Alterações urinárias assintomáticas em portadores do anticorpo para virus da hepatite C

Vários estudos associam a infecção crônica pelo vírus da hepatite C a manifestação extra-hepáticas incluindo glomerulonefrite membranoproliferativa. A prevalência de anormalidades urinárias associadas a esta infecção ainda é controversa. Este trabalho tem por objetivo investigar a prevalência de alterações urinárias assintomáticas em doadores de sangue com anti VHC positivo. No período de Julho de 1997 a Março de 1998 foram avaliados 58 doadores de sangue anti VHC + (41 homens e 17 mulheres; com idade média de 34 anos) e 128 doadores VHC -, considerado grupo controle (93 homens e 35 mulheres; com idade média de 31 anos). Todos foram questionados quanto à história prévia de doenças sistêmicas, do trato urinário, uso de drogas (incluindo AINE e uso de analgésico ), transfusão de sangue, fumo e uso de álcool. O anti VHC foi medido pela técnica de enzima imunoensaio, de segunda geração (Abbot Laboratories). As amostras de urina foram inicialmente rastreadas utilizando-se fitas Combur 10 (Boehringer). A microalbuminuria foi medida pela imunoturbidimetria (Bayer, kit 6813) e a NAG foi medida pela técnica de espectrofotometria ( SIGMA N 9376 ). Hematúria assintomática foi definida como > 5 hemácias no sedimento urinário, proteinúria com o aparecimento de 1+ na fita reagente; cilindrúria quando foi visualizado qualquer tipo de cilindro; microalbuminúria alterada quando foi >16 mg/l; relação microalbuminúria/creatininúria elevada quando >15mg/g e a NAG urinária elevada, quando os valores estavam > 5,6U/g de creatinina na urina. Na análise estatística utilizou-se um nível de significância de 95% (p=0,05). Verificou-se que a cor branca e o sexo masculino predominaram nos dois grupos, o grupo VHC + apresentou uma freqüência significativamente aumentada em relação ao uso de AINE (p= 0,003), álcool p< 0,001), transfusão (p= 0,006) e drogas (p< 0,001) quando comparado ao grupo VHC -. No grupo VHC + as enzimas hepáticas estavam significativamente mais elevadas (AST p=0,004, ALT p< 0,001), assim como as médias de GGT (p= 0,004). Concluímos que o grupo anti VHC + em estudo não apresenta anormalidades urinárias significativas comparativamente com o grupo controle. A prevalência de hematúria e proteinúria nos grupos VHC + e VHC – foi respectivamente 12,0% x 9,4% e 5,4% x 5,5%, sem diferença estatística entre os dois grupos. As médias da microalbuminúria nos dois grupos foram respectivamente (10,5 + 22,5 ; 8,7 +12,2 , p= 0,560) e a da NAG (3,2 + 1,8 ; 4,2 + 3,3 , p= 0,232). Constatamos uma associação estatisticamente significativa entre a presença de hematúria e proteinúria (p= 0,011), proteinúria e cilindrúria ( p< 0,001), proteinúria e índice de elevado de microalbuminúria ( p<0,001), NAG e índice de microalbuminúria elevado (p= 0,02); hematúria e sexo feminino (p<0,001), níveis de GT elevados nos indivíduos com proteinúria (p=0,038) e uma associação entre NAG e tabagismo ( p=0,004).

Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C virus

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of hepatitis C virus (HCV) in serum. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use. (C) 2004 Elsevier B.V. All rights reserved.

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.

Hibridização reversa e sequenciamento na genotipagem do vírus da hepatite C

INTRODUÇÃO: Os métodos de genotipagem do vírus da hepatite C têm sido muito discutidos. O objetivo deste trabalho foi comparar as metodologias de hibridização reversa e sequenciamento direto para a genotipagem do vírus da hepatite C. MÉTODOS: Noventa e uma amostras de plasma de pacientes assistidos na Faculdade de Medicina de Botucatu da Universidade Estadual Paulista foram utilizadas. A genotipagem por hibridização reversa foi realizada utilizando o kit comercial INNO-LiPA® v.1.0. O sequenciamento direto foi efetuado em sequenciador automático utilizando protocolos in house. RESULTADOS: A genotipagem por sequenciamento direto mostrou-se eficiente na resolução dos resultados inconclusivos pelo kit comercial. O kit mostrou resultados errôneos em relação à subtipagem viral. Além disso, a genotipagem por sequenciamento direto revelou um erro do kit com relação à determinação genotípica questionando a eficiência do método também para a identificação do genótipo viral. CONCLUSÕES: A genotipagem realizada por meio de sequenciamento direto permite uma maior acurácia na classificação viral quando comparada à hibridização reversa.

Genotoxicity assessment of water soluble fractions of biodiesel and its diesel blends using the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of a novel kit (TF-test) for the diagnosis of intestinal parasitic infections

Intestinal parasitic infections are currently a source of concern for Public Health agencies in developing and developed countries. Since three ovum-and-parasite stool examinations have been demonstrated to provide sensitive results, we designed a practical and economical kit (TF-Test) that is now commercially available (Immunoassay Com. Ind. Ltda., S (a) over tildeo Paulo, Brazil). This kit allows the separate collection of three fecal specimens into a preservative solution. The specimens are then pooled, double-filtered, and concentrated by a single rapid centrifugation process. The TF-Test was evaluated in four different laboratories in a study using 1,102 outpatients and individuals living in an endemic area for enteroparasitosis. The overall sensitivity found using the TF-Test (86.2-97.8%) was significantly higher (P<0.01) than the sensitivity of conventional techniques such as the Coprotest (NIL Comercio Exterior Ltda, São Paulo, Brazil) and the combination of Lutz/Hoffman, Faust, and Rugai techniques (De Carli, Diagnostico Laboratorial das Parasitoses Humanas. Metodos e Tecnicas, 1994), which ranged from 48.3% to 75.9%. When the above combined three specimen technique was repeated with three specimens collected on different days, its sensitivity became similar (P > 0.01) to that of the TF-Test. The kappa index values of agreement for the TF-Test were consistent (P < 0.01), being higher and ranking in a better position than conventional techniques. The high sensitivity, cost/benefit ratio, and practical aspects demonstrate that the TF-Test is suitable for individual diagnosis, epidemiological inquiries, or evaluation of chemotherapy in treated communities. (C) 2004 Wiley-Liss, Inc.

Cholinesterase measurements with an automated kit

Background the Test-mate kit determines acetylcholinesterase (AChE, EC 3.1.1.7) and hemoglobin content of a drop of blood, displaying enzyme activities normalized to 25degreesC. Previous models produced inconsistent results at different temperatures. This report focuses on the current model, ChE 400, and two instruments of a previous OP model.Methods AChE activities were determined by the Ellman assay, using the three kits and a 96-well microplate reader Temperatures ranged from 10 to 37degreesC. Fetal bovine serum was the source of AChE.Results Normalized activities decreased below 20degreesC in the ChE model and below 25 C in the OP models. Activities of the same serum sample differed between the three Test-mate kits, ranging from 1.03 to 1.49 mumoles/min/ml. Percent errors were greater than with the microplate reader at all temperatures.Conclusions Neither we nor the manufacturer recommend the current Test-mate model for fieldwork. Nevertheless, there have been field measurements with Test-Mate kits, and we recommend that an enzyme activity standard be run in parallel with their use. (C) 2002 Wiley-Liss, Inc.

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n=72) using a vitrification kit for bovine embryo or slow frozen (n=69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n=92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p<0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p<0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p<0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48h of culture. © 2012 Blackwell Verlag GmbH.

Análise comparativa das metodologias de hibridização reversa e sequenciamento direto para a genotipagem do vírus da hepatite C

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tumor venéreo transmissível espontâneo canino: a inserção do transposon line-1 no gene C-MYC e os critérios de malignidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antígeno plaquetários humanos (HPA) em portadores do vívus da hepatite c (HCV)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The Enhanced Tool-Kit of Two Groups of Wild Bearded Capuchin Monkeys in the Caatinga: Tool Making, Associative Use, and Secondary Tools

The use of stones to crack open encapsulated fruit is widespread among wild bearded capuchin monkeys (Cebus libidinosus) inhabiting savanna-like environments. Some populations in Serra da Capivara National Park (Piaui, Brazil), though, exhibit a seemingly broader toolkit, using wooden sticks as probes, and employing stone tools for a variety of purposes. Over the course of 701.5 hr of visual contact of two wild capuchin groups we recorded 677 tool use episodes. Five hundred and seventeen of these involved the use of stones, and 160 involved the use of sticks (or other plant parts) as probes to access water, arthropods, or the contents of insects` nests. Stones were mostly used as ""hammers""-not only to open fruit or seeds, or smash other food items, but also to break dead wood, conglomerate rock, or cement in search of arthropods, to dislodge bigger stones, and to pulverize embedded quartz pebbles (licking, sniffing, or rubbing the body with the powder produced). Stones also were used in a ""hammer-like"" fashion to loosen the soil for digging out roots and arthropods, and sometimes as ""hoes"" to pull the loosened soil. In a few cases, we observed the re-utilization of stone tools for different purposes (N = 3), or the combined use of two tools-stones and sticks (N = 4) or two stones (N = 5), as sequential or associative tools. On three occasions, the monkeys used smaller stones to loosen bigger quartz pebbles embedded in conglomerate rock, which were subsequently used as tools. These could be considered the first reports of secondary tool use by wild capuchin monkeys. Am. J. Primatol. 71:242-251, 2009. (c) 2008 Wiley-Liss, Inc.

Distribution of human immunodeficiency virus type 1 subtypes in the State of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic (TM) ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male: female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Genotoxicity assessment of water soluble fractions of biodiesel and its diesel blends using the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay

The designation of biodiesel as an environmental-friendly alternative to diesel oil has improved its commercialization and use. However, most biodiesel environmental safety studies refer to air pollution and so far there have been very few literature data about its impacts upon other biotic systems, e.g. water, and exposed organisms. Spill simulations in water were carried out with neat diesel and biodiesel and their blends aiming at assessing their genotoxic potentials should there be contaminations of water systems. The water soluble fractions (WSF) from the spill simulations were submitted to solid phase extraction with C-18 cartridge and the extracts obtained were evaluated carrying out genotoxic and mutagenic bioassays [the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay]. Mutagenic and genotoxic effects were observed, respectively, in the Salmonella/microsome preincubation assay and the in vitro MN test carried out with the biodiesel WSF. This interesting result may be related to the presence of pollutants in biodiesel derived from the raw material source used in its production chain. The data showed that care while using biodiesel should be taken to avoid harmful effects on living organisms in cases of water pollution. (C) 2011 Elsevier Ltd. All rights reserved.

Distribution of human immunodeficiency virus type 1 subtypes in the state of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.