RESUMO
Diagnostic tests for tuberculosis (TB) using interferon gamma (IFN-γ) responses produced by T lymphocytes after stimulation by early secretory antigen target 6 (ESAT-6), culture filtrate protein 10 (CFP-10) or purified protein derivate (PPD) were carried out using ELISA (enzyme-linked immunosorbent assay) in whole blood culture supernatants from children with suspected TB disease (n=21), latent TB infection (LTBI; n=17) and negative controls (NC; n=21) from Recife, Pernambuco, Brazil. The results were analysed using the ROC (receiver operating characteristic) curves and the areas under the curve (AUC) generated varied from 0.5 to 1.0 with higher values indicating increased discriminatory ability. Comparisons of AUCs were made using non-parametric assumptions, and the differences were considered significant if P<0.05. The ROC curve showed a statistical difference (P = 0.015) between the LTBI and NC groups with an AUC of 0.731, TB disease and NC (AUC=0.780; P=0.002) and a group with TB (latent infection+disease, n=38) and NC (AUC=0.758; P = 0.001) when the antigen used was ESAT-6. No statistical difference was found between the groups when CFP-10 or PPD was used. In conclusion, the ESAT-6 test may be the most appropriate for diagnosis of childhood TB, both LTBI and TB disease, when associated with epidemiological and clinical data, especially in endemic areas such as Brazil.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Tuberculose/diagnóstico , Adolescente , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Brasil/epidemiologia , Criança , Pré-Escolar , Doenças Endêmicas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interferon gama/metabolismo , Masculino , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tuberculose/epidemiologia , Tuberculose/imunologiaRESUMO
Human visceral leishmaniasis (HVL) is endemic in the tropical and sub-tropical regions of Africa, Asia, the Mediterranean, Southern Europe and South and Central America, with approximately 500,000 new cases reported annually. As dogs are considered to be the major reservoirs for HVL, the accurate diagnosis of disease in these animals is important. Diagnosis of canine visceral leishmaniasis (CVL) is performed mainly by direct parasitological methods that can yield false-negative results, either because of the very low number of Leishmania spp. organisms in clinical samples (bone marrow and lymph nodes) or because morphological identification is difficult. In addition, these methods are invasive. Conventional serological techniques are limited by cross-reactivity with other parasitic diseases and because several technical procedures have not been standardised. The development of polymerase chain reaction based approaches and immunoassays based on the use of recombinant antigens aimed at improving the sensitivity and specificity of CVL diagnosis is discussed.
Assuntos
Doenças do Cão/diagnóstico , Leishmania/isolamento & purificação , Leishmaniose Visceral/veterinária , Animais , DNA de Protozoário/análise , Doenças do Cão/sangue , Cães , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Valor Preditivo dos TestesRESUMO
Molecular techniques based on the detection of genomic sequences by reverse transcription (RT)-PCR, nested PCR, or real-time PCR have made possible the rapid diagnosis of dengue virus (DENV) infections, and these approaches have been accepted by clinical laboratories as the new standard method for the detection of dengue virus in acute-phase serum samples. One of these PCR-based assays, the two-step RT nested PCR (RT-NPCR) technique is used routinely in laboratories worldwide. In the present study, the two-step RT-NPCR as described by Lanciotti et al. [Lanciotti, R.S., Calisher, C.H., Gubler, D.J., Chang, G.J., Vorndam, A.V., 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J. Clin. Microbiol. 30, 545-551] was adapted to a novel single-tube nested PCR (STNPCR) format, which is less prone to cross-contamination and reduces reaction cost and time. When standards for each dengue serotype were tested, the detection limit of the STNPCR was at least 10 copies for DENV-1 and 100 copies for DENV-2 and DENV-3, whereas the detection limit for the two-step RT-NPCR was 100 copies for each serotype. Sera from 22 patients with confirmed DENV-3 infections and from 14 healthy individuals were then tested in the STNPCR format using the system described by Lanciotti et al. as the reference standard. The results indicated a sensitivity of 75.9% (CI 95%, 60.3-91.4) and a specificity of 100% for the RT-STNPCR. Although RT-STNPCR was less sensitive than the conventional two-step RT-NPCR for the detection of virus in serum samples, it was still adequately sensitive, and the advantages associated with a single-tube format may outweigh the somewhat lower assay sensitivity, making it useful for diagnosis in the field.
Assuntos
Vírus da Dengue/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sorotipagem/métodos , Primers do DNA , DNA Complementar , Dengue/virologia , Vírus da Dengue/classificação , Humanos , RNA Viral , Sensibilidade e EspecificidadeRESUMO
Pathogens causing tuberculosis and other chronic infectious diseases of major public health importance commonly have complex mechanisms involved in their persistence in the host despite specific and sometimes strong immune responses. These diseases are also associated with the lack of efficient vaccines, difficult therapeutics and a high mortality rate among susceptible individuals. Here, we will review features of the host immune response that contribute to the occurrence of disease. In addition, we propose that the immune responses observed in tuberculosis cannot be interpreted solely on the basis of a Th1-Th2 counter-regulatory paradigm since there is growing evidence that natural regulatory T cells may play an important role in the regulation of host immune responses against Mycobacterium tuberculosis. Thus, the development of more effective vaccines against this bacterial disease should take into account the role of natural regulatory T cells in the progression to severe disease and persistence of infection. Finally, new treatments based on manipulation of regulatory T cells should be investigated.
Assuntos
Mycobacterium tuberculosis/imunologia , Linfócitos T Reguladores/microbiologia , Tuberculose/imunologia , Humanos , Imunidade Celular/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Pathogens causing tuberculosis and other chronic infectious diseases of major public health importance commonly have complex mechanisms involved in their persistence in the host despite specific and sometimes strong immune responses. These diseases are also associated with the lack of efficient vaccines, difficult therapeutics and a high mortality rate among susceptible individuals. Here, we will review features of the host immune response that contribute to the occurrence of disease. In addition, we propose that the immune responses observed in tuberculosis cannot be interpreted solely on the basis of a Th1-Th2 counter-regulatory paradigm since there is growing evidence that natural regulatory T cells may play an important role in the regulation of host immune responses against Mycobacterium tuberculosis. Thus, the development of more effective vaccines against this bacterial disease should take into account the role of natural regulatory T cells in the progression to severe disease and persistence of infection. Finally, new treatments based on manipulation of regulatory T cells should be investigated.
Assuntos
Humanos , Mycobacterium tuberculosis/imunologia , Linfócitos T Reguladores/microbiologia , Tuberculose/imunologia , Imunidade Celular/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , /imunologiaRESUMO
This study evaluated the performance of the EIE-leishmaniose-visceral-canina-Bio-Manguinhos (EIE-LVC) kit and to compare it with that of the IFI-leishmaniose-visceral-canina-Bio-Manguinhos (IFI-LVC) kit. Four groups of dogs were studied: group 1 (G1), dogs with clinical signs indicative of CVL and testing positive for the parasite (n = 25); group 2 (G2), dogs with only a presumed diagnosis of CVL (n = 62); group 3 (G3), dogs that had never lived in an area where CVL is endemic and never received a blood transfusion (n = 16); group 4 (G4), dogs carrying other parasites: such as babesiosis (n = 4), ehrlichiosis (n = 6) and demodicosis (n = 1). G1 and G3 were used for the calculation of sensitivity and specificity, respectively. The EIE-LVC showed a sensitivity of 72% (IC 95%: 50.4-87.1%) and a specificity of 87.5% (IC 95%: 60.4-97.8%). The value of the kappa index was 0.975 (CI 95%: 0.926-1.024), which represents an excellent fit. For IFI-LVC, the sensitivity was 68.0% (CI 95%: 46.4-84.3%) and the specificity 87.5% (CI 95%: 60.4-97.8%). When the tests were conducted in parallel, sensitivity was 92.0% (CI 95%: 72.5-98.6%) and specificity 75.0% (CI 95%: 47.4-91.7%). However, when conducted consecutively, the tests showed a sensitivity of 48.0% (CI 95%: 28.3-68.2%) and a specificity of 100.0% (CI 95%: 75.9-99.4%). The analysis of clinically suspected dogs using IFI-LVC and EIE-LVC kits in parallel, revealed that 26/62 animals were positive. Cross-reaction was observed in a dog with demodicosis. These results lead to the following conclusions: (1) the performance of the EIE-LVC kit is not statistically different from the IFI-LVC and (2) the kits must be used in parallel if higher sensitivity is required, reducing the number of false-negative results.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doenças do Cão/diagnóstico , Leishmania donovani/imunologia , Leishmaniose Visceral/veterinária , Kit de Reagentes para Diagnóstico/veterinária , Animais , Estudos de Casos e Controles , Reações Cruzadas , Cães , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/veterinária , Reações Falso-Negativas , Técnicas Imunoenzimáticas/métodos , Técnicas Imunoenzimáticas/veterinária , Leishmaniose Visceral/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
During its life cycle, the flat worm Schistosoma mansoni is exposed to diverse environmental conditions and changes its morphological form. Each change calls for distinct patterns of gene expression. In order to understand the regulation of gene expression, it is necessary to identify regulatory elements in the promoter region of genes, and DNA transacting factors that control transcription. Zinc finger protein domains are responsible for transcription regulation of diverse genes in a wide range of organisms and are also involved in the promotion of protein-protein interactions. A transcript homologous to zinc finger gene sequences was isolated from a S. mansoni adult worm cDNA library and named SmZF1. It codes for a protein of 164 amino acids presenting three C(2)H(2) type zinc finger motifs. The recombinant SmZF1 protein was expressed and used on electrophoretic mobility shift assays to investigate the binding specificity of SmZF1 for DNA and RNA oligonucleotides. Our results demonstrated that SmZF1 binds both ds and ss DNA oligonucleotides, with an apparent preference for the specific D1-3DNA oligonucleotide, and also binds RNA oligonucleotides with lower affinity. Although we found that SmZF1 recognises DNA and RNA oligonucleotides not containing putative target sites, SmZF1 binds preferentially to sequence specific sites. Furthermore, unrelated oligonucleotides are not able to abolish this interaction. In silico studies identified putative SmZF1 binding sites in the complete genome of three model organisms and in partial genome sequences of S. mansoni. Six Drosophila genes presented these binding sites in their promoter region, indicating that they might be controlled by transcription factors containing zinc fingers motifs. Taken together, these results suggest that SmZF1 acts as a putative transcription factor of S. mansoni.
Assuntos
Proteínas de Helminto/genética , Ácidos Nucleicos/genética , Schistosoma mansoni/genética , Fatores de Transcrição/genética , Dedos de Zinco/genética , Animais , Sequência de Bases , DNA de Helmintos/genética , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Regulação da Expressão Gênica/genética , Oligonucleotídeos/genética , Regiões Promotoras Genéticas/genética , RNA de Helmintos/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genéticaAssuntos
Primers do DNA/química , Reação em Cadeia da Polimerase/métodos , Animais , Biomphalaria/genética , DNA de Protozoário/genética , Dessecação , Contaminação de Equipamentos/prevenção & controle , Humanos , Camundongos , Plasmodium/genética , Reação em Cadeia da Polimerase/instrumentação , Reprodutibilidade dos Testes , Schistosoma mansoni/genética , Esquistossomose mansoni/diagnóstico , Solubilidade , Moldes GenéticosRESUMO
Polymerase chain reaction (PCR)-based assays targeting the small-subunit rRNA were developed and evaluated, allowing for the simultaneous diagnosis of Plasmodium falciparum and Plasmodium vivax DNA in human blood samples. The PCR methods and quantitative buffy coat (QBC) were compared in 402 patients. The heminested PCR method showed a sensitivity of 97.4%, which was superior to the sensitivity of the QBC method (91.7%, P < 0.05), to simple PCR (84.6%, P < 0.001), and to PCR with digoxigenin labeling (PCR-DIG) (88.5%, P < 0.001). The PCR-DIG and QBC analyses were more sensitive than simple PCR (P < 0.003 and P < 0.05, respectively). There was no significant difference between the sensitivities of the QBC assay and the PCR-DIG assay. The specificity for the 3 PCR-based methods was 100%, superior to the specificity calculated for the QBC assay (88.95%, P < 0.009). The frequency of a positive result in groups from endemic areas but without detectable parasitemia increased, in order, from simple PCR, QBC test, PCR-DIG, to heminested PCR. An association between a positive PCR result and a history of malaria was also found. Taken together, these data suggest that this technology could be further developed to screen people with oligoparasitemia and to monitor malaria treatment.
Assuntos
DNA de Protozoário/análise , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Animais , Sequência de Bases , Criança , Pré-Escolar , Feminino , Humanos , Malária Falciparum/sangue , Malária Vivax/sangue , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Plasmodium falciparum/genética , Plasmodium vivax/genética , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
In this communication the authors analyzed the pattern of expression of IFN-gamma as a surrogate type 1 response in different clinical forms of schistosomiasis in response to stimulation involving T-cell dependent and T-cell independent pathways, to investigate which pathways were functional in human schistosomiasis, and to further characterize the nature of Th1 response impairment in this parasitic disease.