. The work performed led to additional tasks such as a detailed characterization of laser oscillator seeding the laser chain and the detection and cancelling of additional sources of dispersion.

A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Introduction Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. Methods We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. Results The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. Conclusions The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

A rapid and simple method to detect ESBL in Enterobacter cloacae based on MIC of cefepime

INTRODUCTION: The aim of this study was to identify a rapid and simple phenotypic method for extended-spectrum β-lactamase (ESBL) detection in Enterobacter cloacae. METHODS: A total of 79 consecutive, non-repeated samples of E. cloacae were evaluated. Four phenotypic methods were applied for ESBL detection, results were compared to multiplex polymerase chain reaction (PCR) as the gold standard reference method: 1) ceftazidime and cefotaxime disks with and without clavulanate, both with boronic acid added; 2) disk approximation using cefepime and amoxicillin/clavulanate; 3) ESBL screening by minimum inhibitory concentration (MIC) ≥ 16µg/mL and 4) by MIC ≥ 2µg/mL for cefepime. RESULTS: Method 4 showed the best combination of sensitivity (100%) and specificity (94%). CONCLUSIONS: MIC ≥ 2µg/mL for cefepime would be very useful for the phenotypic detection of ESBL in samples of E. cloacae.

Hepatitis E virus infection in Brazil: results of laboratory-based surveillance from 1998 to 2013

INTRODUCTION:Data on hepatitis E virus (HEV) in Brazil are limited. We analyzed 15 years of HEV surveillance data in a major clinical laboratory in São Paulo, Brazil.METHODS:The seroprevalence of HEV of 2,271 patients subjected to anti-HEV tests from 1998 to 2013 were analyzed.RESULTS:HEV seroprevalence was 2.1%, and the anti-HEV IgM positivity rate was 4.9%. Six hepatitis E patients were identified.CONCLUSIONS:HEV seroprevalence and detection rates appear to have increased in recent years. Hepatitis E should be investigated further and included in the differential diagnosis of hepatitis in Brazil.

Indicators of inflammation and cellular damage in chronic asymptomatic or oligosymptomatic alcoholics: correlation with alteration of bilirubin and hepatic and pancreatic enzymes

Biochemical and hematimetric indicators of inflammation and cell damage were correlated with bilirubin and hepatic and pancreatic enzymes in 30 chronic male alcoholics admitted into psychiatric hospital for detoxification and treatment of alcoholism. Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and total bilirubin were altered, respectively, in 90%, 63%, 87%, 23% and 23% of the cases. None of the indicators of inflammation (lactic dehydrogenase, altered in 16% of the cases; alpha-1 globulin, 24%; alpha-2 globulin, 88%; leucocyte counts, 28%) was correlated with alterations of bilirubin or liver enzymes. Lactic dehydrogenase was poorly sensitive for detection of hepatocytic or muscular damage. Alterations of alpha-globulins seemed to have been due more to alcohol metabolism-induced increase of lipoproteins than to inflammation. Among indicators of cell damage, serum iron, increased in 40% of the cases, seemed to be related to liver damage while creatine phosphokinase, increased in 84% of the cases, related to muscle damage. Hyperamylasemia was found in 20% of the cases and significantly correlated with levels of bilirubin, alkaline phosphatase and gamma-glutamyltransferase. It was indicated that injuries of liver, pancreas, salivary glands, and muscle occurred in asymptomatic or oligosymptomatic chronic alcoholics.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Implementation of membrane processes for improvement of the detection of food contaminants

The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities (http://www.em3e.eu)

Damage propagation in composite Materials meso-mechanical models

Composite materials have a complex behavior, which is difficult to predict under different types of loads. In the course of this dissertation a methodology was developed to predict failure and damage propagation of composite material specimens. This methodology uses finite element numerical models created with Ansys and Matlab softwares. The methodology is able to perform an incremental-iterative analysis, which increases, gradually, the load applied to the specimen. Several structural failure phenomena are considered, such as fiber and/or matrix failure, delamination or shear plasticity. Failure criteria based on element stresses were implemented and a procedure to reduce the stiffness of the failed elements was prepared. The material used in this dissertation consist of a spread tow carbon fabric with a 0°/90° arrangement and the main numerical model analyzed is a 26-plies specimen under compression loads. Numerical results were compared with the results of specimens tested experimentally, whose mechanical properties are unknown, knowing only the geometry of the specimen. The material properties of the numerical model were adjusted in the course of this dissertation, in order to find the lowest difference between the numerical and experimental results with an error lower than 5% (it was performed the numerical model identification based on the experimental results).

Genetic algorithm for shipping route estimation with long-range tracking data : automatic reconstruction of shipping routes based on the historical ship positions for maritime safety applications

Ship tracking systems allow Maritime Organizations that are concerned with the Safety at Sea to obtain information on the current location and route of merchant vessels. Thanks to Space technology in recent years the geographical coverage of the ship tracking platforms has increased significantly, from radar based near-shore traffic monitoring towards a worldwide picture of the maritime traffic situation. The long-range tracking systems currently in operations allow the storage of ship position data over many years: a valuable source of knowledge about the shipping routes between different ocean regions. The outcome of this Master project is a software prototype for the estimation of the most operated shipping route between any two geographical locations. The analysis is based on the historical ship positions acquired with long-range tracking systems. The proposed approach makes use of a Genetic Algorithm applied on a training set of relevant ship positions extracted from the long-term storage tracking database of the European Maritime Safety Agency (EMSA). The analysis of some representative shipping routes is presented and the quality of the results and their operational applications are assessed by a Maritime Safety expert.

Detection of road accident accumulation zones with a visual analytics approach

Nowadays, road accidents are a major public health problem, which increase is forecasted if road safety is not treated properly, dying about 1.2 million people every year around the globe. In 2012, Portugal recorded 573 fatalities in road accidents, on site, revealing the largest decreasing of the European Union for 2011, along with Denmark. Beyond the impact caused by fatalities, it was calculated that the economic and social costs of road accidents weighted about 1.17% of the Portuguese gross domestic product in 2010. Visual Analytics allows the combination of data analysis techniques with interactive visualizations, which facilitates the process of knowledge discovery in sets of large and complex data, while the Geovisual Analytics facilitates the exploration of space-time data through maps with different variables and parameters that are under analysis. In Portugal, the identification of road accident accumulation zones, in this work named black spots, has been restricted to annual fixed windows. In this work, it is presented a dynamic approach based on Visual Analytics techniques that is able to identify the displacement of black spots on sliding windows of 12 months. Moreover, with the use of different parameterizations in the formula usually used to detect black spots, it is possible to identify zones that are almost becoming black spots. Through the proposed visualizations, the study and identification of countermeasures to this social and economic problem can gain new grounds and thus the decision- making process is supported and improved.

Survey and dynamic behaviour of the Our Lady of Conception Church, Portugal

The Our Lady of Conception church is located in village of Monforte (Portugal) and is not in use nowadays. The church presents structural damage and, consequently, a study was carried out. The study involved the survey of the damage, dynamic identification tests under ambient vibration and the numerical analysis. The church is constituted by the central nave, the chancel, the sacristy and the corridor to access the pulpit. The masonry walls present different thickness, namely 0.65 m in the chancel, 0.70 m in the sacristy, 0.92 in the central nave and 0.65 m in the corridor. The masonry walls present 8 buttresses with different dimensions. The total longitudinal and transversal dimensions of the church are equal to 21.10 m and 14.26 m, respectively. The survey of the damage showed that, in general, the masonry walls are in good conditions, with exception of the transversal walls of the nave, which present severe cracks. The arches of the vault presents also severe cracks along the central nave. As consequence, the infiltrations have increased the degradation of the vault and paintings. Furthermore, the foundations present settlements in the Southwest direction. The dynamic identification test were carried out under the action of ambient excitation of the wind and using 12 piezoelectric accelerometers of high sensitivity. The dynamic identification tests allowed to estimate the dynamic properties of the church, namely frequencies, mode shapes and damping ratios. A FEM numerical model was prepared and calibrated, based on the first four experimental modes estimated in the dynamic identification tests. The average error between the experimental and numerical frequencies of the first four modes is equal to 5%. After calibration of the numerical model, pushover analyses with a load pattern proportional to the mass, in the transversal and longitudinal direction of the church, were performed. The results of the analysis numerical allow to conclude that the most vulnerable direction of the church is in the transversal one and the maximum load factor is equal to 0.35.