Incidence of tuberculous and non-tuberculous mycobacteria, differentiated by multiplex PCR, in clinical specimens of a large general hospital

OBJECTIVE: To determine the incidence of Mycobacterium tuberculosis complex and non-tuberculous mycobacterial isolates in the routine setting of a large general hospital using an "in-house" multiplex polymerase chain reaction method and to establish a paradigm for the definitive identification of mycobacteria isolated using semi-automated equipment. METHODS: Established tests, including polymerase chain reaction restriction enzyme analysis, PNB, and NAP inhibition tests as the gold standard, showed 100% agreement with an IS6110/hsp65 multiplex polymerase chain reaction when used to identify stock strains (n = 117). RESULTS: In a subsequent study, 8,790 clinical specimens producing 476 isolates were evaluated with multiplex PCR and also showed 100% agreement in identification using PRA-polymerase chain reaction as the gold standard. The application of this technique to routine analysis was demonstrated in this study. A method was established with the initial application of multiplex PCR for all positive liquid cultures and the subsequent identification of non-tuberculous mycobacteria by polymerase chain reaction restriction enzyme analysis. In total, 77% of isolates belonged to the Mycobacterium tuberculosis complex, and 23% were non-tuberculous mycobacteria. CONCLUSIONS: Several non-tuberculous mycobacterial species were identified, primarily M. avium, but other potentially pathogenic species were also frequently observed, including M. fortuitum, M. abscessus, and M. kansasii. The expeditious communication of these data to the clinical staff was fundamental for the diagnosis of clinical cases. Even in settings where tuberculosis is of major importance, the incidence of non-tuberculous mycobacteria infection is substantial

Comparação imunoquimica dos anti-soros produzidos em coelhos a partir do extrato de cerdas e da hemolinfa de lagartas da mariposa

Orientador: Stephen HysloDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O contato com lagartas de mariposas do gênero Lonomia pode resultar em envenenamento grave, com distúrbios da coagulação e hemorragia. Em casos fatais, têm sido associado à falência renal e hemorragia intracerebral. Atualmente a soroterapia com o anti-soro eqüino produzido contra o extrato de cerdas da Lonomia oblíqua é o tratamento mais eficaz para o envenenamento por esta espécie. Neste trabalho, foram produzidos anti-soros em coelhos contra o extrato de cerdas e a hemolinfa e examinou-se sua imunorreatividade usando as técnicas de ELISA ("enzyme-linked immunosorbent assay"), immunoblot e imunoeletroforese. No ELISA, ambos os anti-soros reagiram igualmente com o extrato de cerdas e com a hemolinfa. Esta imunorreatividade era igual a do antisoro comercial. Os três anti-soros também mostraram reatividade semelhante no imunoblot. O fracionamento, por gel filtração, do extrato de cerdas e da hemolinfa, demonstrou que possuem perfis de eluição qualitativa muito semelhantes e que as imunorreatividades das frações com os três anti-soros eram muito parececidas. Todos os anti-soros neutralizaram a atividade coagulante do extrato de cerdas em plasma humano citratado. Estes resultados indicam que o extrato de cerdas da L. oblíqua e hemolinfa compartilham antígenos comuns e que o anti-soro produzido em coelhos e em cavalos reagem igualmente com as amostras biológicas. Os resultados também sugerem que a hemolinfa poderá servir como fonte de antígenos na produção de anti-soro comercialAbstract: Caterpillars of saturnid moths belonging to the genus Lonomia can cause severe and sometimes fatal envenoming, the main symptoms of which are coagulation disturbances, hemorrhaging, renal failure and intracranial hemorrhaging. The administration of antiserum raised against a spicule extract from Lonomia oblíqua is the only effective treatment for systemic envenomations. In this work, we raised antisera in rabbits against a spicule extract and hemolymph and examined their cross-reactivity by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunoelectrophoresis. In ELISA, the rabbit antisera and the commercial antiserum showed similar dilution curves and cross-reactivities with the spicule extract and hemolymph. Considerable similarity was also seen in the immunoblot patterns with ali three antisera. The immunoelectrophoretic profiles were also alike. Fractionation of the spicule extract and hemolymph by gel filtration yielded qualitatively similar elution profiles which cross-reacted extensively with the three antisera. Ali antisera neutralized the coagulant activity of the spicule extract in human citrated plasma. These results indicate that L. oblíqua spicule extracts and hemolymph share many antigens and that antisera raised in rabbits and horses show similar cross-reactivities with these biological samplesMestradoMestre em Farmacologi

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