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.

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 percent 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.

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.

Excretory/secretory antigens (ES) from in-vitro cultures of Taenia crassiceps cysticerci, and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis.

Antigens were obtained from cysticerci of the ORF strain of Taenia crassiceps, by culture of cysts in protein-free hybridoma medium (PFHM). Budding of new vesicles was observed after 24-48 h. Excretory/secretory (ES) antigens (peptides of <20 kDa) were recovered in the medium after culture for 48 h. SDS-PAGE analysis of vesicular-fluid (VF) antigens (obtained by rupturing T. crassiceps cysticerci in PFHM) and the ES antigens indicated partial homology between the two preparations. ES peptides of 18- and 14-kDa were recognized by polyclonal antibodies produced in rabbits immunized either with the VF antigens or with a total-antigen preparation of T. solium cysticerci. Antibodies present in samples of serum or cerebrospinal fluid (CSF) from patients with neurocysticercosis also reacted with ES peptides. An anti-ES monoclonal antibody detected antigens in the CSF from 10 patients with neurocysticercosis, showing the antigenic homology of the ES antigens with those of T. solium cysticerci in human infections.

Serodiagnosis of human cysticercosis by using antigens from vesicular fluid of Taenia crassiceps cysticerci.

Neurocysticercosis (NC), caused by the presence of Taenia solium metacestodes in tissues, is a severe parasitic infection of the central nervous system with universal distribution. To determine the efficiency of enzyme-linked immunosorbent assay (ELISA) and immunoblot with antigens of T. crassiceps vesicular fluid (Tcra) compared to standard techniques (indirect immunofluorescence test [IFT] and complement fixation test [CFT]) using T. solium cysticerci (Tso) for the serodiagnosis of NC, we studied serum samples from 24 patients with NC, 30 supposedly healthy individuals, 76 blood bank donors, 45 individuals with other non-NC parasitoses, and 97 samples from individuals screened for cysticercosis serology (SC). The sensitivity observed was 100% for ELISA-Tso and ELISA-Tcra, 91.7% for the IFT, and 87.5% for the CFT. The specificity was 90% for ELISA-Tso, 96.7% for ELISA-Tcra, 50% for IFT, and 63.3% for CFT. The efficiency was highest for ELISA-Tcra, followed by ELISA-Tso, IFT, and CFT. Of the 23 samples from SC group, which were reactive to ELISA-Tso and/or ELISA-Tcra, only 3 were positive to immunblot-Tcra (specific peptides of 14- and 18-kDa) and to glycoprotein peptides purified from Tcra antigen (gp-Tcra), showing the low predictive value of ELISA for screening. None of the samples from the remaining groups showed specific reactivity in immunoblot-Tcra. These results demonstrate that ELISA-Tcra can be used as a screening method for the serodiagnosis of NC and support the need for specific tests for confirmation of the results. The immunoblot can be used as a confirmatory test both with Tcra and gp-Tcra, with the latter having an advantage in terms of visualization of the results.

Antigen-specific suppression of cultured lymphocytes from patients with neurocysticercosis.

The biological parasite-host interactions involved in neurocysticercosis (NC) are of a complex nature. A lymphoproliferation assay was performed using mononuclear cells from 11 patients with NC, who were classified according to the alterations obtained by imaging examinations. Antigen extracts from the membrane and/or scolex of Taenia solium and from the vesicular fluid of Taenia crassiceps were used. Mononuclear cells from patients with NC showed antigen-specific suppression when compared with a control group. The patients presenting calcified cysts showed higher suppression when compared with patients in the active phase of disease. The antigen in the vesicular fluid of T. crassiceps seems to play a suppressor role in vitro, completely inhibiting cell proliferation induced by the mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen.

Cysticercus antigens in cerebrospinal fluid samples from patients with neurocysticercosis.

Antigens were detected in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis (NC) by enzyme-linked immunosorbent assay (ELISA) using polyclonal sera of rabbit anti-Taenia solium cysticerci (anti-Tso) and anti- Taenia crassiceps cysticerci vesicular fluid (anti-Tcra or anti-Tcra <30 kDa). A group of NC patients (n = 174) were studied (NC), including 40 patients in different phases of the disease. ELISAs carried out with the anti-Tso, anti-Tcra, and anti-Tcra <30 kDa showed sensitivities of 81.2, 90, and 95.8% and specificities of 82, 98, and 100%, respectively. The 14- and 18-kDa low-molecular-weight peptides were only detected in CSF samples from patients with NC by immunoblotting with anti-Tso and anti-Tcra sera. Because of the importance of the diagnosis and prognosis of cysticercosis, the detection of antigens may contribute as an additional marker to the study and clarification of the parasite-host relationship.

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.

Total IgE detection in paired cerebrospinal fluid and serum samples from patients with neurocysticercosis.

Neurocysticercosis (NC), the presence of Taenia solium metacestodes in tissues, is the most frequent and severe parasitic infection of the central nervous system. We investigated the presence of total IgE by an automated chemiluminescence assay in 53 paired cerebrospinal fluid (CSF) and serum samples from patients with NC (P) and in 40 CSF samples from individuals with other neurological disorders as the control group (C). Total IgE concentration ranged from 1.2 to 6.6 IU/ml (mean = 1.4 IU/ml, standard deviation-sd = 1.1 IU/ml) in 28.3% of CSF samples from the P group, a value significantly higher than for the C group ( pound1.0 IU/ml). The serum samples from the P group showed concentrations ranging from 1. 0 to 2330.0 IU/ml (mean = 224.1 IU/ml, sd = 452.1 IU/ml), which were higher than the normal value cited by the manufacturer (<100.0 IU/ml) in 32.1% of the samples. A significant difference was observed in CSF samples from the P and C groups (p = 0.005) and in serum samples from the P group compared to the normal value (p = 0. 005), with sera showing more frequent abnormal results.

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci.

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.

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.

Neurocysticercosis: detection of IgG, IgA and IgE antibodies in cerebrospinal fluid, serum and saliva samples by ELISA with Taenia solium and Taenia crassiceps antigens.

We assayed samples of cerebrospinal fluid (CSF), serum and saliva from patients with neurocysticercoses, control group and individuals with other parasitoses, by ELISA with Taenia crassiceps vesicular fluid antigen (Tcra) and Taenia solium total antigen (Tso) for the detection of antibodies. The sensitivity for IgG-Tcra was 100% for CSF and serum, and 32.0% for saliva; and for IgG-Tso 100% for CSF, 80.0% for serum and 24.% for saliva. Specificity was 100% for CSF and 80.0% for serum with both antigens, and 100% for saliva with Tcra and 87.5% with Tso. The sensitivity and specificity for IgA-Tcra was, respectively, 40.0% and 100% for CSF, 36.0% and 97.1% for serum, and 4.0% and 90.0% for saliva. IgE detection showed 24.0% sensitivity and 97.1% specificity for serum, with no detection in CSF samples. The search for antibodies revealed the presence of IgG > IgA > IgE in CSF, serum and saliva samples, with IgG being present in all phases of the disease, while IgA/IgE were more frequent in the inactive form.

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.

Specific Taenia crassiceps and Taenia solium antigenic peptides for neurocysticercosis immunodiagnosis using serum samples.

Neurocysticercosis (NC), i.e., the presence of the larval form of Taenia solium in tissues, is the most frequent and severe infection involving the central nervous system. Paired serum and cerebrospinal fluid (CSF) samples from patients with NC, CSF and serum samples from a control group, and serum samples from patients with other parasitoses were studied by enzyme-linked immunosorbent assay (ELISA) and by immunoblotting with Taenia crassiceps vesicular fluid antigen (Tcra) and Taenia solium total saline antigen (Tso) for the detection of immunoglobulin G antibodies. ELISAs carried out with the Tso and Tcra antigens showed 94.1 and 95.6% sensitivities, respectively, for the detection of antibodies in CSF and 70.6% and 91.2% sensitivities, respectively, for the detection of antibodies in serum, with 100% specificity for the detection of antibodies in CSF and 80% specificity for the detection of antibodies in serum for both antigens. On the basis of the reactivities of the peptides in the samples analyzed, the peptides of

Analysis of cells in cerebrospinal fluid from patients with neurocysticercosis by means of flow cytometry.

The events of the cellular immune response in neurocysticercosis (NC) are not fully understood. Studies of the CD3, CD3/CD4, CD3/CD8, CD45/CD19, and CD45/CD56 molecules and activation-related CD69 molecule in cells from the cerebrospinal fluid (CSF) and peripheral blood (PB) of patients with NC may provide a better elucidation of the inflammatory and immunological events occurring in this disease. Seven patients with NC and 3 individuals with other disorders were evaluated by a three-color flow cytometric method. CD69 was detected in a higher percentage of cells in all CSF samples from patients, but not in PB or CSF from the control group. The percentage of CD3+ cells did not differ significantly in CSF and PB cells from patients and controls. The predominance of CD3+CD8+ cells was observed in CSF from one patient and in PB from 2 patients, who were in stage III of the disease (inflammatory process). The percentage of CD45+CD19+ cells was higher in CSF than in PB from patients who presented anti-cysticercus antibodies in CSF. The percentage of CD45+CD56+ cells in CSF was higher than in PB, but this rate was similar to reference values reported by other authors. Our data suggest that the cytometric method applied to a larger number of CSF samples may provide a better understanding of the cell-mediated immune response involved in NC.

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.

Human neurocysticercosis. IgE in cerebrospinal fluid.

The detection of IgE is technically difficult because of its reduced concentrations in serum, and even lower concentrations in cerebrospinal fluid (CSF). In the present investigation we studied 86 CSF samples using an immunoenzymatic method with an anti-IgE-alkaline phosphatase conjugate and a fluorigenic substrate. The samples were from three groups: A) 29 patients with neurocysticercosis (NC), B) 36 patients with different neurologic disorders (neurosyphilis, neurotuberculosis, meningitis, tumors, hemorrhage) and C) 21 discharged individuals who had been hospitalized for bacterial meningitis. The results obtained were: A) 0.05 to 3.00 IU/ml (0.76 +/- 0.79), B) 0.00 to 1.50 IU/ml (0.23 +/- 0.34) and C) 0.05 to 1.25 IU/ml (0.34 +/- 0.34). The present results suggest that IgE appears to play a role in the pathogeny of NC and that efforts should be made to standardize a test for the detection of specific IgE antibodies.

Hemagglutination test for the diagnosis of human neurocysticercosis: development of a stable reagent using homologous and heterologous antigens.

A hemagglutination (HA) test was standardized using formalin- and tannin-treated gander red blood cells sensitized with a total salt extract of C. cellulosae (HA-Cc) and an antigenic extract of Cysticercus longicollis (HA-Cl) vesicular fluid. A total of 61 cerebrospinal fluid (CSF) samples were assayed, 41 from patients with neurocysticercosis and 20 from a control group which were, respectively, reactive and non-reactive to ELISA using C. cellulosae. The CSF samples from the control group did not react and 35 (85.4%) and 34 (82.9%) CSF samples from patients were reactive to the HA-Cc and HA-Cl tests, respectively. The reagents ready for use were stable up to 6 months when stored at 4 degrees C in 50% glycerol. The present results confirm that the reagent using Cysticercus longicollis stabilized with glycerol can be used as an alternative in the immunological diagnosis of neurocysticercosis.