Serodiagnosis of human cysticercosis by using antigens from vesicular fluid of Taenia crassiceps cysticerci.

Neurocysticercosis (NC), caused by the presence of Taenia solium metacestodes in tissues, is a severe parasitic infection of the central nervous system with universal distribution. To determine the efficiency of enzyme-linked immunosorbent assay (ELISA) and immunoblot with antigens of T. crassiceps vesicular fluid (Tcra) compared to standard techniques (indirect immunofluorescence test [IFT] and complement fixation test [CFT]) using T. solium cysticerci (Tso) for the serodiagnosis of NC, we studied serum samples from 24 patients with NC, 30 supposedly healthy individuals, 76 blood bank donors, 45 individuals with other non-NC parasitoses, and 97 samples from individuals screened for cysticercosis serology (SC). The sensitivity observed was 100% for ELISA-Tso and ELISA-Tcra, 91.7% for the IFT, and 87.5% for the CFT. The specificity was 90% for ELISA-Tso, 96.7% for ELISA-Tcra, 50% for IFT, and 63.3% for CFT. The efficiency was highest for ELISA-Tcra, followed by ELISA-Tso, IFT, and CFT. Of the 23 samples from SC group, which were reactive to ELISA-Tso and/or ELISA-Tcra, only 3 were positive to immunblot-Tcra (specific peptides of 14- and 18-kDa) and to glycoprotein peptides purified from Tcra antigen (gp-Tcra), showing the low predictive value of ELISA for screening. None of the samples from the remaining groups showed specific reactivity in immunoblot-Tcra. These results demonstrate that ELISA-Tcra can be used as a screening method for the serodiagnosis of NC and support the need for specific tests for confirmation of the results. The immunoblot can be used as a confirmatory test both with Tcra and gp-Tcra, with the latter having an advantage in terms of visualization of the results.

Molecular markers in acute and chronic phases of human toxoplasmosis: determination of immunoglobulin G avidity by Western blotting.

We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of Toxoplasma gondii infection.

Serological diagnosis of Chagas' disease: a potential confirmatory assay using preserved protein antigens of Trypanosoma cruzi.

The diagnosis of Chagas' disease relies mostly on data provided by immunologic tests, but inconclusive results often require elucidation, especially in blood banks. When six different types of Trypanosoma cruzi epimastigote antigens were studied by an immunoblotting assay (IBA), a preserved protein antigen (Ag PP) was found to present the most interesting immunochemical features because of its high reactivity with anti-T. cruzi antibodies. Thus, the IBA with Ag PP (PP IBA) was assessed with panels of coded and noncoded serum samples prepared in different laboratories, including the Brazilian Reference Laboratory for Chagas' Disease. It was found that serum samples from patients proved (clinically, eletrocardiographically, serologically, and epidemiologically) to have Chagas' disease consistently recognized 12 bands (140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, and 18 kDa) of Ag PP. In contrast, sera from nonchagasic patients, including patients with mucocutaneous leishmaniasis, were negative or reacted weakly, and one serum sample did not have more than five different bands. These bands were 78, 57, 46, 35, 27, 23, 20, or 18 kDa. A criterion was adopted to interpret the results obtained in the PP IBA. The criterion considered positive a serum sample recognizing all 12 bands and considered negative a serum sample that did not recognize any of the bands except the eight nonspecific bands mentioned above. The PP IBA indicated maximum sensitivity and specificity as well as high positive and negative predictive values. The data demonstrate that the PP IBA discriminates chagasic from nonchagasic infections and seems to be applicable as a confirmatory assay for elucidating inconclusive results obtained by standard serology.

Significado clínico dos anticorpos antinucleares na artrite reumatóide juvenil / Clinical significance of antinuclear antibodies in juvenile rheumatoid arthritis

Os anticorpos antinucleares (AAN) têm sido demonstrados em freqüência extremamente variável na artrite reumatóide juvenil (ARJ), dependendo do substrato empregado e da seleçäo dos pacientes. Além disso, a associaçÝo entre estes anticorpos e a atividade ou a gravidade da doença näo está bem estabelecida. Objetivo: Determinar a freqüência dos AAN e possível associaçöes com diferentes parâmetros clínicos da ARJ, como duraçäo, atividade e gravidade da doença. Material e métodos: 86 pacientes com ARJ foram estudados quanto à presença de AAN (pesquisados pelo método de imunofluorescência indireta em células HEp-2); 32 pacientes com lúpus eritematoso sistêmico juvenil e 52 crianças saudáveis constituíram os grupos-controles. A atividade da doença foi definida segundo uma escala de dois pontos, enquanto a gravidade foi avaliada pelo grau de capacidade funcional. Resultados: AAN foram encontrados em 36 (42 por cento) pacientes com ARJ, sendo mais prevalentes no tipo de início oligoarticular do que no sistêmico (p<0,05). Uma associaçäo com a duraçäo, atividade ou a gravidade da doença näo foi evidenciada (p>0,05), embora uma tendência tenha sido observada em pacientes do tipo de início poliarticular, em atividade de doença (P=0,054). O padräo pontilhado fino foi o mais freqëntemente observado (64 por cento), conquanto outros padröes, como o centrossômico e o do corpo intermediário, também tivessem sido notados. Conclusäo: Anticorpos antinucleares foram encontrados em menor freqüência que aquela descrita na maioria dos trabalhos da literatura para a ARJ, nÝo apresentando associaçäo com o sexo, duraçäo, atividade ou gravidade de doença. Embora o padräo de fluorescência mais freqüente tenha sido o pontilhado, a presença de padröes inusitados de fluorescência nuclear sugere que AAN na ARJ possam apresentar especificidades ainda näo definidas, que poderäo futuramente ser melhor caracterizadas

C2 deficiency in blood donors and lupus patients: prevalence, clinical characteristics and HLA-associations in the Brazilian population.

The objective of the present study was to investigate the prevalence, clinical characteristics, and HLA association of C2 deficiency in the Brazilian population. The frequency of C2 deficiency profile (C2Q degree profile) was 2.2% among 1503 blood donors and 6.6% among 166 patients with systemic lupus erythematosus (SLE). A higher incidence of clinical manifestations possibly related to immune complex disease was observed among blood donors with C2Q degree profile and their relatives with C2Q degree profile when compared to the normal C2 relatives. The comparison of clinical and laboratory features between SLE patients with C2Q degree profile and those with normal C2 revealed earlier disease onset, higher frequency of oral ulcerations and lower frequency of anti-native DNA antibodies in the first group. The HLA study conducted on 18 individuals with C2Q degree profile (11 blood donors and 7 SLE patients) confirmed the previously reported association with the antigens HLA-A25, B18 and DR2, supporting the concept that probably most C2 deficiency cases, throughout the world, are due to a single mutation in the C2 gene in linkage disequilibrium with the A25B18DR2 haplotype.

Development of an enzyme linked immunosorbent assay (ELISA) for detection of anti-native DNA antibodies using DNA from "Crithidia luciliae" as substrate

DNA de cinetoplasto de Crithidia luciliae (kDNA) foi isolado e purificado para desenvolvimento de ensaio imunoenzimático para detecçao de anticorpos anti-DNA nativo. O kDNA foi obtido por lise das células com dodecil sulfato de sódio (SDS) e pronase E e purificado por ultracentrifugaçao sobre soluçao de sacarose a 20 por cento e extraçao protéica. A pureza do kDNA foi verificada pela razao de densidades ópticas a 260 e 280nm e por eletroforese em gel de agarose, após digestao com as enzimas de restriçao HindIII e HaeIII. DNA de timo de vitelo, DNA plasmidial bacteriano e DNA cromossômico de Micrococcus lysodeikticus foram também empregados como substrato para ELISA. Foram testados 158 soros de pacientes atendidos no ambulatório da Disciplina de Reumatologia da Escola Paulista de Medicina, UNIFESP, sendo 65 com lúpus eritematoso sistêmico (LES), 30 com artrite reumatóide, 27 com esclerose sistêmica e 36 com outras doenças reumáticas auto-imunes. Foram também testados 105 soros obtidos de pacientes que estavam sendo investigados quanto a apresentarem LES e 30 soros de controles sadios. Nossos resultados mostraram que o kDNA pode ser purificado e utilizado com sucesso como substrato alternativo em ELISA. Entretanto, o mesmo nao ocorre com DNA cromossômico obtido de M. lysodeikticus, mostrando a necessidade de avaliaçao cuidadosa da fonte de DNA para detecçao de anticorpos anti-DNA, em testes imunoenzimáticos

Padronizaçäo da técnica de imunofixaçäo na detecçäo e identificaçäo de paraproteínas séricas / Standartization of immunofixation technique for detection and identification of seric paraproteins

O diagnóstico e seguimento das paraproteinemias requer a identificaçäo e tipagem de paraproteínas (PP). A imunoeletroforese (IEF) é o método comumente usado embora demorado e pouco sensível. A técnica de imunofixaçäo (IF) é superior por ser mais sensível, rápida e de fácil interpretaçäo, particularmente no reconhecimento de PP presentes em baixa concentraçäo no soro e/ou urina. Consiste de fase eletroforética, seguida de fixaçäo, quando o anti-soro é colocado sobre o gel, precipitando a proteína. Objetivo. Este estudo objetiva padronizar a técnica de IF e compará-la à IEF. Métodos. Foram estudados os soros de 28 pacientes, sendo 25 portadores de mieloma múltiplo e 3 com hipergamaglobulinemia policlonal, comparados com 6 indivíduos normais. Todos foram submetidos à eletroforese (EF) em gel de agarose, à IEF e à IF. Resultados. O principal problema na padronizaçäo da IF foi a determinaçäo da diluiçäo que estabelecesse proporçäo ideal entre antígeno e anticorpo. A concentraçäo sérica ideal da PP, neste estudo, variou de 28 a 35 g/dL. A PP foi detectada e caracterizada por ambas as técnicas em 21 (84 por cento) dos indivíduos e näo detectada por nenhuma delas em 2 (8 por cento). Em outros 2, somente a IF conseguiu identificar a PP. Näo houve banda monoclonal à EF e à IEF que näo fosse identificada pela IF. Conclusäo. Nossos resultados permitem concluir que a IF é mais sensível que a IEF e deve ser incorporada à rotina de diagnóstico

[Standardization of immunofixation technique for the detection and identification of serum paraproteins]. / Padronização da técnica de imunofixação na detecção e identificação de paraproteínas séricas.

Diagnosis and follow up of paraproteinaemias require identification and typing of paraproteins. Immunoelectrophoresis is the most commonly used method, though a lengthy one and with low sensitivity. Immunofixation is more sensitive, faster and of easier interpretation, specially when monoclonal proteins are present in low concentration in the serum and/or urine. Immunofixation includes two steps. The first is electrophoresis; the second is immunofixation of the separated antigen by use of antiserum. The latter step is accomplished by layering the antiserum over the agarose gel immediately after electrophoretic separation of the proteins resulting in antigen/antibody precipitation. PURPOSE--The objective of this study is to standardize the technique of immunofixation and compare it to immunoelectrophoresis. METHOD--The serum of 28 patients (25 with multiple myeloma and 3 with polyclonal hypergammaglobulinaemia) was analysed and compared to 6 normal subjects. All were submitted to electrophoresis on agarose gel, immunoelectrophoresis and immunofixation. RESULTS--Dilution of the serum to produce a concentration suitable for immunofixation is critical. In our study the correct paraprotein concentration was 28 to 35 g/dl. Both methods detected and identified the paraprotein in 21 (84%) of the samples and in 2 (8%) it was not detected at all. In two of the samples, only immunofixation was able to detect and identify the paraprotein. There was not any monoclonal band observed either through the electrophoresis or immunoelectrophoresis that was not detected by the immunofixation. CONCLUSION--These results show that immunofixation is more sensitive than immunoelectrophoresis and therefore should be incorporated into diagnosis routine.

[Serum antigliadin antibodies in the diagnosis and follow-up of celiac disease]. / Anticorpo sérico antigliadina no diagnóstico e seguimento da doença celíaca.

Between July 1985 and June 1990, we prospectively investigated 236 children suspected of having malabsorption syndrome. Each patient had a xylose absorption test and small intestinal biopsy. Blood samples were collected to AGA assay. The aim of the study was to evaluate the use of antigliadin antibodies test, IgG and IgA, in screening celiac disease for intestinal biopsy and in the monitoring of gluten-free diet and challenge in celiac patients. Twenty patients were diagnosed with celiac disease confirmed by three small intestinal biopsies; 12 patients were suspected of having celiac disease, with two biopsies, before and one year after a gluten-free diet; 106 patients had environmental enteropathy; 45 patients had protracted diarrhea and 56 children had failure to thrive with no gastrointestinal symptoms. The AGA test was considered a reliable test in screening for biopsy and in the differential diagnosis between celiac disease and other causes of malabsorption syndrome. The IgG AGA test had high sensitivity (90.4%) and the IgA AGA test had high specificity (92.1%) in screening for celiac disease. In the follow-up of the celiac patients the antibody levels were significantly higher during gluten containing diet than after gluten avoidance being thus a reliable test to evaluate dietary compliance.

Critical appraisal of the pattern of antinuclear antibodies (ANA) testing requested over a six month period at the "Escola Paulista de Medicina" (EPM), São Paulo.

In order to evaluate the pattern of ANA testing solicitation, 506 patients with ANA testing requested from July 1st. 1988 to December 31st, 1988, had their charts reviewed. These patients, randomly selected, were regularly attending the outpatient clinic at the "Escola Paulista de Medicina" (EPM). 289 patients were followed up at the Rheumatology Division (group A) and 217 patients at other clinical divisions at EPM (group B). The diseases that most frequently motivated the request for ANA test were: group A--SLE (22.5%), RA (18.0%), undefined arthropathies (6.2%), PSS and CREST (5.9%) and Raynaud phenomena (5.5%); and group B--rheumatic diseases (24.4%), nephropathies (17.1%), neuropathies (16.6%), dermopathies (7.8%), hemopathies (4.6%), pneumopathies (4.2%) and ophthalmopathies (3.7%). The positivity of ANA test in groups A and B was 32.9% and 17.5% respectively. 94 SLE patients were clinically diagnosed. The positivity of ANA and anti-dsDNA tests in this group was respectively 85.1% and 26.6%. The sensitivity and specificity of 1982 ARA revised criteria were 94.7% and 99% respectively. The likelihood ratio (LR) of a positive or a negative test was established for this population. LR of a positive test was 6.5 while for a negative test it was 0.17. The ANA test, although lacking specificity, has been commonly requested by different specialties in order to practically rule-out the diagnosis of some connective rheumatic diseases. Immunofluorescence technique (IF) using antibodies conjugated with fluorochromes. was first described by Coons et al. in 1941. This method has been used as an important diagnostic tool in routine laboratory tests ever since.(ABSTRACT TRUNCATED AT 250 WORDS)

Cartilage-hair hypoplasia syndrome: immunological evaluation of two cases.

PURPOSE: Immunological evaluation of patients with cartilage-hair hypoplasia. TYPE: Prospective and retrospective studies. PLACE: Division of Allergy, Clinical Immunology and Rheumatology-Dept. of Pediatrics-"Escola Paulista de Medicina". PATIENTS: Two children with cartilage-hair hypoplasia syndrome. METHODS: Clinical and immunological evaluation. Humoral immunity (immunoglobulin levels, poliovirus antibodies, etc.) and T cell immunity (in vitro cultured lymphocytes stimulated with PHA, Con A and PWN, total T cell and subset determination) were studied. RESULTS: Cellular immunodeficiency and hypogammaglobulinemia were observed in one patient and normal values in the other. CONCLUSIONS: Immunological evaluation (cellular and humoral) should be performed in all patients with cartilage-hair hypoplasia.

Hereditary angioedema type II--a study of two families.

Hereditary angioedema (HA) is caused by a quantitative or qualitative deficiency of C1 esterase inhibitor (C1 INH). We present a study of nine patients with HA belonging to two different families. The symptoms started before 10 years of age in most cases (78%). Facial edema (lips, eyes) and of the extremities (feet, hands) were the most frequent complaints. Three patients presented edema of the glottis and one of them underwent a tracheostomy twice. Laboratory tests, outside the acute crisis, revealed low levels of C4 in all patients. The serum levels of C1 INH were normal in seven patients; however, functional activity was not observed in any of them. After the use of a modified androgen (danazol), control of symptoms was observed in all patients, although functional activity was re-established in only five patients.

Common variable immunodeficiency: a clinical and laboratory evaluation of 15 cases.

The clinical and laboratory data for 15 patients with common variable immunodeficiency (CVI) (5 females and 10 males aged 3 years and 6 months to 40 years at first examination) were evaluated. The age of onset of infectious signs and symptoms ranged from 6 months to 35 years. Recurrent pulmonary infections predominated (86.6%), followed by chronic diarrhea (46.6%). Approximately 60% of the patients with pulmonary complaints presented chronic sequelae (bronchiectasis). Two developed a polymyositis-like picture. No neoplasms were observed. All patients presented immunoglobulin levels below 300 mg/dl and absence of antibody responses to poliovirus and to hemagglutinin. Two patients were negative when tested for autoimmunity. Cell immunity tested by the lymphoproliferative response in the presence of phytohemagglutinin was normal in 11 patients and depressed in 4. A decrease in the helper T population and inversion of the OKT4/8 ratio occurred in 13. Cimetidine treatment (1200 mg/day) applied to 5 patients for 4 weeks did not produce any clinical or laboratory improvement. Gamma globulin is the treatment of choice for these patients.

Magnetic resonance imaging in a patient with X-linked agammaglobulinemia and chronic meningoencephalitis.

The case of a boy with congenital agammaglobulinemia is reported. In spite of regular immunoglobulin replacement therapy (fresh plasma transfusion from family donors--20 ml/kg/month), he developed chronic meningoencephalitis (ME). Besides characteristic clinical signs of ME, he also presented at cerebrospinal fluid analysis pleocytosis with lymphocyte predominance and class II cytomorphology, and delta and theta waves in the EEG. Computerized tomography showed dilatation of the ventricles and marked cortical fissures (sulci). Magnetic resonance imaging showed a disease affecting white and gray matter. After diagnosis of ME, replacement therapy with Sandoglobulin (700 mg/kg every 2 weeks) was started. His condition gradually worsened, and coma and death occurred after a follow-up of 18 months. The etiological agent could not be identified.

Avidez de anticorpos IgG especificos como marcadores de infeccao primaria recente pelo Toxoplasma gondii / Avidity of especific IgG antibodies as markers of recent primary infection caused by Toxoplasma gondii

A caracterizacao de infeccao primaria recente pelo Toxoplasma gondii se apoia principalmente na presenca, no soro, de anticorpos especificos IgM. Para fins diagnosticos de toxoplasmose aguda, ou de contagio recente, a possibilidade de outros marcadores e altamente desejavel. Um marcador de infeccao recente atualmente referido e a baixa afinidade ou avidez de anticorpos especificos IgG. Para avaliacao do novo marcador, titularam-se os soros contra poliantigenos do T.gondii pelo teste imunoenzimatico (ELISA), antes e apos tratamento dos complexos antigeno-anticorpo formados, com solucao de ureia 6 M como agente dissociante. O deslocamento de anticorpos de baixa avidez foi indicado por uma queda de titulos, calculada em porcentagem em relacao aos titulos iniciais. Foram estudados 69 soros, 23 de cada um dos 3 perfis sorologicos sucessivos, observados na infeccao, e que a caracterizam respectivamente como recente, em fase de transicao e cronica. Os perfis foram determinados segundo os resultados de uma bateria de testes, incluindo os de imunofluorescencia IgG e IgM, de captura de anticorpos IgM e de hemaglutinacao. Para os soros de infeccao cronica a queda observada foi de 3 por cento ñ 3 por cento, de 34 por cento ñ 12 por cento para toxoplasmose recente e de 12 por cento ñ 9 por cento para a fase de transicao...

[Avidity of specific IgG antibodies as markers of recent primary infection caused by Toxoplasma gondii]. / Avidez de anticorpos IgG específicos como marcadores de infecção primária recente pelo Toxoplasma Gondii.

For serologically characterizing a recent primary toxoplasma infection, the low avidity of IgG specific antibodies was studied. Avidity was evaluated as the decrease of IgG antibody titers in ELISA after treating plates with 6 M urea, as a dissociating solution of low avidity antigen-antibody complexes. Sixty nine serum samples were studied, presenting characteristic patterns of recent, transitional or chronic toxoplasmosis. Serological patterns were determined according to results of IgG and IgM immunofluorescence, IgM-capture, and hemagglutination tests. Twenty three serum samples from each of the referred patterns I, II and III were titrated. For chronic toxoplasmosis infections, which presented a serological pattern III, observed decrease of titers was 3% +/- 3%. For pattern I recent toxoplasmosis sera it was 34% +/- 12%, and for transition pattern II, 12% +/- 9%. Thus, a low avidity of IgG specific antibodies can be applicable for the diagnosis of a recent toxoplasmosis infection.

A simple radial immunohemolysis assay for the measurement of functional complement C2 activity / O ensaio simples de imunohemolise radial para medir da atividade funcional do complemento 2

This study describes a simple radial immunohemolysis method for determining the hemolytic activity of the second component of complement (C2) in human serum. The assay is based on the recovery of hemolytic activity of normal serum which has been pretreated to anactivate endogenous C2 and thenmixed with test serum containing an unknown amount of C2. The pretreated serum, designated R2 reagent, is obtained by heating normal human sera under carefully standardized conditions of temperature, time, volume and type of test tube. R2 reagent is incorporated into agarose together with hemolysin-sensitized erythrocytes, and spread om a plate. The test serum is placed in wells cut in the agarose and, after appropriate incubation, the diameters of the hemolytic areas are measuremed.The area of hemolysis is directly proportional to the logarithm of the serum concentration.As a standard for C2 functional activity, dilutions of a pool of normal sera are tested on the same plate. The method is specific for C2 and can deted as little as 20% of the C2 in normal serum (abouth 6 *g C2 protein/ml). The error in reproducibility is about 3% of the mean.in normal serum, the lower confidence limit of the distribution of the C2 values (based on a sample of 80 individuals) corresponded to 70 % of undiluited serum. This method is sultable for use in clinical laboratories since it is simple, rapid quantitative ans inexpensive, and does not require special equipement

A simple radial immunohemolysis assay for the measurement of functional complement C2 activity.

1. This study describes a simple radial immunohemolysis method for determining the hemolytic activity of the second component of complement (C2) in human serum. The assay is based on the recovery of hemolytic activity of normal serum which has been pretreated to inactivate endogenous C2 and then mixed with test serum containing an unknown amount of C2. 2. The pretreated serum, designated R2 reagent, is obtained by heating normal human sera under carefully standardized conditions of temperature, time, volume and type of test tube. 3. R2 reagent is incorporated into agarose together with hemolysin-sensitized erythrocytes, and spread on a plate. The test serum is placed in wells cut in the agarose and, after appropriate incubation, the diameters of the hemolytic areas are measured. The area of hemolysis is directly proportional to the logarithm of the serum concentration. As a standard for C2 functional activity, dilutions of a pool of normal sera are tested on the same plate. 4. The method is specific for C2 and can detect as little as 20% of the C2 in normal serum (about 6 micrograms C2 protein/ml). The error in reproducibility is about 3% of the mean. In normal serum, the lower confidence limit of the distribution of the C2 values (based on a sample of 80 individuals) corresponded to 70% of undiluted serum. 5. This method is suitable for use in clinical laboratories since it is simple, rapid, quantitative and inexpensive, and does not require special equipment.