[Human leptospirosis in Belo Horizonte City, Minas Gerais, Brazil: a geographic approach]. / Leptospirose humana no município de Belo Horizonte, Minas Gerais, Brasil: uma abordagem geográfica.

A retrospective study on human leptospirosis, was done in Belo Horizonte in 1995, using geographic processing resources. Thirty suspected and 19 confirmed cases of leptospirosis were mapped in the area. The majority of confirmed cases (68.4 +/- 13%) were located in North, Northeast and West of the city. The main foci of disease were found in slums and other poor areas: 73.7 +/- 12% of the confirmed cases and 26.7 +/- 12% of suspected cases. Ninety-five percent +/- 6% of the confirmed cases were found in the outskirts of the city where there was a population increase and inadequate infrastructure. It was observed that 50 +/- 14% of the suspected cases and 42 +/- 14% of the confirmed cases were found in areas of high concentration of water resources. Suspected (83.3 +/- 10%) and confirmed cases (79 +/- 11%) occurred in lower altitude areas of the city (750 to 1,000m) and 78 +/- 12% of the individuals had been in contact with contaminated water and/or animals.

Leptospirose humana no município de Belo Horizonte, Minas Gerais, Brasil: uma abordagem geográfica / Human leptospirosis in Belo Horizonte City, Minas Gerais, Brazil: a geographic approach

A retrospective study on human leptospirosis, was done in Belo Horizonte in 1995, using geographic processing resources. Thirty suspected and 19 confirmed cases of leptospirosis were mapped in the area. The majority of confirmed cases (68.4 +/- 13%) were located in North, Northeast and West of the city. The main foci of disease were found in slums and other poor areas: 73.7 +/- 12% of the confirmed cases and 26.7 +/- 12% of suspected cases. Ninety-five percent +/- 6% of the confirmed cases were found in the outskirts of the city where there was a population increase and inadequate infrastructure. It was observed that 50 +/- 14% of the suspected cases and 42 +/- 14% of the confirmed cases were found in areas of high concentration of water resources. Suspected (83.3 +/- 10%) and confirmed cases (79 +/- 11%) occurred in lower altitude areas of the city (750 to 1,000m) and 78 +/- 12% of the individuals had been in contact with contaminated water and/or animals.

Uma análise retrospectiva da leptospirose humana no município de Belo Horizonte em 1995 foi realizada usando recursos de geoprocessamento.Trinta casos suspeitos e 19 casos confirmados foram localizados na área. A predominância de casos confirmados foi registrada nas regiões norte, nordeste e oeste (68,4 ± 13%). Nas áreas de favelas e bolsões de pobreza foram identificados os principais focos da doença, 73,7 ± 12% dos casos confirmados e 26,7 ± 12% dos casos suspeitos. Na periferia, onde ocorreu um aumento populacional, localizaram-se 95 ± 6% dos casos confirmados, sendo o local com maior carência de infra-estrutura básica. Na distribuição espacial dos principais cursos d'água do município, observou-se que 50 ± 14% dos casos suspeitos e 42 ± 14% dos casos confirmados estavam localizados nas áreas com maior concentração de redes fluviais. Casos suspeitos (83,3 ± 10%) e confirmados (79 ± 11%) ocorreram em partes altimétricas mais baixas da cidade (750 a 1.000m) e 78 ± 12% dos indivíduos tiveram contato com água e / ou animais contaminados.

Effects of hydrostatic pressure on the Leptospira interrogans: high immunogenicity of the pressure-inactivated serovar hardjo.

The Hardjoprajitno strain of Leptospira interrogans serovar hardjo was subjected to different hydrostatic pressures. Complete inactivation occurred when the leptospires were treated with 2 kbar for 60 min. Electron microscopy showed dislocation of the outer membrane, partial loss of the helical shape and extrusion of the axial filament from the cytoplasmic cylinder of the pressurized leptospires. When the pressure-treated leptospires were inoculated into rabbits they were highly immunogenic. The sera of these animals presented a titer of 2048 in the microscopic serum agglutination reaction. Fluorescence measurements indicated that the action of pressure on the leptospires might have resulted from perturbation on membrane protein components, permitting the binding of the fluorescent probe bis (8-anilinonaphthalene-1-sulfonate) (Bis-ANS). This is the first report of the use of hydrostatic pressure to inactivate pathogenic bacteria with the potential to lead to a vaccine.

In vitro human immune reactivity of fast protein liquid chromatography fractionated Paracoccidioides brasiliensis soluble antigens.

Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl. The FPLC system was able to resolve 7 fractions, enumerated from 0 to VI, according to the elution on the NaCl gradient. The analysis of each fraction on SDS-PAGE showed that fractions 0 to V were constituted by multiple protein bands with molecular mass ranging from 18 to 114 kDa. Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide. Sera from PCM patients presenting different clinical forms contained antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA. Further studies were designed to investigate the capacity of these fractions to induce cell proliferation. It was demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononuclear cells, while fraction 0 induced the lowest proliferative response among patients with PCM, in either acute, acute treated, or chronic forms.

Random amplified polymorphic DNA (RAPD) analysis of Paracoccidioides brasiliensis isolates.

Five Paracoccidioides brasiliensis isolates of humans origin were analyzed using three arbitrary primers (3301, 3304 and 3307 of 10, 9 and 10 oligonucleotídes respectively) in random amplified polymorphic DNA (RAPD) analysis. The analysis of the complex RAPD profiles obtained were carried out using the Dice similarity coefficient that distinguished the isolated Pb 02 from the others (Pb 18, Pb 192, Pb 265 and Pb SN). The results revealed limited intraspecific genomic variations in these P. brasiliensis isolates and indicate that RAPD can be useful for analysis of P. brasiliensis genome for characterization or differentiation within this genus.

Model of in vitro granulomatous hypersensitivity in human paracoccidioidomycosis.

With the purpose of studying the immunological components of granulomatous hypersensitivity in patients infected with Paracoccidioides brasiliensis, we used the model of in vitro granuloma formation developed for schistosomiasis studies, that correlates with in vivo granulomatous reactivity occurring around eggs trapped in organs of infected donors. In this case, granuloma formation can be determined examining cellular reactivity manifested as multiple cell layers surrounding antigen-conjugated polyacrilamide beads. Our results showed that peripheral blood mononuclear cells (PBMC) obtained from acute treated and chronic paracoccidioidomycosis patients proliferate and generate in vitro granulomas in response to P. brasiliensis antigens (PbAg). In contrast, no proliferation or granuloma formation were observed when PBMC from acute non-treated patients were used. These studies demonstrate the feasibility of investigating granulomatous hypersensitivity in P. brasiliensis-infected patients by using an in vitro granuloma model.

Production and characterization of monoclonal antibodies to the EDTA extract of Leptospira interrogans, serovar icterohaemorrhagiae.

Monoclonal antibodies (MABs) were produced against an ethylenediaminetetraacetate (EDTA) extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgG1 and IgG2b). The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature. Immunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. The MAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection.

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars.

Primers proposed for the diagnosis of the pathogenic spirochete Leptospira spp. (C. Gravekamp, H. V. D. Kemp, M. Franzen, D. Carrington, G.J. Schoone, G.J.J.M. Van Eys, C. O. R. Everard, R.A. Hartskeel, and W.J. Terpstra, J. Gen. Microbiol. 139:1691-1700, 1993) have been found to produce complex serovar-specific patterns under low-stringency PCR conditions. Such patterns obtained by low-stringency PCR, which maintain the specific band as an internal control, offer, an approach to the standardized identification of Leptospira serovars in clinical laboratories.

Pesquisa de aglutininas antileptospira em diferentes grupos profissionais na cidae de Londrina, Paraná / Anti-Leptospira agglutinins research in different professional groups in the city of Londrina, Paraná

Realizou-se um levantamento soroepidemiológico na cidade de Londrina - Paraná, para leptospirose, em 49 indivíduos näo expostos a risco, 75 trabalhadores da limpeza pública, 55 indivíduos com atividade em ambiente hospitalar e 29 trabalhadores do departamento de Agua e Esgoto. Dos 208 soros analisados pela reaçäo de soroaglutinaçäo microscópica, 28,4 por cento apresentaram aglutininas antileptospira. A maior positividade foi encontrada nos soros dos trabalhadores da limpeza pública (46,7 por cento)

[Anti-Leptospira agglutinins in different professional groups in the city of Londrina, Paraná]. / Pesquisa de aglutininas antileptospira em diferentes grupos profissionais na cidade de Londrina, Paraná.

Serum samples were obtained from 208 individuals in Londrina, Paraná, Brazil. The serum were analysed for leptospiral agglutinins by agglutination microscopic tests and 28.4% were positive. The highest positivity was found for the serum of garbage collector (46.7%).

Isolation and biological activities of endotoxin from Leptospira interrogans.

Endotoxins extracted with ethylenediaminetetraacetate (EDTA) from Leptospira interrogans serovars icterohaemorrhagiae and canicola and Leptospira biflexa serovar patoc were tested for various biological activities characteristic of endotoxins. The presence of lipopolysaccharide biological activity was demonstrated by the Limulus amoebocyte lysate test, pyrogenicity in rabbits, complement interaction inhibiting the erythrocyte lysis, and chicken-embryo lethality. The lipopolysaccharides did not induce the local Shwartzman reaction. The lipopolysaccharides of serovars icterohaemorrhagiae and canicola were immunogenic in rabbits and were cytotoxic to chicken-embryo fibroblasts.

Chemical and biological properties of endotoxin from Leptospira interrogans serovars canicola and icterohaemorrhagiae.

1. Endotoxin-like activity was extracted with phenol-chloroform-petroleum either (PCP) from Leptospira interrogans serovars icterohaemorrhagiae and canicola. Chemical analysis of leptospiral cells obtained from the PCP extract indicated the following distribution of lipopolysaccharide (LPS), protein and polysaccharide in mg/ml: 3.0, 4.5 and 1.0 for icterohaemorrhagiae and 3.3, 5.6 and 1.5 for canicola. 2. The preparations presented several biological activities: positive Limulus test (1.0 pg/ml) for icterohaemorrhagiae and canicola PCP extract and 0.5 pg/ml for E. coli O111:B4 LPS, lethality for chicken embryos (LD50 45, 25 and 1.0) for icterohaemorrhagiae, canicola and E. coli O111:B4 LPS, pyrogenicity in rabbits with an average increase in rectal temperature of 0.6 degrees C, 0.9 degrees C and 2.2 degrees C for canicola, icterohaemorrhagiae and E. coli O111:B4 LPS, reacted with complement inhibiting the lysis of sheep red blood cells, 62%, 75% and 90% for 2.0 micrograms/ml of icterohaemorrhagiae, canicola PCP extract and E. coli O111:B4 LPS. The PCP extract showed no cytotoxicity on chicken embryo fibroblasts and epithelial cells. 3. These results demonstrate that Leptospira endotoxin activity is similar to E. coli O111:B4 lipopolysaccharide.

Chemical and biological properties of endotoxin from leptospira interrogans serovars canicola and icterohaemorrhagiae

Endotoxin-like activity was extracted with phenol-chloroform-petroleum ether (PCP) from Leptospira interrogans serovars icterohaemorrhagiae and canicola. Chemical analysis of leptospiral cells obtained from the PCP extract i9ndicated the following distribution of lipopolysaccharide (LPS), protein and polysaccharide in mg/ml:3.0, 4.5 anmd 1.0 for icterohaemorrhagiae and 3.,3, 5.6 and for canicola. The preparations presented several biological activities: positive Limulus test (1.0 pg/ml) for icterohaemorraahagiae and canicola PCP extract and 0.5 pg/ml for E. coli 0111:B4 LPS, lethality for chicken embryos (LD 50 45, 25 and 1.0) for ecterohaemorrhagiae, canicola and E. coli 0111:B4 LPS, pyrogenicity in rabbits with an average increase in rectal temperature of 0.6 grade C, 0.9 grade C and 2.2 grade C for canicola, icterohaemorrhagiae and E. coli 0111: B4 LPS, reacted with complement inhibiting the lysis of sheep red blood cells 62%, 75% and 90% for 2.0 ug/ml of icterohaemorrhagiae, canicola PCP extract and E. coli 0111:B4 LPS. The PCP extract showed no cytotoxicity on chicken embryo fibroblasts and epithelial cells

Isolation and characterization on partially purified leptospiral antigens

Extrato metilico de Leptospira interrogans sorovar canicola foi purificado por precipitacao com acetona ou acetona e cloroformio. A antigenicidade nao foi alterada por aquecimento ou tratamento com pepsina e pronase, entretanto foi perdida quando o antigeno foi tratado com acido periodico. Analise quimica revelou a presenca de 40 por cento de carboidrato (22 por cento de metilpentose, 28 por cento de hexose), 4 por cento de proteina, 20 por cento de lipide e 2,7 por cento de fosfato. Reacao de fixacao de complemento realizada com soros de pacientes com leptospirose apresentou concordancia com a reacao de aglutinacao microscopica.

Isolation and characterization of partially purified leptospiral antigens.

The methanol extract of Leptospira interrogans serovar canicola was purified by precipitation with acetone or acetone and chloroform. The antigenicity of the antigen was not altered by heating or treatment with pepsin and pronase. However the antigenicity was lost when the antigen was treated with periodic acid. Chemical analysis revealed the presence of 40% carbohydrate (22% methylpentose, 28% hexoses), 4% protein, 20% lipid and 2.7% phosphate. The complement fixation test with sera from patients with leptospirosis agreed with the microscopic agglutination reaction.

The use of methanol extract of Leptospira interrogans in complement fixation tests for leptospirosis.

Methanol extracts were obtained from L. interrogans serovars icterohaemorrhagiae and canicola and L. biflexa serovar patoc. Human sera from 167 normal individuals and 40 patients with different infectious diseases tested by complement fixation tests showed negative reactions. Sera from 100 patients with a suspicion of leptospirosis were tested by complement fixation tests and microscopic agglutination reactions. Agreement of 84% was found for those two reactions. Positive microscopic agglutination tests at a dilution 1:20-1:400 with negative complement fixation tests were observed in 5% of patients and negative microscopic agglutination with complement fixation tests in the range of 1:20-1:1280 were observed in 11% of the cases.