Influence of bacteria upon cytopathic effect and erythrophagocytosis of different axenic strains of Entamoeba histolytica

At this moment, the duality of species suggested for E. histolytica is being considered for discussion. In order to contribute to settling this question, we investigated the possibility of conversion of avirulent ameba to virulent ones, as well as, the possibility of increasing virulence of virulent strains, by means of association with bacteria. Five strains of E. histolytica were employed, two of them regarded as avirulent and three virulent ones. Amebas were associated with the bacteria Escherichia coli 055 and 0115, previously demonstrated as capable to modify the pathogenic behavior of E. histolytica. Changes in virulence of amebas were assessed by cytopathic effect upon cultured mammal cells and erythrophagocytosis. The virulence of pathogenic strains was significantly increased after bacteria association in opposition to what was observed for nonpathogenic ones, which were not influenced by bacteria association.

Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immuno-enzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Intestinal spirochetosis: first cases reported in Brazil and the use of immunohistochemistry as an aid in histopathological diagnosis

Colonization of the colon and rectum by intestinal spirochetes is detected for the first time in Brazil in 4 of 282 (1.41%) patients who had undergone sigmoidoscopy and/or colonoscopy with a histopathological diagnosis of chronic non specific-colitis. This frequency is probably understimated, since surgically obtained specimens were not considered in the present study. Histopathological diagnosis was performed using routine stains like hematoxylin-eosin which showed the typical, of 3-µm thick hematoxyphilic fringe on the brush border of the surface epithelium, and by silver stains like the Warthin-Starry stain. Immunohistochemical procedures using two, polyclonal, primary antibodies, one against Treponema pallidum and the other against Leptospira interrogans serovar copenhageni serogroup Icterohaemorrhagiae cross-reacted with spirochetal antigen/s producing a marked contrast of the fringe over the colonic epithelium, preserving the spiral-shaped morphology of the parasite. In one case with marked diarrhea, immunohistochemistry detected spirochetal antigen/s within a cell in an intestinal crypt, thus demonstrating that the infection can be more widely disseminated than suspected using routine stains. Immunohistochemical procedures, thus, greatly facilitate the histological diagnosis of intestinal spirochetosis and may contribute to a better understanding of the pathogenesis of the disease. Transmission and scanning electron microscopy performed in one case showed that the spirochete closely resembled the species designated as Brachyspira aalborgi.

Virulence parameters in the characterization of strains of Entamoeba histolytica

Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic disease

Use of doxycycline for leptospirosis after high-risk exposure in São Paulo, Brazil

A clinical trial pilot study, double-blinded, randomized, and controlled with a placebo to assess the effectiveness of oral doxycycline (200 mg, single dose) in preventing leptospirosis after high exposure to potentially contamined water was performed in São Paulo, SP, Brazil. Confirmed cases were defined as those with leptospira IgM antibody and symptoms; asymptomatic cases were those presenting with IgM antibodies but no symptoms; and suspected cases were individuals with symptoms but no IgM antibody. Forty subjects were given doxycycline and 42 were given placebo. In the drug-treated group there were 2 confirmed cases, 11 asymptomatic cases, and 6 suspected cases. In the placebo group there were 5 confirmed, 6 symptomatic, and 5 suspected cases. Even though we found a protective association of doxycycline for confirmed leptospirosis cases (RR = 2.3) and seroconversion only (RR = 2.0), the association was not statistically significant because of the small number of individuals enrolled in this pilot study. We observed that the 22% of the volunteers already had IgM antibodies to leptospirosis at the first sampling. Finally, the attack rate to confirmed, asymptomatic, and suspected cases of Leptospirosis was 8.5%, 22%, and 13%, respectively, in this population.

Virulence Factors IN Fungi OF Systemic Mycoses

Pathogenic fungi that cause systemic mycoses retain several factors which allow their growth in adverse conditions provided by the host, leading to the establishment of the parasitic relationship and contributing to disease development. These factors are known as virulence factors which favor the infection process and the pathogenesis of the mycoses. The present study evaluates the virulence factors of pathogenic fungi such as Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Paracoccidioides brasiliensis in terms of thermotolerance, dimorphism, capsule or cell wall components as well as enzyme production. Virulence factors favor fungal adhesion, colonization, dissemination and the ability to survive in hostile environments and elude the immune response mechanisms of the host. Both the virulence factors presented by different fungi and the defense mechanisms provided by the host require action and interaction of complex processes whose knowledge allows a better understanding of the pathogenesis of systemic mycoses.

CONSTITUTIVE MELANIN IN THE CELL WALL OF THE ETIOLOGIC AGENT OF LOBO'S DISEASE

Lobo's disease is a chronic granulomatous disease caused by the obligate pathogenic fungus, whose cell walls contain constitutive melanin. In contrast, melanin does not occur in the cell walls of Paracoccidioides brasiliensis when stained by the Fontana-Masson stain.

Paracoccidioides brasiliensis: A MYCOLOGIC AND IMMUNOCHEMICAL STUDY OF TWO STRAINS

The authors conducted a mycologic, immunochemical and molecular biology study on two strains of Paracoccidioides brasiliensis, one of them, called IBIÁ, isolated from soil in the municipality of IBIÁ (Minas Gerais) by Silva-Vergara et al. (l996,1998)20,21, and the other, BAT, cultivated from a human case of paracoccidioidomycosis in Ribeirão Preto (São Paulo/Brazil) by Freitas da Silva (l996)6. Both strains showed cotton-like (M) and yeast-like (Y) forms and were pathogenic for testicularly inoculated guinea pigs, producing granulomatous and/or suppurative orchitis. Immunochemically was demonstrated the presence of gp43 by double immunodiffusion, immunoelectrophoresis and immunoblotting.

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Relevant prevalence of Mycoplasma hominis and Ureaplasma urealyticum serogroups in HIV-1 infected men without urethritis symptoms

M. hominis and U. urealyticum are the better-known mycoplasma species pathogenic to the human genitourinary tract, causing mainly urethritis, bacterial vaginosis and pregnancy complications. In HIV-infected patients, the prevalence and role of these species is still not well known. The aim of this work was to determinate the prevalence of these species in this group of male patients (HIV group), in comparison to a group of men with clinical symptoms of urethritis (STD group). M. hominis was isolated from 7.5% patients (8/106) and U. urealyticum from 18.9% patients (20/106) from the HIV group, being among these 62.5% and 85% in significant concentrations, respectively. In the STD group these rates were 0.9% (1/110) for M. hominis and 13.6% (15/110) for U. urealyticum, being 100% and 93.3% in significant concentrations, respectively. We could demonstrate infection rates by these mycoplasma species in the HIV group as high as the one found in the STD one, what may indicate the occurrence of opportunistic infections in our population. This fact is discussed here because in immunosuppressed patients, specially M. hominis has been reported causing severe infections, even systemically.

Borrelia burgdorferi antibodies in dogs from Cotia county, São Paulo State, Brazil

Dogs sera samples collected from Cotia County, São Paulo were tested using indirect immunoenzymatic test (ELISA) in order to study Lyme disease serology in dogs. ELISA method was standardized and G39/40 North American strain of Borrelia burgdorferi was used as antigen. Positive results were confirmed employing the Western blotting technique. Because of the possibility of cross-reactions, sera were also tested for different serological strains of Leptospira interrogans and L. biflexa using microscopic sera agglutination test. Twenty-three of 237 (9.7%) serum samples were positive in the ELISA; 20 of them (86.9%) were confirmed by the Western blotting, what suggests that Cotia may be a risk area for Lyme disease. Although 4 samples (1.7%) were positive for Lyme disease and leptospirosis, no correlation was found between the results (X² = 0.725; p = 0.394) what suggests absence of serological cross reactivity.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Liver histology in co-infection of hepatitis C virus (HCV) and Hepatitis G virus (HGV)

As little is known about liver histology in the co-infection of hepatitis C virus (HCV) and hepatitis G virus (HGV), HGV RNA was investigated in 46 blood donors with hepatitis C, 22 of them with liver biopsy: co-infection HCV / HGV (n = 6) and HCV isolated infection (n = 16). Besides staging and grading of inflammation at portal, peri-portal and lobular areas (Brazilian Consensus), the fibrosis progression index was also calculated. All patients had no symptoms or signs of liver disease and prevalence of HGV / HCV co-infection was 15.2%. Most patients had mild liver disease and fibrosis progression index, calculated only in patients with known duration of infection, was 0.110 for co-infection and 0.130 for isolated HCV infection, characterizing these patients as "slow fibrosers". No statistical differences could be found between the groups, although a lesser degree of inflammation was always present in co-infection. In conclusion co-infection HCV / HGV does not induce a more aggressive liver disease, supporting the hypothesis that HGV is not pathogenic.

Clinical-epidemiological study of leptospirosis in humans and reservoirs in Yucatán, México

A leptospirosis clinical-epidemiological study was made in humans and reservoirs in the state of Yucatán, México. Interviews and serological analyses were made on 400 persons from an open population, 439 probable cases of leptospirosis and 1060 animal reservoirs (cows, pigs, dogs, rats and opossums). IgM Leptospira DipstickTM and Microscopic Agglutination Test (MAT) was used to detect human antibodies to leptospiras and serovar respectively. Leptospirosis incidence in humans was 2.2/100,000 inhab. in 1998, 0.7/100,000 in 1999 and 0.9/100,000 in 2000. Overall seroprevalence was 14.2%, relatively unchanged from seroprevalences observed 20 years ago. Highest seropositivity was found in people over 56 years of age, predominating males over females. Predominant serovars in the open population were tarassovi, hardjo, pomona and panama. Leptospirosis cases were most frequent in rural areas, and the anicteric course predominated over the icteric. The panama, icterohaemorrhagiae and pomona serovars predominated in both anicteric and icteric courses. Dogs, pigs and rodents had the highest seropositivity among the reservoirs. Contact with rodents and natural water sources were significant factors (p £ 0.05). Human cases (74%) occurred during the rainy season. It is concluded that leptospirosis is still a serious illness with important clinical and epidemiological implications in the state of Yucatán, Mexico.

Penicillin at the late stage of leptospirosis: a randomized controlled trial

There is evidence that an early start of penicillin reduces the case-fatality rate of leptospirosis and that chemoprophylaxis is efficacious in persons exposed to the sources of leptospira. The existent data, however, are inconsistent regarding the benefit of introducing penicillin at a late stage of leptospirosis. The present study was developed to assess whether the introduction of penicillin after more than four days of symptoms reduces the in-hospital case-fatality rate of leptospirosis. A total of 253 patients aged 15 to 76 years with advanced leptospirosis, i.e., more than four days of symptoms, admitted to an infectious disease hospital located in Salvador, Brazil, were selected for the study. The patients were randomized to one of two treatment groups: with intravenous penicillin, 6 million units day (one million unit every four hours) for seven days (n = 125) and without (n = 128) penicillin. The main outcome was death during hospitalization. The case-fatality rate was approximately twice as high in the group treated with penicillin (12%; 15/125) than in the comparison group (6.3%; 8/128). This difference pointed in the opposite direction of the study hypothesis, but was not statistically significant (p = 0.112). Length of hospital stay was similar between the treatment groups. According to the results of the present randomized clinical trial initiation of penicillin in patients with severe forms of leptospirosis after at least four days of symptomatic leptospirosis is not beneficial. Therefore, more attention should be directed to prevention and earlier initiation of the treatment of leptospirosis.