Central nervous system virion detection in acute measles: histopathological, ultrastructural and pathogenetic aspects

Histopathological and ultrastructural studies of 23 patients who died with clinical diagnosis of measles were carried out. In 12 cases viral nucleocapsids were searched by electron microscopy and detected in 100% of the cases in the lungs and in 50% of the cases in the central nervous system. They were mostly intranuclear. Histopathological changes associated to neurological alterations and the detection of virion are discussed in relation to acute and delayed clinical manifestations.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Enzyme linked immunosorbent assay for rubella antibodies: a simple method of antigen production. A preliminary report

A simple method of rubella antigen production by treatment with sodium desoxycholate for use in enzyme immunoassay (IMT-ELISA) is presented. When this assay was compared with a commercial test (Enzygnost-Rubella, Behring), in the study of 108 sera and 118 filter paper blood samples, 96.9% (219/226) overall agreement and correlation coefficient of 0.90 between absorbances were observed. Seven samples showed discordant results, negative by the commercial kit and positive by our test. Four of those 7 samples were available, being 3 positive by HI.

New analytical methodologies for doping control – detection of anabolic androgenic steroids in human urine

Dissertation submitted to Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa in fulfilment of the requirements for the degree of Doctor of Philosophy (Biochemistry - Biotechnology)

Spatial metrics and Landsat data for urban landuse change detection: case of Addis Ababa, Ethiopia.

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

DNA nanoprobes for molecular detection

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Biológica – especialidade Engenharia Genética, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Multilayer Perceptron Neural Networks Training Through Charged System Search and its Application for Non-Technical Losses Detection

The non-technical loss is not a problem with trivial solution or regional character and its minimization represents the guarantee of investments in product quality and maintenance of power systems, introduced by a competitive environment after the period of privatization in the national scene. In this paper, we show how to improve the training phase of a neural network-based classifier using a recently proposed meta-heuristic technique called Charged System Search, which is based on the interactions between electrically charged particles. The experiments were carried out in the context of non-technical loss in power distribution systems in a dataset obtained from a Brazilian electrical power company, and have demonstrated the robustness of the proposed technique against with several others natureinspired optimization techniques for training neural networks. Thus, it is possible to improve some applications on Smart Grids.

Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Histoplasmin reaction. Comparison of a polysaccharide antigen to the filtrate antigen

This work was planned by taking into account all the knowledge accumulated from the immunological study of paracoccidioidomycosis. It aimed at comparing a polysaccharide antigen from Histoplasma capsulatum to a classic histoplasmin with the help of intradermal tests of delayed type of hypersensitivity. Tests were applied to 115 individuals in Santo Amaro, a town in the state of São Paulo. Positive results using classic histoplasmin were obtained in 46.0% cases whereas positive results using the polysaccharide antigen at its hightest concentration were obtained in 51.30% cases. The major conclusion in this investigation is that it is possible to use the polysaccharide antigen as histoplasmin instead of the filtrate antigen

Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended

Desenvolvimento de Anticorpos Plásticos para um Biomarcador do Cancro

O trabalho descrito compreende o desenvolvimento de um anticorpo plástico (MIP, do inglês Molecularly Imprinted Polymer) para o antigénio carcinoembrionário (CEA, do inglês Carcinoembriogenic Antigen) e a sua aplicação na construção de dispositivos portáteis, de tamanho reduzido e de baixo custo, tendo em vista a monitorização deste biomarcador do cancro do colo-retal em contexto Point-of-Care (POC). O anticorpo plástico foi obtido por tecnologia de impressão molecular orientada, baseada em eletropolimerização sobre uma superfície condutora de vidro recoberto por FTO. De uma forma geral, o processo foi iniciado pela electropolimerização de anilina sobre o vidro, seguindo-se a ligação por adsorção do biomarcador (CEA) ao filme de polianilina, com ou sem monómeros carregados positivamente (Cloreto de vinilbenziltrimetilamónio, VB). A última fase consistiu na electropolimerização de o-fenilenodiamina (oPD) sobre a superfície, seguindo-se a remoção da proteína por clivagem de ligações peptídicas, com o auxílio de tripsina. A eficiência da impressão do biomarcador CEA no material polimérico foi controlada pela preparação de um material análogo, NIP (do inglês, Non-Imprinted Polymer), no qual nem a proteína nem o monómero VB estavam presentes. Os materiais obtidos foram caracterizados quimicamente por técnicas de Infravermelho com Transformada de Fourier (FTIR, do inglês, Fourier Transform Infrared Spectroscopy) e microscopia confocal de Raman. Os materiais sensores preparados foram entretanto incluídos em membranas poliméricas de Poli(cloreto de vinilo) (PVC) plastificado, para construção de sensores (biomiméticos) seletivos a CEA, tendo-se avaliado a resposta analítica em diferentes meios. Obteve-se uma boa resposta potenciométrica em solução tampão de Ácido 4-(2-hidroxietil)piperazina-1-etanosulfónico (HEPES), a pH 4,4, com uma membrana seletiva baseada em MIP preparada com o monómero carregado VB. O limite de deteção foi menor do que 42 pg/mL, observando-se um comportamento linear (versus o logaritmo da concentração) até 625 pg/mL, com um declive aniónico igual a -61,9 mV/década e r2>0,9974. O comportamento analítico dos sensores biomiméticos foi ainda avaliado em urina, tendo em vista a sua aplicação na análise de CEA em urina. Neste caso, o limite de deteção foi menor do que 38 pg/mL, para uma resposta linear até 625 pg/mL, com um declive de -38,4 mV/década e r2> 0,991. De uma forma geral, a aplicação experimental dos sensores biomiméticos evidenciou respostas exatas, sugerindo que os biossensores desenvolvidos prossigam estudos adicionais tendo em vista a sua aplicação em amostras de indivíduos doentes.

Detection of mycoplasmas in urethral swabs from HIV-1 infected patients and control individuals using culture techniques and polymerase chain reaction

The objective of the present study was to determine the prevalence of certain mycoplasma species, i.e., Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma penetrans, in urethral swabs from HIV-1 infected patients compared to swabs from a control group. Mycoplasmas were detected by routine culture techniques and by the Polymerase Chain Reaction (PCR) technique, using 16SrRNA generic primers of conserved region and Mycoplasma penetrans specific primers. The positivity rates obtained with the two methods were comparable. Nevertheless, PCR was more sensitive, while the culture techniques allowed the quantification of the isolates. The results showed no significant difference (p < 0.05) in positivity rates between the methods used for mycoplasma detection.

DETECTION OF HTLV-I ANTIBODIES AND DNA IN BLOOD SAMPLE OF A PATIENT WITH MYELOPATHY IN NIGERIA

We describe a case of human T-lymphotropic virus type I associated myelopathy in a 50-year old woman in Nigeria. The patient presented with progressive loss of tone to the two lower limbs and later inability to walk. The HTLV-I antibody presence in the plasma collected from the patient was repeatedly detected by enzyme immunoassays (Abbott HTLV-I EIA and Coulter SELECT-HTLV I/II) and confirmed by Western blot technique. In addition, HTLV-I DNA was amplified from the genomic DNA isolated from the peripheral blood mononuclear cells of the patient by the polymerase chain reaction technique. This finding is significant being the first report of association of HTLV-I with myelopathy in Nigeria.