Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate

The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.

Demographic and anthropometrical analysis and genotype distribution of chronic hepatitis C patients treated in public and private reference centers in Brazil

Hepatitis C virus (HCV) infection is a serious public health problem, since 80 percent to 85 percent of HCV carriers develop a persistent infection that can progress into liver cirrhosis and hepatocarcinoma. Considering that the response of hepatitis C patients to combination therapy with interferon and ribavirin depends on HCV characteristics as well as on host features, we made a retrospective analysis of demographic and anthropometrical data and HCV genotype distribution of chronic hepatitis C patients treated in public and private reference centers in Brazil. The medical records of 4,996 patients were reviewed, 81 percent from public and 19 percent from private institutions. Patients' median age was 46 years, and there was a higher prevalence of male (62 percent) and white patients (80 percent). The analysis of HCV-infecting strains showed a predominance of genotype 1 (64 percent) over genotypes 2 and 3. The patients' mean weight was 70.6 kg, and 65 percent of the patients weighed less than 77kg. Overweight and obesity were observed in 37.8 percent and 13.6 percent of the patients, respectively. Since a body weight of 75 kg or less has been considered an independent factor that significantly increases the odds of achieving a sustained virological response, the Brazilian population seems to have a more favorable body weight profile to achieve a sustained response than the American and European populations. The finding that 65 percent of chronic hepatitis C patients have a body weight of 77 kg or less may have a positive pharmacoeconomic impact on the treatment of genotype 1 HCV patients with weight-based doses of peginterferon.

Interferon-alpha receptor 1 mRNA expression in peripheral blood mononuclear cells is associated with response to interferon-alpha therapy of patients with chronic hepatitis C

Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/á-actin-mRNA ratio (mean ñ SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 ñ 0.15; N = 5; P < 0.05) compared to non-responders (0.35 ñ 0.17; N = 12) and controls (0.30 ñ 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

In vitro-induced antibody production in chronic hepatitis C virus infection

The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV) antibody production (IVIAP) in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60 percent males) with HCV infection (anti-HCV and HCV-RNA positive). Alanine aminotransferase (ALT) was elevated in 89 percent of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52 percent) and cirrhosis in 11/52 (21 percent) patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14 percent of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540), with 71 percent sensitivity and 94 percent specificity for the detection of more advanced disease stages (grades 3 and 4). These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott(r)), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95 percent) sera from patients in the study group and negative in 38 of the 39 (97 percent) sera from those in the control group, showing an accuracy of 96 percent. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Alteraçoes morfológicas da glândula supra-renal na síndrome da imunodeficiência adquirida / Adrenal gland morphological alterations in the acquired immunodeficiency syndrome

Na síndrome da imunodeficiência adquirida (AIDS) pode-se verificar o acometimento da supra-renal por efeito citopático direto pelo HIV, por infecções oportunistas ou neoplasias. Estes achados poderiam variar de acordo com a procedência do paciente, devido às doenças peculiares à região. Neste trabalho avaliou-se o comprometimento da supra-renal em quatorze pacientes que morreram de AIDS no Hospital Escola, em Uberaba. Treze eram do sexo masculino e treze brancos. A idade foi de 29,9 ± 7,8 anos e o índice de massa corporal foi de 19 ± 4,1kg/m2. Os fragmentos de supra-renal obtidos nas necropsias foram analisados em microscópio de luz. Encontramos inflamação em 100% dos casos, identificando-se o agente etiológico em oito (58,1%) casos. O Citomegalovírus foi identificado em sete casos, o Cryptococcus sp e o Herpes simplex em dois e o Histoplasma sp em um caso, estes achados são semelhantes aos da literatura. Em um caso, encontramos calcificação do parênquima e em outro, flebite da veia central. Em alguns casos que apresentavam lesão não foi possível identificar o agente etiológico, talvez em decorrência do efeito citopático direto pelo HIV ou devido a toxicidade das drogas utilizadas no tratamento da AIDS e das infecções oportunistas.

In the acquired immunodeficiency syndrome (AIDS), the adrenal glands are subject to opportunistic infections, neoplasm or direct cytopathic effect by HIV. It is know that the incidence and type of adrenal involvement vary according to the patient's place of origin. In this paper we evaluate adrenal involvement in fourteen patients that died from AIDS in the University Hospital of Uberaba, Brazil. The group studied was comprised of thirteen males and thirteen whites. The age was 29.9 +/- 7.8 years, and the body mass index was 19.0 +/- 4.1 kg/m2. Adrenal specimens obtained from autopsies were analyzed by light microscopy. Inflammation was found in 100% of the cases and the etiologic agent(s) was (were) identified in eight (58.1%) patients. Cytomegalovirus was identified in seven cases, Cryptococcus sp and Herpes simplex in two and Histoplasma sp in one case, these pathologic findings were similar to literature. We also found parenchymal calcification and adrenal central vein phlebitis in one case each. Injury was found in some cases without identified infections agent. This fact could be due to the direct cytopathic effect by HIV, or due to toxicity of drug therapy used during treatment of AIDS and opportunistic infections.

Significance of the presence of antibodies against hepatitis C virus in asymptomatic blood donors

In order to determine the significance of anti-hepatitis C virus (anti-HCV) antibodies in blood donors, 46 consecutive asymptomatic individuals were recruited at the blood bank of Hospital Sao Paulo, Sao Paulo, Brazil. They were submitted to an interview to collect epidemiological data and to clinical examination and blood samples were obtained for biochemical, serological and virological analysis. All patients were followed for a minimum period of six months and those with abnormal mean alanine aminotransferase (ALT) levels were submited to a liver biopsy after giving informed consent. Hepatitis C virus RNA (HCVRNA) was detected by the polymerase chain reaction (PCR) in 22/46 (47.8 per cent) patients and this finding was associated with parenteral risk factors (P = 0.03) and ethanol abuse (P - 0.03). HCVRNA positivity was also associated with abnormal levels of ALT (P<0.001) and gamma-glutamyl transpeptidase (gamma-GT) (P=0.01). Abnormal ALT levels were a good marker of viremia, with 86.4 per cent sensitivity and 79.2 per cent specificity. Twenty-three patients with elevated mean ALT levels were submitted to a liver biopsy and histopathological changes were observed in 17 of them (73.9 per cent). HCVRNA positivity was associated with severe forms of hepatic disease (chronic hepatitis and cirrhosis). These results indicate the need for a judicious evaluation of all anti-HCV-positive blood donors, including clinical examination, biochemical tests and liver histology when ALT is persistently elevated.

Alfafetoproteína nos tumores de fígado e hepatopatias benignas / Alphafetoprotein in hepatic tumours and benign liver diseases

A alfafetoproteína (AFP) é uma proteína oncofetal que se eleva nä só no carcinoma hepatocelualr, mas também em metástases hepáticas e hepatopatias benignas. Objetivo. O conhecimento das alteraçöes de AFP, nestas doenças, facilitará a interpretaçäo dos resultados frente à suspeita de carcinoma hepatocelular. Casuística e Métodos. Os autores estudaram AFP sérica por enzima imunoensaio (Abbott, normal até 15ng/mL) em 4 grupos: 1) hepatite aguda (HA), n=24;2) hepatopatia crônio (HC), viral ou alcoólica, n = 81; 3) metástase hepática (MH), n = 29: e 4) carcinoma hepatocelular (CHC), n = 15. Resultados Observaram os seguintes resultados em percentuais de AFP menor que 15ng/mL nos 4 grupos: 75 por cento HA; 86,4 por cento HC; 79,3 por cento MH; 6,6 por cento CHC. Entre 15 e 100: HA 25 por cento; HC 8,6; MH 20,6 por cento e CHC 20 por cento. Acima de 100ng/mL: 73,3 por cento CHC e 4,9 por cento HC. Discussäo. A medida que aumentaram o limite de corte, diminuiu a sensibilidade e aumentaram a especificidade diagnóstica para hepatocarcinoma. Os resultados mostraram que a AFP se eleva em hepatopatias benignas (HA e HC) e em MH, porém, níveis acima de 100ng/mL ocorrem com muito maior freqüência no CHC apresentaram AFP elevada, possivelmente porque a doença apresentava-se em estádios avançados

Hepatite pelo vírus A em adulto: ocorrência de formas prolongadas e polifásicas da doença / Hepatitis A in adults: occurrence of prolonged and polyphasic forms of the disease

Os autores estudaram a ocorrência de formas prolongadas e/ou polifásicas da infecçao, em 84 pacientes adultos com diagnóstico de hepatite aguda pelo vírus A. Foram prolongada de doença (duraçao superior a quatro meses) foi observada em 8/84 (9,5 por cento) dos pacientes. Padrao polifásico foi observado em 12/84 (14,3 por cento) dos casos. Observou-se ainda que indivíduos com mais de 18 anos tiveram tendência a demorar mais tempo para normalizar as transaminases, quando comparados com pacientes entre 12 e 18 anos, o mesmo ocorrendo com homens, quando comparados às mulheres. Entretanto, em todos os casos observou-se normalizaçao do quadro, em períodos que chegaram a ser tao longos quanto nove a dez meses de evoluçao.

Phyllanthus amarus no tratamento da infecçäo crônica pelo vírus da hepatite B: crença ou realidade? / Phyllantus amarus in the treatment of chronic infection caused by the hepatitis B virus: belief or reality?

Estudos recentes têm demostrado eficácia do extrato de um vegetal, o Phyllanthus amarus, no tratamento dos portadores crônicos do vírus da hepatite B. Por tratar-se de substância facilmente encontrada em nosso meio e pela ausência de publicaçöes sobre seu uso para esta virose, na literatura nacional, revisäo do assunto foi realizada. Entretanto, os bons resultados referidos nao têm sido repetidos por alguns pesquisadores e, até o momento, a análise dos dados disponíveis revela controvérsias. Falhas metodológicas nesses protocolos, seja com respeito aos critérios de inclusäo, seja na discussäo dos resultados, poderiam explicar a situaçäo do problema. Conclui-se que novos estudos deveräo ser conduzidos, até que se possa estabelecer o real papel do Phyllanthus amarus no tratamento dos portadores crônicos do vírus de hepatite B.

Surto de hepatite aguda pelo vírus A: avaliaçäo epidemiológica, clínica e laboratorial / Outbreak of acute hepatitis by virus A: epidemiologic, clinical and laboratory evaluation

Os autores analisam as características epidemiológicas, clínicas e sorológicas de um surto de hepatite aguda ocorrido na cidade de Valparaiso, SP. O agente etiológico foi identificado como sendo o vírus A da hepatite e a doença incidiu sobretudo em crianças, com cerca de 80% dos casos abaixo de 10 anos de idade. Foram analisadas amostras de soro de 37 comunicante dos casos índices, sendo constatada evidência de infecçäo aguda pelo vírus A em 9/37 (24,3%) dos comunicantes, que se apresentavam totalmente assintomáticos, evidenciando a alta incidência de formas anictéricas e assintomáticas da doença

Estudo histoquímico do músculo esquelético no alcoolismo crônico / Histochemical study of the skeletal muscle in chronic alcoholism

Vinte e dois pacientes alcoólatras crônicos foram submetidos a exame clínico neurológico e biópsia muscular. Eles apresentavam graus variáveis de fraqueza muscular proximal (cinturas escapular e pélvica), tendo um deles evoluído com quadro agudo de miopatia (avaliaçäo pelo 'Manual Muscle Test', MMT). A principal alteraçäo histológica observada é melhor evidenciada pela coloraçäo da ATPase: atrofia muscular (95,3%), predominando nas fibras do tipo II A (71,4%) e, em 76% dos casos, alteraçäo da imagem em mosaico à custa de agrupamentos de fibras musculares de mesmo tipo histoquímico ('type-grouping'). Secundariamente, em 63% dos casos, observa-se proliferaçäo mitocondrial e conseqüente acúmulo lipídico intra-sarcoplasmático. No caso de instalaçäo aguda da fraqueza muscular, o substrato anátomo-patológico é completamente diferente: presença de miosite, predominantemente intersticial, caracterizada por infiltrado linfoplasmocitário e numerosas imagens de necrose tipo degeneraçäo cérea de Zencker. Baseando-se em critérios histológicos, nossos dados sugerem que: a principal gênese da fraqueza muscular observada em pacientes alcoólatras crônicos tem natureza neurogência (polineuropatia alcoólica); a atuaçäo tóxica direta do etanol sobre o músculo esquelético está intimamente relacionada ao metabolismo mitocondrial; a chamada miopatia aguda alcoólica tenha etiologia inflamatória, do tipo viral

Hepatite tóxica causada por ketoconazol: relato de quatro casos / Ketoconazole causing toxic hepatitis: report of 4 cases

Säo descritos quatro casos de hepatite tóxica por ketoconazol. Todas eram mulheres e tomavam a droga de oito a 16 semanas (200mg/dia). O quadro clínico, em todas, foi superponível ao de uma hepatite viral. A normalizaçäo clínico-laboratorial ocorreu entre 60 e 90 dias após a suspensäo da droga, näo tendo havido óbito. Em uma das pacientes, o ketoconazol foi reintroduzido após normalizaçäo clínico-laboratorial, tendo ocorrido nova hepatite tóxica após 15 dias; a segunda suspensäo da droga foi seguida de cura da hepatite

Production of anti-HBsAg monoclonal antibodies

The production of monnoclonal antibodies against the HBsAg is reported. Balb/c mice immunized against a commercial vaccine were used. Upon fusion of spleen cells from an animal having a high titer with the SP2/0 myeloma cell line, we obtained 6 stable cell lines, all of the IgG1 subclass. They showed a wide range of specificities against the classical HBsAg subtypes. These monoclonal antibodies can be used as the basis for the development of new methods for the screening and study of the hepatitis B virus

Marcadores sorológicos do virus da hepatite B e anticorpo delta em homossexuais masculinos brasileiros / Serological markers for hepatitis B virus and delta antibody in Brazilian male homosexuals

Os marcadores sorológicos do vírus B da hepatite B foram estudados em um grupo de homens homossexuais em Säo Paulo, Brasil. Encontraram-se 23% de positividade para o AgHBs nos 26 homossexuais, sendo que 81% apresentavam positividade para alguns dos marcadores do vírus B. Anticorpo anti-o näo foi detectado em nenhum caso. No grupo controle, encontrou-se 1% de positividade para o AgHBs entre 500 homens estudados

Niveis sericos de acido urico em alcoolatras. / Serum uric acid levels in alcoholics

Os autores estudaram 283 alcoolatras e 100 individuos de um grupo controle.Observaram elevacao dos niveis sericos de acido urico em 91 alcoolatras (32,1%), dos quais 62 estavam em abstencao etilica ha 7 ou mais dias (grupo I) e 29 ingeriram bebidas alcoolicas nos 7 dias que precederam a coleta de sangue (grupo II) para o estudo da uricemia. A media dos valores da uricemia, no grupo I, foi de 6,48 +/- 0,27 e no grupo II, 7,55 +/- 0,68, diferenca estatisticamente significante.Ressaltam a importancia do etanol como causa de elevacao serica de acido urico e comentam a respeito de seu mecanismo