Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients.

OBJECTIVE: To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. METHODS: Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCR amplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. RESULTS: Active or latent TB was found in 65 patients, TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was 26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. CONCLUSIONS: Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specific technique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.

Desempenho da técnica nested PCR na detecção específica do complexo Mycobacterium tuberculosis em amostras sanguíneas de pacientes pediátricos / Performance of nested PCR in the specific detection of Mycobacterium tuberculosis complex in blood samples of pediatric patients

Objetivo: Avaliar o desempenho da técnica nested PCR (nPCR) para detectar o complexo Mycobacterium tuberculosis em amostras de sangue de pacientes com suspeita de TB para sua possível utilização como uma ferramenta auxiliar no diagnóstico laboratorial da doença em crianças. Métodos: Detecção do complexo M. tuberculosis em amostras de sangue usando como alvo a sequência de inserção IS6110 do DNA genômico do bacilo. Foram avaliados 120 pacientes, menores de 15 anos de idade, de ambos os sexos, provenientes de hospitais públicos do Recife (PE), no período entre janeiro de 2003 e agosto de 2005. O diagnóstico de TB foi realizado pelo médico assistente do serviço de saúde de acordo com os critérios da Sociedade Torácica Americana. A nPCR amplificou um fragmento de 123 pb com oligonucleotídeos externos (IS1/IS2) e, na reação subsequente, com oligonucleotídeos internos (IS3/IS4), gerando um amplicon de 81 pb. Resultados: A TB ativa ou latente esteve presente em 65 pacientes, foi descartada em 28 suspeitos e 27 não tinham a doença (controles). A sensibilidade da nPCR foi de 26,15%, sendo significativamente maior na forma extrapulmonar (55,56%) em relação à pulmonar (18,18%), e a especificidade foi de 92,73%. Conclusões: Diante das dificuldades diagnósticas da TB infantil e do baixo número de casos estudados,a nPCR em sangue demonstrou ser uma técnica rápida e específica, mas com baixa sensibilidade. Para saber a suareal utilidade no diagnóstico de formas paucibacilares, sobretudo as extrapulmonares, novas pesquisas devem ser desenvolvidas com uma casuística maior de crianças e com outros espécimes biológicos além do sangue.

Objective: To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. Methods: Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCRamplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. Results: Active or latent TB was found in 65 patients,TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. Conclusions: Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specifictechnique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.

Cytokine profile associated with chronic and acute human schistosomiasis mansoni.

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma (IFN-gamma) was evaluated in 43 schistosomiasis patients with different clinical forms. Whole-blood cultures cytokine production in response to soluble egg antigen (SEA), soluble worm adult preparation (SWAP), mitogens, neutralizing antibodies or recombinant IL-13 were measured by ELISA. After SWAP stimulation, chronic patients, particularly hepatointestinals, produced higher levels of IL-4 in comparison with acute patients, suggesting the presence of a type 2 cytokine profile in these patients. Following SEA and SWAP stimulation, hepatosplenic (HS) patients showed increased levels of IFN-gamma when compared with acute patients, indicating that HS disease in humans is associated with a type 1 cytokine response. The mechanisms of immune regulation are apparently different between the clinical stages of the disease, some of which are antigen-specific.

Cytokine profile associated with chronic and acute human schistosomiasis mansoni

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma (IFN-³) was evaluated in 43 schistosomiasis patients with different clinical forms. Whole-blood cultures cytokine production in response to soluble egg antigen (SEA), soluble worm adult preparation (SWAP), mitogens, neutralizing antibodies or recombinant IL-13 were measured by ELISA. After SWAP stimulation, chronic patients, particularly hepatointestinals, produced higher levels of IL-4 in comparison with acute patients, suggesting the presence of a type 2 cytokine profile in these patients. Following SEA and SWAP stimulation, hepatosplenic (HS) patients showed increased levels of IFN-³ when compared with acute patients, indicating that HS disease in humans is associated with a type 1 cytokine response. The mechanisms of immune regulation are apparently different between the clinical stages of the disease, some of which are antigen-specific.

Development of a multiplex single-tube nested PCR (MSTNPCR) assay for Vibrio cholerae O1 detection.

A multiplex nested PCR method for detection of Vibrio cholerae O1 using a single tube was developed (MSTNPCR). Firstly, single-tube nested PCR (STNPCR) with primers directed to ctxA gene was standardized, and its detection limit was compared to simple PCR and two-step nested PCR. Secondly, primers directed to rfbN gene were added to the reaction. The detection limit of the multiplex reaction was determined using V. cholerae O1 DNA and V. cholerae O1 grown in alkaline peptone water (APW). STNPCR was shown to be approximately 100-fold more sensitive than simple PCR and 10 times less sensitive than two-step nested PCR. This drawback is compensated by a lower risk of cross-contamination. The addition of a second target did not impair the detection limit of STNPCR (as little as 1 pg of V. cholerae O1 DNA detected). MSTNPCR could specifically detect up to three V. cholerae O1 cells or colony forming units (cfu) directly from the APW growth. A diagnostic kit consisting of a set of microtubes having the inner primers fixed onto the inside of the tube cap and a set of tubes containing the reaction mixture was evaluated for stability, and it proved to be stable for five months at -20 degrees C. Therefore, MSTNPCR would be useful in the detection of V. cholerae O1 directly from environmental waters in cholera endemic areas and in complementing the identification of toxigenic strains isolated by culture.

[Regulations, conflicts and ethics of medical research in developing countries]. / Regulamentações, conflitos e ética da pesquisa médica em países em desenvolvimento.

The patient is the subject who may meet the needs and interests of medical investigators for their research. This intrinsic conflict becomes more evident and shows particularities in the context of research projects involving clinical trials in developing countries. Target populations in these countries have inadequate access to health services, little understanding of risks involved in a study and also less capacity to claim judicially in the event of damage. In general, the discussions on ethics in research in industrialized countries are characterized by a biomedical approach of disease, and a neoliberal vision of economy and trade. In fact, most biomedical research has been directed to benefit already privileged communities. Therefore, it is necessary to minimize the risk of exploitation of developing countries populations. The present study provides an outlook of ethical protection for human research in developing countries.

Studies on the production and regulation of interleukin, IL-13, IL-4 and interferon-gamma in human Schistosomiasis mansoni.

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma was evaluated in different clinical forms of human schistosomiasis. The mechanisms of immune regulation are apparently different in the various clinical stages of the disease, some of them being antigen specific.

Studies on the production and regulation of interleukin, IL-13, IL-4 and interferon-gamma in human Schistosomiasis mansoni

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma was evaluated in different clinical forms of human schistosomiasis. The mechanisms of immune regulation are apparently different in the various clinical stages of the disease, some of them being antigen specific

Specific situations related to acute schistosomiasis in Pernambuco, Brazil

The present work reports on two epidemiological episodes resulting in acute schistosomiasis involving wealthy persons living in the State of Pernambuco, Brazil. The authors discuss the epidemiological, clinical and serologic characteristics of the acute infections and also the way in which the conditions for transmission occurred

Epidemia de esquistossomose aguda na praia de Porto Galinhas, Pernambuco, Brasil / An outbreak of acute schistosomiasis at Porto de Galinhas beach, Pernambuco, Brazil

Notifica a ocorrência de um surto de esquistossomose aguda na praia de Porto de Galinhas, Pernambuco, Brasil, quando 662 pessoas tiveram diagnóstico positivo para Schistosoma mansoni. A infecçäo humana em massa ocorreu no feriado de 7 de setembro, quando chuvas pesadas provocaram a enchente do rio Ipojuca que invadiu as residências. A maioria dos casos agudos foi representada por famílias de residentes locais que tiveram exposiçäo diária às cercárias durante três semanas, até que as águas baixassem. O inquérito malacológico aponta para a possibilidade dos caramujos vetores terem sido introduzidos na localidade com os aterros realizados em quintais, onde vieram a se reproduzir em criadouros peridomiciliares. A infecçäo dos moluscos se deu a partir de vazamentos de fossas. Os resultados da clínica e do laboratóro apontam para cerca de 62 por cento de casos agudos. Estudos complementares estäo sendo realizados, para a melhor compreensäo deste evento epidemiológico de significativa magnitude para os órgäos de saúde pública e impacto para a populaçäo afetada.

Doenças sexualmente transmissíveis em pacientes infectados com HIV/AIDS no Estado de Pernambuco, Brasil / Sexually transmitted diseases in patients infected with HIV/AIDS in the State of Pernambuco, Brazil

Foram estudados retrospectivamente 399 pacientes diagnosticados como infecçäo por HIV. Os pacientes infectados predominavam na faixa etária de 20 a 40 anos (73,4 por cento), sendo 75 por cento do sexo masculino. A razäo entre pacientes do sexo masculino e feminino näo diferiu quando foram consideradas infecçäo pelo HIV (assintomática) e AIDS. Os casos de infecçäo pelo HIV sem AIDS se concentram no grupo etário de 20-29 anos enquanto que a AIDS predomina na faixa de 30-39 anos. Dentre os pacientes infectados com HIV, apenas 0,8 por cento eram hemofílicos, 3,5 por cento usavam drogas injetáveis e 4,8 por cento foram hemotransfundidos nos últimos 5 anos. 33 por cento eram heterossexuais, 11 por cento bissexuais, 23 por cento homossexuais e 33 por cento näo declararam a inclinaçäo sexual. Sífilis foi a associaçäo mais freqüente (8,8 por cento), seguido de herpes (5,8 por cento) e candidíase genital (4,3 por cento). Nossos resultados sugerem que existe uma associaçäo entre candidíase genital e AIDS, embora isso näo tenha sido observado para as outras DSTs estudadas

Partial protection of mice against Trypanosoma cruzi after immunizing with the TcY 72 antigenic preparation

A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype) was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1) induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2) significant DTH after infection of epimastigotes in mice footpads: (3) peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection: (4) the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

A simple PCR-based procedure for plague diagnosis

Triturados de bacos de animais infectados experimentalmente com Y. pestis, suspensos em salina foram fervidos e os sobrenadantes usados diretamente para amplificacao do PCR sem previa extracao do DNA. O limiar de deteccao pode ser aumentado por uma segunda etapa de amplificacao (Nested-PCR). Nao houve infectados usados como controle

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page). Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted

Effects of betamethasone on the course of experimental infection with Trypanosoma cruzi

Foram estudados os efeitos da betametasona administrada na fase pós-aguda imediata de uma infecçäo pelo T. cruzi em camundongos. O tratamento consistiu de 30 doses diárias de 0,15 mg de betametasona, a partir de 42§ dia de infecçäo, näo havendo aparecimento de novos surtos de parasitemia. No tempo de duraçäo do experimento (7 meses) näo houve diferença entre as lesöes histopatológicas dos animais tratados e dos näo tratados. O grupo experimental apresentou uma maior mortalidade acumulada no 75§ dia de infecçäo, o que pode ser atribuído a infecçöes bacterianas associadas. Por outro lado, camundongos albinos "outbred", infectados com baixo inóculo, näo se apresentaram como bom modelo de doença de Chagas, já que näo desenvolveram lesöes importantes nem na fase aguda nem após 7 meses de infecçäo. Em conclusäo, o tratamento imunosupressivo prolongado, após a fase aguda de uma infecçäo mínima com a cepa Y do T. cruzi näo tem influência sobre o curso da infecçäo, pelo menos no que tange ao agravamento da mesma

Modificaçäo da técnica da bolsa de ar para mensuraçäo da quimiotaxia in vivo / Modification of the air pouch technique for evaluation of chemotaxis in vivo

Propöem algumas modificaçöes a um método de mensuraçäo da resposta quimiotáxica in vivo, que se baseava na observaçäo da resposta leucocitária à introduçäo de quimiotáxicos, em uma bolsa de ar produzida no tecido conjuntivo de camundongos. As modificaçöes introduzidas foram a utilizaçäo de anestesia leve, facilitando a produçäo da bolsa e a imobilizaçäo dos animais em posiçäo vertical, permitindo a localizaçäo da área exposta à atividade quimiotáxica e tornando a técnica mais simples. As citadas modificaçöes tornaram o método mais reprodutível e eficiente, permitindo o emprego de amostras menores no ensaio