Effect of b-Propiolactone treatment on the complement activation mediated by equine antisera

Reduction of complement activation through an alteration of the Fc fragment of immunoglobulins by b-propiolactone treatment was carried out in equine antisera raised against rabies virus, Bothrops venoms and diphtherial toxin. Results were evaluated by means of an anaphylactic test performed on guinea-pigs, and compared to the ones obtained with the same sera purified by saline precipitation (ammonium sulfate), followed or not by enzymatic digestion with pepsin. Protein purity levels for antibothropic serum were 184.5 mg/g and 488.5 mg/g in b-propiolactone treated and pepsin-digested sera, respectively. The recovery of specific activity was 100% and 62.5% when using antibothropic serum treated by b-propiolactone and pepsin digestion, respectively. The antidiphtherial and anti-rabies sera treated with b-propiolactone and pepsin presented protein purity levels of 5,698 and 7,179 Lf/g, 16,233 and 6,784 IU/g, respectively. The recovery of specific activity for these antisera were 88.8%, 77.7%, 100% and 36,5%, respectively. b-propiolactone treatment induced a reduction in complement activation, tested "in vivo", without significant loss of biological activity. This treatment can be used in the preparation of heterologous immunoglobulins for human use.

Trypanosoma cruzi: chemotherapy with benznidazole in mice inoculated with strains from Paraná State and from different endemic areas of Brazil

Strains of Trypanosoma cruzi from different geographical areas have shown different levels of susceptibility to trypanocidal drugs. The susceptibility in vivo to benznidazole was investigated in eighteen strains of T. cruzi. Twelve were isolated from chronic chagasic patients from different Chagas’ disease endemic areas. The other six strains were isolated from the northwestern region of Paraná state; two of them from patients three from triatomines (Triatoma sordida) and one from wild reservoir (Didelphis sp.). To test drug the infected mice were divided into two groups of twenty. One group was treated with benznidazole for twenty consecutive days and the other group was used as untreated control. The treatment began after detection of the infection by direct blood examination or haemoculture. The control of cure was done through haemoculture and indirect immunofluorescence test. The drug eliminated the inflammatory lesions of the skeletal muscle of mice considered cured and from the heart of most of them. Moreover, the inflammatory lesions were reduced in treated but not cured animals. The T. cruzi strains studied showed a gradient of drug susceptibility that varied from 0% to 100%. Ten strains were considered sensitive to the treatment (61 to 100% of cure), one strain was partially sensitive (50% of cure) and seven strains were considered resistant to the treatment (0 to 40% of cure). This variation was observed both in strains of T. cruzi isolated from domestic and sylvatic cycles

EPIDEMIOLOGICAL SURVEY ON CANINE POPULATION WITH THE USE OF IMMUNOLEISH SKIN TEST IN ENDEMIC AREAS OF HUMAN AMERICAN CUTANEOUS LEISHMANIASIS IN THE STATE OF RIO DE JANEIRO, BRAZIL

A survey for canine tegumentary leishmaniasis (CTL) has been carried out between 1986 and 1993 in seven endemic localities for American cutaneous leishmaniasis in the State of Rio de Janeiro. 270 dogs have been examined for their clinical aspects, the development of delayed hypersensitivity (DHS) with Immunoleish antigen and with immunofluorescent antibody research of IgG (IF). 28.2% of them had ulcer lesions and 3.3% had scars. The lesions consisted of single (39.5%) and mucocutaneous lesions (31.6%), multiple cutaneous (25.0%) and mucocutaneous lesions associated with cutaneous ulcers (4.0%). Twelve (15.8%) isolates from biopsies were analyzed by zimodeme and schizodeme and identified as L. (V.) braziliensis. The overall prevalence of canine infection that was evaluated with the skin test was of 40.5% and with IF it was of 25.5%. Both tests showed a high positive rate with relation to the animals with mucosal lesions, as in the case of human mucocutaneous leishmaniasis. The comparison of the two tests showed the skin test to have a better performance although there was no statistical difference (p>0.05) between them. The proportional sensitivity and specificity was of 84.0% and 74.0%, respectively. The Immunoleish skin test and IF are useful tools to be employed in CTL field epidemiological surveys.

THE PERSISTENCE OF LEPTOSPIRAL AGGLUTININS TITERS IN HUMAN SERA DIAGNOSED BY THE MICROSCOPIC AGGLUTINATION TEST

The persistence of agglutinins detected by MAT has created some problems to the interpretation of the results. The aim of this study was to examine the data of serology from 70 patients with serologically confirmed diagnosis of leptospirosis by during 3-13 months after being affected with leptospires in order to elucidate the interpretation of the persistence of agglutinins detected by MAT. Sixty-one patients sera (87.14%) had titers equal or greater than 800. Of these, two individuals maintained titers of 800 thirteen months after the onset. This study showed that only one sample of sera with high titers is not reliable to determine the time at which infection occurred.

PREVALENCE OF ANTI- Toxocara ANTIBODIES IN A RANDOM SAMPLE OF INPATIENTS AT A CHILDREN'S HOSPITAL IN VITÓRIA, ESPÍRITO SANTO, BRAZIL

In the streets of Vitória, in the State of Espírito Santo, Brazil, are large number of stray dogs, many of which are infected with Toxocara canis, suggesting a high risk for human infection. In order to investigate the prevalence of Toxocara infection in children in Espírito Santo we studied the prevalence of anti-Toxocara antibodies in 100 random inpatients over one year of age, at the Children's Hospital N.S. da Glória, the reference children's hospital for the State.All the sera were collected during the period between October 1996 and January 1997. The mean age was 6.6±4.1 yrs. (1 to 14 yrs., median 6yrs.) and there were patients from all of the different wards of the hospital. Sixty-eigth patients came from the metropolitan area of Vitória and the other 32 from 17 other municipalities. The anti-Toxocara antibodies were investigated by ELISA-IgG using a secretory-excretory antigen obtained from second stage larvae. All sera were adsorbed with Ascaris suum antigen before the test. Thirty-nine sera (39%) were positive, predominantly from boys, but the gender difference was not statistically significant (boys:25/56 or 44.6%; girls:14/44 or 31.8%; p=0.311). The prevalence of positive sera was higher, but not statistically significant, in children from the urban periphery of metropolitan Vitória (formed by the cities of Vitória, Cariacica, Vila Velha, Serra and Viana) than in children from 17 other municipalities (44.1% and 28.1% respectively, p=0.190). Although the samples studied do not represent all children living in the State of Espírito Santo, since the Children's Hospital N.S. da Glória admits only patients from the state health system, it is probable that these results indicate a high frequency of Toxocara infection in children living in Espírito Santo. Further studies of population samples are necessary to ascertain the prevalence of Toxocara infection in our country.

TOXOCARIASIS: SEROLOGICAL DIAGNOSIS BY INDIRECT ANTIBODY COMPETITION ELISA

Toxocariasis is caused by infection of man by Toxocara canis and Toxocara cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE) employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody, in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%).

Desenvolvimento de um método potenciométrico para deteção de salmonella typhimurium

A Salmonella é um microrganismo responsável por grande parte das doenças alimentares, podendo por em causa a saúde pública da área contaminada. Uma deteção rápida, eficiente e altamente sensível e extremamente importante, sendo um campo em franco desenvolvimento e alvo de variados e múltiplos estudos na comunidade cientifica atual. Foi desenvolvido um método potenciométrico para a deteção de Salmonellas, com elétrodos seletivos de iões, construídos em laboratório com pontas de micropipetas, fios de prata e sensores com composição otimizada. O elétrodo indicador escolhido foi um ESI seletivo a cadmio, para redução da probabilidade de interferências no método, devido a pouca abundancia do cadmio em amostras alimentares. Elétrodos seletivos a sódio, elétrodos de Ag/AgCl de simples e de dupla juncão foram também construídos e caracterizados para serem aplicados como elétrodos de referência. Adicionalmente otimizaram-se as condições operacionais para a analise potenciométrica, nomeadamente o elétrodo de referencia utilizado, condicionamento dos elétrodos, efeito do pH e volume da solução amostra. A capacidade de realizar leituras em volumes muito pequenos com limites de deteção na ordem dos micromolares por parte dos ESI de membrana polimérica, foi integrada num ensaio com um formato nao competitivo ELISA tipo sanduiche, utilizando um anticorpo primário ligado a nanopartículas de Fe@Au, permitindo a separação dos complexos anticorpo-antigénio formados dos restantes componentes em cada etapa do ensaio, pela simples aplicação de um campo magnético. O anticorpo secundário foi marcado com nanocristais de CdS, que são bastante estáveis e é fácil a transformação em Cd2+ livre, permitindo a leitura potenciométrica. Foram testadas várias concentrações de peroxido de hidrogénio e o efeito da luz para otimizar a dissolução de CdS. O método desenvolvido permitiu traçar curvas de calibração com soluções de Salmonellas incubadas em PBS (pH 4,4) em que o limite de deteção foi de 1100 CFU/mL e de 20 CFU/mL, utilizando volumes de amostra de 10 ƒÊL e 100 ƒÊL, respetivamente para o intervalo de linearidade de 10 a 108 CFU/mL. O método foi aplicado a uma amostra de leite bovino. A taxa de recuperação media obtida foi de 93,7% } 2,8 (media } desvio padrão), tendo em conta dois ensaios de recuperação efetuados (com duas replicas cada), utilizando um volume de amostra de 100 ƒÊL e concentrações de 100 e 1000 CFU/mL de Salmonella incubada.

Proposal of abolition of the skin sensitivity test before equine rabies immune globulin application

An epizootic outbreak of rabies occurred in 1995 in Ribeirão Preto, SP, with 58 cases of animal rabies (54 dogs, 3 cats and 1 bat) confirmed by the Pasteur Institute of São Paulo, and one human death. The need to provide care to a large number of people for the application of equine rabies immune globulin (ERIG) prevented the execution of the skin sensitivity test (SST) and often also the execution of desensitization, procedures routinely used up to that time at the Emergency Unit of the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (EU-UHFMRP-USP), a reference hospital for the application of heterologous sera. In view of our positive experience of several years with the abolition of SST and of the use of premedication before the application of antivenom sera, we used a similar schedule for ERIG application. Of the 1489 victims of animal bites, 1054 (71%) received ERIG; no patient was submitted to SST and all received intravenously anti-histamines (anti-H1 + anti-H2) and corticosteroids before the procedure. The patients were kept under observation for 60 to 180 minutes and no adverse reaction was observed. On the basis of these results, since December 1995 ERIG application has been decentralized in Ribeirão Preto and has become the responsibility of the Emergency Unit of the University Hospital and the Central Basic Health Unit, where the same routine is used. Since then, 4216 patients have received ERIG (1818 at the Basic Health Unit and 2398 at the EU-UHFMRP), with no problems. The ideal would be the routine use of human rabies immune globulin (HRIG) in public health programs, but this is problematic, because of their high cost. However, while this does not occur, the use of SST is no longer justified at the time of application of ERIG, in view of the clinical evidence of low predictive value and low sensitivity of SST involving the application of heterologous sera. It is very important to point out that a negative SST result may lead the health team to a feeling of false safety that no adverse reaction will occur, but this is not true for the anaphylactoid reactions. The decision to use premedication, which is based on knowledge about anaphylaxis and on the pharmacology of the medication used, is left to the judgment of health professionals, who should always be prepared for eventual untoward events.

Advantages of the rapid HIV-1 test in occupational accidents with potentially contaminated material among health workers

In occupational accidents involving health professionals handling potentially contaminated material, the decision to start or to continue prophylactic medication against infection by Human Immunodeficiency Virus (HIV) has been based on the ELISA test applied to a blood sample from the source patient. In order to rationalize the prophylactic use of antiretroviral agents, a rapid serologic diagnostic test of HIV infection was tested by the enzymatic immunoabsorption method (SUDS HIV 1+2, MUREX®) and compared to conventional ELISA (Abbott HIV-1/ HIV-2 3rd Generation plus EIA®). A total of 592 cases of occupational accidents were recorded at the University Hospital of Ribeirão Preto from July 1998 to April 1999. Of these, 109 were simultaneously evaluated by the rapid test and by ELISA HIV. The rapid test was positive in three cases and was confirmed by ELISA and in one the result was inconclusive and later found to be negative by ELISA. In the 106 accidents in which the rapid test was negative no prophylactic medication was instituted, with an estimated reduction in costs of US$ 2,889.35. In addition to this advantage, the good correlation of the rapid test with ELISA, the shorter duration of stress and the absence of exposure of the health worker to the adverse effects of antiretroviral agents suggest the adoption of this test in Programs of Attention to Accidents with Potentially Contaminated Material.

A rapid latex agglutination test for the detection of anti-cysticercus antibodies in cerebrospinal fluid (CSF)

Simple and rapid latex-based diagnostic tests have been used for detecting specific antigens or antibodies in several diseases. In this article, we present the preliminary results obtained with a latex agglutination test (LAT) for diagnosing neurocysticercosis by detection of antibodies in CSF. A total of 43 CSF samples were assayed by the LAT: 19 CSF samples from patients with neurocysticercosis and 24 CSF samples from patients with other neurologic disorders (neurosyphilis, n = 8; neurotoxoplasmosis, n = 3; viral meningitis, n = 4, chronic headache, n = 9). The LAT exhibited 89.5% sensitivity and 75% specificity. The use of LAT seems to be an additional approach for the screening of neurocysticercosis with advantage of simplicity and rapidity. Further studies could be performed using purified antigens and serum samples.

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.

Determinação das propriedades mecânicas à tração de adesivos estruturais frágeis e dúcteis

A crescente evolução na tecnologia das juntas coladas conferiu um potencial atractivo às ligações adesivas, com aplicações nas mais variadas indústrias. Isto deve-se não só aos aspetos económicos, tais como a melhoria da cadência de produção mas também à resistência mecânica que estas proporcionam. A possibilidade de ligar facilmente materiais distintos, a distribuição mais uniforme das tensões, a melhor resistência à fadiga e a elevada capacidade de amortecimento de vibrações estão entre as principais vantagens da utilização deste tipo de ligação. Estas propriedades transformam as juntas coladas numas das preferidas no momento de seleção de meios de união. O trabalho desenvolvido nesta dissertação enquadra-se no âmbito das ligações adesivas e tem como principais objetivos a produção de uma ferramenta para a produção de provetes de adesivo, assim como a determinação das propriedades mecânicas à tração dos mesmos para testar o desempenho do molde fabricado. Para tal, utilizou-se um adesivo frágil (Araldite® AV 138), um dúctil (Araldite® 2015) e um muito dúctil (SikaForce® 7888). Paralelamente é selecionado o método mais adequado na obtenção destes provetes, designadamente escolhendo entre a moldação em molde aberto e a injeção em molde fechado. Com vista à obtenção dos provetes, foi projetado e construído um molde em aço. Recorrendo à máquina de tração Shimadzu AG – X 100, realizaram-se os respetivos ensaios de tração, para a determinação de todas as propriedades mecânicas dos adesivos. Para efeitos de comparação de resultados foram utilizados dois tipos de extensómetros, um mecânico e um ótico. Os resultados experimentais permitiram observar que a presença de vazios afetou especialmente a deformação de rotura e a tensão de rotura. Detetaram-se pequenas discordâncias, comparativamente com os estudos publicados, de algumas características mecânicas obtidas dos diversos adesivos utilizados. Constatou-se também um ligeiro desfasamento entre os valores adquiridos com os dois tipos de extensómetros utilizados.

Evaluation of a rapid dipstick test, Malar-CheckTM, for the diagnosis of Plasmodium falciparum malaria in Brazil

The present study was carried out to evaluate the Malar-CheckTM Pf test, an immunochromatographic assay that detects Plasmodium falciparum Histidine Rich Protein II, does not require equipment, and is easy and rapid to perform. In dilution assays performed to test sensitivity against known parasite density, Malar-CheckTMwere compared with thick blood smear (TBS), the gold standard for diagnosis. Palo Alto isolate or P. falciparum blood from patients with different parasitemias was used. The average cut-off points for each technique in three independent experiments were 12 and 71 parasites/mm³ (TBS and Malar-CheckTM, respectively). In the field assays, samples were collected from patients with fever who visited endemic regions. Compared to TBS, Malar-CheckTMyielded true-positive results in 38 patients, false-positive results in 3, true-negative results in 23, and false-negative result in 1. Malar-CheckTMperformed with samples from falciparum-infected patients after treatment showed persistence of antigen up to 30 days. Malar-CheckTM should aid the diagnosis of P. falciparum in remote areas and improve routine diagnosis even when microscopy is available. Previous P. falciparum infection, which can determine a false-positive test in cured individuals, should be considered. The prompt results obtained with the Malar-CheckTM for early diagnosis could avoid disease evolution to severe cases.

Clinical-epidemiological study of leptospirosis in humans and reservoirs in Yucatán, México

A leptospirosis clinical-epidemiological study was made in humans and reservoirs in the state of Yucatán, México. Interviews and serological analyses were made on 400 persons from an open population, 439 probable cases of leptospirosis and 1060 animal reservoirs (cows, pigs, dogs, rats and opossums). IgM Leptospira DipstickTM and Microscopic Agglutination Test (MAT) was used to detect human antibodies to leptospiras and serovar respectively. Leptospirosis incidence in humans was 2.2/100,000 inhab. in 1998, 0.7/100,000 in 1999 and 0.9/100,000 in 2000. Overall seroprevalence was 14.2%, relatively unchanged from seroprevalences observed 20 years ago. Highest seropositivity was found in people over 56 years of age, predominating males over females. Predominant serovars in the open population were tarassovi, hardjo, pomona and panama. Leptospirosis cases were most frequent in rural areas, and the anicteric course predominated over the icteric. The panama, icterohaemorrhagiae and pomona serovars predominated in both anicteric and icteric courses. Dogs, pigs and rodents had the highest seropositivity among the reservoirs. Contact with rodents and natural water sources were significant factors (p £ 0.05). Human cases (74%) occurred during the rainy season. It is concluded that leptospirosis is still a serious illness with important clinical and epidemiological implications in the state of Yucatán, Mexico.