AN ELISA SUITABLE FOR THE DETECTION OF RABIES VIRUS ANTIBODIES IN SERUM SAMPLES FROM HUMAN VACCINATED WITH EITHER CELL-CULTURE VACCINE OR SUCKLING-MOUSE-BRAIN VACCINE

An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.

Chronic hepatitis C virus infections in Brazilian patients: association with genotypes, clinical parameters and response to long term alpha interferon therapy

The present study assessed the clinical significance of hepatitis C virus (HCV) genotypes and their influence on response to long term recombinant-interferon-alpha (r-IFN-a) therapy in Brazilian patients. One hundred and thirty samples from patients previously genotyped for the HCV and with histologically confirmed chronic hepatitis C (CH-C) were evaluated for clinical and epidemiological parameters (sex, age, time of HCV infection and transmission routes). No difference in disease activity, sex, age or mode and time of transmission were seen among patients infected with HCV types 1, 2 or 3. One hundred and thirteen of them were treated with 3 million units of r-IFN-a, 3 times a week for 12 months. Initial response (IR) was significantly better in patients with genotype 2 (100%) and 3 (46%) infections than in patients with genotype 1 (29%) (p < 0.005). Among subtypes, difference in IR was observed between 1b and 2 (p < 0.005), and between 1b and 3a (p < 0.05). Sustained response (SR) was observed in 12% for (sub)type 1a, 13% for 1b, 19% for 3a, and 40% for type 2; significant differences were found between 1b and 2 (p < 0.001), and between 1b and 3a (p < 0.05). Moreover, presence of cirrhosis was significantly associated with non response and response with relapse (p < 0.05). In conclusion, non-1 HCV genotype and lack of histological diagnosis of cirrhosis were the only baseline features associated with sustained response to treatment. These data indicate that HCV genotyping may have prognostic relevance in the responsiveness to r-IFN-a therapy in Brazilian patients with chronic HCV infection, as seen in other reports worldwide.

Investigating habitat association of breeding birds using public domain satellite imagery and land cover data.

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

A clinical, epidemological, laboratorial, histological and ultrasonographical evaluation of anti-HCV EIA-2 positive blood donors

Between 1992 and 1997, 790 blood donors with anti-HCV EIA-2 strongly reagent (relationship between the sample optical density/cut-off > 3) detected at the blood bank serological screening, were evaluated in ambulatory environment. They were all negative for Chagas disease, syphilis, hepatitis B (HBsAg) and AIDS. Blood samples were collected at the first ambulatorial evaluation, for hemogram, biochemical tests and new serological tests for HCV (anti-HCV EIA-2). In blood samples of 226 repeatedly reagent anti-HCV EIA-2 blood donors, supplementary "immunoblot" test for HCV (RIBA-2) was used. In 209 donors, the presence of HCV-RNA was investigated by the PCR test. The abdominal ultrasonography was realized in 366 donors. In 269 patients liver biopsy was performed for the histopathological study. The follow-up of blood donors showed that 95.6% were repeatedly EIA-2 reagent, 94% were symptomless and denied any hepatitis history, with only 2% mentioning previous jaundice. In 47% of this population at least one risk factor has been detected for the HCV transmission, the use of intravenous drugs being the main one (27.8%). Blood transfusion was the second factor for HCV transmission (27.2%). Hepatomegaly was detected in 54% of the cases. Splenomegaly and signs of portal hypertension have seldom been found in the physical examination, indicating a low degree of hepatic compromising in HCV. Abdominal ultrasound showed alterations in 65% of the subjects, being the steatosis the most frequent (50%). In 83.5% of the donors submitted to the liver biopsy, the histopathological exam showed the presence of chronic hepatitis, usually classified as active (89%) with mild or moderate grade in most of the cases (99.5%). The histopathological exam of the liver was normal in 1.5% of blood donors. The RIBA-2 test and the HCV-RNA investigation by PCR were positive in respectively 91.6 and 75% of the anti-HCV EIA-2 reagent donors. The HCV-RNA research was positive in 82% of the RIBA-2 positive subjects, in 37.5% of the indeterminate RIBA-2 donors and in 9% of the negative RIBA-2 donors. Chronic hepatitis has also been observed in 50% of the histopathological exams of the anti-HCV EIA-2 reagent donors which were indeterminate RIBA-2. Among 18 blood donors with minimal changes histopathological exam 11 (61%) were HCV-RNA positive. Our blood donors anti-HCV reagent generally had clinical, laboratorial and histopathological features observed in patients with chronic HCV hepatitis and a high proportion could be identified in interviews and medical evaluation realized in blood blanks. Generally, these HCV infected donors are identified and discharged only by the serological tests results.

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in São Paulo, Brazil

The respiratory viruses are recognized as the most frequent lower respiratory tract pathogens for infants and young children in developed countries but less is known for developing populations. The authors conducted a prospective study to evaluate the occurrence, clinical patterns, and seasonal trends of viral infections among hospitalized children with lower respiratory tract disease (Group A). The presence of respiratory viruses in children's nasopharyngeal was assessed at admission in a pediatric ward. Cell cultures and immunofluorescence assays were used for viral identification. Complementary tests included blood and pleural cultures conducted for bacterial investigation. Clinical data and radiological exams were recorded at admission and throughout the hospitalization period. To better evaluate the results, a non- respiratory group of patients (Group B) was also constituted for comparison. Starting in February 1995, during a period of 18 months, 414 children were included- 239 in Group A and 175 in Group B. In Group A, 111 children (46.4%) had 114 viruses detected while only 5 children (2.9%) presented viruses in Group B. Respiratory Syncytial Virus was detected in 100 children from Group A (41.8%), Adenovirus in 11 (4.6%), Influenza A virus in 2 (0.8%), and Parainfluenza virus in one child (0.4%). In Group A, aerobic bacteria were found in 14 cases (5.8%). Respiratory Syncytial Virus was associated to other viruses and/or bacteria in six cases. There were two seasonal trends for Respiratory Syncytial Virus cases, which peaked in May and June. All children affected by the virus were younger than 3 years of age, mostly less than one year old. Episodic diffuse bronchial commitment and/or focal alveolar condensation were the clinical patterns more often associated to Respiratory Syncytial Virus cases. All children from Group A survived. In conclusion, it was observed that Respiratory Syncytial Virus was the most frequent pathogen found in hospitalized children admitted for severe respiratory diseases. Affected children were predominantly infants and boys presenting bronchiolitis and focal pneumonias. Similarly to what occurs in other subtropical regions, the virus outbreaks peak in the fall and their occurrence extends to the winter, which parallels an increase in hospital admissions due to respiratory diseases.

Desenvolvimento e aplicação de um biossensor para monitorização de creatinina, um biomarcador associado a doenças emergentes

Este trabalho descreve o desenvolvimento de um material sensor para creatinina por impressão molecular em estrutura polimérica (MIP) e a sua aplicação no desenvolvimento de um dispositivo de natureza potenciométrica para a determinação da molécula alvo em fluidos biológicos. A creatinina é um dos biomarcadores mais utilizados no acompanhamento da doença renal, já que é um bom indicador da taxa de filtração glomerular (TFG). Os materiais biomiméticos desenhados para interação com a creatinina foram obtidos por polimerização radicalar, recorrendo a monómeros de ácido metacríclico ou de vinilpiridina e a um agente de reticulação apropriado. De modo a aferir o efeito da impressão da creatinina na resposta dos materiais MIP à sua presença, foram também preparados e avaliados materiais de controlo, obtidos sem impressão molecular (NIP). O controlo da constituição química destes materiais, incluindo a extração da molécula impressa, foi realizado por Espectroscopia de Raman e de Infravermelho com Transformada de Fourrier. A afinidade de ligação entre estes materiais e a creatinina foi também avaliada com base em estudos cinéticos. Todos os materiais descritos foram integrados em membranas selectivas de elétrodos seletivos de ião, preparadas sem ou com aditivo iónico lipófilo, de carga negativa ou positiva. A avaliação das características gerais de funcionamento destes elétrodos, em meios de composição e pH diferentes, indicaram que as membranas com materiais impressos e aditivo aniónico eram as únicas com utilidade analítica. Os melhores resultados foram obtidos em solução tampão Piperazine-N,N′-bis(2- ethanesulfonic acid), PIPES, de pH 2,8, condição que permitiu obter uma resposta quasi-Nernstiana, a partir de 1,6×10-5 mol L-1. Estes elétrodos demonstraram ainda uma boa selectividade ao apresentaram uma resposta preferencial para a creatinina quando na presença de ureia, carnitina, glucose, ácido ascórbico, albumina, cloreto de cálcio, cloreto de potássio, cloreto de sódio e sulfato de magnésio. Os elétrodos foram ainda aplicados com sucesso na análise de amostras sintéticas de urina, quando os materiais sensores eram baseados em ácido metacrilico, e soro, quando os materiais sensores utilizados eram baseados em vinilpiridina.

Experimental infection and horizontal transmission of Bartonella henselae in domestic cats

In order to study B. henselae transmission among cats, five young cats were kept in confinement for two years, one of them being inoculated by SC route with B. henselae (10(5) UFC). Only occasional contact among cats occurred but the presence of fleas was observed in all animals throughout the period. Blood culture for isolation of bacteria, PCR-HSP and FTSZ (gender specific), and BH-PCR (species-specific), as well as indirect immunofluorescence method for anti-B. henselae antibodies were performed to confirm the infection of the inoculated cat as well as the other naive cats. Considering the inoculated animal, B. henselae was first isolated by blood culture two months after inoculation, bacteremia last for four months, the specific antibodies being detected by IFI during the entire period. All contacting animals presented with bacteremia 6 months after experimental inoculation but IFI did not detect seroconversion in these animals. All the isolates from these cats were characterized as Bartonella (HSP and FTSZ-PCR), henselae (BH-PCR). However, DNA of B. henselae could not be amplified directly from peripheral blood by the PCR protocols used. Isolation of bacteria by blood culture was the most efficient method to diagnose infection compared to PCR or IFI. The role of fleas in the epidemiology of B. henselae infection in cats is discussed.

Long term evaluation of etiological treatment of Chagas disease with benznidazole

The aim of this article is to present an investigation of cure rate, after long follow up, of specific chemotherapy with benznidazole in patients with both acute and chronic Chagas disease, applying quantitative conventional serological tests as the base of the criterion of cure. Twenty one patients with the acute form and 113 with one or other of the various chronic clinical forms of the disease were evaluated, after a follow up period of 13 to 21 years, for the acute, and 6 to 18 years, for the chronic patients. The duration of the acute as well as the chronic disease, a condition which influences the results of the treatment, was determined. The therapeutic schedule was presented, with emphasis on the correlation between adverse reactions and the total dose of 18 grams, approximately, as well as taking into consideration precautions to assure the safety of the treatment. Quantitative serological reactions consisting of complement fixation, indirect immunofluorescence, indirect hemagglutination, and, occasionally, ELISA, were used. Cure was found in 76 per cent of the acute patients but only in 8 per cent of those with chronic forms of the disease. In the light of such contrasting results, fundamentals of the etiological therapy of Chagas disease were discussed, like the criterion of cure, the pathogenesis and the role of immunosuppression showing tissue parasitism in long standing chronic disease, in support of the concept that post-therapeutic consistently positive serological reactions mean the presence of the parasite in the patient's tissues. In relation to the life-cycle of T. cruzi in vertebrate host, there are still some obscure and controversial points, though there is no proof of the existence of resistant or latent forms. However, the finding over the last 15 years, that immunosuppression brings about the reappearance of acute disease in long stand chronic patients justifies a revision of the matter. Facts were quoted in favor of the treatment of chronic patients.

Development of a piezoelectric biosensor based on PVDF films

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Determinação das propriedades mecânicas à tração de adesivos estruturais frágeis e dúcteis

A crescente evolução na tecnologia das juntas coladas conferiu um potencial atractivo às ligações adesivas, com aplicações nas mais variadas indústrias. Isto deve-se não só aos aspetos económicos, tais como a melhoria da cadência de produção mas também à resistência mecânica que estas proporcionam. A possibilidade de ligar facilmente materiais distintos, a distribuição mais uniforme das tensões, a melhor resistência à fadiga e a elevada capacidade de amortecimento de vibrações estão entre as principais vantagens da utilização deste tipo de ligação. Estas propriedades transformam as juntas coladas numas das preferidas no momento de seleção de meios de união. O trabalho desenvolvido nesta dissertação enquadra-se no âmbito das ligações adesivas e tem como principais objetivos a produção de uma ferramenta para a produção de provetes de adesivo, assim como a determinação das propriedades mecânicas à tração dos mesmos para testar o desempenho do molde fabricado. Para tal, utilizou-se um adesivo frágil (Araldite® AV 138), um dúctil (Araldite® 2015) e um muito dúctil (SikaForce® 7888). Paralelamente é selecionado o método mais adequado na obtenção destes provetes, designadamente escolhendo entre a moldação em molde aberto e a injeção em molde fechado. Com vista à obtenção dos provetes, foi projetado e construído um molde em aço. Recorrendo à máquina de tração Shimadzu AG – X 100, realizaram-se os respetivos ensaios de tração, para a determinação de todas as propriedades mecânicas dos adesivos. Para efeitos de comparação de resultados foram utilizados dois tipos de extensómetros, um mecânico e um ótico. Os resultados experimentais permitiram observar que a presença de vazios afetou especialmente a deformação de rotura e a tensão de rotura. Detetaram-se pequenas discordâncias, comparativamente com os estudos publicados, de algumas características mecânicas obtidas dos diversos adesivos utilizados. Constatou-se também um ligeiro desfasamento entre os valores adquiridos com os dois tipos de extensómetros utilizados.

Diagnosis of pulmonary pneumocystosis by microscopy on wet mount preparations

We have compared the searching of the presence of "honeycomb" structures by direct microscopy on wet mount preparations with the direct immunofluorescence (DIF) for the diagnosis of Pneumocystis carinii pneumonia (PCP) in 115 bronchoalveolar (BAL) fluids. The samples belonged to 115 AIDS patients; 87 with presumptive diagnosis of PCP and 28 with presumptive diagnosis other than PCP. The obtained results were coincident in 114 out of 115 studied samples (27 were positive and 87 negative) with both techniques. A higher percentage of positive results (32.18%) among patients with presumptive diagnosis of PCP with respect to those with presumptive diagnosis other than PCP (3.57%) was observed. One BAL fluid was positive only with DIF, showed scarce and isolated P. carinii elements and absence of typical "honeycomb" structures. The searching for "honeycomb" structures by direct microscopy on wet mount preparations could be considered as a cheap and rapid alternative for diagnosis of PCP when other techniques are not available or as screening test for DIF. This method showed a sensitivity close to DIF when it was applied to BAL fluids of AIDS patients with poor clinical condition and it was performed by an experienced microscopist.

Role of ß-lactamase operon on mecA expression in Staphylococcus aureus

RESUMO: Os Staphylococcus aureus resistentes à meticilina (MRSA, do inglês “methicillin-resistant Staphylococcus aureus”) são um dos principais agentes responsáveis por infeções hospitalares. Os MRSA são resistentes a praticamente todos os antibióticos β-lactâmicos devido a dois mecanismos principais: produção de β-lactamase (bla), codificada pelo gene blaZ, e produção de uma proteína de ligação à penicilina (PBP2a, do inglês “penicillin binding protein 2”), codificada pelo gene mecA. Estes dois genes são regulados por sistemas homólogos, constituídos por um sensor-transdutor (BlaR1 e MecR1) e um repressor (BlaI e MecI), de tal modo que ambos os sistemas são capazes de co-regular os genes mecA e blaZ, embora com eficiências de indução muito diferentes. De facto, a indução mediada pelo sistema mecI-mecR1 é tão lenta que se acredita que este sistema não está funcional na maioria das estirpes MRSA. No entanto, dados recentes do nosso laboratório, demonstram a ausência de relação entre a presença do gene mecI e o nível de resistência à meticilina em estirpes MRSA epidémicas, e também que, o fenótipo de resistência da grande maioria das estirpes não é perturbado pela sobre-expressão em trans do repressor mecI. Curiosamente, as duas estirpes em que a expressão da resistência foi afetada pela sobre-expressão do mecI são negativas para o locus da β-lactamase, o que sugere que este locus pode interferir diretamente com a repressão do gene mecA mediada pelo MecI. Nesta tese de mestrado esta hipótese foi explorada usando estratégias de biologia molecular e ensaios fenotípicos da resistência aos -lactâmicos. Os resultados obtidos demonstram que a presença do plasmídeo nativo da β-lactamase não só anula a repressão mediada pelo MecI, como também aumenta o nível de resistência das estirpes parentais. Várias hipóteses foram então formuladas para explicar estas observações. Dados preliminares, em conjunto com evidências experimentais publicadas, sugerem que o BlaI forma hetero-dímeros com o MecI que, após a indução, são inativados eficientemente pelo BlaR1. Em conclusão, estes resultados apresentam novas perspetivas para o mecanismo de regulação do mecA e para uma nova importante função do operão da β-lactamase para o fenótipo das estirpes MRSA.-------------------ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and is also emerging in the community. MRSA is cross-resistant to virtually all β-lactam antibiotics and has acquired two main resistance mechanisms: production of β-lactamase (bla), coded by blaZ, and production of penicillin binding protein 2a (PBP2a), coded by mecA. Both genes are regulated by homologous sensor-transducers (BlaR1 and MecR1) and repressors (BlaI and MecI), and coregulation of mecA and blaZ by both systems has been demonstrated, although with remarkable different efficiencies. In fact, induction of mecA by mecI-mecR1 is so slow that it is believed it is not functional in most MRSA strains. However, recent data from our laboratory has unexpectedly demonstrated that not only there is no correlation between the presence of mecI gene and the resistance level in epidemic MRSA strains, but also that for most strains there were no significant changes on the resistance phenotype upon the mecI overexpression in trans. Interestingly, the two strains in which mecI overexpression affected the resistance expression were negative for the bla locus, suggesting that this locus may interfere directly with the MecI-mediated repression of mecA and account for those puzzling observations. In this master thesis we have explored this hypothesis using molecular biology strategies and phenotypic analysis of -lactam resistance. The data obtained demonstrate that the presence of a wild-type plasmid containing the bla locus not only disrupts the MecImediated repression, but also significantly enhances the expression of resistance. Several preliminary hypotheses were formulated to explain these observations and preliminary data, together with published evidence, support the working model that BlaI forms functional hetero-dimers with MecI, which upon induction are readily inactivated by BlaR1. These results provide new insights into the regulatory mechanism(s) of mecA and open new perspectives for the role of β-lactamase operon in the MRSA phenotype.

Vir sapiens dominabitur astris: astrological knowledge and practices in the Portuguese medieval court (king João I to king Afonso V)

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em História Medieval

Characterization of Nitric Oxide Reductase (NOR) from pseudomonas nautica, a study on biologic nitric oxide reduction

Dissertation submitted to obtain the phD degree in Biochemistry, specialty in Physical- Biochemistry, by the Faculdade de Ciências e Tecnologia from the Universidade Nova de Lisboa