Heterologous antigen extract in ELISA for the detection of human IgE anti-Strongyloides stercoralis

Strongyloides ratti larval extract was used for the standardization of ELISA to detect genus-specific IgE in human strongyloidiasis. Forty serum samples from monoinfected patients shedding S. stercoralis larvae (Group I), 40 from patients with other intestinal parasites (Group II), and 40 from copronegative healthy subjects (Group III) were analyzed. Genus-specific IgE levels (ELISA Index: EI) were significantly higher in the group I (EI = 1.43) than groups II (EI = 0.70) and III (EI = 0.71), showing positivity rates of 55%, 2.5% and 0%, respectively. Similarly, sera from copropositive patients had significantly higher levels of total IgE (866 IU/mL) as compared to those from group II (302 IU/mL) and III (143 IU/mL). A significant positive correlation was found between levels of Strongyloides specific-IgE and total IgE in sera from patients with strongyloidiasis. In conclusion, S. ratti heterologous extract showed to be a useful tool for detecting genus-specific IgE by ELISA, contributing for a better characterization of the immune response profile in human strongyloidiasis.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

Evaluation of an in-house specific immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioMérieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.

Teste de Inclinação em Idosos com Síncope de Causa Desconhecida: Experiência da Estimulação Farmacológica com Nitroglicerina

Está demonstrado o papel do teste de inclinação na avaliação de doentes com síncope de causa não esclarecida. Nos idosos, a prevalência de episódios sincopais aumenta, associa-se a pior prognóstico e, frequentemente, implica o recurso a múltiplos exames de diagnóstico. Objectivos: Avaliar a utilidade do teste de inclinação com recurso a nitroglicerina sub-lingual como agente provocativo em idosos com síncope de etiologia desconhecida. Métodos: Foram estudados 46 doentes consecutivos com > 65 anos (56,5% do sexo feminino; 74 ± 6 anos) por síncope de etiologia não esclarecida. Em 25 doentes(54%) não havia evidência de patologia cardíaca, tendo os restantes cardiopatia hipertensiva (14 doentes) ou isquémica (7 doentes). O protocolo incluiu massagem do seio carotídeo em decúbito a após ortostatismo passivo. O teste de inclinação foi efectuado sob monitorização contínua do electrocardiograma e tensão arterial, com inclinação a 70º durante 20 minutos. Na ausência de síncope, administrou-se 500 mcg de nitroglicerina sub-lingual com monitorização por mais 20 minutos. O teste de inclinação foi considerado positivo quando houve reprodução da sintomatologia acompanhada de bradicardia e/ou hipotensão arterial (respostas cardio-inibitória, vasodepressora ou mista). Considerou-se haver hipotensão ortostática nos casos com queda da tensão arterial sistólica > 20 mmHg ou da tensão arterial diastólica > 10 mmHg nos 3 minutos após ortostatismo; nos doentes com sintomas associados à descida gradual, paralela, da tensão arterial sistólica e diastólica durante o teste de inclinação passivo, admitiu-se um perfil do tipo disautonómico; nos doentes com descida gradual da tensão arterial após nitroglicerina, considerou-se haver resposta exagerada aos nitratos. Resultados: Ocorreu reprodução de sintomas em 34 doentes (73,9%): 19,6% no teste de inclinação passivo (resposta neurocardiogénica vasodepressora – 3 doentes, hipersensibilidade do seio carotídeo – 1 doente, hipotensão ortostática – 1 doente, perfil disautonómico – 4 doentes) e 54,3% após nitroglicerina (resposta neurocardiogénica vasodepressora – 12 doentes, mista – 5 doentes , cardio-inibitória – 2 doentes e resposta exagerada aos nitratos – 6 doentes). A síncope foi neurocardiogénica em 47,8% (teste passivo – 13,6%, pós-nitroglicerina – 86,4%). Em 16,2% dos doentes submetidos a nitroglicerina ocorreu resposta exagerada aos nitratos. Não se registaram complicações durante o exame. Conclusões: O teste de inclinação em idosos com síncope de etiologia não esclarecida: 1. contribui para o diagnóstico diferencial no estudo etiológico da síncope, 2. quando potenciado pela nitroglicerina associa-se a um aumento significativo do número de respostas positivas, e permite identificar um número considerável de doentes com resposta exagerada aos nitratos.

Utilização da Nitroglicerina na Fase Activa do Teste de Inclinação: Haverá Diferenças nos Idosos?

Está demonstrado o papel do teste de inclinação na avaliação de doentes com síncope de causa não esclarecida, sendo utilizado como a técnica gold standard para o diagnóstico da síncope neurocardiogénica, em particular com o recurso a agentes provocativos farmacológicos de modo a melhorar a acuidade diagnóstica. A estimulação com nitroglicerina sub-lingual é, regra geral, bem tolerada e permite aumentar a sensibilidade do teste reduzindo a sua duração. Neste sentido, tem-se verificado também o alargamento da sua aplicação em doentes idosos. Objectivos: Avaliar, numa população referenciada por síncope de etiologia desconhecida, a utilidade do teste de inclinação com recurso a nitroglicerina sub-lingual e comparar o tipo de respostas obtidas nos doentes idosos com o dos restantes doentes. Métodos: Estudámos 158 doentes submetidos a teste de inclinação com utilização de nitroglicerina como agente provocativo. Foram considerados doentes com idade <65 anos (Grupo A, n=74) e ≥65 anos (Grupo B, n=84). O teste de inclinação foi efectuado segundo o protocolo “italiano”, sob monitorização contínua do electrocardiograma e da tensão arterial (Task Force Monitor; CNSystems). Foram incluídos somente doentes assintomáticos na fase passiva do teste. O teste foi considerado positivo para resposta neurocardiogénica quando houve reprodução da sintomatologia acompanhada de bradicardia e/ou hipotensão arterial e as respostas foram definidas como cardio-inibitória, vasodepressora ou mista. Uma descida gradual e paralela da tensão arterial após administração de nitroglicerina seguida de síncope foi considerada como resposta exagerada aos nitratos. Resultados: Não se verificam diferenças na distribuição por sexos entre os grupos. O teste de inclinação foi positivo em 57% do Grupo A e 51% do Grupo B (p=NS), tendo ocorrido resposta exagerada aos nitratos em 11% e 16%, respectivamente (p=NS). Relativamente ao tipo de respostas neurocardiogénicas, as vasodepressoras foram mais frequentes no Grupo B (53% versus 24%; p=0,001) e as mistas tendencialmente mais frequentes no Grupo A (59% versus 40%; p=0,07), sem diferenças significativas no que se refere à resposta do tipo cardio-inibitória (17% nos Grupo A versus 7% no Grupo B; p=NS). Conclusões: Numa população com síncope de etiologia não conhecida, o teste de inclinação potenciado pela nitroglicerina: a) contribui de modo significativo para o esclarecimento diagnóstico com utilidade idêntica em idosos e nos restantes doentes. B) associa-se a maior incidência de resposta neurocardiogénica vasodepressora nos idosos embora com uma taxa semelhante de respostas exageradas aos nitratos.

A simple and cheaper in house varicella zoster virus antibody indirect ELISA

We have developed a cheaper an simple in house indirect ELISA that uses the live attenuated VZV vaccine as a coating antigen. The alternative ELISA had an agreement of 94% when compared with a commercial VZV ELISA kit. Moreover, our ELISA proved to be more reliable than the kit when assessing true negative samples. By adding a standard serum, we were able to produce results in international units per millilitre. Also, the addition of an extra step with 8M urea allowed the assessment of VZV IgG avidity without excessive costs. The cost per sample to test VZV IgG was 2.7 times cheaper with our ELISA, allowing the testing of many samples without the burden of production of VZV antigen in the laboratory.

Dot-ELISA for the diagnosis of neurocysticercosis

The aim of the present study was to standardize and evaluate dot-Enzyme linked immunosorbent assay (Dot-ELISA), a simple and rapid test for the detection of cysticercus antibodies in the serum for the diagnosis of neurocysticercosis (NCC). The antigen used in the study was a complete homogenate of Cysticercus cellulosae cysts obtained from infected pigs and dotted on to nitrocellulose membrane. Test sera were collected from the patients of NCC, and control sera from patients with other diseases and healthy students and blood donors of the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Pondicherry, during a study period from 2001 to 2003. Dot-ELISA detected antibodies in 14 of 25 (56%) in clinically suspected cases of NCC, 13 of 23 (56.5%) in CT/MRI proven cases of NCC and 2 of 25 (8%) each in non-cysticercal CNS infection controls and healthy controls. The test showed a sensitivity of 56.25%, specificity of 92%, positive predictive value of 87.09%, and negative predictive value of 70.76%. Results of the present study shows that the Dot-ELISA as a simple test can be used in the field or poorly equipped laboratories for diagnosis of NCC .

Incidência de "Falsos-Positivos" em Idosos com Fibrilhação Auricular Paroxística Submetidos a Teste de Inclinação

The autonomic nervous system (ANS) plays a role as a modulator in the pathogenesis of paroxysmal atrial fibrillation (PAF). The clinical pattern of vagally mediated PAF has been observed mainly in young patients. Neurocardiogenic responses during orthostatic stress are related to autonomic reflexes in which the vagal influence predominates. AIM: To evaluate the susceptibility of elderly patients with PAF to activation of vasovagal syncope mechanisms. METHODS: We performed passive head-up tilt testing (HUT) in 34 patients (62% women, aged 72 +/- 7 years), with > or = 1 year of clinical history of PAF--19 without structural heart disease, 11 with hypertensive heart disease and 4 with coronary artery disease (who had no previous myocardial infarction, had undergone myocardial revascularization, and had no documented ischemia) (PAF group), and compared the results with those obtained in a group of 34 age-matched patients (53% women, aged 74 +/- 6 years), who underwent HUT due to recurrent syncope (Sc group). In this group, 21 had no documented heart disease and none had a clinical history of AF. There was no diabetes, congestive heart failure or syncope in the PAF group. After a supine resting period, the subjects were tilted at 70 degrees for 20 minutes while in sinus rhythm. No provocative agents were used to complement the HUT. ECG and blood pressure were continuously monitored (Task Force Monitor, CNSystems). The test was considered positive when syncope or presyncope occurred with bradycardia and/or arterial hypotension. Abnormal responses were classified as cardioinhibitory, vasodepressor or mixed. RESULTS: HUT was positive in seven patients of the PAF group--vasodepressor response in five and mixed in two (20.5% of the total; 26.3% of those without heart disease)--and in eight patients (vasodepressor in six and mixed in two) of the Sc group (p=NS). During HUT, three patients of the PAF group had short periods of self-limited PAF (in one, after vasodepressor syncope). There were no differences in gender distribution, age or heart disease. No cardioinhibitory responses or orthostatic hypotension were observed. CONCLUSION: In elderly patients with PAF, a significant number of false positive results during passive HUT may be expected, suggesting increased vasovagal reactions despite aging. This suggests that ANS imbalances may be observed in this population.

Alterações da Actividade Autonómica Durante o Teste de Inclinação em Doentes com Fibrilhação Auricular Paroxística: Análise com Wavelets

The autonomic nervous system (ANS) is known to be an important modulator in the pathogenesis of paroxysmal atrial fibrillation (PAF). Changes in ANS control of heart rate variability (HRV) occur during orthostatism to maintain cardiovascular homeostasis. Wavelet transform has emerged as a useful tool that provides time-frequency decomposition of the signal under investigation, enabling intermittent components of transient phenomena to be analyzed. AIM: To study HRV during head-up tilt (HUT) with wavelet transform analysis in PAF patients and healthy individuals (normals). METHODS: Twenty-one patients with PAF (8 men; age 58 +/- 14 yrs) were examined and compared with 21 normals (7 men, age 48 +/- 12 yrs). After a supine resting period, all subjects underwent passive HUT (60 degrees) while in sinus rhythm. Continuous monitoring of ECG and blood pressure was carried out (Task Force Monitor, CNSystems). Acute changes in RR-intervals were assessed by wavelet analysis and low-frequency power (LF: 0.04-0.15 Hz), high-frequency power (HF: 0.15-0.60 Hz) and LF/HF (sympathovagal) were calculated for 1) the last 2 min of the supine period; 2) the 15 sec of tilting movement (TM); and 3) the 1st (TT1) and 2nd (TT2) min of HUT. Data are expressed as means +/- SEM. RESULTS: Baseline and HUT RR-intervals were similar for the two groups. Supine basal blood pressure was also similar for the two groups, with a sustained increase in PAF patients, and a decrease followed by an increase and then recovery in normals. Basal LF, HF and LF/ HF values in PAF patients were 632 +/- 162 ms2, 534 +/- 231 ms2 and 1.95 +/- 0.39 respectively, and 1058 +/- 223 ms2, 789 +/- 244 ms2 and 2.4 +/- 0.36 respectively in normals (p = NS). During TM, LF, HF and LF/HF values for PAF patients were 747 +/- 277 ms2, 387 +/- 94 ms2 and 2.9 +/- 0.6 respectively, and 1316 +/- 315 ms2, 698 +/- 148 ms2 and 2.8 +/- 0.6 respectively in normals (p < 0.05 for LF and HF). During TF1, LF, HF and LF/ HF values for PAF patients were 1243 +/- 432 ms2, 302 +/- 88 ms2 and 7.7 +/- 2.4 respectively, and 1992 +/- 398 ms2, 333 +/- 76 ms2 and 7.8 +/- 0.98 respectively for normals (p < 0.05 for LF). During TF2, LF, HF and LF/HF values for PAF patients were 871 +/- 256 ms2, 242 +/- 51 ms2 and 4.7 +/- 0.9 respectively, and 1263 +/- 335 ms2, 317 +/- 108 ms2 and 8.6 +/- 0.68 respectively for normals (p < 0.05 for LF/HF). The dynamic profile of HRV showed that LF and HF values in PAF patients did not change significantly during TM or TT2, and LF/HF did not change during TM but increased in TT1 and TT2. CONCLUSION: Patients with PAF present alterations in HRV during orthostatism, with decreased LF and HF power during TM, without significant variations during the first minutes of HUT. These findings suggest that wavelet transform analysis may provide new insights when assessing autonomic heart regulation and highlight the presence of ANS disturbances in PAF.

Serodiagnosis of neurocysticercosis in patients with epileptic seizure using ELISA and immunoblot assay

Sera from 88 patients from Santa Catarina and São Paulo states of Brazil, with epileptic seizures who underwent cerebral computed tomography (CT) were analyzed for the detection of antibodies to T. solium cysticercus by ELISA and Immunoblot (IB) with the following antigens: Taenia solium cysticercus total saline (Tso), Taenia crassiceps cysticercus vesicular fluid (Tcra-vf) and T. crassiceps cysticercus glycoproteins (Tcra-gp). ELISA carried out with Tso, Tcra-vf and Tcra-gp antigens showed 95%, 90% and 80% sensitivities, respectively, and 68%, 85% and 93% specificities, respectively. In the epileptic patients group, ELISA positivity was 30%, 51% and 35% with Tso, Tcra-vf and Tcra-gp antigens respectively. Considering the IB as the confirmatory test, the positivity was 16% (14/88) in the epileptic patients total group and 22% (12/54) in the epileptic patients with positive CT and signals of cysticercosis. We found a significant statistical correlation among ELISA or IB results and the phase of the disease when any antigens were used (p < 0.05). We emphasize the need to introduce in the laboratory routine the search for neurocysticercosis (NC) in patients presenting with epileptic seizures because of the high risk of acquiring NC in our region and its potential cause of epilepsy.

Teste de Inclinação no Diagnóstico e Tratamento da Síncope

A síncope neurocardiogénica é uma entidade com prevalência e incidência significativas. Apesar da baixa mortalidade relacionada com esta entidade, são significativas as implicações na qualidade de vida dos doentes. A metodologia para o seu diagnóstico está hoje claramente estabelecida nas recomendações internacionais e o teste de inclinação é um elemento útil na abordagem diagnóstica, em particular na síncope recorrente de causa desconhecida e frequente. Vários protocolos têm vindo a ser descritos nos últimos anos, inicialmente passivos e posteriormente com introdução de agentes provocativos, dos quais os nitratos sub-linguais são os mais largamente aceites, pela simplicidade e bons resultados da sua utilização. O recurso ao teste de inclinação na avaliação da terapêutica está limitado por problemas relacionados com a reprodutibilidade, que contudo estão na base de uma forma de tratamento – o treino de ortostatismo (tilt training) – embora com taxas de sucesso variáveis. Na presente revisão, iremos abordar os vários aspectos relacionados com a aplicação do teste de inclinação na prática clínica.

Detecção remota em meio urbano: teste com classificadores supervisionados a nível do pixel sobre imagens de alta resolução espacial aplicados numa lógica hierárquica de classes

Dissertação de Mestrado em Gestão do Território, Área de Especialização em Detecção Remota e Sistemas de Informação Geográfica

SleepLab V.2.0: plataforma integrada de teste de algoritmos para classificação não supervisionada do sono

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica

Dot-Elisa for evaluation of hydatid cyst wall, protoscoleces and hydatid cyst fluid antigens in the serodiagnosis of cystic echinococcosis

The aim of the present study is to evaluate cyst wall and protoscolex as an alternate source of antigen in serodiagnosis of cystic echinococcosis (CE). A total of 90 blood samples, 30 each of confirmed CE cases, disease controls and healthy controls were collected. Dot-ELISA using cyst wall, protoscolex and cyst fluid were used to demonstrate anti-hydatid antibodies. The sensitivity of Dot-ELISA using cyst wall, protoscolex and cyst fluid was 96.66%, 86.66% and 93.33% respectively and the specificity of the assay was 70% for Dot-ELISA using cyst fluid, protoscolex and cyst wall antigens. Results of the present study show that cyst wall and protoscolex can also be an useful source of antigen in detection of hydatid antibodies in the serodiagnosis of CE.