Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

ELEVATED ALANINE AMINOTRANSFERASE (ALT) IN BLOOD DONORS: AN ASSESSMENT OF THE MAIN ASSOCIATED CONDITIONS AND ITS RELATIONSHIP TO THE DEVELOPMENT OF HEPATITIS C

The determination of aminotranferases levels is very useful in the diagnosis of hepatopathies. In recent years, an elevated serum ALT level in blood donors has been associated with an increased risk of post-transfusion hepatitis (PTH). The purpose of the study was to research the factors associated with elevated ALT levels in a cohort of voluntary blood donors and to evaluate the relationship between increased ALT levels and the development of hepatitis C (HCV) infection. 166 volunteer blood donors with elevated ALT at the time of their first donation were studied. All of the donors were questioned about previous hepatopathies, exposure to hepatitis, exposure to chemicals, use of medication or drugs, sexual behaviour, contact with blood or secretions and their intake of alcohol. Every three months, the serum levels of AST, ALT, alkaline phosphatase, gamma glutamyl transpeptidase, cholesterol, triglyceride and glycemia are assessed over a two year follow-up. The serum thyroid hormone levels as well as the presence of auto-antibodies were also measured. Abdominal ultrasound was performed in all patients with persistently elevated ALT or AST levels. A needle biopsy of liver was performed in 9 donors without definite diagnostic after medical investigation. The presence of anti-HCV antibodies in 116 donors were assayed again the first clinical evaluation. At the end of follow-up period (2 years later) 71 donors were tested again for the presence of anti-HCV antibodies. None of donors resulted positive for hepatitis B or hepatitis C markers during the follow-up. Of the 116 donors, 101 (87%) had persistently elevated ALT serum levels during the follow-up. Obesity and alcoholism were the principal conditions related to elevated ALT serum levels in 91/101 (90.1%) donors. Hypertriglyceridemia, hypercholesterolemia, hypothyroidism and diabetes mellitus also were associated with increased ALT levels. Only 1/101 (0.9%) had mild chronic active non A-G viral hepatitis and 3/101 (2.9%) had liver biopsy with non-specific reactive hepatitis. The determination of ALT levels was not useful to detect donors infected with HCV at donation in Brazil, including the initial seronegative anti-HCV phase.

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Environmental straits of Cryptococcus neoformans variety grubii in the city of Santos, SP, Brazil

This study involved a total of 116 samples, 79 taken from pigeon droppings and 37 of atmospheric air taken close to accumulations of excrement. Cryptococcus neoformans var. grubii was isolated from 11 (13.9%) of these samples. Other species of Cryptococcus were also isolated from these samples, such as C. albidus (12.6%) and C. laurentii (8.9%). C. neoformans was not isolated from the air samples, though C. albidus (5.4%) was. All the strains of C. neoformans were found to belong to the A serotype (C. neoformans var. grubii). In regard to the studies with the antifungal agents 5-fluorocytosine, fluconazole, itraconazole, amphotericin B and voriconazole, by means of the microdilution method (EUCAST), we point out that one sample demonstrated resistance to fluconazole, this being especially significant because this is an environmental strain.

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36% of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

Potential immunological markers for diagnosis and therapeutic assessment of toxocariasis

In human toxocariasis, there are few approaches using immunological markers for diagnosis and therapeutic assessment. An immunoblot (IB) assay using excretory-secretory Toxocara canis antigen was standardized for monitoring IgG, IgE and IgA antibodies in 27 children with toxocariasis (23 visceral, three mixed visceral and ocular, and one ocular form) for 22-116 months after chemotherapy. IB sensitivity was 100% for IgG antibodies to bands of molecular weight 29-38, 48-54, 95-116, 121-162, >205 kDa, 80.8% for IgE to 29-38, 48-54, 95-121, > 205 kDa, and 65.4% for IgA to 29-38, 48-54, 81-93 kDa. Candidates for diagnostic markers should be IgG antibodies to bands of low molecular weight (29-38 and 48-54 kDa). One group of patients presented the same antibody reactivity to all bands throughout the follow-up study; in the other group, antibodies decayed partially or completely to some or all bands, but these changes were not correlated with time after chemotherapy. Candidates for monitoring patients after chemotherapy may be IgG antibodies to > 205 kDa fractions, IgA to 29-38, 48-54, 81-93 kDa and IgE to 95-121 kDa. Further identification of antigen epitopes related to these markers will allow the development of sensitive and specific immunoassays for the diagnosis and therapeutic assessment of toxocariasis.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Comentários sôbre caso de transmissão da doença de chagas por transfusão de sangue e longo período de incubação

Relatam os autores caso de transmissão da doença de Chagas por transfusão de sangue, com longo período de incubação, de 116 dias. Salientam as decorrências de caráter prático relacionadas com essa verificação.

Prevalência sorológica da doença de chagas e correlação sorológico-eletrocardiográfica em populações não selecionadas do município de Encruzilhada do sul, Rio Grande do Sul

No mês de julho de 1974, no plano de estudo da endemia chagásica na Zona Sul do Rio Grande do Sul, foram colhidas 360 amostras de sangue e registrado igual número de eletrocardiogramas entre as populações rurais de 10 localidades do Município de Encruzilhada do Sul (R. G. do Sul). Dez amostras não puderam ser utilizadas por material insuficiente, anticomplementaridade etc. Das 350 submetidas à fixação de complemento, 104 reagiram positivamente para a Doença de Chagas (29,71%). Dos 350 eletrocardiogramas analisados, 116 apresentaram alterações de vários tipos (33,14%), sendo que 41 pertencem aos 104 indivíduos com sorologia positiva (39,42%) e 75 aos 246 indivíduos com sorologia negativa (30,46%) A diferença percentual de 8,94% entre os dois grupos não se mostrou estatisticamente significativa, Portanto a positividade sorológica parece não ser fator condicionante de um maior índice de alterações eletrocardiográficas, a exemplo do que acontece em outras zonas endêmicas. O Autor compara os resultados atuais com aqueles obtidos em 1954 por Brant e Cols. no mesmo Município, para concluir que neste intervalo de tempo houve um substancial aumento na prevalência sorológica e nas alterações eletrocardiográficas, sem que contudo se delineasse um maior índice de alterações nos indivíduos com positividade sorológica.

Epidemia de hepatite veiculada por gamaglobulina contaminada

Os autores estudaram uma epidemia de Hepatite B em uma instituição localizada no bairro de São Cristovão na cidade do Rio de Janeiro, Brasil. Ocorreu um total de 35 casos: 30 entre os funcionários da instituição e cinco entre familiares de funcionários. Todos os casos foram considerados de hepatite aguda benigna; apenas dois não tiveram icterícia. Clinicamente, chamou-lhes a atenção um importante envolvimento articular: artralgias intensas, com grande restrição dos movimentos, durante o período de incubação e que regrediram completamente logo que a icterícia tornou-se evidente. A distribuição cronológica foi típica de infecção simultânea em fonte única. A investigação epidemiológica revelou que essa fonte foi um produto terapêutico derivado do sangue humano: gamaglobulina. As injeções foram feitas com seringas descartáveis e, ironicamente, com o objetivo de prevenir hepatite. Em dois dias de aplicação, cerca de 120 funcionários receberam a injeção de gamaglobulina, entre os quais 27 desenvolveram hepatite. O período de incubação médio foi de 116 dias. 0 Ag HB, testado em 26 pacientes, foi positivo em 12 (46,2%); oito pacientes com tempo de doença inferior a duas semanas, foram todos positivos. O surto epidêmico ocorreu em condições que caracterizaram um estudo experimental, não planejado, em seres humanos. Os autores estudaram, além do surto epidêmico de São Cristovão, oito casos vinculados ao serviço hospitalar onde trabalhavam. Esses casos apresentaram características que os distinguiram dos casos de hepatite comumente all tratados. Todos os pacientes eram de elevada classe social, com o antecedente de terem tomado injeção de gamaglobulina, da mesma procedência daquela que originou o surto epidêmico de São Cristovão. Nesses casos o período de incubação médio foi de 106 dias. Seis dos oito pacientes foram testados para o Ag HB, que foi positivo em cinco. Onze partidas da gamaglobulina foram testadas, em laboratórios de referência da Organização Mundial de Saúde, encontrando-se o Ag HB em seis (54,5%J. Os autores concluem que houve alguma falha grave no processo de preparo da gamaglobulina em apreço. E por isto deve-se ter reservas quanto á noção difundida de que esse medicamento está isento do risco de transmitir o vírus hepatite.

Diagnóstico de infecção pelo Trypanosoma cruzi em gambas, naturalmente infectados, pela reação de imunofluorescência indireta

Em 116 gambás capturados no município de Bambuí-MG, foi realizada a Reação de lmunofluorescência Indireta (RIU) para a pesquisa de anticorpos circulantes arcri-Trypanosoma cruzi. 44(37,9%) foram reativos para T. cruzi, com titulos variando de 1:10 a 1:320. O parasito Joi demonstrado em exames parasitológicos em 43 (97,7%) desses animais. A RIFI apresentou indices de co-positividade (97,7%), co-negatividade (98,6%) e concordância (98,3%), quando comparada com o xenodiagnóstico e hemocultura. Considerando-se a diluição 1:20 como título discriminante de reagente e não reagente, a RIFI é indicada como um dos métodos ae diagnóstico da infecção chagásica em gambás. A RIFI em eluato de sangue dessecado (ESD) apresentou indicés de Lo-positividcide(78,2%) e concordância (76,0%) baixos e aiscordãncias marcantes em relação aos resultados não reagentes.

Estudo da freqüência de ovos de helmintos intestinais em sanitários de uso público de Sorocaba, SP

Foram pesquisados 405 sanitários sendo 11 de praças, 2 da estação ferroviária, 4 da estação rodoviária, 55 de bares e restaurantes, 146 de escolas estaduais, 116 de escolas municipais, 8 de escolas particulares, 36 de postos de saúde, 16 de centros esportivos, 8 de orfanatos e 3 de shopping. Foram usadas lâminasde microscopia comfita adesiva. O material foi coletado pela colagem da fita nos seguintes elementos: 4 campos do assento, maçanetas interna e externa, trinco, registro de torneira e descarga (botão epuxador). Dos 405 sanitários pesquisados, 22 (5,43%) estavam contaminados. Foram encontrados ovos de: Ascaris lumbricoides, ancilostomídeos, Enterobius vermicularis, Taenia sp e Hymenolepis nana, sendo que em 2 sanitários foram encontrados, concomitantemente, ovos de 2 parasitas.

Prevalência dos marcadores sorológicos dos vírus da hepatite B e D em crianças das tribos Caiabietxucarramãe do parque indígena do Xingu, Brasil central

O Parque Indígena do Xingu (PIX) está localizado no estado do Mato Grosso, na região de transição de cenado ao sul e da floresta Amazônica ao norte. Dados de literatura mostram que a população adulta apresenta elevada prevalência de marcadores do vírus da hepatite B (HBV). O presente estudo visa determinar a prevalência dos marcadores do HBV e do HDV na população indígena do PIX de zero a 14 anos, e investigar a forma de transmissão do HBV na região. Entre as 17 tribos existentes no PIX escolheu-se os Caiabi e os Txucairamãe que diferem em seus hábitos de vida e habitam a região Norte do parque com características de clima, vegetação e fauna semelhantes ás da região Amazônica. Avaliaram-se 222 crianças (116 Txucarramãe e 106 Caiabi) e 33 mulheres em idade fértil. A pesquisa de marcadores sorológicos para HBV e HDV foi feita por técnica imunoenzimãtica. A prevalência global dos marcadores sorológicos nas crianças foi: HBsAg 4,5%; anti-HBs 39,6%; anti- HBc 44,1%; presença de algum marcador do HBV 47,3% e anti-HDV 0,0%, enquanto que nas mulheres em idade fértil foi: HBsAg 12%, todas anti-HBe positivas. A infecção pelo HBV ocoireu mais precocemente entre os Txucarramãe, quando se considerou algum marcador do HBV (p < 0,001). No PIX, área de alta endemicidadepara infecção pelo HBV, a transmissão do vírus ocorre provavelmente de maneira horizontal e não vertical como seria de se esperar, características culturais, condições de habitação e presença de insetos hematófagos são importantes determinantes na sua transmissão. Apesar de não ter sido detectado nenhum caso com hepatite Delta, o PIX é uma área propícia para a sua disseminação.

Retrospective study of 151 patients with cutaneous leishmaniasis treated with meglumine antimoniate

We retrospectively analyzed a series of 151 cases of cutaneous leishmaniasis treated between 1967 and 1982. One-hundred-and-thirty-nine (92%) patients presented with active lesions and were treated with daily doses of meglumine antimoniate: 81 adults received a 5-ml vial IM and 58 children received 1 to 5ml. Forty-five (32.4%) patients underwent continuous treatment with meglumine antimoniate for 25 to 116 days without rest intervals, and 94 (67.6%) intermittent treatment with 2 to 5 series of meglumine antimoniate. Intermittent series could include schedules of daily IM applications for 10 to 25 days each and intervals varying from 10 to 60 days. Antimony dose was calculated for 66 (47.5%) patients and ranged from 3.9 to 28.7 Sb5+/kg/day. Of these, 35 patients received >10mg and 31 patients <10mg Sb5+/kg/day. Median time of healing was longer for lesions on the legs and feet - 67.5 days versus 48.7 days (p < 0.001) for other sites. However, there were no significant differences in the median time of healing between adults and children, intermittent and continuous regimens or high and low antimony doses. Fifty-one patients were reassessed 5 to 14 years after treatment and showed no evidence of disease. These results support further investigation (clinical trials) on treatment using low doses of antimony.