Antibodies against Entamoeba histolytica in individuals with intestinal amoebiasis presenting cysts and/or trophozoites in the feces

Serum samples were obtained from 154 individuals infected with Entamoeba histolytica (78 symptomatic and 76 asymptomatic). Twelve had trophozoites in the feces whereas 142 had only cysts. The sera were used to test the existence of antibodies anti-Entamoeba histolytica employing the Indirect Hemagglutination (IHA), Indirect Immunofluoresccnce (IFAT), Complement Fixation Reaction (CFR) and Counterimmunoelectrophoresis (CIEP). For those individuals with trophozoites in their feces, 75.0 were positive by IHA and IFAT, 83.0 by CFR and 41.7 by CIEP. In individuals who had only cysts, positive results by the same tests were respectively, 5.6%, 12.0%, 19.0% and 5.6%. The difference in relation to the tilers of antibodies detected through IHA, IFAT, CFR and CIEP and in relation to the presence of trophozoites or cysts in the feces was significative for four immunological reactions when X², was employed (P < 0.05).

UltramicroELISA para la detección de anticuerpos IgM anti M. leprae

La disponibilidad del sistema Ultramicroanalítico (SUMA) y de un antígeno especie-específico del M. leprae obtenido mediante síntesis química, permitió la normalización y validación de un ultramicroELISA para la detección de anticuerpos IgM específicos a esta micobacteria. El análisis de 433 sueros de banco de sangre y 265 sueros usados para validar el método y clasificados en un grupo control de donantes de banco de sangre (100), un grupo de pacientes tuberculosos (50), un grupo de enfermos de lepra (65) y un grupo de contactos de estos enfermos (50), mostró la especificidad del ensayo para evidenciar la infección con el M. leprae. Los resultados obtenidos del estudio adicional de 140 muestras de suero de contactos de enfermos estuvieron estrechamente correlacionados (r = 0,98) con los resultados obtenidos por la técnica de microELISA convencional. La utilización del SUMA no solo permite un notable ahorro de reactivos si no además facilita la lectura, cálculo, validación y almacenamiento automático de los resultados.

Modulation of parasitemia and antibody responce to Trypanosoma cruzy by cyclophosphamide in Calomys callosus (Rodentia, Cricetidae)

Calomys callosus a wild rodent, previously described as harboring Trypanosoma cruzi, has a low susceptibility to infection by this protozoan. Experiments were designed to evaluate the contribution of the immune response to the resistance to T. cruzi infection exhibited by C. calossus. Animals were submitted to injections of high (200 mg/kg body weight) and low (20 mg/kg body weight) doses of cyclophosphamide on days -1 or -1 and +5, and inoculated with 4 x 10³ T. cruzi on day O. Parasitemia, mortality and antibody response as measured by direct agglutination of trypomastigotes were observed. Two hundred mg doses of cyclophosphamide resulted in higher parasitemia and mortality as well as in suppression of the antibody response. A single dose of 20 mg enhanced antibody levels on the 20th day after infection, while an additional dose did not further increase antibody production. Parasitemia levels were not depressed, but rather increased in both these groups as compared to untreated controls. Passive transfer of hyperimmune C. callosus anti-T. cruzi serum to cyclophosphamide immunosuppressed animals resulted in lower parasitemia and mortality rates. These results indicate that the immune response plays an important role in the resistance of C. callossus to T. cruzi.

Associantion between leprosy and hepatitis B infecion: a survey in Goiânia, Central Brazil

This investigation presents the results of Hepatitis B virus screening among leprosy patients conducted in Central Brazil as a preliminary information for a HBV vaccination programme. The main objectives were to assess the seroprevalence of HBV serum markers among lepromatous patients and to analyse institutionalization as risk factor for HBV infection in this population. Two groups of lepromatous patients were studied, 83 outpatients and 171 institutionalized ones. Screening for HBV serum markers included the detection of HBsAg, anti-HBc by radioimmune assay (RIA). The prevalence of carrier state (HBsAg) was 4.8% and 8.8% among outpatients and institutionalized, respectively, (p>0.05). Seroprevalence of exposure (all markers) was statistically significant different between outpatients (16.9%) and institutionalized ones (50.3%). Institutionalized patients had an almost four fold risk of HBV infection when compared to the outpatients, and the highest risks were among patients with more than 21 years of residence in the colony, after adjusting for age and sex.

Pre-meal tomato (Lycopersicon esculentum) intake can have anti-obesity effects in young women?

The effect of pre-meal tomato intake in the anthropometric indices and blood levels of triglycerides, cholesterol, glucose, and uric acid of a young women population (n=35, 19.6 ± 1.3 years) was evaluated. During 4 weeks, daily, participants ingested a raw ripe tomato (~90 g) before lunch. Their anthropometric and biochemical parameters were measured repeatedly during the follow-up time. At the end of the 4 weeks, significant reductions were observed on body weight (-1.09 ± 0.12 kg on average), % fat (-1.54 ± 0.52%), fasting blood glucose (-5.29 ± 0.80 mg/dl), triglycerides (-8.31 ± 1.34 mg/dl), cholesterol (-10.17 ± 1.21 mg/ dl), and uric acid (-0.16 ± 0.04 mg/dl) of the participants. The tomato pre-meal ingestion seemed to interfere positively in body weight, fat percentage, and blood levels of glucose, triglycerides, cholesterol, and uric acid of the young adult women that participated in this study.

Prevalências do HBsAg, do anti-HBc e do anti-HCV na população de candidatos a doadores de sangue do Hemocentro-Campinas

Em 29833 doadores pesquisados encontramos prevalência de 1,52% para o HBsAg e de 11% para o anti-HBc. A co-positividade anti-HBc/anti-HBs em 2783 doadores HBsAg negativos/anti-HBc positivos foi de 81,9%. A prevalência para o HBsAg é baixa nos doadores de Campinas, enquanto o anti-HBc apresenta-se com prevalência elevada quando comparado a outros países. A pesquisa do anti-HCV, em doadores de sangue de Campinas, mostrou prevalência de 2,6% para este marcador, que é bem maior que as observadas nos EUA e Europa. Cerca de 36% dos doadores anti-HCV positivos são anti-HBc reagentes, permitindo inferir, que estas duas viroses acometem simultânea ou sequencialmente os doadores de sangue brasileiros.

Hepatites pós-transfusionais na cidade de Campinas, SP, Brasil: II. Presença dos anticorpos anti-HBc e anti-HCV em candidatos a doadores de sangue e ocorrência de hepatites pós-transfusionais pelo vírus C nos receptores de sangue ou derivados

Pesquisamos os anticorpos anti-HBc e anti-HCV em amostras de soros provenientes de 799 candidatos a doadores, que tiveram suas unidades de sangue ou derivados transfundidas a 111 receptores. O anti-HBc e o anti-HCV foram reagentes, em respectivamente 9 e 2,1% dos doadores testados. Observamos que entre os 111 receptores, 44 haviam recebido pelo menos uma unidade anti-HBc positiva e 67 haviam sido transfundidos somente com unidades anti-HBc negativas. Houve um risco 4,5 vezes maior de aquisição de hepatite por vírus C pelos receptores que receberam pelo menos uma unidade anti-HBc positiva Se a pesquisa do anti-HBc fosse realizada na triagem sorológica dos doadores de sangue, cerca de 56% dos casos de HVC nos receptores saiam evitados. A população de receptores que recebeu pelo menos uma unidade anti-HCV reagente, apresentou um risco 29 vezes maior de adquirir esta hepatite, quando comparada aos receptores transfundidos com todas as unidades anti-HCV negativas. A realização do teste para a pesquisa do anti-HCV na triagem dos doadores de sangue, preveniria 79% dos casos de HVC pós-transfusionais. Os candidatos a doadores brasileiros parecem ser acometidos simultânea ou sequencialmente, pelos vírus B e C das hepatites, pois, 44,4% dos doadores anti-HCV positivos, também foram anti-HBc positivos. A realização dos testes para as pesquisas dos anticorpos anti-HBc e anti-HCV, nas triagens hemoterápicas, está indicada para prevenir a transmissão de hepatites pós-transfusionais, em nosso meio.

Immunodiagnosis of hydatid disease: evaluation of antigens from hydatid cyst fluid and the vesicularfluid of Taenia crassiceps metacestode

The specificity and sensitivity of the enzyme immunoassay (EIA), presently used in South America areas where hydatidosis caused by Echinococcus granulosus is endemic, was compared to two alternative EIA. One of these uses an hydatid antigen of different prepraration and the other vesicular fluid of Taenia crassiceps cisticerci (VFCC). The effect of previous neutralization in the serum sample of antibodies anti-normal ovine or murine sera and anti-phosphorylcholine on the diagnostic efficiency of these EIA was studied. The frequency of distribution of the titers obtained with normal sera, hydatid sera positive to DD5 test and hydatid sera negative to DD5 test in three EIA systems was analyzed. Results showed a significant decrease of sensitivity of the EIA using VFCC when compared to these EIA using hydatid antigens. This makes inconvenient the use of VFCC for the immunodiagnosis of hydatid disease. No significant differences between the two EIA using hydatid antigens were observed. SDS-PAGE analysis showed remarkable differences between the VFCC and the hydatid antigens composition and some differences among these latters probably due to manufacturing procedures.

Total serum IgE and parasite: specific IgG and IgA antibodies in human strongyloidiasis

Total serum IgE, and Strongyloides - specific IgG and IgA antibodies were studied in 27 patients with parasitologically proven strongyloidiasis. Clinical manifestations in this case series were investigated by a restrospective study of the patient's records. Total serum IgE levels were elevated (greater than 250 IU/ml) in 59% of the patients (mean concentration = 1364 IU/ml). Parasite - specific IgG and IgA antibodies were detected by ELISA in the serum of 23 (85.2%) and 21 (77.8%) patients, respectively. Elevated serum IgE and clinical manifestations were not useful indexes of the presence of strongyloidiasis. On the other hand, our results support the view that serologic tests, particularly ELISA for detecting Strongyloides - specific IgG antibodies, can be usefully exploited for diagnostic purposes in strongyloidiasis.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p< 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log 10) and the log 10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log 10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

Detection of hepatitis B virus DNA by the polymerase chain reaction in anti-HBE positive chronic hepatitis B patients

Detection of HBV-DNA by PCR was compared with other serological markers (HBsAg, HBeAg and anti-HBe) in a series of49 Chronic Hepatitis B patients, including 12 with a spontaneous clearance of HBsAg. None of these HBsAg negative cases were PCR positive, but 33/37 (89.2%) HBsAg positive cases were PCR positive (p < 0.0001). Among HBsAg positive samples, nine cases were HBeAg positive and anti-HBe negative, all of them PCR positive. Other 3 patients were HBeAg and anti-HBe positive and these cases were also found PCR positive. A third group included 21 patients anti-HBe positive and HBeAg negative: 19 of them were PCR positive and 2 were PCR negative. The last 4 cases were HBeAg and anti-HBe negative, two of them were PCR positive. The detection of anti-HBe viremic cases in the present series suggest that preC variants could occur in our country. In conclusion, the integrated phase o f chronic hepatitis B seems to be less frequent than it was assumed, when only HBeAg or dot blot hybridization techniques were used. The new term "low replication phase" might favorably replace the former "integrated phase".

Immune reconstitution after autologous hematopoietic stem cell transplantation

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

Seasonal variation of anti-resa/Pf155 Plasmodium falciparum antibodies in three localities from the state of Amapá, Brazil

Anti-RESA/Pf155 antibodies were assayed in sera of individuals from three localities (Laranjal do Jari, Vila Padaria and Vila Paraíso) in the State of Amapá, Brazil, during the long-rains and short-rains seasons. All of these had negative blood smears for malaria. Most of the sera collected were positive in Indirect Fluorescent Antibody (IFA) with P. falciparum parasites, with no seasonal variation. A high percentage of these sera (62% to 100%) was RESA positive by Modified Indirect Fluorescent Antibody (MIFA), with a significant (p < 0.05) increase of geometric mean titers during the short-rains season, when the transmission of the disease is highest. ELISA with three repetitive RESA peptides (EENV)3 (4x3), (EENVEHDA)2 (8x2) and (DDEHVEEPTVA)2(11x2) did not reveal statistically significant seasonal variations, although a small enhancement of positivity was observed in V. Padaria (15.3 to 38.8%) in the short-rains season with the 8x2 peptides, and with 4x3 and 8x2 peptides in V. Paraíso, with a decrease in 11x2. MIFA titers appeared to be correlated mainly to the peptide 4x3 and it was the immunodominant in the three localities.

Dot-ELISA-IgM in saliva for the diagnosis of human leptospirosis using polyester fabric-resin as support (Preliminary Report)

In order to improve the diagnosis of human leptospirosis, we standardized the dot-ELISA for the search of specific IgM antibodies in saliva. Saliva and serum samples were collected simultaneously from 20 patients with the icterohemorrhagic form of the disease, from 10 patients with other pathologies and from 5 negative controls. Leptospires of serovars icterohaemorrhagiae, canicola, hebdomadis, brasiliensis and cynopteri grown in EMJH medium and mixed together in equal volumes, were used as antigen at individual protein concentration of 0.2 µg/µl. In the solid phase of the test we used polyester fabric impregnated with N-methylolacrylamide resin. The antigen volume for each test was 1µl, the saliva volume was 8 µl, and the volume of peroxidase-labelled anti-human IgM conjugate was 30 µl. A visual reading was taken after development in freshly prepared chromogen solution. In contrast to the classic nitrocellulose membrane support, the fabric support is easy to obtain and to handle. Saliva can be collected directly onto the support, a fact that facilitates the method and reduces the expenses and risks related to blood processing.