Biological and morphological characteristics of Schistosoma mansoni from Ribeira Valley, State of São Paulo, Brazil: I - susceptibility of Biomphalaria tenagophila snail to sympatric S. mansoni strain

In the São Paulo State, Brazil, where the Biomphalaria tenagophila is the intermediate host, the Ribeira Valley is an important endemic schistosomiasis mansoni area. During last eleven years there has been intense control measures focusing on schistosomiasis. The efforts have been concentrated in the municipalities of Pedro de Toledo and Itariri. We determined the susceptibility of B. tenagophila to sympatric strain of S. mansoni, both recently isolated from Itariri field. In 1988, this strain was isolated and maintained in the experimental model: Swiss mice - sympatric B. tenagophila. The second generation of the worm was evaluated. The snail were divided in the three groups of 60 snails each. One group was exposed to 1 miracidium and other to 10. The third group was the control. The mortality and the shedding of cercariae were checked during 78 days. After that, the positive snails were observed until they ceased to shed cercariae. The exposed molluscs showed mortality rates of 23% and 31% and infection indexes were of 8% and 60% to 1 and 10 miracidia respectively. The mortality was of 22% in the control group. The periods of shedding cercariae in the two groups were 82 and 104 days. We can conclude that B. tenagophila is an effective intermediate host to the sympatric strain of S. mansoni sympatric strain

Mediterranean spotted fever and identification of new agents of rickettsioses in Portugal : epidemiological determinants, host and microbial features in portuguese patients

Resumo: a febre botonosa, também conhecida por febre escaro-nodular (FEN) é uma doença endémica nos Países da bacia do Mediterrâneo, África, Médio Oriente, Índia e Paquistão. O agente etiológico responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservatório é o ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros países da bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para além dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição da terapêutica específica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infecção por R.conorii.Face a estas questões foi avaliada e comparada a epidemiologia, manifestações clínicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposição de manifestações clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inoculação é significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau prognósitco da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunitária à infecção por R. conorii e as interacções hospedeiro - agente etiológico têm sido elucidados com base em modelos animais. Poucos estudos têm sido efectuados em doentes e nenhum estudo prévio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras moléculas envolvidas na resposta imunitária às rickettsioses. Foi avaliado o nível de expressão génica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela técnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam níveis estatisticamente significativos, mais elevados de expressão génica de interferão (IFN-γ, Tumor necrosis factor (TFN-α, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limitação do crescimento intracelular das rickettsias, através da degradação do triptofano. Seis dos 23 doentes apresentaram ainda niveis de expressão elevados de óxido nítrico indutível (iNOS)que actua como microbicida. Encontrou-se uma correlação positiva entre a expressão de RNAm de TNF-α, γ, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunitária tipo Th1, i.e. com papel protector na resposta à infecção.Verificou-se também que os valores de expressão genética do RNAm de IL-10, estavam inversamente correlacionados com a expressão do RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre a resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão génica do RNAm de IL-10 estavam inversamente correlacionados com a expressão RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre uma resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta dissertação é ainda descrita uma nova rickettsiose presente em Portugal, causada pela bactéria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detecção do DNA em biopsia de pele. A presença deste agente foi ainda corroborada pela detecção em paralelo do mesmo agente no ixodídeos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de existência de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-α, IFN-γ, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.

In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy

BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables such as the concentrations of biomass, plasmid, carbon sources (glucose and glycerol) and acetate. In order to achieve robust models able to predict the performance of plasmid production processes, independently of the composition of the cultivation medium, cultivation strategy (batch versus fed-batch) and E. coli strain used, three strategies were adopted, using: (i) E. coliDH5 cultures conducted under different media compositions and culture strategies (batch and fed-batch); (ii) engineered E. coli strains, MG1655endArecApgi and MG1655endArecA, grown on the same medium and culture strategy; (iii) diverse E. coli strains, over batch and fed-batch cultivations and using different media compositions. PLS models showed high accuracy for predicting all variables in the three groups of cultures. CONCLUSIONNIR spectroscopy combined with PLS modeling provides a fast, inexpensive and contamination-free technique to accurately monitoring plasmid bioprocesses in real time, independently of the medium composition, cultivation strategy and the E. coli strain used.

Susceptibility of Biomphalaria tenagophila and Biomphalaria glabrata from the same region to two Brazilian strains of Schistosoma mansoni

B. tenagophila snails from Ouro Branco, MG, showed positivity for S. mansoni, with infection rates of 5%, 10%, (SJ strain), and 1% (LE strain) using a pool of miracidia. The mollusks were found to be susceptive from the 3rd generation reared in laboratory onwards. The B. tenagophila (OB, MG) when individually exposed to 10 miracidia, showed infection rate of 2% for LE strain. B. glabrata snails from Gagé, MG, showed a positivity rate of 58% for S. mansoni (LE strain), under experimental conditions. The B. tenagophila from Cabo Frio, RJ and B. glabrata from Belo Horizonte, MG used as a control for SJ strain showed infection rates of 47% - 85% and 36% respectivily. For the LE strain, B. glabrata (BH, MG) used as control showed infection rate of 40% - 75%.

Utilização de métodos ópticos de correlação de imagem para a determinação da tenacidade à fractura de adesivos estruturais

Mestrado em Engenharia Mecânica

Characterization of a Trypanosoma cruzi strain isolated from a non-endemic area in Northeast Brazil

A cepa WSL (Wild São Lorenço) de T. cruzi, isolada de um cobaio proveniente de São Lorenço da Mata (Nordeste do Brasil) foi caracterizada através da análise do seu comportamento morfobiológico e perfil isoenzimático. Para o estudo do comportamento morfobiológico, tripomastigotas sanguíneos (1 x 10 5) da cepa WSL foram inoculados por via intraperitonal em camundongos albinos Swiss. Como controle a cepa Y (Tipo I) foi usada. Durante o curso da infecção os seguintes parâmetros foram analisados: parasitemia, mortalidade, morfologia dos parasitas no sangue periférico e tropismo tissular. O perfil isoenzimático foi analisado em relação às enzimas ALAT, GPI e PGM usando como controle de referência as cepas Peruana (Tipo I), 21SF (Tipo II) e Colombiana (Tipo III). A cepa WSL apresentou as seguintes características biológicas: 1) multiplicação lenta e pico parasitêmico entre 21 - 25 dias pós-infecção; 2) mortalidade de 3,3% 40 dias pós-infecção; 3) predominância de formas largas no sangue periférico e 4) miotropismo com predominante envolvimento cardíaco. A análise isoenzimática mostrou um padrão de zimodema 2 (Z2) que corresponde às cepas biológicas Tipo II. Os resultados mostram que a cepa WSL apresenta baixa virulência e patogenicidade.

Resistance of Streptococcus pneumoniae to antimicrobials in São Paulo, Brazil: clinical features and serotypes

To study resistance to antimicrobials, serotypes and clinical features of S. pneumoniae in S. Paulo, Brazil, 50 patients with a positive culture were evaluated: 7 were considered carriers and 43 had pneumococcal infections. Pneumonia and meningitis were the most commom infections. Mortality was 34% and underlying diseases were present in 70%. Relative resistance to penicillin occurred in 24% and complete resistance was not detected. Resistance to tetracycline was 32% and to sulfamethoxazole/trimethoprim 32%; one strain had intermediate susceptibility to erythromycin; no resistance was present for chloramphenicol, rifampin or vancomycin. Resistance to at least one of the drugs tested occurred in 62%. Results by the E-test for penicillin were similar to those by the agar dilution method. There were 24 different serotypes and 74% of the strains belonged to the 23-valent vaccine including all the penicillin-resistant strains. In this study S. pneumoniae caused severe infections and presented a high resistance rate to commonly used antimicrobials. Routine surveillance of resistance and the use of vaccination, as well as the restriction of inappropriate use of antimicrobials, are recommended in São Paulo, Brazil.

RESISTANCE TO OXAMNIQUINE OF A Schistosoma mansoni STRAIN ISOLATED FROM PATIENT SUBMITTED TO REPEATED TREATMENTS

A strain of Schistosoma mansoni (R1) was isolated from patient previously submitted to four treatments with oxamniquine, and to another one with praziquantel. The results obtained with chemotherapeutic test, by using oxamniquine in mice infected with the strains R1 and LE (standard), showed an evident resistance to the drug in worms of the strain R1. Thus, at the dose of 250 mg/kg oxamniquine, all mice (17) infected with the LE strain did not show surviving worms, whereas 12 out of 17 mice infected with the R1 strain presented surviving worms. At the dose of 200 mg/kg, the LE strain showed recovery rates of 1.06% and 20.58%, whereas the R1 strain presented 18.57% and 61.14%, for male and female worms, respectively. At the dose of 100 mg/kg, the recovery of male worms was 2.6% for the LE strain, and 29.9% for the R1 strain. At the same dose, the recovery of females did not show statistically significant differences between the two strains (LE = 76.38%, R1 = 79.12%). Praziquantel showed similar antischistosomal activity against both studied strains, when administered at the dose of 500 mg/kg

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

LARVICIDAL ACTIVITY OF Bacillus sphaericus 2362 AGAINST Anopheles nuneztovari, Anopheles darlingi AND Anopheles braziliensis (DIPTERA, CULICIDAE)

In this present study, preliminary data was obtained regarding the mortality rate of the Amazonian anophelines, Anopheles nuneztovari, Anopheles darlingi and Anopheles braziliensis when subjected to treatment with Bacillus sphaericus strain 2362, the WHO standard strain. Initially, experiments were conducted to test the mortality rate of the three species of anopheline larvae. The third larval instar of An. nuneztovari and the second and third larval instars of An. darlingi proved to be the least susceptible. In other experiments, the same three mosquito species were tested with the standard strain 2362, An. nuneztovari was the least susceptible to this insect pathogen, while An. braziliensis was the most susceptible. This latter species showed a difference in the level of LC50 concentration, when compared to the former, of 2.4, 2.5 and 1.8 in readings taken 24, 48 and 72 hours after exposure to the bacillus.

Hepatic regeneration after partial hepatectomy in mice infected with Schistosoma mansoni, at the acute and chronic phases of the disease

Outbred male albino mice normal or infected with 30 cercariae of Schistosoma mansoni (LE strain) were submitted to 65% hepatectomy during the acute (70 days) and chronic phase (160 days) phases of the disease. A group of the infected animals was treated with 400 mg/kg of oxamniquine during the acute phase before hepatectomy. Non-infected, infected and treated but not hepatectomized animals were kept as controls. Hepatic regeneration was evaluated by incorporation of tritiated thymidine, intraperitoneally injected into non-hepatectomized and hepatectomized animals, 24 hours after surgery. The results showed that removal of 65% of the hepatic parenchyma, during the acute phase, led to a statistically significant increase of thymidine incorporation, when compared with the uninfected hepatectomized controls. This phenomenon was not observed at the chronic phase. Treatment with oxamniquine administered during the acute phase led to a decrease in thymidine incorporation rate 160 days after infection (90 days after treatment) and 24 hours after hepatectomy. The data suggest that infection with S. mansoni represents a considerable stimulus for the regenerative capacity of the liver during the acute, but not the chronic phase of disease.

Estimation of infection rate in epidemic models with multiple populations

Dissertação para obtenção do Grau de Mestre em Matemática e Aplicações Especialização em Actuariado, Estatística e Investigação Operacional

Mucosal leishmaniasis ("espundia") responsive to low dose of N-methyl glucamine (Glucantime ®) in Rio de Janeiro, Brazil

Response to treatment with antimonial drugs varies considerably depending on the parasite strain involved, immune status of the patient and clinical form of the disease. Therapeutic regimens with this first line drug have been frequently modified both, in dose and duration of therapy. A regimen of 20 mg/kg/day of pentavalent antimony (Sb5+) during four weeks without an upper limit on the daily dose is currently recommended for mucosal disease ("espundia"). Side-effects with this dose are more marked in elderly patients, more commonly affected by this form of leishmaniasis. According to our experience, leishmaniasis in Rio de Janeiro responds well to antimony and, in cutaneous disease, high cure rates are obtained with 5 mg/kg/day of Sb5+ during 30 to 45-days. In this study a high rate of cure (91.4%) employing this dose was achieved in 36 patients with mild disease in this same geographic region. Side-effects were reduced and no antimony refractoriness was noted with subsequent use of larger dose in patients that failed to respond to initial schedule.