Dot-dye-immunoassay for the diagnosis of schistosomiasis mansoni

A new serological assay dot-dye-immunoassay (dot-DIA) was evaluated for the diagnosis of schistosomiasis mansoni. This method consist of four steps: (a) biding of antigens to a nitrocellulose membrane (NC); (b) blocking of free sites of the NC; (c) incubation in specific primary antibody; (d) detection of primary antibody reactivity by color development using second antibody coupled to textile dyes. Sera from 82 individuals, 61 with Schistosoma mansoni eggs in the stool and 21 stool negative were tested by ELISA, dot-ELISA, and dotDIA. A high level of agreement between the methods tested was observed for all sera tested: ELISA x dot-ELISA: 95.1%, ELISA x dot-DIA: 92.7% and dot-ELISA x dot-DIA: 97.6%. In this study, dot-DIA proved to be a feasible, sensitive, rapid and practical test for the diagnosis of shcistosomiasis.

Malaria seroepidemiology: comparison between indirect fluorescent antibody test and enzyme immunoassay using bloodspot eluates

Blood sampling on filter paper is a current practice seroepidemiological studies by indirect fluorescent antibody test (IFAT). There is, however, scant comparative information about the use of bloodspot eluates for detection of malarial IgG antibodies simultaneously by IFAT and enzyme immunoassay (ELISA). Here we report data obtained by both serological methods done on 219 bloodspot eluate samples collected in a rural community in Brazilian Amazon Basin (Alto Paraíso, Ariquemes municipality) where malaria is endemic. Plasmodium falciparum and P. vivax thick smear antigens were used in the IFAT; a detergent-soluble P. falciparum antigen was prepared for ELISA. Substantial agreement of results (Kappa coefficient k = 0.686) was observed when P. falciparum antigen was used in both tests, and IFAT titers were found to be strongly correlated ELISA antibody units (Spearman correlation coeficient rs = 0.818, p < 0.0001). Only moderate agreement (k = 0.467) between IFAT with P. vivax antigen and ELISA with P. falciparum antigen was observed. Spearman correlation coefficient value between quantitative results (IFAT titers and ELISA antibody units) in this case was numerically lowe (rs = 0.540, p < 0.0001). Our results suggest that, with P. falciparum antigen, both IFAT and ELISA performed on bloodspot eluates are equivalent for seropidemiological purposes.

An outbreak of diarrhoea associated with rotavirus serotype 1 in a day care nursery in Rio de Janeiro, Brazil

Faeces from 17 children less than 1.6 years old 15 adultsmore than 22 years old were collected during an outbreak of gastroenteritis in aday care nursery and screened for the presence of adenovirus and rotavirus by enzyme immunoassay (EIARA) and other viruses by electron microscopy (EM) and polycrylamide gel electrophoresis (PAGE). Ten samples (58.8 per cent) from childrenand one (6.7 per cent) from adults were positive for rotavirus and all samples were negative for bacteria and parasites. No other viruses were observed in EM. An enzyme immunoassay test using monoclonal antibodies (MAb-EIA) to determine the subgroup(s) and the serotype(s) of rotavirus was performed and the results showedthat all positive samples belong to serotype 1, subgroup II of group A rotaviruses. In PAGE test all samples had the same profile and the 10 and 11 dsRNA segments corresponed to the "long" profile of group A of rotaviruses. These results corroborated the MAbEIA results and indicate a sole source of infection. The majorsymptoms observed were: vomiting (60 per cent), fever (70 per cent) and diarrhoea (100 per cent). In previous years (1989 to 1991) we observed only rotavirus serotype 2 in this same day care nursery, but no outbreak was reported.

Compressed collagen gel: a novel scaffold for human bladder cells.

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo-tissue level. This controlled, cell-independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell-seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation.

Poor value of pulsed-field gel electrophoresis to investigate long-term scale epidemiology of methicillin-resistant Staphylococcus aureus.

Pulsed-field gel electrophoresis (PFGE) is widely used for epidemic investigations of methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we evaluated its use in a long-term epidemiological setting (years to few decades, country to continent level). The clustering obtained from PFGE patterns after SmaI digestion of the DNA of 20 strains was compared to that obtained using a phylogenetic typing method (multiprimer RAPD). The results showed that the analysis of small PFGE bands (10-85kb) correlates better with multiprimer RAPD than the analysis of large PFGE bands (>85-700kb), suggesting that the analysis of small bands would be more suitable for the investigation of long-term epidemiological setting. However, given the technical difficulties to obtain a good resolution of these bands and the putative presence of plasmids among them, PFGE does not appear to be a method of choice for the long-term epidemiology analysis of MRSA.

Non-covalent and covalent protein labeling in two-dimensional gel electrophoresis.

Over the past decades, several sensitive post-electrophoretic stains have been developed for an identification of proteins in general, or for a specific detection of post-translational modifications such as phosphorylation, glycosylation or oxidation. Yet, for a visualization and quantification of protein differences, the differential two-dimensional gel electrophoresis, termed DIGE, has become the method of choice for a detection of differences in two sets of proteomes. The goal of this review is to evaluate the use of the most common non-covalent and covalent staining techniques in 2D electrophoresis gels, in order to obtain maximal information per electrophoresis gel and for an identification of potential biomarkers. We will also discuss the use of detergents during covalent labeling, the identification of oxidative modifications and review influence of detergents on finger prints analysis and MS/MS identification in relation to 2D electrophoresis.

The in vivo performance of magnetic particle-loaded injectable, in situ gelling, carriers for the delivery of local hyperthermia.

We investigated the use of in situ implant formation that incorporates superparamagnetic iron oxide nanoparticles (SPIONs) as a form of minimally invasive treatment of cancer lesions by magnetically induced local hyperthermia. We developed injectable formulations that form gels entrapping magnetic particles into a tumor. We used SPIONs embedded in silica microparticles to favor syringeability and incorporated the highest proportion possible to allow large heating capacities. Hydrogel, single-solvent organogel and cosolvent (low-toxicity hydrophilic solvent) organogel formulations were injected into human cancer tumors xenografted in mice. The thermoreversible hydrogels (poloxamer, chitosan), which accommodated 20% w/v of the magnetic microparticles, proved to be inadequate. Alginate hydrogels, however, incorporated 10% w/v of the magnetic microparticles, and the external gelation led to strong implants localizing to the tumor periphery, whereas internal gelation failed in situ. The organogel formulations, which consisted of precipitating polymers dissolved in single organic solvents, displayed various microstructures. A 8% poly(ethylene-vinyl alcohol) in DMSO containing 40% w/v of magnetic microparticles formed the most suitable implants in terms of tumor casting and heat delivery. Importantly, it is of great clinical interest to develop cosolvent formulations with up to 20% w/v of magnetic microparticles that show reduced toxicity and centered tumor implantation.

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical `riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott®), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95%) sera from patients in the study group and negative in 38 of the 39 (97%) sera from those in the control group, showing an accuracy of 96%. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Sample preparation for two-dimensional gel electrophoresis.

The choice of sample preparation protocol is a critical influential factor for isoelectric focusing which in turn affects the two-dimensional gel result in terms of quality and protein species distribution. The optimal protocol varies depending on the nature of the sample for analysis and the properties of the constituent protein species (hydrophobicity, tendency to form aggregates, copy number) intended for resolution. This review explains the standard sample buffer constituents and illustrates a series of protocols for processing diverse samples for two-dimensional gel electrophoresis, including hydrophobic membrane proteins. Current methods for concentrating lower abundance proteins, by removal of high abundance proteins, are also outlined. Finally, since protein staining is becoming increasingly incorporated into the sample preparation procedure, we describe the principles and applications of current (and future) pre-electrophoretic labelling methods.

Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females), Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.

Epidemiological features of rotavirus infection in Goiânia, Goiás, Brazil, from 1986 to 2000

A total of 2,605 faecal specimens from children up to 10 years old with or without diarrhoea were collected. Samples were obtained from 1986 to 2000 in hospitals, outpatient clinics and day-care centers in Goiânia, Goiás. Two methodologies for viral detection were utilized: a combined enzyme immunoassay for rotavirus and adenovirus and polyacrylamide gel electrophoresis. Results showed 374 (14.4%) faecal specimens positive for Rotavirus A, most of them collected from hospitalized children. A significant detection rate of rotavirus during the period from April to August, dry season in Goiânia, and different frequencies of viral detection throughout the years of study were also observed. Rotavirus was significantly related to hospitalization and to diarrhoeal illness in children up to 24 months old. This study reinforces the importance of rotavirus as a cause of diarrhoea in children and may be important in regards to the implementation of rotavirus vaccination strategies in our country.