Toxocariasis/cysticercosis seroprevalence in a long-term rural settlement, São Paulo, Brazil.

Seroprevalence of Toxocara and Taenia solium and risk factors for infection with these parasites were explored in a long-term rural settlement in São Paulo state, Brazil. An ELISA for the detection of anti-Toxocara IgG and IgE and anti-T. solium cysticerci was standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis and by vesicular fluid antigen from Taenia crassiceps cysticerci (VF). For cysticercosis, the reactive ELISA samples were assayed by Western blot using 18 kDa and 14 kDa proteins purified from VF. Out of 182 subjects, 25 (13.7%) presented anti-Toxocara IgG and a positive correlation between total IgE and the reactive index of specific anti-TES IgE (P=0.0265) was found amongst the subjects found seropositive for anti-Toxocara IgG. In these individuals 38.0% showed ocular manifestations. The frequency of anti-T. solium cysticerci confirmed by Western blot was 0.6%. Seropositivity for Toxocara was correlated with low educational levels and the owning of dogs. Embryonated eggs of Toxocara spp. were found in 43.3% of the analysed areas.

Cationic supported lipid bilayers for antigen presentation.

Polystyrene sulfate (PSS) particles (301 nm mean diameter) were covered with single cationic dioctadecyldimethylammonium bromide (DDA) bilayers and used for antigen adsorption and presentation. The antigen was a mixture of purified 18/14 Taenia crassiceps proteins (18/14-Tcra). Firstly, the DDA/PSS assembly was characterized at 1mM NaCl and 5 x 10(9) PSS particles/mL over a range of DDA concentrations (0.001-1mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.01 mM DDA is enough to produce homodisperse and cationic bilayer-covered particles. Secondly, under these experimental conditions, 18/14-Tcra adsorption isotherms onto biomimetic particles or aluminium hydroxide (Al(OH)3) yield limiting adsorption of 0.36 and 1.32 mg protein/mg biomimetic particles or Al(OH)3, respectively. Finally, in mice, superior humoral and cellular immunoresponse from serum IgG and footpad swelling was obtained for antigen/biomimetic particles in comparison to conventional Al(OH)3. Cationic bilayer-covered particles are a novel, highly organized and, possibly, general immunoadjuvant for antigen presentation and subunit vaccine design.

Low fractional excretion of urine sodium in acute renal failure due to sepsis.

A low fractional excretion of sodium (FENa) of less than 1% was present in two patients who had acute renal failure due to sepsis. Both patients had bacteremia and had undergone major abdominal and vascular surgery. Prerenal azotemia due to volume depletion was not present as adequate central filling pressures were maintained with a Swan-Ganz catheter. Interstitial nephritis and obstructive uropathy were carefully ruled out. Acute renal failure due to sepsis should be included among the other conditions recently reported with a low FENa. This is of great importance as errors in fluid management are possible in this high-risk patient population when much reliance is placed on the interpretation of a low FENa of less than 1%.

Evaluation of IgG, IgM, and IgA antibodies to mycoplasma penetrans detected by ELISA and immunoblot in HIV-1-infected and STD patients, in São Paulo, Prazil.

The aim of the present study was to determine the frequency of IgG, IgA, and IgM antibodies to Mycoplasma penetrans in HIV-1-infected patients and in patients with sexually transmitted diseases. We tested serum samples from 106 HIV-1-positive patients and 110 individuals with clinical symptoms of urethritis. ELISA and the immunoblot test were performed using M. penetrans lipid associated membrane proteins as antigen. By ELISA, we found a higher frequency (P < 0.05) of IgG against M. penetrans in HIV-1-infected and STD patients (25.5 and 17.3%) than in controls (1.2%), as well as a higher frequency of IgA (P < 0.05) (15.1 and 17.3% compared to 1.2%). For IgM, no differences were observed (P >/= 0.05) (3.8, 9.1, and 5. 8%, respectively). When the frequencies of IgG, IgM, and IgA antibodies of the HIV-1-infected patients were compared taking into account the CD4/CD8 cell ratios < 0.3 and >/= 0.3, no significant differences were observed between the two groups (13.3, 10, and 20%, compared to 20, 0, and 5%, respectively) (P > 0.05), possibly due to the low number of samples on which we could perform T-cell counts (53/106). The M. penetrans peptide of 38 kDa, considered immunodominant, was recognized in immunoblot by 51.8% of positive sera by ELISA for IgG, 50.0% for IgM, and 75% for IgA in the AIDS patients group, and by 47.4, 60.0, and 75.0%, respectively, in the sexually transmitted disease group. Cross-reactions in immunoblot for IgG were observed in sera from individuals infected with Mycoplasma pneumoniae and Mycoplasma hominis, and cross-reactions in immunoblot for IgA were observed in sera from individuals infected with M. hominis; all of them were ELISA negative to M. penetrans.

Immunoblot with cerebrospinal fluid from patients with neurocysticercosis using antigen from cysticerci of Taenia solium and Taenia crassiceps.

A comparative study was conducted on membrane (M) and vesicular fluid (VF) from cysticerci of Taenia solium (Tso) obtained from naturally infected swine and the Taenia crassiceps ORF strain (Tc) maintained by experimental infection of female BALB/c mice. The study was carried out using immunoblotting to detect antibodies in cerebrospinal fluid (CSF) from patients with neurocysticercosis. No reactivity was observed in the 32 samples from a control group. Of the 23 CSF fluid samples from patients with neurocysticercosis, 22 (95.6%) were reactive in the M-Tso blot and 21 (91.3%) were reactive in the other three blots (VF-Tso, M-Tc, and VF-Tc). Immunodominant peptides in each antigen were 98-92 kD, 56-52 kD, and 72-68 kD in M-Tso; 72-68 kD, 120 kD, 155 kD, 98-94 kD, 76 kD, and 115-108 kD in VF-Tso: 72 kD, 62 kD, and 42 kD in M-Tc; and 72-68 kD and 95-92 kD in VF-Tc. The cross-reactivity observed in the immunoblots performed on CSF samples from patients with neurocysticercosis indicates that the parasites share important epitopes present at sufficient concentrations for use in immunologic tests.

Immunodiagnosis of human leptospirosis by dot-ELISA for the detection of IgM, IgG, and IgA antibodies.

A dot-ELISA was evaluated using antigen obtained from Leptospira interrogans cultures of the serovars brasiliensis, canicola, cynopteri, hebdomadis, and icterohaemorrhagiae for the detection of human IgM, IgG, and IgA. Single serum samples from 63 patients with the icterohemorrhagic form of leptospirosis in the acute phase, collected 3-14 days (mean = 7 days) after the onset of symptoms were tested. Ten patients were examined during convalescence and followed up for a period of 4-12 months. For a control group, serum samples from 10 apparently healthy individuals with no clinical or epidemiologic history of leptospirosis, and from 38 patients with nonleptospiral illnesses were used. In the acute phase, IgM antibodies were detected in 62 (98%) of 63 patients and IgG and IgA were observed in 70% and 76% of them, respectively. For the admission serum samples, the predictive value negative of the dot-ELISA was 98% for IgM, 72% for IgG, and 76% for IgA detection. All 10 patients followed-up during convalescence showed IgM antibodies up to the sixth month, decreasing to 57% by the 10th month, and persisting in only one of six patients during the 11th and 12th months of follow-up. Immunoglobulin G was detected in six patients up to the fourth month and in two of six individuals up to the end of follow-up. Immunoglobulin A was observed in all patients up to the end of the first month, decreasing progressively up to the sixth month, and was no longer detected in any patients from seventh to the 12th months of follow-up. The dot-ELISA can be used as an important laboratory screening test, especially when detecting IgM antibodies. It proved to be effective in the diagnosis of human leptospirosis, and appears to have advantages in terms of yield, time, and case of execution and low cost.

Immunodiagnosis of human leptospirosis using saliva.

When the enzyme-linked immunosorbent assay for the detection of specific immunoglobulin M class antibodies was applied to paired saliva and serum samples from 40 patients with leptospirosis, positivity was 87.5% and 100%, respectively. No positive result was obtained with any saliva or serum sample from 60 individuals used as controls. These results suggest the alternative use of saliva for diagnosis and for epidemiological studies of human leptospirosis.

Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and cross-reactivities in human infections and immunized animals.

Helminth antigens were investigated in the search for accessible heterologous antigens capable to discriminate different helminthiases, by the enzyme linked immunosorbent assay (ELISA) and the immunoblot assay (IB). Antigens used were: Taenia solium cysticercus total saline (Tso); Taenia crassiceps cysticercus vesicular fluid (Tcra-VF); T. crassiceps cysticercus glycoproteins (Tcra-GP and Tcra-(18-14)-GP); Toxocara canis larva excretory-secretory (TES); Schistosoma mansoni adult total saline (Sm) and Echinococcus granulosus hydatid fluid (Eg). The assayed sera were from patients with: cysticercosis (n = 18); toxocariasis (n = 40); schistosomiasis (n = 19) and hydatidosis (n = 50) with proven clinical and laboratory diagnosis, and sera from rabbits immunized with Tso, Tcra-VF, TES and Eg. Cross-reactivity occurred mostly between infections caused by Taenia and Echinococcus or in immunized rabbits, by ELISA. Moreover, the cross-reactivity among helminthiases was found with the use of antigens belonging to phylogenetically related parasite species, Eg, Tso and Tcra-VF, by sharing same antigenic components. Lower cross-reactivities were obtained by IB technique, when only peptides were considered as antigens, and the use of T. crassiceps purified glycoproteins demonstrated high sensitivity and specificity in the diagnosis of human cysticercosis, similarly to that using homologous antigen (Tso) by the same technique.

The relationship between salivary cortisol concentrations and anxiety in adolescent and non-adolescent pregnant women.

The main purpose of the present study was to determine the relationship between salivary cortisol concentrations and self-report anxiety in 50 adolescent and 178 non-adolescent women during the last month of pregnancy. The subjects were randomly selected from a previous study involving women who attended antenatal care from September 1997 to August 2000 at 17 health services in Southeast Brazil. Salivary cortisol was measured with an enzyme immunoassay kit, and anxiety was assessed by the State-Trait Anxiety Inventories (STAI) of Spielberger. After saliva collection, the participants completed the STAI. Mean concentrations of cortisol for both pregnant adolescents (14.17 +/- 6.78 nmol/l) and non-adolescents (13.81 +/- 8.51 nmol/l) were similar (P = 0.89). Forty-three percent of the pregnant adolescents and 30.5% of the non-adolescents felt anxious at the time of being questioned (State Anxiety Inventory (SAI) scores >40; P = 0.06). Cortisol concentrations in adolescents were negatively related to the SAI scores (r = -0.39; P = 0.01) which assess a temporary condition of anxiety. There was a statistically significant difference in mean cortisol concentrations between adolescents with low (<=40) and high (>40) SAI scores (P = 0.03, t-test), but no differences for non-adolescents. The negative relationship between salivary cortisol concentrations and anxiety scores in adolescents may be due to puberty-related hormone differences during this period of life. Pregnant adolescents may possess unique biological or psychological characteristics compared to adults and non-pregnant adolescents. Thus, we need to know more about the hypothalamic-pituitary-adrenocortical axis of pregnant adolescents.

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) for Chagas' disease serodiagnosis.

1. Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) was standardized and evaluated for the diagnosis of Chagas' disease, in comparison with the conventional serological tests indirect immunofluorescence (IFI), passive hemagglutination (PHA) and complement fixation (CF). 2. A total of 236 serum samples positive and negative for the serodiagnosis of Chagas' disease were studied. The group included 50 serum samples serologically positive for leishmaniasis and 36 positive for malaria. 3. The best diagnostic performance of DIG-ELISA was observed when serum samples were diluted to 1:8 and a diameter of zero mm (no color) was taken as the cut-off. Under these conditions, the relative indices of sensitivity, specificity and agreement were 100%. High positive correlation coefficients were obtained between DIG-ELISA and IFI (r1 = 0.9010), PHA (r2 = 0.8943) and CF (r3 = 0.8269). 4. We conclude that DIG-ELISA provides an alternative technique for screening chagasic infections, as well as for seroepidemiological surveys mainly because it is simple, easy to carry out and does not require expensive equipment.

Performance of the ELISA test for swine cysticercosis using antigens of Taenia solium and Taenia crassiceps cysticerci.

Studies were conducted to evaluate antigens of Taenia solium (Tso) and Taenia crassiceps (Tcra) cysticerci in the ELISA test for the diagnosis of swine cysticercosis. The samples analyzed were cysticercosis positive and negative control sera and heterologous sera. Four antigens were assayed: vesicular fluid (VF) and crude (T) Tcra and scolex (S) and crude (T) Tso. All antigens showed good performance, but VF-Tcra was the best followed by T-Tcra. Sensitivity rates of ELISA were respectively, in 2nd and 3rd standard deviation cut-offs, 96.0 and 80.0% for the VF antigen and specificity of 97.5 and 100.0%. Cross-reactivity was verified only for hidatidosis and ascaridiosis. Due to the high performance observed, the ELISA test using Tcra antigens should be recommended for the diagnosis of swine cysticercosis.

Cross-reactivity of anti-Taenia crassiceps cysticerci immune antibodies with Taenia solium antigens.

A comparative study of antibody production was carried out using BALB/c mice immunized with 20 or 50microg vesicular fluid (VF)-Tcra (Taenia crassiceps) antigens, and gel of <30kD or eluate from <30kD peptides. Good IgM, IgA and IgG levels were detected by ELISA-Tcra and the antibodies presented reactivity with the <20kD peptides when tested by immunoblotting-Tcra. The antibodies from animals immunized with 20 and 50microg presented high anti-Tso cross-reactivity in ELISA (IgG>>IgM and IgA). All groups presented IgG antibodies identifying the 12kD Tso-peptide.

Hamburger meat identification by dot-ELISA.

The use of low cost meats to adulterate meats and meat products has been reported. Appropriate methods of analysis then are needed in order to detect this practice. The dot-ELISA method was used to identify the meat of different animal species and to detect adulteration of hamburgers. Antisera to bovine, chicken, swine and horse albumin were produced and they could detect the meat extract of the homologous species at concentrations as low as 0.6%. Thus, the anti-albumin antisera could identify bovine, chicken, swine and horse meat with adequate specificity and sensitivity both in isolation and when added to hamburger. Commercial samples of bovine, chicken and swine hamburgers showed no adulteration with bovine, chicken, swine or horse meats. Our expectation of hamburger adulteration was not confirmed.

Detection of mycoplasmas in urethral swabs from HIV-1 infected patients and control individuals using culture techniques and polymerase chain reaction.

The objective of the present study was to determine the prevalence of certain mycoplasma species, i.e., Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma penetrans, in urethral swabs from HIV-1 infected patients compared to swabs from a control group. Mycoplasmas were detected by routine culture techniques and by the Polymerase Chain Reaction (PCR) technique, using 16SrRNA generic primers of conserved region and Mycoplasma penetrans specific primers. The positivity rates obtained with the two methods were comparable. Nevertheless, PCR was more sensitive, while the culture techniques allowed the quantification of the isolates. The results showed no significant difference (p < 0.05) in positivity rates between the methods used for mycoplasma detection.

[An evaluation of the ELISA-IgM test in the early diagnosis of human leptospirosis]. / Avaliação do teste ELISA-IgM no diagnóstico precoce da leptospirose humana.

Thirty-seven sera samples from patients with leptospirosis icterohaemorrhagic form were studied with a time interval of 2 to 12 days between the beginning of the symptoms and the collection blood samples. It was isolated leptospira of 5 patients' hemocultures (13.5%) and from 4 of these the etiological agent pertained to the serogroup Icterohaemorrhagiae serovar copenhageni. Thirty-five of them (94.6%), including the four patients whose the etiological agent was isolated, showed reactivity in the enzyme linked immunosorbent (ELISA) IgM test. By this way, it was demonstrated that this test is important for a rapid diagnosis of human leptospirosis, even in the beginning of the disease, when there is still leptospiraemic phase.

[The positivity index of the immunoenzyme reaction (ELISA) for cysticercosis in the cerebrospinal fluid (CSF) and in the serum of epilepsy patients]. / Indice de positividade da reação imunoenzimática (ELISA) para cisticercose no líquido cefalorraquidiano (LCR) e no soro de pacientes com epilepsia.

Fifty patients with epilepsy seen in three Londrina Neurological Services, in Paraná, were studied. The positivity prevalence of the enzyme-linked immunosorbent assay (ELISA) for cysticercosis in the cerebrospinal fluid (CSF) and in the serum of these patients was 34.0% and 20.0%, respectively. There was statistically significant difference when these two rates were compared with the reaction positivity in the CSF and the serum in the control group, formed by individuals without neurological diseases. There was no association between the type of seizure (generalized or partial) and the positivity index of ELISA for cysticercosis in the CSF. A greater number of patients with positive ELISA for cysticercosis in the rural area dwellers was found, in relation with the urban area dwellers. From the obtained results in our study we came to the following conclusions: 1. The high positivity ELISA rates for cysticercosis in the CSF and in the patients serum with epilepsy indicate that neurocysticercosis is an important seizure cause in Londrina, PR. 2. The positivity prevalence of ELISA for cysticercosis in CSF was greater in epileptic patients from the rural area than the ones from the urban area. 3. There was no association between the type seizure (generalized or partial) and the ELISA cysticercosis positivity rate in the CSF. 4. The high positivity prevalence of ELISA in the CSF and in the epileptic patients serum in Londrina indicates the priority of performing epidemiologic inquiry to establish the real cysticercosis prevalence in the city. ELISA may be used with this finality due to its high sensibility, its low cost and its simple performance.

[The incidence of hepatitis B markers in pregnant women at their first consultation in metropolitan-area health centers, São Paulo, Brazil]. / Frequência de marcadores de hepatite B em gestantes de primeira consulta em centros de saúde de área metropolitana, São Paulo, Brazil.

Hepatitis B is a severe disease when acquired during the neonatal period. The identification of the infected pregnant women allows prevention of newborn infection by active and passive immunization soon after birth. We studied pregnant women in their first visit to eight different primary medical centers in Butantan, a subdistrict of S. Paulo city. 477 samples were tested for anti-HBc. From 44 (9.2%) anti-HBc positive samples, 2 (0.4%) were HBsAg positive and 37 (7.7%) were anti-HBs positive. A risk factor for hepatitis B could only be detected in 8 (18.9%) of the 44 anti-HBc positive samples.

DOT-ELISA for detection of anti-Cysticercus cellulosae antibodies in human cerebrospinal fluid using a new solid-phase (resin-treated polyester fabrics). Preliminary report.

A dot enzyme-linked immunosorbent assay (DOT-ELISA) was developed to detect specific antibodies in cerebrospinal fluid (CSF) for human neurocysticercosis immunodiagnosis, with Cysticercus cellulosae antigen dotted on a new solid-phase. This was represented by sheets of a synthetic polyester fabric impregnated with a polymerized resin (N-methylol-acrylamide). A very stable preparation was thus obtained, the antigen being covalently bound by cross-linking with free N-methylol groups on the resin. Since robust, no special care was necessary for handling the solid-phase. The test could be performed at room-temperature. From 30 CSF samples assayed, 14 were positive, from a group of 15 cases of neurocysticercosis, with titers from 1 to 128; 15 other samples, from normals or other neurological diseases, were all negative. Test characteristics seem to indicate it as adequate for epidemiological surveys. A more detailed study on sensitivity, specificity, reproducibility and the use in serum samples is being conducted.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens.

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens.

We describe the production of the potential monoclonal antibodies (MoAbs) using BALB/c mice immunized with vesicular fluid (VF)-Tcra (T. crassiceps) antigen. Immune sera presented anti-VF-Tcra (<20kD) IgG and IgM antibodies with cross-reactivity with T. solium (Tso) antigen (8-12, 14, and 18 kD). After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.