QUESTIONNAIRES IN THE SCREENING FOR Schistosoma mansoni INFECTION: A STUDY OF SOCIO DEMOGRAPHIC AND WATER CONTACT VARIABLES IN FOUR COMMUNITIES IN BRAZIL

The use of questionnaires has been recommended for identifying, at a lower cost, individuals at risk for schistosomiasis. In this study, validity of information obtained by questionnaire in the screening for Schistosoma mansoni infection was assessed in four communities in the State of Minas Gerais, Brazil. Explanatory variables were water contact activities, sociodemographic characteristics and previous treatment for schistosomiasis. From 677, 1474, 766 and 3290 individuals eligible for stool examination in the communities, 89 to 97% participated in the study. The estimated probability of individuals to be infected, if they have all characteristics identified as independently associated with S.mansoni infection, varied from 15% in Canabrava, to 42% in Belo Horizonte, 48% in Comercinho and 80% in São José do Acácio. Our results do not support the hypothesis that a same questionnaire on risk factors could be used in screening for S.mansoni infection in different communities.

Toxicity screening of Diclofenac, Propranolol, Sertraline and Simvastatin using Danio rerio and Paracentrotus lividus embryo bioassays

Early life-stage bioassays have been used as an alternative to short-term adult toxicity tests since they are cost-effective. A single couple can produce hundreds or thousands of embryos and hence can be used as a simple high-throughput approach in toxicity studies. In the present study, zebrafish and sea urchin embryo bioassays were used to test the toxicity of four pharmaceuticals belonging to different therapeutic classes: diclofenac, propranolol, simvastatin and sertraline. Simvastatin was the most toxic tested compound for zebrafish embryo, followed by diclofenac. Sertraline was the most toxic drug to sea urchin embryos, inducing development abnormalities at the ng/L range. Overall, our results highlight the potential of sea urchin embryo bioassay as a promising and sensitive approach for the high-throughput methods to test the toxicity of new chemicals, including pharmaceuticals, and identify several drugs that should go through more detailed toxicity assays.

Screening pentachlorophenol degradation ability by environmental fungal strains belonging to the phyla Ascomycota and Zygomycota

Pentachlorophenol (PCP) bioremediation by the fungal strains amongst the cork- colonising community has not yet been analysed. In this paper, the co- and direct metabolism of PCP by each of the 17 fungal species selected from this community were studied. Using hierarchical data analysis, the isolates were ranked by their PCP bioremediation potential. Fifteen isolates were able to degrade PCP under co-metabolic conditions, and surprisingly Chrysonilia sitophila, Trichoderma longibrachiatum, Mucor plumbeus, Penicillium janczewskii and P. glandicola were able to directly metabolise PCP, leading to its complete depletion from media. PCP degradation intermediates are preliminarily discussed. Data emphasise the signiWcance of these fungi to have an interesting potential to be used in PCP bioremediation processes.

AN ELISA SUITABLE FOR THE DETECTION OF RABIES VIRUS ANTIBODIES IN SERUM SAMPLES FROM HUMAN VACCINATED WITH EITHER CELL-CULTURE VACCINE OR SUCKLING-MOUSE-BRAIN VACCINE

An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Evaluation of Etest and macrodilution broth method for antifungal susceptibility testing of Candida sp strains isolated from oral cavities of AIDS patients

A comparison of the Etest and the reference broth macrodilution susceptibility test for fluconazole, ketoconazole, itraconazole and amphotericin B was performed with 59 of Candida species isolated from the oral cavities of AIDS patients. The Etest method was performed according to the manufacturer's instructions, and the reference method was performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines. Our data showed that there was a good correlation between the MICs obtained by the Etest and broth dilution methods. When only the MIC results at ± 2 dilutions for both methods were considered, the agreement rates were 90.4% for itraconazole, ketoconazole and amphotericin B and 84.6% for fluconazole of the C. albicans tested. In contrast, to the reference method, the Etest method classified as susceptible three fluconazole-resistant isolates and one itraconazole-resistant isolate, representing four very major errors. These results indicate that Etest could be considered useful for antifungal sensitivity evaluation of yeasts in clinical laboratories.

Sistema de apoio à decisão do rastreio do cancro da mama

O estudo do rastreio do cancro da mama funciona não só como um benefício, mas também como um método que pode causar controvérsias nas mulheres consideradas saudáveis antes de se submeterem ao rastreio. Assim, a necessidade do utente adquirir conhecimento sobre as vantagens e desvantagens do rastreio do cancro da mama é fundamental para decidir se, se submete ou não, a este rastreio. A decisão do paciente é crucial na decisão final e as consequências já devem ser do seu conhecimento. Com este estudo, pretendeu-se o desenvolvimento de um Sistema de Informação (SI) na Web, que funcione como auxiliar no processo de tomada de decisão para a realização do rastreio do cancro da mama. A decisão do componente do SI é fundamental, com o objetivo de auxiliar os utilizadores no processo de tomada de decisão para a realização do rastreio do cancro da mama. O desenvolvimento da plataforma “Decidir”, apresenta-se numa aplicação crucial no domínio médico da decisão do rastreio do cancro da mama, onde referências às suas controvérsias e benefícios são fundamentais para um melhor cuidado de saúde. Para esse efeito foram implementadas duas ferramentas para ajudar na decisão de realizar ou não o rastreio do cancro da mama. A primeira ferramenta resulta na decisão pessoal de cada indivíduo (paciente/profissionais de saúde), que pode ser variável, conforme um conjunto de proposições relevantes no aspeto pessoal e clínico para a tomada de decisão do rastreio nesta área.

Diagnosis of pulmonary pneumocystosis by microscopy on wet mount preparations

We have compared the searching of the presence of "honeycomb" structures by direct microscopy on wet mount preparations with the direct immunofluorescence (DIF) for the diagnosis of Pneumocystis carinii pneumonia (PCP) in 115 bronchoalveolar (BAL) fluids. The samples belonged to 115 AIDS patients; 87 with presumptive diagnosis of PCP and 28 with presumptive diagnosis other than PCP. The obtained results were coincident in 114 out of 115 studied samples (27 were positive and 87 negative) with both techniques. A higher percentage of positive results (32.18%) among patients with presumptive diagnosis of PCP with respect to those with presumptive diagnosis other than PCP (3.57%) was observed. One BAL fluid was positive only with DIF, showed scarce and isolated P. carinii elements and absence of typical "honeycomb" structures. The searching for "honeycomb" structures by direct microscopy on wet mount preparations could be considered as a cheap and rapid alternative for diagnosis of PCP when other techniques are not available or as screening test for DIF. This method showed a sensitivity close to DIF when it was applied to BAL fluids of AIDS patients with poor clinical condition and it was performed by an experienced microscopist.

Field evaluation of the whole blood immunochromatographic test for rapid bancroftian filariasis diagnosis in the northeast of Brazil

This study evaluated the whole blood immunochromatographic card test (ICT card test) in a survey performed in Northeastern Brazil. 625 people were examined by the thick blood film (TBF) and ICT card test. Residents of a non-endemic area were also tested by the whole blood card test and Og4C3. The sensitivity of the ICT card test was 94.7% overall, but lower in females than males, based on the reasonable assumption that TBF is 100% specific. However, since TBF and other methods have unknown sensitivity, the true specificity of the card test is unknown. Nevertheless, it is possible to estimate upper and lower limits for the specificity, and relate it to the prevalence of the disease. In the endemic area, the possible range of the specificity was from 72.4% to 100%. 29.6% of the card tests performed in the non-endemic area exhibited faint lines that were interpreted as positives. Characteristics of the method including high sensitivity, promptness and simplicity justify its use for screening of filariasis. However, detailed information about the correct interpretation in case of extremely faint lines is essential. Further studies designed to consider problems arising from imperfect standards are necessary, as is a sounder diagnostic definition for the card test.

Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2′-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

New molecularly-imprinted polymer for carnitine and its application as ionophore in potentiometric selective membranes

Carnitine (CRT) is a biological metabolite found in urine that contributes in assessingseveral disease conditions, including cancer. Novel quick screening procedures for CRT are therefore fundamental. This work proposes a novel potentiometric device where molecularly imprinted polymers (MIPs) were used as ionophores. The host-tailored sites were imprinted on a polymeric network assembled by radical polymerization of methacrylic acid (MAA) and trimethylpropane trimethacrylate (TRIM). Non-imprinted polymers (NIPs) were produced as control by removing the template from the reaction media. The selective membrane was prepared by dispersing MIP or NIP particles in plasticizer and poly(vinyl chloride), PVC, and casting this mixture over a solid contact support made of graphite. The composition of the selective membrane was investigated with regard to kind/amount of sensory material (MIP or NIP), and the need for a lipophilic additive. Overall, MIP sensors with additive exhibited the best performance, with near-Nernstian response down to ~ 1 × 10− 4 mol L− 1, at pH 5, and a detection limitof ~ 8 × 10− 5 mol L− 1. Suitable selectivity was found for all membranes, assessed by the matched potential method against some of the most common species in urine (urea, sodium, creatinine, sulfate, fructose and hemoglobin). CRT selective membranes including MIP materials were applied successfully to the potentiometric determination of CRT in urine samples.

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples

XIX Meeting of the Portuguese Electrochemical Society - XVI Iberic Meeting of Electrochemistry

Oriented Tailoring of Plastic Antibodies for Prostate Specific Antigen and Application of the Imprinted Material as Ionophore in Potentiometric Detection

Prostate Specific Antigen (PSA) is the biomarker of choice for screening prostate cancer throughout the population, with PSA values above 10 ng/mL pointing out a high probability of associated cancer1. According to the most recent World Health Organization (WHO) data, prostate cancer is the commonest form of cancer in men in Europe2. Early detection of prostate cancer is thus very important and is currently made by screening PSA in men over 45 years old, combined with other alterations in serum and urine parameters. PSA is a glycoprotein with a molecular mass of approximately 32 kDa consisting of one polypeptide chain, which is produced by the secretory epithelium of human prostate. Currently, the standard methods available for PSA screening are immunoassays like Enzyme-Linked Immunoabsorbent Assay (ELISA). These methods are highly sensitive and specific for the detection of PSA, but they require expensive laboratory facilities and high qualify personal resources. Other highly sensitive and specific methods for the detection of PSA have also become available and are in its majority immunobiosensors1,3-5, relying on antibodies. Less expensive methods producing quicker responses are thus needed, which may be achieved by synthesizing artificial antibodies by means of molecular imprinting techniques. These should also be coupled to simple and low cost devices, such as those of the potentiometric kind, one approach that has been proven successful6. Potentiometric sensors offer the advantage of selectivity and portability for use in point-of-care and have been widely recognized as potential analytical tools in this field. The inherent method is simple, precise, accurate and inexpensive regarding reagent consumption and equipment involved. Thus, this work proposes a new plastic antibody for PSA, designed over the surface of graphene layers extracted from graphite. Charged monomers were used to enable an oriented tailoring of the PSA rebinding sites. Uncharged monomers were used as control. These materials were used as ionophores in conventional solid-contact graphite electrodes. The obtained results showed that the imprinted materials displayed a selective response to PSA. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8X10-11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed smaller sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum samples, with percentage recoveries of 106.5% and relatives errors of 6.5%.