Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specifics probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

An oligonucleotide probe derived from kDNA minirepeats is specific for Leishmania (Viannia)

Sequence analysis of Leishmania (Viannia) kDNA minicircles and analysis of multiple sequence alignments of the conserved region (minirepeats) of five distinct minicircles from L. (V.) braziliensis species with corresponding sequences derived from other dermotropic leishmanias indicated the presence of a sub-genus specific sequence. An oligonucleotide bearing this sequence was designed and used as a molecular probe, being able to recognize solely the sub-genus Viannia species in hybridization experiments. A dendrogram reflecting the homologies among the minirepeat sequences was constructed. Sequence clustering was obtained corresponding to the traditional classification based on similarity of biochemical, biological and parasitological characteristics of these Leishmania species, distinguishing the Old World dermotropic leishmanias, the New World dermotropic leishmanias of the sub-genus Leishmania and of the sub-genus Viannia.

Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenothypical gentamicin resistance amplification (frequencies of 10-3 to 10-5, compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromossomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

Ultrastructural features of the midgut epithelium of females Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae)

A morphological study of the midgut of Lutzomyia intermedia, the primary vector of cutaneous leishmaniasis, in southeast Brazil, was conducted by light, scanning and transmission electron microscopy. The midgut is formed by a layer of epithelium of columnar cells on a non-cellular basal lamina, under which there is a musculature, which consists of circular and longitudinal muscular fibers. A tracheolar network is observed surrounding and penetrating in the musculature. Females were examined 12, 24, 48, 72 h and 5 days following a blood meal and were analyzed comparatively by transmission electron microscopy with starved females. In starved females, the epithelium of both the anterior and posterior sections of the midgut present whorl shaped rough endoplasmic reticulum. The posterior section does not present well-developed cellular structures such as mitochondria. Observations performed at 12, 24, 48 and 72 h after the blood meal showed morphological changes in the cellular structures in this section, and the presence of the peritrophic matrix up to 48 h after the blood meal. Digestion is almost complete and a few residues are detected in the lumen 72 h after blood feeding. Finally, on the 5th day after the blood meal all cellular structures present the original feature resembling that seen in starved sand flies. Morphometric data confirmed the morphological observations. Mitochondria, nuclei and microvilli of midgut epithelial cells are different in starved and blood fed females. The mitochondria present a similar profile in the epithelium of both the anterior and posterior section of the midgut, with higher dimension in starved females. The cell microvilli in the posterior section of the midgut of starved females are twice the size of those that had taken a blood meal. We concluded that there are changes in the midgut cellular structures of L. intermedia during the digestion of blood, which are in agreement with those described for other hematophagous diptera.

Anionic Sites, Fucose Residues and Class I Human Leukocyte Antigen Fate During Interaction of Toxoplasma gondii with Endothelial Cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocytte antigen (HLA) before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containig vacuoles.

Antibody isotype responses to egg antigens in human chronic Schistosomiasis mansoni before and after treatment

In the present communication we analyzed the levels of IgG1, IgG2, IgG3, IgG4 and IgE isotypes to soluble egg antigen of Schistosoma mansoni by ELISA in individuals from an endemic area for schistosomiasis in Northeast Brazil. The analysis was performed before and after treatment to evaluate the age-dependent pattern, and to identify differences in the reactivities to antigens. Our results suggest that schistosomiasis treatment would not interfere with this sort of immune response.

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagelar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.

Circulating filarial antigen in the hydrocele fluid from individuals living in a bancroftian filariasis area - Recife, Brazil: detected by the monoclonal antibody Og4C3-assay

The purpose of this study was to examine the circulating filarial antigen (CFA) detected by the monoclonal antibody (mAb) Og4C3-ELISA in paired samples of serum and hydrocele fluid from 104 men with hydrocele, living in an endemic area of Wuchereria bancrofti. Nocturnal blood specimens were filtered and examined for microfilariae (MF) and ultrasound was used in order to identify the presence of adult worms (the filaria dance sign - FDS) in the lymphatic vessels of the scrotal area. Four groups were selected according to their parasitological status: group I - 71 MF- and FDS-; group II - 21 MF+ and FDS+; group III - 10 MF- and FDS+ and group IV- 2 MF+ and FDS-. CFA was identified simultaneously (fluid and serum) in 11 (15.5%), 21 (100%), 3 (30%), and 1 (50%) in groups I, II, III, and IV, respectively. In despite of high CFA+ level (antigen Og4C3) units/ml, the Geometrical Mean (GM) = 2696) in the sera of these 36/104 paired samples, when compared to the hydrocele fluid, (GM = 1079), showed a very good correlation between the CFA level in the serum and CFA level in the fluid (r = 0.731). CFA level in the serum of the 23 microfilaremics (groups II and IV) was extremely high (GM = 4189) and was correlated with MF density (r = 0.442). These findings report for the first time the potential alternative use of the hydrocele fluid to investigate CFA using the mAb Og4C3-ELISA.

Ecology of Haemagogus sp. and Sabethes sp. (Diptera: Culicidae) in relation to the microclimates of the Caxiuanã National Forest, Pará, Brazil

This study was conducted in a meteorological tower located in the Caxiuanã Forest (municipality of Melgaço, Pará, Brazil) with the aim of assessing the vertical stratification of species of Haemagogus and Sabethes, potential vectors of the yellow fever virus. To investigate the role of microclimates in mosquito stratification, bimonthly collections were conducted at ground level (0 m), 8 m, 16 m and 30 m (canopy level), with the aid of entomological nets and suction tubes, from July 2005-April 2006. A total of 25,498 mosquitoes were collected; specimens of Sabethes sp. and Haemagogus janthinomyswere found mostly at heights of 16 m and 30 m while Hg. leucocelaenus was most frequently observed at ground level. The largest number of vector species was collected during the rainiest months, but this difference between seasons was not statistically significant. However, the number of Hg. janthinomys was positively correlated with variations in temperature and relative humidity.

Construção de um Vê de Gowin para análises de produções acadêmicas de Enfermagem

O artigo apresenta uma ferramenta da Aprendizagem Significativa denominada V de Gowin e a possibilidade de seu uso em um processo de desempacotamento de produções acadêmicas de Enfermagem. Objetiva construir uma proposta de alteração da ferramenta com a utilização dos elementos que compõem a trajetória processual da Teoria da Intervenção Práxica da Enfermagem em Saúde Coletiva (TIPESC) e analisar uma dissertação produzida nesta área de conhecimento. Trata-se de um estudo descritivo e exploratório que apresenta o V de Gowin, propondo alterações no seu domínio metodológico. Constatou-se a possibilidade de utilização para análises de produções acadêmicas de enfermagem deste instrumento heurístico que já se mostrou útil para apoiar processos de ensino e aprendizagem e analisar produções acadêmicas na área, pela capacidade de captar e interpretar qualquer fenômeno articulado aos processos de produção e reprodução social.

Conciliação entre Vida Profissional e Familiar em Cabo Verde. O Papel do Estado e das Famílias

O mundo contemporâneo tem demonstrado, nas últimas décadas, que as sociedades são confrontadas e atravessadas por transformações profundas das estruturas sociais a grande dimensão, gerando mutações na organização do trabalho e da vida familiar, adquirindo formas diversas, consoante os contextos e locais. O forte aumento da participação das mulheres no mercado de trabalho teve um impacto decisivo na reconfiguração familiar, tanto na esfera pública como na esfera privada, com consequências muito expressivas nas formas de articulação entre a vida profissional e familiar. Tanto o modelo do homem provedor foi posto em causa, como as formas actuais de divisão familiar de trabalho revelam assimetrias entre os casais e a evolução do modelo de duplo emprego. Esta investigação, analisa a problemática da conciliação vida familiar e trabalho na sociedade Cabo-verdiana, debruçando-se especificamente sobre as estratégias que os indivíduos utilizam, tendo em conta dois agentes: o Estado e a família. A metodologia de pesquisa utilizada baseou-se na análise documental de âmbito qualitativo e quantitativo e a técnica de recolha de informação, em entrevistas de tipo semi-directivo, a dez casais, casados ou vivendo maritalmente, com filhos e que trabalham

Jogos e Brincadeiras no Ensino Básico Integrado: Um Estudo da Ocupação do Tempo de Recreio na Escola “13 de Janeiro”, da Cidade da Praia

O presente trabalho tem por objectivo compreender a relação entre a prática dos jogos e das brincadeiras e o tempo de recreio, na Escola Básica Integrada “13 de Janeiro” de Palmarejo. Para isso, adoptámos a pesquisa quantitativa, que foi feita através das tabelas, do programa (Word 2007). A recolha e a análise de dados, submeteu-se a uma amostra de 36 alunos e 12 professores, num universo de 847 alunos e 25 professores. Através dos resultados, constatámos que os alunos brincam e jogam sempre em grupo. Esses grupos, escolhem muitas vezes, os jogos praticados por muitos alunos. O que os leva, a procurar os grupos que realizam os jogos da actualidade. Notou-se a frequência da brincadeira de palmadinha no (1º, 2º e 3º ano de escolaridade); o jogo 35 no (4º,5º e 6º ano) e futebol (6º ano). Os jogos eram realizados em diferentes espaços. Os alunos da 3ª fase e do 2º ano ocupavam, mais vezes, a placa as proximidades da mesma; Os da 2ª fase, ocupavam espaço à frente das suas salas e os alunos do 1º ano, ocupavam à frente da cozinha. Esses espaços não são os mais desejados pelos alunos. Alguns da 1ª e 2ª fase, preferem brincar e jogar à frente da cozinha, enquanto, a maioria dos alunos da 3ª fase, brincavam, onde preferem sempre. A fundamentação foi sustentada pelos autores Huizinga, Nallin, Piaget, Souza, Cruz, Vygotsky, Sá Fernandes, Delalande, Marques et all, Pellegrini & Perlmutter, Caillois, Liempd, Suarez, etc. Contudo, os jogos e as brincadeiras, fazem do recreio, o mundo, onde todas mostram o que são “crianças”. E sem esses, acaba-se a alegria. Por isso, os professores devem levar em conta, o recreio. Para isso, a Educação Física terá de ser a “alma gémea” dos mesmos.

Uma Metodologia para a Seleção de Sistemas de Cogeração em Plataformas Offshore

As reservas mundiais provadas de gás natural totalizavam 143 trilhões de m3 em 1999. Deste total, 403 bilhões estão no território brasileiro, sendo 151 bilhões de m3 em terra e 252 bilhões no mar. A Figura 1 mostra a distribuição da produção nacional, sendo quase metade do total extraído na Bacia de Campos, RJ. Pode-se dizer que a cogeração aplicada a plataformas de petróleo não representa uma novidade, já há alguns anos até mesmo os navios utilizam a produção conjunta de trabalho mecânico e calor a partir da queima de um mesmo combustível. Entretanto, existem múltiplas configurações possíveis para atendimento de certas demandas térmicas e elétricas e a escolha da mais adequada pode representar uma sensível diferença em termos de aproveitamento energético.