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.

Structure of the gene encoding a putative Schistosoma mansoni tegumental antigen precursor.

In order to obtain the complete gene encoding the putative precursor of a 15-kDa Schistosoma mansoni tegumental antigen (Sm15), two cDNAs (A70 and A184) and two fragments of independent genomic clones were subcloned and sequenced. The collated sequence contains 4700 nucleotides and represents the full length open reading frame of the gene, encoding a protein of 1032 amino acids with a calculated molecular mass of 116,900. Thus, the gene encodes a much longer protein than that identified in the tegumental membranes, suggesting that it encodes a precursor that is subsequently highly processed. A 964-bp region composed of 5 closely related repeats was found to be present within the translated frame. The predicted protein is highly acidic and there is no indication of hydrophobic domains that may represent transmembrane regions or indicate attachment of a GPI anchor. The coding region has no homologies in the currently available data bases. In the 5' non-transcribed area a copy of the SM alpha repeat family is present. The coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression.

Structure of Sm25, an antigenic integral membrane glycoprotein of adult Schistosoma mansoni.

Sm25 is the principal antigen recognised by antibodies from mice protectively vaccinated with isolated tegumental membranes of adult Schistosoma mansoni. The full-length amino acid sequence of this protein has been deduced from the sequence of two cDNAs, one isolated by screening a cDNA library and the other, including the 5' end of the gene, amplified directly from adult worm RNA using the polymerase chain reaction. The predicted sequence represents a nascent polypeptide of Mr 21,500. Following cleavage of a predicted signal sequence, the Mr of the resulting polypeptide is 17,600. The polypeptide contains 2 potential sites for N-linked glycosylation and a hydrophobic domain at the C-terminus that could facilitate membrane association. Analysis of the mature gene product confirmed that Sm25 is an N-glycosylated integral membrane protein and that the Mr of the deglycosylated polypeptide is between 15,000 and 20,000.

Mapping of the N terminus of the Schistosoma mansoni tegumental antigen Sm15 to its predicted precursor protein.

Sm15 is a major Schistosoma mansoni 15 kDa tegumental antigen, resulting from the proteolytic processing of a larger precursor. The amino terminus of Sm15 was identified by direct amino acid sequencing, and the antigen was tentatively mapped to the segment spanning amino acids 362-497 of the precursor. This will allow subsequent studies to elucidate the possible immunological role of proteolytic processing in schistosomiasis.

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.

Development of vaccines against human parasitic diseases: tools, current status and perspectives.

Vaccines against malaria, leishmaniasis and schistosomiasis are in the most advanced stages of development of all vaccines for human parasitic diseases. Despite the remarkable progress made in identifying and producing protective antigens, at present there are no generally accepted vaccines against parasitic diseases. Vaccines for malaria and leishmaniasis have been taken to clinical trials while vaccines for schistosomiasis are in Phase I/II trials. This review will focus on the most promising antigenic preparations, emphasising the tools, present status and perspectives for development of vaccines against malaria, leishmaniasis and schistosomiasis.

Development and optimization of polymerase chain reaction-based malaria diagnostic methods and their comparison with quantitative buffy coat assay.

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.

Identification of surface-exposed Yersinia pestis proteins by radio-iodination and biotinylation.

When whole cells (stationary phase) of Yersinia pestis strain EV76 were radiolabelled with Iodogen and 131I, 16 major and 10 minor surface-exposed outer membrane proteins (OMPs) were identified. Labelling with N-hydroxysuccinimidyl 6-biotinylamino-hexanoate (biotin X-NHS) resulted in a complex protein profile detectable after blotting and developing with peroxidase-conjugated avidin. Y. pestis cell fractionation revealed that biotin X-NHS labelled not only OMPs but also proteins of inner cell compartments. Therefore, radiolabelling was the more reliable technique for identifying the OMPs of Y. pestis.

The use of non-specific immunopotentiators in experimental Trypanosoma cruzi infection.

The effects of levamisole, isoprinosine and Corynebacterium parvum on Trypanosoma cruzi (Y strain) experimental infection of mice were studied. In prophylactic treatment these drugs reduced the peak of parasitaemia, and had no apparent effect on mortality rate or on histopathological and electrocardiographic findings. Levamisole and isoprinosine had no effect when used after infection. Electrocardiograms were obtained from all chronic chagasic mice. The most frequent changes were left atrial overload and first degree atrio-ventricular block. These findings became more frequent the longer the animals survived. The net effect of the non-specific immunopotentiators seems to depend on several factors: host immune state, severity of infection, dose and timing of drug administration. This probably explains the variable published results and the paradoxical findings of different laboratories.

Diagnosis of Wuchereria bancrofti infection by the polymerase chain reaction using urine and day blood samples from amicrofilaraemic patients.

A sensitive and specific polymerase chain reaction (PCR) based on a highly repeated deoxyribonucleic acid (DNA) sequence (188 bp; SspI repeat) was tested for the detection of Wuchereria bancrofti DNA in blood and urine samples collected during the day from individuals in Coque, Recife, Brazil, an endemic area for W. bancrofti. All microfilaraemic individuals were also positive by PCR, irrespective of the samples used. The PCR system was capable of detecting W. bancrofti DNA in amicrofilaraemic individuals: c. 93% were positive by PCR when day blood samples were used and 59.7% when urine samples collected at 07:00 were used. Thus, nocturnally periodic W. bancrofti infection can be detected in blood samples collected during the day, which is convenient for large-scale screening. In addition, non-invasive urine collection provided suitable samples for PCR, which is clearly advantageous for preliminary mass diagnosis.

Dynamics of the antibody response in patients with therapeutic or spontaneous cure of American cutaneous leishmaniasis.

The antigen specificity and the level of the antibody response were analysed in Perambuco State, Brazil, in sera collected in 1995-96 from 58 patients with clinical American cutaneous leishmaniasis (ACL), 25 ACL patients with apparent cure after chemotherapy with meglumine antimonate, and 10 ACL patients with spontaneous cure. Assessment was by immunoblot analysis, ELISA and indirect immunofluorescence, with Leishmania (Viannia) braziliensis antigens, with a particular interest in evaluating whether the dynamics of the antibody response could be useful to monitor clinical cure. A clear decrease of IgG antibody reactivity was noticed after clinical healing, for all of the antigens analysed, with the exception of the 19 kDa antigen, whose recognition frequency in fact increased in the spontaneously cured patients, suggesting that this antigen may play a role in protective immunity against cutaneous leishmaniasis. The recognition frequencies of the most frequently recognized antigens (27 and 30 kDa antigens) diminished approximately 2-fold in patients clinically healed, suggesting that they could be useful as a marker of cure of ACL. In addition, some of the healthy individuals living in endemic areas presented the same immunoblotting pattern of reactivity observed in active ACL, possibly representing asymptomatically infected individuals.

Vaccines against human parasitic diseases: an overview.

There are no vaccines currently available to control the major human parasitic diseases, although there is evidence of acquired immunity and resistance to reinfection in most of the parasitic infections. The present manuscript concentrates on the vaccines for parasitic diseases that are in the most advanced stages of development: malaria, leishmaniasis and schistosomiasis. Vaccines for malaria and leishmaniasis have been taken to clinical trials while vaccines for schistosomiasis are being considered for Phase I (assessment of safety and immune responsiveness in volunteers). We have attempted to give a factual account of the present status of knowledge of vaccines against human parasitic diseases, emphasizing both the successes and setbacks. We do not intend to cover the enormous literature in the field but have concentrated on the most promising antigenic preparations. Finally, some new approaches for the development of vaccines are discussed including nucleic acid vaccines and the use of cytokines as adjuvants.

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 leukocytes into the area was monitored. The PMN leukocyte response of 15-day infected mice was lower than that of uninfected mice (P less than 0.001): The 15-day infected mice also showed a lower PMN leukocyte response when compared to 5-day infected mice (P less than 0.001). These data suggest that the chemotaxis defect developed gradually during the acute phase of infection.

A strategy to sequence repetitive DNA based on partial restriction enzyme cleavage.

The strategy to sequence repetitive DNA described in this article is based on partial restriction enzyme cleavage. It is an alternative to using nested deletion with exonuclease III or similar enzymes in which progressively more remote regions of the target DNA are brought into range for sequencing by universal primers.

A monoclonal antibody against blood forms of Trypanosoma cruzi lyses the parasite in vitro and inhibits host cell invasion.

Monoclonal antibodies (mAbs) of the IgM isotypes were produced from mice immunized with blood forms of Trypansoma cruzi Y strain. Characterization of the epitope recognized by one of the mAbs, 164C11, as well as the effects of this mAb on complement-mediated lysis and host cell invasion are reported. Immunocytochemical analysis showed that the mAb was reactive with various strains of T. cruzi (Y, WSL, and Colombiana) as well as other trypanosomatids. The mAb 164C11 demonstrated a high complement-mediated lytic activity against bloodstream trypomastigotes, being more effective than chronic mouse serum. A protein with an apparent molecular weight of 72 kDa was detected by this mAb on all developmental stages of T. cruzi. Studies using periodate and endoglycosidase treatments suggested that the epitope is not a carbohydrate and seems to be located on the parasite membrane. In addition, preliminary results are presented, suggesting that the 72-kDa protein is involved in adhesion/or internalization of bloodstream trypomastigotes.

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

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.