Susceptibility to Mycobacterium tuberculosis infection in HIV-positive patients is associated with CARD8 genetic variant.

HLA and other genetic variants, playing an important role in innate and adaptive immunity, are known to influence tuberculosis (TB) development in HIV-1-positive (HIV+) patients. Because inflammasome genes contribute to HIV-1 susceptibility, we investigated the possible association between polymorphisms in inflammasome genes with HIV-1 and Mycobacterium tuberculosis coinfection (HIV+TB+) in a case/control cohort of Brazilian individuals. Nineteen single-nucleotide polymoprhims in 8 inflammasome genes (NLRP1, NLRP3, AIM2, CARD8, CASP1, IL1B, IL1R, and HSP90) were analyzed in HIV+TB+ Brazilian patients (from Recife, Pernambuco). CARD8 rs6509365 polymorphism was associated with HIV+TB+ (P = 5 × 10(-5)), suggesting a predisposing role of this variant in M. tuberculosis susceptibility in HIV+ subjects (odds ratio = 2.45). This effect is even strong when this allele is combined to CARD8 rs2043211 single-nucleotide polymoprhim. The results of this study support the novel association between CARD8 gene and HIV+TB+ coinfection, demonstrating that inflammasome genetics could influence HIV-1 infection and the development of opportunistic infection.

A infecção por Mycobacterium tuberculosis e micobactérias nãotuberculosas na infância: um desafio diagnóst / Childhood Mycobacterium tuberculosis and nontuberculous mycobacter: a challenge on the diagnosis

Objetivos: Avaliar a tuberculose e micobaterioses na infância por meio de uma revisão bibliográfica criteriosae crítica, com ênfase na epidemiologia, aspectos clínicos e ferramentas diagnósticas. Métodos: A pesquisa foirealizada nas bases de dados Lilacs, SciELO, PubMed, durante o período de 2000 a 2009. Quarenta e seis artigos foram considerados e onze diretrizes e manuais nacionais e internacionais. Os descritores utilizados foram: criança, infância, tuberculose, micobactérias atípicas, biologia molecular e diagnóstico. Resultados: As crianças portadoras da infecção pelo Mycobacterium tuberculosis são consideradas por diversos autorescomo “órfãos da tuberculose”. Várias características dificultam o estabelecimento do diagnóstico definitivoda tuberculose e micobacterioses na infância, entre elas as clínicas com lesões não extensivas, o caráterpaucibacilar, as formas latentes, a dificuldade de coleta de espécimes clínicos, as falhas das técnicas dabaciloscopia e cultura e a prática rotineira dos países em desenvolvimento de investigar a etiologia tuberculosa após falência terapêutica para patógenos habituais. Conclusões: A detecção da micobactéria permanece como confirmação diagnóstica e a oportunidade de investigação do perfil de sensibilidade, favorecendo o tratamento efetivo para qualquer faixa etária independente de seu papel na transmissão da doença. Nesse cenário, assumem maior importância, novas estratégias diagnósticas, entre elas as técnicas de biologia molecular com a promessa de melhor sensibilidade, especificidade e pronta detecção.

Objectives: To evaluate the tuberculosis and mycobacteriosis in childhood by a careful and critical literature review, with emphasis on epidemiology, clinical features and diagnostic devices. Methods: The study wasmade based on the following databases: LILACS, SciELO and PubMed, between the period of 2000 to 2009. Forty-six papers and eleven national and international guidelines/manuals were considered. The keywords used were: child, childhood, tuberculosis, atypical mycobacteria, molecular biology and diagnosis. Results: Children infected by Mycobacterium tuberculosis were considered by several authors as “orphans of tuberculosis”. To define final diagnosis of tuberculosis and mycobacterial infections is difficult in childhood because of several characteristics such as: the clinical with nonextensive lesions, the paucibacillary nature, the latent forms, the difficulty of collecting clinical specimens, the failures of the smear and culture techniques and routine practice of developing countries to investigate the etiology of tuberculosis after therapeutical failures for usual pathogens. Conclusions: The detection of mycobacteria remains usual to confirm the diagnosis and the opportunity to investigate the sensitivity profile. This promotes effective treatment for all age groups independent of their role in the disease transmission. In this environment, new diagnostic strategies including the interferon-gamma dosage and the molecular biology techniques can provide better sensitivity, specificity and ready detection.

Preliminary results in the immunodiagnosis of tuberculosis in children based on T cell responses to ESAT-6 and PPD antigens.

The aim of this work was to study the difference in interferon gamma (IFN-gamma) production by T lymphocytes after early secretory antigen target 6 (ESAT-6) or purified protein derivate (PPD) stimulation in whole blood culture supernatants from children with suspected tuberculosis (TB) disease (n = 21), latent TB infection (n = 16) and negative controls (NC) (n = 22) from an endemic area in Brazil. The concentration of IFN-gamma (pg/ml) was measured by enzyme linked immunosorbent assay and the differences in the IFN-gamma levels for each group were compared and evaluated using an unpaired Student's t-test; p values < 0.05 were considered significant. Measurement of IFN-gamma levels after ESAT-6 stimulation raised the possibility of early diagnosis in the latent TB group (p = 0.0030). Nevertheless, the same group showed similar responses to the NC group (p > 0.05) after PPD stimulation. The IFN-gamma assay using ESAT-6 as an antigenic stimulus has the potential to be used as a tool for the immunodiagnosis of early TB in children.

Preliminary results in the immunodiagnosis of tuberculosis in children based on T cell responses to ESAT-6 and PPD antigens

The aim of this work was to study the difference in interferon gamma (IFN-gamma) production by T lymphocytes after early secretory antigen target 6 (ESAT-6) or purified protein derivate (PPD) stimulation in whole blood culture supernatants from children with suspected tuberculosis (TB) disease (n = 21), latent TB infection (n = 16) and negative controls (NC) (n = 22) from an endemic area in Brazil. The concentration of IFN-gamma (pg/ml) was measured by enzyme linked immunosorbent assay and the differences in the IFN-gamma levels for each group were compared and evaluated using an unpaired Student's t-test; p values < 0.05 were considered significant. Measurement of IFN-gamma levels after ESAT-6 stimulation raised the possibility of early diagnosis in the latent TB group (p = 0.0030). Nevertheless, the same group showed similar responses to the NC group (p > 0.05) after PPD stimulation. The IFN-gamma assay using ESAT-6 as an antigenic stimulus has the potential to be used as a tool for the immunodiagnosis of early TB in children.

Development of a single tube hemi-nested PCR for genus-specific detection of Plasmodium in oligoparasitemic patients.

Primers targeting the Plasmodium small-subunit (SSU) rDNA were designed to amplify DNA from P. vivax, P. falciparum, P. malariae, and P. ovale, using conventional PCR, two-step nested PCR (HNPCR), and single tube hemi-nested PCR (STHNPCR). The limit of detection of parasite DNA for the conventional PCR, HNPCR, and STHNPCR were 10 pg, 0.01 pg, and 0.1 pg, respectively, indicating that the STHNPCR is 100-fold more sensitive than conventional PCR, and only 10 times less sensitive than HNPCR. In addition, the detection limit was also defined using blood from a patient infected with P. falciparum. Using the saponin method, the detection limit of the conventional PCR, HNPCR, and STHNPCR were 70, 0.7, and 0.07 parasites/microl, respectively. Finally, the three techniques were evaluated using blood from 30 patients receiving antimalarial treatment, and negative by microscopy and conventional PCR. The HNPCR could still detect specific DNA in 16/30 patients, whereas STHNPCR detected parasite DNA in 10/30 patients, but the difference was not statistically significant. No significant correlation was found between presence of clinical manifestations and presence of parasite DNA, detected by either HNPCR or STHNPCR. We conclude that these sensitive molecular diagnostic systems can be used for the diagnosis of asymptomatic oligoparasitemic patients.