Immunological diagnosis of tuberculosis based on recombinant antigens ESAT-6 and CFP-10 in children from an endemic area in northeast Brazil.

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.

Diagnosis of canine visceral leishmaniasis: biotechnological advances.

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.

Single-tube nested PCR using immobilized internal primers for the identification of dengue virus serotypes.

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.

Immune factors and immunoregulation in tuberculosis.

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.

Canine visceral leishmaniosis: a comparative analysis of the EIE-leishmaniose-visceral-canina-Bio-Manguinhos and the IFI-leishmaniose-visceral-canina-Bio-Manguinhos kits.

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.

Nucleic acid binding properties of SmZF1, a zinc finger protein of Schistosoma mansoni.

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.

Immune factors and immunoregulation in tuberculosis

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.

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

No presente estudo tres tecnicas para isolamento de fracoes enriquecidas em membrana externa de Y. pestis foram avaliadas. As tecnicas utilizadas foram: centrifugacao em gradiente de densidade em sacarose e solubilizacao diferencial com Sarkosyl ou Triton X-100. A analise por eletroforese em gel de poliacrilamida na presenca de dodecil sulfato de sodio (SDS-PAGE) das membranas externas extraidas pelos diferentes metodos evidenciou perfis proteicos semelhantes. A determinacao das atividades de NADH-desidrogenase e succinato-desidrogenase (enzimas de membrana interna) indicou que todas as preparacoes estudadas eram adequadas a estudos analiticos. Obteve-se evidencias preliminares sobre o possivel uso de perfis proteicos de membrana externa na identificacao de variantes geograficos entre isolados selvagens de Y. pestis

Efeitos de imunopotenciadores nao especificos na infeccao experimental pelo Schistosoma mansoni. I. Levamisole / Effects of non-specific immunopotentiators in experimental Schistosoma mansoni infection. I. Levamisole

Os efeitos do levamisole nas alteracoes histopatologicas, resistencia do hospedeiro e quimiotaxia "in vitro" foram estudados na infeccao experimental pelo Schistosoma mansoni em camundongos da linhagem C57B1/10. O tratamento profilatico resultou em um aumento no numero de vermes adultos obtidos pela perfusao e tambem em uma taxa de mortalidade maior (p<0,05). As alteracoes histopatologicas (figado e intestino) foram similares em todos os grupos. Uma reducao significante da quimiotaxia "in vitro" ocorreu em camundongos controles infectados, assim como naqueles submetidos a tratamento profilatico com levamisole. A atividade quimiotatica atingiu os mesmos niveis dos camundongos controles normais (nao-infectados e nao-tratados com levamisole), quando o esquema curativo foi usado. O levamisole parece aumentar a susceptibilidade de camundongos da linhagem C57B1/10 a infeccao pelo S. mansoni quando administrado antes da infeccao e normaliza a atividade quimiotatica, quando dado apos a infeccao.

In vivo leukocyte chemotaxis during the development of acute experimental Trypanosoma cruzi infection

Five and 15 days after T. cruzi infection, staphylococcal protein A was injected into a connective tissue air pouch of mice and the migration of polymorphonuclear leukocyrtes into the area was monitored. The PMN leukocyte response of 15-day infected mice was lower than of uninfected mice (P < 0.001): The 15 - day infected mice also showed a lower leukocyte response when compared to 5 - day infected mice (P < 0.001). These data suggest that chemotaxis defect development gradually during the acute phase of infection

Immunochemical localization of the fraction-1 antigen, a virulence determinant of Yersinia pestis

The distribution of Yersina pestis Fraction-1 (F1) antigen was analyzed in cells grown at 28§C and 37§C. Fractionation of Y. pestis cells followed by analysis in SDS-polyacrylamide gel electrophoresis indicated that the mature form of the F1 antigen is localized in the extracellular matrix and in the cytoplasm. Localization of the F1 antigen was confirmed by immunoblots and a single peptide with a molecular weight of 17,000 daltons was recognized. Electron microscopy of Y. pestis cells labeled with colloidal gold-conjugated antibodies corroborated the extracellular matrix and cytoplasm dual location of the F1 antigen