Chagas' disease: an algorithm for donor screening and positive donor counseling

Classical serological screening assays for Chagas' disease are time consuming and subjective. The objective of the present work is to evaluate the enzyme immuno-assay (ELISA) methodology and to propose an algorithm for blood banks to be applied to Chagas' disease. Seven thousand, nine hundred and ninety nine blood donor samples were screened by both reverse passive hemagglutination (RPHA) and indirect immunofluorescence assay (IFA). Samples reactive on RPHA and/or IFA were submitted to supplementary RPHA, IFA and complement fixation (CFA) tests. This strategy allowed us to create a panel of 60 samples to evaluate the ELISA methodology from 3 different manufacturers. The sensitivity of the screening by IFA and the 3 different ELISA's was 100%. The specificity was better on ELISA methodology. For Chagas disease, ELISA seems to be the best test for blood donor screening, because it showed high sensitivity and specificity, it is not subjective and can be automated. Therefore, it was possible to propose an algorithm to screen samples and confirm donor results at the blood bank.

Comparative study of lymphocytes from individuals that were vaccinated and unvaccinated against the pandemic 2009-2011 H1N1 influenza virus in Southern Brazil

ABSTRACTINTRODUCTION:While no single factor is sufficient to guarantee the success of influenza vaccine programs, knowledge of the levels of immunity in local populations is critical. Here, we analyzed influenza immunity in a population from Southern Brazil, a region with weather conditions that are distinct from those in the rest of country, where influenza infections are endemic, and where greater than 50% of the population is vaccinated annually.METHODS:Peripheral blood mononuclear cells were isolated from 40 individuals. Of these, 20 had received the H1N1 vaccine, while the remaining 20 were unvaccinated against the disease. Cells were stimulated in vitro with the trivalent post-pandemic influenza vaccine or with conserved major histocompatibility complex I (MHC I) peptides derived from hemagglutinin and neuraminidase. Cell viability was then analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)]-based colorimetric assay (MTT), and culture supernatants were assayed for helper T type 1 (Th1) and Th2-specific cytokine levels.RESULTS:Peripheral blood lymphocytes from vaccinated, but not unvaccinated, individuals exhibited significant proliferation in vitro in the presence of a cognate influenza antigen. After culturing with vaccine antigens, cells from vaccinated individuals produced similar levels of interleukin (IL)-10 and interferon (IFN)-γ, while those from unvaccinated individuals produced higher levels of IFN-γ than of IL-10.CONCLUSIONS:Our data indicate that peripheral blood lymphocytes from vaccinated individuals are stimulated upon encountering a cognate antigen, but did not support the hypothesis that cross-reactive responses related to previous infections can ameliorate the immune response. Moreover, monitoring IL-10 production in vaccinated individuals could comprise a valuable tool for predicting disease evolution.

Human schistosomiasis mansoni: studies on in vitro granuloma modulation

Infection with Schistosoma mansoni induces humoral and T cell mediated responses and leads to delayed hipersensitivity that results in granulomatous inflamatory disease around the parasite eggs. Regulation of these responses resulting in a reduction in this anti-egg inflamatory disease is appsrently determined by idiotypic repertoires of the patient, associated with genetic background and multiple external factors. We have previously reported on idiotype/anti-idiotype-receptor transactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive idiotypes develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We repport here on experiments wich extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA idiotypes and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA idiotype reactive T cells were capable of regulating autologous in vitro granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the present of intact (F(ab')2 immunoglobulin molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA sensitized individuals. These data support the hypothesis that anti-SEA cross reactive idiotypes are important in regulating granulomatous hypersensitivy in chronic intestinal schistosomiasis patients and these cross-reactive idiotypes appear to play a major role in cell-cell interactions which result in the regulation of anti-SEA cellular immune responses.

ESTUDO DA GLICOSILAÇÃO DO CICLO-HEXANOL COM DIFERENTES DERIVADOS DE D-GLICOSAMINA E PROMOTORES DE GLICOSILAÇÃO PELO MÉTODO DE KOENIGS-KNORR

We report herein a study on the glycosylation of cyclohexanol with four D-glucosamine-based peracetylated glycosyl chlorides bearing different substituents at C-2 and three glycosylation promoters, silver carbonate, silver triflate and mercury II chloride/mercury II oxide, by the Koenigs-Knorr method. Under the conditions studied, glycosylation was successful only when 3,4,6-tri-O -acetyl-2-deoxy-2-phthalimido-α-D-glucopyranosyl chloride was used as the glycosyl donor, with silver carbonate proving the best promoter. In order to investigate the influence of the nature of the halogen at C-1, we also carried out the glycosylation of cyclohexanol with 3,4,6-tri-O -acetyl-2-deoxy-2-phthalimido-α-D-glucopyranosyl bromide, a more reactive glycosyl donor. As expected, the yield with the bromide derivative was higher with the three promoters and, again, silver carbonate was the most efficient promoter. Finally, to illustrate the well-known efficient procedure for conversion of the phtalimido group at C-2 to the corresponding acetamido group, cyclohexyl 3,4,6-tri-O -acetyl-2-deoxy-2-phtalimido-β-D-glucopyranoside was converted into cyclohexyl 2-deoxy-2-acetamido-β-D-glucopyranoside in two steps, namely, hydrazinolysis of the phtalimido group followed by chemoselective acetylation of the free amino group by treatment with acetic anhydride in methanol, at 77% overall yield.

Variability of the 3' terminal of the polymerase gene of Grapevine leafroll-associated virus 3 isolates from Vale do São Francisco, Brazil

Many viral diseases, including leafroll, which is of great economic importance, affect grapevines (Vitis spp.). A complex of eight viruses [Grapevine leafroll-associated virus (GLRaV) -1 to 8] is associated with this disease. The objective of this study was to compare the variability of the 3' terminal region of the polymerase gene of three isolates of GLRaV-3 (Grapevine leafroll-associated virus-3), from Submédio do Vale do Rio São Francisco (Petrolina-PE) with that of other isolates available at the GenBank, including an isolate from North America and another from Southern Brazil. The viral RNA was extracted from three infected ELISA reactive plants and a fragment of 340 bp was amplified, by RT-PCR, using primers that recognize that portion of the polymerase gene found between nucleotides 8267 and 8606. The three isolates from Vale do Rio São Francisco named Pet-1, Pet-2 and Pet-3, showed similarities ranging from 98% and 94%, respectively to the isolates from North America (AF037268) and Southern Brazilian (AF438411). Considering the whole genome, the main variation found was one amino acid change at position 2766 (F2766Y). These preliminary data indicate the existence of a natural variation among GLRaV-3 isolates from grapevines. This could be due to the vegetative propagation and long cycle of the plant, associated with the error-prone nature of RNA-dependent RNA polymerase.

Security issues during Lula's administration: from the reactive to the assertive approach

Brazil's security agenda during Lula's administration was not homogeneous through the two mandates: the first tenure (2002-2006) revealed a reactive approach towards security topics, while the second one (2006-2010) was more assertive. More specifically, the shift occurred in terms of both its geographical scope - once it incorporated global issues in a more systematic way -, and instruments through which the security agenda was exercised, given the multilateral initiative of Unasur's CDS

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

The diagnostic importance of species specific and cross-reactive components of Taenia solium, Echinococcus granulosus, and Hymenolepis nana

Sera from patients infected with Taenia solium, Hymenolepis nana and Echinococcus granulosus were tested against homologous and heterologous parasite antigens using an ELISA assay, and a high degree of cross-reactivity was verified. To identify polypeptides responsible for this cross reactivity, the Enzyme Linked Immunoelectro Transfer Blot (EITB) was used. Sera from infected patients with T.solium, H.nana, and E.granulosus were assessed against crude, ammonium sulphate precipitated (TSASP), and lentil-lectin purified antigens of T.solium and crude antigens of.H.nana and E.granulosus. Several bands, recognized by sera from patients with T.solium, H.nana, and E.granulosus infections, were common to either two or all three cestodes. Unique reactive bands in H.nana were noted at 49 and 66 K-Da and in E.granulosus at 17-21 K-Da and at 27-32 K-Da. In the crude cysticercosis extract, a specific non glycoprotein band was present at 61-67 K-Da in addiction to specific glycoprotein bands of 50, 42, 24, 21, 18, 14, and 13 K-Da. None of the sera from patients with H.nana or E.granulosus infection cross reacted with these seven glycoprotein bands considered specific for T.solium infection.

ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

Correlation between turbidimetric and nephelometric methods of measuring C-reactive protein in patients with unstable angina or non-ST elevation acute myocardial infarction

OBJECTIVE: To evaluate the performance of the turbidimetric method of C-reactive protein (CRP) as a measure of low-grade inflammation in patients admitted with non-ST elevation acute coronary syndromes (ACS). METHODS: Serum samples obtained at hospital arrival from 68 patients (66±11 years, 40 men), admitted with unstable angina or non-ST elevation acute myocardial infarction were used to measure CRP by the methods of nephelometry and turbidimetry. RESULTS: The medians of C-reactive protein by the turbidimetric and nephelometric methods were 0.5 mg/dL and 0.47 mg/dL, respectively. A strong linear association existed between the 2 methods, according to the regression coefficient (b=0.75; 95% C.I.=0.70-0.80) and correlation coefficient (r=0.96; P<0.001). The mean difference between the nephelometric and turbidimetric CRP was 0.02 ± 0.91 mg/dL, and 100% agreement between the methods in the detection of high CRP was observed. CONCLUSION: In patients with non-ST elevation ACS, CRP values obtained by turbidimetry show a strong linear association with the method of nephelometry and perfect agreement in the detection of high CRP.

High-Sensitivity C-Reactive Protein as a Predictor of Cardiovascular Events after ST-Elevation Myocardial Infarction

Background: The association between high-sensitivity C-reactive protein and recurrent major adverse cardiovascular events (MACE) in patients with ST-elevation myocardial infarction who undergo primary percutaneous coronary intervention remains controversial. Objective: To investigate the potential association between high-sensitivity C-reactive protein and an increased risk of MACE such as death, heart failure, reinfarction, and new revascularization in patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention. Methods: This prospective cohort study included 300 individuals aged >18 years who were diagnosed with ST-elevation myocardial infarction and underwent primary percutaneous coronary intervention at a tertiary health center. An instrument evaluating clinical variables and the Thrombolysis in Myocardial Infarction (TIMI) and Global Registry of Acute Coronary Events (GRACE) risk scores was used. High-sensitivity C-reactive protein was determined by nephelometry. The patients were followed-up during hospitalization and up to 30 days after infarction for the occurrence of MACE. Student's t, Mann-Whitney, chi-square, and logistic regression tests were used for statistical analyses. P values of ≤0.05 were considered statistically significant. Results: The mean age was 59.76 years, and 69.3% of patients were male. No statistically significant association was observed between high-sensitivity C-reactive protein and recurrent MACE (p = 0.11). However, high-sensitivity C-reactive protein was independently associated with 30-day mortality when adjusted for TIMI [odds ratio (OR), 1.27; 95% confidence interval (CI), 1.07-1.51; p = 0.005] and GRACE (OR, 1.26; 95% CI, 1.06-1.49; p = 0.007) risk scores. Conclusion: Although high-sensitivity C-reactive protein was not predictive of combined major cardiovascular events within 30 days after ST-elevation myocardial infarction in patients who underwent primary angioplasty and stent implantation, it was an independent predictor of 30-day mortality.

Correlation between C-Reactive Protein in Peripheral Vein and Coronary Sinus in Stable and Unstable Angina

Background: High sensitivity C-reactive protein (hs-CRP) is commonly used in clinical practice to assess cardiovascular risk. However, a correlation has not yet been established between the absolute levels of peripheral and central hs-CRP. Objective: To assess the correlation between serum hs-CRP levels (mg/L) in a peripheral vein in the left forearm (LFPV) with those in the coronary sinus (CS) of patients with coronary artery disease (CAD) and a diagnosis of stable angina (SA) or unstable angina (UA). Methods: This observational, descriptive, and cross-sectional study was conducted at the Instituto do Coração, Hospital das Clinicas, Faculdade de Medicina, Universidade de São Paulo, and at the Hospital Beneficência Portuguesa de Sao Paulo, where CAD patients referred to the hospital for coronary angiography were evaluated. Results: Forty patients with CAD (20 with SA and 20 with UA) were included in the study. Blood samples from LFPV and CS were collected before coronary angiography. Furthermore, analysis of the correlation between serum levels of hs-CRP in LFPV versus CS showed a strong linear correlation for both SA (r = 0.993, p < 0.001) and UA (r = 0.976, p < 0.001) and for the entire sample (r = 0.985, p < 0.001). Conclusion: Our data suggest a strong linear correlation between hs-CRP levels in LFPV versus CS in patients with SA and UA.

Sex Differences in High Sensitivity C-Reactive Protein in Subjects with Risk Factors of Metabolic Syndrome

Abstract Background: Metabolic syndrome (MetS) is associated with a higher risk of all-cause mortality. High-sensitivity C-reactive protein (hsCRP) is a prototypic marker of inflammation usually increased in MetS. Women with MetS-related diseases present higher hsCRP levels than men with MetS-related diseases, suggesting sex differences in inflammatory markers. However, it is unclear whether serum hsCRP levels are already increased in men and/or women with MetS risk factors and without overt diseases or under pharmacological treatment. Objective: To determine the impact of the number of MetS risk factors on serum hsCRP levels in women and men. Methods One hundred and eighteen subjects (70 men and 48 women; 36 ± 1 years) were divided into four groups according to the number of MetS risk factors: healthy group (CT; no risk factors), MetS ≤ 2, MetS = 3, and MetS ≥ 4. Blood was drawn after 12 hours of fasting for measurement of biochemical variables and hsCRP levels, which were determined by immunoturbidimetric assay. Results: The groups with MetS risk factors presented higher serum hsCRP levels when compared with the CT group (p < 0.02). There were no differences in hsCRP levels among groups with MetS risk factors (p > 0.05). The best linear regression model to explain the association between MetS risk factors and hsCRP levels included waist circumference and HDL cholesterol (r = 0.40, p < 0.01). Women with MetS risk factors presented higher hsCRP levels when compared with men (psex < 0.01). Conclusions: Despite the absence of overt diseases and pharmacological treatment, subjects with MetS risk factors already presented increased hsCRP levels, which were significantly higher in women than men at similar conditions.

Polypeptides reactive with antibodies eluted from the surface of Babesia bovis-infected erythrocytes

A technique was sought that would enable identification of surface-exposed parasite antigens on Babesia bovis-infected erythrocytes (BbIE) that are not detectable by surface-specific immunoprecipitations. Antibodies which bind to the surface of BbIE were recovered from intact cells using a low pH wash procedure. The eluted antibodies were then used in conventional immunoprecipitation assays to identify parasite-synthesized polypeptides carrying epitopes that are exposed on the surface or are cross reactive with shuch epitopes. The results of these experiments support our previous data, obtained using a surface-specific immunoprecipitation technique, in the identification of a repertoire of parasite-derived antigens on the surface of infected erythrocytes (Allred et al., 1991). In addition, two polypeptides of Mr 68,000 and 185,000 were identified wich react strongly with the eluted antibodies but wich are not detected by surface-immunoprecipitation. These data illustrate the potential of this approach for identification of parasite polypeptides wich carry epitopes exposed on, or cross-reactive with exposed epitopes of the infected erythrocyte surface.