Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

Variability in Galactomannan detection by platelia Aspergillus EIA™ according to the Aspergillus species

Here we investigate the extent to which different Aspergillus species release galactomannan (GM) in vitro. Marked variability was observed in GM reactivity between and within Aspergillus species, with A. terreus strains showing the highest GM indexes. The in vivo significance of these findings remains to be determined.

Software development for estimation of optical clearing agent’s diffusion coefficients in biological tissues

The study of chemical diffusion in biological tissues is a research field of high importance and with application in many clinical, research and industrial areas. The evaluation of diffusion and viscosity properties of chemicals in tissues is necessary to characterize treatments or inclusion of preservatives in tissues or organs for low temperature conservation. Recently, we have demonstrated experimentally that the diffusion properties and dynamic viscosity of sugars and alcohols can be evaluated from optical measurements. Our studies were performed in skeletal muscle, but our results have revealed that the same methodology can be used with other tissues and different chemicals. Considering the significant number of studies that can be made with this method, it becomes necessary to turn data processing and calculation easier. With this objective, we have developed a software application that integrates all processing and calculations, turning the researcher work easier and faster. Using the same experimental data that previously was used to estimate the diffusion and viscosity of glucose in skeletal muscle, we have repeated the calculations with the new application. Comparing between the results obtained with the new application and with previous independent routines we have demonstrated great similarity and consequently validated the application. This new tool is now available to be used in similar research to obtain the diffusion properties of other chemicals in different tissues or organs.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

A learning approach to swarm-based path detection and tracking

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Desenvolvimento de software com integração contínua

A integração contínua é uma prática no desenvolvimento de software que já existe há algum tempo mas ainda não é muito conhecida nem usada. Esta prática no desenvolvimento de software surgiu com a programação extrema e tem evoluído ao longo dos últimos anos, adaptando-se às novas tecnologias. O estudo aqui apresentado pretende essencialmente mostrar a real importância e o valor acrescido que a integração contínua pode trazer a um projeto de desenvolvimento de software. O trabalho aqui exposto surge no âmbito de um projeto interno, realizado pelo autor na empresa Konkconsulting, cuja finalidade se prende com o conhecimento mais aprofundado da integração contínua e com o levantamento das necessidades e criação de soluções, de modo a conseguir a sua utilização nos produtos que irão ser desenvolvidos pela empresa. Nesta dissertação, é proposto um conjunto de ferramentas para responder às necessidades imediatas da empresa na implementação de integração contínua num dos seus projetos. Estas ferramentas devem ser de simples e de fácil utilização, de modo a ajudar os programadores durante os desenvolvimentos e responder às necessidades da utilização da integração contínua em um projeto a ser desenvolvido pela empresa, mas ao mesmo tempo, podendo ser facilmente incorporadas em futuros projetos.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

Reconfiguração e desenvolvimento do sistema de controlo de um AGV

O veículo guiado automaticamente (AGV) adquirido pelo Departamento de Engenharia Mecânica (DEM) tem vindo a ficar obsoleto devido ao hardware, que nos dias de hoje começa a dar sinais de falhas bem como falta de peças de substituição, e ao software, sendo o PLC (Programmable Logic Controller) usado muito limitado quanto às suas funções de controlo, ficando as principais tarefas de controlo do AGV a cargo de placas eletrónicas de controlo. Para promover o controlo autónomo do AGV, foi decidido retirar toda a parte de hardware que detinha o controlo do mesmo e passou a ser um novo PLC, com maior capacidade de processamento, a executar todo o tipo de controlo necessário ao funcionamento do mesmo. O hardware considerado apenas incluí, de forma resumida, os motores responsáveis pelo movimento e direção, placa de controlo de potência dos motores, placa de interface entre as saídas digitais do PLC e as entradas da placa de controlo de potência dos motores e os demais sensores necessários à deteção de obstáculos, fins de curso da direção, sensores dos postos de trabalho e avisadores de emergência. Todo o controlo de movimento e direção bem como a seleção das ações a executar passou a ficar a cargo do software programado no PLC assim como a interação entre o sistema de supervisão instalado num posto de controlo e o PLC através de comunicação via rádio. O uso do PLC permitiu a flexibilidade de mudar facilmente a forma como as saídas digitais são usadas, ao contrário de um circuito eletrónico que necessita de uma completa remodelação, tempo de testes e implementação para efetuar a mesma função. O uso de um microcontrolador seria igualmente viável para a aplicação em causa, no entanto o uso do PLC tem a vantagem de ser robusto, mais rápido na velocidade de processamento, existência de software de interface de programação bastante intuitivo e de livre acesso, facilidade de alterar a programação localmente ou remotamente, via rádio, acesso a vários protocolos de comunicação robustos como Modbus, Canbus, Profinet, Modnet, etc., e acesso integrado de uma consola gráfica totalmente programável. iv É ainda possível a sua expansão com adição de módulos de entradas e saídas digitais e/ou analógicas permitindo expandir largamente o uso do AGV para outros fins. A solução está a ser amplamente testada e validada no Laboratório de Automação (LabA) do Departamento de Engenharia Mecânica do ISEP (Instituto Superior de Engenharia do Porto), permitindo a otimização dos sistemas de controlo de direção bem como a interatividade entre o PLC e o programa de interface/supervisão do posto de trabalho.

TEMPORAL TRENDS IN THE DETECTION RATES OF HEPATITIS B IN THE SANTA CATARINA STATE, BRAZIL

Hepatitis B is a serious public health problem. The state of Santa Catarina presents areas of high endemicity. The aim of this study was to describe temporal trends in detection rates of hepatitis B in the period from 2002 to 2009 in Santa Catarina and in its regions. A time series study was carried out. Crude rates were calculated and standardized by age using the direct method. Annual variation percentages were estimated by Joinpoint regression. There were two distinct and significant trends in Santa Catarina. From 2002 to 2006 a significant increase of 5.9% per year was observed. From 2006, there was a significant decrease of 6.4% per year. In this same period the southern and far-western regions had significant increases of 15.9% and 4.6% and significant decreases of 7.5% and 4.8%, respectively. Greater Florianópolis and Northeast also showed significant increases until 2006, of 15.4% and 17.4%, respectively. In the following period, non-significant decreases of 5.8% and 9.8% respectively were observed. Foz do Rio Itajaí and Planalto Serrano showed non-significant increases up to half of the studied period of 21.1% and 12.0%, respectively and after, significant decreases of 21.5% and 18.0%, respectively. Vale do Itajaí showed a significant decrease of 9.7%; Planalto Norte showed a non-significant decrease of 0.6% and Midwest a non-significant increase of 2.7% per year, in the period from 2002 to 2009.

A análise digital de vídeo e software exploratório no ensino da Física

Relatório para obtenção do Grau de Mestre em Ensino da Física e da Química

Urban land cover change detection analysis and modeling spatio-temporal Growth dynamics using Remote Sensing and GIS Techniques: A case study of Dhaka, Bangladesh

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Evaluation of thermal remote sensing for detection of thermal anomalies as earthquake precursors: a case study for Malatya-Pütürge-Doganyol (Turkey) Earthquake, July 13, 2003

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.