Evaluation of the enzyme-linked-immuno-electro-diffusion-assay (ELIEDA) for the diagnosis of Schistosoma mansoni infection with low worm burden

An immunoprecipitation technique, ELIEDA (enzyme-linked-immuno-electro-diffusion assay), was evaluated for the diagnosis of Schistosoma mansoni infection with low worm burden. One hundred of serum samples from patients excreting less than 600 eggs per gram of feces (epg), with unrelated diseases and clinically healthy subjects were studied. In patients with egg counts higher than 200 epg, the sensitivities of IgM and IgG ELIEDA were 1.000 and 0.923, respectively, not differing from other Serologic techniques, such as indirect hemaglutination (IHAT), immunofluorescence (IFT) tests and immuno-electrodiffusion assay (IEDA). However in patients with low egg counts (< 100 epg), the IgG ELIEDA provided better results (0.821) than IgM ELIEDA (0.679), showing sensitivity that did not differ from that of IgG IFT (0.929), but lower than that of IgM IFT (0.964). However, its sensivity was higher than that found with IHAT (0.607) and IEDA (0.536). The specificity of IgG ELIEDA was comparable to that of other techniques. The data indicate that IgG ELIEDA might be useful for the diagnosis of slight S. mansoni infections, and the cellulose acetate membrane strips can be stored for further retrospective studies.

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.

Plasma levels of tumor necrosis factor-alpha in patients with visceral leishmaniasis (Kala-Azar). Association with activity of the disease and clinical remisson following antimonial therapy

Evaluation of TNF-alpha in patients with Kala-azar has drawn increasing interest due to its regulatory role on the immune system, in addition to its cachetizing activity. The objective of this study was to examine the association between plasma levels of TNF-alpha, measured by immunore-activity (ELISA) and bioactivity (cytotoxicity assay with L-929 cells), and clinical manifestations of visceral leishmaniasis. Plasma samples from 19 patients with Kala-azar were obtained before, during and at the end of antimonial therapy. TNF-alpha determinations was done by using the cytotoxicity assay (all patients) and the enzyme-linked immunoassay (ELISA - 14 patients). A discrepancy between results obtained by ELISA and cytotoxicity assay was observed. Levels of circulating TNF-alpha, assessed by ELISA, were higher in patients than in healthy controls, and declined significantly with improvement in clinical and laboratory parameters. Plasma levels before treatment were 124.7 ± 93.3 pg/ml (mean ± SD) and were higher than at the end of therapy 13.9 ± 25.1 pg/ml (mean ± SD) (p = 0.001). In contrast, plasma levels of TNF-alpha evaluated by cytotoxicity assay did not follow a predicted course during follow-up. Lysis, in this case, might be not totally attributed to TNF-alpha. The discrepancy might be attributed to the presence of factor(s) known to influence the release and activity of TNF-alpha.

Presence of Circulating Levels of Interferon-g, Interleukin-10 and Tumor Necrosis Factor-a in Patients with Visceral Leishmaniasis

Experimental murine L. major infection is characterized by the expansion of distinct CD4+ T cell subsets. The Th1 response is related to production of IFN-g and resolution of infection, whereas Th-2 response with production of IL-4 and IL-10 and dissemination of infection. The objective of this study was to measure the circulating levels of IFN-g, IL-10 and TNF-a in patients with visceral leishmaniasis (VL) before, during and at the end of therapy and to examine the association between cytokine levels and activity of VL. Fifteen patients with VL were evaluated. The cytokine determinations were done by using the enzyme-linked immunoassay (ELISA) before, during and at the end of therapy. At baseline, we detected circulating levels of IFN-g in 13 of 15 patients (median = 60 pg/ml); IL-10 in 14 of 15 patients (median = 141.4 pg/ml); and TNF-a in 13 of 14 patients (median = 38.9 pg/ml). As patients improved, following antimonial therapy, circulating levels of IL-10 showed an exponential decay (y = 82.34 e–0,10367x, r = –0.659; p < 0.001). IFN-g was no longer detected after 7/14 days of therapy. On the other hand, circulating levels of TNF-a had a less pronounced decay with time on therapy, remaining detectable in most patients during the first seven days of therapy (y = 36.99-0.933x, r = –0.31; p = 0.05). Part of the expression of a successful response to therapy may, therefore, include reduction in secretion of inflammatory as well as suppressive cytokines. Since IL-10 and IFN-g are both detected prior to therapy, the recognized cellular immune depression seen in these patients may be due to biological predominance of IL-10 (type 2 cytokine), rather than lack of IFN-g (type 1 cytokine) production.

hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory

More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.

Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

Gold nanoparticle to antibody conjugates for diagnosis applications: molecular interactions and immunoassay development

Thesis for the Master degree in Structural and Functional Biochemistry

A disposable glass-based immunosensor for monitoring the cancer biomarker CEA in urine

NanoPT 2014 International Conference, in Portugal, on February 12-14. Poster presentation based on topic Nanobio/Nanomedicine

Measuring the cytochrome c nitrite reductase activity—practical considerations on the enzyme assays

Hindawi Publishing Corporation Bioinorganic Chemistry and Applications Volume 2010, Article ID 634597, 8 pages

Seroprevalence for hepatitis E virus (HEV) infection among volunteer blood donors of the Regional Blood Bank of Londrina, State of Paraná , Brazil

A cross-sectional study was carried out among 996 volunteer blood donors enrolled from May 1999 to December 1999 to determine the seroprevalence of hepatitis E virus (HEV) infection among volunteer blood donors of the Regional Blood Bank of Londrina, State of Paraná, Brazil, and to evaluate whether the rate of seroprevalence of IgG anti-HEV antibodies is associated with sociodemographic variables and with seropositivity for hepatitis A virus (HAV) infection. All participants answered the questionnaire regarding the sociodemographic characterisitcs. Serum samples were tested for IgG antibodies to HEV (anti-HEV) by an enzyme linked immunoassay (ELISA). All serum samples positive for anti-HEV IgG and 237 serum samples negative for anti-HEV were also assayed for IgG anti-HAV antibodies by ELISA. Anti-HEV IgG was confirmed in 23/996 samples, resulting in a seroprevalence of 2.3% for HEV infection, similar to previous results obtained in developed countries. No significant association was found between the presence of anti-HEV IgG antibodies and the sociodemographic variables including gender, age, educational level, rural or urban areas, source of water, and sewer system (p > 0.05). Also, no association with seropositivity for anti-HAV IgG antibodies was observed (p > 0.05). Although this study revealed a low seroprevalence of HEV infection in the population evaluated, the results showed that this virus is circulating among the population from Londrina, South Brazil, and point out the need of further studies to define the clinical and epidemiological importance of HEV infection and to identify additional risk factors involved in the epidemiology and pathogenesis of this infection in this population.

A potentiometric magnetic immunoassay for rapid detection of Salmonella typhimurium

Potentiometric detection with homemade polymeric membrane microelectrodes was coupled to a magnetic sandwich immunoassay for Salmonella typhimurium determination. Cadmium and sodium ion selective electrodes were used respectively as indicator and pseudo-reference electrodes and were prepared in pipette tips to allow potentiometric measurements in microliter sample volumes. In the proposed method, the concentration of S. typhimurium was proportional to the amount of cadmium released upon dissolution of a CdS nanoparticle labeled to the secondary detection antibody. The limit of detection was 2 cells per 100 μL. The immunomagnetic assay with potentiometric detection is suitable for sensitive and rapid (average total time per assay of 75 minutes) detection of S. typhimurium in milk samples. The proposed method is easy to perform, safe, sensitive, and low cost and has potential for in situ analysis.

Prevalence of hepatitis C virus infection in quilombo remnant communities in Central Brazil

In order to determine the prevalence of hepatitis C virus (HCV) infection in quilombo remnant communities in Central Brazil, 1,007 subjects were interviewed in all 12 communities existing in Mato Grosso do Sul State, Central Brazil. Blood samples were collected and sera were tested for anti-HCV by enzyme-linked immunosorbent assay. Positive samples were retested for confirmation using a line immunoassay and were also subjected to HCV RNA detection. The prevalence of HCV infection was 0.2%. This finding shows a low prevalence of HCV infection in quilombo remnant communities in Central Brazil.

High occurrence of Entamoeba histolytica in the municipalities of Ariquemes and Monte Negro, State of Rondônia, Western Amazonia, Brazil

Introduction: Entamoeba histolytica infections were investigated in residents of the Ariquemes and Monte Negro municipalities in Rondônia State, Brazil. Methods: Stool samples of 216 individuals were processed by the spontaneous sedimentation method and analyzed by microscopy for detection of the E. histolytica/E. dispar complex, followed by the immunoassay method using an enzyme-linked immunosorbent assay-based kit for the E. histolytica stool antigen. Results: E. histolytica/E. dispar cysts were present in 61% (50/82) and 44% (59/134) of the samples from Ariquemes and Monte Negro respectively, with a significant difference in the occurrence of infection between the two populations [p < 0.05; χ2 = 5.2; odds ratio = 2.0 (1.1 - 3.6)]. The E. histolytica antigen detection rate was 36.6% (30/82) for stool samples from Ariquemes, and 19.4% (26/134) for stool taken from the residents of Monte Negro. The rate of the occurrence of amoebiasis was significantly higher in the population from Ariquemes [p < 0.05; χ2 = 7.8; odds ratio = 2.4 (1.2 - 4.7)]. Discussion: Due to the high occurrence of E. histolytica infected residents diagnosed in the region and the unavailability in local clinics of a test to distinguish between the two Entamoeba species, physicians should consider treating E. histolytica/E.dispar infections. Conclusion: The results indicate that E. histolytica infection is highly endemic in the studied areas.