Genotipagem utilizando a sequencia de inserção IS6110 e "spoligotyping" de Mycobacterium tuberculosis isolados de pacientes infectados pelo HIV, em Moçambique, Africa

Orientador: Marcelo de Carvalho RamosTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: O M. Avium é um microrganismo oportunista e sua infecção é feeqüentemente encontrada em pacientes com aids no Brasil, apesar do largo uso da quimioterapia antiretroviral altamente efetiva. Este estudo documenta a relevância desse problema. Dentro de uni número significante de pacientes (n=39) infectados com o M. avium, os isolados puderam ser recuperados de uma variedade de espécimes clínicos. Todos os isolados (n=45) foram tipados pela técnica de RFLP usando a seqüência 1S1245. A maioria dos pacientes (n=35) eram infectados pelo HIV. Somente duas cepas não puderam ser tipadas por causa da ausência da seqüência detectável pela 1S1245. Nas 43 cepas restantes os "blots" apresentaram de 6 a 23 bandas. Uma média de 17 seqüências foram observadas para cada cepa. Para alguns pacientes, mais de um isolado pode ser recuperado. Em dois pacientes deste grupo com doença disseminada, o M. avium pode ser recuperado mais de uma vez. De cada paciente, pelo menos duas amostras com diferentes genótipos foram recuperadas de locais estéreis, indicando que eles tinham infecções policlonais. Esses achados têm sido relatados por outros autores. Em um estudo recente, SAAD et aI., 2000, demonstrou que isolados de infecções policlonais e diferentes "fmgerprints" podem apresentar diferentes suscetibilidade antimicrobiano. Quatro "clusters" de pacientes puderam ser identificados. O maior "cluster" foi composto de oito pacientes. Estes resultados indicam que um mecanismo de transmissão recente ocorreu. A fonte de contaminação desses microrganismos não pode ser determinada. Assim, a transmissão pessoa a pessoa não apresentou uma importância significativa na transmissão desse microrganismo. Nós supomos que esses pacientes adquiriram o microrganismo de fontes hospitalares como água, alimento ou mesmo do ambienteAbstract: not informed.DoutoradoCiencias BasicasDoutor em Clínica Médic

Analise do polimorfismo genetico de cepas de Mycobacterium avium isoladas de pacientes atendidos no hospital de clinicas da Unicamp, utilizando a sequencia de inserção IS1245

Orientadores : Maria Cecilia Barisson Villares, Marcelo de Carvalho RamosDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A tuberculose é a principal causa de morte no mundo entre as doenças infecciosas, produzindo 2 milhões de óbitos ao ano. Estima-se que um terço da população mundial esteja infectada pelo Mycobacterium tuberculosis, ou seja, 1,7 bilhões de pessoas. Em países africanos, o problema da tuberculose e da aids adquiriu proporções de grande magnitude. Em Moçambique, o coeficiente de incidência é de 460 casos/100.000 habitantes/ano, com mortalidade de 129 casos/100.000 habitantes/ano. Este estudo documenta a relevância desse problema. Duzentos e trinta e sete isolados clínicos de Mycobacterium tuberculosis foram caracterizados por IS6110-RFLP e ¿Spoligotyping¿, com o objetivo de avaliar a biodiversidade do bacilo da tuberculose na cidade de Maputo, Moçambique, África, onde há uma elevada incidência de tuberculose. Os resultados obtidos com o uso da técnica IS6110-RFLP exibiram uma considerável variabilidade genotípica, ou seja, cerca de 70,5% dos exemplares analisados corresponderam a genótipos únicos. A interpretação desse achado deveria ser feita juntamente com o estudo epidemiológico clássico, bem como a ampliação do número de casos analisados. Porém, a grande variabilidade genotípica nesse cenário em que a transmissão da doença é intensa, dado o número elevado de casos, indica, provavelmente, a situação endêmica em si, bem como o tempo prolongado em que isso se dá. Além disso, a grande mobilidade interna, ocorrida depois das intensas guerras civis, trazendo para a cidade indivíduos de diversos outros locais, pode também ter contribuído para essa diversidade. Encontramos também uma alta freqüência de cepas com baixo número de cópias (20,2% da IS6110). Todos os isolados foram também submetidos ao ¿Spoligotyping¿. Essa técnica foi mais discriminatória para os isolados com menos de 6 cópias da IS6110 do que o RFLP e menos discriminatório para os isolados com mais de 6 cópias da IS6110. As famílias mais freqüentemente encontradas no conjunto das cepas foram: LAM, EAI, T e Beijing, distribuídas entre os 35 agrupamentos, com 193 isolados. Em ambos os agrupamentos, tanto os derivados da técnica ¿IS6110-RFLP¿, quanto o ¿Spoligotyping¿, não houve nenhuma correlação com a resistência à isoniazida, e mesmo com os isolados multirresistentesAbstract: Tuberculosis is the leading cause of death among infectious diseases in the world, with approximately two million deaths each year. Among Sub-Saharan African countries, tuberculosis and AIDS represents an enormous problem. In Mozambique, the incidence of tuberculosis is as high as 460 cases/100,000 inhabitants/year, with a mortality rate of 129 people/100,000 inhabitants/year. The present study re-enforces the importance of this problem. A sample of 237 clinical strains of Mycobacterium tuberculosis isolated from HIV-infected patients, living in the city of Maputo, Mozambique, was genotipically characterized by IS6110-RFLP and Spoligotyping. IS6110-RFLP showed a considerable variability (70.5%) corresponding only to genotypes. Limitations in sampling, and scarce epidemiological data, may account for lack of definition of risk factors for TB transmission in this study. A high frequency of strains exhibiting low IS6110 copy number (n=20,2%) was observed. These strains were further genotyped by Spoligotyping. The most frequently found families, characterized with the use of this technique were: LAM, EAI, T and Beijing. These isolates (n=193) could be grouped in 35 clusters. Spoligotyping was more discriminatory for isolates exhibiting less than six IS6110 copies than RFLP. No correlation was found between resistance to INH, or even multidrug-resistance and clusteringMestradoCiencias BiomedicasMestre em Ciências Médica

Peptidase Activities In Rats Treated Chronically With N(omega)-nitro-l-arginine Methyl Ester (l-name).

The chronic treatment of rats with N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) biosynthesis, results in hypertension. This inhibition of NO production results in activation of the renin-angiotensin system, with increased activity of the carboxypeptidase angiotensin I-converting enzyme (ACE). Since chronic NO inhibition increases ACE activity, we hypothesized that this inhibition could also affect the activities of other peptidases involved in cardiovascular functions. To test this possibility, we examined the activities of aminopeptidase M (APM), dipeptidyl peptidase IV (DPP IV), metalloendopeptidase 24.15 (MEP 24.15) and neutral endopeptidase 24.11 (NEP 24.11) in rat brain, heart, kidney, liver, lung and thoracic aorta. Male Wistar rats were treated chronically with L-NAME (80mgkg(-1) per day) administered in the drinking water for 4 weeks and their organs then removed and processed for the determination of peptidase activities. Treatment with L-NAME did not significantly alter the activities of the four peptidases in brain, heart, kidney, liver and lung. In contrast, in aorta, the activity of APM was slightly but significantly reduced whereas those of DPP IV and MEP 24.15 were markedly enhanced; NEP 24.11 was not detected in this tissue. Immunoblotting for DPP IV and MEP 24.15 showed increased expression in aortic tissue. Neither L-NAME (1-100microM) nor the NO donors sodium nitroprusside and 3-morpholinosydnonimine (SIN-1; 1-100microM) had any consistent effect on the activity of recombinant MEP 24.15 or renal DPP IV. The importance of MEP 24.15 in peptide metabolism was confirmed in pentobartibal-anesthetized rats pretreated with the MEP 24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA2), which significantly potentiated the hypotensive response to bradykinin. The altered peptidase activities seen in aorta may contribute to modulating vascular responses in this model of hypertension.68205-1

Infection Of Polypropylene Threads For Face-lifting With Mycobacterium Fortuitum.

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Genetic diversity of the Mycobacterium tuberculosis Beijing family in Brazil and Mozambique and relation with infectivity and induction of necrosis in THP-1 cells

Introduction: The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility. Objective: To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17). Methods: This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells. Results: The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group. Conclusions: These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions951S190S196CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQsem informaçã

Genetic Diversity Of The Mycobacterium Tuberculosis Beijing Family In Brazil And Mozambique And Relation With Infectivity And Induction Of Necrosis In Thp-1 Cells.

The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility. To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17). This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells. The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group. These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions.95 Suppl 1S190-19

Doença pulmonar por Mycobacterium tuberculosis e micobactérias não-tuberculosas entre pacientes recém-diagnosticados como HIV positivos em Moçambique, África Mycobacterium tuberculosis and nontuberculous mycobacterial isolates among patients with recent HIV infection in Mozambique

OBJETIVO: A micobacteriose é frequentemente diagnosticada entre pacientes infectados pelo HIV. Em Moçambique, onde apenas um pequeno número de pacientes encontra-se sob tratamento anti-retroviral, e a tuberculose tem alta prevalência, existe a necessidade de melhor caracterização destes agentes bacterianos, em nível de espécie, bem como de se caracterizar os padrões de resistência às drogas antituberculosas. MÉTODOS: Em uma coorte de 503 indivíduos HIV positivos suspeitos de tuberculose pulmonar, 320 apresentaram positividade para baciloscopia ou cultura no escarro e no lavado brônquico. RESULTADOS: Bacilos álcool-ácido resistentes foram detectados no escarro em 73% dos casos com cultura positiva. De 277 isolados em cultura, apenas 3 mostraram-se tratar de micobactérias não-tuberculosas: 2 Mycobacterium avium e uma M. simiae. Todos os isolados de M. tuberculosis inicialmente caracterizados através de reação em cadeia de polimerase (RCP) do gene hsp65 foram posteriormente caracterizados como tal através de RCP do gene gyrB. Resistência à isoniazida foi encontrada em 14% dos casos; à rifampicina em 6%; e multirresistência em 5%. Pacientes previamente tratados para tuberculose mostraram tendência a taxas maiores de resistência às drogas de primeira linha. O padrão radiológico mais freqüente encontrado foi o infiltrado intersticial (67%), seguido da presença de linfonodos mediastinais (30%), bronquiectasias (28%), padrão miliar (18%) e cavidades (12%). Os pacientes infectados por micobactérias não-tuberculosas não apresentaram manifestações clínicas distintas das apresentadas pelos outros pacientes. A mediana de linfócitos CD4 entre todos os pacientes foi de 134 células/mm³. CONCLUSÕES: Tuberculose e AIDS em Moçambique estão fortemente associadas, como é de se esperar em países com alta prevalência de tuberculose. Embora as taxas de resistência a drogas sejam altas, o esquema isoniazida-rifampicina continua sendo a escolha apropriada para o início do tratamento.<br>OBJECTIVE: Mycobacteriosis is frequently diagnosed among HIV-infected patients. In Mozambique, where few patients are under antiretroviral therapy and the prevalence of tuberculosis is high, there is need for better characterization of mycobacteria at the species level, as well as for the identification of patterns of resistance to antituberculous drugs. METHODS: We studied a sample of 503 HIV-infected individuals suspected of having pulmonary tuberculosis. Of those 503, 320 tested positive for mycobacteria through sputum smear microscopy or culture of bronchoalveolar lavage fluid. RESULTS: Acid-fast bacilli were observed in the sputum of 73% of the individuals presenting positive cultures. Of 277 isolates tested, only 3 were nontuberculous mycobacteria: 2 were identified as Mycobacterium avium and one was identified as M. simiae. Strains initially characterized as M. tuberculosis complex through polymerase chain reaction restriction analysis (PRA) of the hsp65 gene were later confirmed as such through PRA of the gyrB gene. Among the M. tuberculosis isolates, resistance patterns were as follows: to isoniazid, 14%; to rifampin, 6%; and multidrug resistance, 5%. Previously treated cases showed significantly higher rates of resistance to first-line antituberculous drugs. The most common radiological pattern was interstitial infiltrate (in 67%), followed by mediastinal lymph node enlargement (in 30%), bronchiectasis (in 28%), miliary nodules (in 18%) and cavitation (in 12%). Patients infected with nontuberculous mycobacteria presented clinical profiles indistinguishable from those of other patients. The median CD4 lymphocyte count in this group was 134 cells/mm³. CONCLUSIONS: There is a strong association between tuberculosis and AIDS in Mozambique, as expected in a country with a high prevalence of tuberculosis. Although drug resistance rates are high, the isoniazid-rifampin regimen continues to be the appropriate choice for initial therapy