Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails.

Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer

In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found.

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

Quantitative integration of geophysical and hydraulic data : from the local towards the regional scale range

Des progrès significatifs ont été réalisés dans le domaine de l'intégration quantitative des données géophysique et hydrologique l'échelle locale. Cependant, l'extension à de plus grandes échelles des approches correspondantes constitue encore un défi majeur. Il est néanmoins extrêmement important de relever ce défi pour développer des modèles fiables de flux des eaux souterraines et de transport de contaminant. Pour résoudre ce problème, j'ai développé une technique d'intégration des données hydrogéophysiques basée sur une procédure bayésienne de simulation séquentielle en deux étapes. Cette procédure vise des problèmes à plus grande échelle. L'objectif est de simuler la distribution d'un paramètre hydraulique cible à partir, d'une part, de mesures d'un paramètre géophysique pertinent qui couvrent l'espace de manière exhaustive, mais avec une faible résolution (spatiale) et, d'autre part, de mesures locales de très haute résolution des mêmes paramètres géophysique et hydraulique. Pour cela, mon algorithme lie dans un premier temps les données géophysiques de faible et de haute résolution à travers une procédure de réduction déchelle. Les données géophysiques régionales réduites sont ensuite reliées au champ du paramètre hydraulique à haute résolution. J'illustre d'abord l'application de cette nouvelle approche dintégration des données à une base de données synthétiques réaliste. Celle-ci est constituée de mesures de conductivité hydraulique et électrique de haute résolution réalisées dans les mêmes forages ainsi que destimations des conductivités électriques obtenues à partir de mesures de tomographic de résistivité électrique (ERT) sur l'ensemble de l'espace. Ces dernières mesures ont une faible résolution spatiale. La viabilité globale de cette méthode est testée en effectuant les simulations de flux et de transport au travers du modèle original du champ de conductivité hydraulique ainsi que du modèle simulé. Les simulations sont alors comparées. Les résultats obtenus indiquent que la procédure dintégration des données proposée permet d'obtenir des estimations de la conductivité en adéquation avec la structure à grande échelle ainsi que des predictions fiables des caractéristiques de transports sur des distances de moyenne à grande échelle. Les résultats correspondant au scénario de terrain indiquent que l'approche d'intégration des données nouvellement mise au point est capable d'appréhender correctement les hétérogénéitées à petite échelle aussi bien que les tendances à gande échelle du champ hydraulique prévalent. Les résultats montrent également une flexibilté remarquable et une robustesse de cette nouvelle approche dintégration des données. De ce fait, elle est susceptible d'être appliquée à un large éventail de données géophysiques et hydrologiques, à toutes les gammes déchelles. Dans la deuxième partie de ma thèse, j'évalue en détail la viabilité du réechantillonnage geostatique séquentiel comme mécanisme de proposition pour les méthodes Markov Chain Monte Carlo (MCMC) appliquées à des probmes inverses géophysiques et hydrologiques de grande dimension . L'objectif est de permettre une quantification plus précise et plus réaliste des incertitudes associées aux modèles obtenus. En considérant une série dexemples de tomographic radar puits à puits, j'étudie deux classes de stratégies de rééchantillonnage spatial en considérant leur habilité à générer efficacement et précisément des réalisations de la distribution postérieure bayésienne. Les résultats obtenus montrent que, malgré sa popularité, le réechantillonnage séquentiel est plutôt inefficace à générer des échantillons postérieurs indépendants pour des études de cas synthétiques réalistes, notamment pour le cas assez communs et importants où il existe de fortes corrélations spatiales entre le modèle et les paramètres. Pour résoudre ce problème, j'ai développé un nouvelle approche de perturbation basée sur une déformation progressive. Cette approche est flexible en ce qui concerne le nombre de paramètres du modèle et lintensité de la perturbation. Par rapport au rééchantillonage séquentiel, cette nouvelle approche s'avère être très efficace pour diminuer le nombre requis d'itérations pour générer des échantillons indépendants à partir de la distribution postérieure bayésienne. - Significant progress has been made with regard to the quantitative integration of geophysical and hydrological data at the local scale. However, extending corresponding approaches beyond the local scale still represents a major challenge, yet is critically important for the development of reliable groundwater flow and contaminant transport models. To address this issue, I have developed a hydrogeophysical data integration technique based on a two-step Bayesian sequential simulation procedure that is specifically targeted towards larger-scale problems. The objective is to simulate the distribution of a target hydraulic parameter based on spatially exhaustive, but poorly resolved, measurements of a pertinent geophysical parameter and locally highly resolved, but spatially sparse, measurements of the considered geophysical and hydraulic parameters. To this end, my algorithm links the low- and high-resolution geophysical data via a downscaling procedure before relating the downscaled regional-scale geophysical data to the high-resolution hydraulic parameter field. I first illustrate the application of this novel data integration approach to a realistic synthetic database consisting of collocated high-resolution borehole measurements of the hydraulic and electrical conductivities and spatially exhaustive, low-resolution electrical conductivity estimates obtained from electrical resistivity tomography (ERT). The overall viability of this method is tested and verified by performing and comparing flow and transport simulations through the original and simulated hydraulic conductivity fields. The corresponding results indicate that the proposed data integration procedure does indeed allow for obtaining faithful estimates of the larger-scale hydraulic conductivity structure and reliable predictions of the transport characteristics over medium- to regional-scale distances. The approach is then applied to a corresponding field scenario consisting of collocated high- resolution measurements of the electrical conductivity, as measured using a cone penetrometer testing (CPT) system, and the hydraulic conductivity, as estimated from electromagnetic flowmeter and slug test measurements, in combination with spatially exhaustive low-resolution electrical conductivity estimates obtained from surface-based electrical resistivity tomography (ERT). The corresponding results indicate that the newly developed data integration approach is indeed capable of adequately capturing both the small-scale heterogeneity as well as the larger-scale trend of the prevailing hydraulic conductivity field. The results also indicate that this novel data integration approach is remarkably flexible and robust and hence can be expected to be applicable to a wide range of geophysical and hydrological data at all scale ranges. In the second part of my thesis, I evaluate in detail the viability of sequential geostatistical resampling as a proposal mechanism for Markov Chain Monte Carlo (MCMC) methods applied to high-dimensional geophysical and hydrological inverse problems in order to allow for a more accurate and realistic quantification of the uncertainty associated with the thus inferred models. Focusing on a series of pertinent crosshole georadar tomographic examples, I investigated two classes of geostatistical resampling strategies with regard to their ability to efficiently and accurately generate independent realizations from the Bayesian posterior distribution. The corresponding results indicate that, despite its popularity, sequential resampling is rather inefficient at drawing independent posterior samples for realistic synthetic case studies, notably for the practically common and important scenario of pronounced spatial correlation between model parameters. To address this issue, I have developed a new gradual-deformation-based perturbation approach, which is flexible with regard to the number of model parameters as well as the perturbation strength. Compared to sequential resampling, this newly proposed approach was proven to be highly effective in decreasing the number of iterations required for drawing independent samples from the Bayesian posterior distribution.

Mycobacterium bovis: polymerase chain reaction identification in bovine Lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

Parasite persistence in treated chagasic patients revealed by xenodiagnosis and polymerase chain reaction

Polymerase chain reaction (PCR) was compared with xenodiagnosis performed 20 years after trypanocidal chemotherapy to investigate parasite clearance. Eighty-five seropositive individuals for Chagas disease presenting a positive xenodiagnosis were treated with specific drugs; 37 in the acute phase and 48 in the chronic phase. Fifteen chronic assymptomatic patients received a placebo. Treatment in the acute phase led to PCR negative results in 73% of the cases, while xenodiagnosis was negative in 86%. In the chronic phase, PCR was negative in 65% of the patients and 83% led to xenodiagnosis negative results. Regarding the untreated group (placebo), 73% gave negative results by xenodiagnosis, of which 36% were positive by PCR. Individuals that were considered seronegative (n=10), presented unequivocally negative results in the PCR demonstrating the elimination of parasite DNA. Seventeen individuals had their antibodies titers decreased to such a level that the final results were considered as doubtful and 16 of them presented negative PCR. The molecular method represents a clear advantage over conventional techniques to demonstrate persistent infections in Chagas disease patients that underwent chemotherapy.

Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Conflictes i articulacions entre el dret local i el dret estatal a Madagascar

Aquesta recerca analitza els conflictes i les articulacions entre el dret local i el dret estatal que tenen lloc a les comunitats rurals de Madagascar a l’hora de resoldre els conflictes a l’àmbit local. L’anàlisi es porta a terme a partir d’un cas etnogràfic concret que permet descriure aquesta pluralitat jurídica. A la present memòria es descriu el treball realitzat en el recorregut de la investigació i dóna compte de la gestació del projecte fins a l’actualitat, moment en el qual la recerca es troba en el seu darrer punt de redacció de la tesi.

Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood of individuals, eight years after completion of anti-leprosy therapy

Thirty eight patients with indeterminate leprosy (HI), at least 4 to 6 years after discharge from multibacillary (MB) or paucibacillary (PB) schemes of anti leprosy multidrug therapy (MDT), were submitted to traditional diagnostic procedures for leprosy and to polymerase chain reaction (PCR) analysis of different clinical samples for detection of Mycobacterium leprae DNA. No significant difference was observed for any of the parameters analyzed between PB or MB schemes of treatment and no indications were found for more efficient outcome of HI using the MB scheme. Remarkably, 18 (54.5%) of the individuals were PCR positive in at least one of the samples: positivity of PCR was highest in blood samples and four individuals were PCR positive in blood and some other sample. Upon comparison of PCR results with clinical and histopathological parameters, no correlation was found between PCR-positivity and eventual relapse. This is the first report on detection of M. leprae DNA in PB patients, more than half a decade after completion of MDT, suggesting that live bacilli are present and circulating much longer than expected, although reinfection of the individuals can not be excluded. Overall, we feel that because of the high sensitivity of the assay, extreme care should be taken about association of PCR results, efficacy of treatment and disease status.

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.

Comparación de datos de expresión génica del servidor local con datos de una base de datos remota

En la presente memoria se detalla con precisión las diversas fases del trabajo para construir una aplicación web en el servidor http://revolutionresearch.uab.es que permite enriquecer los clusters de la microarray del usuario con información biomédica de una base de datos remota. Los clusters de origen estadístico (o no) de la microarray del usuario se enriquecen a partir de cruzar sus genes marcadores con la base de datos de genes marcadores de microarrays (base de datos remota) con clusters basados en información biomédica. La base de datos de genes marcadores de microarrays ha sido obtenida a partir de la base de datos de GEO Profiles del NCBI.

Triatomines Involved in Domestic and Wild Trypanosoma cruzi Transmission in Concepción, Corrientes, Argentina

An entomological and serological survey was performed in three localities of the Department of Concepción, Province of Corrientes, Argentina in 1998 and 1999, to identify triatomines species involved in domestic and wild transmission of Chagas disease. Triatomines were collected by man/hour capture in 32 houses randomly selected and 44 nearby outdoor ecotopes. Trypanosoma cruzi infection in triatomines was assessed by direct microscopic observation (400x) of feces and polymerase chain reaction. Serological techniques used for people were Indirect Hemagglutination Test and Indirect Fluorescent Test. Triatomines were collected in 28.1% of the houses and 31.8% of the wild biotopes. Triatoma infestans (Klug 1834) was exclusively found indoors and T. cruzi infected 60% of them. Triatoma sordida (Stål 1859) was mainly found in extradomestic ecotopes where trypanosome infection rate reached 12.7%. Serological study of 98 local people showed that 29.6% were seroreactive; most of their houses were closed to wild biotopes colonized by T. sordida. Results indicate that there is an active T. infestans mediated transmission of Chagas disease in this zone that yields important human prevalence and that the populations of T. sordida in wild biotopes not only sustain the wild T. cruzi cycle but also represent an actual risk for people living in the area.

Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways.

BACKGROUND: Activation of coagulation and fibrinolysis play a role in the pathophysiology of experimental arthritis. Objective: To determine the extent of activation of the coagulation and fibrinolytic pathways in different joint diseases in humans and to ascertain the factors that may influence fibrin deposition within the joint. METHODS: Plasma from normal subjects (controls, n= 21) and plasma and synovial fluid samples from patients with rheumatoid arthritis (RA; n = 64), osteoarthritis (OA; n = 29), spondyloarthropathy (SpA; n = 22) and crystal arthritis (CA; n = 25) were analyzed for the levels of TF (tissue factor) and tissue factor pathway inhibitor (TFPI) activities, thrombin-antithrombin III (TAT) complexes, and F1 + 2 (thrombin fragment), fibrin d-dimer and thrombin-activated fibrinolysis inhibitor (TAFI) antigenic levels. The measurements were analyzed by pairwise correlation with each other as well as with standard parameters of inflammation [C-reactive protein (CRP), joint leukocyte count]. Inter-group comparisons were performed to look for disease-specific differences. RESULTS: Compared with healthy controls, patients with joint diseases had higher levels of TAT, F1 + 2 and d-dimers in their plasma. In the synovial fluid, TF activity, TAT, d-dimers, and TAFI were significantly higher in inflammatory arthritides than in OA. The levels were highest in RA patients. In the plasma, TF activity was correlated with TAT and d-dimer levels with CRP, TFPI, and TAT. In the synovial fluid, TF activity correlated with plasma CRP levels, synovial fluid leukocyte count, and synovial TAT and TAFI levels. In addition, synovial d-dimers correlated with CRP, and synovial TAFI levels were correlated with synovial F1 + 2 and TAT. CONCLUSIONS: Activation of the coagulation and fibrinolytic cascades in the joint and in the circulation is evident in both inflammatory and degenerative joint diseases. Within the joint, inflammatory mechanisms leading to TF-mediated activation of the coagulation pathway and subsequent fibrin deposition is the most likely explanation for the observed findings. In the plasma, the link between inflammation (CRP increase) and TF activation is weak, and a non-TF-mediated mechanism of coagulation activation could explain these findings. RA is characterized by significantly higher levels of TAT in the synovial fluid and plasma than other arthritides. Although fibrinolytic activity is linked to inflammation, the increased amounts of TAFI in the joint, particularly in RA, may explain why fibrin formation is so prominent in this condition compared with other joint diseases.

A Heminested Polymerase Chain Reaction for the Detection of Brazilian Rabies Isolates from Vampire Bats and Herbivores

A heminested-PCR (hn-PCR) using primers to the nucleoprotein-coding gene in a nested set was evaluated in the detection of Brazilian strains of rabies virus (RV). A representative number of RV nucleoprotein sequences belonging to genotype 1 were aligned. Based on such alignment, primers were directed to highly conserved regions. All 42 clinical samples positive by both fluorescent antibody and mouse inoculation tests were also positive by the hn-PCR. Brain tissue that had been left to decompose, obtained from an experimentally inoculated mouse was tested by hn-PCR and yielded positive results. In conclusion, primers designed here were capable of amplifying Brazilian RV isolates obtained from a rural epidemiological cycle.