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Effects of metaperiodate and urea solutions on the serological diagnosis of human sporotrichosis using an indirect ELISA test.

ABSTRACT: Sporotrichosis is an infection of the skin caused by traumatic inoculation of the fungus Sporothrix schenckii. Definitive diagnosis relies on direct visualization of the fungus or its isolation on culture medium, although both have low sensitivity. Alternatively, the detection of the antibody response offers a more rapid alternative for diagnosis. Although the available immunoassays possess good sensitivity and specificity, cross-reactivity is still a problem. This study aimed to evaluate the effect of sodium metaperiodate and 6 M urea solutions on the serological diagnosis of sporotrichosis using an enzyme-linked immunosorbent assay (ELISA) test. Ninety-six-well plates were sensitized with exoantigens from the yeast phase of S. schenckii. Sera of patients with confirmed sporotrichosis, sera of patients with paracoccidioidomycosis, and sera of individuals with a sporotrichin-negative skin test were tested. Two strategies were used; the first consisted of treating the antigen with sodium metaperiodate solution for different incubation times, and the second consisted of treating the serum with 6 M urea solution for different incubation times. ROC curve analysis revealed that the best discrimination parameters were obtained using 6 M urea solution incubated for 5 min and serum dilution at 1/600. The use of 6 M urea solution improves the performance of the ELISA test in the diagnosis of sporotrichosis.

SUBMITTER: Coelho LML

PROVIDER: S-EPMC6863317 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Effects of metaperiodate and urea solutions on the serological diagnosis of human sporotrichosis using an indirect ELISA test.

Coelho Letícia Maria Leomil LML Grisolia Julianne Caravita JC Lúcia Maria M Boczar Míriam M Ferreira Eric Batista EB Nogueira Denismar Alves DA Chavasco Jorge Kleber JK de Camargo Zoilo Pires ZP Lopes-Bezerra Leila Maria Bezerra Leila Maria Lopes LML Coelho Luiz Felipe Leomil LFL Malaquias Luiz Cosme Cotta LCC

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20181213 1

Sporotrichosis is an infection of the skin caused by traumatic inoculation of the fungus Sporothrix schenckii. Definitive diagnosis relies on direct visualization of the fungus or its isolation on culture medium, although both have low sensitivity. Alternatively, the detection of the antibody response offers a more rapid alternative for diagnosis. Although the available immunoassays possess good sensitivity and specificity, cross-reactivity is still a problem. This study aimed to evaluate the ef ...[more]

PMID: 30637654

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Project description:BackgroundA novel porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for PDCoV antigen and antibody detection.ResultsThe nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize mice for polyclonal, hyperimmune sera and monoclonal antibody (mAb) production. The resulting mAbs were evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. The same antigen was used to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 629 known negative serum samples and 311 known positive samples were evaluated for each assay. The enzyme linked immunosorbent assay (ELISA) showed diagnostic sensitivity (DSe) of 96.1% and diagnostic specificity (DSp) of 96.2%. The fluorescent microsphere immunoassay (FMIA) showed a DSe of 95.8% and DSp of 98.1%. Both ELISA and FMIA detected seroconversion of challenged pigs between 8-14 days post-infection (DPI). An indirect fluorescent antibody (IFA) test was also developed using cell culture adapted PDCoV for comparative purposes.ConclusionThese new, specific reagents and serological assays will allow for improved diagnosis of PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.

Project description:The precision by which animal diseases are diagnosed affects our ability to make informed decisions with regards to animal health management, from a clinical and economic perspective. Lameness is a major health condition in dairy cattle. The underlying causes of lameness include bovine digital dermatitis (BDD), which is reported as one of the main causes of infectious lameness in dairy cattle. Presently, the gold standard for BDD diagnosis in dairy cattle is visual inspection of lifted hooves-a labour intensive and subjective method. Research has suggested that Treponema spp. are the main pathogens associated with the establishment of BDD. We explored the potential of indirect enzyme-linked immunosorbent assay (ELISA) as a diagnostic serological tool in the identification of cows at different stages of BDD. Additionally, we evaluated the predictive power of this diagnostic tool on the future occurrence of BDD lesions. A total of 232 cows from three farms were used in the study. Serum samples and hoof health data were collected at three time points: ~ 30 days pre-calving, around calving, and approximately 30 days post-calving. The mean absorbance from the ELISA test was compared across different clinical presentations of BDD as assessed by visual inspection of the hooves according to the M-stage classification system. A transition model was developed to estimate the probability of lesion occurrence in time t + 1 based on the spectrophotometer (absorbance) reading in time t. The mean absorbance reading for both IgG1 and IgG2 anti-Treponema antibodies was associated with disease presence-apart from M4.1 lesions, animals with no lesions had a lower mean when compared to animals with lesions regardless of the score. Additionally, the mean absorbance reading of animals with active lesions was higher when compared to animals with no lesions. However, the anti-Treponema antibody assays failed to identify disease presence in a consistent manner. Moreover, indirect ELISA readings were not a predictor of the future occurrence of BDD lesions. In conclusion, although the levels anti-Treponema antibodies were associated with disease presence, the ELISA test failed to detect disease unequivocally and had no predictive value in the future occurrence of BDD lesions.

Dataset Information

Effects of metaperiodate and urea solutions on the serological diagnosis of human sporotrichosis using an indirect ELISA test.

Publications

Effects of metaperiodate and urea solutions on the serological diagnosis of human sporotrichosis using an indirect ELISA test.

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