Characterization of organic compounds and drugs in sewage sludge aiming for agricultural recycling.

The application of sewage sludge in soils can bring benefits to agricultural productivity, however, the risks arising from this application must be measured and carefully evaluated. Persistent organics compounds and drugs can be present in the sewage sludge and if applied to the soil, they can cause severe risks to the soil biota and contamination of groundwater. This work determined 174 persistent organic compounds and drugs in sludge samples from a wastewater treatment plant using chromatographic methods. The drugs ciprofloxacin, enrofloxacin and diclofenac were quantified, and values varied according to sampling period. For persistent organic compounds, cresols were the most abundant compounds in sewage sludge. With the analyses made of both the elutriate and the filtrate, it was possible to verify the potential for soil retention or leaching that each compound can present with the application of sewage sludge in the agriculture.

Diagnostic usefulness of brief versions of Alcohol Use Disorders Identification Test (AUDIT) for detecting hazardous drinkers in primary care settings.

OBJECTIVE: The aim of this study was to evaluate the diagnostic usefulness of the brief versions of the Alcohol Use Disorders Identification Test (AUDIT) for detecting hazardous drinkers and to compare it with that of the full-AUDIT in primary care settings. METHOD: Five hundred patients were randomly selected in a primary care center. An interview on quantity-frequency was administered for assessment of weekly alcohol intake. The standard used for classification of hazardous drinkers was a weekly alcohol consumption of 280 g for men and 168 g for women. Cut-off points were 8 for the full-AUDIT, 1 for the AUDIT-3 (third item), 3 for the AUDIT-C (items 1, 2 and 3), 5 for the AUDIT-PC (items 1, 2, 4, 5 and 10) and 3 for the modified Fast Alcohol Screening Test (m-FAST; items 3, 5, 8 and 10). Sensitivity, specificity, positive and negative predictive values, and areas under the receiver operating characteristic (AUROC) curves were measured. RESULTS: Diagnostic usefulness of the questionnaires for detecting hazardous drinkers was for the full- AUDIT: 81.4% sensitivity, 94.6% specificity and 0.97 AUROC curve; for the AUDIT-3: 83.1% sensitivity, 90.9% specificity and 0.89 AUROC curve; for the AUDIT-C: 100% sensitivity, 79.4% specificity and 0.97 AUROC curve; for the AUDIT-PC: 98.3% sensitivity 90.9% specificity and 0.97 AUROC curve; and for the m-FAST: 79.7% sensitivity, 93.7% specificity and 0.93 AUROC curve. CONCLUSIONS: The AUDIT-C and AUDIT-PC show a higher sensitivity, lower specificity and a similar AUROC curve than the full-AUDIT, thus allowing their use as screening instruments that are as reliable as the original test for detecting hazardous drinkers. The AUDIT-3 and m-FAST, when compared with the full-AUDIT, performed less well, therefore limiting their use for this purpose.

Characterisation of a Trypanosoma cruzi acidic 30 kDa cysteine protease.

A novel proteolytic activity was identified in epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Epimastigotes showed enzyme activity to be 2-fold higher than amastigotes and trypomastigotes. The protease that displays this activity was purified from epimastigote forms by a four step chromatographic procedure: Diethylaminoethyl-Sephacel, Phenyl-Sepharose, Phenyl-Superose, and Concanavalin A Sepharose columns. The purified enzyme is a glycoprotein that migrates as a 30 kDa protein in 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), under reducing conditions. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. The inhibition pattern of the purified 30 kDa protease showed that it belongs to the cysteine-protease class. In addition to the synthetic substrate, the purified protease hydrolysed bovine serum albumin (BSA) and human type I collagen. The N-terminal amino acid sequence of the protease shows similarity to the mammalian cathepsin B protease.

Trypanosoma cruzi: endocytosis and degradation of specific antibodies by parasite forms.

Specific human IgG antibodies bound to a Trypanosoma cruzi envelope were internalized by antigen receptor-mediated endocytosis. Ferritin conjugated antibodies and horseradish peroxidase (HRP) conjugated IgG were found inside parasite cytoplasmic vesicles. Nonspecific IgG that did not bind to the external membrane was not internalized by the parasite. The ratio of 3H-protein A labeled: specific IgG internalization by parasites in the exponential growth phase (95% epimastigotes) was much smaller than that of parasites in the late stationary growth phase (38% trypomastigotes). Antibodies bound to the latter parasite forms almost disappeared from their outer membranes after 12 hr incubation at 27 degrees C. Results of experiments in which membrane bound antibodies were removed by an excess of pronase showed that only small amounts of radiolabeled IgG were found inside the parasites. The fate of immunoglobulins that vanished from external membrane receptors and did not accumulate inside the cells was explained by experiments in which the supernatants of IgG-3H-protein A labeled parasites were precipitated with trichloroacetic acid (TCA). In these, membrane-bound antibodies were taken in and degraded by the parasites as increased amounts of free radiolabel appeared in the supernatants as functions of incubation time and parasite stage.

Effect of immunotoxins against Trypanosoma cruzi.

Immunotoxins were constructed with IgG antibodies against Trypanosoma cruzi surface antigens by hybridization with abrin (ITA) and ricin (ITR) A chains. The biological activity of the hybrid macromolecules was tested on the parasite forms. Motility of parasite forms was lost in vitro after incubation with ITR. In general, killing of the parasite with ITR was more efficient than with ITA. Inhibition of protein synthesis after incubation with either ITR or ITA, measured by 3H-leucine incorporation, confirmed the parasite immobilization experiments. The lethal effect was potentiated when the immunotoxins were used in the presence of 2.5 mM ammonium chloride. T. cruzi antibodies specific to cell surface antigens are excellent drug carriers that can be delivered to the target cell. However, ITR and ITA did not reduce parasitemia or increase survival of mice infected with T. cruzi.

Human IgG1 and IgG4: the main antibodies against Triatoma infestans (Hemiptera: Reduviidae) salivary gland proteins.

The Triatoma infestans salivary gland proteins (TSGP) can induce local and systemic hypersensitivity reactions in humans. IgG antibodies against TSGP were present in higher levels in sera of Chagas disease patients, and in individuals living in triatomine-infested areas than in controls living in triatomine-free areas. TSGP-specific IgG1 was found in sera of Chagas patients, and of individuals living in triatomine-infested rural areas, and uniquely specific IgG4 was present in sera of Chagas patients living in triatomine-infested areas, reactive against TSGP. Unique specificities were not detected in sera of individuals reacting against the ubiquitous mosquito Culex quinquifasciatus saliva proteins (CSGP). In conclusion, IgG1 reactive against TSGP is the main antibody present in individuals living in the triatomine-infested study areas. Also, IgG4 is found in the sera of insect-transmitted Chagas disease patients living in study areas.

Possible integration of Trypanosoma cruzi kDNA minicircles into the host cell genome by infection.

Infection with Trypanosoma cruzi is known to induce the division of peritoneal macrophages in BALB/c mice. We have demonstrated, by cytogenetic analysis, that accessory DNA elements are associated with the metaphase macrophage chromosomes of such infected macrophages. The identification of these accessory DNA elements with T. cruzi DNA is strongly supported by the association of 3H-label with some chromatids in macrophages previously infected with T. cruzi which had been labelled with 3H-methyl-thymidine. The karyotyping consistently showed preferential associations of T. cruzi DNA with chromosomes 3, 6 and 11. A conclusive demonstration of the parasite origin of the integrated DNA came from fluorescein in situ hybridization studies using specific parasite DNAs as probes. In order to determine the identity of the inserted DNA and to investigate the nature of the integration mechanism, Southern blot analyses were performed on DNA extracted from both uninfected and infected (but parasite-free) macrophages. Hybridizations of BamHI, EcoRI and TaqI digests of DNA from T. cruzi-infected host cells all revealed the presence of a 1.7-kb DNA fragment when probed with kDNA. The covalent association of kDNA with that of the host was confirmed by the fact that AluI and Hinf-I digests of DNA from infected host cells produced a number of bands, in a size range of 0.8-3.6 kb, which hybridized with kDNA minicircles. None of these bands was found in DNA purified from cell-free preparations of the parasite and thus it must be concluded that they represent insertion fragments between parasite and host cell DNA. These results strongly suggest that kDNA minicircles from T. cruzi have been integrated into the genome of the host cell following infection.

Malignant, non-Hodgkin's lymphomas in Trypanosoma cruzi-infected rabbits treated with nitroarenes.

Use of 2-nitroimidazole, 5-nitrofuran and 5-nitroimidazole compounds in T. cruzi-infected rabbits resulted in a reduction in duration of parasitaemia in comparison with untreated, infected rabbits. The chronic myocarditis associated with Chagas' disease was not, however, prevented in nitroarene-treated rabbits; lymphocytic infiltrates associated with cardiac cell lysis, in the absence of parasites in situ, were present in both treated and untreated rabbits. The carcinogenic effect of each trypanocidal nitroarene used in this study was also assessed. Administration of nitroarenes to rabbits resulted in the appearance of solid tumours in 37.8 per cent of animals that received drug therapy. Untreated, control rabbits in this series did not show tumour growth. Furthermore, malignant, mixed-cell type, non-Hodgkin's lymphomas were seen in 32.4 per cent of the treated rabbits. It seems that a direct relationship could be present between the presence of the nitro group, the trypanocidal cytotoxicity and the prevalence of tumours. Benznidazole cleared up parasitaemias in the shortest time and was associated with 41.6 per cent of lymphoma growths, whereas MK-436 required twice as much time to clear blood parasites, and showed lymphomas in 25 per cent of experimental rabbits. The demonstration of a high prevalence of malignant tumours in addition to the chronic myocarditis of Chagas' disease in nitroarene-treated rabbits is important since indiscriminate use of such compounds currently used to treat T. cruzi infections in man could increase the risk of lymphoma.

Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (viannia) braziliensis.

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

[Insertion of Trypanosoma cruzi DNA in the genome of mammal host cell through infection]. / Inserção de DNA de Trypanosoma cruzi no genoma de célula hospedeira de mamífero por meio de infecção.

Trypanosoma cruzi chromatin was observed in macrophage chromosome spreads obtained at different periods of infections in BALB/c mice. Immunofluorescent studies showed that genetic materials associated with the chromosomes were T. cruzi products. In situ hybridization showed the protozoon DNA insertion in the host cell genome. In addition, selective 3H-DNA insertion in chromosomes 3, 6 and 11 was observed, which suggested that transxenogene rearrangement may take place in T. cruzi infections of mammals.

Diversity of Trypanosoma cruzi stocks and clones derived from Chagas disease patients: I--Behavioral characterization in vitro.

In this study, we isolated Trypanosoma cruzi from chronic Chagas heart disease and from megaesophagus patients. The parasite stock hSLU239 (heart disease) yielded clones h1 and h2, whereas stock mSEU142 (megaesophagus) yielded clones m1, m2, m3 and m4. The parasite growth kinetics, doubling time and differentiation in axenic liquid medium showed broad behavioral diversity. It was shown that a particular pattern of behavior for a parental stock could not necessarily be assigned for subsequent clones. This study indicates that i) each Chagas disease patient is infected with several T. cruzi populations; ii) clonal lines derived from patient samples may have different biological characteristics from the original isolate; and that iii) additional behavioral and/or molecular markers are required for further characterization of Trypanosoma cruzi stocks and clones derived from Chagas disease patients in order to identify correlations with pathology.

[The use of 4 immunological exams for the determination of Chagas disease prevalence in streetsweepers of the City Sanitation Service in the Federal District]. / Emprego de quatro exames imunológicos na determinação da prevalência da doença de Chagas nos garis do Serviço de Limpeza Urbana do Distrito Federal.

Seropositivity for Trypanosoma cruzi infection was studied in 368 street-sweepers of the SLU, Federal District, Brazil, with the aid of haemaglutination, immunofluorescence and, also, a delayed-type skin test to the parasite T12E antigen. It showed 32.1%, 42.1% and 38.6% positive results, respectively for each assay. Among these, however, only 47% were positive with each of three exams performed. In addition, 19.7% were positive with two out of three exams performed. The remaining 33.3% sera yielded one positive result out of three exams employed and were submitted to the immunoblot assay. This analysis confirmed 3 cases (37.5%) positive by hemmaglutination, 3 (11.5%) positive by skin test, and 1 (3.7%) positive by immunofluorescence. At the end of the analysis, it was shown that 129 (35%) individuals yielded at least two positive assays and, therefore, they should be considered as T. cruzi-infected individuals.

[Morphometric study of the intrapetrous passage of the facial nerve in adult temporal bones and its application to the study of the pathogenesis of Bell facial paralysis]. / Estudio morfométrico del recorrido intrapetroso del nervio facial en temporales adultos y su aplicación en la patogenia de la parálisis facial de Bell.

After a morphometric study, done in adult temporals, of the inner diametre of the fallopian bony canal, of the thickness of both the epineural space and the nerve trunck (in petrous, tympanic and mastoid segments) and the like with the geniculate ganglion and the pyramidal bend, the AA. thrown out the conclusion that when a generalized edema of the nerve is expected the labyrinthine segment or the distal portion of the mastoidal segment are the sites to be found most precocious or heaviest compressed.

Parasite prolyl oligopeptidases and the challenge of designing chemotherapeuticals for Chagas disease, leishmaniasis and African trypanosomiasis.

The trypanosomatids Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp. cause Chagas disease, leishmaniasis and human African trypanosomiasis, respectively. It is estimated that over 10 million people worldwide suffer from these neglected diseases, posing enormous social and economic problems in endemic areas. There are no vaccines to prevent these infections and chemotherapies are not adequate. This picture indicates that new chemotherapeutic agents must be developed to treat these illnesses. For this purpose, understanding the biology of the pathogenic trypanosomatid- host cell interface is fundamental for molecular and functional characterization of virulence factors that may be used as targets for the development of inhibitors to be used for effective chemotherapy. In this context, it is well known that proteases have crucial functions for both metabolism and infectivity of pathogens and are thus potential drug targets. In this regard, prolyl oligopeptidase and oligopeptidase B, both members of the S9 serine protease family, have been shown to play important roles in the interactions of pathogenic protozoa with their mammalian hosts and may thus be considered targets for drug design. This review aims to discuss structural and functional properties of these intriguing enzymes and their potential as targets for the development of drugs against Chagas disease, leishmaniasis and African trypanosomiasis.

A black hole nova obscured by an inner disk torus.

Stellar-mass black holes (BHs) are mostly found in x-ray transients, a subclass of x-ray binaries that exhibit violent outbursts. None of the 50 galactic BHs known show eclipses, which is surprising for a random distribution of inclinations. Swift J1357.2-093313 is a very faint x-ray transient detected in 2011. On the basis of spectroscopic evidence, we show that it contains a BH in a 2.8-hour orbital period. Further, high-time-resolution optical light curves display profound dips without x-ray counterparts. The observed properties are best explained by the presence of an obscuring toroidal structure moving outward in the inner disk, seen at very high inclination. This observational feature should play a key role in models of inner accretion flows and jet collimation mechanisms in stellar-mass BHs.

[Severe hydroureteronephrosis associated to asymptomatic giant anterior sacral meningocele: a case report and review of the literature]. / Ureterohidronefrosis severa asociada a meningocele sacro anterior gigante asintomático: presentación de un caso y revisión de la literatura.

We present the case of a woman with Marfan's syndrome presenting with a clinical picture of acute gastroenteritis in whom severe bilateral hydroureteronephrosis associated to a neurogenic bladder and a giant anterior sacral meningocele was diagnosed incidentally. The importance of this case lies in the fact that the patient was asymptomatic despite the significant visceral repercussions already occurring that led to questioning of whether MRI follow-up would still be advisable even in the absence of symptoms.

The diagnostic usefulness of AUDIT and AUDIT-C for detecting hazardous drinkers in the elderly.

We compare the diagnostic usefulness of the Alcohol Use Disorders Identification Test (AUDIT) and the AUDIT alcohol consumption questions (AUDIT-C) for detecting hazardous drinkers between the populations over and less than 65 years in primary care settings. To assess weekly alcohol intake an interview on quantity-frequency was administered to 602 patients. Hazardous drinking was defined as a level of consumption of 280 g of alcohol per week for men and 168 g for women. The participants received AUDIT, AUDIT-C and CAGE questionnaires. Gamma-glutamyltransferase (GGT), mean corpuscular volume (MCV), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values were also determined. Average weekly alcohol intake among the population aged 65 and older was 83 g, and 10% were hazardous drinkers. In this age group, the sensitivities of AUDIT and AUDIT-C for detecting this type of drinkers were 67% and 100%, whereas specificities were 95% and 81% respectively. In the younger patient group, the sensitivities were 84% and 100% and the specificities 95% and 79% respectively. In conclusion, both AUDIT and AUDIT-C perform well at detecting hazardous drinkers in the group older than 65 years and that their sensitivities and specificities are comparable to those in younger ages.

Chagas' disease. Immunotoxin inhibition of Trypanosoma cruzi release from infected host cells in vitro.

Virulent tissue culture-derived Trypanosoma cruzi trypomastigotes were readily killed by immune IgG-ricin A chain conjugates (ITR) in vitro. Forty micrograms of ITR immobilized 10(6) trypomastigotes after 48 hours of incubation at 37 degrees C. ITR showed antibody specificity and 125I-labeled anti-T. cruzi IgG bound to parasitized host cells 9-fold more than to nonparasitized host cells. The degree of specificity was evaluated further in experiments in which 10 micrograms of ITR showed 78% inhibition of [3H]thymidine incorporation by T. cruzi. In contrast, nonimmune IgG-ricin A chain conjugate neither immobilized nor inhibited [3H]thymidine incorporation by the parasite. Furthermore, 20 micrograms of ITR significantly inhibited T. cruzi trypomastigote release from infected host cells and thus prevented reinfection of other cells in vitro.