Crohn's disease environmental factors in the developing world: A case-control study in a statewide catchment area in Brazil.

AIM: To identify environmental risk factors associated with the development of Crohn's disease (CD) in order to re-assess the hygiene hypothesis. METHODS: A hospital-based, case-control study was carried out with CD patients (n = 145) and controls (n = 163) representing a socioeconomically diverse statewide catchment area in Brazil. Controls were recruited from caregivers of patients seen in different outpatient clinics at the same hospital. A multi-item survey with 94 questions regarding family history of CD, perinatal and childhood circumstances, living conditions, tobacco use and familial socioeconomic status was carried out by interviewers. RESULTS: On the univariate analysis, predictive variables for CD included being male, under age of 40, a high education level, urban dweller, smaller family size, exposure to enteric pathogens and user of treated water (P < 0.005). On the multivariate analysis, variables significantly associated with CD were male gender (OR = 2.09), under age 40 (OR = 3.10), white (OR = 2.32), from a small family in childhood (OR = 2.34) and adulthood (OR = 3.02), absence of viral infections in childhood (OR = 2.23), exposure to enteric pathogens (OR = 2.41), having had an appendectomy (OR = 2.47) and prior or current smoker (OR = 2.83/1.12). CONCLUSION: Most variables supporting the "hygiene hypothesis" are associated with the development of CD but are not independent predictors of the diagnosis.

A systematic review of East African-Indian family of Mycobacterium tuberculosis in Brazil

ABSTRACT Introduction: The Mycobacterium tuberculosis East African-Indian (EAI) spoligotyping family (belonging to lineage 1, Indo-Oceanic, defined by the region of deletion RD239) is distributed worldwide, but is more prevalent in Southeast Asia, India, and East Africa. Studies in Latin America have rarely identified EAI. In this study, we describe the occurrence of the EAI family in Brazil. Methods: EAI was identified in a systematic literature review of genetic diversity studies pertaining to M. tuberculosis in Brazil, as well as in a survey conducted in Salvador, Bahia, located in the northeastern region of this country. Results: The EAI6-BGD1 spoligotyping family and the EAI5 Spoligotype International Type (SIT) 1983 clade were the most frequently reported, with wide distribution of this particular clade described in Brazil. The distribution of other EAI spoligotyping patterns with broader worldwide distribution was restricted to the southeastern region of the country. Conclusions: EAI may be endemic at a low frequency in Brazil, with some clades indicating increased fitness with respect to this population.

Mycobacterium tuberculosis-induced neutrophil ectosomes decrease macrophage activation.

BACKGROUND: The existence of ectosome-like microvesicles released by neutrophils was proposed a few decades ago. Other studies revealed that the innate immune response during mycobacterial infection is accompanied by an intense migration of neutrophils to the site of infection, which may be important during the acute phase of tuberculosis. We found that the ectosomes derived from infected neutrophils are biologically active and can influence the survival of Mycobacterium tuberculosis within macrophages. METHODS: Mycobacteria were cultured on supplemented Middlebrook-7H9 broth. All strains were grown to the exponential phase and quantitated by serial dilution. Human neutrophils and macrophages were infected with mycobacteria. Ectosomes from neutrophils were isolated post-infection and characterized by transmission electron microscopy and flow cytometry. To determine whether these microvesicles influenced mycobactericidal activity, mycobacteria-infected macrophages were treated with isolated ectosomes. RESULTS: Ectosomes were released from neutrophils infected with mycobacteria. These ectosomes were derived from neutrophil plasma membrane and a small proportion stained with PKH26. These microvesicles, when incubated with infected macrophages, influenced antimycobacterial activity. CONCLUSIONS: This is the first study to demonstrate that ectosomes that are shed from infected neutrophils influence mycobactericidal activity in macrophages in vitro, suggesting that these microvesicles have biological significance. Nevertheless, major gaps in our knowledge of microvesicle biology remain.

Identificação de Mycobacterium bovis em cepas micobacterianas isoladas espécimes clínicos humanos em um complexo hospitalar na cidade do Rio de Janeiro / Identification of Mycobacterium bovis among mycobacterial isolates from human clinical specimens at a university hospital in Rio de Janeiro, Brazil

Entre 2005 e 2006, 8.121 espécimes clínicos enviados ao Laboratório de Micobactérias do Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax, no Rio de Janeiro, RJ, foram inoculados em meio Löwenstein-Jensen contendo glicerol e piruvato. Desses espécimes, 79 isolados de micobactérias tiveram crescimento somente em meio com piruvato, sendo selecionados para a identificação presuntiva de Mycobacterium bovis. Esses isolados foram submetidos à identificação por testes bioquímicos, amplificação por PCR com primers específicos (Rv0577 e Rv1510) e teste de suscetibilidade à pirazinamida. Todas as cepas apresentaram padrões fenotípicos e genotípicos de M. tuberculosis, não sendo detectado M. bovis.

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Löwenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

Genotyping of Mycobacterium leprae from Brazilian leprosy patients suggests the occurrence of reinfection or of bacterial population shift during disease relapse.

We performed genotyping of Mycobacterium leprae present in skin biopsy samples that were collected during the first and the second disease occurrences from eight leprosy patients, seven of whom were diagnosed as suffering from disease relapse. Sequence analysis of part of the M. leprae rpoB, folP1, gyrB and gyrA genes did not show genetic change that supported the presence of drug-resistant bacilli. However, we observed a synonymous nucleotide change at position 297 of gyrA among five of these patients, one presenting C to T (CgyrAT) and four presenting T to C (TgyrAC) at this position. Additional genotyping by analysis of the four short tandem repeats GAA, GTA9, AT17 and TA18 showed that the gyrA single nucleotide polymorphism change was accompanied by a change in short tandem repeat genotype. Our data suggest that leprosy relapse in these patients, living in an area endemic for leprosy, could be caused by M. leprae with a genotype different from the one that caused initial disease.

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques

Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and ethionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100 percent, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis.

O ressurgimento da tuberculose e o impacto do estudo da imunopatogenia pulmonar / The resurgence of tuberculosis and the impact of the study of pulmonary immunopathogenesis

O ressurgimento da tuberculose como uma das doenças contagiosas que mais assola a humanidade deu-se após uma falsa impressão de que se caminhava para o seu controle antes do final do Século XX. Nos últimos dez anos, em associação com centros de pesquisas norte-americanos e europeus, nosso grupo na Universidade Federal do Rio de Janeiro tem estudado diversos aspectos relacionados com a patogenia da forma pulmonar, a mais importante por conta de sua freqüência e importância que tem no ciclo de transmissão. Nossa hipótese é que o estabelecimento da infecção latente e o desenvolvimento da forma ativa dependem de um desequilíbrio entre citocinas ativadoras e desativadoras da função microbicida dos macrófagos. A despeito da presença de mecanismos habitualmente protetores, como de moléculas nos macrófagos que denotam ativação celular e de moléculas comprometidas com a proteção contra a tuberculose, como o óxido nítrico e o interferon-g, a tuberculose progride. Um dos motivos é a presença no sítio de infecção de moléculas como a interleucina-10 e o TGF-b, que tem capacidade de desativar macrófagos previamente ativados. Existem evidências que a micobactéria secreta proteínas capazes de induzir a expressão de interleucina-10, agindo assim para burlar os mecanismos de defesa. Indivíduos suscetíveis teriam mais capacidade de responder a estas moléculas da micobactéria, devido a mutações genéticas que facilitam a produção de interleucina-10. A compreensão destes mecanismos poderá representar avanços na prevenção e descoberta de novos alvos terapêuticos para o controle da tuberculose.

Interferons de tipo I (type-I IFNs) e infecção micobacteriana / Interferon type I and mycobaterial infection

Introdução: após a infecção por Mycobacterium tuberculosis, a grande maioria dos adultos controlam a proliferação micobacteriana intracelular. Diversos fatores (idade, estress, denutrição, alcoolismo, infecções intercorrentes e câncer) têm sido correlacionados com a reativação da tuberculose. Durante as infecções virais, o IFN-α/β é produzido em grande quantidade. Métodos: estudo do efeito da estimulação direta dos macrófagos humanos pelo IFN-α sobre a multiplicação micobacteriana intracelular in vitro. Resultados: o pré-tratamento dos monócitos/macrófagos humanos com o IFN-α, aumentou de modo significativo o crescimento micobacteriano intracelular. Conclusão: nossos resultados são compatíveis com a idéia de que o aumento da produção do IFN-α, poderia contribuir para a realização da tuberculose latente nos pacientes portadores de infecções virais agudas ou crônicas.

Introduction: following infection with Mycobacterium tuberculosis, most adults are able to control mycobacterial proliferation. In a minority of individuals, a variety of factors (aging, stress, malnutrition, alcoholism, intercurrent infections, and cancer), can subsequently blunt this acquired resistance, resulting in the development of overt disease. IFN-α/β are copiously produced during viral infections. Methods: we evaluated the effect of INF-α/β on mycobacterial growth in human macrophages in vitro. Results: after pretreatment of human monocytes with IFN-α striking loss of mycobacteriostatic activity of these cells was observed. Conclusion: these results are compatible with the possibility that the secretion of IFN-/β could directly promote mycobacterial growth in patients harboring these organisms.

Disruption of the gene homologous to mammalian Nramp1 in Mycobacterium tuberculosis does not affect virulence in mice.

Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterial Nramp homolog may therefore be involved in Mycobacterium tuberculosis virulence. Here, we investigated this possibility by inactivating the M. tuberculosis Nramp1 gene (Mramp) by allelic exchange mutagenesis. Disruption of Mramp did not affect the extracellular growth of bacteria under standard conditions. However, the Mramp mutation was associated with growth impairment under conditions of limited iron availability. The Mramp mutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or in Nramp1(+/+) and Nramp1(-/-) congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental and Mramp mutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate that Mramp does not contribute to the virulence of M. tuberculosis in mice.

Alpha/beta interferon impairs the ability of human macrophages to control growth of Mycobacterium bovis BCG.

Administration of alpha/beta interferon (IFN-alpha/beta) to mice infected with Mycobacterium tuberculosis has been shown to increase mycobacterial growth. Because IFN-alpha/beta has direct pleiotropic effects on the differentiation and functional activities of macrophages, we evaluated the effect of IFN-alpha/beta on mycobacterial growth in human monocytes/macrophages in vitro. Monocytes cultured at optimal cell density could control the growth of M. bovis BCG, as assessed both by measurement of luciferase activity expressed by a mycobacterial reporter strain and by counting of CFU. In contrast, unrestrained mycobacterial growth was observed when monocytes were treated with alpha interferon (IFN-alpha) 3 days prior to or concomitant with infection. This striking loss of mycobacteriostatic activity was observed with IFN-alpha and IFN-beta and was induced in both freshly isolated monocytes and culture-derived macrophages. Pretreatment of monocytes with IFN-alpha modified cellular morphology and reduced viability following culture, but neither was observed for culture-derived macrophages, indicating that the effects of IFN-alpha on mycobacteriostatic activity and cell differentiation and death could be dissociated. These results are compatible with the possibility that the secretion of IFN-alpha/beta could directly promote mycobacterial growth in patients harboring these organisms.

Efeitos adversos hepáticos durante o tratamento para tuberculose em hospital geral referência para Aids, Rj, Brasil / Hepatic adverse efects anti-tuberculosis tratment in a geral hospital, Rio de Janeiro, Brasil

Introdução: a frequência de efeitos adversos hepáticos e os fatores associados com a sua ocorrência em um hospital universitário referência para Aids e tuberculose não é completamente conhecida. Métodos: foi realizado um estudo tipo caso-controle com o objetivo de medir prevalência de efeitos hepáticos adversos (EAH) em pacientes sob tratamento medicamentoso anti-tuberculose (TB) e de fatores associados à sua ocorrência. Resultados: foram analisados 588 prontuários médicos de pacientes que fizeram uso de esquema anti-TB com isoniazida, rifampicina e pirazinamida, acrescido ou não de etambutol, atendidos no período de Janeiro de 1994 a dezembro de 1995. EAH foi observado em 40 (6,8%) casos. Foram pareados 200 casos para o grupo controle. Na análise univariada dos grupos caso e controle não houve diferença estatisticamente significativa entre a ocorrência de EAH e os seguintes parâmetros: a idade, gênero esquema inicial de tratamento anti-TB, a história prévia de hepatopatia e/ou presença de alcoolismo. Entretanto, a ocorrência de EAH esteve significamente associada a hospitalização no momento do diagnóstico da TB, a presença de síndrome de imunodeficiência adquirida (SIDA/AIDS), a forma disseminada da TB, a apresentação radiográfica atípica da TB pulmonar, a sorologia positiva para hepatite a vírus B e/ou C, e a evolução clínica desfavorável ou tratamento. Na análise multivariada, somente a hospitalização e o diagnóstico de SIDA permaneceram associados significantemente a EAH. Conclusões: em um hospital Universitário, referência para AIDS e TB, a presença de Aids, de imagem radiológica de TB pulmonar atípica e a TB disseminada estão associados a uma maior taxa de EAH.

Introduction: the frequency of hepatic adverse effects and factors associated with its occurrence in an Universitary Hospital, reference for AIDS and tuberculosis (TB) is not completely known. Methods: a case-control study was conducted to assess the prevalence of hepatic adverse effects of patients using anti-TB treatment and the factors associated with its occurrence. Results: 588 medical charts of TB patients receiving anti-TB treatment with isoniazid, ripampin, pirazinamide, with or without ethambutol attendent betwee January, 1994 and December, 1995 were analyzed. HAE was observed in 40 (6.8%) patients. Two-hundred patients were included as control group. Using univariate analysis to evaluate case and control groups no statistically difference was found between HAE and the following variables: age, gender, anti-TB treatment, liver disease in the past and/or alcohol abuse. However, HAE was significantly associated with hospitalization at the time that TB diagnosis was made, immunosupression, disseminated TB, radiographic atypical presentation of pulmonary TB, seropositivy for B and C hepatitis and clinical unfavorable evolution. In multivariate analysis only hospitalization and Aids were associated to HAE. Conclusion: in a University Hospital, reference for TB and AIDS, the presence of Aids, radiographic atypical presentation of pulmonary TB and disseminated form of TB were associated with higher prevalence of HAE.

Phenotypes of lung mononuclear phagocytes in HIV seronegative tuberculosis patients: evidence for new recruitment and cell activation

Mycobacterium tuberculosis preferentially resides in mononuclear phagocytes. The mechanisms by which mononuclear phagocytes keep M. tuberculosis in check or by which the microbe evades control to cause disease remain poorly understood. As an initial effort to delineate these mechanisms, we examined by immunostaining the phenotype of mononuclear phagocytes obtained from lungs of patients with active tuberculosis. From August 1994 to March 1995, consecutive patients who had an abnormal chest X-ray, no demonstrable acid-fast bacilli in sputum specimens and required a diagnostic bronchoalveolar lavage (BAL) were enrolled. Of the 39 patients enrolled, 21 had microbiologically diagnosed tuberculosis. Thirteen of the 21 tuberculosis patients were either HIV seronegative (n = 12) or had no risk factor for HIV and constituted the tuberculosis group. For comparison, M. tuberculosis negative patients who had BAL samples taken during this time (n = 9) or normal healthy volunteers (n = 3) served as control group. Compared to the control group, the tuberculosis group had significantly higher proportion of cells expressing markers of young monocytes (UCHM1) and RFD7, a marker for phagocytic cells, and increased expression of HLA-DR, a marker of cell activation. In addition, tuberculosis group had significantly higher proportion of cells expressing dendritic cell marker (RFD1) and epithelioid cell marker (RFD9). These data suggest that despite recruitment of monocytes probably from the peripheral blood and local cell activation, host defense of the resident lung cells is insufficient to control M. tuberculosis.

Perfil de saúde respiratória de diferentes grupos profissionais em uma amostra de funcionários da UFRJ / Profile respiratory health of different professional groups in a sample of employees from UFRJ

A aplicação de um questionário padronizado para sintomas e antecedentes respiratórios a 100 funcionários da Universidade Federal do Rio de Janeiro, de ambos os sexos e de diversos grupos etários, resultou na demostração de um ou mais sintomas de 36%, sendo a tosse em 18%, expectoração em 21%, chiado em 25% e dispnéia em 5%. Essa frequência, com exceção da dispnéia, é maior que a encontrada no exterior, e semelhante à observada em estudos de outras regiões do país. Com exceção do chiado, ouve correlação com o tabagismo e a presença de antecedentes cardiorespiratórios. Só o chiado pareceu ter relação com o tipo de ocupação

Colesterol e outros fatores de risco cardiovasculares nos servidores da UFRJ. Prevalência e influência de variáveis sociais / Cholesterol and other cardiovascular risk factors in the employees of the Universidade Federal do Rio de Janeiro. Prevalence and influence of social variables

Objetivo: Investigar a presença de fatores de risco cardiovascular em funcionários da Universidade Federal do Rio de Janeiro. Casuística e Métodos: Duzentos e cinqüenta indivíduos foram selecionados randomicamente de uma populaçäo de 4.651 homens na faixa de 35 a 64 anos. Foram convocados por carta para consulta na qual um questionário visando informaçöes quanto ao grau de escolaridade, nível de renda, ocupaçäo, tabagismo e tratamento anti-hipetensivo foi aplicado. Pressäo arterial, altura e peso corporal foram medidos por pessoal previamente treinado. Foi realizada dosagem de colesterol sérico total e de sua fraçäo HDL. Resultados: Duzentos e nove indivíduos (83,6%) atenderam a convocaçäo. Näo houve diferença significativa entre presentes e faltosos em relaçäo a idade, nível de escolaridade e ocupaçäo. As médias e desvio padräo do colesterol sérico, HDL-colesterol, pressäo arterial sistólica (PAS) e diastólica (PAD) foram respectivamente: 210 ñ 21,9 mg/dl; 41,1 ñ 12,4 mg/dl; 129,7 ñ 24,0 e 80,4 ñ 17,7. A prevalência de hipertensäo arterial, hipercolesterolemia e tabagismo foi de 30, 22% e 38%. A prevalência da associaçäo destes três fatores de risco foi de 4%. Apenas a PAS e a PAD foram estatisticamente diferentes quando comparadas por estratos de renda e escolaridade. Houve forte associaçäo positiva do índice de massa corporal (peso/altura2) com o colesterol sérico e com os níveis de pressäo arterial. Conclusäo: A despeito do alto nível de escolaridade e renda, para padröes brasileiros, na amostra estudada, a prevalência de fatores de risco cardiovascular foi bastante alta, sendo mais importante nos indivíduos acima do peso ideal e nos socialmente menos favorecidos. Indicadores mais precisos dos hábitos alimentares e de status sócio-econômico-cultural säo necessários para o planejamento de açöes de saúde. O controle da obesidade parece ser uma meta desejável no controle paralelo dos fatores de risco a ela associados

Purpose: To investigate the presence of cardiovascular risk factors in employees of the Universidade Federal do Rio de Janeiro. Patients and Methods: A survey of 250 men age ranging from 35 to 64 years, randomly selected among 4651 employees of URFJ, was done. They were invited by letter to a consultation at which previously trained personnel applied a questionnaire to investigate about their education level, income, occupation, smoking habit, and use of anti-hypertensive drugs. They also had their blood pressure, height and weight measured and underwent a blood sample collection for serum cholesterol and HDL-cholesterol analysis. This procedure followed a standardized protocol. Results: Two hundred and nine subjects (83.6%) were examined. The characteristics of age, education, and ocupation were similar for responders and nonresponders. The mean (± standard deviation) for total cholesterol, HDL-cholesterol, systolic and diastolic blood pressure were respectively: 210 ± 21.9 mp/dl; 41.1 ± 12.4 mg/dl; 129.7 ± 24.0 and 80.4 17.7. The prevalence of hypertension, hypercholesterolemia and smoking were: 30%, 22% and 38%. The prevalence of these risk factors together was 4%. Only SBP and DBP were statistically different when analyzed by income or education strata. There was a strong positive correlation between body mass index and serum cholesterol and blood pressure levels. Conclusion: In spite of the high education level and income of this sample for brazilian standards, the prevalence of risk factors for cardiovascular diseases is very high, especially for the overweight and the socially less favoured. More precise indicators of dietary habits and socio-economic and cultural status are necessary to develop adequate interventional strategies. The control of obesity seems to be a desirable goal in achieving the concurrent control of associated risk factors.