Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Identification of a genetic marker associated with the resistance to Schistosoma mansoni infection using random amplified polymorphic DNA analysis

In schistosomiasis, the host/parasite interaction remains not completely understood. Many questions related to the susceptibility of snails to infection by respective trematode still remain unanswered. The control of schistosomiasis requires a good understanding of the host/parasite association. In this work, the susceptibility/resistance to Schistosoma mansoni infection within Biomphalaria alexandrina snails were studied starting one month post infection and continuing thereafter weekly up to 10 weeks after miracidia exposure. Genetic variations between susceptible and resistant strains to Schistosoma infection within B. alexandrina snails using random amplified polymorphic DNA analysis technique were also carried out. The results showed that 39.8% of the examined field snails were resistant, while 60.2% of these snails showed high infection rates.In the resistant genotype snails, OPA-02 primer produced a major low molecular weight marker 430 bp. Among the two snail strains there were interpopulational variations, while the individual specimens from the same snail strain, either susceptible or resistant, record semi-identical genetic bands. Also, the resistant character was ascendant in contrast to a decline in the susceptibility of snails from one generation to the next.

RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

Disagreement between ultrasound and magnetic resonance imaging in the identification of schistosomal periportal fibrosis

Abdominal ultrasound (US) has been widely used in the evaluation of patients with schistosomiasis mansoni. It represents an important indirect method of diagnosis and classification of the disease, and it has also been used as a tool in the evaluation of therapeutic response and regression of fibrosis. We describe the case of a man in whom US showed solid evidence of schistosomal periportal fibrosis and magnetic resonance imaging revealed that periportal signal alteration corresponded to adipose tissue which entered the liver togheter with the portal vein.

Identification of a novel 45-kDa cell surface molecule involved in activation of the human Jurkat T cell line.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

First detection of a human astrovirus type 8 in a child with diarrhea in Belém, Brazil: comparison with other strains worldwide and identification of possible three lineages

This study describes the genetic relationships of the first human astrovirus type-8 (HAstV-8) detected in Belém-Brazil, during a public hospital-based study. This strain was compared with other HAstV-8 strains identified elsewhere which have sequences available at GeneBank. The regions ORF1a (primers Mon348/Mon340) and ORF2 (primers Mon269/Mon270) were analyzed by nucleotide sequencing and a high similarity rate was observed among the Belém strain and other HAstV-8 strains. In ORF1a, homology values of 93-100% were detected, and in ORF2 96-99%. Considering the sequence variation (7%) observed in ORF2 region, it was suggested that HAstV-8 strains could be divided in three different lineages.

Isolation and identification of bovine tuberculosis in a Brazilian herd (São Paulo)

Mycobacterium was verified in animals from a Brazilian dairy herd, a total of 42 samples from 30 cows were submitted to culture and the isolated strains were analyzed by two polymerase chain reaction (PCR), the first specific for species belonging to the Mycobacterium complex (MTBC) and the other for differentiating M. tuberculosis from M. bovis. Twenty seven samples (64.3%) from 18 animals (60%) were positive for mycobacteria by culture, including samples from 15 retrofaryngeal lymphnodes (55.5%), 9 prescapular lymphnodes (33.3%), 2 lungs (7.4%), and 1 liver (3.7%). All isolated colonies were confirmed by PCR to contain MTBC organisms, and were identified as M. bovis by the same methodology.

Isolation and molecular identification of Leishmania ( Viannia ) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes

Leishmania (Viannia) peruviana was isolated from 1/75 Lutzomyia peruensis captured during May 2006 in an endemic cutaneous leishmaniasis region of the Peruvian Andes (Chaute, Huarochiri, Lima, Peru). Sand fly gut with promastigotes was inoculated into a hamster and the remaining body was fixed in ethanol. L. (Viannia) sp. was determined by polymerase chain reaction (PCR), and Leishmania species through molecular genotyping by PCR-restriction fragment length polymorphism analyses targeting the genes cpb and hsp70, resulting L. (V.) peruviana. The infected sand fly appeared 15 days after the rains finished, time expected and useful real time data for interventions when transmission is occurring.

Géopatrimoines des trois Chablais : identification et valorisation des témoins glaciaires

Cette recherche s'intéresse aux témoins glaciaires des Chablais dans quatre de leurs dimensions : géopatrimoine, connaissance objective, inventaire de géosites et valorisation. En 2009, débutait le projet 123 Chablais, pour une durée de quatre ans. Ce projet, élaboré dans le cadre du programme Interreg IV France-Suisse, avait pour but de dynamiser le développement économique local en s'appuyant sur les patrimoines régionaux. Le géopatrimoine, identifié comme une de ces ressources, faisait donc l'objet de plusieurs actions, dont cette recherche. Le premier objectif de ce travail était d'acquérir une vision synthétique des témoins glaciaires locaux et d'en améliorer la connaissance, notamment, leur chronologie. Une série de datations par isotopes cosmogéniques produits in situ a permis de compléter des données disponibles pour deux stades glaciaires bien identifiés: le dernier maximum glaciaire (LGM) et un stade de retrait du glacier du Rhône (le stade de Monthey). Le second objectif était centré sur les processus d'identification et d'inventaire des géosites glaciaires. L'analyse des résultats de l'inventaire a fait apparaître quelques caractéristiques du patrimoine glaciaire chablaisien, discret, diversifié, et comportant deux spécificités exploitables dans le cadre d'une médiation scientifique : son statut de « berceau de la théorie glaciaire » et ses liens étroits avec des activités de la vie quotidienne, en tant que matière première, support de loisir ou facteur de risque. Finalement, cette recherche a débouché sur l'élaboration d'une exposition itinérante sur le patrimoine glaciaire des Chablais.

Identification and functional characterization of a monofunctional peroxisomal enoyl-CoA hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90,6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.

Identification of hepatitis C virus subtype 2c by sequencing analysis in patients from Córdoba, Argentina

In Argentina, most information on hepatitis C virus (HCV) genotype distribution comes from studies carried out in Buenos Aires (east province). In order to identify HCV subtypes in central Argentina, nucleotide sequencing of core region was performed in samples from 36 patients living in Córdoba, the second most populated province of Argentina. The sequence analysis identified subtype 2c as the most prevalent (50%), followed by subtype 1b (33%) and to a lesser extent by subtypes 1a (11%), 3a (3%) and 4a (3%). This is the first report of circulation of HCV subtype 2c in this region of Argentina and also such high prevalence has never been found before in the genotype distribution of South America.

More about the role of 2,6-dichlorophenol in tick courtship: identification and olfactometer bioassay in Amblyomma cajennense and Rhipicephalus sanguineus

This study aimed to identify 2,6-dichlorophenol (2,6-DCP) in Amblyomma cajennense and to evaluate its role in A. cajennense and Rhipicephalus sanguineus courtship. Hexanic extract from attractive females was purified by solid phase extraction and the phenol was identified by the single ion monitoring method using GC/MS. In an olfactometer, the responses of A. cajennense and R. sanguineus males to females, control rubber septa or rubber septa impregnated with 2,6-DCP at 50, 500, and 5000 ng, respectively, were studied. 2,6-DCP was identified in A. cajennense extract and the males oriented themselves toward the concentration of 500 ng. These septa and the females were recognized as copula partners. The septa treated with 2,6-DCP did not attract and were not even recognized by the R. sanguineus males, whereas the females were recognized. Due to the presence of 2,6-DCP in A. cajennense and the results of biological bioassays, it was concluded that this compound acts as an attractant and mounting sex pheromone in this tick, but it does not play any role in R. sanguineus courtship.

Molecular identification of Rickettsia felis in ticks and fleas from an endemic area for Brazilian Spotted Fever

Rickettsioses are arthropod-borne diseases caused by parasites from the Order Rickettsiales. The most prevalent rickettsial disease in Brazil is Brazilian Spotted Fever (BSF). This work intends the molecular detection of those agents in ectoparasites from an endemic area of BSF in the state of Espírito Santo. A total of 502 ectoparasites, among them Amblyomma cajennense, Amblyomma dubitatum (A. cooperi), Riphicephalus sanguineus, Anocentor nitens and Ctenocephalides felis, was collected from domestic animals and the environment and separated in 152 lots according to the origin. Rickettsia sp. was detected in pools of all collected species by amplification of 17kDa protein-encoding gene fragments. The products of PCR amplification of three samples were sequenced, and Rickettsia felis was identified in R. sanguineus and C. felis. These results confirm the presence of Rickettsia felis in areas previously known as endemic for BSF, disease caused by Rickettsia rickettsii. Moreover, they show the needing of further studies for deeper knowledge of R. felis-spotted fever epidemiology and differentiation of these diseases in Brazil.